Figure 2. USV locomotor activity during spontaneous morphine withdrawal on P7 and P14 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). A. P7 USV Distance: The effect of Morphine Treatment on USV distance was dependent on Time (β = −0.020, SE = 0.0026, t(1488) = −7.67, ****p < 0.0001), where morphine-withdrawn pups traveled a greater distance than saline-control pups from 1 – 5 min of the recording session (all **p ≤ 0.0062). B. P7 Total USV Distance: There were no interactions (all p ≥ 0.57). Morphine Treatment was associated with a greater distance traveled during USV recordings associated with spontaneous opioid withdrawal (β = 0.68, SE = 0.14, t(165) = 4.98, ****p < 0.0001). C. P7 Average USV Velocity: There were no interactions (all p ≥ 0.54). There was no effect of Morphine Treatment on average velocity (β = −0.00097, SE = 0.0025, t(167) = −0.39, p = 0.70). SAL, n = 78 (44F, 34M); MOR, n = 82 (43F, 39M). D. P14 USV Distance: There was no Morphine Treatment x Time interaction on USV distance (β = 0.005, SE = 0.014, t(1981) = 0.37, p = 0.71). E. P14 Total USV Distance: There were no interactions (all p ≥ 0.078). Overall, Morphine Treatment was associated with a greater distance traveled during USV recordings associated with spontaneous opioid withdrawal (β = 4.48, SE = 1.69, t(134) = 2.65, **p = 0.0091). F. P14 Average USV Velocity: There were no interactions (all p ≥ 0.63). Morphine Treatment was associated with increased average velocity (β = 0.0083, SE = 0.0025, t(133) = 3.39, ***p = 0.00092). SAL, n = 67 (37F, 30M)

MOR, n = 62 (29F, 33M)