Reduction of motor learning–induced spine formation after multiple exposures to ketamine–xylazine (KX) at P14 to P18. (A) Experimental design. (B) In vivo time-lapse imaging of the same dendritic segments over 2 days (pre- and posttraining) in the primary motor cortex of 1-month-old animals that received saline or KX injections at various ages. Filled and empty arrowheads indicate dendritic spines that were formed and eliminated between the two views. Asterisks indicate dendritic filopodia. Scale bar, 2 μm. (C) Percentage of dendritic spines formed over 2 days in saline- and KX-treated mice. All groups showed significant increase in spine formation after rotarod training. Motor learning–induced increase in spine formation was significantly lower in mice that received three injections of KX at P14 to P18 as compared with saline-injected mice. (D) Percentage of spines eliminated over 2 days in saline- and KX-treated mice. There was no significant difference between saline- and KX-treated groups. (E) The total number of spines increased over 2-day motor training in all groups. Motor learning–induced increase in total spine number was significantly reduced in mice with three KX injections during P14 to P18 as compared with saline-injected mice. Comparisons of means of KX-treated group relative to saline-treated group were carried out using two-tailed unpaired Student t tests. Data are presented as mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001.