Fig. 1. Motor learning induces branch-specific spine formation.

(A) Transcranial two-photon imaging in the primary motor cortex of awake, head-restrained mice before and after rotarod motor training. (B and C) The percentage of dendritic spines formed (B) and eliminated (C) over time after one session of rotarod training (20 trials). Motor training progressively increased new spine formation over the course of 6 to 48 hours. No significant difference in the rate of spine elimination was observed within 48 hours after training. The number of animals is indicated on each column. (D) An example of two sibling apical tuft branches with different degrees of spine formation 24 hours after training. Filled arrowheads indicate newly formed dendritic spines and open ones indicate eliminated spines over a 24-hour interval. Asterisks indicate dendritic filopodia. (E) Motor training–induced spine formation was significantly different between sibling branches (15 trained mice and 8 control mice). (F) No significant difference in spine elimination between sibling branches. (G) Classification of sibling dendritic branches to HFBs and LFBs on the basis of the spine formation rate relative to each other. (H) Motor training significantly increased the rate of spine formation on HFBs 24 hours after training. (I) The average of measured difference in spine formation between HFBs and LFBs was statistically larger (P < 0.0001) for sibling branches (red circle) than for randomly paired branches (box plot of results from 100 simulations of random pairing). The simulation was performed to test the null hypothesis that learning-induced spine changes are distributed randomly across all branches. (J) There was no significant difference in spine elimination between HFBs and LFBs 24 hours after training. (K and L) Mice were first trained to run forward on an accelerating rotarod and, 12 hours later, to run either forward (F-F) or backward (F-B). Correlation of spine formation rate on individual branches between 0–12 hours and 12–24 hours. The correlation was positive when animals were subjected to the same forward training [(K) n = 6 mice] and negative when the animals were trained with a backward running task [(L) n = 8 mice]. (M) Experimental designs are shown in (K) and (L). Sibling branches were classified as HFBs and LFBs on the basis of the degree of spine formation induced by the initial forward training from 0 to 12 hours. There is a significant increase in spine formation on LFBs than on HFBs after backward training, not after forward running or no training, from 12 to 24 hours. Data are presented as means ± SEM. *P < 0.05. **P < 0.01. ****P < 0.0001, nonparametric test.