Fig. 3

Increasing KCC2 induces a sustained increase in spine density and a more rapid rate of spine formation during rotarod learning. a Schematic diagram of experimental protocol. Imaging and rotarod training began 3 weeks after withdrawing Dox from the diet. The same dendrites were imaged over 5 consecutive days. From days 1 to 3, rotarod training was performed in the morning prior to imaging later that day. b, c Representative single dendrites of pyramid neurons from WT (b) and KCC2-overexpressing (c) mice during repeated in vivo imaging before and during rotarod training. Red arrowheads indicate spines that newly appeared compared with the previous day’s image (newly formed spines), while blue arrowheads indicate those that disappeared (eliminated spines). Scale bars in each image in b and c, 5 μm. d Spine density measured at each imaging day was consistently higher in KCC2-overexpressing mice compared with that in WT mice. e The relative number of new spines that formed during the 1st day of rotarod learning was significantly greater in KCC2-overexpressing mice, but not in WT mice. However, subsequent new spine formation rates were not different. The graph plots the percentage of spines in a dendrite that was not present at the previous day’s imaging (7 KCC2 over-expressing mice, 23 dendrites; 7 WT mice, 18 dendrites; one-way ANOVA followed by Tukey’s test, 0 day vs. 1 day KCC2 mice, *p < 0.05). f The proportion of spines that disappeared (spine elimination) across the rotarod training period was not significantly different between WT and KCC2-overexpressing mice. g The spine turnover rate (numerical addition of formation and elimination) was significantly increased following 1 day of rotarod training for KCC2-overexpressing mice (one-way ANOVA, Tukey’s test, **p < 0.01), but not for WT mice—nor was it significantly different for WT or KCC2 mice at 2 and 3 days