Dendritic Spine Plasticity Analysis

Plasticity of dendritic spines was analyzed as described before [26 (link),27 (link),28 (link)]. In total, 7–10 dendrites per animal with a length of ~15–60 µm were obtained for analysis. Spines of all shapes with a length of >0.4 µm were counted manually using Zen 2011 Black Edition (Zeiss, Oberkochen, Germany). Due to resolution limitations, spines emerging laterally from the shaft (xy-plane) but not from the z-plane were taken into consideration. Spines that did not change their position (<0.5 µm) on the dendritic segment between two imaging time points (one week) were counted as stable. Spines were counted as lost when their length was less than 0.4 µm and gained when their length was greater than 0.4 µm. In current study, mice were imaged once per week; therefore, we counted spines as transient when they appeared at one imaging time point (>0.4 µm length) but were lost at the next time point (<0.4 µm length) one week later, as already used previously [17 (link)]. The spine turnover rate was calculated as follows: (N_gained + N_lost)/(2 × N_total)/I_T. N_gained represents the number of newly gained spines at one time point, N_lost is the number of depleted spines, N_total is the number of all spines at one time point, and I_T is the days between two imaging sessions [26 (link),28 (link)].