Fig. 2: Regulation of dendritic spine pruning but not formation by Drd2.

a The strategies to generate SR-Drd2 (control) and SR-Drd2^+/− rats. The SR-Drd2 rats were generated by crossing Drd2-Cre rats with Ai14 (Rosa26-LSL-tdTomato) rats. The SR-Drd2^+/− rats were generated by crossing SR-Drd2 rats with Drd2^+/− rats. b The genotypes of different lines of rats. c Decreased DRD2 protein levels in the ACC of SR-Drd2^+/− rats. The homogenates of ACC from SR-Drd2 and SR-Drd2^+/− rats were subjected to western blot and probed with anti-DRD2 and anti-GAPDH antibodies (top). Quantification of DRD2/GAPDH (bottom). **P = 0.0022, two-sided t-test, n = 3 for each group, data were normalized to controls. Data are presented as mean values ± SEM. d Unchanged DRD1 protein levels in the ACC of SR-Drd2^+/− rats. The homogenates of ACC from control and SR-Drd2^+/− rats were subjected to western blot and probed with anti-DRD1 and anti-GAPDH antibodies (top). Quantification of DRD1/GAPDH (bottom). NS not significant, P = 0.4812, two-sided t-test, n = 4 for each group, data were normalized to controls. Data are presented as mean values ± SEM. e Schematic diagram showing the dendrites of Drd2-positive neurons filled with biocytin in layer 5 of ACC. We analyzed the spines of the secondary basal dendrites. f Representative dendritic spines of Drd2-positive neurons from different aged control and SR-Drd2^+/− rats. Scale bar, 5 μm. g Deficient synaptic pruning but not synapse formation of Drd2-positive neurons in SR-Drd2^+/− rats. The spine densities of Drd2-positive neurons in different aged control and SR-Drd2^+/− rats were quantified. NS not significant, P (2w) = 0.9718, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 32 dendrites from 5 control rats, n = 37 dendrites from 5 SR-Drd2^+/− rats. P (3w) = 0.9831, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 19 dendrites from 4 control rats, n = 26 dendrites from 5 SR-Drd2^+/− rats. P (4w) = 0.6744, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 13 dendrites from 3 control rats, n = 22 dendrites from 4 SR-Drd2^+/− rats. *P = 0.0133, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 12 dendrites from 3 control rats, n = 23 dendrites from 4 SR-Drd2^+/− rats. ***P < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 28 dendrites from 5 control rats, n = 24 dendrites from 4 SR-Drd2^+/− rats. Data are presented as mean values ± SEM. h Line chart of the data in panel g. The statistical results are same as panel g. *P = 0.0133, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 12 dendrites from 3 control rats, n = 23 dendrites from 4 SR-Drd2^+/− rats. ***P < 0.0001, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 28 dendrites from 5 control rats, n = 24 dendrites from 4 SR-Drd2^+/− rats. Data are presented as mean values ± SEM. i Increased densities of mushroom-like and stubby spines in Drd2-positive neurons from SR-Drd2^+/− rats, compared with control rats. The densities of different types of dendritic spines in Drd2-positive neurons from 8-week-old control and SR-Drd2^+/− rats were quantified. NS not significant, P = 0.5398, **P = 0.0068, ***P = 0.0008, two-way ANOVA followed by Sidak’s multiple comparisons test, n = 28 dendrites from 5 control rats, n = 24 dendrites from 4 SR-Drd2^+/− rats. Data are presented as mean values ± SEM.