Fig. 4: Activation of mTOR signaling by Drd2^+/− in synaptic pruning.

a DRD2 recruits β-arrestin/PP2A complex to inhibit AKT-mTOR-S6 signaling pathway. b Increased AKT-mTOR-S6 signaling in the deep layers of ACC from 3–4-week-old SR-Drd2^+/− rats, compared with control rats. The homogenates from deep layers of ACC were subjected to western blot and probed with the indicated antibodies. c Quantification of p-AKT/AKT in panel b. ***P = 0.0009, two-sided t-test, n = 5 for each group, data were normalized to controls. d Quantification of p-mTOR/mTOR in panel b. *P = 0.0249, two-sided t-test, n = 5 for each group, data were normalized to controls. e Quantification of p-S6/S6 in panel b. *P = 0.0291, two-sided t-test, n = 5 for each group, data were normalized to controls. f Quantification of AKT/GAPDH in panel b. NS not significant, P = 0.1459, two-sided t-test, n = 5 for each group, data were normalized to controls. g Quantification of mTOR/GAPDH in panel b. NS not significant, P = 0.1793, two-sided t-test, n = 5 for each group, data were normalized to controls. h Quantification of S6/GAPDH in panel b. NS not significant, P = 0.2099, two-sided t-test, n = 5 for each group, data were normalized to controls. Data are presented as mean values ± SEM. i Experimental design. Control and SR-Drd2^+/− rats received daily injection of rapamycin (1 μM in 0.5 μl per side) or Veh (0.5 μl saline per side) into layer 5 of ACC between 3- and 4-week-old age, and the spine densities and mEPSC of Drd2-positive neurons were analyzed at 8-week-old age. j Diagram showing the injection of Veh or Rap into layer 5 of ACC in control and SR-Drd2^+/− rat. The dashed line of the sagittal section diagram indicates the position of the coronal section. The rectangle indicates the brain regions of ACC. k Nissl staining to determine the injection sites. Scale bar, 1 mm. l Rapamycin rescued the increased spine densities of Drd2-positive neurons from SR-Drd2^+/− rat. Shown were the representative spines of Drd2-positive neurons from different groups of mice. m Quantification of spine densities from Drd2-positive neurons in panel l. **P (SR-Drd2 + Veh vs SR-Drd2^+/− + Veh) = 0.0013, **P (SR-Drd2^+/− + Veh vs SR-Drd2^+/− + Rap) = 0.0019, mixed-effects analysis followed by Sidak’s multiple comparisons test. n = 18 dendrites from 3 SR-Drd2 rats treated with Veh, n = 17 dendrites from 3 SR-Drd2^+/− rats treated with Veh, n = 17 dendrites from 3 SR-Drd2 rats treated with Rap, n = 19 dendrites from 3 SR-Drd2^+/− rats treated with Rap. Data are presented as mean values ± SEM. n Quantification of densities of different types of spines from Drd2-positive neurons in panel l. *P (mushroom) = 0.0211, **P (mushroom) = 0.0041, *P (stubby, SR-Drd2 + Veh vs SR-Drd2^+/− + Veh) = 0.0421, *P (stubby, SR-Drd2^+/− + Veh vs SR-Drd2^+/− + Rap) = 0.0435, two-way ANOVA followed by Turkey’s multiple comparisons test. n = 18 dendrites from 3 SR-Drd2 rats treated with Veh, n = 17 dendrites from 3 SR-Drd2^+/− rats treated with Veh, n = 17 dendrites from 3 SR-Drd2 rats treated with Rap, n = 19 dendrites from 3 SR-Drd2^+/− rats treated with Rap. Data are presented as mean values ± SEM. o Rapamycin rescued the increased mEPSC frequency of Drd2-positive neurons from SR-Drd2^+/− rat. Shown were the representative mEPSC traces of Drd2-positive neurons from different groups of rats. p Quantification of mEPSC frequency of Drd2-positive neurons in panel o. ***P < 0.0001, two-way ANOVA followed by Turkey’s multiple comparisons test. n = 22 neurons from 4 SR-Drd2 rats treated with Veh, n = 23 neurons from 4 SR-Drd2^+/− rats treated with Veh, n = 18 neurons from 4 SR-Drd2 rats treated with Rap, n = 21 neurons from 4 SR-Drd2^+/− rats treated with Rap. Data are presented as mean values ± SEM. q Cumulative plots of mEPSC frequency. r Rapamycin did not affect mEPSC amplitude of Drd2-positive neurons in control and SR-Drd2^+/− rats. Shown were the quantification of mEPSC frequency of Drd2-positive neurons in panel o. NS not significant, P = 0.8442, SR-Drd2 + Veh vs SR-Drd2^+/− + Veh, P = 0.6968, SR-Drd2^+/− + Veh vs SR-Drd2^+/− + Rap, two-way ANOVA followed by Turkey’s multiple comparisons test. n = 22 neurons from 4 SR-Drd2 rats treated with Veh, n = 23 neurons from 4 SR-Drd2^+/− rats treated with Veh, n = 18 neurons from 4 SR-Drd2 rats treated with Rap, n = 21 neurons from 4 SR-Drd2^+/− rats treated with Rap. Data are presented as mean values ± SEM. s Cumulative plots of mEPSC amplitude.