Fig. 3: Rasd2 overexpression in the NAcc attenuates depression-like behavior and activates the DRD2-cAMP-PKA-DARPP-32 signaling pathway in mice.

a Schematic of experimental design and timeline. b Schematic diagram of microinjection of lentivirus vectors into mouse NAcc. Duration of immobility (c), climbing (d), and swimming (e), and the number of fecal boli (defecation) (f) in the forced swim test. N = 10, 11 mice in ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: duration of immobility, t₍₁₉₎ = 2.100, p = 0.0493; duration of climbing, t₍₁₉₎ = 2.130, p = 0.0465; duration of swimming, t₍₁₉₎ = 3.216, p = 0.0046; defecation, t₍₁₉₎ = 2.590, p = 0.0180. Representative tracks (g) and heatmaps (h) displaying mouse movement in the open field test. Total distance moves (i), line crosses (j), rearing (k), and center entries (l) in the open field test. N = 10, 11 mice for ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: distance, t₍₁₉₎ = 0.8095, p = 0.4282; line crosses, t₍₁₉₎ = 1.413, p = 0.1737; rearing, t₍₁₉₎ = 0.5033, p = 0.6206; center entries, t₍₁₉₎ = 0.5485, p = 0.5897. m Graph represents sucrose preference (%) before (Day 0) and after (Day 23) 5-d UMS. N = 10, 11 mice for ov-control and ov-Rasd2 groups, respectively. Two-way ANOVA (factor 1: time; factor 2: treatment): F_{time×treatment (1, 38)} = 0.9972, p = 0.3243; F_{time (1, 38)} = 21.83, p < 0.0001; F_{treatment (1,38)} = 9.783, p = 0.0034. Tukey’s HSD post hoc test: after treatment, p_{(ov-control vs. ov-Rasd2)} = 0.0288; in the ov-Rasd2 mice, p_{(baseline vs. test)} = 0.0011. n Duration of immobility in the tail suspension test. N = 10, 11 mice in ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: t₍₁₉₎ = 3.015, p = 0.0071. o Rasd2 overexpression in the NAcc increased the GluA1 expression in the mPFC after 5-d UMS. N = 4, 5 mice in ov-control and ov-Rasd2 groups, respectively. Two-tailed unpaired t-test: t₍₇₎ = 2.391, p = 0.0481. p Rasd2 overexpression activated the DRD2-cAMP-PKA-DARPP-32 signaling pathway in the NAc. N = 4-5 mice. Two-tailed unpaired t-test: RASD2, t₍₈₎ = 4.255, p = 0.0028; DRD1, t₍₈₎ = 0.5450, p = 0.6006; DRD2, t₍₇₎ = 2.591, p = 0.0359; cAMP, t₍₇₎ = 2.692, p = 0.0310; PKA, t₍₇₎ = 3.887, p = 0.0060; DARPP-32, t₍₇₎ = 2.689, p = 0.0311. q Rasd2 overexpression increased the co-expression of RASD2 and DRD2 (scale bar, 20 μm). N = 4 mice per group. Two-tailed unpaired t-test: t₍₂₀₎ = 2.270, p = 0.0344. The data are expressed as mean ± s.e.m. *p < 0.05 and **p < 0.01. 5-d UMS 5-day unpredictable mild stress, OFT open field test, FST forced swimming test, TST tail suspension test, WB western blotting, IF immunofluorescence, GluA1 glutamate receptor subunit 1, DRD1 dopamine D1 receptor, DRD2 dopamine D2 receptor, cAMP cyclic adenosine monophosphate, PKA protein kinase A, DARPP-32 dopamine and cAMP-regulated phosphoprotein, 32 kDa, NAcc NAc core.