Fig 2: The interaction between mRNAs and miRNAs. miRNAs targeted to mRNAs that encode Gad1, VGAT and GAT-3 were predicted by using three miRNAs target prediction databases, in which the principle of miRNAs target prediction includes seed match, conservation, free energy and site accessibility. The interactive networks of miRNAs and mRNAs associated with GABA release and uptake are made by using Cytoscape software. Green symbols denote mRNAs that are actually down-regulated in the NAc tissue. Red symbols denote miRNAs. Their negative regulation of mRNAs by miRNAs is represented by light blue line

Fig 3: Molecular mechanisms are involved in the impaired GABA synthesis, uptake and release in the medial prefrontal cortices (mPFCs) of chronic unpredictable mild stress (CUMS)-induced depression mice. (a, b) The expression and relative quantity of proteins glutamate decarboxylase-67 (GAD-67), vesicular GAT (VGAT) and GABA transporter (GAT-3) were studied by western blot. (a) The expressions of GAD-67, VGAT and GAT-3 from a control mouse versus a CUMS-induced depression mouse, where internal control is performed with ß-actin. (b) The normalized ratios of proteins (GAD-67, VGAT and GAT-3) to ß-actin from control mice (dark-gray bars, n=8) versus CUMS-induced depression mice (light-gray bars, n=8). The relative ratios for control mice are normalized to be one. (c, d) The expressions and the relative quantity of messenger RNAs (mRNAs) for GAD-67, VGAT and GAT-3 were studied using high-throughput sequencing and quantitative real-time RT-PCR (qRT-PCR). (c) The relative values of mRNAs for GAD-67, VGAT and GAT-3 in control mice (dark-gray bars, n=8) and CUMS-induced depression mice (light-gray bars, n=8), which are analyzed using high-throughput mRNA sequencing. The relative expression levels of mRNAs in CUMS-induced depression mice are normalized to that in controls based on NOIseq package method. Diverge probability larger than 0.8 is thought as significant difference (#P?0.8). (d) The relative values of mRNAs for GAD-67, VGAT and GAT-3 in control mice (dark-gray bars, n=8) versus CUMS-induced depression mice (light-gray bars, n=8), which are carried out by qRT-PCR. ß-actin was set as internal control. The relative values for control mice were normalized to be one. (e, f) The expressions and the relative quantity of mRNAs for miRNA-15b-5p, miRNA-144-3p, miRNA-582-5p and miRNA-879-5p were studied using high-throughput sequencing and qRT-PCR. (e) The relative values of miRNA-15b-5p, miRNA-144-3p, miRNA-582-5p and miRNA-879-5p from control mice (dark-gray bars, n=8) versus CUMS-induced depression mice (light-gray bars, n=8), which are analyzed using high-throughput miRNA sequencing. (f) The relative values of miRNA-15b-5p, miRNA-144-3p, miRNA-582-5p and miRNA-879-5p from control mice (dark-gray bars, n=8) versus CUMS-induced depression mice (light-gray bars, n=8), which are studied by qRT-PCR of miRNAs. U6 was set to be internal control. The relative values for control mice were normalized to be one.*P<0.05, **P<0.01 and ***P<0.001.

Fig 4: Correlation between miRNAs and their target mRNAs expression in the NAc tissue. The relationships between miRNAs and its corresponding target prediction were assessed by Pearson's correlation coefficients. A, shows the correlation between Gad1 mRNA and miR-144-3p expression (r = -.8749; P < .001). B, shows the correlation between VGAT mRNA and miR-144-3p (r = -.9267; P < .001). C, shows the correlation between GAT-3 mRNA and miR-144-3p (r = -.9028; P < .001). D, shows the correlation between GAT-3 mRNA and miR-15b-5p (r = -.8632; P < .001). E, shows the correlation between GAT-3 mRNA and miR-879-5p (r = -.8575; P < .001). F, shows the correlation between VGAT mRNA and miR-582-5p (r = -.8227; P < .001). The data of qRT-PCR of miRNAs and mRNAs were analysed in 10-14 per group. Blue dots indicate congregation of the control group, and red dots indicate congregation of the depression group

Fig 5: Valproate acid increases GABAergic synapses and the frequency of mIPSCs. (A,B) Western blot analysis and data summary show that the relative intensities of GAD67 (A) and VGAT (B) to GAPDH were significantly increased in VPA-reared tadpoles compared to Ctrl. GAD67, N = 6; VGAT, N = 6. N represents the number of independent experiments. *p < 0.05, **p < 0.01. (C) Representative traces were showing the recordings of mIPSCs from Ctrl and VPA-treated animals. Scale bar = 10 pA, 0.1 s. (D–F) The data shows that the frequency of mIPSCs was significantly increased in VPA-reared tadpoles compared to control tadpoles (D). **p < 0.01. The cumulative probability plot shows that the inter-event-interval of mIPSCs was significantly decreased in VPA-treated tadpoles compared to ctrl (E). Kolmogorov–Smirnov test, **p < 0.01. The data shows that the amplitude of mIPSCs was not altered in VPA-reared tadpoles compared to control tadpoles (F). Ctrl, N = 24, n = 5; VPA, N = 24, n = 4. N represents the number of neurons and n represents the number of tadpoles.