Fig 2: Activation of synaptic proteins in the hippocampus. Western blot analysis of synaptic protein expression levels of (A) PSD95, (B) Syn1, (C) GluR1 and (D) BDNF in the hippocampus. SPS group decreased synaptic protein levels. Stimulation of HT8 increased the synaptic protein expression in the hippocampus. (Cropped blots are displayed; Full-length blots are presented in Supplementary Figure, labeled Fig. 3) **p < 0.01, *p < 0.05 vs. Nor group, ##p < 0.01, #p < 0.05 vs. SPS group. n = 3 each. Nor, normal; SPS, single prolonged stress; HT8, acupuncture group with the HT8; CP, control point acupunctured group; Flu, fluoxetine.

Fig 3: Ang II enhances synaptic connectivity of R312H primary neurons.A Representative images showing DIV14 primary cortical neurons of the indicated genotypes co-stained with Syn-1 (green), PSD-95 (red) to visualize functional synapses (arrows), and DAPI (blue), in control or incubated in the presence of 0.7 µM Ang II. B Magnifications are taken from the images on the left. Scale bars 50 µm for small magnification images and 10 µm for large magnification images. C Average number of dendritic spines for the cells of the indicated genotypes incubated in the absence/presence of 0.7 µM Ang II. For a single neuron, the number of spines was counted over a single dendrite for a continuous length of ~100 µm. N = 20 neurons/genotype from: WT: 4 pups; HETERO: 6 pups; HOMO: 6 pups. D Number of functional synapses in co-cultures of primary neurons of the indicated genotypes. Cells were co-stained with SYN-1, PSD-95 and DAPI and functional synapses were identified by SYN-1 and PSD-95 signals co-localizations. N = WT (cnt.-Ang): 8–9 cultures; HETERO: 13–14 cultures; HOMO: 8–7 cultures obtained from: 3 WT, 5 HETERO and 4 HOMO pups. Two technical replicates/culture. Data were analyzed with ImageJ/Fiji software and normalized to WT in control conditions. *P < 0.05; ***P < 0.001, and ****P < 0.0001 (one-way ANOVA, Tukey’s post hoc).

Fig 4: Synaptic connectivity is hindered in R312H cortices.A Representative images of SYN-1 (green), PSD-95 (red), and DAPI (blue) co-staining in the cortices of the indicated genotypes. Functional synapses were identified as SYN-1 and PSD-95 signals co-localizations (arrows). Scale bar 25 µm. B Quantification of functional synapses, given as the number of SYN-1 and PSD-95 co-localizations. Data are normalized to WT. Sections were analyzed with ImageJ/Fiji software. N = 5 brains/genotype. Two technical replicates/brain. C Western blots and densitometric quantification of SYN-1 protein in crude brain lysates of the indicated genotypes. N = 3 brains/genotype. D Western blots and densitometric quantification of SYN-1 protein phosphorylated at S605 (pSYN-1) in crude brain lysates of the indicated genotypes. N = 3 brains/genotype. E Western blots and densitometric quantification of PSD-95 protein in crude brain lysates of the indicated genotypes. N = 3 brains/genotype. The westerns in C–E were carried out using the same bran lysates. Loading controls were Bradford assay and actin (C). *P < 0.05, **P < 0.01, and ****P < 0.0001 (one-way ANOVA, Tukey’s post hoc).

Fig 5: Antidepressant-like effects of MCH in stressed mice. (A) Schematic representation of the STR paradigm. (B) Total weight gain was divided by (C) total food intake to calculate (D) total FER. (E) TST was administered during the 6-min test period and immobility time was recorded. (F) Immobility, (G) climbing, and (H) swimming behaviors in mice were observed during the last 4 min test period and climbing or swimming time was recorded. (I) Concentration of corticosterone was measured using an ELISA kit. The phosphorylation levels of (J) mTOR, (K) PSD95, GluR1 and Syn1 in the PFC were measured by immunoblotting. The amount of phosphorylated proteins were normalized to that of total proteins and presented as bar graphs (right panel). Data represent the means±SEM. ***p<0.001, **p<0.01, *p<0.05 vs. Nor group, ###p<0.001, ##p<0.01, #p<0.05 vs. STR group. Nor, normal; STR, stress; Flu, fluoxetine; FST, forced swim test; TST, tail suspension test.