Fig 2: The s1R agonist ACR16 increased Arc gene expression. The expressions of synapsin1 (Syn1), spinophilin (Ppp1r9b), s1R (Sigmar1), subunit 1 NMDAR (Grin 1), and the immediate-early genes Arc and c-fos were quantified in rat hippocampal primary cell cultures using qPCR. Individual data were normalized as percent change and represented in the violin plots as median percent change with respect to control conditions ± (Min–Max). Six independent experiments were performed for each experimental condition and for each time point. Statistical analysis was performed using Student’s t-test. Comparisons were made with control cultures and between treatments and *(p < 0.05) is used to indicate significant differences to control cultures. Note that only ACR16 (70 nM) was found to increase Arc expression at 6 h

Fig 3: Quantitative proteome and phosphoproteome of the rat neurointermediate lobe.A, principal component analysis (PCA) of the NIL proteome and phosphoproteome in control (blue, n = 6) and water-deprived (WD) rats (red; n = 6). B, volcano plot of WD versus control NIL proteome showing 276 upregulated (p-value <0.05, red) and 573 downregulated (p-value <0.05, blue) proteins. C, volcano plot of WD versus control NIL phosphoproteome showing 746 hyperphosphorylation (p-value <0.05, red) and 755 hypophosphorylation (p-value <0.05, blue) events. D, global ?Ps analysis of phosphoproteins between control and WD rats in the NIL. Numbers of hyperphosphorylated (Hyper) and hypophosphorylated (Hypo) peptides are shown. Dotted lines, ?Ps = ±0.4. E, Venn diagram showing 151 proteins in common with changes at the proteome and phosphoproteome level in response to WD. F, phospho raw abundance for S67-p SYN1, S426-p SYN2, and S426-p SYN2 in the NIL according to LC-MS/MS between control (n = 6) and WD (n = 6) rats. Western blotting analysis of S67-p SYN1 (normalized against SYN1), S426-p SYN2 (normalized against SYN2), and S847-p NOS1 (normalized against NOS1) in control (n = 5) and WD NILs (n = 5 for S847-p NOS1 and n = 4 for S67-p SYN1 and S426-p SYN2). G, immunohistochemistry against S67-p SYN1 and arginine vasopressin (AVP), S426-p SYN2 and AVP, and S847-p NOS1 and NOS1 in the pituitary gland of control rats showing the anterior pituitary (AP), intermediate lobe (IL), and posterior pituitary (PP). Images are representative of n = 4. Scale bar represents 75 µm. H, immunohistochemistry against S67-p SYN1, S426-p SYN2, and S847-p NOS1 in the PP of control and WD rats. Images are representative of n = 4. Scale bar represents 25 µm. ?Ps, phosphorylation state change; LC-MS/MS, Nano-LC mass spectrometry; NIL, neurointermediate lobe.