Fig 2: Buspirone prevents PACAP and VIP neuropeptide dysregulations in the brain of rotenone-treated mice. Real-time qPCR analyses of PACAP and VIP mRNA expression in the midbrain (A,B), striatum (F,G), prefrontal cortex (K,L), amygdala (P,Q), and hippocampus (U,V) following the administration of rotenone (10 mg/kg) and/or buspirone (1, 3, or 10 mg) daily for 21 days. Fold-changes were calculated using the ??Ct method after normalization to s18 (ribosomal protein s18 gene), the housekeeping gene. Each data point represents the mean value from n = 5–8 mice per each group. Representative Western blots and densitometry of PACAP and VIP protein expression in the midbrain (C–E), striatum (H–J), prefrontal cortex (M–O), amygdala (R–T), and hippocampus (W–Y). Protein expression was normalized to GAPDH, the loading control. Western blots are cropped and removed lanes depicting drug-treatment controls are included in Supplementary Figure S6. Densitometric results are expressed as means ± S.E.M from n = 5–8 mice per each group. * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001, compared to vehicle-treated controls; or # p < 0.05, ## p < 0.01, ### p < 0.001, or #### p < 0.0001, compared to rotenone (10 mg/kg)-treated mice, as determined by an ANOVA followed by Sidak’s post-hoc test. PACAP, pituitary adenylate cyclase-activating peptide; VIP, vasoactive intestinal peptide; s18, ribosomal protein s18 gene; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; kDa, Kilodalton.

Fig 3: Scatterplots and regression lines showing the relationship between dysregulated miRNAs and the expression profile of VIP/PACAP system components following exposure to LPS. Only significant correlations are shown. Positive correlations were found between (A) rno-miR-155 and Vip; (B) rno-miR-145-5p and Adcyap1r1 and (C) rno-miR-340-5p and Vipr1 mRNAs. Inverse relationships were found between (D) rno-miR-155 and Vipr1; (E) rno-miR-29a and Vipr1; (F) rno-miR-21 and Vipr2 and (G) rno-miR-146a and Vipr2. Coefficients of determination (r2) and related p values are shown in each panel.

Fig 4: Time-course gene expression profiling of (A) Vip; (B) Adcyap1; (C) Adcyap1r1; (D) Vipr1 and (E) Vipr2 mRNAs in RT4 SCs treated with LPS at the indicated concentration. Relative transcript levels were measured by quantitative real-time PCR analyses. Amplifications were performed using selected primers optimized for qPCR analyses (<150 bp length) recognizing fragments within the CDS of the gene of interest (for details refer to Table 1 below). Results are presented as mean fold changes of time 0 (control) ± SEM.-fold changes of each gene were obtained after normalization to the endogenous ribosomal protein S18 (reference gene) and then calculated using the comparative ??Ct method. Baseline expression levels of control were set to 1. Bar graphs depicted show the mean results obtained from two independent determinations using two separate batches of cells each time (n = 4). * p < 0.05, ** p < 0.01 or * p < 0.001, as determined using ANOVA and Dunnett’s post-hoc comparisons.

Fig 5: Expression of PACAP, VIP and related receptors, PAC1, VPAC1 and VPAC2 in the corpus striatum of 2- and 7-month-old WT and NZBWF1 mice. Depicted are real-time qPCR results of (A) PACAP, (B) VIP, (C) PAC1, (D) VPAC1 and (E) VPAC2 gene expression. Relative changes in mRNA levels were determined using the ?CT method and were normalised to the ribosomal subunit S18 which was used as a housekeeping gene. (F,I) Representative Western blots and related densitometry of (G) PACAP, (H) VIP, (J) PAC1, (K) VPAC1 and (L) VPAC2 protein expression. Densitometric analyses were performed using the ImageJ software and were normalised to GAPDH bands, here used as loading control. Data represent the mean of (n = 5) 2 mo WT, (n = 7) 2 mo NZBWF1, (n = 4) 7 mo WT and (n = 9) 7 mo NZBWF1 mice. Results are expressed as mean ± S.D. * p = 0.05, *** p = 0.001 or **** p = 0.0001 vs. WT at the corresponding age, as determined by two-way ANOVA followed by Tukey post hoc test. PACAP = pituitary adenylate cyclase-activating peptide; VIP = vasoactive intestinal peptide; PAC1 = PACAP receptor 1; VPAC1 = VIP receptor 1; VPAC2 = VIP receptor 2.