Fig 2: Epigenetic changes in Avp gene promoter in aging. Methylation status of the Avp promoter in the SON of control and dehydrated adult and aged male rats. (A) Relative mRNA expression of Dnmt1, Dnmt3a, Tet1, Tet2, and Tet3 was determined by qRT-PCR. (B) Diagram showing 7 CpG sites on the Avp promoter that were examined by colony-based PCR. (C) Representative tile diagrams showing the methylation status of 7 CpG sites for individual clones of the Avp promoter extracted from the SON. (D) Percentage of global methylation on this region of the Avp promoter in control and dehydrated adult and aged rats. (E) Percentage methylation of individual CpG sites on the Avp promoter in control and dehydrated adult and aged rats. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by the 2-way analysis of variance with Tukey's post hoc test. #, p < 0.05 by unpaired t-test. Abbreviation: SON, supraoptic nucleus.

Fig 3: Peptide analysis in aged rats. Adult and aged male rats were subjected to dehydration for 3 days, and NIL peptide measures were performed by MALDI-TOF MS. (A–E) Signal intensity of peptides in NILs was measured by MALDI-TOF MS in individual tissue extracts from adult and aged rats in basal and dehydrated states (n = 11–16). (A) Control and dehydrated peptide profiles are easily classified by PCA according to PC1 (∼60% of variance) in adult rats; PCA plot is shown for the first 3 PCs. (B) Loading plot indicates that a small subset of peptides contributes to differences between control and dehydrated adult rats, among which are masses matching AVP, acetylated alpha-MSH, and di-acetylated alpha-MSH. (C) The AVP and OT signal decreases significantly, while the alpha-MSH signals and proSAAS (221-237) increase significantly with dehydration in adult rats. (D, E) The effect of age on peptide signals in the NIL. (D) The OT signals decreased, while the signals of POMC-derived peptides increased in aged compared to adult rats. (E) Peptide signals in the NIL of adult and aged rats subjected to 3 days of dehydration. The AVP-copeptin signal increased, whereas the proSAAS signal decreased with age. (F) Peptides uniquely altered as a function or aging or by just dehydration. (G) Relative RNA expression of proSAAS and OT in the SON of adult and aged rats. *p < 0.05 by unpaired t-test or peptide names beginning with * by Kruskal-Wallis tests. Abbreviations: AVP, arginine vasopressin; DH, dehydrated; NIL, neurointermediate lobe; PCA, principal component analysis; POMC, proopiomelanocortin.

Fig 4: Group means for oxytocin (OT) and arginine vasopressin (AVP) measured in plasma and saliva before and after Experiment 1. Using between-subjects design, dogs were assigned to conditions consisting of 10 min of affiliative interaction with a human (HAI, N = 19) or 10 min of rest without social contact with the experimenter (Control, N = 19). Dogs in the HAI condition exhibited significant increases in both salivary and plasma OT, and a significant decrease in plasma AVP. Dogs in the control group exhibited no significant changes in salivary or plasma OT, but showed increases in salivary AVP across time. Error bars represent the within-subjects standard error (Cousineau, 2005) and should be interpreted with regard to within-group, but not between group differences. The results of planned between-group, and within-group contrasts are reported in Table 4. Non-transformed concentration means: Salivary OT – HAI group: pre = 630 pg/mL, post = 874 pg/mL; Control group: pre = 563 pg/mL, post = 642 pg/mL; Plasma OT – HAI group: pre = 18.75 pg/mL, post = 19.83 pg/mL; Control group: pre = 20.11 pg/mL, post = 19.84 pg/mL; Salivary AVP – HAI group: pre = 605 pg/mL, post = 546 pg/mL; Control group: pre = 546 pg/mL, post = 724 pg/mL; Plasma AVP – HAI group: pre = 6.45 pg/mL, post = 5.64 pg/mL; Control group: pre = 6.12 pg/mL, post = 5.89 pg/mL.

Fig 5: Effect of aging on the rat AVP system. Adult and aged male rats were subjected to dehydration for 3 days and AVP measures were performed. (A) Body weights were recorded before and after dehydration (n = 11–14). (B) Plasma osmolality was measured (n = 19–23) by freezing point depression. The effect of aging on Avp expression was examined at the transcriptional level in control and dehydrated adult and aged rats. (C, D) The RNA expression level of Avp (both heteronuclear [hnAvp] and mature form) was examined by qRT-PCR (n = 6). AVP measures were performed on NILs and plasma extracts. (E) AVP content in NILs was measured by ELISA (n = 9–10). (F) Plasma AVP level was determined by radioimmunoassay (n = 9). *, p < 0.05; ***, p < 0.001 by the 2-way analysis of variance with Bonferroni post hoc test. #, p < 0.05 by unpaired t-test. Abbreviations: AVP, arginine vasopressin; DH, dehydrated; NIL, neurointermediate lobe.