Fig 2: Age-dependent FOS and FOSB immunoreactivities in the paraventricular nucleus of the hypothalamus (PVN). Representative images depicting the PVN area of 1-month-old (1M) and 24M acute restraint stress (ARS) exposed rats. Note that most of the CRF (white) immunoreactive (ir) cells contain both FOS (green) and FOSB (red) as also shown by the merged image on the right in 1M animals. The immunosignal for both FOS and FOSB declined with age as represented by images of 24M rats. Histogram (A) shows the number (N) of CRF and FOS-containing cells in control (open bars) and ARS exposed (gray columns) rats in eight age groups. Panel (B) demonstrates the N of cells where CRF and FOSB co-exist. Chart (C) demonstrates the aging-related change in the number of CRF cells which contain both FOS and FOSB. Significant differences according to the Tukey’s post hoc tests were highlighted by asterisks (p < 0.05, n = 4–8). (D) A scatter plot of the Spearman’s correlation test illustrates the age-related decline of the N of CRF-FOS labeled cells in ARS. Boxed areas are depicted in higher magnification insets in the right bottom corner of the respective images. 3rd, third ventricle. Scale bar 100 µm.

Fig 3: FOS and FOSB immunoreactivities in the central nucleus of amygdala (CeA). Representative images depicting the CeA area of 2-months-old (2M) control and chronic variable mild stress (CVMS) exposed rats compared with 24M rats upon CVMS. Note that CRF (white) cells are not immunoreactive (ir) for FOS (green). In contrast, in young rats, CeA-CRF cells show FOSB (red) immunoreactivity both in control rats and in animals upon CVMS exposure as also shown by the merged image on the right. The FOSB immunoreactivity declined with age as represented by images of 24M rats, and by histogram (A) that shows the number (N) of CRF and FOSB co-expressing cells in control (open bars) and CVMS exposed (dark columns) rats in six age groups. Scatter plot (B) of the Spearman’s correlation test illustrates the age-related decline of the N of CRF-FOSB labeled cells in CVMS. Boxed areas are depicted in higher magnification insets in the right bottom corner of the respective images. st, stria terminalis. Scale bar 100 μm.

Fig 4: Depiction of tyrosine hydroxylase (TH)‐positive and TH‐negative connections between the medial supramammillary nucleus (SuMM) and selected appetite/reward areas. Regions in blue show increased levels of Fos expression after consumption of sweetened condensed milk (SCM), the region shown in yellow show no change after consumption of SCM. DMH, dorsomedial nucleus of the hypothalamus; LHA, lateral hypothalamic area; SuM, supramammillary nucleus; VMH, ventromedial nucleus; VTA, ventral tegmental area

Fig 5: Expression of Fos immunoreactivity in the medial supramammillary nucleus (SuMM) and appetite‐reward related areas in response to the voluntary consumption of sweetened condensed milk (SCM). Data are the mean ± SEM. **P < 0.01, ***P < 0.001. A‐D, Representative images of Fos (arrowheads) and tyrosine hydroxylase (TH) (arrows) immunohistochemistry within SuMM in control (A, B) and SCM‐fed rats (C, D) in coronal sections at bregma −4.5 mm. E, Number of cells expressing Fos per brain section in SuMM for control (n = 8) and SCM (n = 6) groups (P = 0.0007, Mann‐Whitney test). F, Percentage of SuMM TH‐immunoreactive cells expressing Fos‐positive nuclei per brain section for control (n = 8) and SCM (n = 6) groups (P = 0.0007, Mann‐Whitney test). G and H, Representative images of Fos expression in the lateral hypothalamic area (LH) (bregma −1.72 mm), dorsomedial nucleus of the hypothalamus (DMH) (K, L; bregma −3 mm) and ventral tegmental area (VTA) (O, P; bregma −5.04 mm) in control and SCM‐fed rats. I, M, and Q, Number of cells expressing Fos per brain section for control and SCM groups in the LHA (I; control n = 7, SCM n = 6; P = 0.0012, Mann‐Whitney test), DMH (M; control n = 8, SCM n = 6; P = 0.0013, Mann‐Whitney test) and VTA (Q; control n = 8, SCM n = 4; P = 0.0081, Mann‐Whitney test). J, N, and R, Linear regression plots and coefficients (with significance indicated as *P ≤ 0.05, **P ≤ 0.01 or ***P ≤ 0.001) showing the correlation between Fos expression in the SuMM and Fos expression in the LHA (J; r 2 = 0.7562, P = 0.0001), DMH (N; r 2 = 0.434, P = 0.0104) and VTA (R; r 2 = 0.5827, P = 0.0039). Scale bars represent 200 µm (G, H, K, L, O, P), 50 µm (A, C) and 25 µm (B, D). 3V, third ventricle; AH, anterior hypothalamic nucleus; EA, sublenticular extended amygdala; f, fornix; fr, fasciculus retroflexus; LH, lateral hypothalamic area; ml, medial lemniscus; OC, optic tract; PB, parabrachial pigmented nucleus; pm, principal mammillary tract; PVN, paraventricular nucleus; SNR, substantia nigra; SuM, supramammillary nucleus; VMH, ventromedial nucleus