Fig 2: Distribution of H1R in the rat VMN.(A–F) Confocal microscopy images of dual immunofluorescence labeling of ERa (green, A and D) and H1R (red, B and E) in adult female rats. ERa-ir neurons are abundant in the vl VMN, but few are present in the dm VMN (A). (B) The highest density of H1R expression was found in the vl VMN. Merged images showed the coexistence of abundant ERa-ir and H1R-ir in the VMN (C and F). (D–F) Medium magnification images of representative neural groups within the vl VMN (area with a dotted line), allowing identification of cellular localization of ERa and H1R. (G and H) Detection of H1R immunoreactivities in VMN with (H) or without (G) control peptide. Preabsorption of H1R antibody with its peptide eliminated H1R immunoreactivities (H). 3V, Third ventricle; VMN, ventromedial nucleus of the hypothalamus; vl, ventrolateral part of the VMN; dm, dorsomedial part of the VMN; Me, median eminence; ARC, arcuate nucleus. Scale bars, 500 µm (A–C), 50 µm (D–H).

Fig 3: Quantitative evaluation of normalized H1R protein expression in the vl VMN and dm VMN.H1R protein expression levels were quantified for males (n = 5), OVX females (oil injected controls, n = 5), OVX females with EB (5 µg EB, 24 h or 48 h post-injection, n = 5 each), and OVX females with EB+ progesterone (5 µg EB, followed by 500 µg progesterone, n = 5). In the vl VMN, the H1R protein level tended to be increased by EB in OVX females (not statistically significant) and decreased by progesterone. In the dm VMN, hormonal manipulation did not affect H1R protein expression. Data are presented as the mean ±SEM. **, P<0.01. Data are expressed as the ratios of H1R protein to tubulin protein in the vl VMN and dm VMN regions. Since the immunoblotting were obtained independently for each region (from the respective immunoblotting membranes for the vl VMN and dm VMN), data were not to be compared between the two regions.

Fig 4: Subcellular localization of ERa and H1R in vl VMN neurons.(A, B, C, D, E) Representative images of dual immunofluorescence labeling of ERa (green) and H1R (red), obtained by confocal microscopy. Higher magnification images of individual cells in panel A are shown in B (area with a dotted line), and C, D and E (arrowheads). C–E are confocal images showing that ERa is exclusively localized in nuclei (green) and excluded from nucleoli (dark spot), while H1R is localized in the cell membrane (red). Panel E is a merge of the images from panels C and D. (F, G, H) Visualization of the cellular localization of ERa-ir in VMN neurons. ERa immunostaining (green) were observed in the nucleus defined by PI staining (red). Panel H is the merged image for panel F and G. ERa; ERa immunoreactivities, PI; propidium iodide. Scale bars, 5 µm (A–E), 10 µm (F–H).

Fig 5: Quantitative evaluation of normalized H1R mRNA expression in the vl VMN and dm VMN.Normalized H1R mRNA expression levels were quantified for males (n = 5), OVX females (oil injected controls, n = 5), OVX females with EB (5 µg EB, 24 h or 48 h post-injection, n = 5 each), and OVX females with EB + progesterone (5 µg EB, followed by 500 µg progesterone, n = 5). In the vl VMN, H1R mRNA was significantly increased by EB in OVX females and decreased by progesterone. In the dm VMN, hormonal manipulation did not affect H1R mRNA expression. Data represents the absolute quantities of H1R mRNA compared with GAPDH mRNA amount. Data are presented as the mean ±SEM. **, P<0.01; *, P<0.05.