Fig 2: Subcellular localization of ERα and H1R in vl VMN neurons.(A, B, C, D, E) Representative images of dual immunofluorescence labeling of ERα (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 ERα 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 ERα-ir in VMN neurons. ERα immunostaining (green) were observed in the nucleus defined by PI staining (red). Panel H is the merged image for panel F and G. ERα; ERα immunoreactivities, PI; propidium iodide. Scale bars, 5 µm (A–E), 10 µm (F–H).

Fig 3: 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.

Fig 4: Distribution of H1R in the rat VMN.(A–F) Confocal microscopy images of dual immunofluorescence labeling of ERα (green, A and D) and H1R (red, B and E) in adult female rats. ERα-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 ERα-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 ERα 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 5: Sampling of VMN subregion tissues.Quantitative analysis of H1R mRNA and H1R protein in the two subregions of the VMN was performed after selective dissection of the vl VMN and dm VMN using a stainless steel needle. The dotted lines show the locations of the VMN and vl and dm subregions in the rat basal hypothalamus. (A) Representative photograph of a coronal brain section after sampling (left side). The vl VMN and dm VMN were selectively dissected with the needle (inner diameter 900 µm). An outline of the rat basal hypothalamus used for identification and selective dissection of the VMN subregions. 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.