Osmotic regulation of estrogen receptor-beta in rat vasopressin and oxytocin neurons

Abstract

The vasopressin (VP) magnocellular neurosecretory cells (MNCs) in the supraoptic and paraventricular (PVN) nuclei are regulated by estrogen and exhibit robust expression of estrogen receptor (ER)-beta. In contrast, only approximately 7.5% of oxytocin (OT) MNCs express ER-beta. We examined the osmotic regulation of ER-beta mRNA expression in MNCs using quantitative in situ hybridization histochemistry. Hyper-osmolality induced via 2% hypertonic saline ingestion significantly decreased, whereas sustained hypo-osmolality induced via d-d-arginine VP and liquid diet increased ER-beta mRNA expression in MNCs (p < 0.05). Thus, the expression of ER-beta mRNA correlated inversely with changes in plasma osmolality. Because hyper-osmolality is a potent stimulus for VP and OT release, this suggests an inhibitory role for ER-beta in MNCs. Immunocytochemistry demonstrated that the decrease in ER-beta mRNA was translated into depletion of receptor protein content in hyper-osmotic animals. Numerous MNCs were positive for ER-beta in control animals, but they were virtually devoid of ER-beta-immunoreactivity (IR) in hyper-osmotic animals. The osmotically induced decrease in ER-beta expression was selective for MNCs because ER-beta-IR remained unaltered in PVN parvocellular neurons. Plasma estradiol and testosterone were not correlated with ER-beta mRNA expression after osmotic manipulation, suggesting that ER-beta expression was not driven by ligand availability. Expression of FOS-IR in MNCs with attenuated ER-beta-IR, and the absence of FOS-IR in parvocellular neurons that retain ER-beta-IR suggest a role for neuronal activation in the regulation of ER-beta expression in MNCs. Thus, osmotic modulation of ER-beta expression in MNCs may augment or attenuate an inhibitory effect of gonadal steroids on VP release.

Figure 1.

Change in body weight in the hyper-osmolality, iso-osmolality, and hypoosmolality groups during the acclimation and experimental periods.

Figure 2.

Representative photomicrographs from in situ hybridization histochemistry of 12-μm-thick coronal rat brain sections. A, Autoradiogram of tissue section hybridized with ³⁵S-labeled antisense riboprobe for ER-β mRNA. Note the specific hybridization signals for ER-β mRNA in the bed nucleus of the stria terminalis (BNST) and SON. B, Autoradiogram of anatomically comparable section hybridized with ³⁵S-labeled sense-riboprobe for ER-β mRNA. No hybridization signal was observed in sections incubated with ER-β sense probes, confirming the specificity of the antisense hybridization.

Figure 3.

Representative photomicrographs from in situ hybridization histochemistry of 12-μm-thick, emulsion-dipped coronal rat brain sections. A-C are dark-field micrographs of anatomically comparable sections through SON hybridized with ³⁵Slabeled antisense riboprobe for ER-β mRNA from hyper-osmotic, iso-osmotic, and hypo-osmotic rats, respectively. D-F are dark-field, high-magnification micrographs of magnocellular supraoptic neurons from hyper-osmotic, iso-osmotic, and hypo-osmotic rats, respectively. Scale bars: A-C, 100 μm; D-F, 50 μm. OC, Optic chiasm.

Figure 4.

Expression of ER-β mRNA in rat magnocellular supraoptic neurons after 72 hr of 2% hypertonic saline ingestion (Hyper) and sustained hypo-osmolality (Hypo). A, Group mean ± SEM specific silver grain counts per neuron from emulsion-dipped sections. B, Group mean ± SEM relative optical density from film autoradiograms. A,*p < 0.05 versus each other (Kruskal-Wallis ANOVA on ranks, followed by Student-Newman-Keuls post hoc test). B,*p < 0.009 versus each other (one-way ANOVA, followed by Student-Newman-Keuls post hoc test).

Figure 5.

Photomicrographs of SON (A-F) and PVN (G-L) stained immunocytochemically for ER-β alone (top row), ER-β with oxytocin-neurophysin (OT) (middle row) or FOS with OT (bottom row) from iso-osmotic control rats (iso-osmotic) compared with rats that received 2% hypertonic saline ingestion (hyper-osmotic) for 72 hr. All three staining procedures were performed on adjacent sections, and the representative micrographs depict sections derived from the same rat. Scale bar, 100 μm. OC, Optic chiasm; 3V, third ventricle.

Figure 6.

Photomicrographs of SON (A-C), PVN (E-G), and nucleus circularis (D, H) from iso-osmotic control rats. A and E are Toluidine-Blue-O-stained sections through supraoptic and paraventricular nucleus. Whereas the SON contains essentially only magnocellular neurons (A), the PVN represents a much more heterogeneous population of cells, comprising MNCs in the pm and diverse groups of parvocellular neurons parceled into the dp, mpv, and mpd (***) regions of the nucleus (E). B, F, Sections stained immunocytochemically for nuclear ER-β followed by cytoplasmic oxytocin-neurophysin (OT). C, G, Anatomically comparable sections stained immunocytochemically for ER-β followed by vasopressin-neurophysin (VP). Note that a greater proportion of ER-β-immunoreactive neurons are colocalized with VP rather than OT in supraoptic and magnocellular paraventricular neurons. The minimal colocalization between ER-β-IR and OT-IR is also observed in many accessory cell groups, including the nucleus circularis (H), whereas ER-β-IR is coexpressed with VP-IR in this nucleus (D). Scale bars: A-C, F, G, 100μm; D, H,50μm; E, 200μm. OC, Optic chiasm; 3V, third ventricle.

Figure 7.

Photomicrographs of neurons in the lateral parvocellular part of PVN. A and B are low-power micrographs from sections stained immunocytochemically for ER-β with oxytocin-neurophysin (OT). C and D are high-power photomicrographs from sections shown in A and B, respectively. Note black nuclear deposits for ER-β within brown cytoplasmic OT staining in double-labeled neurons (black arrowheads). ER-β-IR is also expressed in non-OT-reactive neurons (green arrows). There was no difference in the number of ER-β-positive neurons or the extent of its colocalization with OT after 2% hypertonic saline ingestion for 72 hr. E and F are low-power photomicrographs from sections of lateral PVN stained for FOS and OT. Note a comparable level in FOS staining between iso-osmotic and hyper-osmotic rats. G is an anatomically comparable section of lateral PVN from a euhydrated control rat stained immunocytochemically for ER-β with vasopressin-neurophysin(VP). Note the intense ER-β-IR in these neurons with sparse labeling for VP in this area. Scale bars: A, B, E-G, 100 μm; C, D, 50 μm. 3V, Third ventricle.