Estrogen receptor‐β in oxytocin and vasopressin neurons of the rat and human hypothalamus: Immunocytochemical and in situ hybridization studies

Abstract

Topographical distribution of estrogen receptor-beta (ER-beta)-synthesizing oxytocin (OT) and vasopressin (VP) neurons was studied in the hypothalamic paraventricular and supraoptic nuclei (PVH; SO) of ovariectomized rats. In distinct subregions, 45-98% of OT neurons and 88-99% of VP neurons exhibited ER-beta immunoreactivity that was confined to cell nuclei. Neuronal populations differed markedly with respect to the intensity of the ER-beta signal. Magnocellular OT neurons in the PVH, SO, and accessory cell groups typically contained low levels of the ER-beta signal; in contrast, robust receptor labeling was displayed by OT cells in the ventral subdivision of medial parvicellular subnucleus and in the caudal PVH (dorsal subdivision of medial parvicellular subnucleus and lateral parvicellular subnucleus). Estrogen receptor-beta signal was generally more intense and present in higher proportions of magnocellular and parvicellular VP vs. OT neurons of similar topography. Immunocytochemical observations were confirmed via triple-label in situ hybridization, an approach combining use of digoxigenin-, fluorescein-, and 35S-labeled cRNA hybridization probes. Further, ER-beta mRNA was also detectable in corticotropin-releasing hormone neurons in the parvicellular PVH. Finally, double-label immunocytochemical analysis of human autopsy samples showed that subsets of OT and VP neurons also express ER-beta in the human. These neuroanatomical studies provide detailed information about the topographical distribution and cellular abundance of ER-beta within subsets of hypothalamic OT and VP neurons in the rat. The variable receptor content may indicate the differential responsiveness to estrogen in distinct OT and VP neuronal populations. In addition, a relevance of these findings to the human hypothalamus is suggested.

Distribution of type 1 cannabinoid receptor‐expressing neurons in the septal‐hypothalamic region of the mouse: Colocalization with GABAergic and glutamatergic markers

“…The plasmid was grown in DH5α cells (Invitrogen, Carlsbad, CA, USA), isolated with the QIAfilter Plasmid Maxi kit (Qiagen; Valencia, CA, USA), linearized with Sal I and purified with phenol/chloroform/isoamyl alcohol (PCI), followed by chloroform/isoamyl alcohol (CI) extractions, and then, precipitation with NaCl and ethanol. The linearized transcription template was transcribed with T7 RNA polymerase in the presence of 35 S-UTP (NEN Life Science Products, Boston, MA, USA), to yield antisense transcripts (Hrabovszky et al, 2004). To generate a sense control for specificity testing, the insert was cleaved at an internal ApaI site and a 1 Kb sense transcript was transcribed using the SP6 promoter site.…”

“…Prior to hybridization, the sections were pretreated as described elsewhere (Hrabovszky et al, 2004). Briefly, they were fixed in 4% paraformaldehyde for 15 min, acetylated with 0.25% acetic anhydride in 0.9% NaCl/0.1 M triethanolamine (Sigma; pH 8.0) for 10 min, rinsed in standard saline citrate solution (2XSSC; 1X SSC=0.15 M NaCl/0.015 M sodium citrate, pH 7.0) for 2 min, dehydrated in 70, 80, 95 and 100% ethanol (2 min each), delipidated in chloroform for 5 min, and finally, rehydrated partially in 100%, followed by 95% ethanol (2 min each) and air dried.…”

“…Septal-hypothalamic blocks were dissected and 25-µm-thick coronal sections were cut on a freezing microtome (Leica). (Hrabovszky et al, 2006;Hrabovszky et al, 2004;Hrabovszky et al, 1995). Then, the autoradiographic signal was detected with Kodak NTB emulsion in the same way as in single-labeling experiments.…”

Blockade of central nicotine acetylcholine receptor signaling attenuate ghrelin-induced food intake in rodents

“…The plasmid templates were transcribed in vitro with appropriate RNA polymerases to yield antisense probes, and for control purposes, sense transcripts. The ChAT template was labeled with digoxigenin-11-UTP (Roche Diagnostics Co., Indianapolis, IN, USA), and the GHS-R1A template with 35 S-UTP (NEN Life Science Products, Boston, MA, USA), using reaction conditions detailed elsewhere (Hrabovszky et al, 2004). In duallabeling studies, the two probes were combined in the hybridization buffer.…”

“…and the LDTg of four adult male rats. Prehybridization hybridization and posthybridization procedures have been carried out on slide-mounted sections (Hrabovszky et al, 2004). In dual-labeling experiments, the ChAT probe was visualized first with alkaline phosphatase conjugated anti-digoxigenin antibodies and the BCIP/NBT chromogen system (Roche).…”