Estrogen Receptor Alpha on Catecholaminergic Neurons in the Nucleus Tractus Solitarius

Purpose: Catecholaminergic neurons in the Nucleus Tractus Solitarius (NTS) are involved in Hypothalamic-Pituitary-Adrenal (HPA) axis and cardiovascular response to stress. While many studies have shown that pre-menopausal females are protected against the hypertensive and sympatho-excitatory effects of stress, very little is known about the location of neurons expressing estrogen receptors within the NTS. Our goal was to elucidate whether estrogen receptor alpha (ERα) is expressed on catecholaminergic neurons in the NTS in male and female rats. Methods: Adult male and female Sprague-Dawley rats were transcardially perfused with 4% paraformaldehyde and hindbrains harvested. In coronal sections containing the NTS (40um thick) immunohistochemistry was performed to determine whether NTS catecholaminergic neurons express ERα using monoclonal anti-tyrosine hydroxylase (TH) antibody (1:1000, Millipore) and secondary antibody Alexa Fluor 488 donkey anti-mouse (1:500, Jackson ImmunoResearch) and polyclonal anti-ERα antibody (1:2000-5000, Millipore) and secondary antibody Cy3 donkey anti-rabbit (1:400, Jackson ImmunoResearch). Sections were captured using an Olympus BX41 Fluorescence Microscope and analyzed using ImageJ. The NTS was divided into 2 regions: sections caudal to the area postrema (caudal CAUD) and sections lying below the area postrema (sub-postrema SP), and the number of immunoreactive neurons in each region counted and expressed as an average number of labeled neurons per section±SEM. The number of sections analyzed ranged from 7-11 in CAUD and 3-7 in SP. Results: In male rats, TH in CAUD NTS (n=6) was observed in 26±4 and ERα in 24±4 neurons/section. Co-localization of ERα and TH was observed in 13±2 neurons/section. TH in SP NTS (n=5) was observed in 54±3 and ERα in 34±2 neurons/section. Co-localization of ERα and TH was observed in 15±2 neurons/section. In female rats, TH in CAUD NTS (n=6) was observed in 27±2 and ERα in 30±3 neurons/section. Co-localization of ERα and TH was observed in 17±2 neurons/section. TH in SP NTS (n=6) was observed in 50±4 and ERα in 52±5 neurons/section. Co-localization of ERα and TH was observed in 27±2 neurons/section. At sacrifice, females were in estrus (1), diestrus (2) or proestrus (3). Conclusions: In both males and females, ERα is expressed on a subset of catecholaminergic NTS neurons, as well as non-catecholaminergic neurons. This could provide a substrate for estrogen-mediated cardiovascular protection in females.