Estrogen Receptor Expression in Brainstem Noradrenergic Neurons of the Sheep

Abstract

Noradrenergic neurons are implicated in the estrogen-dependent neural regulation of luteinizing hormone secretion in a variety of mammalian species. The current study has used immunocytochemical methods to determine whether estrogen receptors (ER) are expressed within the brainstem of the ewe and to establish their relationship to noradrenergic neurons. Using a monoclonal mouse antiserum directed against the N-terminal of ERα, four distinct populations of ERα-immunoreactive cells were identified in ovine medulla and pons. The largest population was found in the superficial laminae of the spinal nucleus of the trigeminal nerve, followed by the nucleus tractus solitarius, lateral area postrema, and ventrolateral medulla. Double-labelling immunocytochemistry using antisera directed against the ERα and dopamine-β-hydroxylase revealed that noradrenergic neurons expressing ER immunoreactivity were only found in ventrolateral medulla (A1 cell group) and nucleus tractus solitarius (A2 cell group). No double-labelled cells were identified in the A5, A6, or A7 noradrenergic cell groups. ERs were expressed with a clear rostrocaudal topography within the A1 and A2 populations, with 80–90% of noradrenergic neurons expressing ERα in the caudalmost medulla as compared with less than 5% rostral to the obex. Our findings demonstrate that, as in the rat, the ovine A1 and A2 neurons express ERs in a defined topographical manner, while, dissimilar to the rat, ERα is not synthesized by noradrenergic neurons in the other cell groups. These observations indicate that A1 and A2 noradrenergic neurons in the ovine brainstem are likely to be influenced by circulating estrogens and lay the neuroanatomical foundations for investigating the functional role of these cell populations within the gonadotropin-releasing hormone neuron network of the sheep.

Localization of Estrogen-Receptive Neurons Projecting to the GnRH Neuron-Containing Rostral Preoptic Area of the Ewe

“…The distribution of ER-immunoreactive cells was identical to that reported previously with the ID5 [15, 16]and other ER antibodies [6, 7, 8]in the ewe.…”

“…We have used the retrograde tracing approach described previously by Tillet et al [12]and achieved very similar results in terms of the identification of neurons which project to the rPOA and DBB. The sequential use of an ERα antibody, well characterized in sheep [15, 16], with an FG antiserum has enabled us to define those projection neurons which contain ERα. Dual-labeled neurons were unambiguously identified as those containing brown intracytoplasmic ‘dots’ and a black nucleus.…”

“…Perliminary experiments had shown that FG detection was superior with peroxidase-based immunocytochemistry compared to FG fluorescence following ERα immunostaining with nickel-DAB. Brainstem and forebrain sections first underwent free-floating immunostaining using a monoclonal mouse antibody [14]specific for the N-terminal domain of the human ER (ID5, 1:10, gift of Prof. G. Delsol, Toulouse, now available from DAKO, Copenhagen, Denmark) as described previously in the ovine brain [15, 16]. In brief, following 40 h incubation at 4°C in the primary antibody, sections were washed and placed in biotinylated horse antimouse immunoglobulins (1:400, Vector, 90 min at RT) followed by Vector Elite Kit (1:100, Vector, 90 min at RT) and visualized using the glucose oxidase nickel-DAB procedure.…”

“…Within the brainstem, analysis was undertaken on 5 ewes using the same anteroposterior levels reported previously for ERα immunoreactivity in the ovine caudal medulla [16]. In this case, levels G and H, I and J, and K and L according to Tillet and Thibault [17], were grouped to provide data for the rostral, middle and caudal medulla, respectively.…”

Estrogen Actions in the Central Nervous System1

“…In addition to the cholinergic and serotonergic systems, catecholaminergic systems respond to estrogens, i.e., brainstem catecholaminergic neurons (A6 and to a lesser extent A5 and A7) contain small numbers of ER (219), and estrogen treatment after gonadectomy exerts complex, time-dependent effects on the level of tyrosine hydroxylase mRNA (220). Moreover, recent studies of rats (221) and sheep (222) indicate that A1 and A2 noradrenergic neurons specifically express the ER␣ and show cyclical and estrogendependent patterns of immediate early gene expression (223,224). In the rat locus ceruleus, galanin is coexpressed in many noradrenergic neurons, and estrogen treatment increased the expression of galanin mRNA, leading to the speculation that estrogen treatment might reduce noradrenergic tone in the absence of separate effects on tyrosine hydroxylase expression by enhancing the cosecretion of galanin, which reduces noradrenaline release (225).…”