Estrogen receptor-immunoreactive neurons contain calcitonin gene-related peptide, methionine-enkephalin or tyrosine hydroxylase in the female rat preoptic area.

We have shown in our previous studies that estrogen treatment selectively influences calcitonin gene-related peptide (CGRP)-, methionine-enkephalin (Met-Enk)- and tyrosine hydroxylase (TH)-immunoreactive (IR) intensities in the neurons of the periventricular preoptic nucleus (PPN) and the medial preoptic area (MPA) of the female rat. In the present study, we examined whether estrogen receptor (ER)-IR neurons in the PPN and MPA contain CGRP, Met-Enk, or TH using a double-labeling immunohistochemical method and investigated changes in the number of double-labeling cells upon treatment with estrogen. Brain sections of ovariectomized rats and ovariectomized and estrogen-treated rat were stained using the avidin-biotin-peroxidase complex method followed by the peroxidase-anti-peroxidase method. The sections were first incubated with an anti-ER antibody in conjunction with nickel diaminobenzidine which produces a dark blue reaction product in the nucleus. Subsequently, CGRP, Met-Enk or TH antisera were applied to these sections and the resulting brown diaminobenzidine reaction product in the cytoplasm was examined. Neurons that were double-labeled for ER and CGRP, Met-Enk or TH were investigated in the PPN and MPA. The number of doubly labeled ER/CGRP- and ER/TH-IR neurons was large, whereas the number of ER/Met-Enk-IR neurons was small. These results suggest that ER in the PPN and MPA may be more closely related to the mechanism of changes in CGRP- and TH-IR intensities upon estrogen treatment than that in Met-Enk-IR intensity.