Purpose: Estrogens have many beneficial effects in the brain, which are mediated by the interaction of estrogens with specific receptors (ER). Two nuclear estrogen receptors ERα and ERβ, have been previously identified. Recently, a novel membrane bound G protein-coupled receptor, GPR30, also has been identified; however, the role of this receptor with respect to estrogen effects in brain is still largely unclear. Previously studies have reported estrogen replacement therapy has important effects on neurotransmitter production and release following loss of ovarian function. More recent studies suggest the effects of estrogens on brain chemistry may differ depending on when and how loss of ovarian function occurs. To date, there has been little direct comparison of the effects of estrogen and selective estrogen receptor agonists between surgical and transitional menopause on multiple interacting neurotransmitter (NT) pathways within specific brain regions. Using rat models of surgical and natural menopause, our study aimed to test: (1) whether specific estrogen receptor agonists produce distinct and simultaneous changes in NT pathways within specific regions of the brain; and (2) whether effects differ across two different models of menopause.
Methods: Female Sprague–Dawley adult rats were randomly assigned to receive ovariectomy (OVX) or to undergo selective and gradual ovarian follicle depletion by daily injection of 4-vinylcyclohexenediepoxide (VCD). Following the treatments, 17-β estradiol (E2), propylpyrazole triol (PPT, ERα selective agonist), diarylpropiolnitrile (DPN, ERβ selective agonist), G1 (selective GPR30 selective agonist) or vehicle were administered to the rats via a miniosmotic pump implanted subcutaneously in the dorsal neck region. Agonists were administered for either 1 week or 6 weeks (w) before rats were killed. Trunk blood was collected for determination of serum levels of estradiol, testosterone, and androstenedione using a validated LC-MS/MS assay. Tissues from the hippocampus (HPC), frontal cortex (FCX), and striatum (STR) were dissected and brain homogenates were analyzed by high-performance liquid chromatography (HPLC) with electrochemical detection to detect and quantify monoamines, metabolites and amino acids, including tryptophan (TRP) and tyrosine (TYR); dopamine (DA) and its metabolites, 3-4-dihydroxyphenylalanine (DOPAC) and homovanillic acid (HVA); norepinephrine (NE) and epinephrine (EPI) and serotonin (5-HT) and its metabolite 5-hydroxyindole acetic acid (5-HIAA). Concentrations of these substances in the samples were calculated and expressed as ng/mg protein. Turnover ratios (metabolite/monoamine) were calculated as a measure of activity.
Results: HPC OVX-1W: E2 and all the ER agonists decreased TYR. E2, PPT and G1 decreased TRP. Significant increases in DA, 5-HT, NE were detected in rats receiving DPN. Significant increases in 5-HIAA were detected after G1 treatment relative to controls. HPC OVX-6W: E2, PPT and G1 decreased DA while E2 and PPT decreased TYR. No significant change was detected in the VCD-treated groups. FCX OVX-1W: E2 and all the ER agonists increased DA and decreased both TRP and TYR. No effects were observed at 6 weeks or in VCD-treated rats. STR OVX-1W: E2 increased both DA and 5-HT. A significant increase in 5-HIAA was detected in DPN-treated rats. STR OVX-6W: HVA was significantly increased in rats treated with E2, PPT or DPN. STR VCD-1W: in contrast to HPC and FCX, E2 and all the ER agonists increased HVA. EPI was significantly elevated in rats treated with G1. STR VCD-6W: 5-HIAA was elevated in rats treated with PPT.
Conclusion: This study provides for the first time a detailed description of changes in NT pathways that occur in specific brain regions of two different and clinically relevant menopausal models. It is also the first to evaluate the effects of a selective GPR30 agonist on these neurochemical endpoints and to compare with the effects of selective ERα and ERβ agonists. Collectively, our results demonstrate that estradiol and selective ER agonist have significant and discrete neurochemical effects on the brain, which are region-specific, model-specific, and time-dependent. These differences are functionally important and likely contribute to differences in the effects of ER agonist treatments on behavioral endpoints.
Jeffrey K. Yao– Professor, University of Pittsburgh;Veterans Affairs Pittsburgh Healthcare System
Junyi Li– Ph.D.Student, University of Pittsburgh
Ziv Z. Kirshner– Ph.D.Student, University of Pittsburgh
Doug Nelson– Lab Tech, University of Pittsburgh
George. G. Dougherty– Professor, Veterans Affairs Pittsburgh Healthcare System
Robert B. Gibbs– Professor, University of Pittsburgh