Estrogens greatly affect the activity and connectivity of serotonergic neural cell populations, which extend from clusters of nuclei in the brainstem, termed the raphe nuclei, where estrogen receptor β is the most abundantly expressed estrogen receptor subtype. Estrogenic effects on the raphe nuclei are primarily important for influencing various neuropsychological behaviors, including depression, mood swings and anxiety behaviors. Because of this connection, phases of intense hormone fluctuations for instance during menopause are often associated with several mood disturbances that often reduce the quality of life of menopausal women. Accordingly, long-term use of hormone replacement therapy appeared to be the method of choice for many menopausal women to help alleviate vasomotor symptoms, which may include neuropsychological changes such as depression. However, given the limitations and number of serious health risks attributed to hormone replacement therapy, natural compounds such as phytoestrogens are receiving widespread awareness due to their occurrence in medicinal plant extracts and a wide variety of food items including dietary supplements with respective health claims. Flavonoids, particularly the isoflavones and the naringenin-type flavanones, belong to a group of polyphenolic plant-derived secondary metabolites known to possess estrogen-like bioactivities. Nevertheless, little is known about their transactivational activity and their potential to regulate endogenous gene expression of estrogen responsive genes in the raphe nuclei due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor β. Hence, a raphe nuclei-derived cell line that expresses a functional estrogen receptor β was sought as a model to investigate effects of flavonoids in vitro. In this regard, RN46A-B14 cells derived from embryonic day 13 rat medullary raphe nuclei were primarily used in this study as the main cellular model. Nonetheless, expression of endogenous estrogen receptor β in these cells was not sufficient to pursue downstream investigations of estrogen-dependent activities. To overcome this deficit, a rat raphe nuclei-derived in vitro model that overexpresses a functional estrogen receptor β was initially established (herein termed RNDA cells) by stably transducing its parent cell line, RN46A-B14 cells, with a suitable lentiviral expression vector encoding a human estrogen receptor β gene. The stable expression and the functional characterization of the transgenic receptor was confirmed by Western blot analysis and luciferase reporter gene assays, respectively. The same reporter gene assay was used to scrutinize the transactivational activity of the flavonoids in RNDA cells. Key results revealed that Genistein, Daidzein, Equol, Naringenin and 8-Prenylnaringenin demonstrated high transactivational activity in a concentration-dependent manner by stimulating luciferase expression from an estrogen responsive element-regulated reporter gene construct transiently transfected in RNDA cells. Low transactivational activity was observed in RNDA cells in response to increasing concentrations of 7-(O-prenyl)naringenin-4'-acetate. However, no transactivational activity was noticed in response to 6-(1,1-Dimethylallyl)naringenin in the studied cell model. All effects elicited by the flavonoids were antagonized by the pure estrogen receptor antagonist, Fulvestrant, indicating that all substances act by binding to and activating the transgenic ERβ. Additional effects were observed in RNDA cells in response to a co-treatment of 1 µM of either Genistein or Daidzein, but not Equol, with 10 nM 17β-Estradiol. Slight antagonistic effects were observed in the same studied cell line when either 8-Prenylnaringenin or 7-(O-prenyl)naringenin-4'-acetate, but not Naringenin or 6-(1,1-Dimethylallyl)naringenin, were co-added with 17β-Estradiol. Results from the reporter gene assays were validated on the basis of regulation of mRNA expression of estrogen responsive genes following the global assessment of 17β-Estradiol-induced gene expression in this cell line using a DNA microarray technique. Out of 212 estrogen-regulated genes with at least two-fold change of expression, six were selected according to specific features of estrogenic regulation of expression. The expression of the six selected 17β-Estradiol-regulated genes was validated using quantitative real-time PCR analysis. The regulation of mRNA expression of the selected genes in response to the tested flavonoids was then investigated in RNDA cells. Additionally, because RNDA cells encode a temperature-sensitive mutant of the Simian Virus 40 large T-antigen, their neuronal differentiation is constitutive upon shifting them from conditions promoting proliferation (permissive temperature) to differentiation (non permissive temperature). Hence, the regulation of mRNA expression of the selected genes in response to the tested flavonoids was additionally investigated as RNDA cells differentiate. In RNDA cells grown under proliferative conditions, 17β-Estradiol up-regulated mRNA expression of camello-like 5, sex determining region Y-box 18 and keratin type I cytoskeletal 19. Similar effects were observed in response to 8-Prenylnaringenin, Genistein, Daidzein and Equol. In addition, 17β-Estradiol down-regulated mRNA expression of neurofilament medium polypeptide and zinc finger DHHC-type containing 2. Similar effects were observed in response to 8-Prenylnaringenin, Naringenin, Genistein, Daidzein and Equol. Yet, no effect was observed on the regulation of mRNA expression of solute carrier family 6 member 4 in response to 17β-Estradiol or the flavonoids in RNDA cells grown under proliferative conditions. When RNDA cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards 17β-Estradiol or the flavonoids were observed. These expression studies additionally highlighted some of the genes as indicator genes for RNDA cellular differentiation. The newly established RNDA cell line should prove useful to elucidate basic physiological properties of estrogen receptor β in the raphe nuclei. The present study should serve as the basis to help shed light on molecular and cellular mechanisms following the action of phytoestrogens, endocrine disruptors or other exogenous estrogen receptor ligands in neural cell populations, particularly the raphe nuclei, for further applications within the brain.

Estrogens greatly affect the activity and connectivity of serotonergic neural cell populations, which extend from clusters of nuclei in the brainstem, termed the raphe nuclei, where estrogen receptor β is the most abundantly expressed estrogen receptor subtype. Estrogenic effects on the raphe nuclei are primarily important for influencing various neuropsychological behaviors, including depression, mood swings and anxiety behaviors. Because of this connection, phases of intense hormone fluctuations for instance during menopause are often associated with several mood disturbances that often reduce the quality of life of menopausal women. Accordingly, long-term use of hormone replacement therapy appeared to be the method of choice for many menopausal women to help alleviate vasomotor symptoms, which may include neuropsychological changes such as depression. However, given the limitations and number of serious health risks attributed to hormone replacement therapy, natural compounds such as phytoestrogens are receiving widespread awareness due to their occurrence in medicinal plant extracts and a wide variety of food items including dietary supplements with respective health claims. Flavonoids, particularly the isoflavones and the naringenin-type flavanones, belong to a group of polyphenolic plant-derived secondary metabolites known to possess estrogen-like bioactivities. Nevertheless, little is known about their transactivational activity and their potential to regulate endogenous gene expression of estrogen responsive genes in the raphe nuclei due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor β. Hence, a raphe nuclei-derived cell line that expresses a functional estrogen receptor β was sought as a model to investigate effects of flavonoids in vitro. In this regard, RN46A-B14 cells derived from embryonic day 13 rat medullary raphe nuclei were primarily used in this study as the main cellular model. Nonetheless, expression of endogenous estrogen receptor β in these cells was not sufficient to pursue downstream investigations of estrogen-dependent activities. To overcome this deficit, a rat raphe nuclei-derived in vitro model that overexpresses a functional estrogen receptor β was initially established (herein termed RNDA cells) by stably transducing its parent cell line, RN46A-B14 cells, with a suitable lentiviral expression vector encoding a human estrogen receptor β gene. The stable expression and the functional characterization of the transgenic receptor was confirmed by Western blot analysis and luciferase reporter gene assays, respectively. The same reporter gene assay was used to scrutinize the transactivational activity of the flavonoids in RNDA cells. Key results revealed that Genistein, Daidzein, Equol, Naringenin and 8-Prenylnaringenin demonstrated high transactivational activity in a concentration-dependent manner by stimulating luciferase expression from an estrogen responsive element-regulated reporter gene construct transiently transfected in RNDA cells. Low transactivational activity was observed in RNDA cells in response to increasing concentrations of 7-(O-prenyl)naringenin-4'-acetate. However, no transactivational activity was noticed in response to 6-(1,1-Dimethylallyl)naringenin in the studied cell model. All effects elicited by the flavonoids were antagonized by the pure estrogen receptor antagonist, Fulvestrant, indicating that all substances act by binding to and activating the transgenic ERβ. Additional effects were observed in RNDA cells in response to a co-treatment of 1 µM of either Genistein or Daidzein, but not Equol, with 10 nM 17β-Estradiol. Slight antagonistic effects were observed in the same studied cell line when either 8-Prenylnaringenin or 7-(O-prenyl)naringenin-4'-acetate, but not Naringenin or 6-(1,1-Dimethylallyl)naringenin, were co-added with 17β-Estradiol. Results from the reporter gene assays were validated on the basis of regulation of mRNA expression of estrogen responsive genes following the global assessment of 17β-Estradiol-induced gene expression in this cell line using a DNA microarray technique. Out of 212 estrogen-regulated genes with at least two-fold change of expression, six were selected according to specific features of estrogenic regulation of expression. The expression of the six selected 17β-Estradiol-regulated genes was validated using quantitative real-time PCR analysis. The regulation of mRNA expression of the selected genes in response to the tested flavonoids was then investigated in RNDA cells. Additionally, because RNDA cells encode a temperature-sensitive mutant of the Simian Virus 40 large T-antigen, their neuronal differentiation is constitutive upon shifting them from conditions promoting proliferation (permissive temperature) to differentiation (non permissive temperature). Hence, the regulation of mRNA expression of the selected genes in response to the tested flavonoids was additionally investigated as RNDA cells differentiate. In RNDA cells grown under proliferative conditions, 17β-Estradiol up-regulated mRNA expression of camello-like 5, sex determining region Y-box 18 and keratin type I cytoskeletal 19. Similar effects were observed in response to 8-Prenylnaringenin, Genistein, Daidzein and Equol. In addition, 17β-Estradiol down-regulated mRNA expression of neurofilament medium polypeptide and zinc finger DHHC-type containing 2. Similar effects were observed in response to 8-Prenylnaringenin, Naringenin, Genistein, Daidzein and Equol. Yet, no effect was observed on the regulation of mRNA expression of solute carrier family 6 member 4 in response to 17β-Estradiol or the flavonoids in RNDA cells grown under proliferative conditions. When RNDA cells were shifted to conditions promoting differentiation, changes in cell morphology, in mRNA expression levels and in responsiveness towards 17β-Estradiol or the flavonoids were observed. These expression studies additionally highlighted some of the genes as indicator genes for RNDA cellular differentiation. The newly established RNDA cell line should prove useful to elucidate basic physiological properties of estrogen receptor β in the raphe nuclei. The present study should serve as the basis to help shed light on molecular and cellular mechanisms following the action of phytoestrogens, endocrine disruptors or other exogenous estrogen receptor ligands in neural cell populations, particularly the raphe nuclei, for further applications within the brain.