Rapid Effects Of Estrogen And Bisphenol-A On N-MethyL-D-Aspartate Receptor Activity And Their Mechanisom In Development Rat Hippocampus

There is an increasing attention on the effects of estrogen and xenoestrogen on brain development. The developing brain is particularly sensitive to estrogen and is an important target organ of estrogen. Estrogen can affect neurogenesis, expression of brain-devied neurotrophic factor, synaptic plasticity, and then influenced learning and memory. Bisphenol-A (BPA), known as an environmental endocrine disruptor, can mimic the action of endogenous estradiol to influence the brain developmental process. Besides the classical estrogen action via the regulation of gene transcription in nuclei, estrogen has rapid effects via putative membrane receptors. This study explored the rapid effects of estrogen and BPA on the NMDA receptor activity of the developing hippocampus in rats and the possible mechanism underlying these effects.Methods:Male and female rats at the age of postnatal d 18 were used for the present study. Pups were kept with their mothers. The estradiol benzoate (EB) group was injected subcutaneously with EB (80μg/kg) dissolved in peanut oil. Injection volume was about 0.05 ml. The two BPA groups were given intraperitoneal injection BPA of 500 or 50μg/kg dissolved in normal saline with 10% absolute ethyl alcohol. Injection volume was about 0.2 ml. Estrogen receptor antagonist ICI 182,780 or MEK1/2 inhibitor U0126 was applying 15 min after estrogen or BPA administration in hippocampus in vivo through a stereotactic brain drug delivery system. The control group injected the vehicle. The animals were sacrificed 1h after BPA or EB treatment. And the whole intact hippocampus was then dissected out and stored at -80℃until use. Western blot was used to determine the expression of NMDA receptor subunits NR1, NR2B, ERβ, phosphorylation of NR2B, ERK1/2 and phosphorylation of ERK1/2.Results:1. Western blot analysis showed that,1 h after administration with BPA or EB alone, the expression of NMDA receptor subunits NR1, NR2B and ERP was not affected in hippocampus of the male rat on PND18. However, a significant increase in the levels of phosphorylated NR2B (p-NR2B) was detected 1 h after peripheral exposures to BPA or EB alone (P<0.01). Similar to male, no acute effects of BPA or EB alone on the expressions of NMD A receptor subunits NR1, NR2B and ERP of hippocampus were found, whereas the enhanced level of pNR2B can be observed in the female rat on postnatal d 18, revealing that there was no gender difference in the rapid effects of BPA and EB on NR2B phosphorylation, as well as on the expressions of NMDAR subunits and ERβ.2. In order to examine whether the rapid induction of phosphorylation of NR2B by EB or BPA is mediated through the membrane-associated ER, hippocampus was pretreated with ICI 182,780, an antagonist of ER before BPA administration in the present study. The results showed that pretreatment with ICI 182,780 significantly reduced the increased phosphorylation of NR2B induced by EB or BPA (P<0.01) regardless of male or female, indicating that the increased NR2B phosphorylation level induced by EB or BPA was mediated by the membrane-associated ER. Genomic actions are delayed, requiring at least several hours to be established.3. EB or BPA administration alone didn't affect the expression of ERK1/2, but significantly increased the phosphorylation level of ERK1/2 (P<0.01). It revealed the ability of EB and BPA to stimulate ERK1/2 signaling in hippocampus of the postnatal developing rat. However, co-administration of EB or BPA with ICI 182,780 significantly prevented the induction of ERK1/2 phosphorylation in hippocampus, indicating that ERK1/2 phosphorylation induced by EB or BPA was mediated by membrane-associated ER.4. U0126, a specific inhibitor of MEK1/2, was injected into hippocampus 15 min after EB or BPA treatment. Western blot analyses indicated that the inhibitor of MEK1/2 markedly reduced the EB or BPA induced phosphorylation of NR2B, as well as phosphorylation of ERK1/2. It showed that the rapid increase of phosphorylation NR2B induced by EB or BPA was dependent on the ERK1/2 activation, indicating ERK/MAPK signaling mediated the rapid action of EB and BPA on NR2B phosphorylation in hippocampus.5. Co-injection of BPA (500μg/kg) with EB (80μg/kg) did not influence the expressions of NMDA receptor subunits NR1 and NR2B in hippocampus of the male rat on PND18. While co-injection of BPA (500μg/kg) with EB (80μg/kg), the increase of NR2B phosphorylation, which induced by EB, was inhibited by approximately 50%(P<0.01). The results suggested that BPA and EB can rapidly increase NR2B phosphorylation in hippocampus of the developing rats, while BPA may attenuate the effect of EB on NR2B phosphorylation when BPA was co-treated with EB.Conclusion:Together with these results, we concluded that estrogen and BPA, via membrane-associated ERα/βmediated mechanisms, activated ERK1/2 signaling transduction pathway to modulate the phosphorylation of the NMDA receptor NR2B subunit. Our findings of the current and the previous studies, suggest that acute to the estrogen or BPA during developmental stage may have widespread effects on brain through rapidly affecting dendritic spine density and synaptic plasticity of hippocampal neurons.