| Neurons are the basic structural and functional units of brain, including the cell bodies and apophyses, and apophyses can be divided into axons and dendrites. In the early developmental stages, the dendrites first sprout filopodia from the cell body, when the conditions are mature, the filopodia can develop into dendritic spines, then take part in the formantion of synapses, so dendritic development is an important process in brain development, whose structure and characteristics determine the efficiency of synapic transmission of information. RhoA, Racl and Cdc42 are the upstream molecules which are closely related with the development of dendritic structure, among them Rho A negatively control the growth of axons and dendrites, Racl and Cdc42 are critically important for axon outgrowth and dendritic branching, and they can induce pseudopodia or filopodia from actin-rich surfaces and co-regulate the motility of dendrites in neurons. The estrogen can affect brain development by estrogen receptors (ERs), including the formation of neural circuits, dendritic extension and branching, synapse formation, neurotransmitter and neuropeptide expression, apoptosis and survival of cell.Bisphenol A (BPA) is an endocrine disruptor, which has weak estrogenic activity, and can interfere endogenous estrogen activity by binding to estrogen receptors. Because the endogenous estrogen can affect the dendritic development and synaptogenesis, so we speculate that BPA with the estrogenic activity can disturb the structure of dendritic development. It's well known that NMDA receptor is a dual-gated channel which is controlled by the membrane potential and neurotransmitter, and it plays an important role in synaptic transmission, regulation of synaptic plasticity and learning and memory, so it is the one of most important receptors in the central nervous system. When the estrongen binds to membrane estrogen receptor, it activates the NMDA receptor to regulate dendritic development and synaptic plasticity. MAPK/ERK signal pathway is important in brain, ERK is an important regulator for the structure formation and changes of dendritic spines in the hippocampal neurons. It also found that estrogen could activate ERK2/MAPK signal pathway, and ERK2 activity levels changed with estrogen levels in the rat estrous cycle.In summary, in this study, we researched the developmental impact of BPA on the dendritic structure by in vitro cultured hippocampal neurons that were chronicly exposure to BPA, and explored the possible mechanisms of dendritic development after exposure to BPA by adding MK-801, ICI 182,780 and U0126.Methods:Hippocampal neurons were infected by adding crude AdV-lysate (the recombinant adenovirus encoding enhanced green fluorescent protein (GFP), AdV-EGFP) at day 5 in vitro (DIV5), and observed 24-48h after the infection. We set five groups:control, E2 (10μm) and BPA (10,100,1000 nM BPA,0.01% DMSO as vehicle). We added the drugs at day 6 in vitro (DIV6) and captured images of live cells every 2min for 20min by inverted fluorescent microscope after 24 hours. We measured filopodial motility and dendritic filopodia density and the total length of the dendritic by Image Pro-Plus software.In order to further study whether the ER,NMDAR and ERK1/2 take part in the dendritic development, we used 10 uM ICI 182,780 (estrogen receptor antagonist), MK-801 (NMDA receptor antagonist) and U0126 (MEK1/2 antagonist) to pretreat the neurons for 0.5h, then added the BPA (100nM). The control group (DMSO), BPA (100nM) alone exposed group and the E2(100nM) were set at the same time. In order to analyse molecular mechanisms of dendritic development after exposure to BPA, we used Western Bloting to detect the expression of RhoA, Racl and Cdc42, which are the upstream molecules related to dendritic develoment. To observe whether BPA changed the expression of NMD A receptor NR1, NR2B and their phosphorylated levels and the ERB receptor, we detected their expression by immunocytochemical staining. Quantity one and Image-Pro Plus were used to measured the protien bands and immunohistochemical images, respectively. Finally, SPSS 15.0 software was used to analyse experimental data.Results:1. Exposure to BPA (10-100 nM) after 24h significantly promoted the motility and density of dendritic filopodia (P<0.001) and the total length of the dendritic (P<0.05, P<0.01), and E2 had the same trends. This effect could be inhibited by antagonist of ERs, NMDAR and MEK1/2 (P<0.05, P<0.01 or P<0.001).2. BPA (10～1000 nM) significantly up-regulated the expression of Racl/Cdc42 protein (P<0.01 or P<0.001), and reduced RhoA protein (P<0.05 or P<0.01). Exposure to BPA (100nM), ICI 182,780 and MK-801 inhibited the up-regulation of Racl/Cdc42 (P<0.01 or P<0.05), while ICI182,780, MK-801 and U0126 significantly inhibited the down-regulation of RhoA (P<0.01).3. BPA (100nM) significantly up-regulated the expression of ERβprotein (P<0.001), this effect was inhibited by ICI 182,780 and U0126 (P<0.001).4. BPA (100nM) significantly down-regulated the expression of NMDA receptor NR1 and NR2B (P<0.01 or P<0.001), and this effect was significantly inhibited by ICI 182,780 and U0126 (P<0.01 or P<0.05). BPA(100nM) significantly up-regulated the phosphorylation of NR1 and NR2B (P<0.05), and this effect was significantly inhibited by ICI 182,780 (P<0.05).Conclusions:Chronic exposure to BPA can promoted the motility and density of dendritic filopodia and the total length of the dendritic by affecting RhoA, Racl/Cdc42 which were closely related with the development of dendritic structure. This process may be mediated by the estrogen receptors,and via NMDA receptors activated by the ERK1/2 signaling pathway on the dendritic surface.Then the dendritic morphology was affectde by the upstream molecules of dendritic development. |
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