In Vitro Effects Of Bisphenol A On Synaptic Morphological Plasticity Of Hippocampal Neurons

Bisphenol A (BPA), is an environmental endocrine disruptor widely used in plastic production. BPA can mimic natural hormone action, causing interference to normal endocrine system function. Considering the wide-spreading use of articles containing BPA for daily use, high BPA exposure rate in human has caught the attention of people. Over the past twenty years, a growing body of research shows that low doses of BPA can interfere with estrogen and androgen activity quickly and adversely affect the development of brain, resulting in many physiological and psychological abnormalities. Our prior studies have found that exposure to BPA in the perinatal period influenced a variety of social behavior and sex differentiation of offspring mice; BPA exposure to mice which from adolescence to early adulthood regulated hippocampal synaptic interface structure modification negatively, reducing the expression of PSD-95, with the gradual decline of learning and memory. But acute exposure to BPA in vivo promoted short-term memory of passive avoidance in rats. Further studies showed acute BPA exposure increased the density of the dendritic filopodia and dendritic branch of hippocampal neurons cultured in vitro and promoted the mobility of dendritic. Therefore, it is speculated that acute or chronic exposure to BPA may have different effects on the structure and function of neuron.Synaptic plasticity refers to the ability to change synapses forms, increase or decrease in the number and adjust function under certain condition, including synaptic transmission performance and changes in synaptic morphology. Its morphological structure changes mainly manifests in the dendritic spines, whose growth was closely related to the actin cytoskeleton remodeling. The dynamic change of depolymerization and/or polymerization of actin dendritic is the base of spines growth, movement and form changing. Along with the further research on synapses, tons of studies suggest that actin cytoskeleton plays an important role in synaptic morphological plasticity. PSD-95(post synaptic density protein95) is a representative of scaffold protein in the postsynaptic density (PSD) that can integrate signal transduction molecules and regulate synaptic activity. Synapsin I is located in the presynaptic membrane, participating in the regulation of Ca2+/calmodulin dependent neurotransmitter release through phosphorylation (p-synapsin I) and the phosphorylation, to participate in the transmission of information between neurons. It is a symbol of synaptogenesis and a reaction of synaptic transmission efficiency.In regard to this, we would investigate the impact on synaptic morphological plasticity of hippocampal neurons after chronic exposure to BPA furtherly in this study, meanwhile, pretreatments with estrogen receptor antagonist, NMDA receptor antagonist and MEK1/2inhibitors before BPA administration to explore the possible mechanisms of chronic exposure to BPA influencing synaptic morphological plasticity.Methods:Primary hippocampal neuron cultures were prepared with SD rats in24hours after birth. Cultured hippocampal neurons were exposed to DMSO (as control),10and100nM BPA from4DIV to10DIV. Then, Rhodamine labeled phalloidin, a F-actin specificity binding protein, was used to stain cultured hippocampal neurons in vitro to show neuronal cytoskeleton. The effects of hippocampal neuron morphology and the expression of F-actin in dendritic spines and dendritic filopodia chronic exposure to BPA were detected with confocal laser scanning microscope.Next, in order to explore the influence of chronic exposure to BPA on synaptic morphological plasticity, immunofluorescent double labelled staining method was used to mark presynaptic protein (synapsin I) and postsynaptic protein (PSD-95) respectively, detecting the impact of BPA on expression of synapsin I and PSD-95. Then synapsin I (green fluorescence) and PSD-95(red fluorescence) co-expression area (yellow fluorescence) was identified as the location of synapses, to count synaptic density. Fluorescence images were analyzed by Meta Morph7.7software.In order to furtherly analyze the molecular mechanism under the effect of BPA on synaptic morphological plasticity, using Western Bloting to detect the p-ERK expression level and estrogen receptor blocker (ICI182,780), NMD A receptor blocker (MK801) and MEK1/2blocker (U0126) were pretreated before BPA administration, in order to explore if the estrogen receptor, NMDA receptor and ERK pathways were involved in the chronic exposure to BPA effect on hippocampal neurons.Results:1. Chronic exposure to10nM BPA for6days decreased the density and the length of dendritic filopodia in hippocampal neuron (P<0.05), and the effect of BPA can be eliminated by estrogen receptor blocker ICI182,780and MEK1/2blocker U0126(P<0.05or P<0.01); chronic exposure to BPA (10-100nM) for6days, significantly reduced the density of dendritic spines and total spike (P<0.05or P<0.01), which can be prevented by estrogen receptor blocker ICI182,780and NMDA receptor blocker MK801.2. Chronic exposure to BPA (10-100nM) for6days, had no obvious influence on the expression of F-actin of dendritic filopodia in hippocampal neuron, while reduced the F-actin expression in dendritic spines (P<0.05or P<0.01). This effect of BPA can be eliminated by estrogen receptor blocker ICI182,780, NMDA receptor blocker MK801and MEK1/2blocker U0126(P<0.05or P<0.01).3. Chronic exposure to BPA (10～100nM) for6days inhibited the expression of synapsin I and PSD-95(P<0.05or P<0.01), and synaptic density was also decreased by10nM BPA (P<0.01), which can be inhibited by estrogen receptor blocker ICI182,780and MEK1/2blocker U0126(P<0.05or P<0.01).4. Chronic exposure to BPA (10～100nM) for6days significantly decreased p-ERK protein expression level(P<0.05or P<0.01).Conclusion:Different from the promotive effect on dendrites filopodia after acute BPA exposure, chronic exposure to bisphenol A inhibited the development of dendrites filopodia and dendritic spines in hippocampal neuron, as well as synaptogenesis. This process may be mediated by the estrogen receptors, causing actin cytoskeleton remodeling through ERK signal pathway, which facilitates morphological changes in neurons and thus affect the synaptogenesis. The influence of BPA on the development of dendritic and synaptogenesis may be one of the reasons caused a variety of behavior changes and memory impairment in vivo.