Ethanol Toxicity During Brain Development: Alterations of Excitatory Synaptic Transmission in Immature Organotypic Hippocampal Slice Cultures
Ethanol Toxicity During Brain Development: Alterations of Excitatory Synaptic Transmission in Immature Organotypic Hippocampal Slice Cultures
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DOI：
10.1111/acer.13006
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发表时间：
2016-04-01
影响因子：
3.2
通讯作者：
Pellegrini-Giampietro, Domenico E.
中科院分区：
文献类型：
--
作者：
Gerace, Elisabetta;Landucci, Elisa;Pellegrini-Giampietro, Domenico E.
发育中的大脑特别容易受到酒精的影响：怀孕期间饮酒会导致新生儿的许多身体，学习和行为障碍。研究表明，未成熟和成熟脑组织对乙醇（EtOH）毒性的敏感性不同，脑结构和功能的损害程度与突触成熟的阶段有关（100至300 mM）2天后（未成熟）或10天（成熟）的体外培养;然后从培养基中除去EtOH，24小时后，通过荧光显微镜、Western印迹、电生理学、和电子显微镜，以探讨EtOH毒性的分子机制，在发展hippocamps.ResultsEtOH撤退引起选择性CA 1锥体细胞损伤的成熟切片，但不是在未成熟的切片。在成熟切片中突触前和突触后蛋白表达的显着增加表明，切片成熟可能与新突触的发育有关。与慢性乙醇孵育7天，并从培养基中去除诱导GluA 1和GluA 2的表达水平显着下降，突触素和GluN 2A的表达显着减少后，观察到乙醇撤出。全细胞膜片钳记录结果显示，乙醇孵育7 d后，未成熟脑片海马CA 1区锥体细胞自发兴奋性突触后电流（sEPSC）频率显著降低，幅度有下降趋势。电子显微镜显示，在不成熟的切片后，慢性暴露于EtOH.ConclusionsThese结果表明，长时间的孵育与乙醇和其随后的撤出从介质中诱导损伤的兴奋性突触传递和可能的不正确形成的神经元电路在发育中的海马体外，这是暗示的机制，可能会导致胎儿酒精谱系障碍的精神发育迟滞。
BackgroundThe developing brain is particularly vulnerable to alcohol: Drinking during pregnancy can lead to a number of physical, learning, and behavioral disorders in the newborn. It has been demonstrated that immature and mature brain tissues display a differential sensitivity to ethanol (EtOH) toxicity and that cerebral structure and function are diversely impaired according to the stage of synaptic maturation.MethodsRat organotypic hippocampal slice cultures were exposed for 7days to EtOH (100 to 300mM) after 2 days (immature) or 10days (mature) of culture invitro; EtOH was then removed from the medium, and 24hours later, slices were analyzed by fluorescence microscopy, Western blotting, electrophysiology, and electron microscopy to explore the molecular mechanisms of EtOH toxicity in the developing hippocampus.ResultsEtOH withdrawal elicited a selective CA1 pyramidal cell injury in mature slices, but not in immature slices. A significant increase in the expression of pre- and postsynaptic proteins in mature slices revealed that slice maturation is presumably associated with the development of new synapses. Incubation with chronic EtOH for 7days and its removal from the medium induced a significant decrease in GluA1 and GluA2 expression levels; a significant reduction in the expression of synaptophysin and GluN2A was observed only after EtOH withdrawal. Whole-cell patch-clamp recordings showed that incubation with EtOH for 7days induced a significant decrease in spontaneous excitatory postsynaptic current (sEPSC) frequency in CA1 pyramidal cells of immature slices and a trend toward a decrease in sEPSC amplitude. Electron microscopy revealed a disorganization of neurotubuli in immature slices after chronic exposure to EtOH.ConclusionsThese results indicate that prolonged incubation with EtOH and its subsequent withdrawal from the medium induce an impairment of excitatory synaptic transmission and possibly an incorrect formation of neuronal circuits in developing hippocampus invitro, which is suggestive of mechanisms that may lead to mental retardation in fetal alcohol spectrum disorders.