Early life traumas lead to neuroprotection by preconditioning mechanisms. To determine which genes and pathways are most likely involved in specific adaptive effects, immature hippocampal cultures were exposed to a single high dose of glutamate (250 μM), NMDA (100 μM), or KA (300 μM) for 48 h (5-7 DIV) based on our prior "two hit" in vitro model of preconditioning. Transcriptome profiling and immunocytochemistry of gene candidates were performed 7 days later when cultured neurons mature (14 DIV). Many genes were up- and down- regulated involving distinct Ca2+-binding protein families, G-coupled proteins, various growth factors, synaptic vesicle docking factors, certain neurotransmitter receptors, heat shock, oxidative stress, and certain anti-apoptotic Bcl-2 gene members that influence neuronal survival. Immunohistochemistry showed a marked decrease in the number of Calb1 and Calm2 positive neurons following NMDA but not after glutamate exposure whereas ryanodine and Cav1.2 voltage gated channel expression was less affected. Survivors had marked increases in Calm2 immunostaining; however, high-density neural clusters observed in controls, were depleted after NMDA and partly diminished after glutamate. While NR1 mRNA expression was decreased in the microarray, specific antibodies revealed selective loss of the NR1C1 splice variant. Calm2 which can inactivate NMDA receptors by binding to C1 but not C2 regions of its NR1 subunit suggests that loss of the C1 splice variant will reduce co-regulation with Calm2 and alter NR1 trafficking, phosphorylation, and NMDA currents following early life NMDA exposure. A dramatic reduction in the density of GABAAα5 and GABAB receptor expressing neurons was observed after NMDA exposure but immunodensity measurements were unchanged as was the expression of the GABA synthesizing enzyme, GAD, suggesting that fast inhibitory neurotransmission and response to benzodiazepines and GABAB-mediated IPSPs may be preserved in matured survivors. Selective upregulation of Chat and CNRIP was detected after glutamate treatment suggesting this condition would decrease cholinergic and excitatory neurotransmission by decreasing Ach content and CB1 interacting protein function. This decrease likely contributes to memory and attention tasks deficits that follow a single early neurological insult. Diverse changes that follow overactivation of excitatory networks of immature neurons appear long-lasting or permanent and are expected to have profound effects on network function and adaptive responses to further insult.

中文翻译：

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将未成熟的海马神经元暴露于兴奋性毒素后，揭示了独特的转录组和蛋白质调控，并诱导了常见的生存信号通路。

早期的生活创伤会通过预处理机制导致神经保护。为了确定哪些基因和途径最有可能参与特定的适应性作用，将未成熟的海马培养物暴露于单一高剂量的谷氨酸（250μM），NMDA（100μM）或KA（300μM）中48 h（5- 7 DIV）基于我们先前的“两次命中”体外预处理模型。当培养的神经元成熟时（14 DIV），在7天后进行基因候选者的转录组分析和免疫细胞化学分析。许多基因被上调和下调，涉及不同的Ca2 +结合蛋白家族，G偶联蛋白，各种生长因子，突触小泡对接因子，某些神经递质受体，热休克，氧化应激和某些抗凋亡Bcl-2基因影响神经元存活的成员。免疫组织化学显示，NMDA后Calb1和Calm2阳性神经元的数量显着减少，但谷氨酸暴露后则没有，而ryanodine和Cav1.2电压门控通道的表达受到的影响较小。幸存者的Calm2免疫染色明显增加。然而，对照组中观察到的高密度神经簇在NMDA后被耗尽，而在谷氨酸后被部分减弱。虽然微阵列中的NR1 mRNA表达下降，但特异性抗体显示NR1C1剪接变体选择性丢失。通过结合到其NR1亚基的C1而不是C2区域可以使NMDA受体失活的Calm2提示，失去C1剪接变体将减少与Calm2的共调节，并改变NMDA暴露于早期生命后的NR1转运，磷酸化和NMDA电流。NMDA暴露后，观察到表达GABAAα5和GABAB受体的神经元密度显着降低，但免疫密度测量未改变，GABA合成酶GAD的表达也未改变，这表明快速抑制性神经传递以及对苯二氮卓类和GABAB介导的IPSP的反应可能保存在成熟的幸存者中。谷氨酸处理后检测到Chat和CNRIP的选择性上调，表明这种情况将通过降低Ach含量和CB1相互作用蛋白功能来降低胆碱能和兴奋性神经传递。这种减少可能会导致记忆和注意任务的不足，而这种不足是在单个早期神经系统损伤之后发生的。