Department of Physiology and Biomedical Sciences, Seoul National University College of Medicine, Seoul, 03080, Korea.
Neuroscience Research Institute, Seoul National University Medical Research Center, Seoul, 03080, Korea.
Mol Brain. 2021 Sep 15;14(1):142. doi: 10.1186/s13041-021-00852-0.
Assessment of neural activity in the specific brain area is critical for understanding the circuit mechanisms underlying altered brain function and behaviors. A number of immediate early genes (IEGs) that are rapidly transcribed in neuronal cells in response to synaptic activity have been used as markers for neuronal activity. However, protein detection of IEGs requires translation, and the amount of newly synthesized gene product is usually insufficient to detect using western blotting, limiting their utility in western blot analysis of brain tissues for comparison of basal activity between control and genetically modified animals. Here, we show that the phosphorylation status of eukaryotic elongation factor-2 (eEF2) rapidly changes in response to synaptic and neural activities. Intraperitoneal injections of the GABA A receptor (GABAR) antagonist picrotoxin and the glycine receptor antagonist brucine rapidly dephosphorylated eEF2. Conversely, potentiation of GABARs or inhibition of AMPA receptors (AMPARs) induced rapid phosphorylation of eEF2 in both the hippocampus and forebrain of mice. Chemogenetic suppression of hippocampal principal neuron activity promoted eEF2 phosphorylation. Novel context exploration and acute restraint stress rapidly modified the phosphorylation status of hippocampal eEF2. Furthermore, the hippocampal eEF2 phosphorylation levels under basal conditions were reduced in mice exhibiting epilepsy and abnormally enhanced excitability in CA3 pyramidal neurons. Collectively, the results indicated that eEF2 phosphorylation status is sensitive to neural activity and the ratio of phosphorylated eEF2 to total eEF2 could be a molecular signature for estimating neural activity in a specific brain area.
评估特定脑区的神经活动对于理解改变的大脑功能和行为的回路机制至关重要。许多快速在神经元细胞中转录的即刻早期基因（IEGs）已被用作神经元活动的标志物。然而，IEGs 的蛋白质检测需要翻译，并且新合成的基因产物的量通常不足以通过 Western blot 检测，这限制了它们在 Western blot 分析脑组织中的应用，无法比较对照和基因修饰动物之间的基础活性。在这里，我们表明真核延伸因子-2（eEF2）的磷酸化状态会迅速响应突触和神经活动而改变。腹腔内注射 GABA A 受体（GABAR）拮抗剂印防己毒素和甘氨酸受体拮抗剂士的宁可迅速使 eEF2去磷酸化。相反，GABAR 的增强或 AMPA 受体（AMPAR）的抑制会在小鼠的海马体和前脑中迅速诱导 eEF2 的磷酸化。海马体主要神经元活动的化学遗传抑制促进了 eEF2 的磷酸化。新的环境探索和急性束缚应激迅速改变了海马体 eEF2 的磷酸化状态。此外，在癫痫发作和 CA3 锥体神经元异常兴奋性增强的小鼠中，基础条件下海马体 eEF2 的磷酸化水平降低。总的来说，这些结果表明 eEF2 的磷酸化状态对神经活动敏感，磷酸化 eEF2 与总 eEF2 的比值可以作为估计特定脑区神经活动的分子特征。
