MAPK Signaling Determines Anxiety in the Juvenile Mouse Brain but Depression-like Behavior in Adults. Mus musculus

MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In these mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype. Our results reveal the differential function of MAPK signaling in juvenile and adult life phases and emphasize the early postnatal period as critical for the determination of anxiety in adults. Moreover, these results validate inducible gene inactivation as new valuable approach, allowing to discriminate between gene function in the adult and the developing postnatal brain. Overall design: Five male Braf-cko, six male homozygous Braf-flox littermates, six male heterozygous CamkII-Cre, and six male wildtype littermates were killed with CO2, the complete hippocampal tissue was prepared, and total RNA was extracted with the Trizol protocol. The integrity and quality of the RNA samples were analyzed with an RNA electrophoresis chip (RNA 6000 Nano Kit, Agilent, Boeblingen, Germany). RNA samples of high integrity and quality (RIN ≥ 7.5) were further processed with the TotalPrep RNA Amplification Kit (Ambion, Austin, TX, USA) and hybridized onto MouseWG-6 v1.1 Expression Bead-Chips (Illumina, San Diego, CA, USA) following manufacturer’s instructions. Data were analyzed using the software R (used packages: beadarray, limma, and vsn).

丝裂原活化蛋白激酶（MAP kinase, MAPK）信号通路已被证实参与大脑发育、长期记忆形成以及抗抑郁药物响应过程。我们借助诱导型Braf敲除小鼠（inducible Braf knockout mice），阐明了神经元MAPK信号通路在情绪行为调控中的全新功能。成年阶段诱导敲除Braf的小鼠，其焦虑行为表现正常，但抑郁样行为增强，这与药理学研究结果相符。与之相反，幼年脑内Braf失活的小鼠，成年后抑郁样行为无异常，但焦虑水平显著降低。在该突变体小鼠中，我们未观察到γ-氨基丁酸能神经传递（GABAergic neurotransmission）的异常，但在齿状回（dentate gyrus）内发现神经元树突分支明显减少。对海马体组织的基因表达分析显示，共有9个下调的MAPK靶基因，它们可能是导致该突变体表型的候选调控因子。本研究结果揭示了MAPK信号通路在幼年与成年生命阶段的差异化功能，并强调出生后早期是决定成年个体焦虑水平的关键时期。此外，本研究验证了诱导型基因失活技术作为一种极具价值的新方法，可用于区分基因在成年个体与发育中出生后脑内的不同功能。
实验整体设计：共纳入4组雄性小鼠：5只Braf条件性敲除（Braf-cko）小鼠、6只纯合Braf-flox同窝对照小鼠、6只CamkII-Cre杂合子小鼠，以及6只野生型同窝对照小鼠。全部小鼠经CO₂安乐死后，分离获取完整海马体组织，采用Trizol法提取总RNA。使用RNA电泳芯片（RNA 6000 Nano试剂盒，安捷伦，德国伯布林根）检测RNA样品的完整性与质量。对于完整性与质量达标（RNA完整性数值RIN ≥7.5）的RNA样品，采用TotalPrep RNA扩增试剂盒（Ambion，美国德克萨斯州奥斯汀市）进行扩增处理，并按照制造商说明书将样品杂交至MouseWG-6 v1.1表达微珠芯片（Illumina，美国加利福尼亚州圣迭戈市）。数据分析采用R软件完成，所使用的R包包括beadarray、limma与vsn。