The translatome of adult cortical axons is regulated by learning in vivo

Local protein synthesis has not been believed to occur in adult forebrain axons. We have used translating ribosome affinity purification (TRAP) to capture mRNA from the distal amygdaloid projections of auditory cortical axons. An eYFP-tagged ribosomal protein (rpL10a) was expressed in the adult rat auditory cortex via a lentiviral vector, and rats were given Pavlovian fear conditioning or control training two hours before collection of auditory cortex and amygdala. The eYFP tag alone was used as an IP control. Polysome extraction and eYFP immunoprecipitation were performed according to published protocols (Heiman et al., 2008 Cell 135:78; Kratz et al. 2014 Genome Res. 24:1396).此前学界普遍认为，成年前脑轴突中不会发生局部蛋白质合成。本研究借助翻译核糖体亲和纯化（translating ribosome affinity purification, TRAP）技术，从听觉皮层轴突的远端杏仁核投射区域捕获mRNA。研究人员通过慢病毒载体，在成年大鼠听觉皮层中表达了带有增强型黄色荧光蛋白（enhanced yellow fluorescent protein, eYFP）标签的核糖体蛋白rpL10a；在收集听觉皮层与杏仁核组织前2小时，对大鼠实施巴甫洛夫恐惧条件反射训练或对照训练，仅携带eYFP标签的载体作为免疫沉淀（immunoprecipitation, IP）对照。多聚核糖体提取与eYFP免疫沉淀实验均按照已发表的实验流程完成（Heiman等，2008，《细胞》，135:78；Kratz等，2014，《基因组研究》，24:1396）。