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). Overall design: Forty animals were injected with eYFP-L10a or eYFP virus (20 per virus). For each virus, half of the animals were given fear conditioning and half control training (10 per group), and cortex and amygdala were collected from each animal. Samples from five animals were pooled for sequencing, resulting in two biological replicates for each of eight samples (eYFP-L10a/eYFP-conditioned/control-cortex/amygdala). Total RNA was sequenced along with TRAPed RNA for a total of 32 samples.