(a) RT-qPCR template locations used to distinguish between Fos mRNA and ecRNA. (b) Modulation of Fos mRNA by KCl, AMPA, NMDA and TTX reveals overall timecourse and activity-dependence of Fos gene transcription. Fos mRNA is upregulated as soon as 45 min after KCl treatment, and peaks at 4 h following stimulation. AMPA and NMDA treatment (1 h) produced dose-dependent increases in Fos mRNA, whereas neuronal silencing with TTX decreased Fos mRNA. (c) In contrast, Fos ecRNA is induced within 30 min of neuronal depolarization with KCl, and peaks at 45 min following KCl treatment. Neuronal stimulation with KCl, AMPA and NMDA induced much lower levels of ecRNA transcription (as compared with mRNA), and neuronal inactivation with TTX did not alter Fos ecRNA transcription. (n=3–6 per group for KCl experiments, 3 per group for AMPA and NMDA experiments, and 6 per group for TTX experiments). (d,e) 4-h pretreatment with the RNAPII-dependent transcription inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) had no effect on Fos ecRNA but blocked mRNA induction after 1-hour KCl treatment, whereas pretreatment with the RNAPIII inhibitor ML-60218 had no effect on Fos mRNA but decreased ecRNA induction after KCl treatment (n=6–12 per group; mRNA one-way ANOVA, F(3,32)=78.86, P<0.0001; ecRNA one-way ANOVA, F(3,32)=66.55, P<0.0001; Tukey's post hoc test for individual comparisons). (f) Left, MeDIP experimental design. Right, DNA methylation decreases 24 h after KCl treatment in the enhancer, promoter and gene body of the Fos locus (n=8, unpaired Student's t-test; t16>2.733 and P<0.015 for each comparison). All data are expressed as mean±s.e.m. Individual comparisons, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.