Activity-dependent dynamics of the transcription factor of cAMP-response element binding protein in cortical neurons revealed by single-molecule imaging

Transcriptional regulation is crucial for neuronal activity-dependent processes that govern neuronal circuit formation and synaptic plasticity. An intriguing question is how neuronal activity influences the spatiotemporal interactions between transcription factors and their target sites. Here, using a single-molecule imaging technique, we investigated the activity dependence of DNA binding and dissociation events of cAMPresponse element binding protein (CREB), a principal factor in activity-dependent transcription, in mouse cortical neurons. To visualize CREB at the single-molecule level, fluorescent-tagged CREB in living dissociated cortical neurons was observed by highly inclined and laminated optical sheet microscopy.Wefound that a significant fraction ofCREBspots resided in the restricted locations in the nucleus for several seconds (dissociation rate constant: 0.42 s^-1). In contrast, two mutant CREBs, which cannot bind to the cAMP-response element, scarcely exhibited long-term residence. To test the possibility that CREB dynamics depends on neuronal activity, pharmacological treatments and an optogenetic method involving channelrhodopsin-2 were applied to cultured cortical neurons. Increased neuronal activity did not appear to influence the residence time of CREB spots, but markedly increased the number of restricted locations (hot spots) where CREB spots frequently resided with long residence times (>1s).Theseresults suggest thatneuronalactivitypromotesCREB-dependenttranscriptionbyincreasing thefrequencyof CREB binding to highly localized genome locations.