ABSTRACT Experience-dependent remodeling of neural circuits enables animals to adapt their behavior to ever- changing environments. Neuronal activity-dependent molecular alterations, elicited by sensory inputs, at all levels from transcription to post-translational modifications are the basis of neural rewiring. Not surprisingly, impaired molecular responses to neuronal activity are a common hallmark of a broad range of cognitive disorders, such as depression, schizophrenia, and autism. Thus, comprehending activity-dependent molecular changes is crucial to understanding the mechanisms of these mental disorders. Over the past several years, extensive studies have made excellent strides in profiling activity-dependent changes in the transcriptome, alternative splicing, and proteome. These studies have provided significant new insights into how the brain integrates sensory inputs and reorganizes specific neural networks. However, a portion of the RNA molecule has escaped genome-wide scrutiny―the 5'-ends. Widely-used RNA-seq methods underrepresent the 5'-ends of RNAs. Existing methods to profile transcription start sites (TSS), however, predominantly represent the 5' ends; therefore, most of the transcriptome is absent in such libraries, making it challenging to connect TSS usage to dynamic mRNA expression. We recently developed a full-length nascent-transcriptome profiling method, Bru-seq-DLAF, which combines sensitive detection of TSS and nascent RNA sequencing. With Bru-seq-DLAF, we have unveiled tens of genes that dynamically change TSS upon reducing or increasing network activity. Most of these TSS led to alterations in polypeptides at their amino termini. Furthermore, the protein isoforms were conserved between mice and humans, indicating that activity-dependent TSS usage may represent a conserved new layer of gene regulation underlying synaptic plasticity. The proposed research goals are to 1) thoroughly characterize this novel phenomenon in mouse models and 2) determine the roles of activity-dependent TSS in synaptic plasticity. We hypothesize that neuronal activity-dependent TSS controls synaptic plasticity by changing the subcellular localization of proteins. Our team is uniquely poised to test this hypothesis with its unified expertise in genomics and synaptic plasticity. A positive outcome of the proposed study is to illuminate activity-dependent TSS selection as an intricate molecular mechanism for synaptic plasticity. To our knowledge, our data represent the first genome- wide characterization of TSS alterations upon extracellular stimuli in animal cells. Furthermore, many genes that undergo activity-dependent TSS selection have been implicated in mental disorders such as schizophrenia and autism spectrum disorders. Thus, the proposed study will improve the genetics of mental disorders and guide future functional studies on disease-associated genes.