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For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo , pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors. [Display omitted] • Establishing an optogenetic-based quantitative proteomic screening platform • Unbiased screening identified that neural activity decreases pPDH • pPDH level negatively correlates with neural activity in vitro and in vivo • pPDH as a novel inverse activity marker (IAM) to track reduced neuronal activity Yang et al. applied an in vitro optogenetics-based proteomic screening platform to identify that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlates with neuronal activity both in vitro and in vivo. pPDH can serve as a trackable endogenous inverse activity marker (IAM) for post hoc identification of inhibited neurons. [ABSTRACT FROM AUTHOR]