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Abstract: γ-Secretase is a multimeric enzyme important for normal cell/neuronal proliferation, differentiation and plasticity. Determining in vivo γ-secretase expression and activity remains a challenge because its subunit proteins can exist in immature and preassembled forms, but may execute cellular roles irrelevant to γ-site cleavage. In this study, we characterized [3H]-L-685,458 as a radiotracer for the detection of active γ-secretase in adult rat brain. In vitro autoradiography indicated that [3H]-L-685,458 binding was saturatable, displaceable by peptidomimetic and small molecule γ-secretase inhibitors, and exhibited rapid association and dissociation kinetics. In cultured hippocampal slices, [3H]-L-685,458 binding density correlated with Aβ reduction following in-dish dosing of this radioligand or a non-radioactive γ-secretase inhibitor. [3H]-L-685,458 binding sites in the adult brain were differentially distributed across regions and laminas, with heavy binding localized to the olfactory glomeruli, hippocampal CA3 and cerebellar molecular layer, and moderate binding in the cerebral cortex, amygdala and selected subcortical regions. All of these regions showed labeling for presenilin-1 N-terminal fragments (PS1-NTFs). A distinct correlation of dense binding sites with abundant presence of PS1-NTFs was verified in hippocampal mossy fiber terminals and olfactory bulb glomeruli, suggestive of a rich expression of γ-secretase in the synapses at these locations that are characteristic of dynamic plasticity. Together, [3H]-L-685,458 is an excellent radiotracer for mapping active γ-secretase complex, and may serve as a useful tool for studying the enzyme in vivo and in vitro. [Copyright &y& Elsevier]