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The dynamic processes of formatting long-term memory traces in the cortex are poorly understood. The investigation of these processes requires measurements of task-evoked neuronal activities from large numbers of neurons over many days. Here, we present a two-photon imaging-based system to track eventrelated neuronal activity in thousands of neurons through the quantitative measurement of EGFP proteins expressed under the control of the EGR1 gene promoter. A recognition algorithm was developed to detect GFP-positive neurons in multiple cortical volumes and thereby to allow the reproducible tracking of 4,000 neurons in each volume for 2 mo. The analysis revealed a contextspecific response in sparse layer II neurons. The context-evoked response gradually increased during several days of training and was maintained 1 mo later. The formed traces were specifically activated by the training context and were linearly correlated with the behavioral response. Neuronal assemblies that responded to specific contexts were largely separated, indicating the sparse coding of memory-related traces in the layer II cortical circuit. I n the mammalian brain, memory traces in cortical areas are poorly understood. In contrast to the medial temporal lobe, particularly the hippocampus, which is involved in the temporary storage of declarative memories (1, 2), the neocortex is believed to store remote memories (3-6). However, remarkably little knowledge regarding the sites and dynamics of remote memory storage has been revealed at the cellular level owing to the complexity of the connections and the large number of neurons within the cortical circuit.In vivo electrophysiological recording of neuronal firing revolutionized neurobiology by linking neuronal activity with animal behavior. The small number of neurons recorded by the electrodes, however, was a limitation, as information coding and decoding may use an army of neurons forming neuronal assemblies (7,8). Efforts to record the activity of larger populations of neurons in cortical volumes have been actively pursued by either increasing the number of electrode probes (7, 9-11) or using calcium indicator-based imaging (12-15) and immediate early gene (IEG)-based reporters (16-18). The expression of IEGs is correlated with the averaged neuronal activation on external stimuli (19,20), implying that the marked neurons are involved in behavior (1,(21)(22)(23)(24)(25). Studies using in vivo imaging of IEGs have revealed cortical coding in the visual cortex and in other cortical areas, reflecting electrical activation in individual neurons (16,17). Among IEGs, the expression of early growth response protein 1 (EGR1, also known as zif268) is associated with high-frequency stimulation and the induction of long-term plasticity during learning (26,27). To measure neuronal activation in cortical circuits during a behavioral task, we used an EGR1 expression reporter mouse line in which the expression of the EGFP protein is under the control of the Egr1 gene promoter. We designed offline recordi...

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