Working memory, the process through which information is transiently maintained and manipulated over a brief period, is essential for most cognitive functions^1,2,3,4. However, the mechanisms underlying the generation and evolution of working-memory neuronal representations at the population level over long timescales remain unclear. Here, to identify these mechanisms, we trained head-fixed mice to perform an olfactory delayed-association task in which the mice made decisions depending on the sequential identity of two odours separated by a 5 s delay. Optogenetic inhibition of secondary motor neurons during the late-delay and choice epochs strongly impaired the task performance of the mice. Mesoscopic calcium imaging of large neuronal populations of the secondary motor cortex (M2), retrosplenial cortex (RSA) and primary motor cortex (M1) showed that many late-delay-epoch-selective neurons emerged in M2 as the mice learned the task. Working-memory late-delay decoding accuracy substantially improved in the M2, but not in the M1 or RSA, as the mice became experts. During the early expert phase, working-memory representations during the late-delay epoch drifted across days, while the stimulus and choice representations stabilized. In contrast to single-plane layer 2/3 (L2/3) imaging, simultaneous volumetric calcium imaging of up to 73,307 M2 neurons, which included superficial L5 neurons, also revealed stabilization of late-delay working-memory representations with continued practice. Thus, delay- and choice-related activities that are essential for working-memory performance drift during learning and stabilize only after several days of expert performance.

工作记忆是在短时间内短暂维持和操纵信息的过程，对于大多数认知功能至关重要^1,2,3,4。然而，长时间尺度下群体水平上工作记忆神经元表征的产生和进化的机制仍不清楚。在这里，为了识别这些机制，我们训练头部固定的小鼠执行嗅觉延迟关联任务，其中小鼠根据延迟 5 秒的两种气味的顺序特性做出决定。在晚期延迟和选择时期次级运动神经元的光遗传学抑制严重损害了小鼠的任务表现。对次级运动皮层 (M2)、压后皮层 (RSA) 和初级运动皮层 (M1) 的大神经元群​​进行介观钙成像显示，当小鼠学习任务时，M2 中出现了许多晚期延迟时期选择性神经元。随着小鼠成为专家，M2 中的工作记忆后期延迟解码准确性显着提高，但 M1 或 RSA 中却没有显着提高。在早期专家阶段，晚延迟时期的工作记忆表征在几天内漂移，而刺激和选择表征则稳定下来。与单平面 2/3 层 (L2/3) 成像相反，对多达 73,307 个 M2 神经元（其中包括浅层 L5 神经元）进行同步体积钙成像，也揭示了通过持续练习，后期延迟工作记忆表征的稳定性。因此，对于工作记忆表现至关重要的延迟和选择相关活动在学习过程中会发生漂移，只有在几天的专家表现后才稳定下来。