Abstract:The organization in the primary auditory cortex (Au1) is critical to the basic function of auditory information processing and integration. However, recent mapping experiments using in vivo two-photon imaging with different Ca 2+ indicators have reached controversial conclusions on this topic, possibly because of the different sensitivities and properties of the indicators used. Therefore, it is essential to identify a reliable Ca 2+ indicator for use in in vivo functional imaging of the Au1, to understand its functional organization. Here, we demonstrate that a previously reported indicator, Cal-520, performs well in both anesthetized and awake conditions. Cal-520 shows a sufficient sensitivity for the detection of single action potentials, and a high signal-to-noise ratio. Cal-520 reliably reported on both spontaneous and sound-evoked neuronal activity in anesthetized and awake mice. After testing with pure tones at a range of frequencies, we confirmed the local heterogeneity of the functional organization of the mouse Au1. Therefore, Cal-520 is a reliable and useful Ca 2+ indicator for in vivo functional imaging of the Au1.