10.3389/fncir.2018.00033.s003 Ruijie Li Ruijie Li Meng Wang Meng Wang Jiwei Yao Jiwei Yao Shanshan Liang Shanshan Liang Xiang Liao Xiang Liao Mengke Yang Mengke Yang Jianxiong Zhang Jianxiong Zhang Junan Yan Junan Yan Hongbo Jia Hongbo Jia Xiaowei Chen Xiaowei Chen Xingyi Li Xingyi Li Image_2_Two-Photon Functional Imaging of the Auditory Cortex in Behaving Mice: From Neural Networks to Single Spines.TIF Frontiers 2018 two-photon Ca2+ imaging auditory cortex behaving mouse dendritic spines cell-attached recordings 2018-04-24 04:27:30 Figure https://frontiersin.figshare.com/articles/figure/Image_2_Two-Photon_Functional_Imaging_of_the_Auditory_Cortex_in_Behaving_Mice_From_Neural_Networks_to_Single_Spines_TIF/6174839 <p>In vivo two-photon Ca<sup>2+</sup> imaging is a powerful tool for recording neuronal activities during perceptual tasks and has been increasingly applied to behaving animals for acute or chronic experiments. However, the auditory cortex is not easily accessible to imaging because of the abundant temporal muscles, arteries around the ears and their lateral locations. Here, we report a protocol for two-photon Ca<sup>2+</sup> imaging in the auditory cortex of head-fixed behaving mice. By using a custom-made head fixation apparatus and a head-rotated fixation procedure, we achieved two-photon imaging and in combination with targeted cell-attached recordings of auditory cortical neurons in behaving mice. Using synthetic Ca<sup>2+</sup> indicators, we recorded the Ca<sup>2+</sup> transients at multiple scales, including neuronal populations, single neurons, dendrites and single spines, in auditory cortex during behavior. Furthermore, using genetically encoded Ca<sup>2+</sup> indicators (GECIs), we monitored the neuronal dynamics over days throughout the process of associative learning. Therefore, we achieved two-photon functional imaging at multiple scales in auditory cortex of behaving mice, which extends the tool box for investigating the neural basis of audition-related behaviors.</p>