![]() In recent years, miniaturized head-mounted optics have been extensively developed for two-photon imaging in awake behaving animals that could be relatively freely-moving ( Helmchen et al., 2001 Flusberg et al., 2005 Zong et al., 2017). Mechanical stabilization is of higher importance particularly in the Z-axis, since the imaging artifacts induced by Z-axis motions cannot be corrected by post-hoc processing. There are two general strategies of reducing motion artifacts: mechanical stabilization and post-hoc image processing. ![]() However, a major difficulty in performing two-photon imaging in awake and behaving mice is the imaging artifacts due to relative motion between the brain and the microscope. To avoid the unwanted side-effects of anesthesia, in vivo two-photon imaging studies in awake and behaving animals have become increasingly important. However, anesthesia greatly reduces overall brain activity ( Berg-Johnsen and Langmoen, 1992 Cheung et al., 2001), shifts several aspects of temporal processing ( Reid and Alonso, 1996), causes synchronization and up-down state oscillations ( Steriade et al., 1993 Sanchez-Vives and McCormick, 2000 Volgushev et al., 2006) and alters persistent activity ( Major and Tank, 2004). To maintain mechanical stability for high-resolution imaging, early in vivo two-photon Ca 2+ imaging studies were performed on rodents under general anesthesia. Two-photon imaging ( Denk et al., 1990) has been widely applied to investigate brain functions at cellular and subcellular resolution in vivo, particularly by using Ca 2+-sensitive fluorescent indicators ( Stosiek et al., 2003 Chen et al., 2013). Understanding how the brain works requires the knowledge of neuronal activities at multiple scales in the living brain during behavior. 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. Furthermore, using genetically encoded Ca 2+ indicators (GECIs), we monitored the neuronal dynamics over days throughout the process of associative learning. Using synthetic Ca 2+ indicators, we recorded the Ca 2+ transients at multiple scales, including neuronal populations, single neurons, dendrites and single spines, in auditory cortex during behavior. 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. Here, we report a protocol for two-photon Ca 2+ imaging in the auditory cortex of head-fixed behaving mice. However, the auditory cortex is not easily accessible to imaging because of the abundant temporal muscles, arteries around the ears and their lateral locations. In vivo two-photon Ca 2+ 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. 4CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.3Brain Research Instrument Innovation Center, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu, China.2Department of Urology, Institute of Urinary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.1Brain Research Center, Third Military Medical University, Chongqing, China.Ruijie Li 1† Meng Wang 1† Jiwei Yao 2† Shanshan Liang 1 Xiang Liao 1 Mengke Yang 3 Jianxiong Zhang 1 Junan Yan 2 Hongbo Jia 3 Xiaowei Chen 1,4 * Xingyi Li 1 *
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