Two-Photon Functional Imaging of the Auditory Cortex in Behaving Mice: From Neural Networks to Single Spines

In vivo two-photon Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ indicators, we recorded the Ca²⁺ transients at multiple scales, including neuronal populations, single neurons, dendrites and single spines, in auditory cortex during behavior. Furthermore, using genetically encoded Ca²⁺ 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.