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

Abstract

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.

Single-neuron representation of learned complex sounds in the auditory cortex

“…One experimental session typically involved 40-100 stimulus events. The total amount of water consumed by an animal on the rig was sufficient to prevent dehydration, which was confirmed by the body weight control (<20% weight loss throughout all the experimental days) 27 .…”

“…It has been known that training experiences could enhance the overall neuronal population responsiveness in the relevant sensory cortex 12 . Thus, we next performed a set of two-photon Ca 2+ imaging experiments using a genetically encoded Ca 2+ indicator, GCaMP6f 19,27 (Supplementary Methods, Supplementary Movie 4), in A1 L2/3 to analyse single-cell responsiveness throughout our auditory training. By using widefield fluorescence imaging ( Fig.…”

“…Pressure (~600 mbar, 3 min) was applied to the glass to load dye solution. Ca 2+ imaging was performed~2 h after dye injection and lasted for up to 8 h 7,27,59 .…”

Non-invasive, opsin-free mid-infrared modulation activates cortical neurons and accelerates associative learning

“…We next employed two-photon Ca 2+ imaging [23][24][25] to visualize live neuronal population activities during MIM application ( Fig. 2a, see also Supplemental Methods).…”

“…Previous studies have reported that the activities of auditory cortical neurons are involved in auditory associative learning for mice 9,10,26 . We therefore tested whether and how MIM application in the auditory cortex might affect the learning course for a sound-licking associative learning task 24 (Fig. 3a).…”