A Protocol for Microprism‐Based Two‐Photon Imaging Of The Lateral Cortex In The Mouse Inferior Colliculus

The inferior colliculus (IC) is a central hub for auditory information processing that receives widespread convergent projections. The IC comprises three main subdivisions: the central nucleus of the IC (ICC), the dorsal cortex (DC), and the lateral cortex (LC). While the ICC receives primarily ascending auditory information, DC and LC receive major cortical and multisensory projections. The LC has repeated molecular motifs that govern its input-output relationships. However, because the LC is buried deep within a sulcus, it is difficult to image in behaving animals, making it challenging to answer questions about its functional organization. Here, we describe a protocol for coupling two-photon microscopy with a microprism to obtain cellular-resolution sagittal views of functional LC maps. We employed this novel approach to investigate neuronal responses to pure tones in relation to LC motifs. This method will not only provide new insights into the auditory system but will also permit imaging of hidden brain regions previously inaccessible by conventional means. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Craniotomy and implantation of the microprism. Basic Protocol 2: Data acquisition from sound-responsive neurons. Basic protocol 3: Confirming the microprism location. Basic Protocol 4: Analyzing the time traces of neuronal responses and generating a best-frequency tuning Map.