In vivo flavoprotein functional imaging of the mouse auditory cortex combined with neuroanatomical tracing

In vivo functional imaging is a powerful tool for identifying the functional organization of the cerebral cortex. Flavoprotein autofluorescence imaging detects the metabolic activity of endogenous flavoprotein in neuronal tissue and thus provides an indirect measure of brain activity. As flavoprotein imaging is minimally invasive and does not require expensive equipment or reagents, it is easily combined with tract tracing methods and serves as a valuable approach for investigating functional organization of the brain. This paper provides a step-by-step protocol for flavoprotein imaging to record cortical activity in response to auditory stimuli, along with a method for injecting neuroanatomical tracers into functionally identified cortical regions. Key factors for obtaining reproducible results include stabile head fixation, a controlled dark imaging environment, maintenance of body temperature, and oxygen saturation monitoring.