In vivo two-photon imaging of neurons and glia in the mouse spinal cord

Two-photon imaging of the nervous system is now used extensively for visualizing brain dynamics and signal activities. To date, scientists have focused on the analysis either of gray matter forebrain structures, such as the cortex and cerebellum, or they have investigated muscle innervation of peripheral nerves. The spinal cord is an ideal structure to use for imaging central nervous system white matter. The dorsal columns formed by myelinated sensory axons are located directly at the surface of the spinal cord underneath the pia mater. This protocol describes a method for imaging neuronal fibers and neighboring glial cells in transgenic mice using cell type-specific fluorescent protein expression and two-photon laser-scanning microscopy (2pLSM). Depending on how the mice are prepared, single imaging can be performed, or the spinal cord can be imaged repetitively over multiple days, with time for the mouse to recover between imaging sessions.