Three-photon in vivo imaging of neurons and glia in the medial prefrontal cortex at unprecedented depth with sub-cellular resolution

The medial prefrontal cortex (mPFC) is important for higher cognitive functions. In vivo recordings of neuronal activity in the mPFC have been achieved via invasive electrical and optical approaches. Recently developed three-photon (3P) in vivo imaging enables deeper tissue penetration than two-photon microscopy. Here, we demonstrate high resolution structural and functional 3P-imaging in the mPFC of the mouse at unprecedented depth. Specifically, we perform Ca2+-imaging in awake head-fixed mice up to a depth of 1700 µm in the mPFC. We record Ca2+-transients in neurons, astrocytes and longitudinally image dendritic spines of layer 5 pyramidal neurons in the mPFC over a week. In addition, we excite red fluorescent microglia with 1650 nm wavelength and monitor their processes’ motility. Thus, we demonstrate that 3P-imaging of the mPFC enables neuronal and glial recordings with subcellular resolution below one millimeter. This will pave the way for novel experiments and discoveries in the mPFC.