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

The medial prefrontal cortex (mPFC) is important for higher cognitive functions, including working memory, decision making, and emotional control.In vivorecordings of neuronal activity in the mPFC have been achieved via invasive electrical and optical approaches. Here we apply low invasive three-photonin vivoimaging in the mPFC of the mouse at unprecedented depth. Specifically, we measure neuronal and astrocytic Ca²⁺-transient parameters in awake head-fixed mice up to a depth of 1700 µm. Furthermore, we longitudinally record dendritic spine density (0.41 ±0.07 µm^-1) deeper than 1 mm for a week. Using 1650 nm wavelength to excite red fluorescent microglia, we quantify their processes’ motility (58.9 ±2% turnover rate) at previously unreachable depths (1100 µm). We establish three-photon imaging of the mPFC enabling neuronal and glial recordings with subcellular resolution that will pave the way for novel discoveries in this brain region.