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 vivo recordings of neuronal activity in the mPFC have been achieved via invasive electrical and optical approaches. Here we apply low invasive three-photon in vivo imaging 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. 

Methods
Deep overview in vivo z-stacks were recorded with depth increments of 1-10 µm, 0.2-0.65 µm/pixel resolution and 2-3 µs pixel dwell time. Z-stacks (spanning 10-30 µm) of dendritic spines on basal dendrites of mPFC LV/VI neurons were imaged in 900-1100 µm depth with 1 µm depth increments, 0.15 µm/pixel resolution and 2 µs pixel dwell time. For the measurement of microglial fine process motility, z-stacks of individual microglia were imaged with 2-3 µm depth increments, 0.16 µm/pixel resolution, 2 µs pixel dwell time and with 5-10 min time-intervals for a period of 30 minutes. Timelapse imaging of astrocytic Ca2+-activity in mPFC in vivo was performed at 3 Hz frame rates with 1 µm/pixel resolution and 2 µs pixel dwell time. Imaging the Ca2+-activity of layer 5 excitatory neurons in mPFC of awake mice was performed at ≥10 Hz frame rates with 1-2 µm/pixel resolution and 2 µs pixel dwell time. Recordings of the Ca2+-activity of dentate gyrus granule cells in the hippocampus of awake mice was performed at 5-10 Hz frame rates with 0.5-1.5 µm/pixel resolution and 2 µs pixel dwell time. Spinal cord in vivo z-stacks were recorded with depth increments of 3 µm, 0.25 µm/pixel resolution and 2-3 µs pixel dwell time.