Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells are derived from the ventricular zone and migrate toward our brain surface. Many genes are involved in this process, including those responsible for neurodevelopmental and psychiatric disorders. We are addressing their functions on these behaviors in this process.

Recently, we have reported that astrocyte progenitors assume two distinct migration modes, erratic and blood vessel-guided migration. These observations were made using a combination of serotype-specific labeling and time-lapse observation methods introduced in this video. To label cells, we utilized in utero electroporation system, which we develop to visualize individual cells with a high signal-to-noise ratio.

This in vivo gene transfer system also enable us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown vectors. Using this experimental system, we aim to observe the cell behaviors of neurons, glia, and blood vessels and elucidate the crosstalk between them. The findings from these studies will contribute to understanding the pathogenesis of neurodevelopmental disorders.

To begin, place the anesthetized mouse on a 38 degree Celsius plate. After making a midline skin incision, place a sterile plastic sheet on the skin, followed by a piece of sterile tissue paper and create a hole in the center. Wet the paper with sterile PBS and carefully draw out the uterine horn through the holes.

Using a micro pipette attached to an aspirator tube assembly, inject one microliter of the plasmid mix into the left or right lateral ventricle. Through the uterine wall, apply expiratory pressure. Pinch the embryo's head with a tweezer-type electrode and deliver electrical pulses using an electroporator.

After placing the uterine horn back into the abdominal cavity, suture the abdominal wall and the skin using a 5-0 silk thread. To begin, obtain the euthanized mouse administered with the appropriate plasmids. For culture dish preparation, add 1.9 milliliters of culture medium to a glass-based dish and carefully put a cell culture insert on it.

Then add 500 microliters of culture medium onto the filter and place it on ice. Remove the embryos and place them in a Petri dish containing ice-cold PBS. Under a microscope, select embryos based on the expression of green fluorescent protein or other fluorescent markers.

After dissecting the brain, place it in molten agarose gel taken in a cryo mold and roll it several times. Once the agarose solidifies at room temperature, place it on ice. Using a vibrating microtome, slice the brains coronally to a thickness of 350 micrometers and arrange them on the filter.

Then remove 600 microliters of culture medium from both inside and outside the filter cup. After five minutes, add 100 microliters of fresh culture medium to the inside of the cell culture insert. For time-lapse imaging, acquire around 10 Z-stack images using a long working distance 20x lens.

Analysis of time-lapse images of mouse brain provided the trajectories of EGFP-positive migrating cells and RFP-positive migrating cells from 2 to 21 hours.