Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

The clarity technique first described in 2013 is a tissue clearing technique that enables the samplings of larger quantities of cells, blood vessels, so even proteins such a single cell resolution in thick brain slices. This technique enables the study of intact microscopic structures by making the entire brain transparent. This protocol is a simple chip and straightforward pipeline for tissue clear.

This technique can be used for clearing tissues in other systems in addition to brain tissues. To begin, seal the tube and cap using parafilm and puncture two holes in the cap. Then transfer nitrogen gas from the tank using a flexible pipe with five millimeter internal diameter and a 19 gauge needle connected at its end.

Start degassing by attaching the pipe to the tube and allowing gas exchange for 30 minutes at room temperature. Detach the pipe and immediately seal the holes with modeling clay. Then transfer the degas sealed tubes to a 37 degree Celsius bath for 3 1/2 hours to polymerize the hydrogel solution.

Take the brain out from the tube. Using laboratory wipes, remove the polymerized hydrogel around the brain ensuring that no residual gel remains attached to the surface of the brain. Divide the brain into thick slices that contain all areas of interest.

Put the slices in the first clearing solution and incubate at 37 degrees Celsius with rotation at 70 RPM for 24 hours. In the meantime, prepare a perforated tube to place the brain. Put the perforated tube in a beaker filled with second clearing solution on a stirring device.

Then place the brain in the beaker and seal it with aluminum foil to prevent bleaching. After the tissue turns transparent, transfer the brain into PBST in incubate at 37 degrees Celsius with rotation at 70 RPM for 24 hours. Replace the solution with fresh PBST and continue the incubation for another 24 hours.

Then transfer the brain to PBS at room temperature for 24 hours. After 24 hours, remove the brain from PBS and transfer it to refractive index matching solution. Incubate the brain at 37 degrees Celsius overnight.

First, place the sample in the middle of the slide. Using hot glue, create walls on the edges of the slide, almost as high as the tissue. Make sure to leave a small gap at one of the corners.

As the hot glue layer approaches the height of the brain slice, apply one to two drops of the refractive index matching solution on the sample to moisten the upper surface and prevent bubble formation. While the hot glue is still liquid, seal the top with a cover slip, placing it as evenly as possible then fill the chamber with the refractive index matching solution. Close the gap with hot glue.

If the hot glue walls extend beyond the borders of the slide, cut the extending edges. If an oil immersion objective is used for imaging, add another two to three millimeters of glue to the walls above the cover slip to retain the immersion solution. After this protocol, the brain tissue will be cleared.

Clear brain slices were prepared using this protocol to visualize populations of astrocytes and neurons in the CA1 region of mouse hippocampus. All astrocytes expressed tdTomato and excitatory neurons expressed H2B-GFP in their nuclei. The chamber prepared for the clarified tissue was optimal for imaging under a two photon or confocal microscope.

Using a two photon microscope, over 300 astrocytes were observed in a cleared section of the CA1 region of the mouse hippocampus. The hippocampal astrocytes in red and pyramidal cells, somata in green were visible and thick, transparent tissue representing the spatial proximity between these two cell types. Using a scanning laser confocal microscope, axonal bundles from excitatory neurons were traced across an entire hemisphere.

Green bundles were traced from the dorsal hippocampus toward the super mamillary bodies. The red bundles were traced from the mamillary bodies toward their origin at the vent hippocampus. Factors, such as temperature, concentrations and incubation time influenced the outcome of the clarity procedure.

Therefore, preciseness in every step of the protocol is vital for the success of achieving clear tissue. Using this technique, we measure the distances between neurons and astrocytes in several brain structures. This protocol allowed analysis of two to three order of magnitude more cells than in previous studies.