Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The overall goal of this procedure is to prepare the Calot Thal cortical slice with four major auditory structures, the inferior colus, the auditory thalamus, the auditory portion of the thalamic reticular nucleus, and the auditory cortex. This is accomplished by first removing the brain from the mouse. The second step is to block the brain in preparation for slicing.

Next, cut the brain to obtain slices containing the Calot thal cortical connection. The final step is to use flavoprotein imaging to assess connectivity. Ultimately, the brain slice containing the carotal cortical connection can be used for a variety of experiments to better understand information processing in the auditory regions of the mid and four brain Visual demonstration of this method is critical as the steps to block the brain before slicing are difficult to learn because it requires a complex double angled cut, which is key to obtaining intact projections.

Demonstrating the technique will be BJ Slater, a graduate student in my laboratory, To prepare the cutting stage for slicing, cut a small piece of 3%auger of approximately one cubic centimeter to be used as a backstop for the brain, and another piece of 1.5 centimeter by 1.5 millimeter by three millimeters to be used as a bump to support the inferior colliculus. Next, glue the backstop onto the stage with cyanoacrylate adhesive, and then glue the bump on the right side of the backstop such that they form an 80 degree angle on a slide marked with two lines at 90 degrees and a diagonal line at 17 degrees from the top left to the bottom right place the brain dorsal side up at the intersecting point of the two lines. Using a razor blade, remove two to four millimeters of the rostral end of the brain to create a flat surface.

Next place the brain coddle side up on the newly created flat surface. Align the dorsal surface of the brain with a horizontal line and the midline of the brain with a vertical line marked on the slide. Then align the razor blade with a 17 degree line.

Tilt the razor at a 30 degree angle and remove approximately three millimeters of the right cortex in a double diagonal cut to mount the brain on the vibrato stage. Place a small piece of filter paper on the ventral side of the brain so that the long dimension is perpendicular to the midline. Carefully apply a small amount of cyanoacrylate adhesive to the area in front of the backstop and to the left of the bump.

Subsequently, place the double diagonally cut brain side onto the glue so that the coddle part of the brain and the hind brain are propped up onto the bump, and the right side of the brain is against the backstop. In this procedure, quickly place the cutting stage in the vibrato, build a stage with cutting solution. Next, align the blade with the top of the brain.

Remove one to 1.5 millimeters from the top of the brain. Then slice and remove more slices of 300 to 500 micrometers while moving deeper. When the inferior colliculus, the medial ate body and the lateral ICT nucleus are all visible, the dentate gyrus should appear as a C shape and the structure should be in alignment.

Obtain about two slices of 600 micrometers. These are the carotal cortical slices. After that, transfer them to the 32 degree Celsius holding chamber.

To acquire the Flavoprotein Autofluorescence image, collect the images at four hertz for 105 seconds. Illuminate the tissue with blue light 470 to 490 nanometers and capture above 515 nanometers ensuring that the image is neither too dark nor blown out. Then use electrical stimulation to activate the tissue to acquire a calcium image.

Collect images at 10 hertz for 25 seconds. Illuminate the tissue with 365 nanometers light and capture fluorescence above 510 nanometers. Again, ensuring the image is neither too dark nor blown out.

Then use electrical stimulation to activate the cells. This image shows the confirmation of carotal cortical connection in the brain slice by Flavoprotein Autofluorescence. Electrical stimulation given at 0.05 hertz to the inferior colus was imaged at four hertz and four a processed to show the power at that stimulation.

Frequency, the auditory thalamus, thalamic, reticular, nucleus, and auditory cortex are synaptically activated. Here is an unconnected slice. Electrical stimulation of the inferior colus with activation was seen only in the auditory thalamus.

The entire pathway was not captured likely due to one of the cuts being slightly out of plane. With Alam cortical projections. This image shows atypical activation of the auditory cortex.

Electrical stimulation of the inferior colus activated the auditory cortex without visible signal. In the auditory thalamus, it is likely that the thalamus was still activated, but it was not on the image surface. After watching this video, you should have a good understanding of how to obtain AAL thalamic cortical slice, and to assess its connectivity.