Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

The overall goal of this procedure is to produce high quality acute brain slices. This is accomplished by first extracting the brain from the animal. The second step is to dissect the brain region of interest and attach it to the slicing stage while keeping it moist with slicing solution.

Next, the brain is submerged in physiological temperature slicing solution. The final step is viome slicing of the brain. Ultimately, the slices can be used in various in vitro electrophysiological experiments.

The main advantage of this technique over existing methods like ice cold slicing, is that this is simpler and produces superior results. Generally, whoever is new to this method will struggle because it requires all steps to be conducted smoothly and rapidly without unnecessary delays. We first had the idea for this method when we needed to get high quality cerebellar nuclear slices from adult animals.

The method will be demonstrated by Le Anri, a student in our laboratory Before starting this procedure. Ensure the tool, tools, and solutions are prepared beforehand. Table one shows the composition of the standard physical solution or SPS in which the brain will be cut after preparation of the SPS working solution.

According to the written protocol, equilibrate the solution with 95%oxygen and 5%carbon dioxide for 20 minutes, while the SPS Equilibrates organize the tools required for slicing. This figure shows the arrangements of the tools used for brain slicing and can be used as a visual reference when setting up the key to identify each item is contained in the written protocol. After equilibration of the SPS working solution is complete, add two milliliters of one molar calcium chloride to the SPS, then decant around 300 milliliters of the solution into a beaker.

Place the beaker containing SPS onto a heater plate or into a water bath to heat to 36 degrees Celsius. If a heater is used, be sure to include a magnetic stir bar and ensure that the solution doesn't overheat as this will cause iron precipitation and render the solution unusable. Next, boil approximately 500 milliliters of purified water in an electric kettle and add this to a beaker containing 300 milliliters of cool purified water to obtain water slightly warmer than 36 degrees Celsius after anesthetizing and decapitating the mouse.

According to institutionally approved protocols, the head is rinsed of excess blood in the beaker of warmed water. Then use small scissors to expose the foram and magnum of the skull and pull back the skin to expose the skull and better visualize the skull sutures to open the skull. Insert the tip of the small scissors through the forearm and magnum.

Immediately turn them towards the lateral side and gently cut along the lateral ledge of the parietal bone up to a location behind the eyes and the frontal parietal suture. Then turn the scissors towards the center of the skull and cut across the midline to the other side of the skull. This diagram shows the complete pattern of cuts to open the skull.

Now use fine forceps to lift the skull from the frontal parietal corner and pull it upwards diagonally to expose the brain, transfer the brain and remaining skull to a Petri dish containing warm gast.SPS. Then use a scalpel and spatula to detach the region of interest from the rest of the brain. Here, a single cut is made through both the midbrain and pons to detach the cerebellum.

The red line on this schematic illustrates the location of the cut. Rinse the cerebellum by placing it into a second small Petri dish containing warm gassed SPS. Then transfer the cerebellum to a piece of wet filter paper.

Use a scalpel to cut the brain brainstem to form a straight wide base to be glued to the cutting stage. The blue line on this diagram shows the area that will be a dear to the cutting stage. Finally, return the cut cerebellum to the small Petri dish containing warm gas SPS, and then proceed to slicing.

First, ensure that the cutting stage is dry, and then apply a small drop of cyanoacrylate glue to the middle of the stage. Then use a sula to lift the brain block from the SPS and dab with a small piece of filter paper to remove any excess liquid from the brain. Once the excess liquid is removed, slide the brain from the spatula onto the drop of glue in a single movement.

Then use a pasta by pet to apply a few drops of SPS to the brain. Place the cutting stage with the brain attached into the slicing chamber. Align the brain block so that the cerebellar cortex is facing the blade.

This ensures that the region of interest is subjected to the least amount of mechanical pressure before being sliced. Now, begin cutting the brain using the microtome according to the manufacturer's instructions, while constantly gassing the SPS in the cutting chamber throughout the slicing procedure. Maintain the temperature of the cutting chamber at 34 to 37 degrees Celsius by filling the external chamber with warmed purified water.

While slicing, gently support each slice with a soft brush to prevent them from folding. Try not to touch the region of interest and do not allow the brush to touch the vibrator blade as this will cause damage to the blade. Using a wide mouthed fire polished glass pasta pipette, transfer each slice from the slicing chamber to a holding chamber containing continuously gassed SPS and to keep at 36 degrees Celsius.

Make sure that the gassing does not result in small bubbles trapped in the chamber that could damage the slices once the brain has been fully sliced. Allow the slices to rest submerged in the holding chamber at 34 to 37 degrees Celsius for at least an hour before starting the experiment. This allows for the degradation of damaged and dying tissue resulting in a cleaner slice surface.

After the hour has passed, allow the recovery chamber temperature to cool down to room temperature. The slices are now ready to be used for experiments. This image shows a horizontal cerebellar slice of obtained with the demonstrated method.

The three major layers of the cerebellar cortex are indicated. GC is the granule layer. PC indicates the bikini cell layer and ml.

The molecular layer white matter is labeled WMA patch clamp electrode is schematically drawn onto a GOGI cell, abbreviated GOC. The scale bar represents 40 microns. This is an example recording from the GOGI cell previously shown.

The recording was obtained with a patch clamp electrode. In current clamp mode. The black color is a representative trace outta six gray traces.

Here a coronal cerebral cortical slice obtained with the demonstrated method is shown. Cortical parametal cells are shown together with a schematic drawing of a patch clamp electrode. This is an example patch clamp recording.

In current clamp mode from the parametal cell shown in the previous image, the trace shows typical behavior of a cortical parametal cell. Please pay attention during this procedure to constantly monitor the temperature.