Generation of Tilted Amygdala Slices from a Mouse Brain

Take a mouse brain immersed in an ice-cold buffer.

Remove the cerebellum and trim the anterior section containing the medial prefrontal cortex, or mPFC.

Attach the tissue to an angled agar block, which is glued onto a vibratome specimen stage.

Attach additional agar blocks to provide stability during slicing. Place the stage inside the vibratome cutting chamber filled with oxygenated ice-cold buffer.

Generate tilted slices containing the amygdala that respond to fear stimuli and neuronal projections from the mPFC, which form synaptic connections in the amygdala and regulate its activity.

The tilted slices expose the projections, allowing the study of the functional connectivity between the mPFC and the amygdala.

Cut the slices in half and place them in a chamber containing oxygenated artificial cerebrospinal fluid at room temperature to stabilize the tissue.

Incubate the slices at the physiological temperature to recover the tissue for downstream assays.

Use a scalpel to cut off the cerebellum. Then, isolate the anterior portion of the brain containing the medial prefrontal cortex. Fill the cutting chamber with ice-cold cutting solution maintained at 4 degrees Celsius using a cooling unit. Oxygenate the solution.

Blot the brain dry with filter paper. Glue the 4% agar block that has been cut at a 35-degree angle to the vibratory stage, and glue the posterior part of the brain tissue onto this block. Glue two additional agar blocks in front of and behind the brain block for stability while slicing.

Place the stage with tissue attached in the cutting chamber, and ensure it is submerged. Begin cutting the acute slices. Cut each slice in half, and transfer to an interface chamber supplied with oxygenated ACSF at room temperature. After cutting the acute amygdala slices, place the interface chamber in a water bath at 36 degrees Celsius, and allow the slices to recover for 35 to 45 minutes.