Generation of Tilted Amygdala Slices from a Mouse Brain

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Preparation of Acute Slices

1. Prepare artificial cerebrospinal fluid (ACSF), cutting solution, tools (scissors, scalpel, forceps, spatula, Pasteur pipette), and agar blocks.
   Note: 1 L of artificial cerebrospinal fluid (ACSF) is required per experiment, and is prepared by dissolving chemicals in double-distilled H₂O as previously published. The cutting solution is prepared by supplementing 200 ml of ACSF with 0.87 ml of 2 M MgSO₄ stock solution.
2. Oxygenate ACSF and cutting solution throughout the experiment with 95% O₂ and 5% CO_2.
3. Deeply anesthetize mouse using a small animal anesthesia machine using isoflurane (3% in oxygen). Check anesthesia depth using limb withdrawal reflex before continuing.
4. Decapitate mouse using large scissors and immediately chill head in ice-cold cutting solution.
5. Open skull by a single midline incision from caudal to rostral and gently push pieces of skull to the sides using forceps. Rapidly remove brain by gently lifting it out of the skull using a small, rounded spatula. Cut off cerebellum with scalpel. Place brain in ice-cold cutting solution.
6. For medial prefrontal cortex or mPFC injection sites: cut off anterior part of the brain (containing mPFC) using a scalpel and put in ice-cold cutting solution until slicing.
7. Remove excess cutting solution with filter paper and glue the posterior part of the brain onto the vibratome stage.
8. For tilted amygdala slices, glue the brain on an agar block (4%) cut at a 35° angle (Figure 1D, middle). For coronal amygdala slices, medial geniculate nucleus/ posterior intralaminar thalamic nucleus (MGm/PIN) injection sites and mPFC injection sites, glue the brain directly on stage (Figure 1D, left and right). Place an additional agar block behind brain for stability while slicing.
9. Place the stage in cutting chamber with ice-cold oxygenated cutting solution that is maintained at 4 °C using a cooling unit. Prepare acute slices of the amygdala (320 µm) using a sapphire blade. Place slices in an interface chamber supplied with oxygenated ACSF at room temperature (RT).
10. After preparation of acute amygdala slices, place the interface chamber in a water bath at 36 °C to recover slices for 35 – 45 min. Subsequently, return the interface chamber to RT.
11. During commencement of step 1.10, cut slices of the injection sites as described before for acute amygdala slices (steps 1.8 – 1.9). Recovery of slices in the water bath is not required here. Optional: To quickly estimate injection site location, observe slices on a stereoscope equipped with a fluorescent lamp and appropriate filter sets.