4.9. Amphetamine, Ketamine and Pilocarpine Treatment

To pharmacologically manipulate dopaminergic and glutamatergic brain circuits, adult male C57BL6J mice were intraperitoneally treated daily for 8 days with either D-amphetamine sulphate (3 mg/kg; TOCRIS, Bristol, UK) or ketamine (30 mg/kg; SIGMA, St. Louis, MO, United States), respectively. Fifteen minutes after the last injection, mice were sacrificed and the striatum, frontal cortex, and hippocampus were rapidly dissected out and frozen at −80 °C until use. To activate the cerebral cholinergic system, another group of mice were treated with pilocarpine (45 mg/kg, i.p.). To reduce the peripheral convulsant consequences of pilocarpine, the procedure was a first injection of lithium (LiCl, 423 mg/kg, i.p.) 20–23 h prior to the administration of methyl-scopolamine (1 mg/kg, i.p.), which was injected 30 min before the final pilocarpine administration. Status epilepticus was stopped after approximately 120 min with valium (10 mg/kg, i.p.) [48]. Ten days after pilocarpine treatment, mice were sacrificed and the hippocampus was rapidly dissected out and frozen at −80 °C until use. For all treatments, control animals were injected with saline solution.

Each brain area was homogenized in 50 mM Tris–HCl (pH 7.5) containing 10% glycerol, 1% Triton X-100, 150 mM NaCl, 100 mM NaF, 5 μM ZnCl₂, 10 mM EDTA, protease inhibitors (phenylmethylsulphonyl fluoride (2 mM), aprotinin (1 μg/mL), leupeptin (1 μg/mL), and sodium orthovanadate (1 mM)). After homogenization, samples were centrifuged at 16,000× g for 15 min at 4 °C, the supernatants were collected, and protein concentration was measured using the Pierce Coomassie Plus Protein Assay (Bradford, Thermo Fisher Scientific, Waltham, MA, USA). To quantify the VPS13A concentration in the supernatants, fifteen micrograms of protein were subjected to Western blot analysis, as explained above. Tubulin was used as a loading control.