Hippocampal slice preparation for patch-clamp recordings

Juvenile (13 to 19 days old) mice were used for all patch clamp recordings. For BAD reconstitution experiments, mice were injected (at postnatal day 1 or 2) in both hemispheres with 200 nl of AAV2/8.CAG.BAD.2A.mCherry. Mice were anesthetized by isoflurane inhalation and decapitated into ice-cold slicing solution (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 7 MgCl₂, 0.5 CaCl₂, 25 D-glucose, 75 sucrose and (osmolality ~340 mmol/kg). The hippocampus was isolated and mounted on a 5% agar cube which was then glued on the stage of a vibrating microtome (Campden Instuments 7000smz, Loughborough, England). Acute hippocampal slices 400 µm thick were cut in the transverse plane. Slices were then incubated at 37°C for 35 min in artificial cerebrospinal fluid (ACSF) containing (in mM): 120 NaCl, 2.5 KCl, 1 NaH₂PO₄, 26 NaHCO₃, 1.3 MgCl₂, 2.5 CaCl₂, 10 D-glucose (~300 mmol/Kg, pH 7.4). Additionally, all patch-clamp recording solutions contained 100 µM picrotoxin and 1 mM kynurenic acid to block fast synaptic transmission. These synaptic blockers were directly dissolved into ACSF immediately before experiments. After recovery, slices were stored at room temperature for 0 to 3 hr before use. All solutions were bubbled continuously with 95% O₂ and 5% CO₂.