Preparation and Transfection of Hippocampal Slices

Hippocampal slices were prepared from P6-P9 C57BL/6N mice as described previously [44 (link)]. Briefly, the animal was deeply anesthetized with isoflurane, after which the animal was quickly decapitated, and the brain removed. The hippocampi were isolated and cut into 350 µm sections in an ice-cold dissection medium (250 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 2 mM NaHCO₃, 4 mM KCl, 5 mM MgCI₂, 1 mM CaCl₂, 10 mM D-glucose, and 248 mM sucrose). The slices were cultured on the membrane inserts (PICM0RG50; Merck, Ireland), placed on the culture medium (50% minimal essential medium, 21% Hank’s balanced salt solution, 15 mM NaHCO₃, 6.25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, 10 mM D-glucose, 1 mM L-glutamine, 0.88 mM ascorbic acid, 1 mg/mL insulin, and 25% horse serum), and incubated at 35°C in 5% CO₂.
The cultured slices at DIV3 were transfected with AAV using a glass pipette (Narishige). The preparation of AAV (titers typically ~5 × 10⁹ genome copies/µl) has been described previously in detail [45 (link),46 (link)]. For sparse labeling, AAVs encoding Clover-CaMKIIα and mCherry-Rem2 were coinfected with a low amount of AAV encoding Cre with the ratio of 100:300:4. On DIV13 or 14, two-photon FLIM-FRET experiment was carried out.