Glass pipette

Hippocampal slices were prepared from postnatal day 6-9 C57BL/6N mice as described previously (Stoppini et al., 1991) . 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; Millipore, Darmstadt, Germany), placed on the culture medium (50% minimal essential medium [MEM], 21% Hank's balanced salt solution, 15 mM NaHCO3, 6.25 mM N-2-hydroxyethylpiperazine-N′-2ethanesulfonic 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.
For the imaging of spine morphology and sLTP, the cultured neurons were transfected by an injection of AAVs using a glass pipette (Narishige, Tokyo, Japan) after 2-6 days in the slice culture. For Ca 2+ imaging, other cultured neurons were transfected with a gene gun (Scientz Biotechnology, Ningbo, China) using 1.6 µm gold particles coated with plasmids after 8-9 days in slice culture. To make the bullets for the gene gun, gold particles (6 mg) and DNA (12 µg) were used in a 30 cm long tube.