Primary hippocampal neurons

Postnatal (P1–P2) pups from Wistar rats were decapitated and the brains were extracted. The hippocampi were isolated, washed in Hank’s balanced salt solution (HBSS; Invitrogen, Darmstadt, Germany) and incubated in enzymatic digestion solution for 1 h at room temperature. After washing in HBSS the hippocampi were incubated in inactivation solution for 15 min. After another washing step in Neurobasal A, neurons were mechanically dissociated by pipetting. In all, 15,000 neurons per well were added to the plating medium (MEM, 10% horse serum, 3.3 mM glucose, 2 mM glutamine) in PLL-coated 96-multiple glass bottom well plates (SensoPlate, Greiner Bio-One International GmbH, Kremsmünster, Austria) and kept at 37 °C, 5% CO₂. After ~ 1 h, when the neurons adhered to the glass bottom, the plating medium was exchanged with 100 μL Neurobasal A and plates were further cultured at 37 °C, 5% CO₂. To maintain healthy cultures, 50 μL of medium was removed every second day and replaced with 50 μL of fresh Neurobasal A. The primary rat hippocampal neuron cultures were prepared with minor modifications from the original protocol⁷⁷. Five days after plating, neurons were infected with an AAV (88e5 TU AAV/5 μl) containing the sequence for NbSyn87 fused to mCherry and NLS sequences (NbSyn87-mCherry-NLS).