Primary neuron culture and transfection

Hippocampi were collected from embryonic day 18–19 rats and were incubated in HBSS containing 2.5% trypsin at 37 °C for 20 min. After rinsing with HBSS 3 times, neurons were dissociated by repeated trituration with a fire-polished Pasteur pipet, and were plated on coverslips coated with poly-l-lysine and laminin. Neurons were cultured in neurobasal medium supplemented with B27 (Invitrogen), 2 mM l-glutamine, 1% (v/v) penicillin/streptomycin (100 U/ml, Gibco), and 2% fetal bovine serum (Gibco) in a 10% CO₂ incubator. For the IRES-mediated expression of epitope (HA or Flag)-tagged aminopeptidase P1 in cultured neurons, the eGFP sequence was PCR amplified from the pEGFP-N1 vector and inserted into the pGW1 vector at the HindIII and KpnI sites. The vector was serially digested with KpnI, Mung bean nuclease, and BglII to yield upstream blunt and downstream sticky ends. The IRES sequence was PCR-amplified from the pIRES2-EGFP vector using 5′ phosphorylated forward primer and the reverse primer containing the recognition sequences for BglII, digested with BglII, and then ligated to the digested pGW1-eGFP vector. In parallel, Xpnpep1 cDNA was amplified from rat hippocampal cDNA using PCR primers containing the SalI recognition sequence (forward primer) and FLAG- or HA-EcoRI sequences (reverse primer). Epitope (HA or FLAG)-tagged Xpnpep1 was inserted into the pGW1-eGFP-IRES vector at the SalI and EcoRI sites. The sequence-verified constructs were transfected into cultured hippocampal neurons at DIV 12 using a calcium phosphate transfection kit (Takara Bio Inc. and Promega), and neurons were fixed with 4% paraformaldehyde/4% sucrose at DIV 14.

Glia-free hippocampal neuron cultures and neuron-free glial cultures were prepared from embryonic day 18–19 mice according to the protocol described above. To establish glia-free neuronal culture, dissociated hippocampal cells were cultivated in serum-free neurobasal medium, and cells were treated with the antimitotic agent AraC (3 μM; Sigma) for 8 days from DIV 12. AraC was then removed from the culture by washing the cells with fresh neuron culture medium, and neurons were harvested at DIV 21. To obtain neuron-free glial cultures, dissociated hippocampal cells were cultured in Dulbecco’s modified Eagle’s medium containing 2.5 mM glucose, 4 mM l-glutamine, 3.7 g/L sodium bicarbonate, 10% (v/v) FBS, 1 mM sodium pyruvate, and 1% (v/v) penicillin/streptomycin. The cell culture medium was replaced with fresh media once every 3 days. To remove neurons, cells were detached with 0.25% trypsin-EDTA at DIV 9 and plated in a new culture dish. The cells were harvested at DIV 19 when they reached 90–100% confluence.