Primary Neuron Culture and Lentiviral Infection.

Cortical neurons were isolated from newborn C57BL/6 mice as described (11, 43). The cells were dissociated by papain (Worthington) digestion and plated on glass coverslips coated with Poly-d-lysine (Sigma). The neurons were maintained in the Neurobasal medium (Life Technologies) supplemented with B-27 (Life Technologies), glutaMAX (Life Technologies), and Ara-C (Sigma) according to Life Technologies’ instructions.

The pLKO.1-based SHC001 plasmid was obtained from University of Colorado Functional Genomics Facility. The plasmid was digested with BamHI and KpnI to remove the puromycin resistance gene (puroR). A DNA fragment encoding GFP, P2A sequence, and the Vc peptide was subcloned into the BamHI and KpnI sites such that their expression was driven by the hPGK promoter. In a control construct, GFP and P2A were subcloned in the same way except that the Vc peptide sequence was omitted. To produce lentiviral particles, lentiviral expression plasmids were transfected into HEK 293T cells along with helper plasmids—pCMV-VSVG, psPAX2, and pAdVAntage. Forty-eight hours after transfection, cell culture supernatants containing lentiviral particles were collected and filtered through 0.45-µm syringe filters (Corning). The supernatants were concentrated by centrifugation at 25,000 rpm for 1 h in a Beckman SW28 rotor. Neurons were infected with the lentiviruses at days in vitro (DIV) 4 and analyzed by electrophysiological measurements at DIV 16–20. Syntaxin-1 expression was measured using the antibodies described above. VAMP2 and Munc18-1 expression levels were detected using anti-VAMP2 antibodies (clone Cl69.1; Synaptic Systems) and anti-Munc18-1 antibodies (clone 31/Munc-18; BD Biosciences), respectively. Anti-α-tubulin antibodies (clone TU-01) were acquired from BioLegend. Neuronal morphology was examined by immunostaining using anti-MAP2 antibodies (M9942; Sigma) as described (43). All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Colorado Boulder.