Cell culture

As heterologous expression system we used tsA-201 cells (SIGMA, RRID:CVCL_2737). This cell line has been eradicated from mycoplasma at ECACC and its identity has been confirmed by STR profiling. They were grown in DMEM (Invitrogen) with 10% fetal bovine serum and 0.2% penicillin/streptomycin, passaged once a week, and incubated in 5% CO₂ at 37°C. Cells were transfected with 0.2–1 μg DNA per plasmid, plated for 24 hr after transfection, and used for experiments the next day. Lipofectamine 2000 (Invitrogen) was the chosen method of transfection. Successfully transfected cells were identified on the basis of green fluorescent protein (GFP) fluorescence.

Neurons were isolated from Sprague Dawley rats (RRID:RGD_5508397), which were handled according to guidelines approved by the University of Washington Institutional Animal Care and Use Committee. Neurons from superior cervical ganglion (SCG) were prepared from 7 to 12 week-old male rats by enzymatic digestion as described previously (Vivas et al., 2013). Isolated neurons were plated on poly-L-lysine (Sigma) coated glass chips and incubated in 5% CO₂ at 37°C in DMEM supplemented with 10% FBS and 0.2% penicillin/streptomycin.

Hippocampal neurons were prepared from newborn (P1) rats. The hippocampi of six pups were isolated and digested as described previously (Moreno et al., 2016). Neurons were plated on poly-D-lysine-coated coverslips at a density of 2 × 10⁵ cells/coverslip and maintained with MEM (Invitrogen) supplemented with 10% horse serum, 2% B27, 25 mM HEPES, 20 mM glucose, 2 mM GlutaMAX, 1 mM sodium pyruvate, and 1% penicillin/streptomycin. Neurons were incubated in 5% CO₂ at 35°C for two weeks before experiments and one-third of the medium was replaced every 5 days.