Rat Hippocampal Neuron Cultures

Hippocampi were dissected from embryonic day (E) 16 to E18 rat embryos obtained from pregnant Sprague-Dawley dams (Envigo). All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Columbia University. Cell dissociation was performed using TripLE Express. Neurons (50,000 to 60,000 per microfluidic chamber) were plated on poly-d-lysine (0.1 mg ml⁻¹; MilliporeSigma)– and laminin (2 μg ml⁻¹; Bio-Techne)–coated substrates and grown in Neurobasal supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 1 mM sodium pyruvate, and antibiotics (50 U ml⁻¹ penicillin-streptomycin). Tripartite microfluidic chambers were produced with Dow Corning Sylgard 184 Silicone Encapsulant Clear Kit (10:1 mix ratio; Ellsworth Adhesives) cured at ~70°C for at least 4 hours following published protocols (19 (link)). Chamber design incorporated two sets of 200-μm-long microgroove barriers to exclude the crossing of cell bodies and dendrites. After 24 hours, the medium was changed to Neurobasal containing 1× B27 and 2 mM l-glutamine. Subsequent medium changes (half volumes) were performed at day in vitro 4 (DIV4) and thereafter every 3 to 5 days. To prevent glial cell proliferation, medium changes included 5-flurodeoxyuridine and uridine (final concentration: 10 mM; MilliporeSigma) after DIV4. Neuronal cultures were grown in a 37°C, 5% CO₂ humidified atmosphere until DIV12 to DIV14. Whenever stated, axonal or cell body compartments were treated with 30 μM ciliobrevin A (R&D Systems), 100 nM emetine (MilliporeSigma), or 10/15 μM nelfinavir (MilliporeSigma). Dovitinib (Selleck Chemicals) was bath-applied at 40 nM 30 min before Aβ₄₂ stimulation. Unless otherwise specified, reagents were purchased from Thermo Fisher Scientific.