Primary Cultured Hippocampal Neuron Protocol

As previously described (Buscemi et al., 2007 (link); Hui et al., 2012b (link)), primary cultured hippocampal neurons were prepared from Sprague-Dawley rats. Pregnant dams at embryonic day 18 were sacrificed by asphyxiation with CO₂. After the fetuses were removed and decapitated, meninges-free hippocampi were isolated, trypsinized, and seeded onto 35-mm² poly-D-lysine coated glass-bottom tissue culture dishes. Neurons grown in Neurobasal™ medium containing L-glutamine, antibiotic/antimycotic and B₂₇ supplement were maintained in an incubator (37°C, 5% CO₂) for 10–14 days, at which time they were taken for experimentation. Neurons were treated with 12 different ART drugs for up to 48 hrs during which time the media was not changed. Measurements of endolysosomal pH, endolysosome sizes, and Aβ levels were conducted using separate dishes of cells. Typically, the purity of the neuronal cultures was more than 95% as determined by immunostaining of neurons with mouse anti-NeuN or goat anti-MAP2 antibodies (Millipore), and of astrocytes with mouse anti-GFAP antibody (Sigma).