Methods for culturing hippocampal networks with different E/I cellular compositions. (A) Hippocampal neurons are dissected from mouse embryos at day 17 of gestation. Females from mouse model GAD II-IRES-Cre are mated with males from mouse model Ai9-tdTomato-lox, yielding GABAergic cells that coexpress tdTomato with GAD II. FACS is used to sort tdTomato-expressing cells (GAD+ population, GABAergic cells) from those not expressing (GAD−). GAD+ and GAD− populations are then seeded at different ratios in microfluidic growth chambers on a glial cell layer. (B) Examples for three different cultures with seeding percentages of 0, 50, and 100% GABAergic cells and corresponding experimentally observed percentages of 4, 49, and 91%. Imaging was performed on a SP5 Leica confocal microscope with the fluorescent measurement overlaid on a transmitted light image.