Astrocyte Modulation of Hippocampal Synaptogenesis

Primary cortical astrocytes were trypsinized from established cultures and sub-cultured in 24 well plates (250,000 cells/mL) for synaptogenesis and electrophysiology experiments, or on the underside of 0.4 μm mesh 6-well plate inserts for protein expression experiments. Wells and inserts were coated with 40 μg/mL PDL. Forty-eight hours prior to co-culture with neurons, astrocytes were serum deprived (DMEM + 0.1% bovine serum albumin and P/S) for 24 hours, and then treated with or without carbachol (0.01, 0.10, 1 mM) for an additional 24 hours. Astrocytes were washed with PBS and incubated in serum-free medium for three hours prior to co-culture with neurons. In some experiments, 30 minutes prior to the addition of carbachol, astrocytes were treated with 10 μM of the acetylcholine receptor antagonists, mecamylamine, gallamine, or 4-DAMP. After treatment washout and medium conditioning, primary hippocampal neurons (12–13 DIC), grown on glass coverslips were inverted over the pre-treated astrocyte monolayer for immunocytochemistry or electrophysiology experiments. Neurons were never in direct contact with astrocytes, nor were they exposed to astrocyte treatments. For Western blot experiments, astrocytes plated on the underside of porous inserts and their medium were transferred to 6-well plates containing primary hippocampal neurons.
To block TSP1 signaling in neurons, after astrocyte carbachol pre-treatment and washout, neurons and astrocytes were pre-treated for half hour with gabapentin (15 or 30 μM) prior to co-culturing; gabapentin remained throughout the co-culture incubation. For all co-culture experiments, astrocytes and neurons were co-incubated for 24 hours.