We used mouse HT22 hippocampal cells (the kind gift of David Schubert, The Salk Institute, La Jolla, CA, USA). This cell line 26 was selected because of its high concentration of glucocorticoid receptors 27 and because the hippocampus expresses relatively high levels of CRH, especially after stimulation 28. HT22 cells were propagated in DMEM (Gibco) supplemented with 10% fetal bovine serum and maintained at 37∞C in a humidified incubator (5% CO2). Cells were seeded at a density of 5×104/well in a 12-well tissue culture plate. When 60–70% confluent, either the -2232 C or -2232G rhCRH pDsRed reporter constructs (1 μg) was co-transfected with a GFP reporter (0.5μg pGlow-TOPO, Invitrogen, Carlsbad, CA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions. Cells were treated with 30μM Forskolin (Sigma) or 30μM Forskolin + Cortisol (Sigma) 20, 27, 29, 12 h following transfection. We selected a low cortisol dose (1 nM) for this study based on reports that this dose produced Type I GR activation, without Type II GR activation or TypeI:Type II dimerization, in the HT22 cell line 30. This
