For BrdU phenotype analysis, every 27th section through the hippocampus was triple-labeled with BrdU (CY3), NeuN (CY5), and GFAP (CY2). All BrdU-IR cells in the SGZ, approximately 20 (naïve: 27 ± 3; nondependent: 20 ± 0.7; dependent: 18 ± 2) BrdU-IR cells from each rat were scanned and analyzed for BrdU/NeuN, BrdU/GFAP or BrdU alone labeling. All labeling was visualized and analyzed using a confocal microscope (LaserSharp 2000, version 5.2, emission wavelengths 488, 568, and 647 nm; Bio-Rad Laboratories). The percent of BrdU-IR cells that were NeuN positive or GFAP positive or GFAP/NeuN negative in relation to the total number of BrdU cells were analyzed from each rat. For calculated phenotype analyses the ratio of BrdU-IR cells that were BrdU/NeuN positive and BrdU/GFAP positive in relation to the total number of BrdU cells were analyzed from each rat. The values for mature neurons, mature glia and other phenotype were computed by multiplying the ratio of double-labeled-IR cells and total number of cells from each rat. All microscopic quantification and analysis were made by an observer blind to the study.
