GC treatment drives neurons but not astrocytes into apoptosis in neonatal and adult rats.

<p><b>A</b>–<b>L</b>, Representative confocal images showing double staining for calbindin-D28K (<b>A</b>, <b>D</b>, <b>G</b>, <b>J</b>) and phosho-H2A.X (<b>B</b>, <b>E</b>, <b>H</b>, <b>K</b>) in the dentate gyrus of control and GC-treated neonatal (<b>A</b>–<b>C, G</b>–<b>I</b>) and adult (<b>D</b>–<b>F, J</b>–<b>L</b>) rats. Hoechst 33342 staining was used to identify cell nuclei and to help delineate the SGZ and GCL. Arrows indicate the representative positive phosphor-H2A.X staining in calbindin-D28K positive neurons. <b>M</b> and <b>O</b>, are representative images showing double-staining of GFAP and phospho-H2A.X in the <i>stratum radiatum</i> of the hippocampal CA3 and CA1 subfields (CA3-r, CA1-r) in GC-treated neonatal (<b>M</b>) and adult (<b>O</b>) rats. Arrowheads indicate GFAP-positive astrocytes that were negative for phospho-H2A.X, an early marker of apoptosis. Arrows indicate the representative phosphor-H2A.X staining in GFAP-negative cells. <b>N</b> and <b>P</b> illustrate the significant increase of apoptosis in calbindin-positive neurons, but not GFAP-labeled astrocytes, in neonatal (<b>N</b>) and adult (<b>P</b>) rats treated with GC (dexamethasone, 200 µg/kg/d on days 1–3, tapering to 100 µg/kg/d on days 4–7). The counts are from all hippocampal subregions displaying positive signal for calbindin (granule cell layer of DG) or GFAP (molecular and polymorphic cell layers of DG, and the <i>strata oriens</i> and <i>radiatum</i> of CA1-CA3). <b>Q</b>, Stacking figure showing that GC treatment does not induce apoptosis in astrocytes in any hippocampal subfield, as indicated by double-staining of GFAP and phopho-H2A.X. The relative numbers (%) of phospho-H2A.X⁺/GFAP⁺ cells relative to total GFAP⁺ cells in each subfield were calculated; each value was used to create the stacking figure in which each column represents the % of apoptotic events in astrocytes in each subfield <i>vs.</i> the total number of apoptotic events in astrocytes in the whole hippocampal formation (100%). o, <i>stratum oriens</i>; m, molecular layer; p, polymorphic cell layer. r, <i>stratum radiatum</i>. * p<0.05 compared to CON. Scale bars: 20 µm.