Astrocytes are spared from GC-triggered apoptosis in primary hippocampal cultures.

<p>Hippocampal cultures were genetically tagged with either Tα-tubulin-GFP or GFAP-GFP plasmids, to identify neurons and astrocytes, respectively. Approximately 50% of astrocytes in typical cultures displayed GR immunoreactivity (<b>A–C</b>, examples shown by arrowheads). After exposure to GC or vehicle, apoptosis in the different cell populations was visualized by TUNEL and Hoechst 33342 histochemistry (<b>D–F and G–I</b>). Solid arrowheads exemplify GFP⁺ cells that entered apoptosis after GC treatment; open arrowheads indicate non-apoptotic GFP-transfected cells. Numerical data (mean ± SD) from analysis of TUNEL staining in either all cells in culture, Tα-tubulin-GFP or GFAP-GFAP sub-populations are depicted in (<b>J</b>). * p<0.05 vs. CON, # p<0.05 vs. DEX. Scale bars: 50 µm in <b>A–C</b> and 20 µm in <b>D–I</b>.</p

Insights into potential mechanisms underlying astrocytic resistance to GC-induced apoptosis.

<p>Astrocytes show less susceptibility to GC- (<b>A</b>) and staurosporine (STA)-induced (<b>B</b>) apoptosis, as compared to neuronal cells. Both neurons and astrocytes respond to GC treatment with a significant increase of ROS (measured by fluorescent DHE nuclear translocation) (<b>C</b>); note that, as compared to neurons, astrocytes generate markedly lower levels of ROS under basal conditions and after GC treatment. The ratios of expression of mRNAs for pro- vs. anti-apoptotic members of the Bcl2 family (<i>bax</i> vs. <i>bcl-X_L</i> and <i>bcl-2</i>) are different in neurons and astrocytes (<b>D and E</b>); mRNA levels were determined by qPCR. Neurons and astrocytes also respond differentially to GC treatment in terms of their activated caspase 3 responses (measured by immunoblotting) (<b>F</b>), with astrocytes showing smaller increases in levels of activated caspase 3. Numerical data are shown as mean ± SD. * p<0.05 vs. neuron CON; # p<0.05 vs. astrocyte CON; + p<0.05 GC-treated neurons vs. GC-treated astrocytes. Scale bars: 25 µm.</p

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.</p

Three-dimensional analysis of a neuron double-labeled with BrdU and Golgi-Cox.

<p>Three-dimensional morphometric analysis of a Golgi-impregnated neuron co-labeled with BrdU (depicted with green dot) and Dapi (staining of nuclei depicted with blue dots) (<b>A</b>) using computer-assisted reconstructions. Neuronal reconstruction was performed using a motorized microscope and Neurolucida software with the automatic AutoNeuron extension module directly from the confocal image (red colour in <b>B</b> and <b>C</b>) and using manual reconstruction (white colour in <b>D</b>). Different colours on <b>C</b> depict distinct dendritic branches and black arrows in <b>B</b> and <b>D</b> depict the differences detected between the automatic and manual reconstructions. Image <b>E</b> depicts a neuronal segment showing all different spine types (mushroom, thin, wide and ramified). Scale bars: 50 µm.</p

The BrdU/Ki67 ratio differs throughout the subpendymal zone.

<p>The SEZ total BrdU/Ki67 ratio is represented for the anterior-posterior <b>(A)</b> and dorsal-ventral axes <b>(B)</b>. For the different dorsal-ventral regions the BrdU/Ki67 ratios were assessed at the anterior, intermediate and posterior levels <b>(C)</b>. The threshold value for statistical significance was set at 0.05 (* p<0.05).</p

Anti-proliferative actions of GC in astrocytes are GR-dependent.

<p><b>A,</b> Representative immunostaining of GFAP in the enriched astrocytic culture. <b>B–E</b>, are representative images showing BrdU incorporation in control (<b>B, C</b>) and GC-treated (dexamethasone, at 10⁻⁵ M for 48 h in medium with charcoal-stripped serum) astrocytes (<b>D, E</b>). Hoechst 33342 counterstaining demonstrates comparable cell densities. BrdU (20 µM) was added to cultures 12 h before fixation. <b>F,</b> shows that the anti-proliferative actions of GC are counteracted by addition of the GR antagonist, RU38486 (10⁻⁵ M). <b>G</b>, Representative Western blots showing GC (dexamethasone; 10⁻⁵ M in medium supplemented with charcoal-stripped serum; 48 h) regulation of various key regulators of the cell cycle in cultured astrocytes; the semi-quantitative (n = 4) data from these immunoblotting experiments are shown in <b>H.</b> Note that while GC treatment downregulates cyclin D1 protein expression, the treatment results in a concomitant increase in the levels of the cell cycle inhibitor, p27. Cyclin E and CDK6 expression levels are not changed after exposing astrocytes to GC. Numerical data represent mean ± SD. * p<0.05 vs. CON, # p<0.05 vs. GC. Scale bar: 50 µm.</p

Enriched astrocytic cultures also respond to GC with moderate activation of caspase 3, but fail to show signs of early- or late-stage apoptosis.

<p>After re-plating, enriched astrocytic cultures were treated with GC (48 h) in medium containing either charcoal-stripped serum (data not shown) or B27 supplement (representative images in <b>A–L</b>). Enriched astrocytes responded to GC treatment DEX with moderately increased immunostaining for activated caspase 3; these cells did not enter late-stage (stage II) apoptosis, as shown by TUNEL (<b>A–F</b>). In contrast, staurosporine (STA) induced a marked activation of caspase 3 and apoptosis (<b>G–I</b>). The immunocytochemical results shown for activated caspase 3 in <b>A–I</b> were confirmed by immunoblotting (J). Staurosporine, but not GC, treatment of enriched astrocytic cultures significantly increased levels of immunoreactive phospho-H2A.X, a marker of early apoptosis, as shown by immunoblotting studies (<b>K</b>). Similarly, astrocytes exposed to STA, but not GC, displayed high molecular weight (HMW) DNA fragments, when lysates where subjected to pulse-field gel electrophoresis (PFGE) (<b>L</b>); all lanes were loaded with DNA from the same number of astrocytes, and arrow indicates 50 kb HMW DNA fragments. Scale bars: 50 µm.</p

PROBE–Preferred Reporting Orientations for Behavioral Experiments.

<p>PROBE–Preferred Reporting Orientations for Behavioral Experiments.</p

<i>In utero</i> glucocorticoid exposure decreases the length of small intestine at 24 hours, 1 and 3 months old.

<p>(A) The length of small intestine is shorter in <i>iuGC</i> rats at 1 and 3 months old, n = 6, 17, 6 CTR and n = 9, 10, 7 <i>iuGC</i>. (B) Stomach length in control and <i>iuGC</i> animals, n = 10, 9, 6 CTR and n = 10, 11, 7 <i>iuGC</i>. (C) The length of colon in control and <i>iuGC</i> animals, n = 10, 9, 6 CTR and n = 10, 11, 7 <i>iuGC</i>. (D) Small Intestine is shorter in <i>iuGC</i> animals at 24 hours of life, n = 4 CTR and n = 13 <i>iuGC</i>. <i>iuGC</i>, <i>in utero</i> glucocorticoid exposed animals. *<i>P</i> <0.05, *** <i>P</i> <0.001.</p

Neural stem and progenitor cells decrease along the subependymal zone dorsal-ventral axis.

<p>A DCX wholemount staining for the lateral wall is represented in <b>(A)</b> (Scale bar = 1 mm). DCX positive cell rates were estimated through the lateral wall for anterior, intermediate and posterior SEZ <b>(B)</b>, dorsolateral and ventral SEZ <b>(C)</b>. BrdU retaining cells were double stained with GFAP and assessed in the dorsolateral and ventral SEZ <b>(D)</b>. The same analysis was performed for proliferating neuroblasts (double BrdU/DCX positive cells) <b>(E)</b>. The images for the BrdU, DCX and BrdU/DCX staining are represented in <b>(F)</b> (Scale bar = 20 µm). LV, lateral ventricle; Str, striatum. All results are expressed as number of positive cells per area (in mm²). The threshold value for statistical significance was set at 0.05 (* p<0.05).</p