Fig. 4.

Ethanol exposure increases cell death in the DG. (A) Photomicrograph of Nissl-stained coronal section of rat hippocampus. Arrow shows pyknotic nucleus in the basal granular layer where precursors are located (6-week survival). (B) Counting of pyknotic nuclei shows a marked increase in the DG of such figures in ethanol-fed rats (40.75 ± 2.9 pyknotic nuclei per DG section, n = 4) as compared with controls (14.67 ± 1.38, n = 4). P ≤ 0.01, Mann–Whitney test (6-week survival). (C) BrdUrd-labeled section of the DG counterstained with Nissl, 6-week survival. Dotted line indicates chosen division between basal layers (including subgranular cell layer) and the outer layers (OL) of the DG. Arrows point to BrdUrd-positive cells in the basal layers, where most of the newly born cells can be found at this survival point. The number of pyknotic cells per mm² (D) is clearly higher in the basal layers of the DG than in the outer layers (ol). (E) TUNEL was used to assess cell death in the DG of ethanol vs. control-fed rats. (Bar = 25 μm.) Positive labeling was observed predominantly in the basal layers (6-week survival). (F) There was a marked increase in the number of TUNEL-positive cells per section in ethanol-fed rats (6.5 ± 0.87, n = 4) as compared with controls (2.5 ± 0.87, n = 4). P ≤ 0.01, Mann–Whitney test (6-week survival). (G) BrdUrd-labeled pyknotic cells in the DG. BrdUrd was administered 3 days before perfusion (BrdUrd in red, nuclear staining in green with Cyquant 1:2,000; BrdUrd protocol 2). (Bar = 10 μm.) (H) Apoptotic nuclei were observed in the basal layers of the DG of ethanol-fed rats at the ultrastructural level (6-week survival). The arrow points to the typical fragmentation of the nuclear membranes in advanced stages of apoptosis. Also note dense chromatin within the periphery of the nucleus. (Bar = 5 μm.)