Fig. 4. TGF-β accelerates 5 mm K+-mediated apoptotic cell death of cerebellar granule neurons in vitro. Granule neurons were cultured and treated as described in Materials and Methods. All neuron preparations presented here were fixed and processed on DIV9. Nomarsky optics of cultures stained for in situ DNA end breaks shows that there is substantially more DNA fragmentation in low K+ than in high K+-grown neurons. The percentages of stained cells increase in low K+-grown neurons exposed to TGF-β2, whereas there is no difference between treated and untreated high K+ cultures.A, Low K+ control; B, low K+ exposed to TGF-β2 (1 ng/ml); E, high K+ control;F, high K+ exposed to TGF-β2 (1 ng/ml). For A andB, magnification is 480×; scale bar (shown inH), 13 μm. For E and F, magnification is 320×; scale bar (shown in H), 20 μm. Visualization under UV illumination of nuclei stained with the fluorescent dye Hoechst 33258 (5 μg/ml) reveals more condensed chromatin in low K+ than in high K+ culture conditions. TGF-β2 accelerates the rate of chromatin condensation in low K+-grown neurons, but it does not affect neurons maintained in high K+ culture conditions. C, Low K+ control;D, low K+ exposed to TGF-β2 (1 ng/ml); G, high K+ control; H, high K+ exposed to TGF-β2 (1 ng/ml). Magnification, 1100×; scale bar (shown in H), 6 μm.