Fig. 2.
Induction of neuronal apoptosis after prolonged morphine treatment. A–C, Micrographs from the superficial spinal cord dorsal horn illustrate apoptotic cells in rats receiving repeated intrathecal saline (A), 10 μg morphine boluses (B), and the combination of 10 μg of morphine and 20 μg of PDC (C) for 7 d. D–F, The TUNEL (red) staining colocalizes with condensed and fragmented nuclei, as shown by the Hoechst staining (blue) and the merged image inF, indicating that the TUNEL staining specifically detected in vivo DNA fragmentation (arrows). Note that all TUNEL-positive cells were colocalized with the Hoechst staining. D′–F′, Note the high-magnification views of these insets (arrows) fromD–F showing nuclear fragmentation and condensed DNA segments. G–I, The TUNEL (red) staining was colocalized with the NeuN staining (green) as shown in I (merged), indicating that the costained apoptotic cells were neurons in the dorsal horn. Note that some TUNEL-positive cells were not colocalized with NeuN, indicating the presence of apoptotic glial cells. J–L, The TUNEL (red) staining was colocalized with the GAD67 staining (green) as shown in L (merged), indicating that the costained apoptotic cells contain the GABA-synthesizing enzyme and are likely to be GABAergic neurons. Images from D–L were taken from rats receiving intrathecal 20 μg morphine boluses or 20 nmol · μl−1 · hr−1morphine infusion for 7 d. Scale bars: A–C, 30 μm; D–L, 15 μm; D′–F′, 5 μm.