20-Hydroxyecdysone Inhibits the Mitotic Activity of Neuronal Precursors in the Developing Mushroom Bodies of the Honeybee, Apis mellifera Dagmar Malun, Ariane D. Moseleit, Bernd Gru ¨ newald Neurobiologie, Institut fu ¨ r Biologie, Freie Universita ¨ t Berlin, Ko ¨ nigin-Luise-Str. 28-30, 14195 Berlin, Germany Received 29 January 2003; accepted 8 April 2003 ABSTRACT: The mushroom bodies (MBs) within the brain of the honeybee, Apis mellifera, are prominent paired neuropil structures consisting of a lateral and a median subunit. The intrinsic MB neurons (Kenyon cells) of each of these subunits are generated in four distinct proliferation centers, each associated with a ca- lyx. Previous BrdU studies revealed that neurogenesis of Kenyon cells starts at the first larval stage (L1) by symmetrical cell division of Kenyon precursor cells, and ceases abruptly at a midpupal stage (P5). In the present work, we confirmed these results using the antiphospho histone H3 mitosis marker to label mitotically active cells in a cell culture system, in histological sections, and in whole-mount brain preparations. To elucidate whether the steroid hormone ecdysone plays a role in the termination of Kenyon cell neurogenesis, we manipu- lated the hormone titer by injecting 20-hydroxyecdysone (20E) into animals of those pupal stages (P0/1, P3, P4) in which neurogenesis of Kenyon cells was still extensive. The effects of 20E were evaluated by determining the number of mitotically active cells in confocal micro- scopic images of squash preparations of the MB prolif- eration centers. In all pupal stages studied, 20E caused a reduction of mitotic activity, indicating its involvement in the cessation of Kenyon cell neurogenesis. © 2003 Wiley Periodicals, Inc. J Neurobiol 57: 1–14, 2003 Keywords: insect; Kenyon cells; ecdysteroid; phospho histone H3 mitosis marker; BrdU INTRODUCTION Steroid hormones play a major role throughout devel- opment and maturation of the nervous system in both vertebrates and invertebrates (for review see Arnold and Gorski, 1984; Weeks and Levine, 1990; Kawata, 1995; Levine et al., 1995). In rats, neurogenesis and apoptosis are regulated by adrenal steroids in the adult dentate gyrus (Gould and Cameron, 1996); in the adult canary brain gonadal steroids control neurogen- esis (Nordeen and Nordeen, 1989). Steroids also reg- ulate the dendritic morphology of mammalian mo- toneurons (Kurz et al., 1986). Classical steroid hormones in insects are ecdysone and ecdysone-like substances, which are collectively called ecdysteroids (Ga ¨de et al., 1997). The roles played by ecdysteroids during ontogenesis are many and diverse, including regulation of molting in embryos, larvae, and nymphs (Truman, 1996), and control of neuronal cell death, cell proliferation, cellular physiology, and structural plasticity of neuronal arbors (for review see Weeks and Levine, 1990). As shown for the moth Manduca sexta, ecdysteroids control neurogenesis within the optic lobes anlagen; proliferation can be turned on and off depending on the ecdysteroid concentration (Champlin and Truman, 1998a,b). Also, ecdysteroids’ involvement in neuronal death was shown for Manduca sexta (Truman and Schwartz, 1984; Fahr- bach and Truman, 1987; Streichert et al., 1997; Hoff- Correspondence to: D. Malun (malun@zedat.fu-berlin.de). Contract grant sponsor: DFG; contract grant number: SFB 515 (D.M. and B.G.). © 2003 Wiley Periodicals, Inc. DOI 10.1002/neu.10251 1