Synaptic Organization in the Adult Drosophila Mushroom Body Calyx FLORIAN LEISS, 1 CLAUDIA GROH, 2 NANCY J. BUTCHER, 2 IAN A. MEINERTZHAGEN, 2 AND GAIA TAVOSANIS 1 * 1 Dendrite Differentiation, Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, 82152 Munich, Germany 2 Neuroscience Institute, Life Science Centre, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1 ABSTRACT Insect mushroom bodies are critical for olfactory associa- tive learning. We have carried out an extensive quantitative description of the synaptic organization of the calyx of adult Drosophila melanogaster, the main olfactory input region of the mushroom body. By using high-resolution confocal mi- croscopy, electron microscopy-based three-dimensional reconstructions, and genetic labeling of the neuronal pop- ulations contributing to the calyx, we resolved the precise connections between large cholinergic boutons of antennal lobe projection neurons and the dendrites of Kenyon cells, the mushroom body intrinsic neurons. Throughout the ca- lyx, these elements constitute synaptic complexes called microglomeruli. By single-cell labeling, we show that each Kenyon cell’s claw-like dendritic specialization is highly en- riched in filamentous actin, suggesting that this might be a site of plastic reorganization. In fact, Lim kinase (LimK) overexpression in the Kenyon cells modifies the shape of the microglomeruli. Confocal and electron microscopy indi- cate that each Kenyon cell claw enwraps a single bouton of a projection neuron. Each bouton is contacted by a number of such claw-like specializations as well as profiles of -aminobutyric acid-positive neurons. The dendrites of dis- tinct populations of Kenyon cells involved in different types of memory are partially segregated within the calyx and contribute to different subsets of microglomeruli. Our anal- ysis suggests, though, that projection neuron boutons can contact more than one type of Kenyon cell. These findings represent an important basis for the functional analysis of the olfactory pathway, including the formation of associa- tive olfactory memories. J. Comp. Neurol. 517:808 – 824, 2009. © 2009 Wiley-Liss, Inc. Indexing terms: mushroom body calyx; microglomerulus; Kenyon cell; projection neuron; F-actin; electron microscopy; dendrite; Drosophila Current key questions in neurobiology are how sensory information is represented in higher brain centers and how associative memories are established. Central to both ques- tions is an understanding of how the underlying neuronal circuits are organized. Odor processing, in particular, has recently been the object of systematic analyses, which reveal a great degree of conservation of olfactory circuit design in both mammals and insects (Hildebrand and Shepherd, 1997; Vosshall and Stocker, 2007). In spite of considerable recent progress in our understanding of odor representations at the level of the primary olfactory centers, the olfactory bulb of mammals or the antennal lobe of insects, the processing of odor information in higher brain centers, including the insect mushroom bodies, remains rather elusive and lacks detailed information on neural circuits. The mushroom bodies in the fruit fly Drosophila melanogaster have for a long time been implicated in the generation and retrieval of olfactory asso- ciative memories (Heisenberg et al., 1985; de Belle and Heisenberg, 1994; Zars et al., 2000). However, detailed con- nections within the mushroom body input region, the calyx, are still not completely elucidated. Drosophila melanogaster has about 1,200 olfactory receptor neurons in the adult an- tenna (Stocker, 1994) and a further 120 in the maxillary palp (Shanbhag et al., 1999), each expressing one of about 60 olfactory receptor molecules (Hallem et al., 2004). Olfactory receptor neurons send their axons to the antennal lobe where, according to the olfactory receptor they express, they segre- gate into 43 genetically and functionally defined glomeruli Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number: SPP1111 Ta 265/2-2 (to G.T.); Grant sponsor: National Sciences and Engi- neering Research Council of Canada; Grant number: DIS-000065 (to I.A.M.); Grant sponsor: Deutscher Akademischer Austausch Dienst (to C.G.). Claudia Groh’s current address is Department of Behavioral Physiology and Sociobiology, Biocenter, University of Wu ¨ rzburg, Am Hubland, 97074 Wu ¨ rzburg, Germany. *Correspondence to: Gaia Tavosanis, Dendrite Differentiation Group, Dept. Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany. E-mail: gaia@neuro.mpg.de Received 6 March 2009; Revised 25 June 2009; Accepted 29 July 2009 DOI 10.1002/cne.22184 Published online August 6, 2009 in Wiley InterScience (www.interscience. wiley.com). The Journal of Comparative Neurology 517:808 – 824 (2009) Research in Systems Neuroscience © 2009 Wiley-Liss, Inc.