MOLECULAR AND SYNAPTIC MECHANISMS Cell types and coincident synapses in the ellipsoid body of Drosophila Alfonso Mart  ın-Pe ~ na, 1,2 Angel Acebes, 1,3 Jos  e-Rodrigo Rodr  ıguez, 1 Valerie Chevalier, 1 Sergio Casas-Tinto, 1 Tilman Triphan, 4,5 Roland Strauss 4,6 and Alberto Ferr us 1 1 Department of Cellular, Molecular and Developmental Neurobiology, Cajal Institute, C.S.I.C., Ave. Dr. Arce 37, E-28002 Madrid, Spain 2 Department of Neurology, McKnight Brain Institute, College of Medicine, University of Florida, Gainesville, FL, USA 3 Center for Biomedical Research of the Canary Islands, Institute of Biomedical Technologies, University of La Laguna, Tenerife, Spain 4 Biozentrum der Universitaet Wuerzburg, Lehrstuhl f€ ur Genetik und Neurobiologie, Wuerzburg, Germany 5 HHMI Janelia Farm Research Campus, Ashburn, VA, USA 6 Department of Zoologie III–Neurobiologie, Johannes Gutenberg-Universitaet Mainz, Mainz, Germany Keywords: central complex, coincidence detectors, development, locomotion control Abstract Cellular ultrastructures for signal integration are unknown in any nervous system. The ellipsoid body (EB) of the Drosophila brain is thought to control locomotion upon integration of various modalities of sensory signals with the animal internal status. However, the expected excitatory and inhibitory input convergence that virtually all brain centres exhibit is not yet described in the EB. Based on the EB expression domains of genetic constructs from the choline acetyl transferase (Cha), glutamic acid decarboxyl- ase (GAD) and tyrosine hydroxylase (TH) genes, we identified a new set of neurons with the characteristic ring-shaped morphol- ogy (R neurons) which are presumably cholinergic, in addition to the existing GABA-expressing neurons. The R1 morphological subtype is represented in the Cha- and TH-expressing classes. In addition, using transmission electron microscopy, we identified a novel type of synapse in the EB, which exhibits the precise array of two independent active zones over the same postsynaptic dendritic domain, that we named ‘agora’. This array is compatible with a coincidence detector role, and represents ~8% of all EB synapses in Drosophila. Presumably excitatory R neurons contribute to coincident synapses. Functional silencing of EB neurons by driving genetically tetanus toxin expression either reduces walking speed or alters movement orientation depending on the targeted R neuron subset, thus revealing functional specialisations in the EB for locomotion control. Introduction Signal integration is a fundamental property of neural systems in eliciting behaviour. However, cellular ultrastructures that could sus- tain this function are unknown in any nervous system. In insects, the central complex (CC) controls locomotion (Strausfeld, 1998; Martin et al., 2001; Ritzmann et al., 2012; Kai & Okada, 2013) and flight (Homberg, 1994; Ilius et al., 2007), following the inte- gration of visual (Ofstad et al., 2011; Kim et al., 2012; Heinze et al., 2013; Weir et al., 2013) and acoustic (Kunst et al., 2011) and olfactory (Zhang et al., 2013; Kong et al., 2010; Wu et al., 2007; however see Krashes & Waddell, 2008) stimuli with other, less well characterised, motivational states. Drosophila CC is com- posed of four centres: protocerebral bridge, fan-shaped body, noduli and ellipsoid body (EB) (Fig 1A; see Strausfeld, 2012; for a com- parative study across Arthropods). The homologous mammalian brain centre is thought to be the striatum and its associated basal ganglia, based on functional, structural and gene expression criteria (Strausfeld & Hirth, 2013). Structural and functional evidence supports the idea that the CC is an integrative centre for locomotion control. Screening for mor- phological brain mutants in Drosophila, eight genes were identified as required for proper CC structure and walking (Strauss & Heisen- berg, 1993). Conversely, in a slow-walking screening, 13% of the total outcome showed anatomical defects in the CC (Strauss, 2002). Concerning the functional evidence, in Drosophila and Apis, CC neurons respond to light stimulation (Bausenwein et al., 1994; Milde, 1988). EB neuron inactivation causes loss of directionality toward visual targets when these become invisible (Neuser et al., 2008). In addition to spatial orientation (Kuntz et al., 2012), the EB is also required for pattern and visual memories (Ofstad et al., 2011), and it is involved in middle-term olfactory memory formation through its projections to and from the mushroom bodies, another integrative brain centre (Zhang et al., 2013). In Schistocerca Correspondence: A. Ferr us, as above. E-mail: aferrus@cajal.csic.es Received 16 September 2013, revised 31 January 2014, accepted 3 February 2014 © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd European Journal of Neuroscience, Vol. 39, pp. 1586–1601, 2014 doi:10.1111/ejn.12537