Distribution of metabotropic receptors of serotonin, dopamine, GABA, glutamate, and short neuropeptide F in the central complex of Drosophila

Neuroscience 208 (2012) 11–26

DISTRIBUTION OF METABOTROPIC RECEPTORS OF SEROTONIN, DOPAMINE, GABA, GLUTAMATE, AND SHORT NEUROPEPTIDE F IN THE CENTRAL COMPLEX OF DROSOPHILA L. KAHSAI,1 M. A. CARLSSON, Å. M. E. WINTHER2* AND D. R. NÄSSEL*

Key words: G-protein– coupled receptor, neurotransmitter, neuropeptide, insect brain, central body.

Department of Zoology, Stockholm University, S-10691 Stockholm, Sweden

The central complex is a prominent and highly ordered set of neuropils located at the midline of the insect brain. Several studies in Drosophila and other insects have shown the significance of the central complex in coordination of various motor functions such as locomotor control (Ilius et al., 1994; Martin et al., 1999, 2001; Strauss, 2002; Kahsai et al., 2010) and sky compass orientation (Vitzthum et al., 2002; Heinze and Homberg, 2008; Sakura et al., 2008). This brain center has also been implicated in certain forms of visual memory and in long-term olfactory learning (Liu et al., 2006; Wu et al., 2007; Neuser et al., 2008; Wang et al., 2008; Pan et al., 2009) as well as in courtship behavior (Joiner and Griffith, 2000; Popov et al., 2005; Wenzel et al., 2005; Sakai and Kitamoto, 2006; Weinrich et al., 2008). The central complex is composed of four interconnected substructures, which are from anterior to posterior: the ellipsoid body (EB), the fan-shaped body (FB), the noduli (NO), and the protocerebral bridge (PB) (Williams, 1975; Strausfeld, 1976; Homberg, 1985; Hanesch et al., 1989; Renn et al., 1999; Young and Armstrong, 2010). These substructures are further connected to two other paired neuropil regions in the protocerebrum: the ventral bodies (VBO) and the lateral triangles (LTR). By means of Golgi silver impregnation and GAL4 enhancer trap studies, it has been demonstrated that the central complex in Drosophila is composed of at least 60 different neuron types (Hanesch et al., 1989; Renn et al., 1999; Li et al., 2009; Young and Armstrong, 2010). The neurons of the central complex are classified into three major types: large field tangential neurons, small field columnar neurons, and pontine neurons. The large-field tangential neurons comprise the ring (R) neurons of the EB and the F-neurons of the FB, and connect a single central complex structure to other midbrain regions. The smallfield columnar neurons integrate two or three substructures, and the pontine neurons connect different layers of one single substructure (Hanesch et al., 1989; Young and Armstrong, 2010). Considerable attention has been given recently to both the morphology and function of the central complex, because of its role as a higher integration center organizing complex orientation behaviors (Heinze and Homberg, 2007, 2008; Neuser et al., 2008; Heinze et al., 2009; Kahsai et al., 2010; Young and Armstrong, 2010). Recently an extensive investigation was made on the distribution of different neuropeptides in relation to certain

Abstract—The central complex is a prominent set of midline neuropils in the insect brain, known to be a higher locomotor control center that integrates visual inputs and modulates motor outputs. It is composed of four major neuropil structures, the ellipsoid body (EB), fan-shaped body (FB), noduli (NO), and protocerebral bridge (PB). In Drosophila different types of central complex neurons have been shown to express multiple neuropeptides and neurotransmitters; however, the distribution of corresponding receptors is not known. Here, we have mapped metabotropic, G-protein– coupled receptors (GPCRs) of several neurotransmitters to neurons of the central complex. By combining immunocytochemistry with GAL4 driven green fluorescent protein, we examined the distribution patterns of six different GPCRs: two serotonin receptor subtypes (5-HT1B and 5-HT7), a dopamine receptor (DopR), the metabotropic GABAB receptor (GABABR), the metabotropic glutamate receptor (DmGluRA) and a short neuropeptide F receptor (sNPFR1). Five of the six GPCRs were mapped to different neurons in the EB (sNPFR1 was not seen). Different layers of the FB express DopR, GABABR, DmGluRA, and sNPFR1, whereas only GABABR and DmGluRA were localized to the PB. Finally, strong expression of DopR and DmGluRA was detected in the NO. In most cases the distribution patterns of the GPCRs matched the expression of markers for their respective ligands. In some nonmatching regions it is likely that other types of dopamine and serotonin receptors or ionotropic GABA and glutamate receptors are expressed. Our data suggest that chemical signaling and signal modulation are diverse and highly complex in the different compartments and circuits of the Drosophila central complex. The information provided here, on receptor distribution, will be very useful for future analysis of functional circuits in the central complex, based on targeted interference with receptor expression. © 2012 IBRO. Published by Elsevier Ltd. All rights reserved. 1 Present address: Division of Biological Sciences and Veterinary, Medical Diagnostic Laboratory, University of Missouri, Columbia, MO 65211, USA. 2 Present address: Department of Neuroscience, Karolinska Institutet, von Eulers väg 3, S-171 77 Stockholm, Sweden. *Corresponding author. Tel: ⫹46-8-52487871 or ⫹46-8-164077; fax: ⫹46-8-325861 or ⫹46-8-167715. E-mail address: [email protected] (Å. M. E. Winther) or dnassel@ zoologi.su.se (D. R. Nässel). Abbreviations: DopR, dopamine receptor; EB, ellipsoid body; FB, fanshaped body; GFP, green fluorescent protein; GPCRs, G-protein– coupled receptors; LTR, lateral triangles; NO, noduli; PB, protocerebral bridge; TH, tyrosine hydroxylase; ventral bodies vGluT, vesicular glutamate transporter.

0306-4522/12 $36.00 © 2012 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2012.02.007

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neurotransmitters in the Drosophila central complex (Kahsai and Winther, 2011). In that study, a number of neuropeptides were mapped to both tangential and columnar neurons. It was also shown that subpopulations of peptideexpressing neurons co-localize a marker for the excitatory neurotransmitter acetylcholine. The mapping of neurotransmitters and neuromodulators is an important first step in unraveling the chemical circuitry in the central complex and forms a basis for designing experiments to understand signal processing in this brain region (see Kahsai et al., 2010). However, little is known about the distribution of receptors of different neurotransmitters and neuropeptides in the central complex. Therefore, in the present study, we have mapped the distribution of six different G-protein– coupled receptors (GPCRs), also known as metabotropic receptors (for classification of GPCRs in Drosophila see Brody and Cravchik, 2000; Hewes and Taghert, 2001; Hauser et al., 2006). We have selected three representatives from the rhodopsin-like family (Class A GPCRs): the serotonin receptors, 5-HT1B, and 5-HT7 (Witz et al., 1990; Saudou et al., 1992; Becnel et al., 2011), one dopamine receptor, dDA1 (Kim et al., 2003), and the only known receptor for short neuropeptide F, sNPFR1 (Mertens et al., 2002; Feng et al., 2003). In addition, we investigated the distribution of the metabotropic GABA receptor (GABABR) (Mezler et al., 2001) and a metabotropic glutamate receptor (DmGluRA) (Parmentier et al., 1996), both from the metabotropic glutamate-like receptor family (Class C GPCRs). It should be noted that none of these GPCRs, except 5-HT7, were previously mapped in any detail to central complex structures, and they were also not localized in relation to their respective ligands in this part of the brain. A study using immunocytochemistry and a 5-HT1BGAL4 driver showed that 5-HT1B is expressed in the mushroom body, pars intercerebralis, certain clock neurons, optic lobes, and dorsal neurons in the adult Drosophila brain (Yuan et al., 2005), and the expression pattern of 5-HT7 was determined with a GAL4 driver in certain brain regions, including the CX (Becnel et al., 2011). The dDA1 has been localized by immunohistochemistry to the mushroom body, central complex and to large neurosecretory cells, but not in detail or in relation to dopaminergic neurons (Kim et al., 2003; Lebestky et al., 2009; Kong et al., 2010). Previous studies on GABA receptor distribution revealed that the GABABR is widely expressed in the adult nervous system, most abundantly in the mushroom body calyces, EB, optic and antennal lobes (Enell et al., 2007; Kolodziejczyk et al., 2008; Root et al., 2008; Okada et al., 2009). The DmGluRA has been reported to be expressed in most neuropils in the adult brain (Devaud et al., 2008) as well as in motor neurons at the neuromuscular junction (Parmentier et al., 1996) and in certain clock neurons (Hamasaka et al., 2007). Here, we compared the distribution patterns of the six GPCRs to those of their respective ligands in the central complex. Our approach was to employ immunocytochemistry in combination with GAL4-directed expression of green fluorescent protein (GFP). For GPCRs, either pro-

moter GAL4 lines or specific antisera were employed, and for the ligands, we used either antisera to the neurotransmitters or their biosynthetic enzymes or GAL4 lines serving as markers for biosynthetic enzymes or vesicular transporters. We found all six GPCRs expressed in specific patterns in the different central complex structures, and their distributions correspond well to those of their ligands in most of the neuropil structures. Although we examined only six GPCRs so far our data suggest that chemical signaling and neuromodulation is very complex in the circuits of the central complex.

EXPERIMENTAL PROCEDURES Fly strains For conventional immunocytochemical experiments, we used 4 – 6 days old adult male and female Drosophila melanogaster (w1118 strain). For comparisons of patterns of GPCR distribution and their ligands, we employed immunocytochemistry on flies expressing GFP driven by different GAL4 lines. The serotonin receptor subtypes 5-HT1BDRO and 5-HT7DRO (in the following referred to as 5-HT1B and 5-HT7) were localized using a 5-HT1B-GAL4 (Yuan et al., 2005), gift of Amita Sehgal (University of Pennsylvania, Philadelphia, PA, USA) and a 5-HT7-GAL4 (Becnel et al., 2011), gift of Charles Nichols (Health Science Center, New Orleans, LA, USA). To visualize GABABR expression, we used a GABABR2-GAL4 (Root et al., 2008), gift of Jing W. Wang (University of California, San Diego, CA, USA). The distribution of the receptor for short neuropeptide F (sNPFR1) was visualized with a promoter GAL4 produced at Janelia Farm (Pfeiffer et al., 2008), obtained from Bloomington Drosophila Stock Center (BDSC), at Indiana University, Bloomington, IN, USA. A tyrosine hydroxylase (TH) promoter GAL4 (Friggi-Grelin et al., 2003), obtained from S. Birmann (CNRS, ESPCI, Chimie ParisTech, France), was used to identify dopamine-producing neurons. Glutamate-producing neurons were visualized with a vesicular glutamate transporter (dvglutCNSIII) GAL4 (Daniels et al., 2008), gift of A. DiAntonio (Washington University, St. Louis, MO, USA). To identify R-neurons (R3 and R4d) of the EB, we employed the enhancer trap GAL4 c232 (Renn et al., 1999), a gift from Paul Taghert (Washington University, St. Louis, MO, USA). The previously given GAL4 lines were crossed with UAS-mcd8-gfp flies, from BDSC to visualize GAL4 expression. All flies were raised under 12-h:12-h light/dark conditions at 25 °C, and fed standard Drosophila food. Immunocytochemistry. For all immunocytochemistry adult Drosophila heads were opened up in 0.01 M phosphate-buffered saline with 0.25% Triton X-100, pH 7.2 (PBS-Tx) and fixed in ice-cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.4 (PB) for 4 h. Following rinsing with 0.1 M PB, brains were either dissected out for whole mount immunocytochemistry or whole heads were incubated overnight in 20% sucrose in 0.1 M PB at 4 °C for cryostat sectioning. Frozen sections, 30 –50-␮m thick, of the head were cut on a Leitz 1720 cryostat at ⫺23 °C and mounted on microscope slides. Incubation with primary antisera was performed for 72 h for whole mount tissues, whereas sections were incubated for 36 h at 4 °C. Tissues or sections were rinsed thoroughly with PBS-Tx and were incubated overnight at 4 °C in secondary antibody. Finally the tissues or sections were rinsed in PBS-Tx and PBS and then mounted in 80% glycerol in PBS. For each experiment, at least 10 adult brains were analyzed. Antisera. For localization of metabotropic receptors, the following antisera were used: a mouse polyclonal antiserum to a protein portion (derived from a 173 bp fragment of the dDA1 cDNA) of the Drosophila D-1 dopamine receptor dDA1, also des-

L. Kahsai et al. / Neuroscience 208 (2012) 11–26 ignated DopR (a gift from K.Y. Han, University Park, PA, USA) at a dilution of 1:200 (Kim et al., 2003), a rabbit antiserum to a portion of the GABABR2 at a dilution of 1:16,000 (Hamasaka et al., 2005), a mouse monoclonal antibody to DmGluRA (obtained from I. Sinning, European Molecular Biology Laboratory, Heidelberg, Germany) at a dilution of 1:10 (Eroglu et al., 2002, 2003; Panneels et al., 2003). The DmGluRA antibody was raised against recombinant receptor protein that was purified to homogeneity (Eroglu et al., 2002). For detection of ligands or markers of ligands, we used: a mouse monoclonal antibody to serotonin (Dako, Clone 5HTH209) at dilution of 1:80 and a rabbit antiserum to Drosophila full length GAD1 (glutamic acid decarboxylase 1) obtained from F. R. Jackson (Tufts University School of Medicine, Boston, MA, USA), a marker for GABA signaling (Jackson et al., 1990; Featherstone et al., 2000) at a dilution of 1:1000 and a rabbit antiserum to sNPF precursor at a dilution of 1:1000 (Johard et al., 2008) kindly provided by Dr. J.A. Veenstra. To visualize synaptic neuropil regions, either a mouse monoclonal antibody nc82 that labels the presynaptic protein Bruchpilot (Hofbauer et al., 2009) was used or a mouse monoclonal anti-synapsin (Klagges et al., 1996), both at a dilution of 1:10 (both from Developmental Studies Hybridoma Bank, Iowa City, IA, USA). For detection of primary antisera, Cy3 or Cy2-conjugated goat anti-rabbit or goat anti-mouse antisera (Jackson ImmunoResearch, West Grove, PA, USA) were used at a dilution of 1:1500. In some experiments we used Alexa 488 or Alexa 546-tagged secondary antibodies (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:1000.

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Imaging Confocal images were collected with a Zeiss laser scanning microscope (LSM 510 META, Zeiss) based on an Axiovert S100 microscope (Zeiss, Jena, Germany) with Argon2/488 nm and HeNe 543 nm lasers. For imaging, 20⫻ dry and 40⫻ and 63⫻ oil immersion objective lenses were used. Images were obtained at an optical section thickness of 0.5– 0.9 ␮m and were stacked by the Zeiss LSM software. Finally, images were imported into Adobe Photoshop CS3 Extended version 10.0 and adjusted for contrast and brightness.

RESULTS Structure of the central complex The central complex is localized in protocerebrum of the brain and composed of four major neuropil structures: the EB, FB, NO, and PB (Fig. 1A). The most anterior substructure of the central complex is the doughnut shaped EB. The FB, located posterior to the EB is the largest substructure in the central complex. It is composed of vertical segments and horizontal layers. Posterior to the FB, between the calyces of the mushroom body (MB) lies the PB. The PB is composed of 16 segments, with 8 segments in each brain hemisphere. The paired NO are mirror images of each other and are located ventral to the FB and posterior to the EB. Each of the noduli is composed of 3 layers.

Fig. 1. Structure of the central complex. (A) Frontal schematic view of the central complex and its substructures; EB, canal (C), FB, noduli (NO), and PB with the accessory neuropils designated lateral triangles (LTR) and ventral bodies (VBO). Schematic drawing altered from Kahsai and Winther, 2011. (B) Whole mount projection showing the expression of the enhancer trap GAL4 line c232. The c232-GAL4 drives expression in circular arborizations in the EB, in the R3 and R4d ring structures. The axons of R3 and R4d neurons run in the RF-tract to the LTR and then enter the EB either from the canal (asterisk) arborizing outward or from the periphery arborizing inward (arrowhead). Two groups of cell bodies (arrows) are observed. Scale bar 20 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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Fig. 2. Distribution of two serotonin receptor types in relation to serotonin in the EB. Expression of 5-HT1B-GAL4 and 5-HT7-GAL4 (GFP, green) and immunolabeling with nc82 and serotonin antisera (both magenta) on whole mount adult Drosophila brain. (A and B) GFP expression of 5-HT1B-GAL4 in combination with nc82 staining (A anterior, B posterior). 5-HT1B-GAL4 expression is observed in a midring of the anterior region of the EB (A, arrow), in the innermost posterior ring (B, R1), in an outer rim of the posterior region (B, arrow) and in the LTR. (C and D) 5-HT7-GAL4 – driven GFP expression and nc82 staining (C anterior, D posterior). 5-HT7-GAL4 expression is detected in a midring in the anterior region of the EB (C, arrowhead), in the outer

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In addition, there are two-paired neuropil structures that are closely linked to the central complex; LTR and the VBO (Fig. 1A). The VBO is sometimes difficult to distinguish because extrinsic fibers contribute largely to this region and thus making it less delineated from adjacent neuropils. Therefore, we have not included the VBO in our analysis of GPCRs and their ligands in the central complex. However, the LTR, with its triangular shape, can easily be distinguished particularly in horizontal sections. To analyze the distribution pattern of the GPCRs in the EB, we employed an enhancer trap GAL4 line c232 that drives GFP expression in R3 and R4d neurons of the EB (Fig. 1B). The cell bodies of R3 and R4d neurons are located in the anterior region of the protocerebrum in two groups. These cell bodies send their axons along the RF tract (fibers of R- and F-neurons) to the LTR where they arborize and then project to the EB (Fig. 1B). In the following, we provide a more detailed description of the EB, since all, but one, of the GPCRs examined in this study are expressed in the EB. As revealed by using a synaptic neuropil marker, nc82, the doughnut-shaped EB is partially wedged into the FB (Fig. 2B2, D2). The EB comprises two major rings, one anterior and one posterior (Fig. 2A2, C2). Each of these major rings can be further subdivided into additional concentric rings (Hanesch et al., 1989; Renn et al., 1999; Young and Armstrong, 2010). Small-field neurons connect the anterior and posterior rings and other central complex structures. Large-field neurons, designated R-neurons, arborize in concentric rings and, thus, make up the ring-like structure of the EB. The R-neurons extend their axons along a prominent tract, the RF-tract, they have dendritic arborizations in the LTR and from where they project to the EB (Fig. 2B–G), (Hanesch et al., 1989; Renn et al., 1999). Based on projection patterns, the R-neurons can be divided into four major types; R1, R2, R3, and R4 (Hanesch et al., 1989). In total, there are more that 100 R-neurons in each hemisphere (Renn et al., 1999; Young and Armstrong, 2010; Becnel et al., 2011). The R1 axons arborize in the innermost ring, R2 and R4 in outer rings, and R3 in both inner and midrings (Hanesch et al., 1989; Renn et al., 1999) (Fig. 2B1, C1). R1-3 project to the EB via the EB canal and arborize outwardly, whereas R4 axons connect to the EB from the periphery of the ring (Hanesch et al., 1989; Renn et al., 1999). Localization of serotonin and 5-HT1B and 5-HT7 receptors in the central complex We used immunocytochemistry and GAL4 expression analysis to study the distribution of receptors and their

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ligands in the central complex. Various receptor-GAL4 lines and the enhancer trap line c232 were employed. Unfortunately most of the receptor, antisera used in this study, do not label cell bodies or thin axonal processes. Thus, it was difficult to identify the cell bodies of the neurons that express the receptor in processes innervating the central complex. In Drosophila, four metabotropic serotonin receptors have been identified that are structurally and pharmacologically similar to mammalian serotonin receptors. These are 5-HT1A, 5-HT1B, 5-HT2, and 5-HT7 (Witz et al., 1990; Saudou et al., 1992; Colas et al., 1995; Blenau and Thamm, 2011). These have also been referred to as 5-HT1ADro, 5-HT1BDro, 5-HT2Dro, and 5-HT7Dro. The different serotonin receptor subtypes are suggested to modulate various behaviors such as circadian behavior and sleep regulation, aggression and larval light responses, as well as regulation of insulin producing cells and development (Colas et al., 1999; Yuan et al., 2005, 2006; Nichols, 2007; Schaerlinger et al., 2007; Johnson et al., 2009; Becnel et al., 2011; Luo et al., 2012). We studied the distribution of two of these receptors, 5-HT1B and 5-HT7, in the central complex. When expressed in mammalian cells, the Drosophila 5-HT1B receptor inhibits adenylate cyclase (Saudou et al., 1992), whereas 5-HT7 activates adenylate cyclase (Witz et al., 1990), similar to their orthologs in mammals (Nichols and Nichols, 2008). We used 5-HT1B-GAL4 and 5-HT7-GAL4 in combination with nc82 immunolabeling to analyze the expression patterns. The specificity of the 5-HT1B-GAL4 has been confirmed using a 5-HT1B antiserum (Yuan et al., 2005). The 5-HT7-GAL4, was recently characterized (Becnel et al., 2011). As revealed by matching to nc82 staining, the 5-HT1BGAL4 drives expression in both the anterior and posterior ring of the EB (Fig. 2A, B). Projections of posterior optical sections reveal 5-HT1B-GAL4 labeling in the posterior and innermost EB-ring and in an outer ring (Fig. 2B). Moreover, the 5-HT1B-GAL4 drives expression in additional rings in the midportion of the EB (Fig. 2A, B). No 5-HT1B labeling was observed in the FB, PB, or NO. A group of 5-HT1BGAL4 –positive neurons were observed in the anterior region of the cortex (Fig. 2I), in a region where R-neurons have been localized (Hanesch et al., 1989; Renn et al., 1999; Young and Armstrong, 2010). However, because the expression of 5-HT1B-GAL4 is intense in numerous neuronal processes throughout the brain, it was difficult to distinguish individual axonal processes to connect them to the LTR and the EB.

rim in the posterior region of the EB (C and D, arrows) and in the innermost ring in the posterior region of the EB (D, R1). (E and F) Expression of 5-HT1B-GAL4 (E) and 5-HT7-GAL4 (F) – driven GFP with labeling with serotonin antibody on cryostat sections of adult Drosophila heads. Serotonin immunolabeling was detected in varicosities in close apposition to the labeling of 5-HT1B-GAL4 in the middle ring of the EB (E3). Superimposed serotonin immunoreactivity and 5-HT7-GAL4 expression were observed in a midring of the EB (F3). Serotonin immunolabeling and expression of both 5-HT1B-GAL4 and 5-HT7-GAL4 are detected in the LTR (E, F). (G) Whole mount projection of 5HT7-GAL4 showing R-neurons projecting along the RF tract and arborizing in the LTR and then on to the EB. (H) Merged images of 5-HT7-GAL4 expression and nc82 immunolabeling showing 5-HT7-GAL4 expressing cell bodies (R) localized between the mushroom body (MB) and the antennal lobe (AL) in the anterior region of the brain. (I) 5-HT1B expressing cell bodies are localized below the heel of the MB. All images are projections of sections, except (H) and (I) that are single sections. a, anterior; p, posterior; c, canal. Scale bars 20 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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The 5-HT7-GAL4 is expressed in the whole anterior ring (Fig. 2C) in contrast to the 5-HT1B-GAL4 that labels the anterior ring of the EB only partially. In the posterior ring, intense 5-HT1B-GAL4 labeling was observed in the innermost ring and in a rim of the outermost ring, resembling R4 innervation (Fig. 2C, D). The 5-HT7-GAL4 drove expression in a group of approximately 30 cell bodies in each brain hemisphere in the anterior cortex with axons running in the RF-tract to the LTR and then on to the EB via the EB canal (Fig. 2G, H). Previously described R-neurons have their cell bodies in this area (Hanesch et al., 1989; Renn et al., 1999; Young and Armstrong, 2010). We did not detect any 5-HT7-GAL4 expression in the other central complex substructures. To examine the distribution of the two serotonin receptor GAL4 lines in relation to neurons expressing serotonin, we applied a mouse monoclonal serotonin antibody to brains expressing GFP directed by the GAL4s. Varicosities displaying serotonin immunoreactivity were detected in close apposition with the expression of 5-HT1B-GAL4 in a midsection of the anterior ring of the EB (Fig. 2E). The labeling of receptor and ligand appeared partly nonoverlapping in some regions. No serotonin immunoreactivity was detected in the outer and innermost rings of the EB where 5-HT1B-GAL4 is expressed. Serotonin immunoreactive varicosities were found superimposed with 5-HT7GAL4 expressing processes in a midportion of the anterior ring and in the outer rim of the posterior EB ring (Fig. 2F). Distribution of the dopamine receptor DopR in relation to tyrosine hydroxylase Mammalian dopamine receptors are classified in two subtypes, D1-like and D2-like, which couples to stimulatory Gs or to inhibitory Go/Gi, respectively (Missale et al., 1998). In Drosophila, three dopamine receptors are well known, two of these, DopR (also known as dDA1) and DopR2 (also known as DAMB), are members of the D1-like receptor family and the third, D2R (also known as Dop2R), belongs to the D2-like family. A fourth likely dopamine receptor, structurally related to ␤-adrenergic–like receptors, activated by both dopamine and ecdysteroids, has also been identified (Srivastava et al., 2005). Functionally, dopamine receptors have been associated with control of locomotor activity, arousal, and learning (Han et al., 1996; Draper et al., 2007; Kim et al., 2007; Andretic et al., 2008; Seugnet et al., 2008; Lebestky et al., 2009; Kong et al., 2010). DopR is expressed in several central complex substructures. Strong labeling has been detected in the EB, FB, and NO (Kim et al., 2003; Lebestky et al., 2009; Kong et al., 2010). Here we confirmed these findings and compared the expression pattern of DopR with that of TH by using a mouse polyclonal DopR antibody and a TH-GAL4 (Fig. 3). Studies have shown that TH, the first and ratelimiting enzyme in the dopamine biosynthetic pathway is expressed in all dopamine-immunoreactive neurons (Nässel and Elekes, 1992; Monastirioti, 1999). The expression of the TH-GAL4 in the EB, FB, NO, and PB has been confirmed by a mouse monoclonal TH antibody (FriggiGrelin et al., 2003; Kahsai and Winther, 2011). To analyze

the expression pattern of DopR in the EB, we used the enhancer trap GAL4 line c232 that drives expression in R3 and R4d neurons (Renn et al., 1999). Immunolabeling of c232 with an antiserum to DopR revealed immunoreactive DopR punctates in the R3 ring of the EB (Fig. 3A). Weak DopR immunoreactivity was also detected in the R4d ring of the EB (Fig. 3A). We then applied the DopR antibody on brains that express GFP directed by TH-GAL4. Punctate DopR immunolabeling was observed in close contact with TH expression in R3 ring of the EB (Fig. 3B). In addition, weakly stained DopR immunoreactive punctates were detected that overlap with the expression of TH in the R4d ring of the EB. In the FB, the dorsal and ventral layers displayed strong DopR immunoreactivity, and weak labeling was observed in central layers (Fig. 3C). Both DopR immunolabeling and TH expression were detected in close contact in the ventral layer of the FB (Fig. 3C). Based on previous immunocytochemical studies on the distribution of TH (Kahsai and Winther, 2011), we suggest that this is layer 3. Previously, we showed that weak TH immunoreactivity is present in the remaining layers (layers 1–2 and 4 –7); however, the TH-GAL4 – directed expression in most of these layers is missing or is too weak to be detected. Thus, combining this study with the former (Kahsai and Winther, 2011) support that both the dopamine marker and the DopR are expressed in all FB layers. However, different labeling intensities may suggest that the amount of receptor and ligand expression varies between the separate layers. Furthermore, in the NO, the DopR antibody labeled all three layers, with the strongest immunoreactivity in layer I and III (Fig. 3D). TH-GAL4 is expressed in layers I and II (Fig. 3D). A horizontally cut section shows an overview of the overlapping of DopR immunolabeling and THGAL4 in the EB and FB (Fig. 3E). In accordance with the study of Kim and others (Kim et al., 2003), we detected no DopR immunoreactivity in the PB. Additionally, we detected TH expression, but no DopR immunoreactivity in the LTR (Fig. 3E). GABABR2 distribution in relation to GAD1 Next, we examined the distribution pattern of a metabotropic GABA receptor, GABABR, in relation to the biosynthetic enzyme of GABA, GAD1. In Drosophila, three types of metabotropic GABA receptors, also known as GABAB receptors, have been identified (Mezler et al., 2001). GABABR1 and GABABR2 dimerize to form a functional unit and mediate their effect through Go/Gi protein activation (Mezler et al., 2001). GABABR3 has not yet been functionally characterized, as this receptor either alone or in combination with the other receptor subtypes, could not be activated by GABA when expressed in heterologous cells (Mezler et al., 2001), although it may have a function in certain clock neurons of Drosophila (Dahdal et al., 2010). In Drosophila, GABAB receptors mediate odor-evoked inhibition in the antennal lobe, and they are involved in the fine-tuning of olfactory behavior, in modulation of the circadian clock, and regulation of insulin-producing cells in the brain (Hamasaka et al., 2005; Wilson and Laurent,

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Fig. 3. Dopamine receptor immunoreactivity in relation to c232-GAL4 and TH-GAL4 in the central complex. The c232-GAL4 was used to visualize R3 and R4d neurons of the EB, and the tyrosine hydroxylase (TH)-GAL4 was used as a marker for dopaminergic neurons. Expression of the GAL4 lines was visualized with GFP (green) in relation to dopamine receptor (DopR) immunoreactivity (magenta). (A) Immunolabeling with DopR antiserum on c232-GAL4 GFP-expressing brains revealed punctates of DopR immunoreactivity in R3, weak immunoreactivity was also observed in the R4d ring. (B–D) DopR immunoreactive punctates were detected in close contact with the expression of TH GAL4 in R3 ring (B, arrowhead) and in R4d ring (B, arrow) of the EB, in a ventral layer of the FB (C, possibly layer 2), and in layer I and weakly in layer II of the NO (D). (E) DopR immunoreactivity and TH-GAL4 expression in a horizontally cut section showing an overview of the FB and the EB. The juxtaposed labeling of DopR and TH-GAL4 in the FB and EB is indicated by arrows. TH-GAL4 expression is also observed in the LTR. All images are projections of optical sections from cryostat sections. D, dorsal; C, central; V, ventral. Scale bars 20 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

2005; Root et al., 2008; Dahdal et al., 2010; Enell et al., 2010; Murmu et al., 2011). The general localization of the GABABR and its relation to GAD1 in Drosophila has previously been described using a GABABR2 antiserum and a GAD1-GAL4 (Hamasaka et al., 2005; Enell et al., 2007). The distribution of GABABR2 immunoreactivity is likely to be indicative of functional GABABR dimers (see Mezler et al., 2001; Hamasaka et al., 2005). GAD1-GAL4 has been shown to be a good marker for GABA, in neuropil struc-

tures such as the antennal lobe, optic lobe, and the EB (Ng et al., 2002; Enell et al., 2007; Kolodziejczyk et al., 2008). Here, we employed GABABR2 and GAD1 antisera in combination with the c232-GAL4 enhancer trap line that drives expression in R3 and R4d neurons. We also compared the receptor-ligand distribution in the central complex, using GAD1 antiserum and a GABABR2-GAL4. Furthermore the GABABR2-GAL4 was used together with the GABABR2 antiserum to confirm the specificity of this GAL4

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line in the central complex. GAD1 immunoreactivity was observed in both posterior and anterior rings of the EB (Fig. 4A1). Immunolabeling of brains expressing GFP under the control of the c232-GAL4 with GAD1 antiserum suggests that GAD1 is expressed in rings corresponding to R3 and R4d, as well as in additional rings of the EB (Fig. 4A). However, no co-localization of c232 and GAD1 signal could be detected in the R3 and R4d neuronal cell bodies (Fig. 4H). This could be either because the GAD1 protein is mainly distributed in the synaptic terminals of the GABAergic neurons, rather than in their cell bodies, or indicate that GABAergic neurons other than those of the c232-GAL4 expressing ones innervate the EB. Weak GABABR2 immunoreactive punctates were identified in rings corresponding to R3 and stronger labeling was observed between R3 and R4d; no labeling could be detected in R4d (Fig. 4B). Thus, a comparison of the labeling patterns of c232-GAL4/anti-GAD1 and c232-GAL4/antiGABABR showed that the expression of GAD1 is slightly more extensive than the receptor distribution in the EB; GAD1 labeling in the EB appears to cover the entire EB, whereas the receptor distribution is more restricted (Fig. 4A, B). Immunolabeling of GABABR2-GAL4 with GAD1 antiserum revealed a close match between GABABR2 expression and GAD1 immunoreactivity in the posterior ring of the EB, between the R3 and R4d rings (Fig. 4C). To confirm the expression of GABABR2-GAL4 in the central complex we used GABABR2 antiserum on brains expressing GABABR2-GAL4 – driven GFP. We found a good agreement between the antiserum staining and GAL4 expression in the EB, PB, and LTR (Fig. 4D–F). However, in the FB, the expression of the GABABR2-GAL4 was more restricted and labeled only a dorsal and a central layer, whereas very weak GABABR2 immunoreactivity was distributed in all layers of the FB (Fig. 4G). No GABABR or GAD1 labeling was detected in the NO. Metabotropic glutamate receptor distribution compared with the expression of a vesicular glutamate transporter There is one known functional metabotropic glutamate receptor in Drosophila, DmGluRA, which couples to Go/Gi type of G-proteins (Parmentier et al., 1996). This receptor is expressed for instance in the neuromuscular junction where it is involved in modulation of excitatory neurotransmission (Bogdanik et al., 2004). DmGluRA is also expressed in certain clock neurons in the brain (Hamasaka et al., 2007). The distribution of DmGluRA in the adult brain is not restricted to clock neurons; in fact, it is expressed in most neuropils except the lobes of the mushroom bodies (Devaud et al., 2008). Here, we compared the distribution pattern of DmGluRA to that of vesicular glutamate transporter (vGluT). vGluT is a protein required for loading of glutamate into synaptic vesicles (Mahr and Aberle, 2006) and has been shown to be a good marker for glutamate (Daniels et al., 2008). We used an antiserum to DmGluRA and a vGluT-GAL4 to analyze their distribution patterns in the central complex. By using a vGluT antibody, the spec-

ificity of the vGluT-GAL4 has been confirmed (Daniels et al., 2008). The vGluT-GAL4 drives expression in the EB, FB, PB, and NO (Daniels et al., 2008; Kahsai and Winther, 2011). Before receptor-ligand analysis, we analyzed the distribution of DmGluRA in the EB. Thus, c232-GAL4 GFP expressing flies were stained with DmGluRA antiserum. We detected punctates of DmGluRA immunoreactivity in rings corresponding to R3 and R4d (Fig. 5A) and in the LTR (Fig. 5B). Immunolabeling of vGluT-GAL4 expressing brains with DmGluRA antiserum revealed overlapping between DmGluRA and vGluT expression in R3 and R4d rings of the EB. Only vGluT-GAL4 expression could be detected in a ring proximal to R3 (Fig. 5B). In the FB, punctates of DmGluRA immunoreactivity was detected in central and ventral layers (Fig. 5C). We observed strong vGluT-GAL4 expression in central layers and weaker in dorsal and ventral. One central layer displayed both strong DmGluRA immunoreactivity and strong GAL4 expression (Fig. 5C). All three layers of the NO expressed DmGluRA and vGluT, with stronger immunoreactivity of DmGluRA in the dorsal part of layer III (Fig. 5D). Both DmGluRA and vGluT labeling were seen in the PB; however, two segments on each lateral side seem to express DmGluRA but not vGluT (Fig. 5E). Distribution of sNPF and its receptor sNPFR1 in the fan-shaped body Only two neuropeptides have been studied functionally in the central complex, sNPF and Drosophila tachykinins, DTKs (Kahsai et al., 2010). That study indicated roles of both peptides in specific aspects of fine tuning of locomotor activity. Both of the known receptors of DTKs, DTKR and NKD, have been mapped to neurons with branches in different layers of the FB (Birse et al., 2006; Poels et al., 2009), but the distribution of the sNPF receptor, sNPFR1 has not been mapped to neuropils of the adult brain of Drosophila. Here we utilized an sNPFR1-GAL4 driver to analyze receptor distribution in the central complex. We compared the distribution of GAL4 expression with neuronal processes immunolabeled with a specific antiserum to the sNPF precursor (Johard et al., 2008). As seen in Fig. 6A, sNPFR1-GAL4 expression can be found in six of the layers (1, 2, and 4 –7) of the FB. In at least four of these, we could also find sNPF-immunolabeled processes (1, 2, 5 and 7). In one layer (4), there is strong GAL4 expression, but no peptide immunolabeling; this set of processes may represent output portions of the receptor-expressing neurons, lacking receptor protein. No sNPFR1 expression was detected in the PB (Fig. 6B), the EB (Fig. 6C), or the NO (not shown). This matches the lack of sNPF in these structures (Kahsai et al., 2010; Kahsai and Winther, 2011). As further support for the fidelity of the sNPFR1-GAL4 expression, we show here the distribution in axon terminations of olfactory sensory neurons in the antennal lobe and in insulinproducing cells of the pars intercerebralis (Fig. 6D); both of

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Fig. 4. Distribution of GABAB receptor in relation to c232-GAL4 and GAD1 in the central complex. (A and B) Localization of GAD1 (A) and GABAB receptor (B) immunoreactivities (magenta) in relation to c232-GAL4 – driven GFP expression. GAD1 was used as a marker to visualize GABA distribution in the central complex. Overlapping of GAD1 and c232 labeling was detected in R3 and R4d rings of the EB (A). No overlap between GABABR immunostaining and c232 expression was detected (B). (C) Immunolabeling was also applied to GABABR2-GAL4-expressing brains with antiserum to GAD1 (magenta), revealing GABABR2-GAL4 expression and GAD1 staining in the posterior region (p) of the EB (C3). (D–F) The expression of GABABR2-GAL4 and GABABR immunoreactivity matched well in the EB (D), PB (E), and LTR (F). (G) Some mismatches between the GABABR-GAL4 and the GABABR antibody were detected in the FB. (H) Single section showing weakly stained GAD1-immunoreactive cell bodies near c232 expressing R-neurons. Images with GAD1 immunolabeling are projections of optical sections from whole mounted brains, images with GABABR immunostaining are projections of optical sections from of cryostat sections. a, anterior; p posterior; c, canal. Scale bars 20 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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Fig. 5. Immunolabeling of glutamate receptor in relation to c232-GAL4 and vGluT-GAL4 in the central complex. R3 and R4d neurons of the EB were visualized using the enhancer trap-GAL4 line c232, and vGluT-GAL4 was used as a marker of glutamate-producing neurons. (A) Immunostaining using glutamate receptor DmGluRA antiserum (magenta) on c232-GAL4 – driven GFP-expressing brains reveals DmGluRA immunoreactive punctates in R3 and R4d rings of the EB and in the LTR. (B) Close apposition of DmGluRA immunoreactivity and expression of vGlutT-GAL4 was observed in an outer ring of the EB, corresponding to the R3 ring. (C) Overlap between DmGluRA immunostaining and vGluT-GAL4 expression was detected in some central and ventral layers of the FB, nonoverlapping labeling was detected in dorsal and in a central layers. (D and E) DmGluRA and vGluT-GAL4 labeling matched well in the three layers of the NO (D) and in the PB (E). Note that in the PB two glomeruli on each side display DmGluRA immunoreactivity but no vGluT-GAL4 expression (brackets). All images are projections of optical sections from cryostat sections. D, dorsal; C, central; V, ventral. Scale bars 20 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

these cell types have been proposed from experimental studies to express the sNPFR1 (Lee et al., 2008; Root et al., 2011).

DISCUSSION In this study, we demonstrated the distribution pattern of six GPCRs in relation to their ligands in the central complex of Drosophila. We found a good match between ligands and GPCRs for serotonin, dopamine, GABA,

glutamate, and the neuropeptide sNPF. However, further receptor types exist for these neurotransmitters (except sNPF): two more serotonin and two further dopamine receptors of GPCR type, as well as ionotropic receptors for GABA and glutamate. Thus this report is providing a first glimpse into neuromodulatory roles of these neurotransmitters in the central complex, but reveals less about possible fast neurotransmission mediated by GABA and glutamate. The findings of this

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Fig. 6. Distribution of the peptide receptor sNPFR1 in the central complex. (A1–A3). Distribution of sNPFR1-GAL4 expression (green) in fan-shaped body compared with immunolabeling with antiserum to sequence of sNPF precursor (␣-sNPFp; magenta). The peptide immunolabeling is seen in five layers (1, 2, 5–7) and the sNPFR1 expression in at least four of these (possibly lacking in layer 6). (B) No sNPFR1 expression was found in the protocerebral bridge (PB). The PB was visualized with antiserum to synapsin (magenta). (C) Also the ellipsoid body (EB; synapsin immunolabeled) is devoid of sNPFR1 expression. (D) As support for the fidelity of the sNPFR1 expression, we show the expression in the insulin-producing cells (IPC) with their axons (arrow) running to tritocerebal neuropil (TC) and in axon terminations of olfactory sensory neurons in the antennal lobes (AL). Scale bars: A, 25 ␮m;, B, 50 ␮m; C, 10 ␮m; and D, 50 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

article are summarized in Tables 1 and 2 and associated Fig. 7. Serotonin signaling in the central complex of Drosophila Immunocytochemical studies have previously localized serotonin to major neuropil structures in the central complex: the EB, the FB, and the NO (Vallés and White, 1988; Kahsai and Winther, 2011). Here, we analyzed the expression pattern of serotonin in relation to two serotonin receptors, 5-HT1B, and 5-HT7, by using serotonin antibody and receptor GAL4 lines. Both receptor GAL4 lines drove expression in the EB and the LTR. However, we could not detect any receptor expression in the FB or NO. Possibly, other serotonin receptor types are expressed in these substructures. One of these, the 5-HT2

receptor, has been reported to be present in the EB, but not been detected in the other central complex substructures (Nichols, 2007). The distribution pattern of the fourth serotonin receptor, 5-HT1A, which belongs to the same family as the 5-HT1B receptor, has not yet been described in detail in the central complex of Drosophila, but reported to be expressed in neurons with branches both in the EB and FB (Luo et al., 2012). Thus, it will be valuable in the future to analyze the expression pattern of 5-HT1A in more detail. It is possible that the receptor protein expression in the FB and NO is considerably lower than in the EB and, thus, difficult to detect. It has been suggested that low amounts of GPCR expression has obstructed immunocytochemical analysis and, thus, limited the information on GPCRs distribution (Nässel, 2009; Nässel and Winther, 2010).

Table 1. Distribution of receptors in substructures of the central complex Receptor

Rings in the EB

Layers in the FB

PB

NO

LTR

5HT1B 5HT7 DopR GABABR DmGluRA sNPFR1

Partially in the anterior EB and in posterior EB Whole anterior EB and in posterior Anterior and posterior EB (R3 and R4d rings) Posterior EB (weak in R3 ring, no labeling in R4d) Anterior and posterior EB (R3 and R4d rings) nd

nd nd Dorsal and ventral FB yes Central and ventral FB Dorsal and ventral FB

nd nd nd yes yes nd

nd nd Layers I, II and III nd Layers I, II and III nd

yes yes nd yes yes nd

yes, detected in neuronal processes; nd, not detected; EB, ellipsoid body; FB, fan-shaped body; PB, protocerebral bridge; NO, noduli; LTR, lateral triangle. Refer to Fig. 7 for structures in central complex.

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Table 2. Substructures of central complex where ligand distribution matches its receptor Receptors/ligandsa

Substructures

Figures

5HT1B/serotonin 5HT7/serotonin DopR/TH

EB EB EB, FB, NO

GABABR/GAD1 DmGluRA/vGluT

EB EB, FB, PB, NO

sNPFR1/sNPF

FB

Figs. 2E1–3 Figs. 2F1–3 Fig. 3B1–3, Fig. 3C1–3, Fig. 3D1–3 Fig. 4C1–3 Fig. 5B1–3, Fig. 5 C1–3, Fig. 5D1–3, Fig. 5E1–3 Fig. 6A1–3

TH, tyrosine hydroxylase; GAD1, glutamic acid decarboxylase1; vGlut, vesicular glutamate transporter, EB, ellipsoid body; FB, fanshaped body; PB, protocerebral bridge; NO, noduli. Refer to Fig. 7 for structures in central complex. a Ligand or other marker for ligand (TH,GAD1,and vGlut).

Although, both 5-HT1B and 5-HT7 receptors are expressed in the EB, some differences in their distribution patterns exist. One major difference lies in the anterior region where 5-HT1B is distributed in the middle ring in parts only, whereas 5-HT7 is expressed over the whole anterior region. In the posterior portion, the distribution of the two receptors is mostly similar; both are expressed in the inner and outermost rings of the EB, with stronger expression of 5-HT7 in the inner ring. This differential distribution of 5-HT1B and 5-HT7 in the EB suggests different functional roles for the serotonin receptor types in the central complex. Certainly this is interesting because it is likely that the 5-HT7 receptor is stimulatory and the 5-HT1B inhibitory (Nichols and Nichols, 2008; Blenau and Thamm, 2011). Distinct roles of serotonin receptor subtypes have been demonstrated previously, for example, both 5-HT1A and 5-HT1B are expressed in the mushroom bodies, and when assayed for baseline sleep, 5-HT1A mutant flies exhibited short and fragmented sleep, whereas sleep in 5-HT1B mutant flies was not affected (Yuan et al., 2005, 2006). In addition, serotonin receptors 5-HT1A, and 5-HT2 were recently shown to modulate different types of aggressive behaviors in Drosophila (Dierick and Greenspan, 2007; Johnson et al., 2009).

We have additionally shown expression of 5-HT1BGAL4 and 5-HT7-GAL4 in relation to serotonin immunolabeling. Varicose serotonin immunoreactive processes were detected superimposed on the 5-HT7 labeling in the middle ring of the EB, suggesting a possible presynaptic expression of 5-HT7. However, no superimposed serotonin processes were seen in the expression pattern of 5-HT1B in the EB. Based on our finding of the distribution of serotonin in relation to 5-HT1B and 5-HT7 receptors and two accounts describing expression of 5-HT1A (Luo et al., 2012) and 5-HT2 (Nichols, 2007), it seems that serotonin targets all four types of serotonin receptors in the EB. The identity of the specific neuron types expressing these serotonin receptors remains to be demonstrated. Dopamine signaling in the central complex By using antiserum to dopamine and TH, the neuronal distribution of dopamine has been shown in Drosophila and other flies (Nässel and Elekes, 1992; Monastirioti, 1999). In addition, TH has been shown to be expressed in the EB, FB, PB, and NO (Friggi-Grelin et al., 2003; Mao and Davis, 2009; Kahsai and Winther, 2011). We compared the expression pattern of TH with that of the stimulatory D1-like receptor DopR and found their distribution to match well in the EB, FB, and NO. In the EB, using the enhancer trap GAL4 line, c232, we identified punctates of DopR in R3 and R4d rings. In the PB and LTR, no DopR immunoreactivity was detected. Here, we suggest the possible presence of other dopamine receptor types or low DopR expression in these neuropil regions. Furthermore, in the NO we detected additional DopR labeling in layer III. Considering the expression of TH in layer II and the small overlap between layer II and III, we suggest a possible case of volume transmission where the dopamine is released in large amount and diffuses to other adjacent neuropil areas. GABA signaling in the central complex GABA and other indicators of GABA signaling, such as the biosynthetic enzyme GAD1 and the vesicular GABA transporter (vGAT), are highly expressed in the EB and

Fig. 7. Schematic representation of the central complex in frontal and horizontal view. This figure shows some of the structures related to in Tables 1 and 2. FB, fan-shaped body; EB, ellipsoid body; No, noduli; D, dorsal layer; C, central layer; V, ventral layer; R3 and R4d, rings of the EB; asterisk, canal of EB.

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sparsely in the FB (Hanesch et al., 1989; Enell et al., 2007). As previously reported, and confirmed in this study, GABABR2 is found strongly expressed in the EB, PB, and LTR and is diffusely and very weakly, distributed in almost all layers of the FB (Enell et al., 2007). As correlated with the c232 enhancer trap GAL4 line, GAD1 immunoreactivity was observed in R3 and R4d rings of the EB. On the other hand, no c232-expressing rings were immunolabeled with antiserum to GABABR2 indicating presence of other types of GABA receptors such as the ionotropic, GABAA receptors. In fact, one of the subunits of GABAA receptors, RDL is widely distributed in the EB (French-Constant et al., 1993; Enell et al., 2007). Because antisera to both GABABR2 and GAD1 were generated in rabbit, we employed a GABABR2-GAL4 to compare the expression pattern of this receptor with that of GAD1 immunostaining. A close match between GABABR2 expression and GAD1 immunostaining was revealed in the posterior ring of the EB and in the LTR, suggesting slow GABAergic inhibition in these regions. However, a mismatch between GAD1 staining and GABABR expression was found in the FB and PB. Varicosities of GAD1 staining were detected in the most ventral layer of the FB, and no staining was seen in the PB. The GABABR2-GAL4 – expressing processes in the FB and PB could represent output regions of neurons that do not express receptor protein (but are visible with the reporter). This is partly supported by our correlation between the expression of GABABR2-GAL4 and the GABABR2 antiserum. We found the GAL4 and antiserum to correspond well in the EB, PB, and LTR, but not in the FB. Overall our results show clear evidence for GABAergic signaling mediated via GABABRs in the EB and LTR. Glutamate signaling in the central complex Reports have shown the distribution of vesicular glutamate transporter (vGluT) in neuronal processes in the EB, FB, PB, and NO of Drosophila (Daniels et al., 2008; Kahsai and Winther, 2011). The distribution of DmGluRA has also been partially demonstrated in the adult Drosophila brain (Hamasaka et al., 2007; Devaud et al., 2008). We found that DmGluRA immunoreactive punctates correspond well with the expression of vGluT in all the major neuropil structures in the central complex. Superposition between DmGluRA immunolabeling and vGluT expression was detected in R3 rings of the EB (as identified by the c232 line), in the central and ventral layer of the FB, in the three layers of the NO and in the PB, indicating glutamatergic transmission in this region. Additional vGluT labeling was seen in rings proximal to the R3 ring of the EB, suggesting glutamate signaling via other types of receptors such as the ionotropic glutamate receptors. sNPF signaling in the central complex In the central complex, sNPF is restricted to several layers of the FB (Nässel et al., 2008; Kahsai et al., 2010; Kahsai and Winther, 2011), and the distribution of the sNPFR1 matches these layers quite well. This receptor is the only GPCR known for sNPF in Drosophila (Mertens et al., 2002;

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Feng et al., 2003). It is not clear what G-protein ␣-subunit that sNPFR1 couples to because only nonhomologous expression systems and a promiscuous G-protein (G␣16) were utilized for receptor assay (Mertens et al., 2002; Feng et al., 2003; Reale et al., 2004). In insulin-producing cells of the Drosophila brain, however, experiments suggested that the sNPFR1 signals via extracellular signal regulated kinase, ERK (Lee et al., 2008). Both the sNPF peptide and receptor distribution is widespread and suggests pleiotropic functions of this signaling system in the fruit fly (see Mertens et al., 2002; Nässel et al., 2008; Nässel and Wegener, 2011). Experimental data indicate that sNPF signaling plays roles in regulation of feeding and growth, regulation of osmotic stress, modulation of olfactory sensitivity, control of locomotor activity (in central complex) and maybe also in modulation of learning and memory in mushroom bodies (summarized in Nässel and Winther, 2010; Nässel and Wegener, 2011; Root et al., 2011). Studies in the central complex where sNPF expression in specific subsets of neurons was diminished by RNA interference show effects on distance walked by the flies and in females a higher speed of walking (Kahsai et al., 2010). Functional implications and concluding remarks Central complex neurons are not only morphologically heterogeneous, but also their content of neurotransmitters and neuropeptides is very diverse. A few studies have attempted to examine the functional roles of neurotransmitters and neuromodulators in the central complex. Recently the peptides DTK and sNPF, expressed in FBneurons, have been shown to modulate spatial orientation and walking activity in Drosophila (Kahsai et al., 2010). Other studies showed that the dopamine receptor DopR expressed in EB-neurons are involved in the regulation of ethanol-induced locomotor activity (Kong et al., 2010) and in arousal (Lebestky et al., 2009). Some aspects of longterm olfactory memory require ionotropic glutamate receptors in the EB (Wu et al., 2007) and GABA-expressing R-neurons of the EB regulate spatial orientation memory (Neuser et al., 2008). Furthermore, several reports using nontargeted pharmacological and genetic manipulations have shown the roles of different neurotransmitters in behaviors that are associated with the central complex. For example, serotonin signaling has been shown to play an important role in place memory (Sitaraman et al., 2008), and dopamine is known to be involved in spontaneous locomotion (Pendleton et al., 2002), as well as locomotion induced by drugs of abuse (Bainton et al., 2000; Pendleton et al., 2002). GABA signaling, via a metabotropic receptor, has been implicated in locomotion and ethanol tolerance (Dzitoyeva et al., 2003). Thus, the central complex organizes and regulates several diverse and complex behaviors, and it is not surprising that neurons in this neuropil region produces multiple neurotransmitters and neuromodulators (Monastirioti, 1999; Nässel and Homberg, 2006; Nässel and Winther, 2010; Boyan and Reichert, 2011; Kahsai and Winther, 2011; Kunst et al., 2011) as this allows for complex chemical signal processing and modulation. Here, we add to the

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complexity and provide a detailed map of six metabotropic receptors in relation to their ligands (see Tables 1 and 2). In addition to the six GPCRs examined, our comparative receptor/ligand data suggest that additional GPCR types are expressed for dopamine and serotonin and also ionotropic receptors for GABA and glutamate, thus, further increasing the base for modulation and plasticity within these circuits. Moreover, three other neuropeptide receptors have been implicated in the Drosophila FB by immunocytochemistry: two DTK receptor types and one proctolin receptor (Johnson et al., 2003; Birse et al., 2006; Poels et al., 2009). Our study provides a starting point for further anatomical studies of this intricate neuropil region. In addition, data presented here can be used for designing experimental work using targeted interference with ligands or receptors in specific central complex neurons followed by behavioral analysis similar to a previous study from this laboratory (Kahsai et al., 2010). Acknowledgments—The research was funded by the Swedish Research Council (D.R.N.), Carl Tryggers Foundation (D.R.N., Å.M.E.W.), Royal Swedish Academy of Science, and Magnus Bergvalls Foundation (Å.M.E.W.). We are grateful to Kyung-An Han, Rob Jackson and Jan Veenstra for providing antisera. The monoclonal antibodies developed by E. Buchner were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA, USA. We also thank Serge Birmann, Aaron DiAntonio, Charles Nichols, Amita Sehgal, Paul H. Taghert, Jing W. Wang, and Bloomington Drosophila stock center for providing different Drosophila lines. This study was supported by grants VR 621-2007-6500 from the Swedish Research Council (D.R.N.), Carl Trygger Foundation (D.R.N. and Å.M.E.W.), Royal Swedish Academy of Science, and Magnus Bergvalls Foundation (Å.M.E.W.).

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(Accepted 7 February 2012) (Available online 11 February 2012)