conjugated to AmOA1 peptide that was used to make antibodies in goat (Fig. 1G). To prepare the preadsorbed antibody, 400 mM of peptide was conjugated to KLH via glutaraldehyde and incubated with working dilution of Ganti-AmOA1. For double immunofluorescence staining with Ganti-AmOA1 and GABA, the agarose brain sections were incubated simulta- neously with both antibodies, then after a thorough wash in PBS, the secondary antibodies F(ab’) 2 fragments of donkey anti-Goat IgG Cy2 and F(ab’) 2 fragments of donkey anti-Rabbit IgG Cy5 were added in dilution 1:200 for incubation overnight. For control of staining, both of the secondary antibodies were incubated with sections that had only one of the primary antibodies, and the staining did not show any cross-reaction between the antibodies (Fig. S1D–F). Procedures with all antibodies were performed at room tem- perature. Images of sections treated with antibodies preincubated with peptide were taken with the intensity of fluorescence gain equal to or greater than images of antibody treated sections. For comparison, images were collected at the same level of gain and intensity. In Fig. 1D, F, G the image was collected at a higher gain because confocal collection at the same gain as shown in Figures 1C and D resulted in such low intensity that the image was not visible. Results Specificity of the anti-AmOA1 antibodies Two different polyclonal antibodies were used in our studies, one raised in rabbit (Ranti-AmOA1) and another raised in goat (Ganti-AmOA1) against two different peptides corresponding to different regions in the cytoplasmic carboxyl terminus of the AmOA1 receptor. Double labeling experiments revealed that these antibodies labeled the same cells and processes in the neuropil of the bee brain (an example of double staining in the antennal lobe is in Figure S1, G1-3). For this reason we do not specify the R or G version of the antibody in the results. Unless otherwise noted in the legends, figures show data from the Ranti- AmOA1 serum. Both antibodies labeled cell body clusters and many processes in the antennal lobe, mushroom body (calyces, pedunculus, alpha, beta and gamma lobes), optic lobe, subesophageal ganglion and central complex (Fig. 1A, C, E, 2). The immunostained cell body clusters and processes were repeatable from one animal to the next when we compared brains of forager bees caught at the entrance of the same colony. Staining in cell bodies and processes ranged from high to low intensity (Fig. 1A,C,E). This variation is consistent with data from in situ hybridization where differences in gene expression in cell bodies were previously reported [43]. Preadsorption of either antibody with the corresponding peptide-G-KLH (concentration of the peptide was 100 or 400 mM) abolished specific labeling (Fig. 1D,G). Furthermore, the rabbit or goat preimmune serum used on adjacent sections at the same concentration as AmOA1 antiserum did not show any staining (Fig. 1B,F). The AmOA1 peptide used for immunization in rabbit shows 87% sequence identity to the fruit fly OAMB receptor isoform A peptide sequence (NH2-DFRFAFKRIICRCFC-OH; amino acids 578–592 of DmOA1 A; AAF55798). Therefore, we used the Drosophila oamb96 mutant line, which contains a deletion spanning exons 2 and 3 and does not produce the DmOA1 A protein in the brain [44], as a control for antibody specificity. We used Ranti- AmOA1 to label sections from brains of wild type Canton S (CS) and oamb96 flies (Figure S1H–K). As expected [40], the Ranti- AmOA1 antibodies labeled cells and neuropils in the mushroom body alpha prime and beta prime lobes as well in the antennal AmOA1 in the Honey Bee Brain lobes of CS female flies (Fig. S1H,J). However, specific labeling in the mushroom body lobe and antennal lobe was absent from the oamb96 sterile female flies (Fig. S1I,K). These data show that the antibodies specifically recognized the Drosophila OAMB receptor isoform A. Neuroanatomical distribution of the AmOA1 immunostaining in the honey bee brain Our description below of AmOA1 distribution in the brain is based on immunostaining of ten honey bee forager brains processed with Ranti-AmOA1 and five honey bee brains processed with Ganti-AmOA1. A schematic view of the overall AmOA1 immunoreactivity of cell bodies and processes in the honey bee brain is shown in Figure 2. This drawing was made from confocal images of frontal sections of bee brain. It summarizes the most typical labeled cell bodies and processes in the central complex, mushroom bodies, antennal lobes, lateral protocerebrum, optic lobes and subesophageal ganglion. We begin each section below with a brief description of the anatomy of that area of the brain. AmOA1 staining in the antennal lobes The primary olfactory neuropil is the antennal lobe, which consists of approximately 160 spherical glomeruli that surround a coarse neuropil in the core (Fig. 2, 3A–C, 5). The principal inputs of the glomeruli are olfactory receptor neurons from the antennal nerve. Glomeruli are also invaded by processes from at least three cell types: (1) two types of projection neurons; uniglomerular projection neurons, which connect the antennal lobe with the mushroom body calyx and the lateral horn via the lateral or medial antennocerebral tract (the l- and m-ACT), and multi- glomerular projection neurons that connect the antennal lobe with the protocerebral lobe and lateral horn via the medio-lateral tract (mlACT); (2) two or more types of local interneurons with axons in the coarse neuropil and dendrites in the glomeruli; and (3) multiglomerular interneurons that express biogenic amines and have cell bodies outside of the antennal lobe, for example VUMmx1 [34,52–54]. All but the latter type of interneuron have cell bodies in one of three groups that lie lateral (LG), dorsal (DG) or mediodorsal (MDG) to the antennal lobe (Fig. 3). Each group (LG, DG, MDG) contains the cell bodies of two or more types of antennal lobe neurons. AmOA1 immunoreactivity was present in all three cell body groups (MDG, DG, and LG) that surround the glomerular neuropil as well as in glomeruli and the coarse (central) area of the antennal lobe neuropils (Fig. 3A, B, C). The olfactory receptor neurons enter from the antennal nerve via four tracts (T1–T4), but only T1 and T2 enter the coarse area in the center of the antennal lobe (Fig. 3A). The olfactory receptor neuron axons were not stained with the AmOA1 antibodies, except for a few scattered beaded processes, which may also be glial cells, in the T1–T2 tracts where they enter the coarse area (Fig. 3A arrow). The glomeruli, as well as the coarse neuropil of antennal lobes, were strongly labeled with AmOA1 antibodies (Fig. 3A–C). Among the different areas of the antennal lobe, labeling in the glomeruli was the most variable both within and between preparations. Immunostaining in some glomeruli revealed a very strongly labeled core region with absence of staining in the surrounding cortex (Fig. 3A double arrow) where most of the olfactory receptor neurons from sensory tracts T1–T3 send their input to the glomeruli [55]. Some glomeruli exhibited an almost complete absence of AmOA1 immunoreactivity, or staining was scattered throughout the entire glomerulus, (Fig. 3B). In other PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e14536