nerves [63,64,78]. It also gives rise to motoneurons that supply the muscles of the mouthparts. AmOA1 is expressed in the cells with small somata located in the lateral and median part of the ganglion. There are also AmOA1 positive processes in the neuropil of the subesophageal ganglion. However, it is difficult to assign a specific function to these AmOA1 positive cells. The lack of AmOA1 staining in the octopaminergic VUM cells suggests that this receptor is not acting as an autoreceptor to regulate the release of octopamine. AmOA1 in the central complex The central complex is widespread in arthropods, and the complexity of its structure is correlated with the complexity of the organism’s behavioral repertoire [66]. Mutation analysis in fruit flies has shown that the central body functions to control locomotor activity by affecting gait and the coordination of left and right sides of the body during walking as well as landmark orientation [79]. In various taxa, including the honey bee, the central complex receives inputs from the optic lobes and ocelli. Information from those modalities are represented, and sometimes segregated, in different subdivisions [65]. In the honey bee, electrophysiological recordings from neurons intrinsic to the central complex revealed that they respond to several stimulus modalities [80]. In the honey bee, octopamine immunoreactivity is found in the protocerebral bridge, in the chiasmal axons reaching forward through the fan shaped body, in the fan shaped body and in the ellipsoid body [32]. The AmOA1 receptor is expressed at relatively high levels in the ellipsoid body in the GABAergic tangential neurons that originate in the frontal part of the brain near the dorsal lobe. The protocerebral bridge stains much less intensely for AmOA1 with only a few labeled fibers running perpendicular to its columnar elements. Given that octopamine plays an important role in arousal [5], that octopamine affects the behavioral profile of the honey bee [16], and that the central complex is involved in control of motor behavior, it is possible that the AmOA1 receptor may be important for modulating arousal levels and coordinating motor behavior in the honey bee. Comparison of AmOA1 receptor and octopamine staining in the brain Several studies have examined the pattern of octopamine- like immunoreactivity in the honey bee brain [30,32]. Although, in general, the staining patterns for octopamine and the AmOA1 receptor overlap, there are regions where this is not the case. Regions where octopamine is present at relatively high levels while anti-AmOA1 antibodies show relatively low levels of staining, such as the lip of the mushroom body calyx [32], may be explained by the presence of other distinct octopamine receptors. Several other putative octopamine receptors exist in the honey bee [38]; however, to date, nothing is known about their expression pattern in the brain. More puzzling, perhaps, is the expression of AmOA1 in regions of the brain that appear to have little or no octopamine. For example, AmOA1 is present in processes in regions of the mushroom body vertical lobe, which do not show octopamine immunoreactivity [32]. These receptors may be responding to octopamine diffusing onto these cells from a relatively distant release site and, thus, acting as a neurohor- mone. Extrasynaptic signaling by a biogenic amine has been well characterized in C. elegans where dopamine acts upon motor neurons that express dopamine receptors, but which are not postsynaptic to dopaminergic neurons [81]. AmOA1 in the Honey Bee Brain Conclusions AmOA1 receptors are expressed on GABAergic neurons located in several regions throughout the brain: the mushroom bodies, the antennal lobes and the central complex. Although it is clear that not all GABAergic neurons express the AmOA1 receptor, these data suggest that octopamine may modulate the activity of some inhibitory circuits via the AmOA1 receptor. Although other octopamine receptors are probably present in the honey bee, they belong to a separate subfamily that most likely is linked to cAMP signaling [38,42]. Thus, the expression pattern of AmOA1 presented here indicates cells that will respond to octopamine by increasing intracellular levels of Ca2+ [43]. This information will be a valuable guide in future studies examining the role of octopamine in specific behaviors. Supporting Information Figure S1 Characterization of anti-AmOA1 antibodies and double staining controls. A: Double immunostaining of the bee antennal lobe with anti-GABA and Ranti-AmOA1 antibodies B, C: Two consecutive sections of the honey bee antennal lobe for control of sequential staining with Ranti-AmOA1 (B) and GABA (C) antisera where the GABA or AmOA1 antibodies were omitted. D-F: Double immunofluorescence staining in the bee antennal lobe with anti-GABA and anti-AmOA1 antibodies from goat (Ganti-AmOA1). The staining reveals that most GABAergic neurons in the lateral group (LG) are positive for AmOA1. Neurons that have low intensity staining with AmOA1 but a high level of staining with anti-GABA are shown by an asterisk. G1-G3: Comparisons of immunostaining of the anti-AmOA1 from rabbit (Ranti-AmOA1) with anti-AmOA1 from goat (Ganti-AmOA1) on the same section of the antennal lobe. Double fluorescence staining in the antennal lobe with antibodies against the AmOA1 receptor from rabbit (G1, magenta, Ranti-AmOA1) and goat (G2, green Ganti-AmOA1), reveal staining in the same cell bodies (lateral cluster, LG) and the same processes in the glomerular neuropil as shown by white in the merged image (G3). H: Control of immunostaining in agarose sections in the wild type (WT) Drosophila brain. The Ranti-AmOA1 antibodies recognized the OAMB receptor in the mushroom body (a/b’ lobes and spur region of the pedunculus). A high level of staining is also observed in the anterior superiormedial protocerebrum (asmpr). I: In the oamb96 mutant, staining in the mushroom body is not present. J: In the antennal lobe (ant lobe) of a wild type fly, the Ranti- AmOA1 antibody recognizes cell bodies surrounding the antennal lobe neuropil and processes in the glomerular neuropil. K: In an oamb96 mutant fly, specific staining in the glomerular neuropil and cells is absent. Scale bars: 25 mm. Found at: doi:10.1371/journal.pone.0014536.s001 (23.11 MB TIF) Acknowledgments The authors are grateful to Prof Nick Strausfeld for allowing us to use his facilities during the initial stages of this work, to Prof Mike Adams and Dr Serge Birman for letting us perform experiments in their labs, and to Dr Birgit Ehmer and to Dr Sheena Brown for reading the first version of the manuscript. We also thank Dr Jurgen Rybak and Danielle Protas for valuable comments on the manuscript. We are grateful to Kim Fondrk and Osman Kaftanoglu for maintaining the honey bee colonies at Arizona State University. Author Contributions Conceived and designed the experiments: IS JM BHS. Performed the experiments: IS JM. Analyzed the data: IS. Wrote the paper: IS JM BHS. PLoS ONE | www.plosone.org 14 January 2011 | Volume 6 | Issue 1 | e14536