Distribution of the Octopamine Receptor AmOA1 in the Honey Bee Brain . . Irina Sinakevitch , Julie A. Mustard , Brian H. Smith* Arizona State University, School of Life Sciences, Tempe, Arizona, United States of America Abstract Octopamine plays an important role in many behaviors in invertebrates. It acts via binding to G protein coupled receptors located on the plasma membrane of responsive cells. Several distinct subtypes of octopamine receptors have been found in invertebrates, yet little is known about the expression pattern of these different receptor subtypes and how each subtype may contribute to different behaviors. One honey bee (Apis mellifera) octopamine receptor, AmOA1, was recently cloned and characterized. Here we continue to characterize the AmOA1 receptor by investigating its distribution in the honey bee brain. We used two independent antibodies produced against two distinct peptides in the carboxyl-terminus to study the distribution of the AmOA1 receptor in the honey bee brain. We found that both anti-AmOA1 antibodies revealed labeling of cell body clusters throughout the brain and within the following brain neuropils: the antennal lobes; the calyces, pedunculus, vertical (alpha, gamma) and medial (beta) lobes of the mushroom body; the optic lobes; the subesophageal ganglion; and the central complex. Double immunofluorescence staining using anti-GABA and anti-AmOA1 receptor antibodies revealed that a population of inhibitory GABAergic local interneurons in the antennal lobes express the AmOA1 receptor in the cell bodies, axons and their endings in the glomeruli. In the mushroom bodies, AmOA1 receptors are expressed in a subpopulation of inhibitory GABAergic feedback neurons that ends in the visual (outer half of basal ring and collar regions) and olfactory (lip and inner basal ring region) calyx neuropils, as well as in the collar and lip zones of the vertical and medial lobes. The data suggest that one effect of octopamine via AmOA1 in the antennal lobe and mushroom body is to modulate inhibitory neurons. Citation: Sinakevitch I, Mustard JA, Smith BH (2011) Distribution of the Octopamine Receptor AmOA1 in the Honey Bee Brain. PLoS ONE 6(1): e14536. doi:10.1371/journal.pone.0014536 Editor: Bjo¨ rn Brembs, Freie Universitaet Berlin, Germany Received June 9, 2009; Accepted December 1, 2010; Published January 18, 2011 Copyright: 2011 Sinakevitch et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by the National Institutes of Health National Center for Research Resources grant RR014166 and National Institute on Deafness and Other Communication Disorders grant DC007997 to BHS; National Institute on Drug Abuse grant DA017694 to JAM; and Fondation pour Recherche Medicale grant UFM20060306548 to IS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: brian.h.smith@asu.edu . These authors contributed equally to this work. Introduction The biogenic amine octopamine acts as a neurotransmitter, neuromodulator and neurohormone in the nervous system of invertebrates [1,2]. Numerous functions have been assigned to octopamine including regulation of lipid versus carbohydrate metabolism in insect flight muscle [3,4], increasing levels of arousal [5–7], modulation of sensory perception [8–12], aggres- sion [13,14], control of locomotion [15–17] and signaling the presence of reward in appetitive olfactory learning [18–21]. In the honey bee, octopamine has also been linked with social behaviors such as nestmate recognition [22], hygienic behavior [23] and division of labor [24,25]. In order to understand the specificity of the action of octopamine in so many different types of behaviors, it will be necessary to describe not only where octopamine is released but also the distribution of different octopamine receptor subtypes in the brain. Several studies have investigated the sites of octopamine release in the insect central and peripheral nervous system [26–33]. In the honey bee brain, different clusters of octopaminergic neurons release octopamine in distributed areas of the brain. For example, many areas receive input from the octopaminergic ventral unpaired median neurons (central VUM neurons) identified in part by cell bodies that lie on the median (midline) ventral part of the subesophageal ganglion [30,32,34,35]. Two of these neurons, VUMmx1 and VUMmd1, have a primary neurite that projects through the midline tract of the subesophageal ganglion and gives rise to two symmetrical secondary axons that project collaterals to the antennal lobes, lateral horn, lateral protocerebrum and to the lip and basal ring of the mushroom body calyces [34,35]. Collectively, these neuropils process many of the different types of sensory stimuli that honey bees are capable of associating with reinforcement. Octopamine also plays an important role in modulating activity in the visual neuropil and the central complex of the honey bee brain. There is a group of six neurons, group G3a [32], located in the medial part of the tritocerebrum that send their axons through the posterior optic tract to provide extensive arborizations in the lobula, medulla and lamina of the compound eyes. Some of these neurons send their collaterals into the optic lobe, the ocelli and central complex. Octopamine application in the optic lobes enhances directional sensitivity of the antennal reflex to visual stimuli [8], and it has modulatory effects on motion-sensitive neurons in the lobula [36]. In addition, the central complex PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e14536