olfactory associative learning in the honey bee [18] suggest that this receptor is an important part of the downstream targets of the associative reinforcement pathway mediated by VUMmx1. One way that AmOA1 may play a role in associative learning is through alteration of interactions in the antennal lobe network when odor is paired with sucrose [71]. In the antennal lobes, antibodies against AmOA1 stain three groups of neurons that are located laterally and dorsally. Double immunostaining with anti- AmOA1 and anti-GABA showed that these AmOA1 positive cells are a subpopulation of the GABAergic neurons. The neurons that stained with anti-AmOA1 but not with GABA might belong to subset of histaminergic local interneurons [72]. Local interneurons in the antennal lobes (LIN; Fig. 9) act to transform olfactory sensory information received from the antennae before it is sent via projection neurons to other areas of the brain. Expression of AmOA1 in the local interneurons provides a mechanism for linking the odor representation with the occurrence of the sucrose reward via release of octopamine from VUMmx1. It is difficult to identify the subtype of neurons from the other two cell clusters in the antennal lobe (the dorsal and dorso-medial cell clusters) that stain with AmOA1. However, they are probably not the uniglomerular projection neurons because staining is absent from their axons and terminals in the lip and basal ring region of the mushroom body calyx, which are the targets of these projection neurons. (This observation will need to be confirmed by AmOA1 staining of uniglomerular PNs that have been intracel- lularly filled, as the receptor might be expressed in the dendrites of these neurons). On the other hand, AmOA1 immunoreactivity was present in the medio-lateral ACT (m-l ACT) and in the posterior lateral protocerebrum. This pattern suggests that some of the labeled neurons from the dorsal and dorso-medial groups may be multiglomerular projection neurons that receive input from several glomeruli [54]. Therefore, AmOA1 may not only affect odor representation via modulation of the local interneurons, but may also act upon the multiglomerular projection neurons that carry odor infor- mation from the antennal lobes to the lateral horn. This partitioning of AmOA1 mediated sensitivity to octopamine fits with a recent hypothesis that proposes different signaling roles for the two different olfactory tracts in the honey bee antennal lobes [54,73]. In the fruit fly, a set of projection neurons that carry olfactory information directly from the antennal lobes to the lateral protocerebrum appear to be important for more stereotypical ‘‘experience-independent’’ behaviors [74]. Given the expression of AmOA1 in the m-l ACT, it would be interesting to determine if this receptor is involved in experience-independent olfactory behavior as well as playing a role in associative learning. AmOA1 expression in the mushroom bodies Immunocytochemical studies suggest that octopamine is likely released in the mushroom body calyces and the c division of the vertical lobe [30,32]. The calyces are innervated by the VUMmx1 and VUMmd1 neurons, which have fine arborizations mostly in the lip and basal ring area, and a few branches are also present in the collar region [30,32,34,35]. AmOA1 immunoreactivity overlapped substantially with VUM-based octopaminergic inputs in the mushroom body calyces. The clawed Kenyon cells express AmOA1 in their cell bodies, dendrites and axons. These cells are of interest for several reasons. First, the axons of clawed Kenyon cells from across the entire calyx converge on the c lobe of the mushroom body, thereby integrating input from different sensory AmOA1 in the Honey Bee Brain modalities [60]. Second, different octopaminergic modulatory pathways, which may carry different types of information, target different regions of clawed Kenyon cells. Octopamine released from VUM neurons in the lip and inner basal ring likely targets dendrites of the different types of Kenyon cells. Octopamine released from VUM neurons probably also targets afferent neurons from visual neuropils and GABAergic neurons in the PCT since these neurons express AmOA1 receptors. In addition, the octopamineric VCBN neuron (nomenclature of [75]; LV from [32]) has a cell body located in the VUM cluster in the subesophageal ganglion, and its axons target axons of the clawed Kenyon cells in the c lobe of the mushroom body. The fact that different types of sensory information from a number of different brain regions converge on clawed Kenyon cells, and that octopamine has been found to be necessary for appetitive olfactory learning [18–21], suggests that the clawed Kenyon cells should be an important subject for future studies. A comparison of GABA immunoreactivity with the localization of staining for AmOA1 suggests that AmOA1 receptors are expressed in a subset of GABAergic feedback neurons (PCT). In the honey bee, most of the GABA immunoreactivity in the calyces arises from a group of feedback or recurrent neurons innervating both input and output areas of the mushroom bodies [68,69]. These neurons change their properties in response to odors after olfactory conditioning and are likely to be involved in memory formation [69]. Our data provide the first evidence that the octopamine released in the mushroom body calyx and lobe may target GABAergic feedback neurons. Possible roles for AmOA1 in visual processing In most preparations, the regions of the mushroom body associated with the visual calyx neuropil (collar and outer basal ring) showed relatively intense staining for AmOA1. We found small populations of class I Kenyon cells [60] that strongly express AmOA1 immunoreactivity in their cell bodies and in axons in both the vertical and medial lobes. These Kenyon cells have their dendrites in the visual calyx neuropil while their axons project through defined tracts in both the medial and vertical lobes to give rise to outputs from the mushroom bodies that represent the basal ring and collar regions. The edge area in the outer zone of the basal ring was especially brightly stained with AmOA1 antibodies. This area corresponds to rosette- shaped Kenyon cells that receive input from the ventral medulla [60,61]. Octopamine has been shown to play various roles in visual processing in insects. In the honey bee, application of octopamine to the lobula enhances the directional antennal response to a moving striped pattern [8]. Furthermore, field potentials in the lobula showed increases in amplitude in the presence of exogenous octopamine [36]. In locusts, the release of octopamine by the PM4 cells (homologs of the honey bee G3A cells) is stimulated by multimodal input from the central brain [6]. This release of octopamine has been implicated in dishabituation of output neurons from the optic lobe [6,76,77]. Our results show that octopamine has multiple targets in the optic lobes, which suggests that the dishabituation may result from broad-based effects on the neural networks in the optic lobes. AmOA1 in the subesophageal ganglion The subesophageal ganglion of the honey bee receives sensory projections from receptors located on the respective mouthparts through the corresponding mandibular, maxillary, and labial PLoS ONE | www.plosone.org 13 January 2011 | Volume 6 | Issue 1 | e14536