antiserum labels a subpopulation of class I Kenyon cell bodies with higher intensity. It is possible to follow their stained axons and dendrites in the outer basal ring zone (Fig. 3D,H); the area at the edge of the dorsal basal ring region is strongly labeled in all preparations. The Kenyon cell dendrites in the collar are less intensely stained than dendrites in the outer basal ring (Fig. 3D,H). The edge of the outer basal ring area receives visual input from axons from the medulla, whereas the collar region receives input from both the medulla and lobula of the compound eyes. The Kenyon cells with dendrites in the collar and basal ring express AmOA1 protein in their cell bodies, dendrites and in their axons that make up the layers in the vertical lobe and medial lobe (Fig. 3E, F). The class II Kenyon cells also exhibited strong immunoreac- tivity in their cell bodies and their axons in the ventral part, or c division, of the vertical lobe (Fig. 3E, H). Some of these AmOA1expressing class II cells have their cell bodies outside of the mushroom body calyces (arrow in Fig. 3H) and their dendrites spread throughout the calyx into all sensory input zones. Their axons are brightly labeled in the c division of the vertical lobe (Fig. 3E). Apart from the Kenyon cells, other types of neurons in the mushroom body lobes also express the AmOA1 receptors. These are extrinsic neurons with large AmOA1 antiserum positive axons in the c division of the vertical lobe. The neurons that enter the vertical lobe just above the c division (arrow in Fig. 3F, also 7F, 7H2) correspond to the feedback neurons that connect mushroom body calyces with mushroom body lobes. The areas of the calyx that receive inputs from the antennal lobe (lip and inner basal ring) stained with somewhat different levels of intensity in different animals (Fig. 1A, 3D, 3H, 7B, 7D2). For example, the lip is more intensely labeled in figure 3H than in 3D. In most preparations, the lip and inner basal ring were less intensely stained compared to subdivisions of the calyx that receive input from visual processing areas (collar and outer basal ring; Fig. 3D, 3G, 7B, 7D2). Especially brightly stained was the edge area in the outer zone of the basal ring (o.w.br in Fig. 3D, 7B). This area corresponds to rosette-shaped Kenyon cells that receive input from the ventral medulla [60,61]. The stained processes in this outer basal ring area may correspond to the endings of afferents from the medulla (Fig. 3G,H). Neurons that connect the medulla to the mushroom body calyx via the anterior superior optical tract (asot) are located in the posterior edge between the mushroom body calyx and the visual neuropil (Fig. 2, arrowhead in 3H). Some of the neurons from this group showed strong AmOA1 immunoreactivity in cell bodies and axons in the asot (Fig. 2), processes in the serpentine layer of the medulla (Fig. 4C), and in the visual input area in the outer basal ring (Fig. 3D,H). There is also staining in some neurons that connect the mushroom bodies with the lobula through the lobula optic tract (lot; Fig. 2). In most preparations, the protocerebral lobe is stained with AmOA1 antibodies at a lower intensity compared the antennal lobe and mushroom body neuropil (Fig. 3I). The few positively stained processes are scattered through the lateral protocerebrum neuropil and in the lateral horn. AmOA1 expression in the optic lobes The optic lobes are neuropilar structures adjacent to the compound eyes. They receive and process input from photore- ceptors originating in the retina (Fig. 2). The optic lobe consists of the lamina, the medulla (the outer and the inner separated by the serpentine layer; Fig. 2, 3H, 4A–C), and the lobula (Fig. 3H). Some photoreceptor cells exhibit intense staining with the AmOA1 receptor antibodies (Fig. 2, cluster 3; arrow in Fig. 4A). AmOA1 in the Honey Bee Brain Lamina monopolar cells express the AmOA1 receptor as illustrated in figure 4A where a cluster of monopolar cell bodies is brightly stained with AmOA1 antibodies (Fig. 2, cluster 4; Fig. 4A arrowhead). The first and second optic chiasmata between the lamina and the medulla, and between the medulla and the lobula, respectively, have AmOA1 immunoreactive fibers (Fig. 4B, C). In the medulla there are columnar cells labeled with AmOA1 antiserum (Fig. 2, cluster 5; Fig. 4C) that may either be intrinsic to the medulla or that may be transmedullary and connect the medulla with the lobula via the 2nd optic chiasma. The AmOA1 antiserum stained processes in the medulla serpentine layer may belong to the cells that connect the medulla to the mushroom body calyces (Fig. 2, cluster 2; Fig. 3H), the cell bodies of which are located between the base of the lateral calyx and the dorso-medial edge of the medulla. Likewise, stained processes in the lobula may belong to cells that connect the optic lobula with mushroom body lobula cells (Fig. 2). AmOA1 staining in the subesophageal ganglion The subesophageal ganglion of the honey bee is composed of the fused mandibular, maxillary and labial neuromeres. These neuromeres receive gustatory sensory information and are involved in the motor control of the mouthparts [64]. There are clusters of small cell bodies located in the median ventral and lateral parts of the subesophageal ganglion that stained strongly with AmOA1 antibodies (Fig. 2, cluster 7; Fig. 4D–F). The VUM cells, characterized by large cell bodies in the median part of the subesophageal ganglion, are not stained in either the cell bodies or in the primary neurites in the median and lateral tracts (Fig. 4D– F). Scattered processes staining with AmOA1 antiserum are present in the subesophageal ganglion neuropil and may belong to the descending and ascending neurons as well as to local interneurons (Fig. 4D–F). Staining was absent in the mandibular labial and maxillary nerves (data not shown). AmOA1 staining in the central complex The central complex is a prominent structure located in the central brain between the two protocerebral hemispheres [65,66]. It comprises four neuropilar regions: the ellipsoid body (the lower division of the central body), the fan shaped body (the upper division of the central body), the paired noduli and the protocerebral bridge. These structures are interconnected by sets of columnar neurons that form a regular projection pattern. Anti-AmOA1 staining in the honey bee central complex is illustrated in Figures 2 and 4G. A group of AmOA1 expressing cells located above the dorsal lobe in the medial protocerebrum (Fig. 2, cluster 8) send their axons to terminate in the ellipsoid body (Fig. 4G), and these axons have a very high level of AmOA1 immunoreactivity. These axons can be seen entering the base of the ellipsoid body in Fig. 4G, and we traced them back to the cell bodies located near the dorsal lobe (Fig. 2, cluster 8; confocal image not shown). The fan shaped body neuropil has a lower level of AmOA1 immunoreactivity relative to the high intensity staining in the ellipsoid body (Fig. 4G). GABAergic local interneurons in the antennal lobes label with AmOA1 antibodies In order to compare the distribution of octopamine and the distribution of AmOA1 in the antennal lobes, we labeled adjacent sections of the same brain with octopamine (Fig. 5A) and AmOA1 antisera (Fig. 5B). The octopamine-like immunoreactive processes from VUM neurons enter in the coarse center of the antennal lobes from the ventro-posterior deutocerebral area and give rise to PLoS ONE | www.plosone.org 7 January 2011 | Volume 6 | Issue 1 | e14536