AmOA1 in the Honey Bee Brain Figure 2. Schematic drawing of AmOA1 immunoreactive neurons and processes in the brain and subesophageal ganglion of the bee made from confocal images of agarose sections of bee brain stained with anti-AmOA1 antibodies. The right hemisphere shows a frontal view of the bee brain with the optic lobe neuropils (Re, retina; La, lamina; Lo, lobula), antennal lobe (al) and summarized mushroom body neuropil; the left hemisphere of the brain shows a more posterior frontal view at the level of the dorsal lobe (dl), pedunculus (ped) and calyces of the mushroom bodies. AN, antennal nerve; DG, dorsal group of antennal lobe neurons; LG, lateral group of antennal lobe neurons; MDG, medial group of antennal lobe neurons. Some neurons that express the AmOA1 receptor could be identified as: 1, PCT (or feedback neuron group); 2, lobula and medulla mushroom body neurons. The cells in this cluster have fibers in the anterior superior optical tract (asot). The arrow shows axons (only the ending of the tract) from these neurons as they enter the medulla serpentine layer. A few AmOA1 positive fibers enter through the lobula optical tract (lot) that connects the lobula and mushroom bodies.; 3, photoreceptor cells in the retina; 4, monopolar cell bodies in the lamina; 5, medulla columnar neurons; 6, photoreceptor cells in the ocelli; 7, a median group of subesophageal intrinsic neurons; 8, ellipsoid body neurons, 9, a group of median neurosecretory cells (MNC) in the pars intercerebralis. Abbreviations: K, Kenyon cell bodies; Ca, calyx of the mushroom bodies; o.w.br, the outer wedge of the basal ring; co, collar; br, basal ring; ped, pedunculus; vl, vertical lobe of the mushroom bodies; ml, medial lobe of the mushroom bodies; c, gamma lobe of the mushroom bodies; PCT, protocerebral-calycal tract; EB, ellipsoid body; FB, fanshaped body; o.Me, outer medulla; i. Me, inner Medulla; es, esophagus; SEG, subesophageal ganglion. Scale bar: 250 mm. doi:10.1371/journal.pone.0014536.g002 glomeruli the cortex area was stained with higher intensity (Fig. 3C). One type of AmOA1 labeled process in the coarse neuropil corresponds to axons from a large group of cells located laterally (LG; Fig. 3B). Among the types of antennal neurons, the LG contains cell bodies of local interneurons [53]. A large fraction of neurons from cluster LG were strongly labeled by AmOA1 antiserum in the cell bodies and axons in the coarse neuropil area. Cell compartments stained with different intensity. The nucleus was not labeled. Bright staining observed in the cytoplasm could be due to labeling of receptors during translation, in the process of being transported to the plasma membrane, during receptor recycling and/or receptors targeted for degradation. The DG and MDG (dorsal and medio-dorsal groups respec- tively, Fig. 3B, C) also contained AmOA1 immunopositive neurons. These neurons may correspond to multi-glomerular projection neurons given the presence of staining in the medial- lateral tract (not shown), which connects the antennal lobes with the protocerebral lobe [54]. AmOA1 staining is absent in the axons of the projection neurons of the m- and l-ACT. In our preparations staining in the target neuropil of these neurons in the lip and inner basal ring zones of the calyx of the mushroom bodies has a low intensity. We have not been able to conclusively assign this weak staining to PN axon endings or to Kenyon cell dendrites (Fig. 3D, H). Double staining experiments with labeled PNs need to be performed in order to confirm these observations. AmOA1 staining in the mushroom bodies In honey bees, each mushroom body consists of paired calyces (lateral and median) connected to a stalk-like structure that forms a peduncle with two (medial and vertical) lobes (Fig. 2). The major components of the mushroom body are intrinsic neurons known as the Kenyon cells [56]. Their axons form the pedunculus and lobes and their dendrites make up the calyces. There are two broad classes of Kenyon cells. The Class I Kenyon cells have cell bodies contained within the calycal cups and their dendrites extend into the inner wall of the calyx. Their axons project through the pedunculus and give rise to two branches, one in each lobe. Class PLoS ONE | www.plosone.org 5 January 2011 | Volume 6 | Issue 1 | e14536