AmOA1 in the Honey Bee Brain Figure 4. AmOA1 immunoreactivity is observed in the visual neuropil (A–C), central complex (D) and subesophageal (SEG) ganglion (E–G). A: In the lamina (La), AmOA1 immunoreactivity was observed in the ends of some photoreceptor cells that originate in the retina (Re; arrow) and in a subset of the monopolar cells (arrowhead). B: AmOA1 expression is seen in processes in the first chiasma (1 o ch) and the lamina neuropil. C: AmOA1 immunoreactive processes are organized in discrete strata in the medulla (Me). The columnar intrinsic neurons are intensely stained. The serpentine layer, which separates the outer medulla (o. Me) from the inner medulla (i. Me), has staining in tangential elements that may belong to medulla neurons that are connected to the calyx of the mushroom bodies. The second optic chiasma (2 o.ch) between the medulla and lobula also contains AmOA1 immunoreactive processes. D: A sagittal section made through the midline of the subesophageal ganglion where the VUM neurons are located in clusters in the maxillar (mx) and mandibular (md) neuromeres is shown. Anterior (a) is to the left and dorsal (d) is on the top. AmOA1 staining is observed in neurons with small cell bodies located in the clusters with the VUM cells; the VUM cells themselves are not stained with anti- AmOA1 antibodies. E, F: Frontal sections of the subesophageal ganglion from two different bee preparations. AmOA1 staining is observed in small cell bodies located in the midline of the ventral part of the subesophageal ganglion. ln, lateral neurite tract. G: The fan-shaped (FB) body is less intensely stained with anti-AmOA1 than the ellipsoid body. Scale bars: 50 mm in A; 25 mm in B,C,G; 70 mm in D,E,F. doi:10.1371/journal.pone.0014536.g004 very fine branches to supply each glomerulus of the antennal lobe. Staining of the adjacent section with AmOA1 antiserum reveals cell bodies clustered in the lateral rind of the antennal lobe with AmOA1 positive profiles in the central coarse area and fine distribution of AmOA1 positive varicosities in each glomerulus (Fig. 5B). These data suggest that octopamine is released parasynaptically, rather than presynaptically to each profile. The cell bodies in the lateral cluster of the antennal lobe labeled with AmOA1 antibodies (Fig. 5B,D1) belong to local interneurons that are known to be GABA positive [67] (Fig. 5C,D2). The anti-GABA antibody stained local interneu- rons in the cell bodies, processes in the coarse area of the antennal lobe, and highly packed processes in each glo- merulus (Fig. 5C, D2). Comparison between the distribution of AmOA1 (Fig. 5B, D1) and GABA (Fig. 5C,D2) revealed that GABAergic local interneurons express the AmOA1 receptor. All co-localized cell bodies and processes are white in the merged image (Fig. 5D3). At higher magnification, the GABA-like immunoreactive neurons have much more elaborate and wide spread branching than AmOA1 staining (Fig. 5E), which could reflect very localized expression of the AmOA1receptor on GABAeric cell processes. In our preparations, the majority of the GABAergic neurons express AmOA1 receptors with a range of staining intensity. However, there are some GABAergic neurons where the staining for AmOA1 is very low or absent (e.g. Fig. 5D, where a group of cells labeled with an asterisk have very low AmOA1 immunore- activity). Comparisons of GABAergic with AmOA1 staining in the cell bodies and processes reveal that not all GABA neurons express the receptors in those areas of the cell (Fig. 5E1-3, Figure S1D–F). Conversely, some AmOA1 positive neurons are not GABAer- gic. The arrow in figure 5D indicates AmOA1 receptor positive neurons in the lateral cluster that are not stained with the GABA antibody. In addition, the AmOA1 receptor is expressed in cell bodies of the medial cluster (DG and MDG) that do not show GABA-like immunoreactivity. PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e14536