AmOA1 in the Honey Bee Brain Figure 6. GABAergic feedback neurons in the protocerebro-calycal tract (PCT) are shown. A: GABA-like immunoreactive neurons with large cell bodies (Cb) are located anteriorly and laterally between the lateral protocerebrum and optic lobes. Each cell sends its primary neurite through the lateral protocerebrum towards the vertical lobe of the mushroom body forming the large GABA-like immunoreactive fiber tract (arrow in A, B). B: After reaching the vertical lobe of the mushroom body, each primary neurite divides into two branches. One branch enters the vertical lobe producing fine arborizations in the dorso-lateral margin of the vertical lobe (vl, arrowhead in A). The other branch follows the GABA-like immunoreactive fibers (arrowheads in B, C) through the protocerebro-calycal tract (PCT) to enter the calyces (C, D). C: Each calyx receives GABA-like immunoreactive fibers that produce fine arborizations in the lip, collar (co) and basal ring (br). D: GABAergic feedback neurons innervate the median (M Ca) and the lateral (L Ca) calyces, pedunculus (ped) and medial lobe (ml). The insert in C illustrates the specificity of the GABA antibodies. This section shows the lack of staining when the GABA antiserum was preadsorbed with 0.01 mM GABA conjugated to carrier protein (BSA) prior to use on the section. Scale bar: 100 mm in A, B; 50 mm in C, D. doi:10.1371/journal.pone.0014536.g006 the vertical and medial lobes (Fig. 7C, D1, G, H1). In our immunofluorescence double labeling with anti-GABA and anti- AmOA1 antibodies, subsets of GABAergic PCT neurons co- localized with the AmOA1 receptor (Fig. 7D,H). The feedback neurons that express AmOA1 immunoreactivity in the calyx are in the lip, collar and outer basal ring area (Fig. 7B, D2). There is low intensity staining in the inner basal ring area, with high intensity of staining at the edge of basal ring zone (double arrowheads in Fig. 7B). The arrows in figure 7F,G show where feedback neurons enter the vertical lobe (the midline of the brain is on the left). Double staining on the same sections with anti-GABA and anti-AmOA1 revealed only a small subpopulation of GABAer- gic feedback neurons in the calyx and vertical lobes co-stained (Fig. 7D3,H3). These subpopulations of GABA and AmOA1 immunoreactive feedback neurons have their endings mostly in the lip, collar and outer layer of the basal ring (Fig. 7D3) while the inner basal ring zone is not stained with AmOA1 antibodies. In the vertical lobe, the axons of feedback neurons that express AmOA1 receptors are mostly in the region occupied by the axons of Kenyon cells with dendrites in the collar and lip zones of the calyx. In addition, there are extrinsic GABAergic neurons that enter the c lobe that also co-stain with anti-AmOA1 (Fig. 7F, H2, H3). The tangential GABAergic neurons of the central complex express AmOA1 We used octopamine antiserum to compare the distribution of octopamine labeling with AmOA1 receptor staining in the central complex of the honey bee on two adjacent sections (Fig. 8A, B). The origin of the octopamine innervations in the fan shaped body and ellipsoid body has several sources: neurons from clusters G4 and G2, and possibly some fibers from the VUM neuron(s) located in the subesophageal ganglion [32]. The section adjacent to the section shown in figure 8A was labeled with AmOA1 antibodies (Fig. 8B), and shows high intensity staining in the ellipsoid body with less intense staining in the fan shaped body. The neurons that stained for AmOA1 belong to the tangential GABAergic neurons whose cells bodies are located in cluster 8 (Fig. 2) and that send their input into the ellipsoid body and fine branches into the fan shaped body (Fig. 8C). The expression of AmOA1 in these GABAergic neurons was confirmed using double immunofluorescence staining with anti-GABA and anti-AmOA1 (Fig. 8D1, 2). The overlap of staining for GABA and AmOA1 confirms that AmOA1 receptors are expressed in the GABAerigic tangential neurons (Fig. 8D3). PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e14536