AmOA1 in the Honey Bee Brain Figure 5. A subset of the GABAergic local interneurons expresses the AmOA1 receptor in the antennal lobe. A: In a frontal section of the antennal lobe, octopamine (OA) immunoreactive processes originating from VUM (ventral unpaired median) neurons invade each glomerulus of the antennal lobe, the middle is on the left. B: In the adjacent antennal lobe section, anti-AmOA1 antibodies revealed staining in the cell body cluster lying laterally and providing AmOA1 positive fibers to the non-glomerular (coarse) area of the antennal lobe. C: GABA antiserum used on a frontal section of the antennal lobe revealed GABA immunolabeled local interneurons (LIN) that have their cell bodies located laterally. These cells project axons into the coarse area of the antennal lobe and have fine arborizations ending in each glomerulus. D: Double staining with anti-AmOA1 and anti- GABA revealed a subset of the lateral group (LG) of AmOA immunoreactive cells and processes (magenta, D1) co-localize with GABA (green, D2). In the merged image (D3) co-localization appears white. Not all lateral group neurons (arrow), or the dorsal and medial-dorsal group neurons (DG and MDG) positive for AmOA1 express GABA. Conversely, not all GABAergic cells express AmOA1 (asterisk). E1-3: Enlarged images of the antennal glomeruli double stained with anti-GABA and anti-AmOA1 antibodies. The enlarged images of the antennal lobe glomeruli reveal that the AmOA1 immunoreactivity is in beaded profiles (E1). The GABA-like neurons (E2) have much more elaborate and widespread branching than AmOA1 staining (E3). Scale bars: 25 mm in A, B, C, 50 mm in D, 15 mm in E. doi:10.1371/journal.pone.0014536.g005 A subpopulation of mushroom body feedback neurons express AmOA1 A group of GABAergic neurons provide feedback from the mushroom body output lobes and pedunculus to the calyces [67,68] (Fig. 6A–D). These GABA stained feedback neurons have their ventral somata clustered in the anterior lateral protocerebral lobe (Fig. 6A). Their primary neurites project dorsally and medially and then bifurcate at the dorso-lateral margin of the vertical lobe. One branch enters the vertical lobe and innervates the medial lobe and pedunculus (Fig. 6A–D; only the beginning of medial lobe is shown in Fig. 6D). The other branch runs outside the mushroom body within the protocerebral-calycal tract (PCT) in a dorsal and posterior direction, bifurcates between the median and lateral calyces and then sends collaterals into inner ring tracts that run between the peduncular stalk and calyx (Fig. 6C,D). Each feedback neuron innervates both the median and the lateral calyx [69]. In order to compare the distribution of the AmOA1 receptor in the mushroom body with the distribution of octopamine, we stained adjacent sections with AmOA1 or octopamine antisera (Fig. 7A,B,E,F). Consecutive sections in Figures 7A and B reveal octopamine and AmOA1 receptor in the lip and outer basal ring zones. The octopamine immunoreactive profiles belong to the VUMmx1 and VUMmd1 neurons, which have their cell bodies in the subesophageal ganglion. The vertical lobe has octopamine immunoreactive processes only in the c lobe and in the neuropil of the protocerebrum that surrounds the vertical lobe (Fig. 7E). Interestingly, AmOA1 expression is more widespread than octopamine in the vertical lobe (Fig. 7F). AmOA1 receptor immunoreactivity is in the arborizations of processes that arrive in the calyx through the PCT tract, which contains GABAergic feedback neurons (Fig. 2, cluster 1, Fig. 6). These GABAergic feedback neurons branch in all areas of the calyx, which includes the lip, collar and basal ring, as well as into PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e14536