receives octopaminergic innervation from neurons located in the posterior of the brain. These neurons connect the protocerebral bridge with the ellipsoid body and lateral protocerebrum [32]. Octopamine acts via binding to different G protein coupled receptors that regulate intracellular levels of cyclic AMP (cAMP) or calcium [37,38]. Four distinct octopamine receptor subtypes have been cloned and characterized from Drosophila [39–41], and homologs of these receptors are found in the honey bee genome sequence [38,42]. The characterized fruit fly octopamine receptors and their putative honey bee orthologs cluster into two classes [38]. One class contains OAMB, and receptors in this group probably act to regulate intracellular calcium levels [39,43]. The other octopamine receptors are closely related to each other and cluster together in a second class. Octopamine receptors in the second group act through the cAMP second messenger pathway, as stimulation with octopamine results in an increase in cAMP [38]. To date, only one octopamine receptor (AmOA1) has been cloned and characterized from honey bee [43]. When expressed in HEK cells, activation of AmOA1 receptors by octopamine leads to oscillations of intracellular Ca2+ levels and a relatively small increase in cAMP levels [43]. AmOA1 is the ortholog of the fruit fly OAMB (also known as DmOA1A or DmOcta) receptor [40,41]. Although OAMB was originally believed to regulate cAMP, recent evidence suggests that, as for AmOA1, activation of this receptor leads to increases in Ca2+ [39]. Down regulation of the expression of AmOA1 via RNA interference significantly reduces olfactory learning [18], suggesting that AmOA1 receptors are an important part of the OA reinforce- ment pathway. Previous work on the distribution of Amoa1 transcript in the honey bee brain suggests that this biogenic amine receptor is widely expressed in many somata throughout the brain [43]. While these studies have provided a wealth of important information, using antibodies against the AmOA1 receptor for immunolabeling can reveal more about the exact locations of the receptor protein thereby suggesting a role for a receptor in specific neuroanatomical pathways. In the present study, we use two polyclonal antibodies generated (in rabbit and goat) against two different peptide sequences in the C-terminus of AmOA1 to examine the distribution of the AmOA1 receptor in the honey bee brain. We describe AmOA1 immunolabeling specifically in the antennal lobes, mushroom body, central complex, optic lobes and subesophageal neuropils of forager honey bees. Furthermore, double immunofluorescence staining using anti- GABA and anti-AmOA1 receptor antibodies revealed that the receptor is expressed in the GABAergic local interneurons in the antennal lobe and in the GABAergic feedback neurons in the mushroom body. Thus we present here for the first time evidence that the AmOA1 receptor is expressed in the inhibitory pathways in the olfactory learning and memory neuropils of the bee brain. Materials and Methods Animals Honey bees (Apis mellifera) used in this study were adult New World Carniolan pollen foragers from colonies maintained at Arizona State University. We used fruit fly stocks to analyze specificity of our antibodies. Fly stocks and crosses were maintained at 25uC on standard corn meal-yeast-agar medium. The following strains were used: wild-type Canton-S; oamb96 mutant with a deletion in the OAMB locus [44] kindly provided by Dr. B. Dickson (Institute of Molecular Pathology, Vienna, Austria). AmOA1 in the Honey Bee Brain Primary antisera Rabbit polyclonal antibodies against AmOA1 (Ranti-AmOA1) were generated against a 15 amino acid peptide (NH2- DFRFAFKSIICKCFC-OH) conjugated to keyhole limpet hemo- cyanin (KLH) via glutaraldehyde by Alpha Diagnostic Interna- tional (San Antonio, TX). AmOA1 antiserum from rabbit was purified by preabsorption with glutaraldehyde treated KLH. The purified antiserum from rabbit 65-4 was used for all the immunocytochemical analyses reported below. Goat polyclonal antibodies against AmOA1 (Ganti-AmOA1) were generated against a different AmOA1 peptide sequence. In this case the synthetic peptide acetyl-AMRNDRSPSYSMQVP QQGC-amide was used, which corresponds to amino acids 547– 564 of AmOA1 (21st Century Biochemicals, Inc., Marlborough, MA). The peptide was analyzed by HPLC and nanospray MS and the sequence confirmed by CID MS/MS (MS CheckTM, 21st Century Biochemicals). The peptide was conjugated to KLH using MBS and dialyzed prior to injection. The fourth bleeds were tested by ELISA and the antibodies were affinity purified using the above peptide covalently attached to cross-linked agarose beads. Octopamine antiserum was obtained from rabbit immunized with octopamine conjugated to bovine serum albumin via glutaraldehyde (BSA) [45]. Its specificity to octopamine has been described [46]. This octopamine antiserum has been used previously to characterize octopamine-like immunoreactivity in the honey bee brain [32]. GABA antiserum (GEMAC; Talence, France) was raised in rabbit using GABA conjugated with glutaraldehyde to BSA, bovine hemoglobin, or poly-L-lysine. The antiserum specificity has been described elsewhere [47–49], and it has already been used to characterize GABA in the honey bee visual system [48]. Immunohistochemistry Honey bee brains and fly brains were removed in fixative containing 2.5% paraformaldehyde (EMS), 1.5% glutaraldehyde (EMS) in 0.1 M sodium cacodylate buffer (pH 7.0), with 1% sodium metabisulfite (SMB, Sigma) and were incubated in the same solution overnight at 4uC. Fixation with glutaraldehyde produces a background of blade marks in agarose sections [50,51]. However, the blade marks produce a regular pattern that is differentiable from the neuropil staining. We found that fixative containing glutaraldehyde worked best for staining tissue sections, probably because the AmOA1 antibody was generated with peptide conjugated to carrier protein via glutaraldehyde. After fixation, whole brains were incubated for 15 minutes in Tris-SMB buffer (0.05 M Tris-HCl, pH 7.5; 0.45% SMB) containing 0.5% NaBH4 to saturate double bonds. After washing (4610 min) in Tris-SMB buffer, brains were embedded in agarose and cut into 60 mm sections. Immunohistochemistry with rabbit primary antibodies: Ranti-AmOA1, anti-octopamine and anti-GABA Sections were washed (6610 min) in Tris-SMB buffer with 0.5% of Triton X100 (Tris-SMB-TX), then preincubated with 5% Normal Swine Serum (Dakopatts a/s, Glostrup, Denmark) for one hour. Next, the brain sections were incubated overnight with primary antibodies: Ranti-AmOA1 at 1:1000, or anti-octopamine or anti-GABA at 1:100 diluted in Tris -SMB-TX. After washing (6610 min) in 0.05 M Tris, pH 7.5, 0.5% Triton X100 (Tris - TX), sections were incubated overnight in goat anti-rabbit IgG antibodies conjugated to either Alexa 488 (Molecular Probes, Eugene, OR) or Cy5 (Jackson Laboratories, West Grove). After a final wash in 0.05 M Tris-HCl buffer pH 7.5, sections were PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e14536