Apis mellifera octopamine receptor 1 (AmOA1) expression in antennal lobe networks of the honey bee (Apis mellifera) and fruit fly (Drosophila melanogaster)

Abstract

Octopamine (OA) underlies reinforcement during appetitive conditioning in the honey bee and fruit fly, acting via different subtypes of receptors. Recently, antibodies raised against a peptide sequence of one honey bee OA receptor, AmOA1, were used to study the distribution of these receptors in the honey bee brain (Sinakevitch et al., 2011). These antibodies also recognize an isoform of the AmOA1 ortholog in the fruit fly (OAMB, mushroom body OA receptor). Here we describe in detail the distribution of AmOA1 receptors in different types of neurons in the honey bee and fruit fly antennal lobes. We integrate this information into a detailed anatomical analysis of olfactory receptor neurons (ORNs), uni- and multi-glomerular projection neurons (uPNs, and mPNs) and local interneurons (LNs) in glomeruli of the antennal lobe. These neurons were revealed by dye injection into the antennal nerve, antennal lobe, medial and lateral antenno-protocerbral tracts (m-APT and l-APT), and lateral protocerebral lobe (LPL) by use of labeled cell lines in the fruit fly or by staining with anti-GABA. We found that ORN receptor terminals and uPNs largely do not show immunostaining for AmOA1. About seventeen GABAergic mPNs leave the antennal lobe through the ml-APT and branch into the LPL. Many, but not all, mPNs show staining for AmOA1. AmOA1 receptors are also in glomeruli on GABAergic processes associated with LNs. The data suggest that in both species one important action of OA in the antennal lobe involves modulation of different types of inhibitory neurons via AmOA1 receptors. We integrated this new information into a model of circuitry within glomeruli of the antennal lobes of these species.

Keywords: G-protein receptors; biogenic amine receptors; learning and plasticity; octopamine; olfactory pathways.

Figure 1

Schematic view of the main olfactory pathways in the honey bee brain (based on Fonta et al., ; Abel et al., ; Strausfeld, ; Sinakevitch et al., , ; Kelber et al., ; Kirschner et al., ; Schröter et al., ; Girardin et al., 2013). Olfactory Receptor Neuron (ORN) axons from the antenna enter through the antennal nerve (AN) into the antennal lobe (ant lobe) and converge onto the outer cortex of glomeruli. Each glomerulus is innervated by processes of several types of neurons. Uniglomerular projection neurons (uPNs magenta and green) have dendritic branches in a single glomerulus and send axons to higher-order brain centers such as the MB calyces (ca), lateral protocerbral lobe (LPL), and lateral horn (LH). There are two uniglomerular antenno-protocerebral tracts, the l-APT (green) and m-APT (magenta), which reflect the segregation of glomeruli into rostral (l-APT) and caudal (m-APT) hemispherical clusters. Kenyon cells (not shown in the figure) are the intrinsic cells that make up the MB. Kenyon cell dendrites form pairs of calyces (ca) with specific zones that receive different types of inputs, the lip, collar (co), and basal ring (br). The lip and br are innervated by uPN axons. The Kenyon cell axons project ventrally and split to form medal (M), vertical (V), and γ lobes, which are the main output regions of the MB. Protocerebral tract neurons (PCTN) receive inputs in the lobes and provide GABAergic feedback to the calyces. Multiglomerular PNs project axons via the medio-lateral protocerebral tracts (ml-APT 1,2) to the LPL and LH. At least two types of local interneurons (LN) interconnect glomeruli within the AL, homogeneous LN_homo (yellow), and heterogeneous LN_het (red). Ventral unpaired median neurons (VUM) have cell bodies in the maxillary (VUMmx1 is shown) and mandibullar neuromeres of the subesophageal (SEG) ganglion and connect gustatory processing in the SEG to all antennal lobe glomeruli, the LPL, LH, and MB calyces. CB, central body; M, medial lobe; V, vertical lobe; γ, gamma lobe; lo, lobula; me, medulla; m, median; l, lateral; r, rostral; c, caudal. Cell types in each side of the brain are bilaterally symmetric, but for clarity different cells are shown in each half. Scale bar: 250 μm.

Figure 2

Apis mellifera. General morphology of the antennal lobe and glomerulus. GABAergic neurons in the honeybee antennal lobe are local interneurons and multiglomerular PNs that branch into LPL and LP. (A) Frontal section of the honeybee antennal lobe immunostained with anti-synapsin antibodies (green) and Rhodamine-dextran labeled olfactory receptors neurons (magenta). (A1) Anti-synapsin (green) shapes the synaptic neuropil of the antennal lobe in all glomeruli, highlighting the potential synaptic connections between different types of neurons. (A2) Rhodamine-dextran injection into the antennal nerve revealed olfactory receptor neuron (ORN) endings (magenta) surrounding each glomerulus to form the cortex layer. The core area of glomerulus is free of ORNs. (A3) The merged images of the cortex area of the ORN endings in the antennal lobe overlapped with the area marked by anti-synapsin (white) indicate that ORNs synapse and receive synapses from other antennal lobe neurons in the cortex rind of glomerulus. Insert in (A2): The schematic of the glomerulus overlaid on the middle section indicated by the image of the projection neuron dendrite (blue, PNs) and ORNs (magenta). The distribution of PN dendrites in the core and cortex of the glomerulus in the section made through the midline of the glomerulus demonstrates that large axons of PNs are in the core area and fine dendrites of PNs in the cortex where they overlap with ORNs: b-the length of the cortex area in the center, a-the length of the glomerulus. (B) Schematic view of the olfactory pathways in the brain of the honey bee where arrows show the site of Rhodamine-dextran or/and neurobiotin injections. The octopaminergic neuron VUMmx1 has a cell body in the subesophageal ganglion (SEG) and carries information along the olfactory pathway from the antennal lobe to the lateral horn (LH), lateral protocerebral lobe (LPL), and the MB calyx (ca). The tracts that carry the uniglomerular PNs are l-APT and m-APT, while the two tracts for multiglomerular mPNs are ml-APT 1,2. (C) Double stainings of uPNs (magenta) and anti-GABA (green) in the antennal lobe demonstrate that uniglomerular PNs are not GABAergic. (C1) Injection into the l-APT and m-APT as indicated in (B) revealed uPNs with dendrites in both core and cortex areas of glomeruli. (C2) Merged images of GABA (green) and uPNs (magenta) indicate that cell bodies of the uPNs are not GABAergic. White labeling in the glomerulus is due to overloaded dye in PNs and not co-localization, as illustrated in the image of the glomerulus with uPN dendrites and anti-GABA staining in the insert of (C2). (D) Double labeling of the l-APT (D1 single image) and anti-GABA (D2 green single image) on the frontal section of the honey bee protocerebrum. The l-APT stained by injection in the antennal lobe (D3) The merged image illustrates that the two axons framing l-APT are GABAergic (white) and connect to the lateral protocerebrum (LH), another l-APT GABAergic fiber originating from antennal lobe branches in the lateral protocerbral lobe (LPL). Double arrows indicate the absence of anti-GABA in the uPN l-APT axons before their entry to the MB calyx. (E) Double staining of the m-APT and ml-APT revealed by injection of dye into LPL (single image E1, magenta in E3) and anti-GABA (green in E1and E2) (E1) uPNs in m-APT tract (E2) Anti-GABA staining manifests only in the section that we identify as the beginning of the ml-APT (E3) The m-APT is not stained with GABA, however a few GABAergic fibers are in the m-APT. These fibers are in the lateral part of the m-APT exiting from the antennal lobe that we identified as ml-APT 1,2(white merge image). (F) GABA staining in the m-APT and the two groups of GABAergic neurons in the frontal section of the antennal lobe are made in the area of the m-APT tract. The axons from the ml-APT are indicated by arrows. (G) GABA staining is in the ml-APT-1 that branches to the ventral part of the LH. (H) Schematic presentation of GABAergic cell clusters made after ten frontal brain sections (35 μm each) stained with GABA. The three groups of neurons identified are MVG, MG, and LG. Four neurons are identified as Giant MVG GABAergic neurons: they have a defined location and large somata (arrows). All figures show the right part of the brain: the middle of the brain is on the left and the lateral on the right. V, vertical lobe; γ, gamma lobe of MB. Scale bar: A, C–H = 35 μm; insert in C2 = 15 μm.

Figure 3

Apis mellifera: The GABAergic processes in glomeruli express AmOA1 (A) Triple labeling of the anti-AmOA1 (green A1) and anti–GABA (magenta A2) in a glomerulus with neurobiotin-injected uPNs (blue A3). (A3) The fibers that enter into the glomerulus are GABAergic co-stained with AmOA1 (white, arrow), non-GABAergic processes co-stained with AmOA1 (green, arrowhead) and uPNs that do not co-localize with AmOA1 (blue, double arrowheads). (B) GABA and AmOA1 staining at higher magnification (C) Triple labeling of a glomerulus (C1) in which anti-AmOA1 (green, C1 and C2 single image) and ORNs were labeled by anterograde staining with Rhodamine-dextran (magenta C1, single image C3), and uPNs are shown by retrograde staining (blue C1, and black and white in a single image C4). (D) The same glomerulus as in (C1) but only ORNs (magenta) and AmOA1(green) are shown (E) illustrates a high magnification of the area shown in the square designating in (D). Arrows show the close proximity of the AmOA1 stained profiles and ORNs. (F) The same glomerulus as in (C1) but only staining of AmOA1 and uPNs are shown. (G) A high magnification of the area shown by the square designated in (F). Arrowheads show AmOA1 in the area that surrounds the uPNs fibers, which might be presynaptic to uPNs. (H) Detail of the antennal lobe labeled with anti-AmOA1 (green, H1) and anterogradely labeled ORNs (magenta H2 in merge image). ORNs do not show the co-expression with AmOA1. (H3) PNs are labeled by injection into the LPL where three glomeruli from the lateral part of the antennal lobe are shown with cell bodies surrounding the antennal lobe. (H4) Merged image where anti-AmOA1 (green) and PNs (magenta) illustrate that there are possible co-localizations of AmOA1 in the cell bodies of subsets of PNs. Scale bars: A,C,D,F = 15 μm; B,E,G = 5 μm; H = 35 μm.

Figure 4

Apis mellifera: The AmOA1 immunostainings in the central brain neuropils (LPL, PL, and MB calyx) are co-localized in the GABAergic neurons but not in uPNs. (A) An injection of a neurobiotin tracer into the antennal lobe revealed all antenna-protocerebral tracts (APTs). (B) The origin of l-APT tracts did not show GABA (magenta, B1) and AmOA1 (green, B2) staining inside of the tract. (B3) The merged image shows white fibers indicating the co-localization of anti-GABA and Anti-AmOA1 in the area surrounded the l-APT. (C) The beginning of the m-APT in a frontal section of the brain in which the anti-GABA (C1, magenta) and anti-AmOA1 (C2, green) are co-localized in the lateral part of the tract (white image merge in C3). The medial part of the m-APT containing axons from uPNs is not GABAergic. (D) The calyx of the MB on the frontal section of the brain with injected subsets of uPNs from l-APT and m-APT (D1, single image) stained with anti-GABA (D2, single image), and anti-AmOA1 (D3, single image). (D1) uPNs ending in the lip (lip) and basal ring (br) of the MB calyx. uPNs enter to the calyx via APT. (D2) Anti-GABA profiles originating from PCT (feedback) neurons that enter to the calyx via PCT (protocerebral tract) can be found in all calyxes. (D3) Anti-AmOA1 immunostained the Kenyon cell (K) as well as a subset of the PCT neuron GABAergic endings as demonstrated in the merged image (D4) (anti-GABA magenta, and anti-AmOA1 green). (D4) The white area in the merged image clearly indicates the distribution of anti-AmOA1 staining within the subset of the GABAergic endings. (D5) The merged image of the uPNs (magenta) and anti-AmOA1 do not show co-localization of AmOA1 within uPNs ending in the calyx. (E) The Lateral Protocerebral Lobe (LPL) with triple staining injected m-APT uPNs dendrites (E1), anti-GABA (E2), and anti-AmOA1 (E3). (E4) Merged images of anti-GABA (magenta) and anti-AmOA1 (green) revealed co-staining in the fibers from ml-APT 1, 2 (arrows) and in the GABAergic processes of the LPL. (E5) There is no evidence for co-localization in merged image of uPNs (magenta) and anti-AmOA1 (green). (F) The anti-AmOA1 stainings only the subset of the GABAergic mPNs in the ml-APT1. (F1) The neurobiotin deposits in the antennal lobe revealed an ml-APT tract that we associated with mPNs. (F2) GABA immunoreactivity in the ml-APT-1. (F3) Anti-AmOA1staining in the ml-APT1. (F4) As shown in merged staining (white) only a few GABAergic fibers (magenta) are co-stained with anti-AmOA1 (green). (F5) The same for the merged image showing injected mPNs fibers (magenta) and anti-AmOA1 (green). Scale bar: A,D–F = 100 μm; B,C = 20 μm.

Figure 5

Apis mellifera. The mPNs branching in the glomeruli as revealed by neurobiotin injection into the lateral protocerebral lobe. (A) Schematic of the honey bee brain that illustrates octopaminergic neurons that branch in the antennal lobe, lateral protocerebral lobe (LPL) and calyx of the MB. Neurobiotin injected into the LPL revealed three groups of neurons named as follow DCLmPNs (B1), DRLmPNs (C1), and VMmPNs (D1). A subpopulation of the neurobiotin injected neurons from DCLmPNs (magenta B1 and B2) were co-labeled with GABA (green). (B3) A higher magnification of the GABA neurons co-stained with anti-AmOA1: arrowheads indicate the GABAergic DCLmPNs neurons that co-labeled with anti-AmOA1. (C1) There are three neurons in the DRLmPNs group that are located dorsally in a lateral cluster of the most caudal part of the antennal lobe. These neurobiotin labeled neurons (C2, magenta) are co-stained with anti-GABA (C3, green single image). (C4) The merged image shows the anti-GABA (green) and neurobiotin (magenta) labeled neurons. (D) An image from the brain section of the antennal lobe where few axons from different cells connect different glomeruli. One neuron we identify from VMmPNs group, it was possible to follow the neurite from cell body and its branching into glomeruli. This neuron (D2, magenta single image) co-localized with GABA (D3, green single image) is shown in the merged image in (D4). (E) The section from the same preparation through most ventral part of the antennal lobe. The branching pattern of the neurons in the glomeruli is in the cortex area. (F) The mPNs ending in the glomeruli cortex revealed spine like (arrows) and bleb-like structures (arrowheads). (G) The anatomy of single uPN revealed by intracellular injection into the cell body on the left, collapse frontal view. (H) The right schematic demonstrates the branching pattern of the uPN and mPN in the antennal glomeruli. uPN has the thick fibers in the core and fine arborization in the cortex (red), mPNs fibers are in outer area of the core and in the inner cortex. Asterisks in (B2,B3) indicated the axons from antennal nerve traveled to mechanosensory and motor center neuropil. Scale bar: B = 20 μm, C1,D2,E = 50 μm, F,G = 15 μm.

Figure 6

Drosophila melanogaster: a subpopulation of GABAergic neurons are co-stained with anti-OA1 antibodies. (A) Schematic of the organization of the Drosophila olfactory system in sagittal view. MP, maxillary palp; ORN, olfactory receptor neuron; SEG, subesophageal ganglion; ant lob, antennal lobe; ca, calyx, ped, pedunculus; v, vertical lobe of MB; A, anterior; P, posterior; Dorsal, Ve- ventral. Broken lines indicate the approximate orientation of sections through antennal lobe (in C,F,D) and lateral protocerbrum (E). (B) Section through the center of one glomerulus labeled with anti-synapsin (green, B1) and anti-GFP in ORNs (magenta, B2) in OR83b-GAL4;UAS-mcd8-GFP flies. The glomerulus has a core area where the ORNs do not branch (B3). (C1) Anti-OA1 stained groups of cells and processes in the glomeruli and the aglomerular neuropile area of the antennal lobe in OR83b-GAL4;UAS-mcd8-GFP (C1, magenta) on the oblique frontal brain cross-section of the antennal lobe. (C2) In the same section, GFP (green) takes up a large percentage, or perhaps all, of the olfactory receptor endings in the antennal lobe glomeruli. (C3) The majority of the sensory neurons terminals do not label with the OA1 antiserum (magenta) with a few exceptions. (D,E) Anti-OA1 antibodies do not label the majority of uniglomerular projection neurons (uPNs). (D1) Here, anti-OA1 labeled clusters of cells surrounding the antennal lobes. These neurons are not projection neurons. GH146-GAL4; UAS-mcd8-GFP projection neurons expressed GFP (green) (D2). In GFP expressing neurons there is no OA1 immunoreactivity as shown in our merged image (D3), co-localization would show as white. (E) Anti-OA1 staining is absent in most uPN axons that leave the antennal lobe via the m-APT and branch in the calyx (ca) of the MB and lateral horn (LH); one exception is the axon shown by two arrowheads (E1). mPNs leave the antennal lobe via the ml-APT, a large portion of the ml-APT fibers are OA1 positive (arrow) in (E1,E3). These fibers also exhibit anti-GABA staining (inserts in E1). Three neurons that have their axons in ml-APT are also labeled with anti-GFP and GABA in GH146-GAL4; UAS-mcd8-GFP (E2 and insert in E2). These neurons are not labeled with anti-OA1 in this brain preparation (E3). (F1) In the same section as shown in (C) anti-OA1 (magenta) labels the cell bodies of laterally located neurons. The scattered, stained processes are in all glomeruli. (F2) The same sections labeled with anti-GABA antibodies; and GABA-like immunoreactivity is found in neurons with cell bodies in lateral and dorso-lateral clusters. These neurons supply GABAergic processes to the glomeruli. The merged image (F3) of the same sections shows the group of GABAergic neurons co-stained with anti-OA1 (white). Anti-OA1 staining is found in the cell bodies and processes in the glomeruli and in the aglomerular area of the antennal lobe. Arrows in (C1) and (D1,F1) indicate lateral neurons cluster (LN) and ventral cluster (VN) of anti-AmOA1 positives neurons. In (D2), the arrowhead indicates thick fibers of the PNs entering the glomerulus that might correspond to the core area of the glomerulus. Scale bar: A = 10 μm, B–F = 20 μm.

Figure 7

Schematic representation of the neural network proposed for a glomerulus in the honey bee (model architecture based on Fonta et al., ; Abel et al., ; Nishino et al., ; Meyer and Galizia, ; Girardin et al., 2013). Octopamine is released into the whole glomerulus in both core and cortex and acts on the GABArgic local interneurons (LNs) and on GABAergic multiglomerular projection neurons (mPNs). Each glomerulus has a uniglomerular PN (uPNs) that branches into both core and cortex areas of the glomerulus. uPNs receive excitatory synapses from ORNs in the cortex area and inhibitory synapses from hetero-multiglomerular local neurons from neighboring glomeruli in the core area. The uPNs also receive inhibitory synapses in the cortex that come from the mPNs. In the glomerulus there are GABAergic and non-GABAergic multiglomerular LNs. For simplicity, only one type of the GABAergic LNs is shown in the glomeruli; LNg (LN1 or heteroLN in Figure 1) that branches in all areas of the glomerulus (cortex and core) where it receives excitatory synapses from ORN in the cortex and inhibitory synapses in the core and sends the inhibitory output into the neighboring glomeruli. Both types of GABAergic neurons (LNs and mPNs) express AmOA1 receptors in the glomerulus. Based on our indirect evidence of AmOA1 and GABA stainings in glomeruli, we hypothesize two possible mPNs connections, one that receives excitatory synapses from ORNs in the glomerulus cortex and makes synapses in the LH as well as another that receives synaptic input from LPL and LH and makes synapses onto the processes in the cortex. Future physiological and anatomical studies will clarify the branching patterns that we proposed for mPNs.