Distribution of the octopamine receptor AmOA1 in the honey bee brain

Abstract

Octopamine plays an important role in many behaviors in invertebrates. It acts via binding to G protein coupled receptors located on the plasma membrane of responsive cells. Several distinct subtypes of octopamine receptors have been found in invertebrates, yet little is known about the expression pattern of these different receptor subtypes and how each subtype may contribute to different behaviors. One honey bee (Apis mellifera) octopamine receptor, AmOA1, was recently cloned and characterized. Here we continue to characterize the AmOA1 receptor by investigating its distribution in the honey bee brain. We used two independent antibodies produced against two distinct peptides in the carboxyl-terminus to study the distribution of the AmOA1 receptor in the honey bee brain. We found that both anti-AmOA1 antibodies revealed labeling of cell body clusters throughout the brain and within the following brain neuropils: the antennal lobes; the calyces, pedunculus, vertical (alpha, gamma) and medial (beta) lobes of the mushroom body; the optic lobes; the subesophageal ganglion; and the central complex. Double immunofluorescence staining using anti-GABA and anti-AmOA1 receptor antibodies revealed that a population of inhibitory GABAergic local interneurons in the antennal lobes express the AmOA1 receptor in the cell bodies, axons and their endings in the glomeruli. In the mushroom bodies, AmOA1 receptors are expressed in a subpopulation of inhibitory GABAergic feedback neurons that ends in the visual (outer half of basal ring and collar regions) and olfactory (lip and inner basal ring region) calyx neuropils, as well as in the collar and lip zones of the vertical and medial lobes. The data suggest that one effect of octopamine via AmOA1 in the antennal lobe and mushroom body is to modulate inhibitory neurons.

Figure 1. Anti-AmOA1 antibodies are specific for the AmOA1 receptor.

Pre- and post-adsorption controls of immunolabeling with anti-AmOA1 antibodies from rabbit (A–D) and from goat (E–G). Consecutive sections of the mushroom body calyx stained with Ranti-AmOA1 serum (A) and pre-immune serum taken from the rabbit before immunization and used at the same dilution as the Ranti-AmOA1 serum (B). The Ranti-AmOA1 serum revealed staining in Kenyon cell bodies (K) with different intensity and in processes of the calyx lip and collar (col). C: Section of the antennal lobe stained with Ranti-AmOA1 that was preadsorbed with KLH treated with glutaraldehyde (KLH-G) alone. D: A section from the same brain stained with Ranti-AmOA1 that was preadsorbed with the AmOA1 peptide conjugated to KLH (KLH-G-OA1pep). E: Sections of the antennal lobe stained with affinity purified anti-AmOA1 antibodies from goat (Ganti-AmOA1). F: pre-immune serum from the goat before immunization used at the same dilution as the Ganti-AmOA1 serum. G: Specific staining from cell bodies and their processes disappeared after pretreatment with the KLH conjugated to the AmOA1 peptide. The confocal images in D, F and G were adjusted to a higher level of intensity then stained sections in C, E to show the image of the antennal lobe. Scale bars: 50 µm.

Figure 2. Schematic drawing of AmOA1 immunoreactive neurons and processes in the brain and subesophageal ganglion of the bee made from confocal images of agarose sections of bee brain stained with anti-AmOA1 antibodies.

The right hemisphere shows a frontal view of the bee brain with the optic lobe neuropils (Re, retina; La, lamina; Lo, lobula), antennal lobe (al) and summarized mushroom body neuropil; the left hemisphere of the brain shows a more posterior frontal view at the level of the dorsal lobe (dl), pedunculus (ped) and calyces of the mushroom bodies. AN, antennal nerve; DG, dorsal group of antennal lobe neurons; LG, lateral group of antennal lobe neurons; MDG, medial group of antennal lobe neurons. Some neurons that express the AmOA1 receptor could be identified as: 1, PCT (or feedback neuron group); 2, lobula and medulla mushroom body neurons. The cells in this cluster have fibers in the anterior superior optical tract (asot). The arrow shows axons (only the ending of the tract) from these neurons as they enter the medulla serpentine layer. A few AmOA1 positive fibers enter through the lobula optical tract (lot) that connects the lobula and mushroom bodies.; 3, photoreceptor cells in the retina; 4, monopolar cell bodies in the lamina; 5, medulla columnar neurons; 6, photoreceptor cells in the ocelli; 7, a median group of subesophageal intrinsic neurons; 8, ellipsoid body neurons, 9, a group of median neurosecretory cells (MNC) in the pars intercerebralis. Abbreviations: K, Kenyon cell bodies; Ca, calyx of the mushroom bodies; o.w.br, the outer wedge of the basal ring; co, collar; br, basal ring; ped, pedunculus; vl, vertical lobe of the mushroom bodies; ml, medial lobe of the mushroom bodies; γ, gamma lobe of the mushroom bodies; PCT, protocerebral-calycal tract; EB, ellipsoid body; FB, fanshaped body; o.Me, outer medulla; i. Me, inner Medulla; es, esophagus; SEG, subesophageal ganglion. Scale bar: 250 µm.

Figure 3. AmOA1 is expressed in the antennal lobes, mushroom body and lateral protocerebrum.

A–C: Example of cell bodies and neuropil labeled with AmOA1 in the antennal lobe. The lateral side of the antennal lobe is on the right. A: In the antennal nerve (AN), the tracts of olfactory receptor neurons (T1–T2) have a few beaded processes positive for staining with AmOA1 antibodies (arrow). The glomeruli surrounding the entrance of the antennal nerve show different patterns of AmOA1 immunoreactivity. The double arrows point out an example of low intensity AmOA1 staining in the glomerular cortex where the endings of the olfactory receptor neurons are located. In contrast, the core region of this glomerulus has a high level of AmOA1 staining. B: Neurons in the lateral group (LG) exhibit a high intensity of AmOA1 immunoreactivity in their cell bodies and in their axons that are in the coarse area (arrow) of the antennal lobes. The cells bodies of the dorsal group (DG) also show intense staining. C: The medial dorsal group (MDG) of antennal neurons was also stained with high intensity with anti-AmOA1 antibodies. D–H: AmOA1 immunoreactivity in the mushroom body. D: One half of a calyx stained with AmOA1 antiserum. Kenyon cell bodies (K) that supply the collar or outer basal ring exhibit differing AmOA1 immunoreactivity. The dendrites of Kenyon cells are labeled with high intensity in the outer wedge basal ring zone of the calyx (o.w.br) whereas less intense staining is observed in the calyx collar (co). Arrowheads indicate endings of extrinsic (feedback) neurons expressing AmOA1 in the calyx lip, the outer basal ring (o.br), and with a few branches in the collar neuropil. The inner basil ring (i. br) show relatively low levels of staining. E: A sagittal section made through the mushroom body vertical (V) and medial (M) lobes. Dorsal is at the top. In the medial and vertical lobes, there are two layers of Kenyon cell axons stained with anti-AmOA1 antibodies (arrowheads). These layers correspond to the basal ring and collar Kenyon cells. The class II clawed Kenyon cells, which have their axons in the ventral part of the vertical lobe (the γ lobe), are also positive for AmOA1 expression. Arrows show the axons of γ lobe Kenyon cells brightly labeled with anti-AmOA1. The cell bodies of these cells lay outside of calyx (see arrow in H). F: A frontal section through the vertical lobe of mushroom body. AmOA1 immunoreactive extrinsic neurons enter and branch in the γ lobe. The arrow indicates an example of AmOA1 immunoreactive neurites of feedback neurons that enter in the vertical lobe. G: A schematic representation of a calycal cross section illustrating calycal regions receiving olfactory (yellow), gustatory and mechanosensory (dark blue), and visual input from the lobula (dark red) and medulla (light blue). H: The AmOA1 immunoreactive cell bodies of neurons located in a cluster between the dorso-medial edge of the medulla (Me) and the lateral calyx are neurons that connect the mushroom body calyx with the medulla. The cells in this cluster have fibers in the anterior superior optical tract (asot). The arrowhead shows axons from these neurons entering the medulla serpentine layer. AmOA1 positive fibers run along the dorsal edge of the lobula (Lo) and enter into the serpentine layer of the medulla. A few AmOA1 positive fibers enter through the lobula optical tract (lot) that connects lobula and mushroom bodies. I: The lateral protocerebrum (LP) exhibits few fibers or stained structures (midline is on the left). There are diffuse processes in the lateral horn (LH) that express AmOA1 in this animal. Scale bar: 50 µm in A,B,C; 25 µm in D,E,F,H; 75 µm in I.

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 µm in A; 25 µm in B,C,G; 70 µm in D,E,F.

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 µm in A, B, C, 50 µm in D, 15 µm in E.

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 µm in A, B; 50 µm in C, D.

Figure 7. A subset of the GABAergic feedback (PCT) neurons in the mushroom body expresses the AmOA1 receptor.

A: Frontal section of the halves of two calyces labeled with octopamine antiserum (OA). Octopamine immunoreactive processes are in the outer and inner basal ring (o. br, i. br), collar (co) and calyx lip. B: The frontal section adjacent to the section shown in A labeled with anti-AmOA1 antibodies. The AmOA1 positive processes are in the lip, collar and basal ring zones of the calyx in large afferent profiles. Note that the Kenyon cell bodies (K) of the outer basal ring and collar are also labeled with anti-AmOA1. The double arrowheads indicate the outer wedge of the basil ring, which shows relatively intense staining. C: GABA-like immunoreactivity in the feedback neurons have ends in all zones of the calyx. D1-3: Double staining for GABA and AmOA1 in frontal sections of the lateral calyx. Not all of the GABAergic feedback neurons exhibit anti-AmOA1 immunolabeling (arrow). The AmOA1 receptor mostly co-localized with feedback neurons that end in the lip and basal ring zone of the calyx. E: Frontal section through the vertical lobe of the mushroom body labeled with octopamine antiserum. The vertical lobe has octopamine immunoreactive processes only in the γ lobe and in the neuropil of the protocerebrum that surrounds the vertical lobe. F: In a section adjacent to the section shown in E, the vertical lobe of the mushroom body labeled with AmOA1 antiserum. The anti-AmOA1 staining is in: i) extrinsic neurons branching in the γ lobe and ii) processes from feedback neurons that enter the lobe laterally (arrow). The axons of Kenyon cells in the γ lobe labeled with low intensity. G: Frontal section of the vertical lobe labeled with GABA antiserum. The feedback neurons exhibit GABA-like immunoreactivity in profiles that enter in the lobe medio-laterally and branch in the lip, collar and basal ring zone of the vertical lobe. The extrinsic GABA-like immunoreactive processes branch in the γ lobe. H1-3: Double staining with anti-GABA and anti-AmOA1 in the section adjacent to the section shown in G. A subset of GABAergic feedback profiles co-localized with anti-AmOA1. Scale bar: 25 µm.

Figure 8. Tangential GABAergic neurons in the central complex express the AmOA1 receptor.

A: A frontal section of the central complex labeled with anti-octopamine (OA). Octopamine in is present in the ellipsoid body and the fan shaped body. B: Frontal section adjacent to the section shown in A labeled with anti-AmOA1 antibodies. The AmOA1 immunoreactivity is in the tangential fibers that invade the ellipsoid body. Note the fan shaped body is less intensely stained than the ellipsoid body. C: GABA-like immunoreactivity in the tangential neurons innervating the ellipsoid body and fan shaped body. D. Section adjacent to section C double stained with GABA antiserum (D1, green) and anti-AmOA1 antibodies (D2, magenta) revealed that GABAergic processes in the ellipsoid body are co-labeled with AmOA1 receptor (white in merged images) in D3. Scale bar: 25 µm.

Figure 9. Schematic view of the visual and olfactory pathways in the honey bee mushroom body calyx.

The figure was made after , , –. co, collar; br, basal ring; LH, lateral horn, l-ACT, lateral antenna-cerebral tract; m-ACT median antenna-cerebral tract; ASOT, anterior superior optic tract; AN, antennal nerve; CB, central body; M, medial lobe; V, vertical lobe; γ, gamma lobe; LIN, local interneurons; VUMmx1, ventral unpaired median neuron in maxillary neuromere; lo-lobula; me-medulla; m, median; l, lateral; r-rostral; c-caudal. Scale bar: 250 µm.