The Biogenic Amine Tyramine and its Receptor (AmTyr1) in Olfactory Neuropils in the Honey Bee (Apis mellifera) Brain

Abstract

This article describes the cellular sources for tyramine and the cellular targets of tyramine via the Tyramine Receptor 1 (AmTyr1) in the olfactory learning and memory neuropils of the honey bee brain. Clusters of approximately 160 tyramine immunoreactive neurons are the source of tyraminergic fibers with small varicosities in the optic lobes, antennal lobes, lateral protocerebrum, mushroom body (calyces and gamma lobes), tritocerebrum and subesophageal ganglion (SEG). Our tyramine mapping study shows that the primary sources of tyramine in the antennal lobe and calyx of the mushroom body are from at least two Ventral Unpaired Median neurons (VUMmd and VUMmx) with cell bodies in the SEG. To reveal AmTyr1 receptors in the brain, we used newly characterized anti-AmTyr1 antibodies. Immunolocalization studies in the antennal lobe with anti-AmTyr1 antibodies showed that the AmTyr1 expression pattern is mostly in the presynaptic sites of olfactory receptor neurons (ORNs). In the mushroom body calyx, anti-AmTyr1 mapped the presynaptic sites of uniglomerular Projection Neurons (PNs) located primarily in the microglomeruli of the lip and basal ring calyx area. Release of tyramine/octopamine from VUM (md and mx) neurons in the antennal lobe and mushroom body calyx would target AmTyr1 expressed on ORN and uniglomerular PN presynaptic terminals. The presynaptic location of AmTyr1, its structural similarity with vertebrate alpha-2 adrenergic receptors, and previous pharmacological evidence suggests that it has an important role in the presynaptic inhibitory control of neurotransmitter release.

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

Figure 1

Controls for immunolabeling with tyramine antiserum. We used three sections of the subesophageal ganglion (SEG) that contain ventral median group cells (VUM). The sections were labeled with one of the following: (A) tyramine antiserum; (B) tyramine antiserum pre-incubated with conjugated tyramine-bovine serum albumin (BSA); (C) tyramine antiserum pre-incubated with octopamine-BSA. Scale bar: 25 μm.

Figure 2

Characterization of anti-AmTyr1 antibodies. (A) Predicted structural model of the Apis mellifera tyramine receptor (NP_001011594.1). Peptides (P1, P2) from the N-terminal region and the loop between helices 4 and 5 used to generate the antibodies are represented by their amino acid sequence (pink). (B) Affinity purified anti-AmTyr1 antibodies against peptide-1 and peptide-2 were tested in western analyses. The relative positions of molecular weight (MW) standards in kDa are indicated. The affinity purified anti-AmTyr1-P1 and anti-AmTyr1-P2 each revealed a band corresponding to an approximate molecular weight of 45 KDa. Preincubation of the anti-AmTyr1 antibodies separately with corresponding peptide conjugates abolished the band. (C) Anti-AmTyr1 immunostainings in the antennal lobe revealed processes in the cortex of glomeruli on frontal section of the brain. (D) In the next consecutive section, staining in glomeruli was not present when the anti-AmTYR1 antibodies were pre-incubated with Keyhole Limpet Hemocyanin (KLH)-conjugated peptide-1 before immunostaining. (E) Expression of AmTyr1gene in brains injected with 70 nl of 100 μM dsiAmTyr1 RNA or dsiScramble 14 h after treatments. AmActin was used as a reference gene. The relative gene expression was calculated using the 2^−ΔΔCt method. The data are expressed as mean ± SE. (F) Anti-AmTyr1 staining in the brain section 18 h after injection dsiAmTyr1 RNA (G) and dsiScr. Arrows in (F,G) indicate injections sites in the frontal sections of the bee brains. (H) Quantification of the average fluorescence intensity value in the box X (Fx), outlined in F in the raw images of brains that were injected with dsiTyr1 and dsiScr. Images were collected with a confocal fluorescent microscope with the same gain settings and intensity level. Relative intensity level of fluorescence dropped to 42 ± 5% (mean ± SE) in the dsiTyr1 injected brains compared to dsiScr brains in the local area of the injections. Scale bar: C,D = 10 μm, F,G = 100 μm.

Figure 3

Tyramine-like immunoreactivity in the honey bee brain using inverted fluorescence images. (A) Schematic representation of the brain (frontal view) with the groups of cell bodies labeled with anti-tyramine antibodies. The left and right halves of the schematic demonstrate the caudal and rostral planes of the brain. The tyramine containing cell groups (magenta) are G2-G6 and VUM. The plane of the sagittal sections on corresponding images (B,E,F,G,J) is indicated by the vertical lines. (B) The sagittal section through the SEG with anti-tyramine labeled groups of median neurons in mandibular (Md), maxillary (Mx) and labial (Lb) neuromeres with their primary neurites in corresponding Md (MdT), Mx (MxT) and Lb (LbT) tracts. (C,D) Frontal sections of SEG made via Md (C) and Mx (D) neuromeres respectively, show corresponding frontal view of VUMmd (C) and VUMmx (D) and ventral paired median (VPM) neurons. The VUM neurons send their primary neurites to the corresponding tracts, and the secondary neurites branch in the deutocerebrum (circle in C,E). (F,G) Two tyramine immunoreactive axons from VUM neurons, one from MdN and one from MxN, innervate the antennal lobe (sagittal sections, front on the left) and give rise to ramifications in glomeruli and aglomerular neuropils of the antennal lobe (G). Asterisk in (F) indicates dorsal lobe. (G) The tyramine immunoreactivity in tract T5-T6 is from unidentified neurons in the tritocerebrum and SEG. (H) These unidentified tyramine immunoreactive neurons enter into the antennal nerve and are running along the top of the antennal nerve in the sagittal view and inside of the nerve (frontal view, insert). (I) The secondary neurites from tyramine immunoreactive VUMmd and mx enter in the lateral antenna-protocerebral tract (l-APT) and innervate the lateral horn (LH) and mushroom body calyx (ca). (J) In the mushroom body calyx, they innervate the basal ring (br) and lip areas, which receive olfactory afferents from the antennal lobe. The mushroom body pedunculus (ped) and lobe are almost free from tyramine immunoreactive innervation (I,J,K) except for a few branches in the γ lobe (K) that might originate from LPM neurons from SEG. The arrow in (C) indicates tyramine immunoreactive fibers running alongside of the esophageal (es) to the corpora cardiaca nerve (NCC). Ant lobe, antennal lobe; ca, calyx of mushroom body; SEG, subesophageal ganglion; VUM, ventral unpaired median neurons; Ant n, antennal nerve; LPL, lateral protocerbral lobe; APL, anterior protocerebral lobe; AOTu, anterior optic tubercule; V, γ, β, vertical lobe of mushroom bodies, KC, Kenyon cell bodies. Scale bar: A = 250 μm, B = 50 μm, C–K = 100 μm.

Figure 4

Anti-AmTyr1 labeled the neuropil in the honey bee brain. (A) In the antennal lobe, the anti-AmTyr1 is in the cortex area of each glomerulus, but not in the glomerular core (c) and not in the aglomerular neuropil (aglom). The anti-AmTyr1 staining is also absent in the antennal nerve (ant nerve) and olfactory neuron axons tract T1. Asterisk shows a subset of the AmTyr1 positive medial group cell bodies. (B) All area of mushroom body calyx (ca) and pedunculus (ped) were labeled with anti-AmTyr1 with various level of intensity. There is a higher density of staining in the pedunculus (ped) of the mushroom body compared to the lip, collar (co) and basal ring (br) area of the calyx. Note: the central complex has anti-AmTyr1 staining in the fan shaped body and ellipsoid body (eb). (C) The mushroom body vertical lobe (V) exhibits high-intensity level anti-AmTyr1 staining in a basal ring (br), collar (co) and lip area of the Kenyon cells (KCs) axons that have dendrites in the corresponding area of a calyx. The illustrations in (A–C) are inverted fluorescence images. γ—gamma lobe of mushroom body, o-ocelli. Scale bar: A = 50 μm, B = 75 μm, C = 50 μm.

Figure 5

Anti-AmTyr1 labeled synapses of the olfactory receptor neuron (ORN) axons in the antennal lobe glomerulus. (A,B) Triple immunofluorescence labeled with anti-AmTyr1 antibodies (magenta), neurobiotin tracer in ORNs (green) and anti-synapsin (blue). (B) Images are higher magnifications of details from corresponding squares indicated in (A). (A1,B1) Anti-AmTyr1 immunostaining expressed in the cortex of glomeruli (magenta). (A2,B2) The ending of the (ORNs, green) revealed by neurobiotin injections into antenna. Anti-AmTyr1 in glomeruli (A1,A3,A5,B1,B3,B5, magenta) is in ORN endings (A2,A3,A5,B2,B3,B5, green) co-labeled with anti-synapsin (blue, A4,A5,B4,B5). The white color in merged images (A3,A5,B3,B5) revealed anti-AmTyr1 co-stained in the ORN together with synapsin. The white arrows in (B1–B5) indicate co-localization with ORN endings by both anti-AmTyr1 and anti-synapsin; yellow arrow shows co-localization anti-AmTyr1 with synapsin but not with neurobiotin. Scale bar: A = 10 μm; B = 2 μm.

Figure 6

Anti-AmTyr1 immunostaining in the antennal nerve. (A) General view of the antennal segments 5 and 6, neurobiotin was injected in the antennal lobe, and the image was obtained by overexposure with the confocal gain to illustrate different types of sensilla (tC–tricoid sensilla type C; Arrow indicates sensilla placodea (p). (B) Details of the ventral area of the antenna at higher magnification (tA, tB1, tricoid sensilla type A and B1 respectively, b-basiconic sensilla). (C) The section via antenna illustrates merged images of the group of ORN cell bodies and various processes labeled with neurobiotin tracer (green) anti-AmTyr1 (magenta), and 4′,6-diamidino-2-pheylindole (DAPI), marking the nucleus, P-indicate the fibers in the sensilla placodea. (D) Images present higher magnifications of details from corresonding squares indicated in (C). (D1) shows of cell bodies and processes labeled with neurobiotin (green) and anti-AmTyr1 staining (magenta, single staining) and nuclei (blue, DAPI). (D2) illustrates only neurobiotin labeled processes (green) and nuclei (DAPI, blue). (D3) illustrates only anti-AmTyr1 (magenta) and nuclei (DAPI). (D4) shows the nuclei staining. The arrow indicates cell bodies that have co-staining with AmTyr1 and neurobiotin. (E) Anti-AmTyr1 (E1 single image, magenta) is in the area of sensilla placodea (p) with dendrites of ORNs labeled with neurobiotin (green E2). The absence of the white staining in merged image (E3) demonstrates that AmTyr1 does not co-label dendrites of labeled ORNs. Scale bar: A = 100 μm; B,E = 20 μm; C = 25 μm; D = 10 μm.

Figure 7

Triple staining with anti-AmTyr1 (magenta) and anti-synapsin (blue) in the mushroom body after neurobiotin injection in uniglomerular projection neurons (uPNs; green, A,B) and subsets of KCs (green, C,D). (A) Anti-AmTyr1 antibodies (magenta, A1) co-label the neurobiotin injected uPNs ending in the calyx (green, A2). Arrows in (A2) show the axon from uPNs entering the basal ring and lip area of the calyx and labeling presynaptic parts of microglomeruli. Images in (B) illustrate at higher magnification microglomeruli indicated by the square in (A1). Arrows in (B) show that single microglomeruli label with anti-AmTyr1 (magenta, B1) in a uPN terminal bouton (B2). The white staining in merged images (A3,B3) indicates co-labeling of anti-AmTyr1 with uPN terminal microglomeruli. The microglomeruli labeled with anti-synapsin as a presynaptic marker (A4,B4, blue). The white staining in merged triple staining image (A5,B5) indicates that anti-AmTyr1 and anti-synapsin are co-labeled in uPN microglomeruli. (C,D) Anti-AmTyr1 labeled microglomeruli (magenta, C1, insert in C1) in the calyx, subsets of KC bodies, the pedunculus (ped, C1). Also, areas of the mushroom body vertical lobe that correspond to KCs with dendrites in the basal ring, lip and collar areas of the calyx have a high level of staining intensity (D1). The (KCs) were injected with neurobiotin in the area indicated in (C) by an ellipse, and in single image staining (green, in C2,D2) the neurobiotin revealed in cell bodies, dendrite in calyx (C2, insert in C2) and in lobe (D2). Only subsets of KCs took up the neurobiotin in this preparation. For the injection of neurobiotin in the area shown by the ellipse in (C), the subsets of KCs that took up the tracer express it in cell bodies, in dendrites in the lip, basal ring and collar (C2, insert in C2), and in the axons of the corresponding area of vertical and gamma lobe (D2). Merged images (C3,D3) show the co-localization of KCs that take up neurobiotin with AmTyr1 in axons but not in the dendrites of the calyx (inserts in C3). (C) AmTyr1 (magenta C1) expression in subsets of mushroom body KC axons (green, C2) but not in the dendrites in the calyx (insert in C). The merged images in (C5) illustrate co-localization of anti-AmTyr1 with anti-synapsin (blue, C4 single staining) in calyx microglomeruli, but not with neurobiotin labeled KC dendrites in calyx (insert in C4,C5 respectively). Anti-AmTyr1 in the mushroom body lobe is in KC axons; these KCs have dendrites in the basal ring and collar areas. In insert (D1)-AmTyr1 (magenta) is in axons of KC labeled with neurobiotin (D2, single image) and co-labeled with synapsin in (D4; single image). In the triple staining image (D5; insert D5) the white color corresponds to co-labeling of synapsin in KC axons and AmTyr1. Note, that not all synapsin labeling processes (single staining image D4, insert D4) express AmTyr1 (single staining image D1, insert D1). Arrows in insert (D1–D5) shows AmTyr1 in the axon of KC co-labeled with synapsin. Scale bar: A,C,D = 50 μm, B = 2 μm.

Figure 8

Schematic view of the neural network proposed for the honey bee antennal lobe (modified from Sinakevitch et al., 2013). Each glomerulus can be defined by three types of neurons that are tuned to a narrow range of odorants: (i) ORN axons that project excitatory branches into the cortex of the glomerulus; (ii) the glomerular uPNs that receive input in both the cortex and core of the glomerulus and project excitatory output branches to the LH and Mushroom body calyx; and (iii) inhibitory hetero-LNs that branch in all areas of the glomerulus (cortex and core), where they receive excitatory output from the cortex and inhibitory from the core. Hetero-LNs also have inhibitory input in the core area of one glomerulus. Hetero-LNs also have two types of neurotransmitter (GABA and Histamine, Dacks et al., 2010). The neurons that interconnect all glomeruli are multiglomerular LNs (containing both GABA and Allatostatin, Kreissl et al., 2010). They have input/output branches in the core area where they inhibit neurons in the core. There are also multiglomerular inhibitory GABAergic mPNs that are not illustrated here. We propose that VUM neurons release both octopamine and tyramine in the antennal lobe, LH and mushroom body calyx. Each glomerulus will respond to the presence of each biogenic amine through specific receptors. Inhibitory LNs (hetero and homo) express AmOA1. The ORN axons express AmTyr1, and the uPNs axons in the LH and Mushroom body calyx also express AmTyr1 receptors. In both cases, AmTyr1 is in a position to regulate excitatory transmission into the respective areas. We hypothesize that the action of octopamine and tyramine released by VUM could be dependent on the ratio of the amines and on the specific target cells that express the receptors. An excess of octopamine in a glomerulus leads to inhibiting the inhibition in the core and simultaneously blocks excitation in neighboring glomeruli via AmOA1 on GABAergic LNs. An excess of tyramine inhibits the release of the excitatory neurotransmitter in the synapses.