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.
Project description: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.
Project description:Feedback plays important roles in sensory processing. Mushroom bodies are believed to be involved in olfactory learning/memory and multisensory integration in insects. Previous cobalt-labeling studies have suggested the existence of feedback from the mushroom bodies to the antennal lobes in the honey bee. In this study, the existence of functional feedback from Drosophila mushroom bodies to the antennal lobes was investigated through ectopic expression of the ATP receptor P2X(2) in the Kenyon cells of mushroom bodies. Activation of Kenyon cells induced depolarization in projection neurons and local interneurons in the antennal lobes in a nicotinic receptor-dependent manner. Activation of Kenyon cell axons in the betagamma-lobes in the mushroom body induced more potent responses in the antennal lobe neurons than activation of Kenyon cell somata. Our results indicate that functional feedback from Kenyon cells to projection neurons and local interneurons is present in Drosophila and is likely mediated by the betagamma-lobes. The presence of this functional feedback from the mushroom bodies to the antennal lobes suggests top-down modulation of olfactory information processing in Drosophila.
Project description:The biogenic amine octopamine is an important neuromodulator, neurohormone and neurotransmitter in insects. We here investigate the role of octopamine signaling in honey bee phototaxis. Our results show that groups of bees differ naturally in their phototaxis. Pollen forgers display a lower light responsiveness than nectar foragers. The lower phototaxis of pollen foragers coincides with higher octopamine titers in the optic lobes but is independent of octopamine receptor gene expression. Increasing octopamine brain titers reduces responsiveness to light, while tyramine application enhances phototaxis. These findings suggest an involvement of octopamine signaling in honey bee phototaxis and possibly division of labor, which is hypothesized to be based on individual differences in sensory responsiveness.
Project description:The anatomical organization of distinct regions in the insect brain often reflects their functions. In the present study, the brain structure of Apolygus lucorum was examined by using immunolabeling and three-dimensional reconstruction. The results revealed the location and volume of prominent neuropils, such as the antennal lobes (AL), optic lobes (OL), anterior optic tubercles (AOTU), central body (CB), lateral accessory lobes (LAL), mushroom lobes, and distinct tritocerebral neuropils. As expected, this brain is similar to that of other insects. One exception, however, is that the antennal lobes were found to be the most prominent neuropils. Their size relative to the entire brain is the largest among all insect species studied so far. In contrast, the calyx, a region getting direct input from the antennal lobe, has a smaller size relative to the brain than that of other species. These findings may suggest that olfaction plays an essential role for A. lucorum.
Project description:In the honeybee brain, two prominent tracts - the medial and the lateral antennal lobe tract - project from the primary olfactory center, the antennal lobes (ALs), to the central brain, the mushroom bodies (MBs), and the protocerebral lobe (PL). Intracellularly stained uniglomerular projection neurons were reconstructed, registered to the 3D honeybee standard brain atlas, and then used to derive the spatial properties and quantitative morphology of the neurons of both tracts. We evaluated putative synaptic contacts of projection neurons (PNs) using confocal microscopy. Analysis of the patterns of axon terminals revealed a domain-like innervation within the MB lip neuropil. PNs of the lateral tract arborized more sparsely within the lips and exhibited fewer synaptic boutons, while medial tract neurons occupied broader regions in the MB calyces and the PL. Our data show that uPNs from the medial and lateral tract innervate both the core and the cortex of the ipsilateral MB lip but differ in their innervation patterns in these regions. In the mushroombody neuropil collar we found evidence for ALT boutons suggesting the collar as a multi modal input site including olfactory input similar to lip and basal ring. In addition, our data support the conclusion drawn in previous studies that reciprocal synapses exist between PNs, octopaminergic-, and GABAergic cells in the MB calyces. For the first time, we found evidence for connections between both tracts within the AL.
Project description:Neuronal plasticity allows an animal to respond to environmental changes by modulating its response to stimuli. In the honey bee (Apis mellifera), the biogenic amine octopamine plays a crucial role in appetitive odor learning, but little is known about how octopamine affects the brain. We investigated its effect in the antennal lobe, the first olfactory center in the brain, using calcium imaging to record background activity and odor responses before and after octopamine application. We show that octopamine increases background activity in olfactory output neurons, while reducing average calcium levels. Odor responses were modulated both upwards and downwards, with more odor response increases in glomeruli with negative or weak odor responses. Importantly, the octopamine effect was variable across glomeruli, odorants, odorant concentrations and animals, suggesting that the octopaminergic network is shaped by plasticity depending on an individual animal's history and possibly other factors. Using RNA interference, we show that the octopamine receptor AmOA1 (homolog of the Drosophila OAMB receptor) is involved in the octopamine effect. We propose a network model in which octopamine receptors are plastic in their density and located on a subpopulation of inhibitory neurons in a disinhibitory pathway. This would improve odor-coding of behaviorally relevant, previously experienced odors.
Project description:Processing of olfactory information in the antennal lobes of insects and olfactory bulbs of vertebrates is modulated by centrifugal inputs that represent reinforcing events. Octopamine release by one such pathway in the honeybee antennal lobe modulates olfactory processing in relation to nectar (sucrose) reinforcement. To test more specifically what role octopamine plays in the antennal lobe, we used two treatments to disrupt an octopamine receptor from Apis mellifera brain (AmOAR) function: (1) an OAR antagonist, mianserin, was used to block receptor function, and (2) AmOAR double-stranded RNA was used to silence receptor expression. Both treatments inhibited olfactory acquisition and recall, but they did not disrupt odor discrimination. These results suggest that octopamine mediates consolidation of a component of olfactory memory at this early processing stage in the antennal lobe. Furthermore, after consolidation, octopamine release becomes essential for recall, which suggests that the modulatory circuits become incorporated as essential components of neural representations that activate odor memory.
Project description:Dopamine (DA) plays a fundamental role in insect behavior as it acts both as a general modulator of behavior and as a value system in associative learning where it mediates the reinforcing properties of unconditioned stimuli (US). Here we aimed at characterizing the dopaminergic neurons in the central nervous system of the honey bee, an insect that serves as an established model for the study of learning and memory. We used tyrosine hydroxylase (TH) immunoreactivity (ir) to ensure that the neurons detected synthesize DA endogenously. We found three main dopaminergic clusters, C1-C3, which had been previously described; the C1 cluster is located in a small region adjacent to the esophagus (ES) and the antennal lobe (AL); the C2 cluster is situated above the C1 cluster, between the AL and the vertical lobe (VL) of the mushroom body (MB); the C3 cluster is located below the calyces (CA) of the MB. In addition, we found a novel dopaminergic cluster, C4, located above the dorsomedial border of the lobula, which innervates the visual neuropils of the bee brain. Additional smaller processes and clusters were found and are described. The profuse dopaminergic innervation of the entire bee brain and the specific connectivity of DA neurons, with visual, olfactory and gustatory circuits, provide a foundation for a deeper understanding of how these sensory modules are modulated by DA, and the DA-dependent value-based associations that occur during associative learning.
Project description:Little information is available regarding the adverse effects of pesticides on natural honey bee populations. This study highlights the detrimental effects of pesticides on honey bee olfaction through behavioural studies, scanning electron microscopic imaging of antennal sensillae and confocal microscopic studies of honey bee brains for calcium ions on Apis cerana, a native Indian honey bee species. There was a significant decrease in proboscis extension response and biologically active free calcium ions and adverse changes in antennal sensillae in pesticide exposed field honey bee populations compared to morphometrically similar honey bees sampled from low/no pesticide sites. Controlled laboratory experiments corroborated these findings. This study reports for the first time the changes in antennal sensillae, expression of Calpain 1(an important calcium binding protein) and resting state free calcium in brains of honey bees exposed to pesticide stress.
Project description:The mushroom bodies are the higher-order integration center in the insect brain and are involved in higher brain functions such as learning and memory. In the social hymenopteran insects such as honeybees, the mushroom bodies are the prominent brain structures. The mushroom bodies are composed of lobed neuropils formed by thousands of parallel-projecting axons of intrinsic neurons, and the lobes are divided into parallel subdivisions. In the present paper, we report a new antigenic marker to label a single layer in the vertical lobes of the European honeybee Apis mellifera. In the brain of A. mellifera, a monoclonal antibody (mAb) 15C3, which was originally developed against an insect ecdysone receptor (EcR) protein, immunolabels a single layer of the vertical lobes that correspond to the most dorsal layer of the ?-lobe. The 15C3 mAb recognizes a single ~200 kDa protein expressed in the adult honeybee brain. In addition, the 15C3 mAb immunoreactivity was also observed in the lobes of the developing pupal mushroom bodies. Since ?-lobe is well known to their extensive reorganization that occurs during metamorphosis in Drosophila, the novel antigenic marker for the honeybee ?-lobe allows us to investigate morphological changes of the mushroom bodies during metamorphosis.