Behavioural pharmacology of octopamine,tyramine and dopamine in honey bees

In the honey bee, responsiveness to sucrose correlates with many behavioural parameters such as age of first foraging, foraging role and learning. Sucrose responsiveness can be measured using the proboscis extension response (PER) by applying sucrose solutions of increasing concentrations to the antenna of a bee. We tested whether the biogenic amines octopamine, tyramine and dopamine, and the dopamine receptor agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN) can modulate sucrose responsiveness. The compounds were either injected into the thorax or fed in sucrose solution to compare different methods of application. Injection and feeding of tyramine or octopamine significantly increased sucrose responsiveness. Dopamine decreased sucrose responsiveness when injected into the thorax. Feeding of dopamine had no effect. Injection of 6,7-ADTN into the thorax and feeding of 6,7-ADTN reduced sucrose responsiveness significantly. These data demonstrate that sucrose responsiveness in honey bees can be modulated by biogenic amines, which has far reaching consequences for other types of behaviour in this insect.     

Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera)

In the honey bee (Apis mellifera), young workers usually perform tasks in the nest while older workers forage in the field. The behavioral shift from nest-task to foraging activity is accompanied by physiological and sensory changes so that foragers can be characterized by a higher juvenile hormone (JH) level, a lower vitellogenin protein titer, and an increased responsiveness to water and sucrose stimuli. JH was hypothesized to be the key mediator of behavioral development, physiology, and sensory sensitivity in honey bee workers. Recent research, however, has shown that JH is controlled by the hemolymph vitellogenin level, which implies that the fat body specific vitellogenin gene can be a key regulator of behavioral change. Here, we show that downregulation of vitellogenin activity by RNA interference (RNAi) causes an increase in the gustatory responsiveness of worker bees. Our observations suggest that vitellogenin is an important regulator of long-term changes in honey bee behavior.     

Changes in neuronal acetylcholinesterase gene expression and division of labor in honey bee colonies

Division of labor in honey bee colonies is highlighted by adult bees making a transition at 2–3 wk of age from working in the hive to foraging for nectar and pollen outside. This behavioral development involves acquisition of new tasks that may require advanced learning capabilities. Because acetylcholinesterase (AChE) hydrolyzes acetylcholine, a major neurotransmitter associated with learning in the insect brain, we searched for changes in AChE expression in the brain during bee behavioral development. Biochemical aspects of the AChE protein were similar in foragers and “nurse” bees that work in the hive tending brood. However, catalytic AChE activity was significantly lower in foragers. Cloning of bee AChE cDNA enabled mRNA analysis, which demonstrated that the forager-related decrease in AChE activity was associated with decreased AChE mRNA levels. This was particularly apparent in the mushroom bodies, a brain region known to be involved with olfactory and visual learning and memory. In addition, treatment with the AChE-inhibitor metrifonate improved performance in an olfactory-learning assay. These findings demonstrate long-term, naturally occurring developmental downregulation of AChE gene expression in the bee brain, and suggest that this genomic plasticity can contribute to facilitated learning capabilities in forager bees.     

PKG in honey bees: Spatial expression,Amfor gene expression,sucrose responsiveness,and division of labor

Division of labor is a hallmark of social insects. In honey bees, division of labor involves transition of female workers from one task to the next. The most distinct tasks are nursing (providing food for the brood) and foraging (collecting pollen and nectar). The brain mechanisms regulating this form of behavioral plasticity have largely remained elusive. Recently, it was suggested that division of labor is based on nutrition‐associated signaling pathways. One highly conserved gene associated with food‐related behavior across species is the foraging gene, which encodes a cyclic guanosine monophosphate (cGMP)‐dependent protein kinase (PKG). Our analysis of this gene reveals the presence of alternative splicing in the honey bee. One isoform is expressed in the brain. Expression of this isoform is most pronounced in the mushroom bodies, the subesophageal ganglion, and the corpora allata. Division of labor and sucrose responsiveness in honey bees correlate significantly with foraging gene expression in distinct brain regions. Activating PKG selectively increases sucrose responsiveness in nurse bees to the level of foragers, whereas the same treatment does not affect responsiveness to light. These findings demonstrate a direct link between PKG signaling in distinct brain areas and division of labor. Furthermore, they demonstrate that the difference in sensory responsiveness between nurse bees and foragers can be compensated for by activating PKG. Our findings on the function of PKG in regulating specific sensory responsiveness and social organization offer valuable indications for the function of the cGMP/PKG pathway in many other insects and vertebrates. J. Comp. Neurol. 522:1786–1799, 2014. © 2013 Wiley Periodicals, Inc.     

Structural plasticity of identified glomeruli in the antennal lobes of the adult worker honey bee

Adult worker honey bees alter their behaviour with age but retain a strong reliance on sensory information from the antennae. The antennae house a diverse array of receptors, including mechanoreceptors, hygroreceptors, olfactory receptors, and contact chemoreceptors, which relay information to the brain. Antennal sensory neurons that project to the antennal lobes of the brain converge onto second-order interneurones to form discrete spheres of neuropil, called glomeruli. The spatial organisation of glomeruli in the antennal lobes of the honey bee is constant, but the central distribution of information from receptors tuned to different sensory modalities is unknown. Here we show that the glomerular neuropil of the antennal lobes undergoes constant modification during the lifetime of the adult worker bee. Changes in morphology are site specific and highly predictable. The total volume of the glomerular neuropil of the antennal lobe increased significantly during the first 4 days of adult life. Each of the five readily identifiable glomeruli examined in this study exhibited a unique pattern of growth. The growth of two of the five glomeruli changed dramatically with the shift to foraging duties. Furthermore, significant differences were identified between the antennal lobes of bees performing nectar-and pollen-foraging tasks. The highly compartmentalized nature of the antennal lobes, the ease with which specific glomeruli can be identified, and the predictability of changes to the antennal lobe neuropil make this an ideal system for examining the mechanisms and behavioural consequences of structural plasticity in primary sensory centres of the brain. © 1996 Wiley-Liss, Inc.     

Biogenic amines and activity levels alter the neural energetic response to aggressive social cues in the honey bee Apis mellifera

Mitochondrial activity is highly dynamic in the healthy brain, and it can reflect both the signaling potential and the signaling history of neural circuits. Recent studies spanning invertebrates to mammals have highlighted a role for neural mitochondrial dynamics in learning and memory processes as well as behavior. In the current study, we investigate the interplay between biogenic amine signaling and neural energetics in the honey bee, Apis mellifera. In this species, aggressive behaviors are regulated by neural energetic state and biogenic amine titers, but it is unclear how these mechanisms are linked to impact behavioral expression. We show that brain mitochondrial number is highest in aggression-relevant brain regions and in individual bees that are most responsive to aggressive cues, emphasizing the importance of energetics in modulating this phenotype. We also show that the neural energetic response to alarm pheromone, an aggression inducing social cue, is activity dependent, modulated by the “fight or flight” insect neurotransmitter octopamine. Two other neuroactive compounds known to cause variation in aggression, dopamine, and serotonin, also modulate neural energetic state in aggression-relevant regions of the brain. However, the effects of these compounds on respiration at baseline and following alarm pheromone exposure are distinct, suggesting unique mechanisms underlying variation in mitochondrial respiration in these circuits. These results motivate new explanations for the ways in which biogenic amines alter sensory perception in the context of aggression. Considering neural energetics improves predictions about the regulation of complex and context-dependent behavioral phenotypes.     

Development of dopamine-immunoreactive neurons associated with the antennal lobes of the honey bee, Apis mellifera.

This report examines the development of the dopaminergic system in the primary antennosensory centres (antennal lobes) of the brain of the honey bee, Apis mellifera, and the effects of dopamine on neurite outgrowth of antennal-lobe neurons in vitro. Antibodies raised against dopamine were used to follow the development of a small population of dopamine-immunoreactive neurons that invade the antennal lobes during metamorphic adult development. Immunopositive somata associated with the antennal lobes were first detected at stage 2 of the nine stages of metamorphic adult development, but processes of these neurons within the antennal-lobe neuropil did not exhibit immunostaining until pupal stage 3. Severe depletion of primary sensory input to the right antennal lobe early in metamorphic adult development or removal of the right antenna from newly emerged bees did not alter the expression of dopamine immunoreactivity in the antennal-lobe neuropil. The presence of dopamine in developing antennal lobes was confirmed by using high performance liquid chromatography with electrochemical detection. Levels of dopamine were significantly higher at pupal stage 4 than at all other stages examined. This surge in dopamine levels coincided with rapid growth and compartmentalisation of the antennal-lobe neuropil. Exogenously applied dopamine (50 microM) enhanced the growth of antennal-lobe neurons in vitro, but only in cells derived from pupae at stage 5 of metamorphic adult development. The early appearance of dopamine-immunoreactive neurons and the effects of dopamine on stage 5 antennal-lobe neurons in vitro support the view that dopamine plays a role in the developing brain of the honey bee.     

Octopamine-like immunoreactivity in the honey bee and cockroach: comparable organization in the brain and subesophageal ganglion

A serum raised against octopamine reveals in cockroaches and honey bees structurally comparable systems of perikarya and their extensive yet discrete systems of arborizations in neuropils. Numerous and prominent clusters of lateral cell bodies in the brain as well as many midline perikarya provide octopamine-like immunoreactive processes to circumscribed regions of the subesophageal ganglion, antennal lobe glomeruli, optic neuropils, and neuropils of the protocerebrum. There is dense octopaminergic innervation in the protocerebral bridge and ellipsoid body of the central complex. The antennal lobes are supplied by at least three octopamine-immunoreactive neurons. In contrast, the mushroom bodies show the fewest immunoreactive elements. In Apis a single axon supplies sparse immunoreactive processes to the calyces' basal ring, collar, and lip. A diffuse arrangement of immunoreactive processes invades all zones of the mushroom body calyces in Periplaneta. These processes derive from an ascending axon ascribed to a dorsal unpaired median neuron at the maxillary segment of the subesophageal ganglion. In both taxa octopamine-immunoreactive processes invade only the gamma lobes of the mushroom bodies, omitting their other divisions. The present observations are discussed with respect to possible roles of octopamine in sensory integration and association.     

Phototactic behaviour correlates with gustatory responsiveness in honey bees (Apis mellifera L.)

The response threshold hypothesis of division of labour in honey bees assumes that individuals differ in their responsiveness to different stimulus modalities. However, previous experiments have shown that responsiveness to gustatory stimuli correlates with responsiveness to odours, pollen and tactile stimuli. Evaluation of these stimuli involves sensory receptors on the antenna. We tested whether responsiveness to gustatory stimuli correlates with responsiveness to visual stimuli in a phototaxis experiment, which is independent of antennal input. Gustatory responsiveness was measured using the proboscis extension response to antennal stimulation with water and different sucrose concentrations. Phototaxis was quantified by measuring the walking times a bee needed to reach light sources of different intensities. Walking behaviour in the darkness was measured to test for differences in locomotor behaviour. The walking time towards a light stimulus, the path length, and the walking speed depended on the intensity of the light stimulus. Responsiveness to visual stimuli correlated significantly with gustatory responsiveness. Bees displaying a high gustatory responsiveness were also very sensitive to light. Locomotor activity did not correlate with gustatory responsiveness. This shows that gustatory responsiveness is a good indicator of sensitivity for visual stimuli, which are not perceived by the antenna.     

Comparison of octopamine-like immunoreactivity in the brains of the fruit fly and blow fly

A serum raised against conjugated octopamine reveals structurally comparable systems of perikarya and arborizations in protocerebral neuropils of two species of Diptera, Drosophila melanogaster and Phaenicia sericata; the latter is used extensively for electrophysiological studies of the optic lobes and their central projections. Clusters of cell bodies in the brain as well as midline perikarya provide octopamine-like immunoreactive processes to the optic lobes, circumscribed regions of the protocerebrum and the central complex, particularly the protocerebral bridge, fan-shaped body, and ellipsoid body. Ventral unpaired median somata provide immunoreactive processes within the subesophageal ganglion and tritocerebrum. Ascending neurites from these cells also supply the antennal lobe glomeruli, regions of the lateral protocerebrum, the mushroom body calyces, and the lobula complex. The mushroom body's gamma lobes contain immunoreactive processes that originate from processes that arborize in the protocerebrum. The present observations are discussed with respect to similarities and differences between two species of Diptera, one of which has neurons large enough for intracellular penetrations. The results are also discussed with respect to recent studies on octopamine-immunoreactive organization in honey bees and cockroaches and the suggested roles of octopamine in sensory processing, learning, and memory.     

An improved method for the separation and detection of biogenic amines in adult Drosophila brain extracts by high performance liquid chromatography

Biogenic amines are critically important neuromodulators in both vertebrates and invertebrates. Quantification of these amines can be difficult, particularly in neural extracts of Drosophila melanogaster that contain interfering electroactive compounds. We have developed a method for the reliable separation and quantification of the biogenic amines dopamine, serotonin, tyramine, and octopamine in Drosophila brain extracts using high performance liquid chromatography with electrochemical detection. Our method obviates the need for complex preparatory procedures or instrumentation, and can reproducibly detect picogram quantities of these amines. By optimizing the composition of the mobile phase and the electrode potential, and by examining common complications in the analysis of biological samples, we have developed a reliable technique for monitoring levels of biogenic amines in the Drosophila brain.     

Metamodulation of the biogenic amines: second-order modulation by steroid hormones and amine cocktails

An evolutionarily conserved feature of neural systems is that they can be modified by neuromodulators. These modulatory chemical signals include the biogenic amines, octopamine (OA), serotonin (5-HT) and dopamine (DA). Such modulation effectively broadens the operational range in which specific neural circuits can function adaptively. This report discusses how these amines are themselves modulated; for example, by the steroid hormone 20-hydroxyecdysone (20-E) or by the addition of a second biogenic amine. Such second-order neuromodulation, termed metamodulation, is discussed in the context of two well-studied invertebrate systems: the tobacco hornworm moth Manduca sexta, a model of neurodevelopment and plasticity, and the medicinal leech Hirudo medicinalis, a long-favored preparation used to study neural circuits at the level of identified neurons. A portion of this article reviews our previous research of M. sexta that shows that the 'preadult' rise in 20-E is both necessary and sufficient for the increased levels of octopamine observed in the adult. Such elevated levels likely play an important role in the production and modulation of adult behaviors. The somatic growth of median octopaminergic neurons and the late expression of OA-immunoreactivity by novel lateral neurons are also demonstrated to be dependent on 20-E. New immunocytochemical results of stained dopaminergic neurons in the larval and adult moth brain are provided as well, and the potential influence of 20-E on the developmental expression of this neuromodulator is presented. Turning attention to the leech, data indicate that the actions of OA are dramatically altered when 5-HT is combined with OA in the bath surrounding the isolated nervous system. Although either OA or 5-HT alone induces fictive swimming behavior, a cocktail of these two amines strongly inhibits the generation of swimming. Subsequent removal of such a mixture induces nearly continuous swimming and constitutes the best swim-inducing stimulus encountered to date. To understand better how these nonadditive effects are achieved, new results are discussed that indicate that the leech brain is the target of metamodulation by the two amines. Both the arthropod and annelid systems presented here highlight the multiple levels of metamodulation that can exist in nervous systems, and the diverse ways that a modulator's actions can become altered over short or long time periods.     

Side-specific operant conditioning of antennal movements in the honey bee

Operant conditioning of antennal movements in honey bees was used to investigate whether learned changes on one antenna influence antennal movements of the contralateral antenna. Conditioning of the right antenna did not alter antennal movements of the left antenna and subsequent conditioning of the left antenna did not abolish the previously learned change in the right antenna. Thus, the antennal systems on each side are largely independent from each other.     

Age-related changes in the number and structure of synapses in the lip region of the mushroom bodies in the ant Pheidole dentata

Behavioral development in the worker caste of many adult ants follows a pattern of task transitions that contribute to the division of labor within colonies. In the ant Pheidole dentata, the number of tasks that minor workers attend to increases as they progress from brood-care activities within the nest to acts outside the nest such as foraging and defense. In this study we investigated synapse maturation in the lip region of mushroom bodies in young and old minor workers because of its potentially crucial role in behavioral development, task performance, and repertoire expansion. As minor workers aged, individual presynaptic boutons enlarged and acquired more synapses and vesicles, but the total number of synapses in the lip region did not change significantly. Glial cell processes occupied less of the synaptic neuropil as ants matured. These findings indicate an expansion and enhancement of efficacy at specific sets of synaptic connections between the projection interneurons and Kenyon cell dendrites and a commensurate loss of other connections as minor workers age and expand their behavioral repertoire.     

The Role of octopamine and tyramine in Drosophila larval locomotion

The characteristic crawling behavior of Drosophila larvae consists of a series of rhythmic waves of peristalsis and episodes of head swinging and turning. The two biogenic amines octopamine and tyramine have recently been shown to modulate various parameters of locomotion, such as muscle contraction, the time spent in pausing or forward locomotion, and the initiation and maintenance of rhythmic motor patterns. By using mutants having altered octopamine and tyramine levels and by genetic interference with both systems we confirm that signaling of these two amines is necessary for larval locomotion. We show that a small set of about 40 octopaminergic/tyraminergic neurons within the ventral nerve cord is sufficient to trigger proper larval locomotion. Using single‐cell clones, we describe the morphology of these neurons individually. Given various potential roles of octopamine and tyramine in the larval brain, such as locomotion, learning and memory, stress‐induced behaviors or the regulation of the energy state, functions that are often not easy to discriminate, we dissect here for the first time a subset of this complex circuit that modulates specifically larval locomotion. Thus, these data will help to understand—for a given neuronal modulator—how specific behavioral functions are executed within distinct subcircuits of a complex neuronal network. J. Comp. Neurol. 520:3764–3785, 2012. © 2012 Wiley Periodicals, Inc.     

A genome-wide inventory of neurohormone GPCRs in the red flour beetle Tribolium castaneum

Insect neurohormones (biogenic amines, neuropeptides, and protein hormones) and their G protein-coupled receptors (GPCRs) play a central role in the control of behavior, reproduction, development, feeding and many other physiological processes. The recent completion of several insect genome projects has enabled us to obtain a complete inventory of neurohormone GPCRs in these insects and, by a comparative genomics approach, to analyze the evolution of these proteins. The red flour beetle Tribolium castaneum is the latest addition to the list of insects with a sequenced genome and the first coleopteran (beetle) to be sequenced. Coleoptera is the largest insect order and about 30% of all animal species living on earth are coleopterans. Some coleopterans are severe agricultural pests, which is also true for T. castaneum, a global pest for stored grain and other dried commodities for human consumption. In addition, T. castaneum is a model for insect development. Here, we have investigated the presence of neurohormone GPCRs in Tribolium and compared them with those from the fruit fly Drosophila melanogaster (Diptera) and the honey bee Apis mellifera (Hymenoptera). We found 20 biogenic amine GPCRs in Tribolium (21 in Drosophila; 19 in the honey bee), 48 neuropeptide GPCRs (45 in Drosophila; 35 in the honey bee), and 4 protein hormone GPCRs (4 in Drosophila; 2 in the honey bee). Furthermore, we identified the likely ligands for 45 of these 72 Tribolium GPCRs. A highly interesting finding in Tribolium was the occurrence of a vasopressin GPCR and a vasopressin peptide. So far, the vasopressin/GPCR couple has not been detected in any other insect with a sequenced genome (D. melanogaster and six other Drosophila species, Anopheles gambiae, Aedes aegypti, Bombyx mori, and A. mellifera). Tribolium lives in very dry environments. Vasopressin in mammals is the major neurohormone steering water reabsorption in the kidneys. Its presence in Tribolium, therefore, might be related to the animal's need to effectively control water reabsorption. Other striking differences between Tribolium and the other two insects are the absence of the allatostatin-A, kinin, and corazonin neuropeptide/receptor couples and the duplications of other hormonal systems. Our survey of 340 million years of insect neurohormone GPCR evolution shows that neuropeptide/receptor couples can easily duplicate or disappear during insect evolution. It also shows that Drosophila is not a good representative of all insects, because several of the hormonal systems that we now find in Tribolium do not exist in Drosophila.     

Cerebrospinal fluid studies in the Rett syndrome: biogenic amines and beta-endorphins.

The etiology of the Rett syndrome (RS) is unknown. Reduced function of biogenic amines has been described. Symptoms of central apnea, hyperventilation, hypothermia, peripheral analgesia, muscle rigidity, myoclonic jerks, hand stereotypy and seizures occur in RS and have been suggested as a result of elevated central beta-endorphins. It was hypothesized that a dysfunctional modulation of endogenous opiate systems and biogenic amines may be present. Cerebrospinal fluid (CSF) from 12 girls with RS was studied for beta-endorphin immunoreactivity, and biogenic amines. Lactates and pyruvate levels were measured. Eleven of the 12 girls had elevated beta-endorphin immunoreactivity in CSF, 4 girls had reduced biogenic amines and 6 girls had elevated pyruvate and lactate levels. Whether the elevated beta-endorphin immunoreactivity is a primary disorder or is a result of secondary feedback mechanisms is unknown. Naltrexone, an antiopioid drug, may reduce symptoms.     

Nonassociative plasticity alters competitive interactions among mixture components in early olfactory processing

Experience‐related plasticity is an essential component of networks involved in early olfactory processing. However, the mechanisms and functions of plasticity in these neural networks are not well understood. We studied nonassociative plasticity by evaluating responses to two pure odors (A and X) and their binary mixture using calcium imaging of odor‐elicited activity in output neurons of the honey bee antennal lobe. Unreinforced exposure to A or X produced no change in the neural response elicited by the pure odors. However, exposure to one odor (e.g. A) caused the response to the mixture to become more similar to that of the other component (X). We also show in behavioral analyses that unreinforced exposure to A caused the mixture to become perceptually more similar to X. These results suggest that nonassociative plasticity modifies neural networks in such a way that it affects local competitive interactions among mixture components. We used a computational model to evaluate the most likely targets for modification. Hebbian modification of synapses from inhibitory local interneurons to projection neurons most reliably produced the observed shift in response to the mixture. These results are consistent with a model in which the antennal lobe acts to filter olfactory information according to its relevance for performing a particular task.     

Distribution and developmental expression of octopamine-immunoreactive neurons in the central nervous system of the leech

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1–6 and posterior ganglia 15–21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7–14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamineimmunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis, Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamineimmunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior. © 1995 Wiley-Liss, Inc.