Gaseous neuromodulator-related genes expressed in the brain of honeybee Apis mellifera

Nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO) are thought to act as gaseous neuromodulators in the brain across species. For example, in the brain of honeybee Apis mellifera, NO plays important roles in olfactory learning and discrimination, but the existence of H2S- and CO-mediated signaling pathways remains unknown. In the present study, we identified the genes of nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), cystathionine beta-synthase (CBS), and heme oxygenase (HO) from the honeybee brain. The honeybee brain contains at least one gene for each of NOS, CBS, and HO. The deduced proteins for NOS, CBS, and HO are thought to contain domains to generate NO, H2S, and CO, respectively, and to contain putative Ca2+/calmodulin-binding domains. On the other hand, the honeybee brain contains three subunits of sGC: sGCalpha1, sGCbeta1, and sGCbeta3. Phylogenetic analysis of sGC revealed that Apis sGCalpha1 and sGCbeta1 are closely related to NO- and CO-sensitive sGC subunits, whereas Apis sGCbeta3 is closely related to insect O2-sensitive sGC subunits. In addition, we performed in situ hybridization for Apis NOS mRNA and NADPH-diaphorase histochemistry in the honeybee brain. The NOS gene was strongly expressed in the optic lobes and in the Kenyon cells of the mushroom bodies. NOS activity was detected in the optic lobes, the mushroom bodies, the central body complex, the lateral protocerebral lobes, and the antennal lobes. These findings suggest that NO is involved in various brain functions and that H2S and CO can be endogenously produced in the honeybee brain.     

Characterization of a metabotropic glutamate receptor in the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis>): implications for memory formation

G-protein-coupled metabotropic glutamate receptors (GPC mGluRs) are important constituents of glutamatergic synapses where they contribute to synaptic plasticity and development. Here we characterised a member of this family in the honeybee. We show that the honeybee genome encodes a genuine mGluR (AmGluRA) that is expressed at low to medium levels in both pupal and adult brains. Analysis of honeybee protein sequence places it within the type 3 GPCR family, which includes mGlu receptors, GABA-B receptors, calcium-sensing receptors, and pheromone receptors. Phylogenetic comparisons combined with pharmacological evaluation in HEK 293 cells transiently expressing AmGluRA show that the honeybee protein belongs to the group II mGluRs. With respect to learning and memory AmGluRA appears to be required for memory formation. Both agonists and antagonists selective against the group II mGluRs impair long-term (24 h) associative olfactory memory formation when applied 1 h before training, but have no effect when injected post-training or pre-testing. Our results strengthen the notion that glutamate is a key neurotransmitter in memory processes in the honeybee.     

Caste-specific postembryonic development of primary and secondary olfactory centers in the female honeybee brain

Eusocial insects are characterized by division of labor among a sterile worker caste and a reproductive queen. In the honeybee both female castes are determined postembryonically by environmental factors, and queens develop substantially faster than workers. Since olfaction plays a crucial role in organizing honeybee behavior and social interactions, we compared the development of primary and secondary olfactory centers in the brain. Age-synchronized queen and worker pupae were raised in incubators at 34.5 degrees C, and their external morphology was characterized for all pupal stages. The development of olfactory synaptic neuropil was analyzed using anti-synapsin immunocytochemistry, f-actin-phalloidin labeling and confocal microscopy. In the antennal lobes of queens olfactory glomeruli formed approximately 4 days earlier than in workers. The adult number of olfactory glomeruli was in a similar range, but the total glomerular volume was slightly smaller in queens. Olfactory and visual subdivisions (lip, collar) of the mushroom-body calyx formed early, whereas the basal ring separated late. Synaptic microglomeruli in the olfactory lip were established approximately 3-4 days earlier in queens compared to workers. We propose that developmental heterochrony results in fewer synapses in olfactory centers (smaller glomeruli, fewer microglomeruli) in queens, which may result in poorer performance on olfactory learning tasks compared to workers.     

Eph家族蛋白在阿尔茨海默症等神经精神疾病中的作用

促红细胞生成素产生肝细胞(erythropoietin-producing hepatomocellular, Eph)受体是受体酪氨酸激酶家族中数量最多的成员。Eph受体与其配体肝配蛋白(Eph receptor-interacting proteins, ephrin)被统称为Eph家族蛋白,通过独特的双向信号传递在调控正常学习和记忆中扮演重要角色。近年大量的研究发现,Eph家族蛋白在多种神经精神疾病中发挥复杂而又重要的作用,主要是通过改变突触效能,参与神经元形态发生和调控基因表达等方式影响上述疾病的进程。然而,目前靶向Eph家族蛋白对阿尔茨海默症(Alzheimer’s disease, AD)、焦虑症及恐惧症等疾病进行治疗的研究却为数甚微。同时,单纯以β样淀粉蛋白为靶点的抗AD药物开发均遭遇瓶颈。因此,探索Eph家族蛋白在上述疾病中的具体作用变得十分迫切。本文综述了Eph家族蛋白在AD、焦虑症和恐惧症中的最新研究进展,旨在为靶向Eph家族蛋白治疗相关疾病提供新的思路。     

Study of nicotinic acetylcholine receptors on cultured antennal lobe neurones from adult honeybee brains

In insects, acetylcholine (ACh) is the main neurotransmitter, and nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee, nAChRs are expressed in diverse structures including the primary olfactory centres of the brain, the antennal lobes (AL) and the mushroom bodies. Whole-cell, voltage-clamp recordings were used to characterize the nAChRs present on cultured AL cells from adult honeybee, Apis mellifera. In 90% of the cells, applications of ACh induced fast inward currents that desensitized slowly. The classical nicotinic agonists nicotine and imidacloprid elicited respectively 45 and 43% of the maximum ACh-induced currents. The ACh-elicited currents were blocked by nicotinic antagonists methyllycaconitine, dihydroxy-β-erythroidine and α-bungarotoxin. The nAChRs on adult AL cells are cation permeable channels. Our data indicate the existence of functional nAChRs on adult AL cells that differ from nAChRs on pupal Kenyon cells from mushroom bodies by their pharmacological profile and ionic permeability, suggesting that these receptors could be implicated in different functions.     

An investigation of the role of dopamine in the antennal lobes of the honeybee,Apis mellifera

Summary The effects of dopamine applied to the antennal lobes of the honeybee are investigated using the proboscis conditioning paradigm (Kuwabara 1957). The percentage of bees responding to a conditioned olfactory stimulus after a single conditioning trial is reduced significantly by the application of dopamine (10^–6 M) to the antennal lobes of the bee brain. Reduction in response levels is significantly smaller in bees conditioned to the olfactory stimulus in multiple conditioning trials prior to treatment with dopamine. The effects of dopamine on the percentage of bees responding to a conditioned olfactory stimulus are blocked by the butyrophenone, haloperidol (10^–5 M). The possible role of dopaminergic interneurones in the antennal lobes of the bee brain is discussed.     

Encoding of plant odour information in insects: peripheral and central mechanisms

Insects are suitable model organisms for studying mechanisms underlying olfactory coding and olfactory learning, by their unique adaptation to host plants in which the chemical senses are essential. Recent molecular biological studies have shown that a large number of genes in insects and other organisms are coding for olfactory receptor proteins. In general, one receptor type seems to be expressed in each neurone. The functional characterisations of olfactory receptor neurones have been extensive in certain insect species, demonstrating a fine-tuning of single neurones to biologically relevant odourants; both insect and plant produced volatiles. Stained neurones of the same functional type have been shown to project in one and the same glomerular unit in the primary olfactory centre, the antennal lobe. This corresponds to molecular biological studies, showing projections in one glomerulus by neurones expressing the same receptor type. Comparison of these findings with physiological and morphological characterisations of antennal lobe neurones has indicated correspondence between input and output of the glomerular units. Examples are presented from studies of heliothine moths. From the antennal lobe, the olfactory information is further conveyed to the mushroom bodies, particularly important for learning, and the lateral protocerebrum, a premotoric area. The three brain areas are regions of synaptic plasticity important in learning of odours, which is well studied in the honeybee but also in species of moths.     

A metallic impregnation technique adapted to study the honeybee Apis mellifera L. brain

In order to visualize the distribution pattern of the neuronal bodies and neurofibrils in the honeybee brain, we adapted a metallic impregnation technique first described for vertebrate nervous system by Ramón y Cajal. The honeybee brain constitution plays a key role in the development of learning and memory capacities. The general characteristics observed in the honeybee brain, stained by metallic impregnation, revealed its anatomical and morphological constitution in agreement with studies of other insect brains using different techniques. Metallic impregnation evidenced the optic lobe neuropils, the ocelli fiber cells, the neuron extensions of the calyces, and the axon bundles that involve the antennal glomeruli, as well as the neuron extensions in the alpha lobe. We also observed that the antennal glomeruli were mainly formed by fibers. The optical lobes were impregnated distinctly in the monopolar neuron bodies and in the fibers. In the mushroom bodies, we observed the lip, collar and calyx basal areas. Based on our results, the metallic impregnation technique is effective to visualize neuronal bodies and neurofibrils; moreover, is simpler and faster than other techniques, offering new insights for the investigation of the invertebrate nervous system.     

Multisensory convergence in the mushroom bodies of ants and bees

The mushroom bodies, central neuropils in the arthropod brain, are involved in learning and memory and in the control of complex behavior. In most insects, the mushroom bodies receive direct olfactory input in their calyx region. In Hymenoptera, olfactory input is layered in the calyx. In ants, several layers can be discriminated that correspond to different clusters of glomeruli in the antennal lobes, perhaps corresponding to different classes of odors. Only in Hymenoptera, the mushroom body calyx also receives direct visual input from the optic lobes. In bees, six calycal layers receive input from different classes of visual interneurons, probably representing different parts of the visual field and different visual properties. Taken together, the mushroom bodies receive distinct multisensory information in many segregated input layers.     

Decreased maternal behavior and anxiety in ephrin‐A5−/− mice

During development of the nervous system, molecular signals mediating cell–cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin‐A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin‐A5 ^?/? mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin‐A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.     

Localization of short-term memory in the brain of the bee, Apis mellifera

ABSTRACT. Fixed honey-bees were conditioned to a scent in a one-trial learning paradigm. In contrast to free-flying colour-conditioned bees, fixed scent-conditioned bees do not show a biphasic time dependence of the conditioned response. Small metal probes were used to cool localized areas of the antennal lobes, alpha-lobes, and calyces of the mushroom bodies of the brain at various times after conditioning. Localized cooling impaired the formation of memory in all three structures. The susceptibility to impairment after conditioning lasted approximately 3 min in the antennal lobes, 7 min in the alpha-lobes, and 10 min in the calyx area. It was possible to determine the influence of the contralateral hemisphere (relative to the learning antenna) by conditioning bees with only one antenna. No contralateral impairment was found in the antennal lobes; there were minor effects in the alphalobes; contralateral cooling led to reductions of the conditioned response only in the calyx area. The temperature dependence of memory impairment was different for the antennal lobes and the mushroom bodies (alpha-lobes and calyces). The latter were most sensitive to cooling at 5°C. No correlation between cooling duration and impairment of memory was found in the antennal lobes, but there was a linear relation between impairment and cooling duration in the alpha-lobes. Brief cooling (5 or 10 s) resulted in significant impairment of memory formation only in the calyx area. A series of control experiments proved that the impairment of memory is due to a reversible block of neural activity. It was possible to show that the impairment is specific for the three neural structures analysed, by cooling the lobula of the optic system at various times after conditioning. Lesions of the brain or application of KCl also resulted in time-dependent reductions of the conditioned response. Cooling the entire animal at various times after conditioning led to similar memory impairment to that resulting from localized cooling of the alphalobes.     

Neuromechanism Study of Insect–Machine Interface: Flight Control by Neural Electrical Stimulation

The insect–machine interface (IMI) is a novel approach developed for man-made air vehicles, which directly controls insect flight by either neuromuscular or neural stimulation. In our previous study of IMI, we induced flight initiation and cessation reproducibly in restrained honeybees (Apis mellifera L.) via electrical stimulation of the bilateral optic lobes. To explore the neuromechanism underlying IMI, we applied electrical stimulation to seven subregions of the honeybee brain with the aid of a new method for localizing brain regions. Results showed that the success rate for initiating honeybee flight decreased in the order: α-lobe (or β-lobe), ellipsoid body, lobula, medulla and antennal lobe. Based on a comparison with other neurobiological studies in honeybees, we propose that there is a cluster of descending neurons in the honeybee brain that transmits neural excitation from stimulated brain areas to the thoracic ganglia, leading to flight behavior. This neural circuit may involve the higher-order integration center, the primary visual processing center and the suboesophageal ganglion, which is also associated with a possible learning and memory pathway. By pharmacologically manipulating the electrically stimulated honeybee brain, we have shown that octopamine, rather than dopamine, serotonin and acetylcholine, plays a part in the circuit underlying electrically elicited honeybee flight. Our study presents a new brain stimulation protocol for the honeybee–machine interface and has solved one of the questions with regard to understanding which functional divisions of the insect brain participate in flight control. It will support further studies to uncover the involved neurons inside specific brain areas and to test the hypothesized involvement of a visual learning and memory pathway in IMI flight control.     

Physiology and morphology of olfactory neurons associating with the protocerebral lobe of the honeybee brain

Physiology and morphology of olfactory neurons associated with the protocerebral lobe around the alpha-lobe of the mushroom body were studied in the brain of the honeybee Apis mellifera using intracellular recording and staining techniques. The responses of neurons to behaviorally relevant odorants (a blend, and components of the Nasonov pheromone, and some other non-pheromonal odors) were recorded. Different response patterns were observed within different neurons, and often within the same neuron, in response to different stimuli. All the neurons stained had innervations in the protocerebral lobe. The cell profiles varied from cells connecting the antennal lobe with both the protocerebral and lateral protocerebral lobes (projection neurons), cells linking the pedunculus of the mushroom body with both the protocerebral and lateral protocerebral lobes (PE1 neurons), cells linking the alpha-lobe and protocerebral lobe with the calyces of the mushroom body (feedback neurons), and cells linking the alpha-lobe and protocerebral lobe with the antennal lobe (recurrent neurons), to cells connecting the protocerebral lobe with the contralateral protocerebrum (bilateral neurons). These findings suggest that the protocerebral lobe acts as an olfactory center associating with other centers, and provides multi-layered recurrent networks within the protocerebrum and between the deutocerebrum and the protocerebrum in honeybee olfactory pathways.     

Distribution of APGWamide-immunoreactivity in the brain and reproductive organs of adult Pygmy squid, Idiosepius pygmaeus

The neuropeptide APGWamide is involved in the control of the reproductive behavior in molluscs. Using immunocytochemistry, we investigated the distribution of APGWa-immunoreactive neurons in the brain and reproductive organs of adult male and female specimen of Idiosepius pygmaeus. The study showed that the APGWamide-immunoreactive neurons and fibers are localized in the dorsal basal and vertical lobes of the supraesophageal mass, the palliovisceral lobe of the posterior subesophageal mass and olfactory lobe of the optic tract in male brains, with the highest number of APGWamide-immunoreactive neurons in the palliovisceral and olfactory lobes. In females, only the palliovisceral and olfactory lobes contained APGWa-immunoreactive neurons. The number of APGWamide-immunoreactive neurons in male I. pygmaeus brain is significantly higher than in females. Furthermore, APGWamide-immunoreactive fibers are localized exclusively in male reproductive organs and mantle muscles. Together these data suggest a role for APGW-amide in the control of male reproduction.     

Eph receptors and neural plasticity

Eph receptor tyrosine kinases are largely known for their involvement in brain development but, as some of these receptor tyrosine kinases are also expressed in adults, their possible role in the mature nervous system has begun to be explored. Evidence for the involvement of Eph receptors in synaptic plasticity, learning and memory is only emerging and needs corroboration. However, it is likely that the actions of Eph kinases in the adult brain will attract significant attention and become a fertile research area, as occurred in the case of the neurotrophins.     

Conservation of novel <Emphasis Type="Italic">Mahya</Emphasis> genes shows the existence of neural functions common between Hymenoptera and Deuterostome

Honeybees have been shown to exhibit cognitive performances that were thought to be specific to some vertebrates. However, the molecular and cellular mechanisms of such cognitive abilities of the bees have not been understood. We have identified a novel gene, Mahya, expressed in the brain of the honeybee, Apis mellifera, and other Hymenoptera. Mahya orthologues are present in Deuterostomes but are absent or highly diverged in nematodes and, intriguingly, in two dipteran insects (fruit fly and mosquito) and Lepidoptera (silk moth). Mahya genes encode novel secretory proteins with a follistatin-like domain (Kazal-type serine/threonine protease inhibitor domain and EF-hand calcium-binding domain), two immunoglobulin domains, and a C-terminal novel domain. Honeybee Mahya is expressed in the mushroom bodies and antennal lobes of the brain. Zebra fish Mahya orthologues are expressed in the olfactory bulb, telencephalon, habenula, optic tectum, and cerebellum of the brain. Mouse Mahya orthologues are expressed in the olfactory bulb, hippocampus, and cerebellum of the brain. These results suggest that Mahya may be involved in learning and memory and in processing of sensory information in Hymenoptera and vertebrates. Furthermore, the limited existence of Mahya in the genomes of Hymenoptera and Deuterostomes supports the hypothesis that the genes typically represented by Mahya were lost or highly diverged during the evolution of the central nervous system of specific Bilaterian branches under the specific selection and subsequent adaptation associated with different ecologies and life histories.     

Identification of proteins whose expression is up- or down-regulated in the mushroom bodies in the honeybee brain using proteomics

To identify protein(s) with different expression patterns in the mushroom bodies (MBs) in the honeybee brain, we compared the protein profiles of MBs and optic lobes (OLs) using proteomics. Two-dimensional gel electrophoresis revealed that five and three spots were selectively expressed in the MBs or OLs, respectively. Liquid chromatography tandem mass spectrometry analysis identified juvenile hormone diol kinase and glyceraldehyde-3-phosphate dehydrogenase as MB- and OL-selective proteins, respectively. In situ hybridization revealed that jhdk expression was upregulated in MB neuron subsets, whereas gapdh expression was downregulated, indicating that MBs have a distinct gene and protein expression profile in the honeybee brain.     

Expression and function of the Eph A receptors and their ligands ephrins A in the rat thymus

Thymus development and function are dependent on the definition of different and graded microenvironments that provide the maturing T cell with the different signals that drive its maturation to a functional T lymphocyte. In these processes, cell-cell interactions, cell migration, and positioning are clues for the correct functioning of the organ. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, has been implicated in all these processes by regulating cytoskeleton and adhesion functioning, but a systemic analysis of their presence and possible functional role in thymus has not yet been conducted. In this regard, the current study combines different experimental approaches for analyzing the expression of four members of the Eph A family and their ligands, ephrins A, in the embryonic and adult rat thymus. The patterns of Eph and ephrin expression in the distinct thymic regions were different but overlapping. In general, the studied Eph A were expressed on thymic epithelial cells, whereas ephrins A seem to be more restricted to thymocytes, although Eph A1 and ephrin A1 are expressed on both cell types. Furthermore, the supply of either Eph A-Fc or ephrin A-Fc fusion proteins to fetal thymus organ cultures interferes with T cell development, suggesting an important role for this family of proteins in the cell mechanisms that drive intrathymic T cell development.     

Acetylcholine,GABA and glutamate induce ionic currents in cultured antennal lobe neurons of the honeybee, <Emphasis Type="Italic">Apis mellifera</Emphasis>

The honeybee, Apis mellifera, is a valuable model system for the study of olfactory coding and its learning and memory capabilities. In order to understand the synaptic organisation of olfactory information processing, the transmitter receptors of the antennal lobe need to be characterized. Using whole-cell patch-clamp recordings, we analysed the ligand-gated ionic currents of antennal lobe neurons in primary cell culture. Pressure applications of acetylcholine (ACh), γ-amino butyric acid (GABA) or glutamate induced rapidly activating ionic currents. The ACh-induced current flows through a cation-selective ionotropic receptor with a nicotinic profile. The ACh-induced current is partially blocked by α-bungarotoxin. Epibatidine and imidacloprid are partial agonists. Our data indicate the existence of an ionotropic GABA receptor which is permeable to chloride ions and sensitive to picrotoxin (PTX) and the insecticide fipronil. We also identified the existence of a chloride current activated by pressure applications of glutamate. The glutamate-induced current is sensitive to PTX. Thus, within the honeybee antennal lobe, an excitatory cholinergic transmitter system and two inhibitory networks that use GABA or glutamate as their neurotransmitter were identified.     

Prepro-tachykinin gene expression in the brain of the honeybee<Emphasis Type="Italic"> Apis mellifera</Emphasis>

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization. The prepro-AmTRP gene is expressed predominantly in the MBs and in some neurons located in the optic and antennal lobes. cDNA microarray studies have revealed that AmTRP expression is enriched in the MBs compared with other brain regions. There is no difference in AmTRP-expressing cells among worker, queen, and drone brains, suggesting that the cell types that express the prepro-AmTRP gene do not change according to division of labor, sex, or caste. The unique expression pattern of the prepro-AmTRP gene suggests that AmTRPs function as neuromodulators in the MBs of the honeybee brain.This work was supported by a Grant-in-Aid from the Bio-oriented Technology Research Advancement Institution (BRAIN)