Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

Morphological changes of the optic lobe from late embryonic to adult stages in oval squids Sepioteuthis lessoniana

The optic lobe is the largest brain area within the central nervous system of cephalopods and it plays important roles in the processing of visual information, the regulation of body patterning, and locomotive behavior. The oval squid Sepioteuthis lessoniana has relatively large optic lobes that are responsible for visual communication via dynamic body patterning. It has been observed that the visual behaviors of oval squids change as the animals mature, yet little is known about how the structure of the optic lobes changes during development. The aim of the present study was to characterize the ontogenetic changes in neural organization of the optic lobes of S. lessoniana from late embryonic stage to adulthood. Magnetic resonance imaging and micro‐CT scans were acquired to reconstruct the 3D‐structure of the optic lobes and examine the external morphology at different developmental stages. In addition, optic lobe slices with nuclear staining were used to reveal changes in the internal morphology throughout development. As oval squids mature, the proportion of the brain making up the optic lobes increases continuously, and the optic lobes appear to have a prominent dent on the ventrolateral side. Inside the optic lobe, the cortex and the medulla expand steadily from the late embryonic stage to adulthood, but the cell islands in the tangential zone of the optic lobe decrease continuously in parallel. Interestingly, the size of the nuclei of cells within the medulla of the optic lobe increases throughout development. These findings suggest that the optic lobe undergoes continuous external morphological change and internal neural reorganization throughout the oval squid's development. These morphological changes in the optic lobe are likely to be responsible for changes in the visuomotor behavior of oval squids from hatching to adulthood.     

Immunocytochemistry of a novel GABA receptor subunitRdl inDrosophila melanogaster

Following our recent cloning of a novel γ-aminobutyric acid (GABA) receptor subunit geneResistance to dieldrin orRdl from the cyclodiene resistance locus inDrosophila melanogaster, we were interested in defining its pattern of expression during development. Here we report the raising of an anti-Rdl polyclonal antibody that recognizes a single protein of the expected 65 kDa size in immunoblots ofDrosophila head homogenates.In situ hybridization usingRdl cDNA probes and the anti-Rdl antibody shows thatRdl message and protein are highly expressed in the developing central nervous system (CNS) of 15–17 h embryos. Interestingly, despite the use of GABA in both the peripheral and CNS of insects,Rdl GABA receptor subunits appear to be confined to the CNS. Detailed immunocytochemistry ofDrosophila brain sections showed particularly strong anti-Rdl antibody staining in the optic lobes, ellipsoid body, fan shaped body, ventrolateral protocerebrum and the glomeruli of the antennal lobes. Results are compared with the distribution of staining observed in the insect CNS with antibodies against GABA itself and synaptotagmin, a synaptic vesicle protein.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Analysis of neural elements in head-mutant Drosophila embryos suggests segmental origin of the optic lobes

We describe the development of 20 sensory organs in the embryonic Drosophila head, which give rise to 7 sensory nerves of the peripheral nervous system (PNS), and 4 ganglia of the stomatogastric nervous system (SNS). Using these neural elements and the optic lobes as well as expression domains of the segment polarity gene engrailed in the wild-type head of Drosophila embryos as markers we examined the phenotype of different mutants which lack various and distinct portions of the embryonic head. In the mutants, distinct neural elements and engrailed expression domains, serving as segmental markers, are deleted. These mutants also affect the optic lobes to various degrees. Our results suggest that the optic lobes are of segmental origin and that they derive from the ocular segment anteriorly adjacent to the antennal segment of the developing head.     

Immunocytochemical demonstration of S-antigen (arrestin) in the brain of the blowfly Calliphora vicina

S-Antigen (arrestin)-immunoreaction can be considered as a marker for retinal and extraretinal photoreceptors in both vertebrate and invertebrate species. The present immunocytochemical study with the blowfly Calliphora vicina revealed S-antigen immunoreaction in retinal photoreceptors and various groups of neurons bilaterally distributed in the optic lobes and in the proto-, deuto- and tritocerebrum. S-Antigen-immunoreactive processes and terminal formations were found in the lower division of the central body complex and in the neuropil of the mushroom body. Also neuropil regions of the optic lobe, the lamina, medulla and lobula displayed S-antigen-immunoreactive fibers which were arranged in different patterns. These immunocytochemical data suggest that extraocular photoreceptors may be located in various parts of the blowfly brain. They provide a structural basis for further experiments which are needed to identify definitely these elements as extraretinal photoreceptors.     

Queen Dominance May Reduce Worker Mushroom Body Size in a Social Bee

The mushroom body (MB) is an area of the insect brain involved in learning, memory, and sensory integration. Here, we used the sweat bee Megalopta genalis (Halictidae) to test for differences between queens and workers in the volume of the MB calyces. We used confocal microscopy to measure the volume of the whole brain, MB calyces, optic lobes, and antennal lobes of queens and workers. Queens had larger brains, larger MB calyces, and a larger MB calyces:whole brain ratio than workers, suggesting an effect of social dominance in brain development. This could result from social interactions leading to smaller worker MBs, or larger queen MBs. It could also result from other factors, such as differences in age or sensory experience. To test these explanations, we next compared queens and workers to other groups. We compared newly emerged bees, bees reared in isolation for 10 days, bees initiating new observation nests, and bees initiating new natural nests collected from the field to queens and workers. Queens did not differ from these other groups. We suggest that the effects of queen dominance over workers, rather than differences in age, experience, or reproductive status, are responsible for the queen–worker differences we observed. Worker MB development may be affected by queen aggression directly and/or manipulation of larval nutrition, which is provisioned by the queen. We found no consistent differences in the size of antennal lobes or optic lobes associated with differences in age, experience, reproductive status, or social caste.     

Manipulating the light/dark cycle: effects on dopamine levels in optic lobes of the honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) brain

This study examines the relationship between cyclical variations in optic-lobe dopamine levels and the circadian behavioural rhythmicity exhibited by forager bees. Our results show that changing the light–dark regimen to which bees are exposed has a significant impact not only on forager behaviour, but also on the levels of dopamine that can be detected in the optic lobes of the brain. Consistent with earlier reports, we show that foraging behaviour exhibits properties characteristic of a circadian rhythm. Foraging activity is entrained by daily light cycles to periods close to 24 h, it changes predictably in response to phase shifts in light, and it is able to free-run under constant conditions. Dopamine levels in the optic lobes also undergo cyclical variations, and fluctuations in endogenous dopamine levels are influenced significantly by alterations to the light/dark cycle. However, the time course of these changes is markedly different from changes observed at a behavioural level. No direct correlation could be identified between levels of dopamine in the optic lobes and circadian rhythmic activity of the honey bee.     

吡虫啉对意大利蜜蜂脑乙酰胆碱受体分布的影响

蜜蜂是自然界主要的授粉昆虫; 新烟碱类杀虫剂(neonicotinoid insecticide)通过结合害虫体内乙酰胆碱受体（nAChR）使害虫致死, 是目前广泛用于田间害虫防控的杀虫剂。本研究以意大利蜜蜂Apis mellifera ligustica和新烟碱类杀虫剂的代表品种吡虫啉为材料, 应用免疫组织化学的方法, 研究了正常成年蜜蜂脑内蘑菇体及视叶nAChR-α7的表达和分布; 分析了亚致死剂量新烟碱类杀虫剂吡虫啉对nAChR-α7表达和分布的影响。结果表明, nAChR-α7在正常蜜蜂脑蘑菇体和视叶中均可检测到, 在蘑菇体中分布相对较少, 但在视叶分布丰富。吡虫啉对nAChR-α7在视叶的表达和分布有显著抑制作用, 但对蘑菇体nAChR-α7的表达没有显著影响。结果提示, 新烟碱类杀虫剂吡虫啉除了文献报道的抑制nAChR的表达外, 还能抑制nAChR-α7的表达量, 这是新烟碱类杀虫剂作用机制的新发现。     

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.     

Ethanol Alters Brain Phospholipid Levels Which Correlate with Altered Brain Morphology

The effects of embryonic exposure on brain phospholipid levels were studied by injecting various concentrations of ethanol into fertile chicken eggs at 0 days of development. At 18 days of development, the levels of total phospholipids and various phospholipid classes were assayed in brain tissue and correlated to neuron densities within the cerebral hemispheres and the optic lobes. Although ethanol concentrations ranging from 0 to 3700 μm/Kg egg wt. failed to influence either total brain weight or total brain phospholipid levels, ethanol-induced changes in the levels of individual phospholipid classes were observed. When injected with 7 μm of ethanol/Kg egg wt., a 2- to 3-fold increase in brain phosphatidylethanolamine (PE) levels were observed with reduced levels of brain phosphatidylcholine (PC) and brain sphingomyelin (SP). When injected with 74 μm of ethanol/Kg egg wt., ethanol-induced increases in brain phosphatidylserine (PS) and PE were observed with ethanol-induced decreases in brain PC and SP. Cell fractionation studies demonstrated ethanol-induced increases in brain PE and PS and ethanol-induced decreases in brain PC and SP in nuclear, mitochondrial, and microsomal membranes. These ethanol-induced alterations in brain phospholipid profiles correlated with ethanol-induced reductions in neuron densities within the cerebral hemispheres and optic lobes.     

Expression of UV-, blue-, long-wavelength-sensitive opsins and melatonin in extraretinal photoreceptors of the optic lobes of hawkmoths

Lepidopterans display biological rhythms associated with egg laying, eclosion and flight activity but the photoreceptors that mediate these behavioural patterns are largely unknown. To further our progress in identifying candidate light-input channels for the lepidopteran circadian system, we have developed polyclonal antibodies against ultraviolet (UV)-, blue- and extraretinal long-wavelength (LW)-sensitive opsins and examined opsin immunoreactivity in the adult optic lobes of four hawkmoths, Manduca sexta, Acherontia atropos, Agrius convolvuli and Hippotion celerio. Outside the retina, UV and blue opsin protein expression is restricted to the adult stemmata, with no apparent expression elsewhere in the brain. Melatonin, which is known to have a seasonal influence on reproduction and behaviour, is expressed with opsins in adult stemmata together with visual arrestin and chaoptin. By contrast, the LW opsin protein is not expressed in the retina or stemmata but rather exhibits a distinct and widespread distribution in dorsal and ventral neurons of the optic lobes. The lamina, medulla, lobula and lobula plate, accessory medulla and adjacent neurons innervating this structure also exhibit strong LW opsin immunoreactivity. Together with the adult stemmata, these neurons appear to be functional photoreceptors, as visual arrestin, chaoptin and melatonin are also co-expressed with LW opsin. These findings are the first to suggest a role for three spectrally distinct classes of opsin in the extraretinal detection of changes in ambient light and to show melatonin-mediated neuroendocrine output in the entrainment of sphingid moth circadian and/or photoperiodic rhythms.This work was partially supported by the Canadian Institute for Advanced Research (A.D.B.) and the National Science Foundation (grant nos. IBN-0082700 and IBN-0346765; A.D.B.).     

Distribution and levels of dopamine and its metabolites in brains of reproductive workers in honeybees

To explore the role of dopamine and its metabolites for change of reproductive states of workers in honeybees (Apis mellifera), brain levels of dopamine relative substances were measured and localized in both normal workers and queenless workers. Dopamine and two possible metabolites of dopamine, N-acetyldopamine (NADA) and norepinephrine were detected in brain extracts. The brain levels of dopamine, NADA and norepinephrine were positively correlated with ovary development. Individuals with high dopamine levels had high levels of NADA or norepinephrine, suggesting that these metabolites might be involved in the change of reproductive sates of workers. Dopamine was distributed mainly in the protocerebrum, whereas NADA was in both the optic lobes and the protocerebrum. Dopamine levels in each distinct brain regions were higher in queenless workers than in normal workers, whereas there was a higher NADA level in the optic lobes in queenless workers than in normal workers. These results suggest that dopamine might be stored and/or released around the protocerebrum and the deutocerebrum, and also diffuse to the optic lobes where dopamine secretory cells are absent, resulting in high NADA levels in the optic lobes. The different manner of level changes of dopamine and its metabolites in each brain region might cause compound behavioural modulations in reproductive workers.     

Differential sensitivity of cholinergic and GABAergic neurons in chick embryos treated intracerebrally with ethanol at 8 days of embryonic age

We have shown that in embryos treated with ethanol in ovo during days 1–3, a critical period of neuroembryogenesis, cholinergic neuronal phenotypic expression is decreased whereas GABAergic and catecholaminergic neuronal populations are increased as assessed by neuronal markers choline acetyltransferse (ChAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) respectively. In this study, ethanol was administered intracerebrally to embryos at embryonic day 8, embryos were sacrificed at day 9 and ChAT and GAD activities assayed separately in cerebral hemispheres and remaining brain (diencephalon-midbrain and optic lobes). We found that ChAT activity was enhanced in the cerebral hemispheres only, whereas GAD activity was decreased in both cerebral hemispheres and remaining brain. We have concluded that the differential responses of neuronal phenotypes to ethanol may reflect compensatory mechanisms to ethanol insult. Moreover, these findings emphasize the vulnerability of the GABAergic neuronal phenotypes to ethanol neurotoxicity during early brain development in the chick.     

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)     

Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier