Changes in brain amine levels associated with the morphological and behavioural development of the worker honeybee

Summary Changes in biogenic amine levels associated with the morphological and behavioural development of the worker honeybee are examined.A significant increase in amine levels in the head of the honeybee is associated with transition from the larval to pupal stage. Adult emergence is also accompanied by a significant increase in 5-HT levels in the brain, but no significant change in brain dopamine (DA) levels. NADA (N-acetyldopamine) levels increase during larval and pupal development, but in contrast to both DA and 5-HT, drop significantly during the transition from pupa to adult.Levels of DA in the brain of nectar and pollen forager bees, presumed to be among the oldest adults sampled, were found to be significantly higher than in nurses, undertakers or food storers. These results suggest that an age-dependent change in amine levels occurs in the brain of the worker bee. In the optic lobes, levels of DA and 5-HT were found to be significantly higher in pollen forager bees than in all other behavioural groups. Significant differences in amine levels in the optic lobes of nectar foragers and pollen foragers indicate that some differences in amine levels occur independent of worker age. The functional significance of differences in brain amine levels and whether or not biogenic amines play a direct role in the control of honeybee behaviour has yet to be established.Abbreviations DA dopamine - 5-HT 5-hydroxytryptamine or serotonin - NADA N-acetyldopamine     

Brain tyramine and reproductive states of workers in honeybees

To explore the role of tyramine in the transformation of reproductive states of honeybee workers, brain levels of tyramine and N-acetyltyramine were measured in both normal and queenless workers. Queenless workers had higher tyramine levels and lower N-acetyltyramine levels than normal workers did. Intermediate reproductive workers that were transferred into a normal colony from a queenless colony had intermediate levels of tyramine and N-acetyltyramine. Elevation of tyramine in the queenless workers occurred at an earlier adult stage than elevation of dopamine. Tyramine levels in intermediate reproductive workers returned to the levels seen in normal workers, but dopamine levels in intermediate reproductive workers remained elevated at the same level as in queenless workers. Thus, brain tyramine may be regulated by the colony condition with or without a queen. Injection of an amine uptake inhibitor, reserpine, depleted tyramine and elevated N-acetyltyramine. Distributions of tyramine and dopamine within the brain were distinctively different, whereas distributions of N-acetyltyramine and N-acetyldopamine were similar, suggesting that each functional amine is stored in specific neurosecretory cells and released to the relevant receptor sites but that metabolism into each N-acetylmetabolite is determined by diffusion.     

Analysis of the conserved neurotrophic factor MANF in the Drosophila adult brain

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor that supports and protects dopaminergic neurons. The Drosophila MANF (DmMANF) null mutant animals die during early development, and DmMANF is required for the maintenance of dopamine positive neurites. The aim of this study was to investigate the role of DmMANF during later developmental stages. Here we report that DmMANF expression in the adult brain is much wider than in the embryonic and larval stages. It is expressed in both glia and neurons including dopaminergic neurons. Clonal analysis showed that DmMANF is not required cell-autonomously for the differentiation of either glia or dopaminergic neurons. In addition, DmMANF overexpression resulted in no apparent abnormal dopaminergic phenotype while DmMANF silencing in glia resulted in prolonged larval stage.     

Age-related changes in dopamine receptor densities in the brain of the honey bee, Apis mellifera

Changes in the levels of binding of ³H-SCH-23390, a vertebrate D1 dopamine receptor ligand, and ³H-spiperone, a vertebrate D2 dopamine receptor ligand were investigated in the brain of the worker honey bee during metamorphic adult development and during the lifetime of the adult bee. Age-related fluctuations in binding levels were markedly different for these two ligands. ³H-SCH-23390 and ³H-spiperone binding sites were present at low levels during metamorphic adult development. After adult emergence, however, ³H-SCH-23390 binding levels, in contrast to those of ³H-spiperone, increased significantly. Within the first 48 h of adult life ³H-SCH-23390 binding reached a level not significantly different from that detected in forager bees. No significant fluctuations in the levels of ³H-spiperone binding were observed during the adult lifetime of the bee. Measurements of dopamine levels in the brains of pupal and adult bees revealed no direct correlation between fluctuations in endogenous amine levels and the amount of binding of either ³H-SCH-23390 or ³H-spiperone. These results provide evidence for subtype-specific patterns of expression of dopamine receptors in the insect brain and show that D1- and D2-like receptors are expressed not only in the adult CNS, but also in the developing brain of the bee. Accepted: 4 June 1997     

Octopamine- and dopamine-sensitive adenylate cyclase in the brain ofLocusta migratoria during its development

Octopamine- and dopamine-sensitive adenylate cyclases were studied in the brain of Locusta migratoria during its metamorphosis. In the adult brain the effects of octopamine and dopamine on adenylate cyclase were additive, suggesting the presence of separate populations of adenylate cyclase-linked receptors for octopamine and dopamine. There are no separate receptors for noradrenaline. Octopamine stimulates adenylate cyclase in both adult and larval brain; however, in adult brain octopamine is more potent than in larval brain. Dopamine stimulates adenylate cyclase activity only in adult brain. The sensitivity of adenylate cyclase to octopamine changes during the development of the animal. Phentolamine and cyproheptadine are potent antagonists of octopamine-stimulated adenylate cyclase, while propranolol has a weak effect. No cytosol factor which would modulate either basal or octopamine-stimulated adenylate cyclase was found. The effect of GTP and octopamine on adenylate cyclase was synergistic in adult brain but not in larval brain, while the effect of GppNHp and octopamine was synergistic in both adult and larval brains.     

Morphological and biochemical characterizations of the great scallop Pecten maximus metamorphosis

In order to characterize Pecten maximus metamorphosis within a hatchery environment, the relationships existing among the various larval rearing parameters, the biochemical composition of the larvae and metamorphosis have been determined. Metamorphosis levels are correlated with the percentages of double ring larvae, as well as with the larval lipid content. A multiple regression incorporating the percentage of double ring larvae and larval lipid content shows that these two combined parameters explain 50 % of the total metamorphosis variance, with an equal relative importance for each of them. In an attempt to identify other possible endogenous markers, the kinetics of biogenic amines were also examined throughout larval and post-larval development. A steady increase in serotonin and dopamine levels was recorded during larval development while a sudden decrease in both molecules was noted during metamorphosis. It is suggested that these two amines may be used as indicators of larval competence for P. maximus metamorphosis.     

N-β-alanyldopamine and N-acetyldopamine occurrence and synthesis in the central nervous system of Manduca sexta (L.)

N-β-Alanyldopamine (NBAD), N-acetyldopamine (NADA), dopamine (DA), and 3,4-dihydroxyphenylalanine (DOPA) were detected in the brain and ganglia of the central nervous system of larval and adult tobacco hornworms, Manduca sexta, by reversed phase HPLC with electrochemical detection. NBAD predominated in larval neural tissue during development of the fifth instar and increased to peak concentrations of 936, 650 and 263 nmol g⁻¹ in the abdominal ganglia, subesophageal plus thoracic ganglia and brain, respectively, at the wandering stage of development. Concentrations of all catecholamines decreased in the pharate pupa and were generally lowest in the adult nerve cord. DA was the second most abundant catecholamine in larval ganglia, but the primary catecholamine in adult ganglia. Relatively high levels of DOPA also occurred in the ganglia of wandering larvae but not at other times. NADA was detected only in the abdominal ganglia of day 3 larvae. N-Acyltransferases that catalyze synthesis of NBAD and NADA from DA also were present in abdominal ganglia, as demonstrated by analysis of in vitro cultures in which exogenous DA stimulated synthesis of both N-acylated catecholamines. Maximal NBAD synthesis occurred in ganglia removed from wandering stage larvae (9.3 nmol g⁻¹ day⁻¹), whereas NADA synthesis was highest in ganglia from pharate pupae (7.3 nmol g⁻¹ day⁻¹). Thus, N-β-alanylation and N-acetylation are competing metabolic reactions for DA in the hornworm's nervous system. The role played by the N-acylated catecholamines in M. sexta neurophysiology is unknown, but these compounds may be storage or inactive forms of the putative neurotransmitter DA.     

Effect of various insecticides on the larval growth and biogenic amine levels of Tribolium castaneum Herbst

Various insecticides reduced larval growth of the red flour beetle (tribolium castaneum Herbst) and various biogenic amines, including octopamine (OA), dopamine (DA), serotonin (5-HT), epinephrine (E), norepinephrine (NE), their precursors and metabolites in the insects were measured by high-performance liquid chromatography coupled with electrochemical detection. Tyrosine occurred in the highest concentration followed by OA, tryptophan and 3,4-dihydroxymandelic acid (DOMA). Tyramine (a precursor of OA in the biosynthetic pathway), synephrine (N-methyl OA), DA, 5-HT, E, NE and their related substances occurred in extremely low quantities compared with OA. The insects were stressed by various insecticides, which resulted in a dramatic change of biogenic amine levels: the OA levels increased, whereas the levels of other biogenic amines and related substances decreased.     

Developmental Changes in Biogenic Amine Levels in the Central Nervous System and the Haemolymph of the Eastern Death's Head Hawk Moth,Acherontia styx (Lepidoptera: Sphingidae)

In an effort to understand the role of biogenic amines in insect development, changes in the levels of octopamine (OA), dopamine (DA), epinephrine (E), norepinephrine (NE), and serotonin (5-HT) in the brain, the optic lobes and the haemolymph of different developmental stages of Acherontia styx were analyzed using HPLC with electrochemical detector. In the brain, OA was the most abundant monoamine. DA, OA, and E levels in larvae peaked around the wandering stage (W). A dramatic increase in DA, 5-HT, and E levels was observed in the brain of the adult as compared to the pupal stage. NE, however, was not detected in the brain of most stages of the insect, except in the brain of 20-day-old pupae and adults. A 3-fold increase in OA levels was observed in the optic lobes of the adult as compared to late pupal stage. No changes were observed for E, DA, and 5-HT. NE was not detected in the optic lobes. In the haemolymph of 5^th instar larvae, OA was also the most abundant amine. Both DA and OA peaked prior to the onset of the W stage. In contrast, E and NE concentrations decreased with development of the 5^th instar larvae. 5-HT was not detected in the haemolymph. Finally, different profiles for amine levels were observed for the two forms of the 5^th instar larvae (green vs brown). These results are interpreted in the light of the role of biogenic amines and their relation to development in the nervous system of lepidopteran insects.     

Metabolic Responses of the Dopaminergic System during Hypoxia in Newborn Brain

The purpose of this review is to describe the relationship between the dopamine and amino acid neurotransmitter systems and cortical oxygen pressure during different levels of cerebral hypoxia using newborn piglets as an animal model, adding new data from our laboratory. The extracellular dopamine increases as the oxygen pressure in the cortex decreases. The relationship between oxygen pressure and dopamine levels is the same whether the hypoxia is induced by reduced F_iO₂ (high-flow hypoxia) or by hypocapnia-induced cerebral vasoconstriction (low-flow hypoxia). Thus it appears that, particularly in mild hypoxia, the extracellular level of dopamine depends primarily on the oxygen concentration in the tissue with minimal influence of parameters such as blood flow and pH. There is no "oxygen reserve" in the brain of newborn piglets and the extracellular levels of dopamine in the striatum increase almost linearly with decrease in oxygen pressure, with even small decreases in oxygen pressure resulting in increased dopamine levels. In contrast, the changes in extracellular concentrations of the excitatory amino acids glutamate and aspartate are variable and transient. In a majority of 2- to 5 day-old piglets even very low oxygen pressures in the brain did not result in significant alterations in the extracellular levels of glutamate and aspartate. These changes in the dopaminergic system may contribute directly and indirectly to the neuronal damage that occurs during hypoxic/ischemic insult and reoxygenation in newborn brain, particularly in the striatum. A variety of mechanisms are discussed by which dopamine, in particular extracellular dopamine, can increase cellular toxicity.     

Drosophila Clueless Is Highly Expressed in Larval Neuroblasts,Affects Mitochondrial Localization and Suppresses Mitochondrial Oxidative Damage

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, in this study we have identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. We show while mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, overall brain morphology appears to be normal. This is explained by our observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu’s specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus Clu’s role in mitochondrial function is not critical during larval development, but is important for pupae and adults.     

Sources of error in neurotransmitter analysis

A compound having fluorescence characteristics similar to those of 5-hydroxytryptamine (5-HT), when reacted with ninhydrin, is present in several invertebrates (Fasciola hepatica, Aplysia californica, Tritonia diomedia, and Hirudo medicinalis). However, this substance is not identical with 5-HT [ Andreini, G. C., Beretta, C., Faustini, R., and Gallina, G. (1970)Experientia26, 166–167]. We confirmed these findings for Fasciola and also observed this substance in Spirometra mansonoides and mouse brain. Using chromatography and amino acid analysis we identified this substance as lysine. While 5-HT is also demonstrable in Spirometra, no 5-HT is present in Fasciola. Although epinephrine and dopamine seem to be present in Spirometra when the hydroxyindole technique of Laverty and Taylor [ Laverty, R., and Taylor, K. M. (1968)Anal. Biochem.22, 269–279] is used, neither amine is detectable in adult Spirometra by mass spectrometric analysis. A relatively high concentration of tyrosine in Spirometra could account for the apparent presence of dopamine. Therefore, lysine, tyrosine, and possibly other substances can be sources of error in the detection of biogenic amines in invertebrates.     

Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease

The study has demonstrated that dopamine induces membrane depolarization and a loss of phosphorylation capacity in dose-dependent manner in isolated rat brain mitochondria during extended in vitro incubation and the phenomena are not prevented by oxyradical scavengers or metal chelators. Dopamine effects on brain mitochondria are, however, markedly prevented by reduced glutathione and N-acetyl cysteine and promoted by tyrosinase present in the incubation medium. The results imply that quinone oxidation products of dopamine are involved in mitochondrial damage under this condition. When PC12 cells are exposed to dopamine in varying concentrations (100-400 μM) for up to 24 h, a pronounced impairment of mitochondrial bio-energetic functions at several levels is observed along with a significant (nearly 40%) loss of cell viability with features of apoptotic nuclear changes and increased activities of caspase 3 and caspase 9 and all these effects of dopamine are remarkably prevented by N-acetyl cysteine. N-acetyl cysteine also blocks nearly completely the dopamine induced increase in reactive oxygen species production and the formation of quinoprotein adducts in mitochondrial fraction within PC12 cells and also the accumulation of quinone products in the culture medium. Clorgyline, an inhibitor of MAO-A, markedly decreases the formation of reactive oxygen species in PC12 cells upon dopamine exposure but has only mild protective actions against quinoprotein adduct formation, mitochondrial dysfunctions, cell death and caspase activation induced by dopamine. The results have indicated that quinone oxidation products and not reactive oxygen species are primarily involved in cytotoxic effects of dopamine and the mitochondrial impairment plays a central role in the latter process. The data have clear implications in the pathogenesis of Parkinson's disease.     

EFFECTS OF ELECTRICAL STIMULATION OF THE NIGROSTRIATAL PATHWAY OF THE RAT ON DOPAMINE METABOLISM

Abstract— Electrical stimulation of the nigrostriatal dopaminergic pathway in rat brain elicited a frequency and current intensity-dependent increased in the formation of homovanillic acid in the basal ganglia. The accumulation of the acid in probenecid-treated animals was constant over 1 h, when maximally stimulated at 25 Hz and 300 μA. Dopamine levels remained unchanged during stimulation. When prior to stimulation the inhibitor of catecholamine synthesis α-methyl- p -tyrosine methyl ester was administered, dopamine levels declined biphasically. Tyrosine and nomifensine, a dopamine uptake inhibitor, and apomorphine had no major effect on the formation of homovanillic acid, whilst α-methyl- p -tyrosine prevented its formation. Our data suggest that dopamine in the striatum is compartmentalized and that the newly-synthesized amine is released and converted to homovanillic acid. Apomorphine decreases dopamine flux only when dopaminergic neurons are at rest. When depolarized neither access of the precursor nor reuptake seem to influence the conversion of dopamine to homovanillic acid.     

CNS mediated inhibition of gastric secretion by bombesin: independence from interaction with brain catecholaminergic, and serotoninergic pathways and pituitary hormones

The effects of hypophysectomy and pharmacologic manipulation of brain biogenic amines on gastric secretion (volume and titratable acidity) and on CNS-mediated inhibition of gastric secretion by bombesin were studied in pylorus-ligated rats. Bombesin (100 ng), given intracisternally (i.c.), reduced the gastric secretory volume by 61%, raised pH values to 5 and virtually suppressed the titratable acidity of gastric secretion. Hypophysectomy did not modify the volume of secretion, lowered the gastric acid concentration by 37% and did not alter the magnitude of bombesin's suppressive effect, suggesting that pituitary-derived substances do not participate in the expression of bombesin's action. Depletion of brain catecholamines by combined administration of the neurotoxic agent 6-hydroxydopamine (400 μg twice, i.c.) and the catecholamine synthesis inhibitor α-methyl-p-tyrosine (250 mg/kg) or blockade of dopamine receptors by haloperidol (25 μg, i.c.), which induced a rise in plasma prolactin levels (indirect evidence of suppression of dopaminergic inhibitory tonus) neither modified gastric secretion nor the antisecretory effect of bombesin. Depletion of brain serotonin by the indolamine neurotoxin 5,6-dihydroxytryptamine (50 μg, i.c.) combined with p-chlorophenylalanine (315 mg/kg), an inhibitor of tryptophane-hydroxylase, did not affect gastric secretion or bombesin's action. Administration of dopamine, serotonin or noradrenaline at 10-μg dose levels i.c. had no effect on gastric secretion. The demonstration that pharmacologic measures designed to interfere with the normal functioning of brain catecholaminergic and serotoninergic systems did not modify gastric secretion is not in favor of their involvement in the brain control of gastric secretion. Moreover, the fact that the potent antisecretory action of bombesin is not mimicked by, nor dependent upon, intact biogenic amine pathways further supports the concept that a direct neuropeptidergic pathway may participate in the CNS regulation of gastric secretion.     

The Role of Dopamine in Drosophila Larval Classical Olfactory Conditioning

Learning and memory is not an attribute of higher animals. Even Drosophila larvae are able to form and recall an association of a given odor with an aversive or appetitive gustatory reinforcer. As the Drosophila larva has turned into a particularly simple model for studying odor processing, a detailed neuronal and functional map of the olfactory pathway is available up to the third order neurons in the mushroom bodies. At this point, a convergence of olfactory processing and gustatory reinforcement is suggested to underlie associative memory formation. The dopaminergic system was shown to be involved in mammalian and insect olfactory conditioning. To analyze the anatomy and function of the larval dopaminergic system, we first characterize dopaminergic neurons immunohistochemically up to the single cell level and subsequent test for the effects of distortions in the dopamine system upon aversive (odor-salt) as well as appetitive (odor-sugar) associative learning. Single cell analysis suggests that dopaminergic neurons do not directly connect gustatory input in the larval suboesophageal ganglion to olfactory information in the mushroom bodies. However, a number of dopaminergic neurons innervate different regions of the brain, including protocerebra, mushroom bodies and suboesophageal ganglion. We found that dopamine receptors are highly enriched in the mushroom bodies and that aversive and appetitive olfactory learning is strongly impaired in dopamine receptor mutants. Genetically interfering with dopaminergic signaling supports this finding, although our data do not exclude on naïve odor and sugar preferences of the larvae. Our data suggest that dopaminergic neurons provide input to different brain regions including protocerebra, suboesophageal ganglion and mushroom bodies by more than one route. We therefore propose that different types of dopaminergic neurons might be involved in different types of signaling necessary for aversive and appetitive olfactory memory formation respectively, or for the retrieval of these memory traces. Future studies of the dopaminergic system need to take into account such cellular dissociations in function in order to be meaningful.     

EFFECTS OF AMINO-OXYACETIC ACID, ETHANOLAMINE-O-SULPHATE AND GABA ON THE CONTENTS OF GABA AND VARIOUS AMINES IN BRAIN SLICES

—The effects of amino-oxyacetic acid, ethanolamine-O-sulphate and γ-aminobutyric acid (GABA) on the contents of GABA, noradrenaline, dopamine and serotonin (5-HT) in slices of rat hypothalamus and midbrain were studied in vitro using a simultaneous fluorimetric assay procedure. Following control incubations the levels of 5-HT were raised, while the levels of the other substances remained steady. Amino-oxyacetic acid caused a reduction in the contents of noradrenaline and 5-HT, but had no effect on either GABA or dopamine. Ethanolamine-O-sulphate both raised the GABA content and lowered the noradrenaline content of slices, while the levels of dopamine and 5-HT were not altered. The presence of GABA in the incubation medium produced complex changes in these levels, depending both on the dose of GABA used and the brain area studied. In the hypothalamus, 0·07 mm -GABA caused an elevation in 5-HT, a drop in noradrenaline, and no change in either GABA or dopamine. With 5 mm -GABA, the noradrenaline level was raised slightly above control values and the endogenous GABA level doubled, while 5-HT and dopamine levels were not different from controls. Similar changes in 5-HT and GABA contents were observed with midbrain slices, but noradrenaline and dopamine were not affected. The possible modes of action of amino-oxyacetic acid and ethanolamine-O-sulphate on the amino acid and amine systems in the brain are discussed.     

Changes of biogenic amine levels in haemolymph during diapausing and non-diapausing status in Pieris brassicae L

During the larval development of Pieris brassicae, photoperiod status induces either direct (non-diapausing) development or diapausing development. Biogenic amines such as dopamine and serotonin and their derivatives may be implicated in the physiological control and adaptation of these insects to different photoperiods. High performance liquid chromatography coupled with electrochemical detection was used to measure biogenic amine concentrations in insect haemolymph in different photoperiod conditions. At the end of the higher photosensitivity phase, dopamine levels were two-fold higher in diapausing (0.914 pmol/microl+/-0.025) as compared to non-diapausing insects (0.415 pmol/microl+/-0.02; P<0.001). Globally, dopamine catabolites are found in higher levels in non-diapausing insects (in prepupa, near seven times more for NADA, and 25 times more for DOPAc), thus indicating a higher dopamine turn-over. Serotonin levels were detected only at the beginning of this instar in diapausing insects but not in direct development insects. During pupal life, dopamine levels were significantly higher in diapausing than in non-diapausing insects (in 3-day pupae, dopamine was 28 times higher), and remained at high levels throughout diapausing life. At the beginning of diapause, serotonin levels were higher in diapausing insects than in non-diapausing insects (in 3-day pupae, serotonin was 13 times higher). These levels decreased in the middle of diapause period, and then increased just before its breaking. Dopamine and serotonin levels presented the typical kinetics of diapause development and may be used as an indicator of the evolution of diapause status.