Octopaminergic Signaling Mediates Neural Regulation of Innate Immunity in Caenorhabditis elegans.

Upon pathogen infection, the nervous system regulates innate immunity to confer coordinated protection to the host. However, the precise mechanisms of such regulation remain unclear. Previous studies have demonstrated that OCTR-1, a putative G protein-coupled receptor for catecholamine, functions in the sensory neurons designated "ASH" to suppress innate immune responses in Caenorhabditis elegans It is unknown what molecules act as OCTR-1 ligands in the neural immune regulatory circuit. Here we identify neurotransmitter octopamine (OA) as an endogenous ligand for OCTR-1 in immune regulation and show that the OA-producing RIC neurons function in the OCTR-1 neural circuit to suppress innate immunity. RIC neurons are deactivated in the presence of pathogens but transiently activated by nonpathogenic bacteria. Our data support a model whereby an octopaminergic immunoinhibitory pathway is tonically active under normal conditions to maintain immunological homeostasis or suppress unwanted innate immune responses but downregulated upon pathogen infection to allow enhanced innate immunity. As excessive innate immune responses have been linked to a myriad of human health concerns, our study could potentially benefit the development of more-effective treatments for innate immune disorders.IMPORTANCE Insufficient or excessive immune responses to pathogen infection are major causes of disease. Increasing evidence indicates that the nervous system regulates the immune system to help maintain immunological homeostasis. However, the precise mechanisms of this regulation are largely unknown. Here we show the existence of an octopaminergic immunoinhibitory pathway in Caenorhabditis elegans Our study results indicate that this pathway is tonically active under normal conditions to maintain immunological homeostasis or suppress unwanted innate immune responses but downregulated upon pathogen infection to allow enhanced innate immunity. As excessive innate immune responses have been linked to human health conditions such as Crohn's disease, rheumatoid arthritis, atherosclerosis, diabetes, and Alzheimer's disease, elucidating octopaminergic neural regulation of innate immunity could be helpful in the development of new treatments for innate immune diseases.

Octopamine enhances the immune responses of freshwater giant prawn, Macrobrachium rosenbergii, via octopamine receptors.

Octopamine (OA) is known to play an important role in regulating insect immune responses. In Macrobrachium rosenbergii (18.0 ± 1.7 g), OA at 25.0 and 250.0 pmol/prawn significantly increased THC, semigranular cells (SGCs) and PO activity in hemocytes per 50 µL hemolymph, hyaline cells, granular cells (GCs) and RBs in hemocytes per 10 µL hemolymph, and RBs per hemocyte, and however, significantly decreased PO activity per granulocyte (GC + SGC), which returned to control levels after 4 h of injection. The significantly increased phagocytic activity and clearance efficiency of prawn received OA for 8 h returned to control levels after 16 h of injection. In addition, the significantly increased glucose and decreased lactate were observed within 1 h of OA injection. In the susceptibility test, prawn received OA at 25.0 or 250.0 pmol/prawn for 2 h then challenged with Lactococcus garvieae at 105 colony-forming units/prawn significantly increased the resistance of prawns by 23.3% and 30.0%, respectively, compared to the saline-challenged control after 144 h of challenge. In addition, the changes on immunocompetence induced by OA were observed to be blocked by adrenoceptors antagonists. These results suggest that OA administration at 250.0 pmol/prawn or less causes the mediate a transient up-regulation in immune and physiologic responses to promote the resistance of M. rosenbergii to L. garvieae, which are thought to be mediated by α- and ß-adrenergic-like octopamine receptors.

Predator exposure-induced immunosuppression: trade-off, immune redistribution or immune reconfiguration?

Although predator exposure increases the risk of wound infections, it typically induces immunosuppression. A number of non-mutually exclusive hypotheses have been put forward to explain this immunosuppression, including: trade-offs between the immune system and other systems required for anti-predator behaviour, redistribution of immune resources towards mechanisms needed to defend against wound infections, and reconfiguration of the immune system to optimize defence under the physiological state of fight-or-flight readiness. We tested the ability of each hypothesis to explain the effects of chronic predator stress on the immune system of the caterpillar Manduca sexta Predator exposure induced defensive behaviours, reduced mass gain, increased development time and increased the concentration of the stress neurohormone octopamine. It had no significant effect on haemocyte number, melanization rate, phenoloxidase activity, lysozyme-like activity or nodule production. Predator stress reduced haemolymph glutathione concentrations. It also increased constitutive expression of the antimicrobial peptide attacin-1 but reduced attacin-1 expression in response to an immune challenge. These results best fit the immune reconfiguration hypothesis, although the other hypotheses are also consistent with some results. Interpreting stress-related changes in immune function may require an examination at the level of the whole organism.

Monoclonal antibody-based immunoassay for analysis of octopamine in housefly.

Octopamine (OA) is one of the biogenic monoamines in the housefly, which acts as an important neurohormone in the physiological process of this pest. In this study, a new hapten of OA was synthesized via aldol condensation. With the hapten, monoclonal antibodies (MAb) were generated and their characterizations were investigated. An indirect competitive enzyme-linked immunosorbent assay (icELISA) based on MAb 3C11-E3 was established, which required simple sample pre-treatments and had low cross-reactivity with OA structural analogise. The half maximal inhibition concentration (IC50) and the detected range (IC20-IC80) of the icELISA were 128 ng/mL and 12-1438 ng/mL, respectively. Average recoveries of OA ranged from 73 to 129% in the housefly.

Octopamine-like immunoreactive neurons in the brain and subesophageal ganglion of the parasitic wasps Nasonia vitripennis and N. giraulti.

Octopamine is an important neuromodulator in the insect nervous system, influencing memory formation, sensory perception and motor control. In this study, we compare the distribution of octopamine-like immunoreactive neurons in two parasitic wasp species of the Nasonia genus, N. vitripennis and N. giraulti. These two species were previously described as differing in their learning and memory formation, which raised the question as to whether morphological differences in octopaminergic neurons underpinned these variations. Immunohistochemistry in combination with confocal laser scanning microscopy was used to reveal and compare the somata and major projections of the octopaminergic neurons in these wasps. The brains of both species showed similar staining patterns, with six different neuron clusters being identified in the brain and five different clusters in the subesophageal ganglion. Of those clusters found in the subesophageal ganglion, three contained unpaired neurons, whereas the other three consisted in paired neurons. The overall pattern of octopaminergic neurons in both species was similar, with no differences in the numbers or projections of the ventral unpaired median (VUM) neurons, which are known to be involved in memory formation in insects. In one other cluster in the brain, located in-between the optic lobe and the antennal lobe, we detected more neurons in N. vitripennis compared with N. giraulti. Combining our results with findings made previously in other Hymenopteran species, we discuss possible functions and some of the ultimate factors influencing the evolution of the octopaminergic system in the insect brain.

The major cockroach allergen Bla g 4 binds tyramine and octopamine.

Bla g 4 is a male cockroach specific protein and is one of the major allergens produced by Blattella germanica (German cockroach). This protein belongs to the lipocalin family that comprises a set of proteins that characteristically bind small hydrophobic molecules and play a role in a number of processes such as: retinoid and pheromone transport, prostaglandin synthesis and mammalian immune response. Using NMR and isothermal titration calorimetry we demonstrated that Bla g 4 binds tyramine and octopamine in solution. In addition, crystal structure analysis of the complex revealed details of tyramine binding. As tyramine and octopamine play important roles in invertebrates, and are counterparts to vertebrate adrenergic transmitters, we speculate that these molecules are physiological ligands for Bla g 4. The nature of binding these ligands to Bla g 4 sheds light on the possible biological function of the protein. In addition, we performed a large-scale analysis of Bla g 4 and Per a 4 (an allergen from American cockroach) homologs to get insights into the function of these proteins. This analysis together with a structural comparison of Blag 4 and Per a 4 suggests that these proteins may play different roles and most likely bind different ligands. Accession numbers: The atomic coordinates and the structure factors have been deposited to the Protein Data Band under accession codes: 4N7C for native Bla g 4 and 4N7D for the Se-Met Bla g 4 structure.

Octopamine-immunoreactive neurons in the brain and subesophageal ganglion of the hawkmoth Manduca sexta.

Octopamine is a neuroactive monoamine that functions as a neurohormone, a neuromodulator, and a neurotransmitter in many invertebrate nervous systems, but little is known about the distribution of octopamine in the brain. We therefore used a monoclonal antibody to study the distribution of octopamine-like immunoreactivity in the brain of the hawkmoth Manduca sexta. Immunoreactive processes were observed in many regions of the brain, with the distinct exception of the upper division of the central body. We focused our analysis on nine ventral unpaired median (VUM) neurons with cell bodies in the labial neuromere of the subesophageal ganglion. Seven of these neurons projected caudally through the ventral nerve cord. Two neurons projected rostrally into the brain (supraesophageal ganglion), and one of these was a bilateral neuron that sent projections to the gamma-lobe of the mushroom body and the lateral protocerebrum. Octopamine-immunoreactive processes from one or more cells originating in the subesophageal ganglion also form direct connections between the antennal lobes and the calyces of the mushroom bodies.

Immunoreactivity against choline acetyltransferase, gamma-aminobutyric acid, histamine, octopamine, and serotonin in the larval chemosensory system of Dosophila melanogaster.

We have studied the distribution of choline acetyltransferase (ChAT), gamma-aminobutyric acid (GABA), histamine, octopamine and serotonin in the larval chemosensory system of Drosophila melanogaster. Colocalization at the confocal level with green fluorescent protein (GFP) or Tau-GFP reporters, expressed in selected P[GAL4] enhancer trap lines, was used to identify the cells making up these neurotransmitters. As in the adult fly, larval olfactory afferents project into the (larval) antennal lobe (LAL), where they synapse onto local interneurons and projection neurons, whereas gustatory afferents terminate essentially in the tritocerebral-subesophageal (TR-SOG) region. We demonstrate that the neuropils of the LAL and the TR-SOG are immunoreactive to ChAT and GABA. In addition, serotonin- and octopamine-immunoreactive fibers are present in the LAL. ChAT immunostaining is localized in subsets of olfactory and gustatory afferents and in many of the projection neurons. In contrast, GABA is expressed in most, and perhaps all, of the local interneurons. Serotonin immunoreactivity in the LAL derives from a single neuron that is situated close to the LAL and projects to additional neuropil regions. Taken together, these findings resemble the situation in the adult fly. Hence, given the highly reduced numbers of odorant receptor neurons in the larva, as shown in a previous study (Python and Stocker [2002] J. Comp. Neurol. 445:374-387), the larval system may become an attractive model system for studying the roles of neurotransmitters in olfactory processing.

Characterization of Drosophila tyramine beta-hydroxylase gene and isolation of mutant flies lacking octopamine.

Octopamine is likely to be an important neuroactive molecule in invertebrates. Here we report the molecular cloning of the Drosophila melanogaster gene, which encodes tyramine beta-hydroxylase (TBH), the enzyme that catalyzes the last step in octopamine biosynthesis. The deduced amino acid sequence of the encoded protein exhibits 39% identity to the evolutionarily related mammalian dopamine beta-hydroxylase enzyme. We generated a polyclonal antibody against the protein product of T beta h gene, and we demonstrate that the TBH expression pattern is remarkably similar to the previously described octopamine immunoreactivity in Drosophila. We further report the creation of null mutations at the T beta h locus, which result in complete absence of TBH protein and blockage of the octopamine biosynthesis. T beta h-null flies are octopamine-less but survive to adulthood. They are normal in external morphology, but the females are sterile, because although they mate, they retain fully developed eggs. Finally, we demonstrate that this defect in egg laying is associated with the octopamine deficit, because females that have retained eggs initiate egg laying when transferred onto octopamine-supplemented food.

Octopamine immunoreactivity in the fruit fly Drosophila melanogaster.

Octopamine has been proposed as a neurotransmitter/modulator/hormone serving a variety of physiological functions in invertebrates. We have initiated a study of octopamine in the fruit fly Drosophila melanogaster, which provides an excellent system for genetic and molecular analysis of neuroactive molecules. As a first step, the distribution of octopamine immunoreactivity was studied by means of an octopamine-specific antiserum. We focused on the central nervous system (CNS) and on the innervation of the larval body wall muscles. The larval octopamine neuronal pattern was composed of prominent neurons along the midline of the ventral ganglion, whereas brain lobes were devoid of immunoreactive somata. However, intense immunoreactive neuropil was observed both in the ventral ganglion and in the brain lobes. Some of the immunoreactive neurons sent peripheral fibers that innervated most of the muscles of the larval body wall. Octopamine immunoreactivity was observed at neuromuscular junctions in all larval stages, being present in a well-defined subset of synaptic boutons, type II. Octopamine immunoreactivity in the adult CNS revealed many additional neurons compared to the larval CNS, indicating that at least a subset of adult octopamine neurons may differentiate during metamorphosis. Major octopamine-immunoreactive neuronal clusters and neuronal processes were observed in the subesophageal ganglion, deutocerebrum, and dorsal protocerebrum, and intense neuropil staining was detected primarily in the optic lobes and in the central complex.

Taurine-like immunoreactivity in octopaminergic neurones of the cockroach, Periplaneta americana (L.).

Taurine (2-aminoethanesulphonic acid) is reported to interact with the octopaminergic system. The distribution of taurine-like immunoreactivity (-LIR) in relation to octopamine-like immunoreactive dorsal unpaired median (DUM) neurones was investigated with the aim of revealing possible colocalization of these two neuromediators. The specificity of the anti-taurine serum used was demonstrated by dot blot immunoassay and by use of preabsorption controls. There was no crossreactivity with octopamine. The specificity of the octopamine antiserum employed has been described elsewhere. Taurine-LIR could be demonstrated in large dorso-median cells in the suboesophageal and the mesothoracic ganglion as well as in the abdominal ganglia. In addition taurine-LIR is distributed in numerous other regions of the ganglia. A comparison of the immunostaining for taurine and octopamine indicates that several of the taurine-like immunoreactive (-LI) neurones are probably members of the octopamine-immunoreactive DUM cell population. These taurine-LI neurones resemble octopamine-LI DUM cells in soma position and size as well as in the projections of their primary neurites. Colocalization of octopamine-LIR and taurine-LIR within the same neuronal element could be shown by alternate immunostaining of consecutive sections. It is probable that all octopamine-LI DUM neurones also exhibit taurine-LIR, and the possible physiological significance of this coexistence is discussed.

Small sets of putative interneurons are octopamine-immunoreactive in the central nervous system of the pond snail, Lymnaea stagnalis.

An antibody raised against conjugated octopamine was applied to map octopamine-containing neurons in the central nervous system of the pond snail Lymnaea stagnalis. A small number of octopamine-like immunoreactive neurones occurs in all ganglia, but the pleural ones. The neurons are located either in small clusters or occur individually. Major concentrations of octopamine-immunoreactive neurons can first of all be found in the buccal, cerebral and pedal ganglia. Varicose arborizations were observed in the neuropiles, but peripheral projections of labelled elements could not be traced. We suggest that a set of octopaminergic interneurons would exist in the Lymnaea brain. Mapping of octopamine-immunoreactive neurons given may also facilitate physiological investigations on octopaminergic neurotransmission in the gastropod nervous system.

Octopamine immunoreactive cell populations in the locust thoracic-abdominal nervous system.

We describe octopamine-immunoreactive somata and their projections in the pro- meso-, meta- and pregenital abdominal-ganglia of locusts. Immunoreactive midline somata were identified as dorsal- and ventral- unpaired median (DUM- and VUM-, respectively) neurones due to their: characteristic large size and positions of somata, primary neurites in DUM-tracts giving rise to T-junctions, and bilaterally projecting axons. In the prothoracic ganglion there are most likely 8 such cells; in the meso- and metathoracic, some 20 each; and in each individual pregenital abdominal ganglion, typically 3. All appear to project to peripheral nerves and their numbers correspond to the number of peripherally projecting DUM-cells identified to date in each ganglion. We suggest that probably all peripherally projecting DUM-cells are octopaminergic in the examined ganglia. Presumptive DUM-interneurones are not octopamine-immunoreactive, but, confirming other studies, are shown to label with an antiserum to gamma-amino butyric acid (GABA). Other octopamine-immunoreactive neurones include a pair of midline, prothoracic, anterior medial cells, not necessarily DUM-cells, and a pair of ventral lateral somata in each thoracic- and the first abdominal ganglion. The latter project intersegmentally in ventral tracts. Intersegmentally projecting octopamine-immunoreactive fibers in dorsal tracts probably arise from a prothoracic DUM-cell, which leaves through suboesophageal nerves, or descending suboesophageal DUM-cells. Thus, the octopamine-immunoreactive system of thoracic and pregenital abdominal ganglia in locust comprises all peripherally projecting DUM-cells and a plurisegmental network.

Specific antisera against the catecholamines: L-3,4-dihydroxyphenylalanine, dopamine, noradrenaline, and octopamine tested by an enzyme-linked immunosorbent assay.

Antisera were raised against L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme-linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catecholamine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the nonconjugated compound was poorly recognized. The nonreduced conjugates of L-DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross-reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L-DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti-NA and anti-OA antibodies, the lateral chain is important.

Radioimmunoassay of metanephrine and normetanephrine for diagnosis of pheochromocytoma.

Sensitive and specific radioimmunoassays of metanephrine and normetanephrine were developed by use of 125I-labeled synephrine and specific metanephrine antibody, and 125I-labeled octopamine and specific normetanephrine antibody. Specific antibody for both metanephrine and normetanephrine was raised in rabbits by immunization with bovine serum albumin conjugated with the corresponding hapten, prepared by the method of Grota and Brown (Endocrinology 1976;98:615). The detection limits of the metanephrine and the normetanephrine radioimmunoassays were 2 and 6 pg/tube, respectively. Mean plasma metanephrine and normetanephrine values for 24 normal subjects were 62 (SD 14) and 100 (SD 40) ng/L, respectively. Mean urinary metanephrine and normetanephrine values for 22 normal subjects were 154 (SD 74) and 217 (SD 109) micrograms/day. For 14 pheochromocytoma patients, plasma metanephrine and normetanephrine values ranged from 29 to 683 and from 28 to 7850 ng/L, and urinary metanephrine and normetanephrine values were 606 to 6630 and 296 to 4800 micrograms/day, respectively. The present methods are simple and suitable for routine tests or for mass screening for pheochromocytoma.

Antisera against catecholamines: specificity studies and physicochemical data for anti-dopamine and anti-p-tyramine antibodies.

Antibodies against dopamine and p-tyramine were raised in rabbits. The two catecholamines were conjugated to albumin by glutaraldehyde. The specificity of the antibodies was established by equilibrium dialysis competition experiments using an immunoreactive tritiated derivative synthesized by coupling dopamine or p-tyramine to N-alpha-acetyl-L-lysine N-methylamide with glutaraldehyde. Hence, these radiolabelled ligands mimicked the antigenic determinant of conjugated immunogens. A comparison of the data obtained showed the high specificity of each antiserum for its hapten coupled by glutaraldehyde. The anti-dopamine antibodies recognized dopamine-glutaraldehyde but not p-tyramine-glutaraldehyde. The opposite occurred for the anti-p-tyramine antibodies. A slight modification of the molecular structure provided the opportunity for a specific response against that molecule. But this difference was more important when related to the hapten region where the antibody affinity was maximal. The cross-reactivity was observed to be more important dopamine and p-tyramine than between dopamine and noradrenaline on the one hand and between p-tyramine and dopamine than p-tyramine and octopamine on the other hand.