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There is ample evidence linking octopamine (OA) and tyramine (TA) to several neurophysiological functions in arthropods. In our laboratory we use the freshwater prawn Macrobrachium rosenbergii to study the neural basis of aggressive behavior. As a first step towards understanding the possible role of these amines and their receptors in the modulation of interactive behaviors, we have cloned a putative octopamine/tyramine receptor. The predicted sequence of the cloned OA/TA(Mac) receptor consists of 1,579 base pairs (bp), with an open reading frame of 1,350bp that encodes a 450 amino acid protein. This putative protein displays sequence identities of 70% to an Aedes aegypti mosquito TA receptor, followed by 60% to a Stegomyia aegypti mosquito OA receptor, 59% and 58% to the migratory locust TA-1 and -2 receptors respectively, and 57% with the silkworm OA receptor. We also mapped the OA/TA(Mac) receptor distribution by in-situ hybridization to the receptor's mRNA, and by immunohistochemistry to its protein. We observed stained cell bodies for the receptor's mRNA, mainly in the midline region of the thoracic and in the abdominal ganglia, as well as diffuse staining in the brain ganglia. For the receptor's protein, we observed extensive punctate staining within the neuropil and on the membrane of specific groups of neurons in all ganglia throughout the CNS, including the brain, the midline region and neuropiles of the thoracic ganglia, and ventral part and neuropiles of the abdominal ganglia. The same pattern of stained cells was observed on the thoracic and abdominal ganglia in both in-situ hybridization and immunohistochemistry experiments. Diffuse staining observed with in-situ hybridization also coincides with punctate staining observed in brain, SEG, thoracic, and abdominal ganglia in immunohistochemical preparations. This work provides the first step towards characterizing the neural networks that mediate octopaminergic signaling in prawn., (Published by Elsevier B.V.)

The kinetic and pharmacological properties of [3H]tyramine and [3H]octopamine binding to membrane preparations of locust (Locusta migratoria migratorioides) brain were studied to characterize the tyramine and octopamine receptors. [3H]Tyramine and [3H]octopamine bind specifically and reversibly to the locust brain membrane with equilibrium achieved after 20 min. The dissociation of [3H]tyramine is monophasic while that of the [3H]octopamine shows a biphasic tendency. Scatchard analysis of the saturation curves reveals a single high affinity binding site for each of tyramine and octopamine. The mean (+/- S.E.M.) values of Kd and Bmax are 6.11 +/- 0.71 nM and 21.45 +/- 3.0 fmol/mg tissue for tyramine and 5.65 +/- 0.91 nM and 15.0 +/- 2.4 fmol/mg tissue for octopamine, respectively. Pharmacological analysis of the binding suggests the presence of both tyramine and octopamine receptors in the locust brain. alpha-Adrenergic agonists and antagonists have a high affinity to the octopamine but not the tyramine receptor whereas dopaminergic drugs have a higher affinity to the tyramine receptor than the octopamine receptor. No highly effective inhibitors of tyramine binding were identified. The serotonergic blockers, mianserin, LSD, BOL are effective blockers for both tyramine and octopamine receptors, whereas the serotonergic antagonist gramine is more active against the octopamine than the serotonin receptor. The results suggest that a G-protein binding mechanism is involved in the expression of both the tyramine and octopamine effects.

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.

A radiochemical-enzymatic assay was utilized to measure endogenous levels of octopamine in the nerve cord and blood of the earthworm. The results show the presence of octopamine in the ventral nerve cord and blood at concentrations of 2.79 +/- 0.5 (means +/- S.D.) ng/mg wet tissue weight and 9.5 +/- 1.6 x 10(-8) M (means +/- S.D.) respectively. The ability of the ventral nerve cord to synthesize octopamine was investigated by incubating the tissue in vitro in radiolabeled percursors. The results show the synthesis of [3H]octopamine from both precursor [3H]tyrosine and [3H]tyramine. In vitro incubation of the isolated ventral nerve cord in physiological concentrations of octopamine revealed modulation of the spontaneous rhythmic contractions of the nerve cord. The data provide direct support for a modulatory role of octopamine in L. terrestris.

The effects of electrolytic lesions of the locus coeruleus (LC) on endogenous octopamine and noradrenaline levels were observed at various temporal intervals. Noradrenaline was assayed from halved tissue samples by high-performance liquid chromatography (HPLC) while octopamine was determined from the other halves of the brain samples by radioenzymatic assay methods accompanied by chromatographic separation. Brains were dissected into 8 regions: cerebellum, anterior and posterior cortex, hippocampus, hypothalamus, medulla/pons, midbrain and striatum. Assays were performed at postlesion intervals of 4, 8, 13 and 18 days. The rates of depletion of noradrenaline at the tested intervals roughly matched those of octopamine in the anterior cortex, posterior cortex and striatum. In the cerebellum, hypothalamus, hippocampus and pons/medulla, levels of octopamine diminished later than those of noradrenaline, at times showing an initial increase within the first week after the LC lesion. In the midbrain however, this pattern was reversed, octopamine levels initially decreasing in parallel with noradrenaline but subsequently recovering to significantly higher values. The results do not unequivocally support the hypothesis that octopamine is present in the brain solely as a 'false transmitter' in noradrenergic neurones. Neither, however, do they provide clear support for earlier contentions that octopamine is likely to play a role in central neurones as a synaptic transmitter, independent of the noradrenergic system.

The release of octopamine and proctolin from the oviduct visceral muscles of the locust, Locusta migratoria, has been investigated. Salines containing elevated potassium concentrations (100 mM) were capable of releasing both octopamine and proctolin, although the proportion of the total store of octopamine released (19%) was much greater than that of proctolin (0.4%). The high potassium-induced release of octopamine was calcium-dependent. Electrical stimulation of the oviducal nerves also resulted in the release of octopamine and proctolin. The release of both substances was frequency-dependent with maximum release of octopamine occurring at about 5 Hz, and maximum release of proctolin at 30 Hz. Neural stimulation was a more effective means of inducing proctolin release than was high-potassium saline, with 6.3% of the total store of proctolin released with 5 min stimulation at 30 Hz. The neurally stimulated release of proctolin was calcium-dependent. In addition, intrasomatic stimulation of the octopaminergic neurons which project to the oviducts resulted in the release of octopamine. The results clearly indicate that both octopamine and proctolin, which have previously been shown to be associated with locust oviducts, are released in a calcium-dependent manner by physiological stimuli. This strengthens the case for octopamine and proctolin as natural regulators of this insect visceral muscle.

According to recent research, a significant relationship seems to exist between brain contents of p-octopamine and two-way avoidance responding in rats. Levels of this amine in hypothalamus and brainstem are higher in rats from the Roman High Avoidance (RHA) strain than in rats from the Roman Low Avoidance (RLA) strain. Intracerebroventricular administration of p-octopamine facilitates avoidance responding. The present paper reports the behavioral and neurochemical effects of p-octopamine administration on rats from the Roman strains. This treatment has no significant effect on cerebral levels of catecholamines, but it temporarily suppresses the strain differences in p-octopamine levels. In parallel, it also suppresses the strain differences in two-way avoidance conditioning, its facilitatory effect being much greater in RLA than in RHA rats. Avoidance behavior appears to be a useful model for the study of octopamine functions in the mammalian brain.

An enzymatic assay for octopamine capable of detecting 50 pg of amine was developed and used to study the distribution of octopamine in regions of the rat central nervous system. The presence of octopamine in the rat pineal organ was confirmed by mass spectrometry; Administration of a monoamine oxidase inhibitor and of tyramine led to increases in CNS octopamine levels while the administration of reserpine intraperitoneally or 6-hydroxydopamine intraventricularly led to decreases in octopamine levels. The results suggest that in the mammalian CNS octopamine is present in neural structures where it may be involved in synaptic function.