Myoinhibitory neuropeptides in the American cockroach.

A large number of myostimulatory neuropeptides from neurohaemal organs of the American cockroach have been described since 1989. These peptides, isolated from the retrocerebral complex and abdominal perisympathetic organs, are thought to be released as hormones. To study the coordinated action of these neuropeptides in the regulation of visceral muscle activity, it might be necessary to include myoinhibitors as well, however, not a single myoinhibitory neuropeptide of the American cockroach has been described so far. To fill this gap, we describe the isolation of LMS (leucomyosuppressin) and Pea-MIP (myoinhibitory peptide) from neurohaemal organs of the American cockroach. LMS was very effective in inhibiting phasic activity of all visceral muscles tested. It was found in the corpora cardiaca of different species of cockroaches, as well as in related insect groups, including mantids and termites. Pea-MIP which is strongly accumulated in the corpora cardiaca was not detected with a muscle bioassay system but when searching for tryptophane-containing peptides using a diode-array detector. This peptide caused only a moderate inhibition in visceral muscle assays. The distribution of Pea-MIP in neurohaemal organs and cells supplying these organs with Pea-MIP immunoreactive material, is described. Additionally to LMS and Pea-MIP, a member of the allatostatin peptide family, known to exhibit inhibitory properties in other insects, was tested in visceral muscle assays. This allatostatin was highly effective in inhibiting spontaneous activity of the foregut, but not of other tested visceral muscles of the American cockroach.

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.

Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Isolation of periviscerokinin-2 from the abdominal perisympathetic organs of the American cockroach, Periplaneta americana.

Using the isolated hyperneural muscle as bioassay, a novel myotropin was isolated from the abdominal perisympathetic organs of Periplaneta americana. This is the second neuropeptide identified from insect perisympathetic organs. Peptide sequence analysis and mass spectrometry yielded the following structure: Gly-Ser-Ser-Ser-Gly-Leu-Ile-Ser-Met-Pro-Arg-Val-NH2. This peptide, named periviscerokinin-2, was confirmed to be amidated by chemical synthesis, bioassay, and comparison of retention times between native and synthetic peptides. A highly specific antiserum was used to determine sites of synthesis in the abdominal ganglia. Besides periviscerokinin-1, periviscerokinin-2 is the only putative myotropic neurohormone from the abdominal perisympathetic organs that is effective in the nanomolar range. This confirms the hypothesis that the neurohormonal system of the ventral nerve cord is remarkably different from that of the brain.

Isolation and structural elucidation of eight kinins from the retrocerebral complex of the American cockroach, Periplaneta americana.

By monitoring the contractile activity of the hindgut of the American cockroach in vitro eight myotropic neuropeptides were isolated from the retrocerebral complex of the American cockroach. Peptide sequence analysis and mass spectrometry yielded the following structures: Arg- Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-1), Asp-Ala-Ser-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-2), Asp-Pro-Ser-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-3), Gly-Ala-Gln-Phe-Ser-Ser-Trp-Gly-NH2 (Pea-K-4), Ser-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 (Pea-K-5), Asp-Pro-Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lem-K-7), Gly-Ala-Asp-Phe-Tyr-Ser-Trp-Gly-NH2 (Lem-K-8) and Ala-Phe-Ser-Ser-Trp-Gly-NH2 (Lom-K). The C-terminal sequence Phe-X-Ser-Trp-Gly-NH2 characterized the peptides as members of the insect kinin family. All structures were confirmed by comparison of retention times between synthetic and natural peptides. The threshold concentration for stimulatory effects of the synthetic peptides on the isolated hindgut was about 10(-9) M and there was no significant difference measured between the different kinin forms. These neuropeptides are the first members of the insect kinin-family isolated from the American cockroach. Their occurrence in the retrocerebral complex suggests a physiological role as neurohormone.

A comparative immunocytochemical study using an antiserum against a synthetic analogue of the corpora cardiaca peptide Pea-CAH-I (MI, neurohormone D) of Periplaneta americana.

An antiserum against the octapeptide Pea-CAH-I, a member of the adipokinetic hormone/red pigment-concentrating hormone family, has been produced for immunocytochemical staining in insects and various other invertebrate species. The anti-Pea-CAH-I serum stains the glandular corpora cardiaca cells of those insect species that synthesize identical or structurally similar peptides. In the corpora cardiaca of species producing peptides with a different C-terminus, these cells remain unstained. Pea-CAH-I-like immunoreactivity has also been found in neurons of the central nervous system of all invertebrate orders studied. The antiserum recognizes the C-terminal sequence Pro-Asn-Trp-NH2 of the Pea-CAH-I molecule as established by enzyme immunoassay. The widespread Pea-CAH-I-like immunoreactivity in all nervous systems of the studied animals probably does not reflect the presence of Pea-CAH-I but the occurrence of peptides carrying similar epitopes.

Octopamine in the developing nervous system of the pond snail, Lymnaea stagnalis L.

Development of the octopaminergic system in the pond snail, Lymnaea stagnalis, was investigated by means of immunocytochemistry and radioenzymatic assay. The earliest octopamine-immunoreactive neurons appear at a late embryonic stage (E85) of development following metamorphosis. At this moment of development, the adult-like pattern of distribution and projection (arborization) characteristics of octopamine-immunoreactive neurone can already be observed. During hatching and postembryonic (juvenile) development the number of labelled neurons increases only within the ventro-medial cell groups of the cerebral ganglia, whereas the extent of varicose axon arborization of the labelled neurons increases gradually. No peripheral projections of the embryonic and postembryonic octopamine-immunoreactive neurons were observed. The postembryonic increase in number of immunoreactive neurons and development of axonal arborization is accompanied by a rapid, exponential enhancement of octopamine content of Lymnaea CNS, as detected radioenzymatically. A possible role of the octopaminergic neurons in the regulation of certain physiological function(s), active only from a late stage of embryonic development, is suggested.

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.

Distribution and developmental expression of octopamine-immunoreactive neurons in the central nervous system of the leech.

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1-6 and posterior ganglia 15-21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7-14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamine-immunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis. Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamine-immunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior.

The role of proctolin in the antenna-heart beat acceleration of Periplaneta americana (L.).

So-called accessorial pulsatile circulatory organs in insects have developed during evolutionary reduction of the vascular system. As such a peripheral organ, the antenna-heart supplies the antennae with haemolymph. In the antenna-heart, the beat rhythm is generated by a myogenic automatism and is controlled neuronally. The electrical stimulation of the antenna-heart nerve produces an immediate heart beat inhibition brought about by octopamine release. Following this heart inhibition, an accelerating effect can be observed, which is based on a peptidergic mechanism. Proctolin-like material was found in both the dilator muscle and the antenna-heart nerve by means of immunocytochemistry. After HPLC-separation of antenna-heart tissue, proctolin was identified by sequencing the bioactive material and subsequent mass spectrometry. Proctolin proved to be extraordinarily effective producing stimulation of the antenna-heart beat rate up to 450%. The threshold concentration is 10(-10) mol.1(-1) and the value of the dissociation constant was fixed to KO = 1.3 x 10(-8) mol.1(-1). The chrono-trophic effect is caused by an increase in the steepness of the rising phase of pacemaker activity. The enhancement of the membrane resistance Rinput indicates a reduction of K(+)-conductance by proctolin. The PI-second messenger system is involved as well. Proctolin was identified and isolated from a real target organ. This fact and the physiological results furnished proof of peptidergic transmitter function of proctolin in the antenna-heart.

Periviscerokinin (Pea-PVK): a novel myotropic neuropeptide from the perisympathetic organs of the American cockroach.

A myotropic neuropeptide was isolated from extracts of 1000 abdominal perisympathetic organs of males of the cockroach, Periplaneta americana. This peptide, termed periviscerokinin, has excitatory actions on the hyperneural muscle of Periplaneta americana. After peptide sequence analysis and mass spectrometry, the structure of this peptide was confirmed by chemical synthesis and bioassay to be Gly-Ala-Ser-Gly-Leu-Ile-Pro-Val-Met-Arg-Asn-NH2. This sequence is different from the other known myotropic peptides in insects. The threshold concentration for stimulatory effects of the synthetic peptide on the isolated hyperneural muscle was about 10(-9) M, suggesting a physiological role as a neurohormone.

Octopamine-like immunoreactivity in the brain and subesophageal ganglion of the honeybee.

The organization of putative octopaminergic pathways in the brain and subesophageal ganglion of the honeybee was investigated with a well-defined polyclonal antiserum against octopamine. Five prominent groups of just over 100 immunoreactive (IR) somata were found in the cerebral ganglion: Neurosecretory cells in the pars intercerebralis innervating the corpora cardiaca via NCC I, one cluster mediodorsal to the antennal lobe, one scattered on both sides of the midline of the protocerebrum, one between the lateral protocerebral lobes and the dorsal lobes, and a single soma on either side of the central body. With the exception of the pedunculi and beta-lobes of the mushroom bodies, varicose immunoreactive fibers penetrate all parts of the cerebral ganglion. Strong labelling was found in the central complex and the protocerebral bridge. Fine networks of labelled processes invade the antennal lobes, the calyces and a small part of the alpha-lobes of the mushroom bodies, the protocerebrum, and all three optic ganglia. In the subesophageal ganglion, one labelled cell body was found in the lateral soma layer of the mandibular segment. Each of the three neuromeres contains a group of six to ten somata in the ventral median parts. Most of the ventral median cells send their neurites dorsally through the midline tracts, whereas the neurites of a few cells follow the ventral cell body neurite tracts. Octopamine-IR was demonstrated in all neuropils that contain pathways for proboscis extension learning in honeybees. Because octopaminergic mechanisms seem to be involved in the behavioral plasticity of the proboscis extension reflex, our study provides anatomical data on the neurochemical organization of an appetitive learning paradigm.

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.

Mapping of octopamine-immunoreactive neurons in the central nervous system of the lobster.

It has been suggested that serotonin and octopamine serve important roles in behavioral regulation in lobsters. In this paper the locations of octopamine-immunoreactive neurons were mapped in wholemount preparations of the ventral nerve cord of 4th stage lobster (Homarus americanus) larvae. Approximately 86 neurons were found, distributed as follows: brain, 12; circumesophageal ganglia, 2; subesophageal ganglion, 38; thoracic ganglia, 6 each; and 4th and 5th abdominal ganglia, 2 each. All the octopamine-immunoreactive neurons are paired and located along the midline. Of the 86 neurons, 28 were identified as neurosecretory, and 26 as intersegmental ascending thoracic, ascending abdominal, or descending interneurons. The neurosecretory system is arranged segmentally and located entirely within the thoracic and subesophageal neuromeres with extensive terminal fields of endings along 2nd thoracic and subesophageal nerve roots. This set of neurons shares the features of central and peripheral endings with 2 pairs of large serotonin-containing neurosecretory neurons found in the fifth thoracic and first abdominal ganglia. The intersegmental neurons include: (1) two cells in the brain and 2 pairs of cells in the 3rd and 4th neuromeres of the subesophageal ganglion, which project to the 6th abdominal ganglion; (2) a segmentally organized group of ascending interneurons found in the subesophageal and in all thoracic ganglia; and (3) pairs of ascending interneurons found in the 4th and 5th ganglia in the abdominal nerve cord. By means of a biochemical assay, the cell bodies of octopamine-immunoreactive neurosecretory cells in the thoracic segment of the nerve cord were found to contain 40-100 fmol of octopamine, while control neurons had none.

A new specific antibody reveals octopamine-like immunoreactivity in cockroach ventral nerve cord.

An antiserum was raised in rabbits immunized with octopamine conjugated to thyroglobulin. The specificity of this antiserum for octopamine is shown by dot blot immunoassay analysis. The antiserum does not crossreact with dopamine, noradrenaline, and serotonin, but slight crossreactivity with the amine tyramine at high concentrations was observed. The tyramine crossreactivity could be eliminated by preabsorption with a tyramine-glutaraldehyde-BSA conjugate. Using this antiserum, we describe the topographical distribution of octopamine-immunoreactive (ir) neuronal elements in wholemounts and paraffin sections of the ventral nerve cord of the American cockroach. The pattern of octopamine immunostaining is completely different from that obtained with an antidopamine serum, and can be blocked by preabsorbing the antioctopamine serum with BSA-conjugated octopamine. Cell bodies and dendritic processes of putatively octopaminergic dorsal (DUM) and ventral (VUM) unpaired median neurons were clearly octopamine-ir in all ganglia examined. The numbers of stained DUM somata in the mesothoracic, metathoracic, and terminal ganglion of females correspond to those of peripherally projecting DUM cells revealed previously by retrograde tracing (Gregory, Philos Trans R Soc Lond [Biol] 306:191, 1984; Tanaka and Washio, Comp Biochem Physiol 91A:37, 1988; Stoya et al., Zool Jb Physiol 93:75, 1989). In addition, various, previously unknown, paired cells with octopamine-like immunoreactivity were found in all ventral ganglia except abdominal ganglia 3-6. Some of these probably project intersegmentally.

The octopaminergic system within the ventral nerve cord of the American cockroach.

Octopamine-immunoreactive neurons within the ventral nerve cord of the cockroach, Periplaneta americana, were mapped with a new anti-octopamine serum. The specificity of this antiserum was demonstrated by dot blot immunoassay and by comparing the immunocytochemical staining patterns obtained after incubation with anti-dopamine and anti-octopamine serum. Putative octopaminergic dorsal and ventral unpaired median (DUM resp. VUM) neurons showed octopamine-like immunoreactivity in all ventral ganglia. The numbers of DUM cells in the mesothoracic, metathoracic and terminal ganglia of females correspond to those previously characterized by retrograde staining 19, 33, 34. It could be shown that besides segmentally projecting there are also intersegmentally projecting DUM neurons within the thoracic ganglia. In addition various, previously unknown, paired octopamine-ir cells were revealed in all ventral ganglia except the abdominal ganglia 2-5.

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.