Distribution of metabotropic receptors of serotonin, dopamine, GABA, glutamate, and short neuropeptide F in the central complex of Drosophila.

The central complex is a prominent set of midline neuropils in the insect brain, known to be a higher locomotor control center that integrates visual inputs and modulates motor outputs. It is composed of four major neuropil structures, the ellipsoid body (EB), fan-shaped body (FB), noduli (NO), and protocerebral bridge (PB). In Drosophila different types of central complex neurons have been shown to express multiple neuropeptides and neurotransmitters; however, the distribution of corresponding receptors is not known. Here, we have mapped metabotropic, G-protein-coupled receptors (GPCRs) of several neurotransmitters to neurons of the central complex. By combining immunocytochemistry with GAL4 driven green fluorescent protein, we examined the distribution patterns of six different GPCRs: two serotonin receptor subtypes (5-HT(1B) and 5-HT(7)), a dopamine receptor (DopR), the metabotropic GABA(B) receptor (GABA(B)R), the metabotropic glutamate receptor (DmGluR(A)) and a short neuropeptide F receptor (sNPFR1). Five of the six GPCRs were mapped to different neurons in the EB (sNPFR1 was not seen). Different layers of the FB express DopR, GABA(B)R, DmGluR(A,) and sNPFR1, whereas only GABA(B)R and DmGluR(A) were localized to the PB. Finally, strong expression of DopR and DmGluR(A) was detected in the NO. In most cases the distribution patterns of the GPCRs matched the expression of markers for their respective ligands. In some nonmatching regions it is likely that other types of dopamine and serotonin receptors or ionotropic GABA and glutamate receptors are expressed. Our data suggest that chemical signaling and signal modulation are diverse and highly complex in the different compartments and circuits of the Drosophila central complex. The information provided here, on receptor distribution, will be very useful for future analysis of functional circuits in the central complex, based on targeted interference with receptor expression.

Neurochemical fine tuning of a peripheral tissue: peptidergic and aminergic regulation of fluid secretion by Malpighian tubules in the tobacco hawkmoth M. sexta.

The actions of various peptides and other compounds on fluid secretion by Malpighian tubules in the tobacco hawkmoth Manduca sexta sexta are investigated in this study. Using a newly developed pharate adult Malpighian tubule bioassay, we show that three tachykinin-related peptides (TRPs), leucokinin I, serotonin (5-HT), octopamine, the cardioacceleratory peptides 1a, 1b and 2c, cGMP and cAMP each cause an increase in the rate of fluid secretion in pharate adult tubules. Whereas the possible hormonal sources of biogenic amines and some of the peptides are known, the distribution of TRPs has not been investigated previously in M. sexta. Thus we performed immunocytochemistry using an anti-TRP antiserum. We show the presence of TRP-like material in a small subset of cells in the M. sexta central nervous system (CNS). The larval brain contains approximately 60 TRP-immunopositive cells and there are approximately 100 such cells in the adult brain including the optic lobes. Every ganglion of the ventral nerve cord also contains TRP-like immunoreactive cells. No TRP-containing neurosecretory cells were seen in the CNS, but endocrine cells of the midgut reacted with the antiserum. We propose the hypothesis that the control in insects of physiological systems by hormones may not always involve tissue-specific hormones that force stereotypical responses in their target systems. Instead, there may exist in the extracellular fluid a continuous broadcast of information in the form of a chemical language to which some or all parts of the body continuously respond on a moment-to-moment basis, and which ensures a more effective and efficient coordination of function than could be achieved otherwise.

A putative tachykinin receptor in the cockroach brain: molecular cloning and analysis of expression by means of antisera to portions of the receptor protein.

Tachykinins constitute a neuropeptide family that mediate their actions via a subfamily of structurally related G-protein-coupled receptors. Two receptors, Drosophila neurokinin receptor (NKD) and Drosophila tachykinin receptor (DTKR), with sequence similarities to mammalian tachykinin receptors have previously been cloned in Drosophila. In this study we have isolated a cockroach (Leucophaea maderae) cDNA clone by screening a brain cDNA library with a degenerate oligonucleotide probe based on a conserved sequence within the seventh transmembrane region of the Drosophila tachykinin receptors. This clone, Leucophaea tachykinin receptor (LTKR), encodes a portion of a putative receptor which could be aligned with the C-terminal half of members of the tachykinin receptor subfamily. In the fifth, sixth and seventh transmembrane regions the deduced amino acid sequence of LTKR exhibits 79% sequence identity to the DTKR receptor and 54% to that of NKD. This suggests that LTKR is orthologous to the DTKR receptor. To study the distribution of the predicted LTKR protein by immunocytochemistry, antisera were raised against synthetic peptides corresponding to a region of the third intracellular loop of LTKR. In the cockroach brain immunoreactive neuronal processes were seen in several synaptic neuropils of the protocerebrum and tritocerebrum as well as in the frontal ganglion. Some immunoreactive neuronal cell bodies were detected in the protocerebrum. Double labeling immunocytochemistry revealed that there is a substantial superposition between distribution of LTKR and processes containing tachykinin-related peptide (TRP). Some brain areas, however, only display TRP immunoreactive processes and no LTKR, suggesting the presence of at least one more TRP receptor type.

Pigment-dispersing factor in the locust abdominal ganglia may have roles as circulating neurohormone and central neuromodulator.

Pigment-dispersing factor (PDF) is a neuropeptide that has been indicated as a likely output signal from the circadian clock neurons in the brain of Drosophila. In addition to these brain neurons, there are PDF-immunoreactive (PDFI) neurons in the abdominal ganglia of Drosophila and other insects; the function of these neurons is not known. We have analyzed PDFI neurons in the abdominal ganglia of the locust Locusta migratoria. These PDFI neurons can first be detected at about 45% embryonic development and have an adult appearance at about 80%. In each of the abdominal ganglia (A3-A7) there is one pair of lateral PDFI neurons and in each of the A5-A7 ganglia there is additionally a pair of median neurons. The lateral neurons supply varicose branches to neurohemal areas of the lateral heart nerves and perisympathetic organs, whereas the median cells form processes in the terminal abdominal ganglion and supply terminals on the hindgut. Because PDF does not influence hindgut contractility, it is possible that also these median neurons release PDF into the circulation. Release from one or both the PDFI neuron types was confirmed by measurements of PDF-immunoreactivity in hemolymph by enzyme immunoassay. PDF applied to the terminal abdominal ganglion triggers firing of action potentials in motoneurons with axons in the genital nerves of males and the 8th ventral nerve of females. Because this action is blocked in calcium-free saline, it is likely that PDF acts via interneurons. Thus, PDF seems to have a modulatory role in central neuronal circuits of the terminal abdominal ganglion that control muscles of genital organs.

Intestinal peptides as circulating hormones: release of tachykinin-related peptide from the locust and cockroach midgut.

Tachykinin-related peptides (TRPs) in the locust Locusta migratoria and the cockroach Leucophaea maderae have stimulatory effects on some muscles that are not innervated by TRP-containing neurons. Thus, these tissues may be affected by circulating TRPs. Here, we have investigated whether the midgut is the source of circulating TRPs. TRP-immunoreactive material in the locust midgut is found only in the endocrine cells of the gut epithelium. In both species of insect, the endocrine cells contain several isoforms of TRPs, as determined by immunocytochemistry and a combination of chromatography (HPLC) and enzyme immunoassay (ELISA). The release of TRPs was investigated by ELISA using isolated midguts of the locust and cockroach. Elevated levels of K(+) in the bathing saline induced the release of TRP from the midgut of both species. To examine the release of TRPs into the circulation in vivo, we measured haemolymph levels of TRPs in fed and starved locusts. The concentration of TRP-immunoreactive material in fed locusts was estimated to be 0.15 nmol l(-1), and this increased approximately fourfold in insects starved for 24 h. In accordance with this observation, the content of TRP-immunoreactive material in the midgut was lower in starved locusts than in fed locusts. Although part of the increased blood concentration of TRPs may be due to reduced blood volume, our data suggest that TRPs are released as hormones from the midgut of the locust and cockroach and that this release may be linked to nutritional status.

Molecular cloning, genomic organization, and expression of a C-type (Manduca sexta-type) allatostatin preprohormone from Drosophila melanogaster.

The insect allatostatins are a diverse group of neuropeptides that obtained their names by their inhibitory actions on the corpora allata (two endocrine glands near the insect brain), where they block the biosynthesis of juvenile hormone (a terpenoid important for development and reproduction). Chemically, the allatostatins can be subdivided into three different peptide groups: the large group of A-type (cockroach-type) allatostatins, which have the common C-terminal sequence Y/FXFGLamide; the B-type (cricket-type) allatostatins, which have the C-terminal sequence W(X(6))Wamide in common; and a single allatostatin that we now call C-type allatostatin that was first discovered in the moth Manduca sexta, and which has a nonamidated C terminus, and a structure unrelated to the A- and B-type allatostatins. We have previously cloned the preprohormones for the A- and B-type allatostatins from Drosophila melanogaster. Here we report on the cloning of a Drosophila C-type allatostatin preprohormone (DAP-C). DAP-C is 121 amino acid residues long and contains one copy of a peptide sequence that in its processed form has the sequence Y in position 4) from the Manduca sexta C-type allatostatin. The DAP-C gene has three introns and four exons and is located at position 32D2-3 on the left arm of the second chromosome. Northern blots show that the gene is strongly expressed in larvae and adult flies, but less in pupae and embryos. In situ hybridizations of larvae show that the gene is expressed in various neurons of the brain and abdominal ganglia and in endocrine cells of the midgut. This is the first publication on the structure of a C-type allatostatin from insects other than moths and the first report on the presence of all three types of allatostatins in a representative of the insect order Diptera (flies).

The distribution of a kinin-like peptide and its co-localization with a CRF-like peptide in the blood-feeding bug, Rhodnius prolixus.

Rhodnius prolixus, a blood-feeding hemipteran insect, ingests large meals which are followed by rapid diuresis to eliminate excess water and salt. In Rhodnius, serotonin and an unidentified peptide(s) [33,34] have been shown to act as neurohormonal diuretic factors. In other insects, two families of diuretic peptides, the corticotropin releasing factor (CRF)-like, and kinin peptides [9], have been identified and sequenced. Recently, we demonstrated the presence of a CRF-like diuretic peptide in the CNS and digestive system of Rhodnius [47] using immunohistochemistry and bioassay. In this study, combining immunohistochemistry and radioimmunoassay (RIA) techniques, we show the presence of leucokinin-like peptide(s) in the CNS and digestive system of Rhodnius 5th instar. Additionally, double-label immunohistochemistry demonstrates that the leucokinin-like and CRF-like peptides are co-localized in the posterior lateral neurosecretory cells of the mesothoracic ganglionic mass (MTGM) and in neurohaemal areas on abdominal nerves one and two, suggesting the possibility of co-release of the peptides into the hemolymph.Partially purified extracts of the CNS and neurohaemal tissue were tested in vitro on Malpighian tubule secretion and cAMP assays. The factors eluting with increasing acetonitrile percentages from Sep-Pak cartridges were assayed in the presence or absence of ketanserin, a serotonin antagonist which blocks the effects of serotonin on Malpighian tubules. The results indicate activity of serotonin and a CRF-like diuretic peptide on Rhodnius Malpighian tubules, but fail to demonstrate activity of the leucokinin-like peptide(s). The rapid diuresis following feeding is a highly coordinated event, requiring the movement of water and salt across the epithelial cells of the crop into the hemolymph, and from the hemolymph across the cells of the Malpighian tubules. The urine then travels along the Malpighian tubules into the hindgut in order to be expelled. The presence of a leucokinin-like peptide(s) in the CNS and digestive system, which co-localizes with a CRF-like peptide(s), suggests that kinins may play a role in the rapid diuresis, although possibly not directly on the Malpighian tubules.

Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species.

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus. Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae, CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria. The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10(-9)-10(-6) M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus. CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10(-9)-10(-6) M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor, however, the CavTKs induced no significant effect on the heart. Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut. In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.

Molecular cloning, genomic organization, and expression of a B-type (cricket-type) allatostatin preprohormone from Drosophila melanogaster.

The insect allatostatins obtained their names because they block the biosynthesis of juvenile hormone (a terpenoid) in the corpora allata (two endocrine organs near the insect brain). Chemically, the allatostatins can be subdivided into three different peptide groups: the A-type allatostatins, first discovered in cockroaches, which have the C-terminal sequence Y/FXFGLamide in common; the B-type allatostatins, first discovered in crickets, which all have the C-terminal sequence W(X)(6)Wamide; and the C-type allatostatins, first discovered in the moth Manduca sexta, which have an unrelated and nonamidated C terminus. We have previously reported the structure of an A-type allatostatin preprohormone from the fruitfly Drosophila melanogaster. Here we describe the molecular cloning of a B-type prepro-allatostatin from Drosophila (DAP-B). DAP-B is 211 amino acid residues long and contains one copy each of the following putative allatostatins: AWQSLQSSWamide (drostatin-B1), AWKSMNVAWamide (drostatin-B2),

Primary commissure pioneer neurons in the brain of the grasshopper Schistocerca gregaria: development, ultrastructure, and neuropeptide expression.

The bilaterally paired primary commissure pioneer neurons in the median domain of the grasshopper brain are large, descending interneurons that uniquely express the TERM-1 antigen, even in the adult. After pioneering the primary interhemispheric brain commissure, these neurons extend TERM-1-immunoreactive collaterals into most parts of the brain except the mushroom bodies. In this report, the authors show that the TERM-1 antigen is located in the cell body cytoplasm of these neurons and not on the membranes. Screening with antisera to insect neuropeptides reveals that an antiserum recognizing peptides of the leucokinin family labels the cell body cytoplasm of the primary commissure neurons. Leucokinin-related peptides are known to modulate motility of visceral muscle, play a role in diuresis, and are likely to be neuromodulators in the insect nervous system. The primary commissure neurons differ ultrastructurally from median neurosecretory cells in that their cell body cytoplasm is more extensive, contains high numbers of mitochondria and extensive endoplasmic reticulum, but does not contain neurosecretory granules. In the adult, the cell somata are enveloped by multiple glia membranes and associated trophospongia. According to these ultrastructural characteristics, the primary commissure pioneers are not classical neurosecretory cells.

Functional roles of neuropeptides in the insect central nervous system.

With the completion of the Drosophila genome sequencing project we can begin to appreciate the extent of the complexity in the components involved in signal transfer and modulation in the nervous system of an animal with reasonably complex behavior. Of all the different classes of signaling substances utilized by the nervous system, the neuropeptides are the most diverse structurally and functionally. Thus peptidergic mechanisms of action in the central nervous system need to be analyzed in the context of the neuronal circuits in which they act and generalized traits cannot be established. By taking advantage of Drosophila molecular genetics and the presence of identifiable neurons, it has been possible to interfere with peptidergic signaling in small populations of central neurons and monitor the consequences on behavior. These studies and experiments on other insects with large identifiable neurons, permitting cellular analysis of signaling mechanisms, have outlined important principles for temporal and spatial action of neuropeptides in outputs of the circadian clock and in orchestrating molting behavior. Considering the large number of neuropeptides available in each insect species and their diverse distribution patterns, it is to be expected that different neuropeptides play roles in most aspects of insect physiology and behavior.

Peptide-induced Ca(2+) movements in a tonic insect muscle: effects of proctolin and periviscerokinin-2.

Although most of the characterized insect neuropeptides have been detected by their actions on muscle contractions, not much is known about the mechanisms underlying excitation-contraction coupling. Thus we initiated a pharmacological study on the myotropic action of the peptides periviscerokinin-2 (PVK-2) and proctolin on the hyperneural muscle of the cockroach Periplaneta americana. Both peptides required extracellular Ca(2+) to induce muscle contraction, and a blockage of sarcolemmal Ca(2+) channels by Mn(2+) or La(3+) inhibited myotropic effects. The peptides were able to induce contractions in dependence on the extracellular Ca(2+) concentration in muscles depolarized with high K(+) saline. A reduction of extracellular Na(+), K(+), or Cl(-) did not effect peptide action. Nifedipine, an L-type Ca(2+)-channel blocker, partially blocked the response to both peptides but to a much lesser extent than contractions evoked by elevated K(+). Using calcium imaging with fluo-3, we show that proctolin induces an increase of the intracellular Ca(2+) concentration. In calcium-free saline, no increase of the intracellular Ca(2+) concentration could be detected. The inhibiting effect of ryanodine, thapsigargin, and TMB-8 on peptide-induced contractions suggests that Ca(2+) release from the sarcoplasmic reticulum plays a major role during peptide-induced contractions. Preliminary experiments suggest that the peptides do not employ cyclic nucleotides as second messengers, but may activate protein kinase C. Our results indicate that the peptides induce Ca(2+) influx by an activation or modulation of dihydropyridine-sensitive and voltage-independent sarcolemmal Ca(2+) channels. Ca(2+)-induced Ca(2+) release from intracellular stores, but not inositol trisphosphate-induced Ca(2+) release, seems to account for most of the observed increase in intracellular Ca(2+). Additionally, both peptides were able to potentiate glutamate-induced contractions at threshold concentrations.

Allatotropin-like neuropeptide in the cockroach abdominal nervous system: myotropic actions, sexually dimorphic distribution and colocalization with serotonin.

Allatotropin (AT) was isolated from the moth Manduca sexta as a peptide stimulating biosynthesis of juvenile hormone in the corpora allata, but has also been shown to be cardioactive in the same species. Here, we have investigated the presence and biological activity of AT-like peptide in the cockroaches Leucophaea maderae and Periplaneta americana with focus on abdominal ganglia and their target tissues. An antiserum to M. sexta AT was used for immunocytochemical mapping of neurons in the abdominal ganglia. A small number of interneurons and efferent neurons were found AT-like immunoreactive (AT-LI) in each of the abdominal ganglia. A prominent sexual dimorphism was detected in the terminal abdominal ganglion: in L. maderae the male ganglion there are approximately 18 AT-LI neurons with cell bodies posteriorly and efferent axons in the genital nerves; in the female ganglion 4-5 AT-LI cell bodies (with efferent axons) were found in the same region. Correlated with the extra efferents in males, the male accessory glands are richly supplied by AT-LI fibers and in females a less prominent innervation was seen in oviduct muscle. A similar dimorphism was seen in abdominal ganglia of P. americana. A sexual dimorphism was also detected in the abdominal ganglia A4-A6 of L. maderae. In each of these ganglia, approximately 8-10 large AT-LI neuronal cell bodies were found along the midline; in females these neurons have significantly larger cell bodies than in males. In both sexes, and both cockroach species, two large dorsal midline neurons were detected in A-5 and 6, which seem to send axons to the hindgut: the rectal pads of the hindgut are supplied by arborizing AT-LI axons. In males and females of both species, efferent AT-LI axons from midline neurons in A3-A6 supply the lateral heart nerves and other neurohemal release sites with arborizations. The efferent midline neurons of females contain colocalized serotonin-immunoreactivity. We tested the in vitro actions of M. sexta AT on muscle contractions in the L. maderae hindgut and the abdominal heart of both species. The frequency of contractions in the hindgut increased dose dependently when applying AT at 5 x 10(-8) to 5 x 10(-6) M (maximal response at 5 x 10(-7) M). Also the frequency of contractions of the heart increased by application of AT (threshold response at 5 x 10(-9) M). This effect was more prominent in males of both species (maximal response was a 35-40% increase in males and 10-20% in females). In conclusion, an AT-like peptide is present in neurons and neurosecretory cells of cockroach abdominal ganglia and seems to play a role in control of contractions in the hindgut and heart and also to have some function in male accessory glands and oviduct.

High environmental salinity induces memory enhancement and increases levels of brain angiotensin-like peptides in the crab Chasmagnathus granulatus.

Previous work on the brackish-water crab Chasmagnathus granulatus demonstrated that an endogenous peptide similar to angiotensin II plays a significant role in enhancing long-term memory that involves an association between context and an iterative danger stimulus (context-signal memory). The present results show that this memory enhancement could be produced by moving crabs from brackish water to sea water (33.0%) and keeping them there for at least 4 days. The possibility that such a facilitatory effect is due to osmotic stress is ruled out. Coincidentally, the level of angiotensin-II-like peptides in crab brain, measured by radioimmunoassay, increases with the length of exposure to sea water, reaching a significantly different level at the fourth day. The presence of angiotensin-II-like immunoreactive material in neural structures of the supraoesophageal and eyestalk ganglia was confirmed by immunohistochemical analysis. The results are interpreted as supporting the hypothesis that exposure to water of high salinity is an external cue triggering a process mediated by angiotensins that leads to enhanced memory in these crabs.

Baratin, a nonamidated neurostimulating neuropeptide, isolated from cockroach brain: distribution and actions in the cockroach and locust nervous systems.

During the purification of tachykinin-related peptides from the brain of the cockroach Leucophaea maderae, a few other peptides were collected in adjacent high-performance liquid chromatography fractions. Edman degradation, mass spectrometry, and chemical synthesis revealed that one of these peptides had the sequence DNSQWGGFA. This nonamidated nonapeptide was designated baratin and appears not to be related to any known insect peptide. Baratin was not found to be bioactive in the L. maderae hindgut or oviduct muscle contraction assay. (Both synthetic nonamidated and amidated baratin were tested.) To screen for possible sites of action, we raised a rabbit antiserum to baratin. We found baratin-immunoreactive (BAR-IR) interneurons throughout the cockroach central nervous system. Some prominent brain neuropils were supplied by BAR-IR neuron processes: the central body, the calyx, and lobes of the mushroom bodies, parts of the optic lobe, and the tritocerebral neuropil. Additionally we found BAR-IR neurosecretory cells in the median neurosecretory cell group with processes supplying the storage lobe of the corpora cardiaca. In each of the thoracic and abdominal ganglia processes of BAR-IR projection neurons and local neurons were seen. The baratin antiserum also labeled neurons in the brain of the locust Locusta migratoria, some of which are similar to those of the cockroach. A prominent system of interganglionic BAR-IR processes was found in the locust subesophageal, thoracic, and abdominal ganglia. This was formed by four large projection neurons with cell bodies in the abdominal ganglia A1-2. The processes of these BAR-IR neurons are distributed dorsally and laterally in each of the ventral nerve cord ganglia. When baratin (10(-6)-10(-4) M) was applied to desheathed abdominal ganglia of locusts and cockroaches, we could monitor bursts of action potentials in neurons with axons in the anterior abdominal nerve (nerve 1), but not in the posterior nerve (nerve 2). In ganglia displaying spontaneous rhythmic firing in units of nerve 1, baratin strengthened the rhythmic pattern. Thus baratin appears to have a role in modulation of motor patterns in abdominal ganglia. The immunocytochemical findings suggest further modulatory actions of baratin in different circuits of the brain and ventral nerve cord.

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.

Proline-specific dipeptidyl peptidase activity in the cockroach brain and intestine: partial characterization, distribution, and inactivation of tachykinin-related peptides.

Proline-specific dipeptidyl peptidase (DPP IV) is an established enzyme known to degrade neuropeptides and peptide hormones in vertebrate tissues. DPP IV cleaves peptides at the Pro2 residue. Because several neuropeptides of the cockroach Leucophaea maderae, such as LemTRP-1 (APSGFLGVRamide), are potential substrates for this peptidase, we investigated the occurrence of proline-specific DPP activity in cockroach tissues. Partly purified DPP activity was characterized from the brain and midgut of L. maderae by using Gly-Pro-4-nitroanilide as a substrate. The highest activity was obtained from the membrane fraction of intestine; about 10 times less activity (per milligram protein) was obtained from brain membranes. A smaller amount of soluble DPP activity could also be identified in both tissues. Gel chromatography of the solubilized intestinal DPP activity revealed a molecular mass of about 75 kDa. The enzyme had a pH optimum of 8.5. Diprotin A (Ile-Pro-Ile) was an efficient competitive inhibitor of the cockroach DPP, whereas other known DPP inhibitors were found to be less potent. When incubated with human and cockroach DPP IV, the cleavage products of LemTRP-1 were AP and SGFLGVRamide (des-AP-LemTRP-1) as determined by mass spectrometry of high-performance liquid chromatography (HPLC)-purified peptide fragments. The AP fragment was biologically inactive and the des-AP fragment had a drastically reduced myostimulatory activity on the hindgut of L. maderae. The blowfly TRP callitachykinin-I (CavTK-I; APTAFYGVRamide) was cleaved in two steps to des-AP-CavTK-I and desAPTA-CavTK-I, showing that cockroach DPP does not only liberate Xaa-Pro, but also Xaa-Ala dipeptides. The fragment desAPTA-CavTK-I was completely inactive on the cockroach hindgut. To compare, LemTRP-3 and CavTK-II, which lack a Pro2, were not cleaved by DPP IV. Enzyme histochemistry for DPP IV was performed on cryostat sections of brain and intestine with Gly-Pro-4-methoxy-2-naphthylamide as the substrate and Fast Blue B as the chromogen. Strong histochemical labeling was seen in specific neuropils of the brain such as the calyces of the mushroom bodies, the antennal glomeruli, and the central body. Also, the inner lining of the midgut (the peritrophic membrane) and the malpighian tubules were strongly labeled by reaction product. In both the brain and intestine, the enzyme-histochemical reaction was inhibited by diprotin A.

Tachykinin-related peptide and GABA-mediated presynaptic inhibition of crayfish photoreceptors.

Off-axis illumination elicits lateral inhibition at the primary visual synapse in crustacea and insects. The evidence suggests that the inhibitory action is presynaptic (i.e., on the photoreceptor terminal) and that the amacrine neurons of the lamina ganglionaris (the first synaptic layer) may be part of the inhibitory pathway. The neurotransmitters and the synaptic mechanisms are unknown. We show by immunocytochemistry that GABA and a tachykinin-related peptide (TRP) are localized in the amacrine neurons of the crayfish lamina ganglionaris. Indirect evidence suggests that GABA and TRP may be colocalized in these neurons. The extensive processes of the amacrine neurons occupy lamina layers containing the terminals of photoreceptors. Application of exogenous GABA and TRP to photoreceptor terminals produces a short-latency, dose-dependent hyperpolarization with a decay time constant on the order of a few seconds. TRP also exhibits actions that evolve over several minutes. These include a reduction of the receptor potential (and the light-elicited current) by approximately 40% and potentiation of the action of GABA by approximately 100%. The mechanisms of TRP action in crayfish are not known, but a plausible pathway is a TRP-dependent elevation of intracellular Ca(2+) that reduces photoreceptor sensitivity in arthropods. Although the mechanisms are not established, the results indicate that in crayfish photoreceptors TRP displays actions on two time scales and can exert profound modulatory control over cell function.

Locustatachykinin isoforms in the locust: distribution and quantification in the central nervous system and action on the oviduct muscle.

The presence of locustatachykinin (LomTK)-like immunoreactivity is demonstrated in the central nervous system (CNS) of Locusta migratoria with the use of a polyclonal antiserum raised against LomTK1. By developing a radioimmunoassay with the same antiserum, we have demonstrated picomolar amounts of LomTK-like material in the tissues of the central nervous system. In contrast, only femptomolar amounts of LomTK-like material are associated with the oviduct tissue. The relative amounts of the different LomTK isoforms in the brain and the abdominal ganglionic chain were examined by separating the native peptides on high-performance liquid chromatography and comparing their retention times to synthetic LomTK standards. The amounts of the different isoforms of LomTK differed between and within the two regions of the central nervous system. However, the ratios of the different isoform amounts were similar between the two regions. The myostimulatory activities of LomTKs 1 to 4 were characterized by using the locust oviduct bioassay. LomTKs 1, 2, and 3 appeared to be more efficacious than LomTK4.

Structure, distribution, and biological activity of novel members of the allatostatin family in the crayfish Orconectes limosus.

In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.