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PubMed2003

Conservation of ecdysis-triggering hormone signalling in insects.

Pre-ecdysis- and ecdysis-triggering hormones (PETH and ETH) from endocrine Inka cells initiate ecdysis in moths and Drosophila through direct actions on the central nervous system (CNS). Using immunohistochemistry, we found Inka cells in representatives of all major insect orders. In most insects, Inka cells are numerous, small and scattered throughout the tracheal system. Only some higher holometabolous insects exhibit 8-9 pairs of large Inka cells attached to tracheae in each prothoracic and abdominal segment. The number and morphology of Inka cells can be very variable even in the same individuals or related insects, but all produce peptide hormones that are completely released at each ecdysis. Injection of tracheal extracts prepared from representatives of several insect orders induces pre-ecdysis and ecdysis behaviours in pharate larvae of Bombyx, indicating functional similarity of these peptides. We isolated several PETH-immunoreactive peptides from tracheal extracts of the cockroach Nauphoeta cinerea and the bug Pyrrhocoris apterus and identified the gene encoding two putative ETHs in the mosquito Anopheles gambiae. Inka cells also are stained with antisera to myomodulin, FMRFamide and other peptides sharing RXamide carboxyl termini. However, our enzyme immunoassays show that these antisera cross-react with PETH and ETH. Our results suggest that Inka cells of different insects produce only peptide hormones closely related to PETH and ETH, which are essential endocrine factors required for activation of the ecdysis behavioural sequence.

Animals↗
PubMed2006

Transcriptional regulation of neuropeptide and peptide hormone expression by the Drosophila dimmed and cryptocephal genes.

The regulation of neuropeptide and peptide hormone gene expression is essential for the development and function of neuroendocrine cells in integrated physiological networks. In insects, a decline in circulating ecdysteroids triggers the activation of a neuroendocrine system to stimulate ecdysis, the behaviors used to shed the old cuticle at the culmination of each molt. Here we show that two evolutionarily conserved transcription factor genes, the basic helix-loop-helix (bHLH) gene dimmed (dimm) and the basic-leucine zipper (bZIP) gene cryptocephal (crc), control expression of diverse neuropeptides and peptide hormones in Drosophila. Central nervous system expression of three neuropeptide genes, Dromyosuppressin, FMRFamide-related and Leucokinin, is activated by dimm. Expression of Ecdysis triggering hormone (ETH) in the endocrine Inka cells requires crc; homozygous crc mutant larvae display markedly reduced ETH levels and corresponding defects in ecdysis. crc activates ETH expression though a 382 bp enhancer, which completely recapitulates the ETH expression pattern. The enhancer contains two evolutionarily conserved regions, and both are imperfect matches to recognition elements for activating transcription factor-4 (ATF-4), the vertebrate ortholog of the CRC protein and an important intermediate in cellular responses to endoplasmic reticulum stress. These regions also contain a putative ecdysteroid response element and a predicted binding site for the products of the E74 ecdysone response gene. These results suggest that convergence between ATF-related signaling and an important intracellular steroid response pathway may contribute to the neuroendocrine regulation of insect molting.

Animals↗
PubMed2006

The regenerative nidi of the locust midgut as a model to study epithelial cell differentiation from stem cells.

A better knowledge of the regulatory mechanisms involved in stem cell proliferation and/or differentiation could reveal new methods for the treatment of some diseases. Most previous studies in the field of stem cell biology have been carried out on cultured isolated cells. In the case of adult tissue stem cells, mesenchymal bone marrow derived cells have been most widely studied, while the undifferentiated stem cells present in the epithelial tissues are less known. In order to advance further our understanding of epithelial tissue stem cells, new in vivo models are required. The present study focuses on the dynamics of a new and simple model of intestinal epithelial regeneration found in the midgut of the migratory locust, Locusta migratoria (Linnaeus 1758). The locust midgut consists of three cell types: columnar cells, endocrine cells and undifferentiated regenerative clustered cells. The undifferentiated epithelial midgut cells give rise to two other cell types and are located in a nest of regenerative cells known as regenerative niche. We have performed single and continuous bromodeoxyuridine (BrdU) administration experiments to study regeneration niches and their cellular dynamics. Immunocytochemistry and immunofluorescence techniques were used to detect the incorporation of BrdU into regenerative niches and the presence of FMRFamide-like immunoreactivity, as a marker for endocrine cell differentiation. Some isolated label retaining cells (LRC) were observed at the niche base 10-15 days after the final BrdU administration. We propose that these cells are the stem cells of this epithelial tissue. We also calculated the length of the cell cycle phases for a subpopulation of transit amplifying cells within the regenerative niche: G1, 2.5+/-0.5 h; S, 5.5+/-0.5 h; G2, 0.75+/-0.25 h; M, 2.5+/-0.5 h. These amplifying cells will give rise to the columnar epithelial non-endocrine lineage. The differentiation of an endocrine cell from a niche stem cell occurs less frequently and thus leads to a lower proportion of endocrine cells as compared with epithelial columnar digestive cells (up to three endocrine cells per niche). Endocrine cell commitment seems to occur very early in the differentiation process within the niche, as double-labelled BrdU and FMRF endocrine cells have never been found. The only exception is the endocrine cells located in the ampullar region of the midgut, some of which show double immunostaining after long-term chronic BrdU injection. In summary, we have characterized a new and simple animal model of epithelial stem cell regeneration that may be useful for understanding the complex biological process that drives tissue renewal from undifferentiated and uncommitted progenitor cells.

Animals↗
PubMed2006

Neural control of the velum in larvae of the gastropod, Ilyanassa obsoleta.

Larval molluscs commonly use ciliated vela to swim and feed. In this study we used immunohistochemistry to demonstrate innervation of velar cilia and muscles by monoaminergic and peptidergic fibres in the caenogastropod, Ilyanassa obsoleta. Photoelectric recordings from pre-oral cilia on isolated pieces of velum revealed that serotonin increased, whereas catecholamines (dopamine and norepinephrine) decreased beat frequency at concentrations of 10(-6) to 10(-9) mol l(-1). Catecholamines also increased the frequency of momentary, isolated arrests of pre-oral cilia, but failed to suppress beating of the post-oral cilia at these concentrations. The neuropeptides, FMRFamide and Leu-enkephalin, did not affect the frequency of ciliary beating or of isolated ciliary arrests, but did induce numerous muscular contractions, which were accompanied by sustained ciliary arrests. In terms of whole animal behaviour, serotonin caused larvae to concentrate toward the top of a water column and to increase feeding, whereas catecholamines caused larvae to concentrate toward the bottom of a water column and decrease feeding. Monoamine analogues which facilitated or opposed the effects of synthetic transmitters on larval behaviour, further suggested that these transmitters are released endogenously to control velar function. Finally, applications of peptides to whole larvae caused increased frequency of locomotory arrests. Together these findings demonstrate several potential roles for the nervous system in controlling larval behaviour in gastropods.

Animals↗
PubMed1991

Receptive fields and properties of a new cluster of mechanoreceptor neurons innervating the mantle region and the branchial cavity of the marine mollusk Aplysia californica.

The rostral LE cluster (rLE) is a new set of mechanoreceptor neurons of the abdominal ganglion innervating the mantle area, the branchial cavity, the gill and the siphon of the marine mollusk Aplysia californica Cooper. We have compared the organization of rLE cell receptive fields with that of three other clusters of sensory neurons in the abdominal ganglion (LE, RE and RF) that we have reanalysed. There is extensive overlap of receptive fields from the four populations of sensory cells, and the most exposed areas of the mantle are the most densely innervated. The sensory threshold is similar for all groups. The action potentials of the LE, rLE and RE neurons are broadened by serotonin and the peptide SCPB and narrowed by dopamine and FMRFamide. The RF group does not show the same kind of sensitivity to these neuromodulators. The synaptic outputs of the LE and rLE neurons undergo similar synaptic depression and homosynaptic and heterosynaptic facilitation. We estimate that 100 mechanoreceptor neurons innervate the entire mantle and siphon skin, gill and branchial cavity of Aplysia. The degree of their convergence onto various interneurons and motor neurons mediating the gill- and siphon-withdrawal reflex and other reflexes is under investigation.

Animals↗
PubMed1994

The effects of myomodulin and structurally related neuropeptides on skeletal neuromuscular transmission in the locust.

1. The modulatory actions of myomodulin A on tension generated in the extensortibiae muscle of the locust hindleg by stimulation of the slow excitatory motoneurone (SETi) depend upon the frequency of stimulation. Myomodulin A has no consistent effect on the tension induced by the fast extensor motoneurone (FETi) or upon the myogenic rhythm present in the extensor. The effects of a range of structurally related neuropeptides have also been assessed. 2. At low frequencies of SETi stimulation (1 Hz and below), the predominant modulatory effects are increases in the amplitude, contraction rate and relaxation rate of twitch tension. At higher frequencies, where twitches summate but tetanus is incomplete (up to 20 Hz), these effects are superimposed upon an increase of maintained tension. 3. The modulatory actions of myomodulin-like peptides show some similarities to and some differences from the modulatory actions of octopamine, proctolin and FMRFamide-like neuropeptides in this preparation, but are likely to be mediated via a distinct set of receptors. 4. The results of the present study, taken together with the localization of myomodulin-like immunoreactivity in specific sets of neurones in the locust nervous system, suggest the presence of a novel modulatory system in insects that uses myomodulin-like neuropeptides. It also indicates that myomodulins, which were first identified in molluscs, may represent another interphyletic family of neuropeptides.

Animals↗
PubMed1997

The aminergic and peptidergic innervation of insect salivary glands

Insect salivary glands are glands associated with nutrient intake whose secretions are generally involved in the digestion and lubrication of food. They are under the control of neuroactive substances and may be innervated from several sources including the suboesophageal ganglion, the stomatogastric nervous system and the unpaired median nerves. Both amines and peptides have been suggested to play roles in the control of insect salivation, as indicated by their association with terminals on salivary glands, their effects in salivary gland bioassays and their ability to alter second messenger levels and ion channel conformations. Serotonin and dopamine appear to be the most prominent amines associated with insect salivary glands. Either one or both of these amines are found associated with the salivary glands of the locust, stick insect, cockroach, cricket, dragonfly, mosquito, adult moth and kissing bug. Their roles, although not fully elucidated, appear to be in the control of salivary secretion. Several peptides, including members of the FMRFamide-related family of peptides, are also found associated with insect salivary glands. Sources of peptidergic innervation are as varied as those for aminergic innervation, but information regarding the physiological role of these peptides is lacking. The relevance of the different levels of complexity of salivary gland innervation, which range from the absence of innervation in some species (blowfly) to the presence of several distinct sources in others (locust, cockroach), is not well understood. This review serves to consolidate what is known of the phenotype of salivary neurones in relation to the control of salivation.

Journal Article↗
PubMed1998

Inhibition of midgut ion transport by allatotropin (Mas-AT) and Manduca FLRFamides in the tobacco hornworm Manduca sexta.

Short-circuit current (Isc) across the posterior midgut of day 2 fifth-instar tobacco hornworms (Manduca sexta) is inhibited by Manduca allatotropin (Mas-AT) and two Manduca FLRFamides (F7G and F7D). Another FLRFamide, F10, and the related molluscan tetrapeptide FMRFamide elicited only a modest inhibition of Isc. Mas-AT, F7G and F7D inhibited the Isc in a dose-dependent manner. Maximal inhibition of Isc by Mas-AT was achieved at a concentration of 50 nmol l-1. At 100 nmol l-1, Mas-AT produced a significantly greater inhibition of Isc than did F7G and F7D. The inhibition caused by Mas-AT was reversed by removing the peptide. Furthermore, the action of Mas-AT could be blocked by preabsorption with its antiserum. When F7G or F7D was added after the Isc had been inhibited by Mas-AT, a further reduction in the Isc was observed. No additive effects were seen when F7G and F7D were added together. In comparison with the anterior and middle regions, the posterior section of the midgut was the most sensitive to these three peptides. The Isc of midguts dissected from feeding fourth instars was inhibited by Mas-AT, F7D and F7G, whereas the Isc of midguts dissected from pharate fifth instars or wandering fifth instars was virtually unaffected by these peptides. Active ion transport across the posterior midgut of the silk moths Hyalophora cecropia and Bombyx mori was unaffected by these peptides.

Amino Acid Sequence↗
PubMed2002

Modulation of ecdysis in the moth Manduca sexta: the roles of the suboesophageal and thoracic ganglia.

The sequential behaviours shown by insects at ecdysis are due to the sequential release of various hormones, but the transition from one phase to the next can be fine-tuned by inhibitory influences. The ecdysis sequence in the moth Manduca sexta was initiated by injecting sensitive animals with the neuropeptide ecdysis-triggering hormone (ETH). Exposure to ETH stimulates the release of eclosion hormone (EH) which, in turn, activates a set of neurons containing crustacean cardioactive peptide (CCAP) by elevating their levels of intracellular cyclic GMP. We characterized a set of non-CCAP containing neurons that also appear to be EH targets because of their response to cyclic GMP at ecdysis. The neurons did not display leucokinin-, diuretic-hormone- or FMRFamide-like immunoreactivity. They are probably the bursicon-containing cells described previously. After release of EH, there is a transient inhibition of the abdominal centers responsible for ecdysis. Transection experiments suggested that this suppression is via descending inhibitory units from the suboesophageal and thoracic ganglia. The duration of this inhibition appears to depend on the levels of cyclic GMP and can be extended by pharmacologically suppressing cyclic GMP breakdown. We further found that brief exposure to CO(2) caused premature ecdysis. Since the CO(2) treatment was effective only after EH release, it probably acts by suppressing descending inhibition. Studies on adult eclosion suggest that CO(2), given at the appropriate time, can uncouple the basic larval motor program from modulatory influences provided by the adult pterothoracic ganglion. CO(2) therefore appears to be a novel and non-invasive tool for studies of ecdysis behavior in insects.

Animals↗
PubMedDate not supplied

The effect of neuropeptides on the ERG of the crayfish Orconectes limosus.

CCAP (Crustacean Cardioactive Peptide), Proctolin, FMRFamide, Met- and Leu-enkephalin, Substance P, RPCH (red pigment concentrating hormone) and PDH (pigment dispersing hormone) were applied to the isolated retina of the crayfish Orconectes limosus. Changes in light sensitivity, measured as changes of the amplitude of the electroretinogram (ERG) were observed after application of RPCH, PDH and CCAP. RPCH caused an increase of the ERG amplitude to 133% of its reference value whereas PDH and CCAP decreased the amplitude to 78% and 30% respectively. A dose-response curve showed that 10(-9) mol/l CCAP produce a half-maximal effect.

Animals↗
PubMed2004

Synaptic strengthening mediated by bone morphogenetic protein-dependent retrograde signaling in the Drosophila CNS.

Retrograde signaling is an essential component of synaptic development and physiology. Previous studies show that bone morphogenetic protein (BMP)-dependent retrograde signaling is required for the proper development of the neuromuscular junction (NMJ) in Drosophila. These studies, moreover, raised the significant possibility that the development of central motor circuitry might similarly be reliant on such signaling. To test this hypothesis, retrograde signaling between postsynaptic motoneurons and their presynaptic interneurons is examined. Postsynaptic expression of an adenylate cyclase encoded by rutabaga (rut), is sufficient to strengthen synaptic transmission at these identified central synapses. Results are presented to show that the underlying mechanism is dependent on BMP retrograde signaling. Thus, presynaptic expression of an activated TGF-beta receptor, thickvien (tkv), or postsynaptic expression of a TGF-beta ligand, glass-bottom boat (gbb), is sufficient to phenocopy strengthening of synaptic transmission. In the absence of gbb, endogenous synaptic transmission is significantly weakened and, moreover, postsynaptic overexpression of rut is unable to potentiate synaptic function. Potentiation of presynaptic neurotransmitter release, mediated by increased postsynaptic expression of gbb, is dependent on normal cholinergic activity, indicative that either the secretion of this retrograde signal, or its transduction, is activity dependent. Thus, in addition to the development of the NMJ and expression of myoactive FMRFamide-like peptides in specific central neurons, the results of the present study indicate that this retrograde signaling cascade also integrates the development and function of central motor circuitry that controls movement in Drosophila larvae.

Adenylyl Cyclases↗
PubMed2007

The role of RFamide peptides in feeding.

In the three decades since FMRFamide was isolated from the clam Macrocallista nimbosa, the list of RFamide peptides has been steadily growing. These peptides occur widely across the animal kingdom, including five groups of RFamide peptides identified in mammals. Although there is tremendous diversity in structure and biological activity in the RFamides, the involvement of these peptides in the regulation of energy balance and feeding behaviour appears consistently through evolution. Even so, questions remain as to whether feeding-related actions represent a primary function of the RFamides, especially within mammals. However, as we will discuss here, the study of RFamide function is rapidly expanding and with it so is our understanding of how these peptides can influence food intake directly as well as related aspects of feeding behaviour and energy expenditure.

Analgesics, Opioid↗
PubMed1988

An acetylcholine-induced potassium current in tail sensory neurons in the pleural ganglion of Aplysia.

Acetylcholine (ACh) induces a hyperpolarization during current clamp and an outward current during voltage clamp in tail sensory neurons of Aplysia kurodai. This response was proved to be produced by a specific increase in membrane permeability toward potassium ions, the cholinergic antagonists, d-tubocurarine chloride (d-TC), and atropine mildly reduced the ACh response, while tetraethylammonium (TEA) most effectively blocked this response. These findings provide evidence that tail sensory neurons have the inhibitory ACh receptor in addition to the known receptors for serotonin (5-HT), small cardioactive peptide B (SCPB), and neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide).

Acetylcholine↗
PubMed2002

Neuropeptide signaling systems - potential drug targets for parasite and pest control.

Current problems of drug resistance in parasites and pests demand the identification of new targets and their exploitation through novel drug design and development programs. Neuropeptide signaling systems in helminths (nematodes and platyhelminths = worms) and arthropods are well developed and complex, play a crucial role in many aspects of their biology, and appear to have significant potential as targets for novel drugs. The best-known neuropeptide family in invertebrates is the FMRFamide-related peptides (FaRPs). Amongst many roles, FaRPs potently influence motor function. The genome sequencing projects of Drosophila melanogaster and Caenorhabditis elegans have revealed unexpected complexity within the FaRPergic systems of arthropods and nematodes, although available evidence for platyhelminths indicates structural and functional simplicity. Regardless of these differences, FaRPs potently modulate motor function in arthropods, nematodes and platyhelminths and there appears to be at least some commonality in the FaRPergic signaling systems therein. Moreover, there is now increasing evidence of cross-phyla activity for individual FaRPs, providing clear signals of opportunities for target selection and the identification and development of broad-spectrum drugs.

Amino Acid Sequence↗
PubMed2000

Synaptic neuropil in nerves of the crustacean stomatogastric nervous system: an immunocytochemical and electron microscopical study.

Patches of peptide-immunoreactive varicosities have been found in nerves of the stomatogastric nervous system (STNS) of decapod crustaceans. In the present study, these patches were examined in detail in the stomatogastric nerve (stn) and in the superior oesophageal nerve (son) of the crayfish Cherax destructor by using whole-mount immunocytochemical techniques combined with confocal microscopy and, in addition, electron microscopy. Double-labeling experiments with antibodies generated against the peptides allatostatin, FMRFamide and proctolin, combined with an antibody generated against the small vesicle protein synapsin, suggest that each patch contains small synaptic vesicles in addition to all three peptides. The neuropil regions of the ganglia of the STNS were also strongly stained by the synapsin antibody. Synapsin-like immunoreactivity was also studied in the crab Cancer pagurus and the lobster Homarus americanus. A similar pattern of staining was found for all three species, but the distribution within the stn varied. In H. americanus, a lightly stained weblike structure was found on the surface of nerves including the inferior oesophageal nerve, the son, and the anterior stn. By using electron microscopy, synapses were found in the core of the stn-son junction of C. destructor, in the same region where the synapsin-like and the peptide staining was localized. In addition, putative neurohemal release sites were found in the peripheral sheath of the stn. The presynaptic profiles found in the core of the stn seem to correspond to the types of presynaptic profiles found in the neuropil of the stomatogastric ganglion. These findings demonstrate that synaptic neuropil is present in the nerves of the STNS of a decapod crustacean.

Animals↗
PubMed2000

Parallel organization in honey bee mushroom bodies by peptidergic Kenyon cells.

Antisera against the neuromodulatory peptides, Phe-Met-Arg-Phe-NH(2)-amide (FMRFamide) and gastrin cholecystokinin, demonstrate that the mushroom bodies of honey bees are subdivided longitudinally into strata. Three-dimensional reconstructions demonstrate that these strata project in parallel through the entire pedunculus and through the medial and vertical lobes. Immunostaining reveals clusters of immunoreactive cell bodies within the calyx cups and immunoreactive bundles of axons that line the inside of the calyx cup and lead to strata. Together, these features reveal that immunoreactive strata are composed of Kenyon cell axons rather than extrinsic elements, as suggested previously by some authors. Sorting amongst Kenyon cell axons into their appropriate strata already begins in the calyx before these axons enter the pedunculus. The three main concentric divisions of each calyx (the lip, collar, and basal ring) are divided further into immunoreactive and immunonegative zones. The lip neuropil is divided into two discrete zones, the collar neuropil is divided into five zones, and the basal ring neuropil is divided into four zones. Earlier studies proposed that the lip, collar, and basal ring are represented by three broad bands in the lobes: axons from adjacent Kenyon cell dendrites in the calyces are adjacent in the lobes even after their polar arrangements in the calyces have been transformed to rectilinear arrangements in the lobes. The universality of this arrangement is not supported by the present results. Although immunoreactive zones are found in all three calycal regions, immunoreactive strata in the lobes occur mainly in the two bands that were ascribed previously to the collar and the basal ring. In the lobes, immunoreactive strata are visited by the dendrites of efferent neurons that carry information from the mushroom bodies to other parts of the brain. Morphologically and chemically distinct subdivisions through the pedunculus and lobes of honey bees are comparable to longitudinal subdivisions demonstrated in the mushroom bodies of other insects, such as the cockroach Periplaneta americana. The functional and evolutionary significance of the results is discussed.

Animals↗
PubMed2000

Comparative pharmacology of feeding in molluscs.

1. This paper reviews the role of transmitters in identified neurons of gastropod molluscs in generating and modulating fictive feeding. 2. In Lymnaea and Helisoma the 3 phase rhythm is generated by sets of interneurons which use acetylcholine for the N1 (protraction) phase, glutamate for the N2 (rasp) phase interneurons. The N3 interneurons are likely to use several different transmitters, of which one is octopamine. 3. In all the species examined, serotonin (5-HT) is released from giant cerebral cells. Other amines, including dopamine and octopamine, are present in the buccal ganglia and all these amines activate or enhance feeding. 4. Nitric oxide (NO), mostly originating from sensory processes, can also activate fictive feeding, but (at least in Lymnaea) may also be released centrally from buccal (B2) and cerebral neurons (CGC). 5. The central pattern generator for feeding is also modulated by peptides including APGWamide, SCP(B) and FMRFamide. 6. There is increasing evidence that most of these transmitters/modulators act on feeding neurons through second messenger systems--allowing them to act as longer-lasting neuromodulators of the feeding network. 7. Many of the transmitters are used in similar ways by each of the gastropods examined so far, so that their function in the CNS seems to have been conserved through evolution.

Acetylcholine↗
PubMed1992

Diversity in tachykinin-like peptides in the insect brain.

When testing a large number of antisera against tachykinins of various vertebrates and one insect (the cockroach Leucophaea maderae) we found three distinct populations of tachykinin-immunoreactive neurons in the blowfly: (1) one recognized by antisera against substance P, (2) another by antisera against the frog peptide kassinin and (3) a third with antisera raised against the cockroach peptide Leucokinin I. As a comparison tests on the cockroach Leucophaea showed that only antisera against neurokinin A (NKA) and leucokinin I gave immunostaining, RIA and immunocytochemical displacement tests demonstrated that the each of the three listed types of antisera was specific for the corresponding antigenic peptide and showed virtually no cross reactivity with the other tachykinin peptides. By immunocytochemistry we have mapped the three populations of tachykinin-immunoreactive neurons in the blowfly CNS. Two constitute unique sets of interneurons that were previously not detected with other antisera, the third, recognized by antisera against substance P, is a subpopulation of the FMRFamide immunoreactive neurons. The leucokinin immunoreactive material in the blowfly seems to be chemically different from that in Leucophaea and also the sets of neurons immunolabeled in the two insect species are to some extent different. The preliminary results presented here indicate the presence of multiple forms of tachykinin-like peptides in the blowfly and that these are distinct from those in the cockroach. From the immunocytochemistry it appears that the insect tachykinins may be involved in a variety of regulatory functions in the CNS and in some cases possibly act as neurohormones.

Amino Acid Sequence↗