Immunocytochemical demonstration of octopamine-immunoreactive cells in the nervous system of Locusta migratoria and Schistocerca gregaria

Summary The distribution of octopamine in the metathoracic ganglion, brain and corpus cardiacum of Locusta migratoria and Schistocerca gregaria was investigated by means of immunocytochemistry with an antiserum against octopamine. The dorsal unpaired median (DUM) cells of the metathoracic ganglion were found to be strongly octopamine-immunoreactive. In the rostroventral part of the protocerebrum a group of seven immunopositive cells was demonstrated. Stained nerve fibres of these cells run into three directions: circumoesophageal connectives, midbrain, and optic lobes. As far as the protocerebrum is concerned, immunoreactive fibres were found in the central body, the protocerebral bridge, and in other neuropile areas. In the optic lobe a dense plexus of immunopositive fibres was found in the lobula and in the medulla. In the brain one other immunopositive cell was demonstrated, situated at the lateral border of the tritocerebrum. Octopamine could not be shown to occur either in the globuli cells of the mushroom bodies or in the dorsolateral part of the protocerebrum, where the perikarya of the secretomotor neurones are located that innervate the glandular cells of the corpus cardiacum. In the nervi corporis cardiaci II, which contain the axons of the neurones that extend into the glandular part of the corpus cardiacum, and in the corpus cardiacum proper no specific octopamine immunoreactivity could be found.     

Serotonin and gastrin/cholecystokinin-like immunorcactive neurons in the larval retrocerebral complex of the blowfly Calliphora erythrocephala

Summary The presence and distribution of neurons immunoreactive against antibodies to serotonin (5-HT) and gastrin/cholecystokinin (gastrin/CCK) has been studied in the larval retrocerebral complex of the blowfly Calliphora erythrocephala, a composite structure which consists of the corpus cardiacum, the corpus allatum, the thoracic gland and a portion of the cephalic aorta. Immunoreactive material was found in all these elements except in the corpus allatum. Six to eight cell bodies in the corpus cardiacum and four to eight cell bodies in the thoracic gland were 5-HT immunoreactive (5-HTi). These 5-HTi cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the cephalic aorta, corpus cardiacum and ventral part of the thoracic gland. Six to eight cell bodies in the corpus cardiacum and four to six cell bodies in the thoracic gland reacted with antibodies against gastrin/CCK. These cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the corpus cardiacum and the cephalic aorta in a pattern resembling that of the 5-HTi fibers. Additional gastrin/CCK-like immunoreactive fibers were shown to come from the central nervous system via the two nervi corporis cardiaci. An electron-microscopical analysis was performed to analyze further the morphological features revealed by the light-microscopic immunocytochemical technique. This confirmed the existence of neurosecretory-like terminals among the gland cells of the thoracic glands and the existence of neurohemal sites in several regions of the larval retrocerebral complex. Some functional aspects of the retrocerebral complex are discussed on the basis of the presented data.     

Serotonin-immunoreactive neurones in the brain of Locusta migratoria innervating the corpus cardiacum

Summary The serotoninergic innervation of the corpus cardiacum (CC) of Locusta migratoria was investigated using two antisera against serotonin. A dense network of immunoreactive nerve fibres was present in the storage lobe of the CC. Immunopositive fibres only sporadically crossed the border between the storage lobe and the glandular lobe of the CC. Immunopositive fibres entered the storage lobe of the CC via the nervus corporis cardiaci I (NCCI); NCCII was immunonegative. Unilateral retrograde fillings of the NCCI with the fluorescent tracer Lucifer yellow, followed by antiserotonin immunocytochemistry, revealed about 20 double-labelled neurones in the anterior part of the pars intercerebralis. The double-labelled neurones were scattered between fluorescent non-immunoreactive neurones. Additionally, 5–7 neurones labelled only with Lucifer yellow were found at the ventrolateral side of the tritocerebrum. No immunopositive neurones were observed in the hypocerebral ganglion. Immunopositive fibres from neurones in the frontal ganglion ran via the recurrent nerve and the neuropile of the hypocerebral ganglion into the paired oesophageal nerve. At most, a few immunopositive nerve fibres occurred in the cardiostomatogastric nerves II, which connect the storage lobe of the CC with the paired oesophageal nerve at the caudal end of the hypocerebral ganglion.     

Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora vomitoria

The Leu-callatostatins are a series of four neuropeptides isolated from nervous tissues of the blowfly Calliphora vomitoria that show C-terminal sequence homology to the allatostatins of cockroaches. The allatostatins have an important role in the reproductive processes of insects as inhibitors of the synthesis and release of juvenile hormone from the corpus allatum. In this study, the distribution of the Leu-callatostatin-immunoreactive neurones and endocrine cells has been mapped in C. vomitoria and, in contrast to the cockroach allatostatins, it has been shown that there is no cytological basis to suggest that the dipteran peptides act as regulators of juvenile hormone. Although occurring in various neurones in the brain and thoracico-abdominal ganglion, there is no evidence of Leu-callatostatin-immunoreactive pathways linking the brain to the corpus allatum, or of immunoreactive terminals in this gland. Three different types of functions for the Leu-callatostatins are suggested by the occurrence of immunoreactive material in cells and by the pathways that have been identified. (1) A role in neurotransmission or neuromodulation appears evident from immunoreactive neurones in the medulla of the optic lobes, and from immunoreactive material in the central body and in descending interneurones in the suboesophageal ganglion that project to the neuropile of the thoracico-abdominal ganglion. (2) Leu-callatostatin neurones directly innervate muscles of the hindgut and the heart. Immunoreactive fibres from neurones of the abdominal ganglion pass by way of the median abdominal nerve to ramify extensively over several areas of the hindgut. Physiological experiments with synthetic peptides show that the Leu-callatostatins are potent inhibitors of peristaltic movements of the ileum. Leu-callatostatin 3 is active at 10^-16 to 10^-13 M. This form or regulatory control over gut motility appears to be highly specific since the patterns of contraction in other regions are unaffected by these peptides. (3) Evidence that the Leu-callatostatins act as neurohormones comes from the presence of varicosities in axons passing through the corpus cardiacum (but not the corpus allatum) and also from material in extraganglionic neurosecretory cells in the thorax. Fibres from these peripheral neurones are especially prominent over the large nerve bundles supplying the legs. There are also a considerable number of Leu-callatostatin-immunoreactive endocrine cells in a specific region of the midgut. The conclusion from this study is that although conservation of the structure of the allatostatin-type of peptides is evident through a long period of evolution it cannot be assumed that all of their functions have also been conserved. Several different types of functions for the Leu-callatostatins of the blowfly are proposed in this study, but there is no evidence to suggest a role in the regulation of juvenile hormone synthesis and release.     

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

The Corpus Cardiacum Neurosecretory Cells of Schistocerca gregaria. Electron Microscopy of Resting and Secreting Cells

The corpora cardiaca of Schistocerca consist of a neurohaemal part containing mainly extrinsic neurosecretory axons coming from the brain, and of a glandular part consisting mainly of intrinsic neurosecretory cells. Some extrinsic axons penetrate into the glandular region, and innervate intrinsic corpus cardiacum neurosecretory cells. The fine structure of the latter has been examined and related to other neurosecretory cell types. Secretion occurs by exocytosis. Omega-profiles are more frequently observed in corpora cardiaca stimulated electrically or by acetylcholine than in the controls.     

Immunocytochemical mapping of gastrin/CCK-like peptides in the neuroendocrine system of the blowfly Calliphora vomitoria (Diptera)

Summary The distribution of gastrin/CCK-like immunoreactive material has been studied in the retrocerebral complex of Calliphora. The material reacts with antisera specific for the common COOH terminus of gastrin and CCK but not with N-terminal antisera. The three thoracic ganglia and the fused abdominal ganglia each contain a specific number of symmetrically arranged immunoreactive cells both dorsally and ventrally in pairs on either side of the midline in a sagittal plane. The neuropil of these ganglia also contains a considerable amount of immunoreactive fibres and droplets. Reconstructed axonal pathways suggest that some of the nerve fibres have their origins within the brain and/or the suboesophageal ganglion. Immunoreactive material may also be seen apparently leaving the thoracic ganglion posteriorly via the abdominal nerves, and there is strong evidence of a neurohaemal organ within the dorsal sheath in the region of the metathoracic and abdominal ganglia. There appears to be a direct correlation between the content of peptidergic material of cells and fibres and the age and diet of the flies. The corpus cardiacum contains COOH-terminal specific gastrin/CCK-like material within the intrinsic cells and in the neuropil. It is present also in the cardiac-recurrent nerve entering the corpus cardiacum anteriorly and in the nerves leaving the gland dorsoposteriorly, the aortic or cardiac nerves. It is not observed, however, in the nerves leaving the corpus cardiacum ventroposteriorly, the so-called oesophageal, gastric or crop-duct nerves. The corpus allatum and the hypocerebral ganglion do not contain immunoreactive material of this type. Gastrin/CCK-like and secretin-like immunoreactive materials appear to co-exist in the cells of the corpus cardiacum and co-existence of gastrin/CCK-like and pancreatic polypeptide like substances occurs within certain cells of the thoracic ganglion.     

Histology of the neurosecretory system and the retrocerebral endocrine glands of the adult migratory grasshopper, Melanoplus sanguinipes (Fab.) (Orthoptera: Acrididae)

Neurosecretory cells of only one type (A, sub type A₂) are seen in adult Melanoplus. Two groups of about 400 cells each are located dorsally in the pars intercerebralis medialis; four cells are located deep within the protocerebrum. We found no neurosecretory cells in other parts of the central or sympathetic nervous systems. In about 10% of the specimens, there was marked asymmetry in the location of the dorsal cell groups, with both of these groups and their axons located in one lobe of the protocerebrum. The nervi corporis cardiaci 1 cross-over in the corpus cardiacum, with the result that material produced by neurosecretory cells on one side of the brain is transported along axons that undergo two chiasmata to the corpus cardiacum of the same side. Stainable secretory material could be traced clearly from the cerebral cells to the corpus cardiacum, and even into the oesophageal nerves from the hypocerebral ganglion. However, stainable neurosecretory material is never present in the corpus allatum or along any of the nerves to this gland.     

Axonal projections within the brain-retrocerebral complex of the cricket,Teleogryllus commodus

Summary The cerebral origins and axonal trajectories of neurons projecting to the retrocerebral complex of the cricket, Teleogryllus commodus, were examined in silver-intensified nickel preparations. Spatially separate groups of somata in the pars intercerebralis (PI) and in the pars lateralis (PL), commonly accepted as neurosecretory loci, were found to give rise to axons which terminate in the nervus corporis allati 2, the corpus allatum, or the corpus cardiacum. Additional findings demonstrated a distinct group of somata from the PI whose axons run in the esophageal nerve (stomatogastric nervous system), nine somata in the subesophageal ganglion with axons projecting into the nervus corporis allati 2, and also a small cluster of tritocerebral perikarya with axons terminating in the corpus cardiacum. Somata residing in the PI and PL were found to be compartmentally organized based upon the retrocerebral destinations of their axons. Possible functional consequences of these results with respect to the insect neurosecretory system are discussed.     

Physiological differentiation of DH-PBAN-producing neurosecretory cells in the silkworm embryo

Embryonic diapause of the silkworm, Bombyx mori, is induced by a neuropeptide hormone, the diapause hormone (DH), which is secreted from a limited number of neurosecretory cells in the subesophageal ganglion (SG) at the maternal generation. We examined the developmental fate of the hormone-producing cell (DH-pheromone biosynthesis activating neuropeptide [PBAN]-producing cell) in the embryonic stage at the level of gene expression and cell biology. The DH-PBAN gene expression started at the histogenesis stage and gradually increased toward hatching. DH is an amidated peptide belonging to FXPRLamide family. The immunoreactive somata against anti FXPRLamide antiserum were found in the SG from blastokinesis. Immunoreactive neural processes with varicosites were also found on the corpus cardiacum and the corpus allatum. The implantation of a part of a developing embryo including the SG into the pupae with the SG removed induced diapause eggs in the progeny. These results were obtained from eggs incubated under diapause-averting conditions as well as diapause-inducing conditions. Thus, a neurosecretory system responsible for biosynthesis of FXPRLamide neuropeptides is established as early as histogenesis, although the system to regulate the secretion of neuropeptide hormones has not been fully formed by that time.     

Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata

Summary A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H₂O₂-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.     

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine.A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor.The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.     

The innervation of the corpus cardiacum of Locusta migratoria: A neuroanatomical study with the use of Lucifer yellow

Summary Neural connections of the corpus cardiacum (CC) in the African locust, Locusta migratoria, were labelled with the fluorescent tracer Lucifer yellow. (1) Unilateral anterograde labelling of the nervus corporis cardiaci I revealed fluorescent fibres in the storage lobe of the CC (CCS). Some fluorescent fibres in the CCS closely approached the ipsilateral border of the glandular lobes of the CC (CCG). Fluorescent fibres also projected into the neuropile of the hypocerebral ganglion via the ipsilateral nervi cardiostomatogastrici I and II, and from there into the oesophageal nerves. (2) Unilateral anterograde labelling of the nervus corporis cardiaci II revealed fluorescent fibres in the CCS and in the ipsilateral CCG. Fluorescent fibres also projected via the ipsilateral nervus corporis allati I into the corpus allatum. (3) Unilateral retrograde labelling of the nervus corporis allati I revealed a distinct fluorescent nerve tract that runs through the CCS and into the nervus corporis cardiaci II. The tract arises from about eight cell bodies in the brain at the rostroventral side of the ipsilateral calyx of the mushroom body. (4) Labelling of the recurrent nerve revealed fluorescent fibres and some fluorescent cell bodies in the hypocerebral ganglion and, via the nervi cardiostomatogastrici I and II, also in the CCS. Fluorescent fibres were also present in the oesophageal nerves.     

In vivo studies on the release of hormones from the corpora cardiaca of locusts

Summary Sectioning of the afferent nerves (NCCl and NCCll) to the locust corpus cardiacum prevents thein vivo release of adipokinetic hormone from the glandular lobes. This failure to release the hormone during flight and the consequent lack of lipid mobilisation brings about an impairment of flight performance which can be corrected by injections of corpus cardiacum extracts. Sectioning of the NCCl and NCCll reduces markedly the activity of the corpora allata. However, the poor flight performance of allatectomised locusts is not related to an inability to mobilise lipid since injections of corpus cardiacum extract which will mobilise fat body lipid in these locusts have no effect on flight performance. The results of individual sectioning of the NCCl and NCCll suggest that a double innervation of the glandular lobes functionsin vivo to control adipokinetic hormone release but that the NCCl alone may control the release of the diuretic hormone.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Peptide immunocytochemistry of neurons projecting to the retrocerebral complex in the blow fly, <Emphasis Type="Italic">Protophormia terraenovae</Emphasis>

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to characterize neurons with somata in the pars intercerebralis (PI), pars lateralis (PL), and subesophageal ganglion (SEG), designated as PI neurons, PL neurons, and SEG neurons, respectively, all of which project to the retrocerebral complex in the blow fly, Protophormia terraenovae. Immunocytochemistry combined with backfills through the cardiac-recurrent nerve revealed that at least two pairs of PI and SEG neurons for each were FMRFamide-immunoreactive. Immunoreactivity against [Arg7]-corazonin, beta-pigment-dispersing hormone (beta-PDH), cholecystokinin8, or FMRFamide was observed in PL neurons. Immunoreactive colocalization of [Arg7]-corazonin with beta-PDH, [Arg7]-corazonin with cholecystokinin8, or beta-PDH with FMRFamide was found in two to three somata in the PL of a hemisphere. Based on their anatomical and immunocytochemical characteristics, PI neurons were classified into two types, PL neurons into six types, and SEG neurons into two types. Fibers in the retrocerebral complex showed [Arg7]-corazonin, beta-PDH, cholecystokinin8, and FMRFamide immunoreactivity. Cholecystokinin8 immunoreactivity was also detected in intrinsic cells of the corpus cardiacum. The corpus allatum was densely innervated by FMRFamide-immunoreactive varicose fibers. These results suggest that PI, PL, and SEG neurons release [Arg7]-corazonin, beta-PDH, cholecystokinin8, or FMRFamide-like peptides from the corpus cardiacum or corpus allatum into the hemolymph, and that some PL neurons may simultaneously release several neuropeptides.     

An electron microscopic study of the corpus cardiacum of adult Drosophila melanogaster and its afferent nerves

The left and right afferent nerves to the corpus cardiacum contain approximately 50 and 90 axons, respectively, when they enter the gland. Both bifurcate within the gland, so that four nerves leave the corpus cardiacum. These, however, contain a total of 90 axons. Of the 50 axons unaccounted for 30 are believed to terminate in the corpus cardiacum and 20 in the corpus allatum. The corpus cardiacum contains 12 intrinsic neurosecretory cells which are disposed laterally with respect to the extrinsic axons. Many of these contain neurosecretory spheres and presumably originate in the brain. The intrinsic corpus cardiacum cells also contain neurosecretory granules, and they produce short cytoplasmic processes which interdigitate with the extrinsic axons.     

A neurosecretory hormone-releasing factor from ovaries of mosquitoes fed blood

In mosquitoes, a hormone (egg development neurosecretory hormone or EDNH), produced by the medial neurosecretory cells and stored in the corpus cardiacum soon after eclosion, is released after a blood meal, and vitellogenesis begins a few hours later. When either the ovaries or the neurosecretory cells and corpus cardiacum are removed before the blood meal, vitellogenin is not synthesized. Therefore, we tested the hypothesis that the release of EDNH from the corpus cardiacum is dependent on the secretion of a releasing factor from the ovaries.Using a bioassay for EDNH in the corpus cardiacum, we found that the gland of an ovariectomized female remained active after blood feeding, and therefore, has not released EDNH. When an ovary was implanted before the blood meal, the corpus cardiacum was inactive, and therefore, had released EDNH. We concluded that the ovaries secrete an EDNH-releasing factor, and that this factor and EDNH must both be in circulation before vitellogenesis can begin. Although releasing factor alone did not stimulate vitellogenesis, it was the rate limiting process that controlled the onset of vitellogenesis.Using a bioassay for the EDNH-releasing factor from the ovaries and using rocket-immuno-electrophoresis, we showed that a Culex ovary, but not an Anopheles ovary, could replaces Aedes ovaries as a source of the releasing factor.In Ae. aegypti, EDNH-releasing factor was required again after oviposition in order to reinitiate the vitellogenic process in females that took a second blood meal. Thus, the releasing factor is part of the mechanism regulating cyclic egg maturation in mosquitoes.     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the insect Leucophaea maderae

Summary Immunocytochemical tests with eight monoclonal antibodies against either bovine or human insulin and seven polyclonal antibodies against bovine insulin were carried out to determine the presence of insulin-like neuropeptides in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Reaction products identified in the brain, subesophageal ganglion, and corpus cardiacum-corpus allatum complex indicate the presence of materials resembling mammalian insulins in its antigenic properties. The immunostaining observed with monoclonal antibodies appears to indicate the occurrence of an insulin-related peptide that shows sequential similarities with parts of both the A- and B-chains of mammalian insulin molecules. These suppositions are supported by the results of dot-blot and two-site time-resolved immunofluorometric assay (TR-IFMA) screenings of fractions of Leucophaea tissue extracts obtained by chromatography. The polyclonal antibodies yielded reaction products in some of the same areas and in additional parts of the neuroendocrine system not visualized by the monoclonal antibodies. Immunoreaction was observed in the following areas: the pars intercerebralis of the protocerebrum, the nervi corporis cardiaci I transporting insulin-like material to the corpus cardiacum, the dorsolateral protocerebral area and the optic lobes, the deutocerebrum, the tritocerebrum, and the subesophageal ganglion. In addition, smaller cell bodies with immunoreactive deposits occur at the border between proto- and deutocerebrum, and in the central area of the protocerebrum. The distribution of reactive material in the corpus cardiacum-corpus allatum complex after use of both groups of antibodies was the same. The fact that polyclonal and monoclonal antibodies yielded reaction products in different cells of the brain suggests either that the two groups of antibodies recognize different epitopes of the same molecule, or that they reveal two different types of immunoreactive molecules related to mammalian insulins. Together with the biochemical data reported by Nagasawa and coworkers (PNAS 83, 1986) the present immunocytochemical analysis has established a closer relationship between mammalian and insect insulins than was previously known.Supported in part by NIH grant NS 2344-02 (B.S.) and SNF grant 11-5082 and 11-7705 (G.N.H.)     

20-Hydroxy-ecdysone as a modulator of electrical activity in neurosecretory cells of Rhodnius prolixus

Ovariectomy of the adult female Rhodnius results in a reduction of the electrical activity of the corpus cardiacum. Injection of 20-hydroxy-ecdysone into ovariectomized animals increases the activity to control values. Incubation of heads isolated from ovariectomized females in 1.0 × 10^?7 M 20-hydroxy-ecdysone elicits a recruitment of large-amplitude spikes and the appearance of bursting activity in the corpus cardiacum of mated females which is characteristic of neurohormone release. Simultaneous recordings from the median neurosecretory cells and the corpus cardiacum demonstrate that the action neurosecretory cells.