Immunocytochemical localization of peptidergic neurons and neurosecretory cells in the neuro-endocrine system of the Colorado potato beetle with antisera to vertebrate regulatory peptides

A large number of antisera to regulatory vertebrate peptides was tested immunocytochemically on the nervous system of the Colorado potato beetle to further characterize the peptidergic cells of the neuro-endocrine system and to reveal cells participating in endocrine control mechanisms. Neurons, neurosecretory cells, axons and axon terminals were revealed by antisera to ACTH, gastrin, CCK, alpha-endorphin, beta-endorphin, gamma 1-MSH, insulin, motilin, human calcitonin, growth hormone, somatostatin, CRF, ovine prolactin and rat prolactin. Together with previously described results these findings demonstrate that at least 19 different peptidergic cell types are present in the Colorado potato beetle. Several of these cell types are identical with the known neurosecretory cells, while others have not been identified before. The functions of the immunoreactive neurons are as yet unclear, although in two cases the localization of these cells gives some clues. Thus the lateral neurosecretory cells, which are immunoreactive with antisera to beta-endorphin and ovine prolactin, may regulate corpus allatum activity, whereas a CRF immunoreactive substance seems to be used as neurotransmitter by antennal receptors. These immunocytochemical findings do not imply that the immunoreactive substances are evolutionarily related to the vertebrate peptides to which the antisera were raised. It is postulated that if the part of the substance recognized by a certain antiserum is functionally important for the insect, which should be so if the insect peptide is evolutionarily related to its vertebrate homologue, the antiserum should reveal homologous cells in different insect species. The consequence of this hypothesis is, that if an antiserum does not reveal homologous neurons in different insect species, the immunologically demonstrated substance is probably of little physiological importance, and will not be related evolutionarily to the vertebrate analogue. The positive immunocytochemical results in the Colorado potato beetle are discussed in relation to these considerations.     

Immunohistochemical localisation of substances reactive to antisera against α-and β-endorphin and met-enkephalin in the brain of Rana temporaria L.

Summary In the brain of Rana temporaria, two distinct systems reactive with - and -endorphin antisera, respectively, and with a met-enkephalin antiserum, have been detected immunohistochemically.Neurons reacting with - and -endorphin antisera are located (1) in the preoptic nucleus, and (2) in the pars ventralis of the tuber cinereum. Immunoreactive nerve fibres of both groups of perikarya end in the infundibular floor near the capillaries and the preoptico-hypophysial tract. Control reactions have shown that the immunoreactivity is suppressed by the corresponding antigens, but also by -LPH. In view of these results the immunoreactive systems examined correspond to an /-endorphin system or a lipotropinergic system.Neurons reacting with the met-enkephalin antiserum are located in the paraventricular organ. Intense immunofluorescence was observed in the infundibular floor. Controls show that the labelling by met-enkephalin antiserum is exclusively suppressed by met-enkephalin.In the pituitary gland, on the other hand, - and -endorphin antisera reveal: 1) the MSH/ACTH-like cells of the pars intermedia and 2) the ACTH-like cells of the pars distalis.Supported by the D.G.R.S.T., Contrat no 77.7.0648     

Immunocytochemical demonstration of proopiomelanocortin- and other opioid-related substances and a CRF-like peptide in the gut of the american cockroach,Periplaneta americana L.

Summary Using the peroxidase-antiperoxidase technique, we showed the presence of peptides which are immunologically resembling mammalian corticotropin releasing hormone (CRF)-, adrenocorticotropic hormone (ACTH)-, -endorphin (-END)-, -melanocyte stimulating hormone (-MSH)-, methionine-enkephaline (met-ENK)- and leucine enkephaline (leu-ENK)- like immunoreactivity in hundreds to thousands of endocrine cells and nerve fibers in the midgut of the American cockroach Periplaneta americana.In the cockroach hindgut no immunoreactive cell bodies could be observed, although nerve fibers were clearly noticed to be recognized by antisera to CRF, ACTH_1–24, ACTH_11–24 and -END.Nothing is exactly known as to the function(s) of the demonstrated materials, but one can speculate that these numerous immunoreactive cells, might have important paracrine and/or endocrine functions in the insect physiology.     

Immunocytochemical evidence for intragranular processing of pro-opiomelanocortin in the melanotropic cells of the rabbit

Summary The immunogold technique, employing antisera with clear-cut specificities, was used to localise different processing stages of pro-opiomelanocortin (POMC) in rabbit melanotropic cells. While the antiserum against ₃-MSH labelled all the secretory granules including intrasaccular condensations in the Golgi apparatus, antisera against -MSH only labelled extra-Golgi secretory vesicles (SV). All extra-Golgi SV were likewise labelled with the three antisera against -MSH used, despite their different specificities for the desacetylated, N-acetylated or C-amidated forms of the peptide. The antibody against -endorphin also labelled the extra-Golgi SV, while only some SV were labelled with the antibody against -endorphin. These results correlate with biochemical data in favour of mainly — if not exclusively — intragranular processing of POMC. Except for ₃-MSH, the cleavage of which could coincide with Golgi packaging of secretory material, other post-translational modifications of the precursor seem to occur when SV are discharged outside the Golgi area. The cleavage of -endorphin appears to be a later step in POMC processing, occurring in some mature SV.     

Cross-reactivity between human and fish pituitary hormones as demonstrated by immunocytochemistry

Summary In this communication we describe the immunocytochemical cross-reactivity between antisera to various human pituitary hormones and specific hormone producing cell types in the pituitary gland of sexually mature male platyfish (Xiphophorus maculatus). Antisera to human pituitary hormones cross-reacted either with cells known to produce corresponding hormones (or hormone subunits) in the platyfish (e.g., ACTH, prolactin, TSH , LH , FSH , TSH ) or with no pituitary cells at all (e.g., LH , FSH ). The one exception was antiserum to human growth hormone which cross-reacted with MSH and ACTH producing cells. The platyfish pituitary is proposed as a test system for immunocytochemically screening antisera for purity and specificity in order to determine their applicability in particular studies.     

Immunoreactive material resembling vertebrate neuropeptides in the corpus cardiacum and corpus allatum of the insect Leucophaea moderae

Summary The presence and differential distribution of substances antigenically related to known vertebrate neuropeptides demonstrated within the corpus cardiacum of the insect Leucophaea are as follows: Of ten mammalian antisera tested, six yielded substantial immunoreactive deposits resembling oxytocin, somatostatin, Substance P, met-enkephalin, bombesin, and neurotensin, respectively. In the remaining four, the reaction was moderate (vasopressin, -endorphin) or marginal (LH-RF, calcitonin). With regard to their regional distribution, these biochemically distinct reaction products seem to fall into two groups: (1) Materials resembling oxytocin, vasopressin, met-enkephalin, -endorphin (and presumably also neurotensin and LH-RF) predominate in the central release area of the organ and are considered to be of extrinsic (cerebral) origin. (2) Substances localized primarily in areas rich in intrinsic glandular cells of the corpus cardiacum, and revealed by antisera raised against somatostatin, Substance P, and bombesin, are judged to be synthesized and stored within this organ. In peptidergic fibers entering the adjacent corpora allata, thus far Substance P-, -endorphin-, and LH-RF-like immunoreactivities have been demonstrated. Some of these new neuropeptides may be contained in classical neurosecretory neurons, formerly identified by less specific methods, others must be assigned to additional peptidergic neurons heretofore unknown.Supported by NSF grant BMS 74-12456 (B.S.). The excellent technical assistance of Mrs. Sarah Wurzelmann is gratefully acknowledged     

Effects of background adaptation on α-MSH and β-endorphin in secretory granule types of melanotrope cells of Xenopus laevis

Placing the clawed toad Xenopus laevis on a black background stimulates the melanotrope cells in the pars intermedia of the pituitary gland to release proopiomelanocortin (POMC)-derived peptides, including -MSH and N-acetyl-endorphin. In this study three types of secretory granules, electron-dense(130 nm Ø), moderately electron-dense ( 160 nm Ø) and electronlucent ( 180 nm Ø), have been identified in these cells. Apparently, only dark granules are formed by the Golgi apparatus and lucent granules release their contents via exocytosis. Immuno-electron microscopy (immunogold double labelling) of glutaraldehyde-fixed and freeze-substituted material shows that desacetyl--MSH and N-acetyl--endorphin coexist in all three granule types. Quantification of immunostaining revealed that immunoreactivities to these peptides are lowest in the dark granules and highest in the light ones. It is proposed that intragranular processing of POMC to immunoreactive desacetyl--MSH and N-acetyl--endorphin involves an increase in granule size and a decrease in granule electron density. Black background-induced activation of the melanotrope cell is reflected by an increase in immunoreactivity of the secretory granules to each of the antisera. This suggests that cell activation stimulates the formation of peptides by intragranular processing of POMC and/or of intermediate POMC-processing products. In addition, cell activation evoked an increase in the percentage of the granule population that reacts with anti-N-acetyl--endorphin, probably by stimulating intragranular acetylation of -endorphin. Apparently, this acetylation is a regulated event that occurs in the cytoplasm, independently from the acetylation of desacetyl--MSH which takes place near the plasmalemma at the time of granule exocytosis.     

Ontogenesis of cells producing polypeptide hormones (ACTH,MSH, LPH,GH, Prolactin) in the fetal hypophysis of the rat: Influence of the hypothalamus

Summary The ontogenesis of cells containing polypeptide hormones (ACTH, MSH, LPH, GH and Prolactin) was investigated in the fetal rat hypophysis by immunohistochemistry using the peroxidase-antiperoxidase complex.Corticotrophs, melanotrophs and lipotropic cells were revealed earlier in the pars distalis than in the pars intermedia. In the pars distalis, cells producing LPH were found in the morning of day 15 of gestation using anti-- or anti--LPH sera, and in the afternoon using anti-- or -endorphin sera. Cells containing -MSH were observed from the afternoon of day 15. The cells stainable with the anti--MSH, anti--(17-39)ACTH and anti--(l-24)ACTH sera appeared on day 16. In the pars intermedia, the cells producing -MSH, MSH, - and -endorphin, and -LPH were observed in the morning of day 17, while cells containing ACTH were only revealed in the afternoon of the same day of gestation. Based on the treatment of serial paraffin sections with various antisera, it was clearly shown that MSH, ACTH, and LPH occur in the same cells located in the pars distalis as in the pars intermedia.The development of the corticotrophs, melanotrophs and lipotropic cells does not require the presence of the fetal hypothalamus or other central nervous structures. The pituitary glands of 21 day-old fetuses encephalectomized on day 16 showed as many reactive cells as those of the littermate controls.The somatotrophs were first revealed in the pars distalis in the afternoon of day 19. The cells producing prolactin were not observed before day 21 of gestation. On some cases GH and prolactin were found together in one cell. The cytodifferentiation of GH and prolactin cells is apparently not under hypothalamic control.     

Presence of β-endorphin-like immunoreactivity in the anterior pituitary gland of rat and man and evidence for the differential localization with ACTH

Summary Two antisera, Y-10 and Y-18 were raised in rabbits against synthetic human -endorphin conjugated to bovine serum albumin and keyhole limpet haemocyanin respectively. Antiserum Y-10 has been shown by radioimmunoassay to be highly specific for human -endorphin with minimal or no cross-reactivity against other pituitary peptides whilst antiserum Y-18 crossreacted on an equimolar basis against -endorphin and -lipotropin. When used in the immunohistochemical procedure, both antisera specifically stained the corticotrophs in human anterior pituitary tissue. A similar effect was observed when antiserum Y-18 was applied to rat anterior pituitary tissue in the immunohistochemical procedure. Y-10 antiserum, on the other hand, stained not only rat corticotrophs but also somatotrophs. The somatotrophin staining could not be attributed to the enkephalins reported to be present in these cells.The non-specific -endorphin antiserum Y-18 was used to stain anterior pituitaries from dehydrated and adrenalectomized rats as well as rats of the Brattleboro strain. In tissues from the three experimental animals, cells that stained positively for -endorphin did not give a positive immunoreaction for ACTH and vice versa in some other sections. It is concluded that under the physiological conditions, formalin fixation of the tissue causes the proopiocortin molecule to be trapped in a conformation such that either ACTH or -endorphin-like determinants are available for reacting with the appropriate antiserum.This work was financed by the Medical Research Council of New Zealand, NIH Research Program Project Grant HD-12303 and by U.S.P.H.S. Grant NS-16304 from NIH. We thank Drs Guillemin, Bloom and Ling for samples of -endorphin and -endorphin antisera     

The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of the bullforg (Rana catesbeiana) pituitary gland

Summary Our recent finding that the number of immunoreactive -subunit cells was invariably greater than the total number of immunoreactive gonadotropin (GTH) and thyrotropin (TSH) cells in the bullfrog (Rana catesbeiana) pituitary gland raises the possibility that the -subunit also exists in pituitary cells other than GTH and TSH cells. The present study demonstrates that there are a considerable number of immunoreactive prolactin (PRL) cells that are also stained with antibody against the -subunit when adjacent sections are immunocytochemically examined. Neither immunoreactive growth hormone nor adrenocorticotropin cells are stained with the antibody against the -subunit. The specificity of the antibody against the -subunit and of that against PRL was demonstrated by preabsorption test, non-competitive binding test, and immunoblot analysis. Double-immunolabeling with gold particles of different sizes for the -subunit and PRL revealed that most of the immunolabeled PRL-secretory granules are also labeled with the -subunit antibody. The gold particles indicating the presence of the -subunit were mostly found in the peripheral zone of the secretory granules.     

Cytological localization of α-MSH,ACTH and β-endorphin in the pars intermedia of the cichlid teleost Sarotherodon mossambicus

Summary The pars intermedia of S. mossambicus contains two different endocrine-cell types. The predominant cell type is lead-haematoxyline-positive and assumed to synthesize MSH and related peptides. The second cell type is PAS positive and its function and product(s) are unknown. Staining of light-microscopic and ultrathin sections with antisera against -MSH, ACTH 1–24 and human -endorphin revealed that only the lead-haematoxyline-positive cells of the pars intermedia react with these antisera, and that the secretory granules of these cells contain compounds that were immunoreactive to all three antisera. These findings are in line with the hypothesis that -MSH, ACTH and endorphins are derived from the same precursor molecule. No specific reaction with one of the antisera could be detected in the PAS positive cells.     

Presence of adrenocorticotropin and β-Endorphin immunoreactivities in the magnocellular neurosecretory system of the rat hypothalamus

Summary Antisera raised against ACTH (1–39), -endorphin and the 16K proopiocortin were used, in association with the immunoperoxidase reaction, to localize positively-staining cell bodies and nerve fibres in the hypothalamus of the rat. Antigens, cross-reactive against anti-ACTH (1–39) serum were detected in a fibre system in the rostro-dorsal hypothalamus situated between the optic chiasm and the third ventricle while immunoreactive 16K-like material was present in fibres localized in the caudal hypothalamus, dorso-lateral to the arcuate nucleus. This latter system was also associated with the appearance of ACTH (1–39) and ACTH (17–39) immunoreactivity.Cells of the arcuate nucleus stained positively for ACTH (1–39), 16K antigen and -endorphin, and on examining adjacent thin sections it was observed that cells that contained 16K antigen-like material, also gave a positive immunoreaction with ACTH (1–39) and -endorphin antisera. In the magnocellular system, cells of the supraoptic (SON) and paraventricular (PVN) nuclei also gave a positive immunoreaction with anti-ACTH (1–39), 16K antigen and -endorphin serum. As in the case of the arcuate nucleus, common cells stained for these three antigens.On the basis of the precursor theory for the synthesis of ACTH, 16K antigen and -endorphin, it was not unexpected to find these three fragments of pro-opiocortin localized together in cells of the arcuate nucleus. That ACTH (1–39), 16K antigen and -endorphin-like materials are present in the magnocellular neurosecretory system would suggest that cells of the SON and PVN are not only involved in the synthesis of neurophysin and the neurohypophysial hormones, but also of some products of the pro-opiocortin molecule. Whether the biochemical nature of the ACTH and -endorphin in cells of the SON and PVN is identical to that of anterior pituitary origin remains to be established, as does the biosynthetic relationship between neurophysin and oxytocin/ vasopressin and these fragments of pro-opiocortin.Drs. M.M. Wilkes, S.S.C. Yen, G. Pelletier, B.A. Eipper and R. Walter are thanked for supplying some of the antisera and antigens used in this study. Thanks also go to Ciba-Geigy Ltd. and Organon Inc. for supplies of ACTH (17–39) and ACTH (1–24) respectively. This work was financed by The Medical Research Council of New Zealand     

Studies on the neurosecretory system of Iphita limbata Stal.

Summary The present paper gives an account of some experiments upon the insect Iphita limbata Stal. (Hemiptera: Pyrrhocoridae). The experiments were carried out in order to find out whether in the adult animal there is a relationship between the activity of the neurosecretory cells and the water balance. Under varying conditions one group of the neurosecretory cells of the pars intercerebralis of the brain, the so called A-cells, show histological differences. It has been seen that under conditions stimulating hydration there is a marked retention of the stainable colloids in the cytoplasm of A cells. This retention probably indicates a relationship between the secretions of A cells and the water balance of the insect.The author is indebted to Mr. N. R. Prabhoo and Miss Maya Menon of this Department for a critical discussion of the paper.     

Correlation of neuronal size and peptide immunoreactivity in the guinea-pig trigeminal ganglion

Summary Frequency and size of guinea-pig trigeminal neurones immunoreactive with antisera to -neo-endorphin(-neo-END), dynorphin A-(DYN), vasoactive intestinal polypeptide-(VIP), somatostatin-(SOM), and substance P-(SP) are reported. Co-localisation of the various peptides to the same ganglion cells was investigated immunocytochemically in consecutive 7-m thick paraffin sections. According to their size, all peptide-immunoreactive neurones belong to the class of small ganglion cells. Within this cell group, SP-immunoreactive neurones appear to be the largest, followed by SOM-, VIP-, -neo-END- and DYN-immunoreactive ganglion cells. The observed differences in size are statistically significant with the exception of that between -neo-END and DYN. This finding correlates well with the observed co-occurrence of the two immunoreactive peptides. All -neo-END-immunoreactive perikarya are also reactive to VIP antisera. These neurones are significantly smaller than those containing VIP-immunoreactivity exclusively. Ganglion cells displaying co-existence of -neoEND- and SP-immunoreactivity or VIP- and SP-immunoreactivity are found too infrequently to allow morphometric analysis. Some non-immunoreactive ganglion cells are shown to be approached by dense baskets of VIP-, -neo-END- or SP-immunoreactive varicose fibres, indicating the presence of intraganglionic modulation sites. The combination of immunohistochemistry and morphometry presented in this study allows the differentiation of diverse populations of primary afferent neurones exhibiting peptide immunoreactivity, most likely reflecting their involvement in different central and peripheral reflex arcs and sensory modalities.     

Localization of melanotropin-like peptides in the central nervous system of two insect species,the migratory locust,Locusta migratoria,and the fleshfly,Sarcophaga bullata

Summary By use of well characterized antisera in the peroxidase-antiperoxidase method, we were able to demonstrateMSH andMSH immunoreactive cells and nerve fibres within the nervous system of adults and larvae ofLocusta migratoria and 3-, 5- and 8-day-old adultSarcophaga bullata. In neither of these insect species, any immunoreaction was obtained with a ₃MSH-antiserum. Double immuno-histochemical stainings revealed thatMSH-like andMSH-like substances are located in different cells. These cells show no immunoreactivity to a number of antisera against other POMC-derivatives (anti-lipotropin, anti-endorphin, anti-ACTH_1–24); thus they appear to containMSH- orMSH-like material in a specific way. The function of the immunologically detected peptides remains to be demonstrated. The distribution of the immunoreactive material suggests that, like in amphibians and other lower vertebrates, the synthesis or release of melanotropins might be under the influence of external stimuli. The present observations support the recently developed concept that even some of the smallest neuropeptides, the melanotropins, have been highly conserved during a long period of evolution.     

Differing immunoreactivities of somatostatin in the cortex and the hypothalamus of the rat

Summary Using an antibody against somatostatin (antiserum F), two somatostatin-immunoreactive systems, (i) a hypothalamic and (ii) an extrahypothalamic cortical system, are demonstrated in the rat. Another antiserum raised against somatostatin (antiserum BS 102) stains only the axons but not the perikarya of the hypothalamic system; the cortical somatostatin system does not react with this antiserum. The electron microscopic findings do not allow decision whether the above-mentioned hypothalamic and cortical neurons possess a common prohormonal form of somatostatin, immunoreactive only with antiserum F. They show, however, that the granules in both neuronal systems differ considerably; in the cortical neurons they measure approximately 65 nm in diameter, in the hypothalamic neurons 90–120 nm in diameter. Thus, both somatostatin systems are different and independent from one another.Supported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr 569/3) and the Stiftung Volkswagenwerk     

Immunocytological detection and localization of a peptide reacting with an α-endorphin antiserum in the corticotropic and melanotropic cells of the trout pituitary (Salmo irideus Gibb)

Summary In the pituitary of the trout, the corticotropic and melanotropic cells display a strong immunocytological reaction with -endorphin antiserum. This reaction persists even when a-endorphin antisera treated with -1-24 ACTH or -MSH are used. In the absence of pharmacological tests on the endorphic potencies of the compounds involved in the immunoreaction, it is not yet clear whether this reaction is due to the presence of an -endorphin-like peptide or simply an immunologically related peptide without the properties of endorphin. However, the presence of such peptides in the fish pituitary is interesting from the comparative point of view.     

NewLeaf Plus® Russet Burbank potatoes: replicase-mediated resistance to potato leafroll virus

Potato leafroll poleovirus and the Colorado potato beetle (Leptinotarsa decemlineata (Say)) are major pests of potato in the USA. The US Department of Agriculture estimates that over 50% of annual insecticide use on potato is applied to control the Colorado potato beetle and aphids that transmit potato leafroll virus (PLRV). To address this issue, Russet Burbank potatoes have been genetically modified for a high level of resistance to infection and the resulting disease symptoms caused by PLRV and to feeding damage caused by the Colorado potato beetle. This resistance was achieved by the expression of the unmodified full-length replicase gene of PLRV and the cry3A insect control protein gene from Bacillus thuringiensis var. tenebrionis. Plant expression constructs containing various modifications of the PLRV replicase gene were produced during the development of this product. The genes in these constructs were a full-length unmodified replicase (open reading frame 2a/2b), an antisense orientation of the full-length cDNA, an open reading frame 1 translation of the full-length gene, and a gene truncation containing the 3 sense coding portion of the replicase gene. Growth chamber experiments demonstrated that transformation of plants with the full-length and 3 sense coding constructs substantially protected these potato plants from infection and disease symptoms caused by PLRV. The Russet Burbank potato expressing the full-length PLV replicase gene and the cry3A gene is a new potato product from NatureMark called NewLeaf Plus®.     

Histochemistry and ultrastructure of adrenergic and acetylcholinesterase-containing nerves supplying follicles and endocrine cells in the guinea-pig ovary

Summary With the use of an anti-human S-100 protein antibody, it was possible to reveal a characteristic cell type in the anterior lobe of the normal human pituitary. These cells, so-called folliculo-stellate cells, were present in all pituitaries studied but their number varied from one gland to another. Immunoreactive cells, isolated or grouped, were arranged close to various secretory granulated cells. Especially by use of double immunoenzymatic labeling, it was evident that these cells are spatially related either to somatotropes, prolactin cells and corticotropes, or to glycoprotein-containing cells. Such immunoreactive cells were rare or absent in pseudo-follicular arrangements of secretory granulated cells. Since it is now possible to identify this cell type by light microscopy and since no reliable functional significance is known, it seems more advisable to term this cell type stellate cell instead of folliculostellate cell.     

Immunohistochemical localization of opioid peptides in the brain of the leech Theromyzon tessulatum

Summary By use of antisera directed against met-enkephalin, leu-enkephalin, dynorphin or -neoendorphin, immunoreactive structures were visualized in the central nervous system and proboscis of the leech Theromyzon tessulatum. Their distribution in the various compartments of the supra- and subesophageal ganglia was mapped. No correspondence could be established between the neurons containing met- or leu-enkephalin-like substances and the different types of neurosecretory cells classically described in Hirudinea. Successive localization of leu- and met-enkephalin on the same section revealed that these two peptides occur in different neurons. Only one cell located in compartment 6 of the supraesophageal ganglion was both dynorphin- and leu-enkephalin-positive. The other dynorphinimmunoreactive cells were not stained with the anti-leuenkephalin serum. The -neoendorphin-immunopositive cells were leu-enkephalin immunonegative and vice versa.