FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

The presence of FMRF-amide, a cardioactive tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas. Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.     

FMRF-amide immunoreactivity in the mammalian gastroenteropancreatic neuroendocrine system

Summary The presence of FMRF-amide, a cardioactiv tetrapeptide, was studied by immunocytochemistry in human and rat gastric antrum and pancreas, and in the ovine, bovine, canine and rabbit pancreas. In human and rat gastric antrum, numerous cells contained FMRF-amide immunoreactive material. By staining of serial sections and by double staining, colocalization of immunoreactivity for gastrin and FMRF-amide was observed in part of the gastrin cells. In the pancreas of these and the other species, immunoreactivity for FMRF-amide was located both in acinar and islet endocrine cells. Colocalization of FMRF-amide and pancreatic polypeptide was found in a proportion of pancreatic polypeptide cells in the pancreas. FMRF-amide immunoreactivity never colocalized with the other neurohormonal peptides which occur in the gastric antrum and the pancreas.Our observations show that neuroendocrine cells occur in the gastric antrum and pancreas which are exclusively immunoreactive or gastrin and for pancreatic polypeptide respectively. In addition cells occur which show immunoreactivity for FMRF-amide as well as for gastrin in the gastric antrum and with antiserum to FMRF-amide as well as for pancreatic polypeptide in the pancreas. It is concluded that FMRF-amide antibodies probably recognize a substance in G and PP cells which is not identical but may be structurally related to gastrin and pancreatic polypeptide.In honour of Prof. P. van Duijn     

Localization of the myomodulin-like immunoreactivity in the leech CNS

The distribution of myomodulinlike immunoreactivity in the leech CNS was determined using an antiserum raised against Aplysia myomodulin. Segmental ganglia contained approximately 60 immunoreactive neurons. In addition, numerous fibers containing immunoreactive varicosities were found throughout the neuropil. Using a combination of Lucifer Yellow injections and immunocytochemistry, we identified neurons including the anterior Pagodas (AP), annulus erector (AE), motor neurons, Leydig, longitudinal muscle motoneurons (L), S cells, and coupling interneurons, all of which are active during the touch-elicited shortening reflex. FMRF-amide-like immunoreactivity in three of these cells (L, AP, and AE) was previously demonstrated. Specific staining for myomodulin was abolished by preadsorption of the antiserum with synthetic myomodulin, but not with FMRF-amide. These results suggest a potential role for myomodulin in both intrinsic and extrinsic modulation of the leech touch-elicited shortening reflex. Further, it is possible that several neurons mediating this reflex contain multiple neuromodulatory peptides. © 1996 John Wiley & Sons, Inc.     

FMRF-amide-like immunoreactive efferent fibers and FMRF-amide suppression of pacemaker neurons in eyes of Bulla

The eyes of certain marine gastropods including Aplysia and Bulla, contain circadian pacemakers, which produce a circadian rhythm of autogenous compound action potential (CAP) activity. The CAPs are generated by the synchronous spike discharge of a distinctive population of retinal pacemaker neurons whose axons convey the CAP activity to the CNS. When CAP activity is recorded from a preparation with eyes attached to the CNS, the CAP activity is modulated by efferent activity. In this study we have identified FMRF-amide-like immunoreactive efferent axons in the optic nerves of Bulla. These axons arborize in the basal retinal neuropil adjacent to the pacemaker neurons and are in a position to make synaptic contacts with their dendrites. Similar immunoreactive fibers are not observed in Aplysia eyes. Exogenous FMRF-amide at micromolar concentrations suppresses ongoing CAP activity in isolated eyes but does not suppress the ERG or phase shift the circadian rhythm of CAP activity. Intracellular recordings from the retinal pacemaker neurons reveal that FMRF-amide hyperpolarizes the membrane potential, suppresses spike discharge, and decreases the input resistance, suggesting that a K conductance is increased. Electrical stimulation of the region of the cerebral ganglion that contains FMRF-amide immunoreactive neurons suppresses ongoing CAP activity. All these results are consistent with the idea that the FMRF-amide immunoreactive central neurons and their axons provide a pathway for efferent modulation of the CAP rhythm generated by the retinal pacemaker neurons.     

An Ultrastructural and Immunocytochemical Study of Gastrodermal Cell Types in Microstomum lineare (Turbellaria,Macrostomida)

The gastrodermal cell types of Microstomum lineare (Turbellaria, Macrostomida) were studied by electron microscopy. Their immunoreactivity (IR) to bovine pancreatic peptide (BPP), FMRF-amide and vasotocin, somatostatin, neurotensin, ACTH, CCK, bombesin, secretin, gastrin/CCK and insulin antisera was tested by light microscopic immunocytochemical methods. In addition to granular club cells and phagocytic cells, neurons and neoblasts occur in the gastroderm of this turbellarian species. This is the first observation of neurons in the gastroderm of a flatworm. Dense-core vesicles (70–100 nm diameter), electron lucent cytoplasm and numerous Golgi complexes characterize the neurons. Unpolarized two-way synapses, neuromuscular junctions and polarized chemical synapses can be observed in the gastroderm. Neoblasts with large nuclei and scanty cytoplasm and differentiating cells containing clusters of basal bodies occur next to the basal lamina of the gastroderm. BPP-like, FMRF-amide-like and vasotocin-like immunoreactivity is demonstrated in the gastroderm. Both BPP and FMRF-amide IR is restricted to the basal cytoplasm of the granular club cells, while a different location for IR to vasotocin antiserum is observed. The status of the neuronal cell in the gastroderm of M. lineare is discussed in relation to endocrine (paracrine) cells and neurons in the gastroderm of invertebrates.     

Isolation and immunochemical characterization of neuropeptides from the midgut of the Colorado potato beetleLeptinotarsa decemlineata

The midgut of the Colorado potato beetle showed endocrine cells immunopositive to monoclonals like MAC-18, MAC-3 and polyclonals to FMRF-amide and AKH-241. Extraction and HPLC-fractionation of the midgut extracts after partial purification and characterization showed immunopositive reaction to the monoclonals and also showed myotropic activity stimulating the potato beetle gut. Molecular mass of the two active peptides isolated were 22 and 26 kDa respectively. Both these peptides could be immunocytochemically demonstrated in the brain neurosecretory cells of the red cotton bug,Dysdercus cingulatus and the castor semilooper,Achaea janata as well.     

Patterns of co-existence of peptides and differences of nerve fibre types associated with noradrenergic and non-noradrenergic (putative cholinergic) neurons in the major pelvic ganglion of the male rat

Summary The pelvic ganglia supply cholinergic and noradrenergic nerve pathways to many organs. Other possible transmitters are also present in these nerves, including peptides. Multiple labelling immunofluorescence techniques were used in this study of the male rat major pelvic ganglion (MPG) to examine: (1) the peptides present in noradrenergic (tyrosine hydroxylase (TH)-positive) and non-noradrenergic (putative cholinergic) neurons, and (2) the types of peptide-containing nerve fibres closely associated with these two groups of neurons. The distribution of the peptide galanin (GAL) within the MPG was also investigated. All of the TH-neurons contained neuropeptide Y (NPY), but none of the other tested peptides. However, many NPY neurons did not contain TH and may have been cholinergic. TH-negative neurons also displayed vasoactive intestinal peptide (VIP), enkephalin (ENK) or GAL. VIP and NPY formed the most common types of putative cholinergic pelvic neurons, but few cells contained both peptides. Many ENK neurons exhibited VIP, NPY or GAL. Varicose nerve terminals surrounding ganglion cells contained ENK, GAL, somatostatin (SOM) and cholecystokinin (CCK). These peptide-immunoreactive fibres were more often associated with the non-noradrenergic (putative cholinergic) than the noradrenergic neurons; two types (SOM and CCK) were preferentially associated with the non-noradrenergic NPY neurons. GAL was distributed throughout the MPG, in small neurons, scattered small, intensely fluorescent (SIF) cells, and both varicose and non-varicose nerve fibres. The nerve fibres were concentrated near the pelvic and penile nerves; most of the varicose fibres formed baskets surrounding individual GAL-negative somata.     

The pharmacological actions of 5-hydroxytryptamine,FMRF-amide and substance P and their possible occurrence in the heart of the snail Helix aspersa L.

The actions of 5-hydroxytryptamine (5-HT), FMRF-amide and substance P were tested on the isolated heart of Helix aspersa. All three compounds were found to produce positive inotropic and chronotropic effects, the order of potency being 5-HT > substance P > FMRF-amide. However, whereas the responses to 5-HT and FMRF-amide were maximal within a few seconds, the response to substance P had a longer latency. Two other similar undecapeptides, eledoisin and physalaemin, were also tested and were found to have very similar actions to those of substance P. The effects of 5-HT and FMRF-amide could be separated using the 5-HT blockers methysergide and ketanserin, which had relatively little effect on the response to FMRF-amide.Immunohistochemical staining was carried out on sections through the Helix auricle and ventricle for 5-HT, FMRF-amide and substance P. Substantial 5-HT-like and substance P-like immunoreactivity was observed, apparently concentrated in nerve endings, but the level of FMRF-amide-like immunoreactivity was considerably lower. The fluorescence produced by all three compounds was evenly distributed throughout the heart tissue. HPLC analysis of heart tissue extract demonstrated a high level of 5-HT (about 8 μg/g wet weight) but a negligible catecholamine content.     

Actions of the molluscan neuropeptide FMRF-amide on neurones in the suboesophageal ganglia of the snail Helix aspersa

The effects of the molluscan neuropeptide FMRF-amide were tested on several neurones in the suboesophageal ganglia of the snail Helix aspersa. Almost all neurones tested responded to the peptide, some being hyperpolarized (H response) and others depolarized (D response). The H response is due primarily to an inward potassium current and may be blocked in 20 microM 4-aminopyridine. The hyperpolarizing actions of FMRF-amide and dopamine may be separated by ergometrine which blocks the response to dopamine but not to FMRF-amide. The D response is due mainly to an inward sodium current but this is not blocked by d-tubocurarine, morphine or TTX. It appears to be mediated by a distinct receptor/ionophore as excitation by ACh and 5-HT are both antagonized by d-tubocurarine. The Leu2-substituted analogue FLRF-amide was found to produce similar H responses to FMRF-amide, but was much less potent at producing D responses. It did, however, produce cross-desensitization of the D response to FMRF-amide, suggesting that it does bind to the FMRF-amide receptor.     

Neurite outgrowth,RGD-dependent,and RGD-independent adhesion of identified molluscan motoneurons on selected substrates

The extracellular matrix (ECM) provides structural support to cells and tissues and is involved in the regulation of various essential physiological processes, including neurite outgrowth. Most of the adhesive interactions between cells and ECM proteins are mediated by integrins. Integrins typically recognize short linear amino acid sequences in ECM proteins, one of the most common being Arginine-Glycine-Aspartate (RGD). The present study investigated neurite outgrowth and adhesion of identified molluscan neurons on a selection of substrates in vitro. Involvement of RGD binding sites in adhesion to the different substrates was investigated using soluble synthetic RGD peptides. The cells adhered to native (i.e., nondenatured) laminin and type IV collagen, but not to native plasma fibronectin. Denaturation of fibronectin dramatically enhanced cell adhesion. Only the adhesion to denatured fibronectin was inhibited by RGD peptides, indicating that denaturation uncovers a RGD binding site in the protein. Laminin as well as denatured fibronectin, but not type IV collagen, induced neurite outgrowth from a percentage of the RPA neurons. These results demonstrate that molluscan neurons can attach to various substrates using both RGD-dependent and RGD-independent adhesion mechanisms. This suggests that at least two different cell adhesion receptors, possibly belonging to the integrin family, are expressed in these neurons. Moreover, the results show that vertebrate ECM proteins can induce outgrowth from these neurons, suggesting that the mechanisms involved in adhesion as well as outgrowth promoting are evolutionarily well conserved. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 37–52, 1998     

Immunocytochemical research on mediators of centrifugal visual neurons in lampreys (Lampetra fluviatilis)]

The aim of this study has been to investigate different neuroactive substances in the lamprey centrifugal visual neurons (CVN) by combining axonal tracing methods and immunocytochemistry. The CVN somata are immunonegative to antibodies recognizing FMRF-amide, LH-RH, 5 HT and TH, but immunopositive to an anti-GABA antiserum (GABA+) in a proportion of 40%. In the retina, the GABA+ axon terminals mainly synapse upon GABA+ and GABA- amacrine cell bodies and dendrites, and on dendrites of GABA- ganglion cells.     

Immunocytochemical Demonstration of Serotonin and Neuropeptides in the Nervous System of Gyrodactylus salaris (Monogenea)

A whole mount immunocytochemical (ICC) method has been used for the investigation of immunoreactivity (IR) to the molluscan cardioactive peptide FMRF-amide, to 9 vertebrate neuropeptides—leu-enkephalin, growth hormone-releasing factor (GRF), urotensin I, urotensin II, bovine pancreatic peptide (BPP), β-endorphin, substance P, secretin and insulin—and to the bioamine 5-HT in the nervous system (NS) of the trematode Gyrodactylus salaris belonging to the taxon Monogenea. Positive results were obtained using antisera to FMRF-amide, leu-enkephalin, urotensin I, GRF and to 5-HT. The present results are the first documentation of the presence of neuroregulatory peptides and a bioamine in the nervous system (NS) of Monogenea. Differences in the distribution pattern of the IR to the different antisera indicate that different subsets of neurons are revealed. In addition, details of the basic neuroanatomical pattern in monogeneans are confirmed by the whole mount ICC method used in this study. Negative results were obtained with antisera to urotensin II, substance P, β-endorphin, secretin, insulin and bovine pancreatic polypeptide (BPP).     

Synthesis of cytochrome oxidase in isolated rat hepatocytes

1. The synthesis of cytochrome oxidase was studied in isolated rat hepatocytes labeled in vitro. Labeled whole cells, isolated mitochondria, microsomes and the post microsomal supernatant were treated with antisera to rat liver holo-cytochrome oxidase, and the subunits were adsorbed onto Sepharose-protein A. 2. Seven peptides, corresponding to subunits of rat liver cytochrome oxidase, were immunoabsorbed from mitochondria isolated from cells labeled in the absence of inhibitors. Two peptides, corresponding to subunits I (45 500 daltons) and II (26 000 daltons), were labeled in mitochondria isolated from cycloheximide-treated cells. Labeling of these peptides was inhibited by chloramphenicol. Peptides I and II correspond to the two most heavily labeled mitochondrial translation products found in submitochondrial particles. Possible explanations for the lack of labeling of a third mitochondrially translated subunit are discussed. Labeling of the five smallest peptides was inhibited by cyclohexamide but not by chloramphenicol. 3. Peptide I appears in the holoenzyme later than the other six peptides after a pulse-chase. It is not labeled in the immunoabsorbed cytochrome oxidase after a 30 min pulse with [35S]-methionine, but appears after a 3 h chase with unlabeled methionine. Labeling of the other subunits showed no further increase after the chase.     

Distribution and localization of immunoreactive FMRFamide-like peptides in the lancelet.

Immunofluorescence was used to study the distribution of FMRFamide (Phe-Met-Arg-Phe-NH2) in premetamorphic larvae and adults of the lancelet, Branchiostoma lanceolatum. In the larvae, FMR-Famide-containing presumably neuronal perikarya and fibers were limited to the anterior third of the dorsal nerve cord. Throughout this region, most of the immunoreactive perikarya and fibers were located ventrolaterally and ventrally within the nerve cord; in addition, in the caudal part of the cerebral vesicle, some of the immunofluorescent cells projected cytoplasmic extensions across the slot-like neural canal. In adult lancelets, immunofluorescence was detected in cells of the Hatschek's pit (a probable homologue of the anterior hypophysis of vertebrates); however, no immunofluorescence was detected in the larval preoral pit, which is the ontogenetic precursor of Hatschek's pit. Moreover, the FMR-Famide-containing elements do not show immunoreactivity to other peptides of the FaRPs family such as pancreatic polypeptide (PP). The results suggest that FMRF-amide may be involved in neuroendocrine functions of lancelets.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

Expression and Electrophysiological Function of Actin in Chick Cerebellar Neurons

Among several monoclonal antibodies obtained by immunizing Balb/c mice with cerebellar synaptic membrane fractions from E20 chick embryos, the antibody, named M35, suppressed Ca-spikes in immature cultured chick cerebellar neurons. M35 immunoprecipitated 43kDa protein from a ¹²⁵I-labeled embryonic crude cerebellar membrane fraction. Immunohistochemically, the M35 antigen was expressed most intensively in Purkinje cells, but its expression was limited to highly motile structures at developmental neuronal remodeling. Electrophysiologically, M35 facilitated current responses to AMPA and inhibited the responses to GABA in cultured cerebellar Purkinje neurons. The several peptides derived from the affinity-purified 43kDa protein were found to have homologous amino acid sequences to non-muscle actins. These results suggest that the antigen recognized by M35 may play an essential role probably as membrane ion channels modulating synaptic functions in not only the development and growth but also the neuronal activity of chick cerebellar Purkinje cells.     

Use of specific antisera for the localisation and quantitation of leucokinin immunoreactivity in the nematode,Ascaris suum

1. The leucokinins (LKs) are a group of eight related peptides isolated from the cockroach, Leucophaea maderae.2. Antisera raised against LK-V, which were specific for the conserved mid to C-terminal region of the LKs, were used to immunostain the parasitic nematode Ascaris suum.3. LK-IR was observed in neurons in the anterior nerve ring, retrovesicular ganglion, and ventral and dorsal nerve cords of the parasite. Immunostaining was specific in that it was abolished by preabsorption of the antiserum with different leucokinins. Some of the LK-immunoreactive neurons were identified on the basis of their morphological similarity with identified neurons in the free-living nematode Caenorhabditis elegans.4. Immunoreactivity towards a number of other peptides, notably neuropeptide F, FMRF-amide, KGQELE and KELTAE, has previously been demonstrated in all LK-immunoreactive neurons, thus confirming the multiple-peptidergic nature of certain nematode nerves.5. LK-IR was demonstrated and quantified in a number of tissues using RIA. Highest amounts were found in extracts of gut; LK-IR was also demonstrated in extracts of body wall, heads and tails, testes, ovaries and pseudocoelomic fluid.6. The distribution of tissue LK-IR did not correlate with the amount of neuronal tissue in the samples.7. Dilution experiments suggested that the LK-IR in the parasite is heterogeneous and that the peptide(s) in some tissues may not be analogous to the insect LKs.8. The LK-IR in the parasite remains to be characterized; however, the results suggest that peptides related to the leucokinins may have a far wider phylogenetic distribution than has hitherto been thought.     

Localization of putative biochemical messengers during Eisenia foetida (Annelida, Oligochaeta) development

This histochemical-immunohistochemical study was performed on the earthworm Eisenia foetida at different developmental stages, to investigate the presence and distribution of cholinergic molecules (AChE, BuChE, alpha-bungarotoxin-binding sites), several biogenic amines (5HT and catecholamines), and some immunologically-related peptides (somatostatin. FMRF-amide, VIP, substance-P, bombesin). The results showed that the pattern of labelling for the markers is different at different stages. In summary, cholinesterases appeared widely distributed in the early embryonic stages. They then were localized in particular areas of the developing nerve and muscle tissues, whereas in newborn and adult earthworms they were restricted to ventral muscular fibers and to some CNS cells. Biogenic amines were constantly present in the embryonic and adult nervous tissues. Immunologically-related peptides were detectable after organogenesis. Our results provide indirect evidence for a role of cholinesterases in regulating early intercellular communications, neurogenesis and myogenesis, and support the hypothesis that some conservative sequences of messenger peptides arose very early in evolution.     

Presence of cells and fibers immunoreactive toward antibodies to different peptides or amine in the digestive tract of the snailHelix aspersa

An immunocytological study of four different parts of the gut of Helix aspersa clearly demonstrates the presence of many cells and fibers immunoreactive toward antibodies directed to vertebrate (α, β-endorphin, α, β-MSH, ACTH 1–24 and ACTH 17–39, met-enkephalin, somatostatin, insulin, glucagon, P.P., serotonin) or invertebrate (FMRF-amide) peptides. These results are evidence of the presence of different substances related to known peptides or amines in the epithelial and connective tissue cells and nerve fibers of the snail gut. Immunocytochemistry may help to elucidate the morpho-functional characteristics of the enteroendocrine cells of H. aspersa.