Neuropeptide F Research Articles

Immunocytochemical distribution of neuropeptide F (NPF) in the gastropod mollusc, Helix aspersa, and in several other invertebrates.

The distribution of neuropeptide F (NPF) immunoreactivity in the snail, Helix aspersa, has been demonstrated by immunocytochemistry using 2 region-specific antisera. One, designated NPF3, was raised against a synthetic N-terminal fragment of Helix aspersa NPF; the other, designated PP221, was raised against the C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP) but cross-reacts fully with the analogous C-terminal region of Helix aspersa NPF. The distribution of NPF immunoreactivity has also been compared with that of FMRFamide using alternate serial sections of Helix aspersa ganglia. Results showed that NPF immunoreactivity was abundant and widespread in the central and peripheral nervous systems and the pattern of immunostaining obtained using both region-specific antisera was similar. Likewise, immunocytochemistry of neural tissues of a congeneric species, Helix pomatia, and 2 prosobranch gastropods, Buccinum undatum and Littorina littorea, produced similar staining patterns with both antisera. However, in the cephalopod mollusc, Loligo vulgaris, and the cestode, Moniezia expansa, positive immunostaining was only obtained with the C-terminal PP antiserum. Immunostaining of alternate serial sections of Helix aspersa ganglia with NPF3, and an antiserum raised to FMRFamide, showed that while a few neurones were immunoreactive with one antiserum only, in the majority, both immunoreactivities were co-localised. NPF thus appears to be an important neuropeptide of widespread distribution in Helix aspersa and the differential immunocytochemical staining obtained using the 2 region-specific antisera would suggest a high degree of primary structural conservation within the gastropod molluscs, but lack of conservation of the N-terminal region of the peptide in other invertebrate groups.

Quantitation and Chromatographic Characterisation of Neuropeptide F (NPF) Immunoreactivity in Molluscan Nervous Tissue Using Region-Specific Antisera

Neuropeptide F (NPF) immunoreactivity has been quantified in extracts of the central nervous system from four gastropod (Helix aspersa, Buccinum undatum, Littorina littorea, and Patella vulgaris) and two bivalve (Mytilus edulis and Pecten maximus) molluscs using two region-specific radioimmunoassays. The first employed an antiserum, NPF3, raised to a synthetic N-terminal fragment of H. aspersa NPF and the second employed an antiserum, PP221, raised to the synthetic C-terminal hexapeptide amide of mammalian pancreatic polypeptide which fully cross-reacts with the analogous region of H. aspersa NPF. NPF immunoreactivity was detected in acidified ethanolic brain extracts of the four gastropod molluscs by both antisera. However, only the C-terminally directed antiserum detected immunoreactivity in brain extracts of the two bivalve molluscs. Reverse-phase HPLC analysis of brain extracts from B. undatum and L. littorea resolved a single NPF immunoreactive peptide which was more hydrophobic than natural H. aspersa NPF chromatographed under the same conditions. Gel permeation chromatography of these NPF immunoreactive peptides indicated that they were of a similar molecular mass to that of H. aspersa NPF. These data suggest that NPF is widely distributed in molluscs with a high degree of structural conservation of N- and C-terminal regions within the gastropod molluscs but significant structural differences within the N-terminal regions of analogous peptides in bivalves.

Immunocytochemical localization of neuropeptide F-Immunoreactivity in the circumoesophageal ganglia of the gastropod mollusc, Helix aspersa using electron microscopy

Neuropeptide F (NPF)-immunoreactivity has been localized by electron microscopy in the central nervous system of the gastropod mollusc, Helix aspersa using a region-specific antiserum in conjunction with post-embedding immunogold labelling. The specific antiserum employed, designated NPF3, was raised to a synthetic N-terminal fragment of H. aspersa NPF. Gold-labelling revealed that NPF-immunoreactivity was found almost exclusively over the contents of dense-cored secretory vesicles within nerve axons in both cerebral and suboesophageal ganglia. Double-labelling of NPF and FMRFamide demonstrated no apparent co-localization in the paired cerebral ganglia. Instead, immunoreactivities of the two neuropeptides were found to have distinct locations within the ganglia. These findings suggest that NPF may be an important neuroregulator in H. aspersa and that in the cerebral ganglia it may be anatomically-distinct from FMRFamide and related peptides.

Distribution and immunochemical characteristics of neuropeptide F (NPF) ( Moniezia expansa)—immunoreactivity in Proteocephalus pollanicola (Cestoda: proteocephalidea)

1. Using immunocytochemical techniques and confocal scanning laser microscopy, the proteocephalidean cestode, Proteocephalus pollanicola from Lough Neagh pollan ( Coregonus autumnalis) was examined for the presence of the native platyhelminth neuropeptide, neuropeptide F (NPF). 2. An antiserum specific for whole-molecule NPF(1–39) ( Moniezia expansa) did not immunostain nerve processes in P. pollanicola. A C-terminally-directed NPF(30–39) ( M. expansa) antiserum immunostained nerve fibres and cell bodies of both the central and peripheral nervous systems, including innervation associated with the female reproductive system. 3. The pattern of immunoreactivity was identical to that obtained using antisera to the C-terminal region of mammalian NPY-superfamily peptides and the invertebrate neuropeptide, FMRFamide. 4. Under radioimmunoassay conditions, only the C-terminally-directed NPF antiserum cross-reacted with the P. pollanicola peptide and detected 58.74 ng g wet weight of NPF-IR in extracts of the worm. 5. Chromatographic characterisation of P. pollanicola NPF-immunoreactivity indicated an apparent molecular weight of 4400–4700 Da, similar to that of NPF ( M. expansa). 6. Further analytical HPLC characterisation identified two molecular forms of P. pollanicola NPF-immunoreactivity, both of which had different retention times from those of NPF ( M. expansa) and NPF ( Artioposthia triangulata). 7. These data suggest that P. pollanicola possesses a neuropeptide which is homologous in its C-terminal region to NPF ( M. expansa) but diners in its mid- to N-terminal region.

Electron immunogold labeling of regulatory peptide immunoreactivity in the nervous system ofMoniezia expansa (Cestoda: Cyclophyllidea)

An electron immunogold-labeling technique was used in conjunction with a post-embedding procedure to demonstrate for the first time the ultrastructural distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF), in the nervous system of the cestode Moniezia expansa. Two axon types, distinguished by their populations of different-sized electron-dense vesicles, were identified. Immunogold labeling demonstrated an apparent homogeneity of PP, FMRFamide and NPF (M. expansa) antigenic sites throughout the larger dense-cored vesicles within the central nervous system. Triple labeling clearly demonstrated the co-localisation of immunoreactivities (IR) for NPF, PP and FMRFamide within the same dense-cored vesicles. The presence of NPF-IR within the vesicles occupying the perikaryon of the neuronal cell body indicated that the peptides had undergone post-translational C-terminal amidation prior to entering the axon. Antigen pre-absorption experiments using NPF prevented labeling with either PP or FMRFamide antisera, and the failure of these antisera to block NPF-IR supports the view that some, if not all, of the PP/FMRFamide-IR is due to NPF-like peptides.

Neuropeptide F-immunoreactivity in the tetrathyridium of Mesocestoides corti (Cestoda: Cyclophyllidea).

The distribution pattern and subcellular localisation of neuropeptide F (NPF) immunoreactivity (IR) in the tetrathyridium stage of Mesocestoides corti were investigated by whole-mount immunocytochemistry in conjunction with confocal scanning laser microscopy (CSLM) and by immunoelectron microscopy using immunogold labeling. Using an antiserum directed to the C-terminal decapeptide amide (residues 30-39) of synthetic NPF (Moniezia expansa), CSLM revealed NPF-IR throughout the central and peripheral nervous systems of parental and dividing tetrathyridia. Ultrastructurally, gold labeling of NPF-IR was confined to the contents of the smaller of the two sizes of electron-dense neuronal vesicle identified.

Neuropeptide F (Moniezia expansa): localization and characterization using specific antisera.

Immunocytochemical techniques used in conjunction with confocal scanning laser microscopy (CSLM) and electron microscopy have been used to demonstrate, for the first time, the distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF) in the cestode, Moniezia expansa. Antisera were raised to intact NPF(1-39) and to the C-terminal decapeptide of NPF(30-39). These antisera were characterized and validated for use in both immunocytochemistry and radioimmunoassay (RIA). NPF immunoreactivity (IR) was detected using both antisera throughout all of the major components of the central and peripheral nervous systems of the worm. The pattern of NPF-IR was found to mirror the IR obtained using a C-terminally directed pancreatic polypeptide (PP) antiserum and FMRFamide antisera; blocking studies using these antisera revealed that FMRFamide and PP antisera cross-react with NPF(M. expansa). RIA of acid-alcohol extracts of the worm measured 114 ng/g using the C-terminal NPF antiserum and 56 ng/g using the whole-molecule-directed antiserum. While the C-terminally-directed NPF antiserum cross-reacts with NPF-related peptides from other invertebrates, the whole-molecule-directed NPF antiserum is specific for NPF(M. expansa). The C-terminal NPF antiserum has potential for use in the identification and purification of NPF analogues from other platyhelminth parasites.

The primary structure of neuropeptide F (NPF) from the garden snail, Helix aspersa

Neuropeptide F (NPF), originally isolated from the sheep tapeworm, Moniezia expansa, consists of 39 amino acid residues terminating in a phenylalaninamide. An analogous neuropeptide has been isolated and sequenced from extracts of circumoesophageal ganglia of the garden snail, Helix aspersa. This neuropeptide exhibits partial primary structural similarity to members of the vertebrate neuropeptide Y (NPY)/pancreatic polypeptide (PP) superfamily. NPF is thus of widespread occurrence in the nervous systems of invertebrates from different phyla and may represent the phylogenetic precursor of the vertebrate NPY/PP superfamily.

Peptide tyrosine phenylalanine: A novel neuropeptide F-related nonapeptide from the brain of the squid, Loligo vulgaris

A novel nonapeptide, sequence YAIVARPRFamide, was isolated from brain extracts of the squid, L. vulgaris. Designated peptide tyrosine phenylalanine (PYF), the peptide shows marked homology with the C-terminal nonapeptides of pancreatic polypeptide and neuropeptide F (NPF) from a number of sources. If PYF is the C-terminal nonapeptide of squid NPF, then it may be derived by a novel processing mechanism involving specific cleavage between two TYR residues. PYF may be a highly truncated, receptor-active variant of NPF.

Neuropeptide F-immunoreactivity in the monogenean parasite Diclidophora merlangi.

The localisation and distribution of neuropeptide F (NPF)-immunoreactivity (IR) in the monogenean fish-gill parasite, Diclidophora merlangi, have been investigated by whole-mount immunocytochemistry interfaced with confocal scanning laser microscopy and, at the ultrastructural level, by indirect immunogold labeling. Using antisera directed to intact synthetic NPF (Moniezia expansa, residues 1-39) or to the C-terminal decapeptide (residues 30-39) of synthetic NPF (M. expansa), immunostaining was found throughout the central (CNS) and peripheral nervous systems (PNS), including the innervation of the reproductive system. Immunoreactivity was found to be more intense using the antiserum to the C-terminal decapeptide fragment of NPF. At the subcellular level, gold labeling of NPF-IR was found exclusively over the contents of dense-cored vesicles that occupied nerve axons of both the CNS and the PNS. The distribution pattern of immunostaining for NPF mirrored exactly that previously documented for the vertebrate pancreatic polypeptide (PP) family of peptides and for FMRFamide. This finding and the results of preabsorption experiments strongly suggest that NPF is the predominant native neuropeptide in D. merlangi and that it accounts for most of the immunostaining previously obtained with PP and FMRFamide antisera.