Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

The action of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides on mammals

First purified 11 years ago from clam ganglia, FMRFamide (Phe-Met-Arg-Phe-NH2) was quickly demonstrated to be cardioactive in several molluscan species. Subsequent discovery that FMRFamide, or FMRFamide-related peptides (FaRPs), were present in mammalian central nervous system and gastrointestinal tract prompted investigations into the effect of FMRFamide on mammals. FMRFamide has now been shown to be cardioexcitatory in mammals, to inhibit morphine-induced antinociception, and to block morphine-, defeat-, and deprivation-induced feeding. It also inhibits colonic propulsive motility, induces behavioral effects when administered intrathecally, and has been reported to have amnesic effects in rodents. A proposal has arisen that a FMRFamide-like substance is an endogenous opioid antagonist and has stimulated a search for such a substance. However, FMRFamide has only weak affinity for opioid receptors and not all the actions of FMRFamide appear to be explained by actions at opioid receptors. Alternative mechanisms have been proposed which suggest that FMRFamide acts as a neuromodulator.     

FMRFamide immunoreactive neurons in the central nervous system of the snail,Achatin a fulica

• 1.1. FMRFamide immunoreactive neurons were detected in the central nervous system of the snail, Achatina fulica.
• 2.2. FMRFamide immunoreactive neurons were found in all the ganglia comprising the central nervous system. In particular, the immunoreactivity was recognized in both the ordinary and giant neurons of the visceral and right parietal ganglia.
• 3.3. In the cerebral and pleural ganglia, FMRFamide immunoreactive neurons were found only in the ordinary neurons. The immunoreactivity was shown to have a tendency to form a group in the cerebral and pedal ganglia.

     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

Visualization of neuropeptides in paraffin-embedded tissue sections of the central nervous system in the decapod crustacean, Penaeus monodon, by imaging mass spectrometry

The distributions of neuropeptides in paraffin-embedded tissue sections (PETS) of the eyestalk, brain, and thoracic ganglia of the shrimp Penaeus monodon were visualized by imaging mass spectrometry (IMS). Peptide signals were obtained from PETS without affecting morphological features. Twenty-nine neuropeptides comprising members of FMRFamide, SIFamides, crustacean hyperglycaemic hormone, orcokinin-related peptides, tachykinin-related peptides, and allatostatin A were detected and visualized. Among these findings we first identified tachykinin-related peptide as a novel neuropeptide in this shrimp species. We found that these neuropeptides were distributed at specific areas in the three neural organs. In addition, 28 peptide sequences derived from 4 types of constitutive proteins, including actin, histones, arginine kinase, and cyclophilin A were also detected. All peptide sequences were verified by liquid chromatography-tandem mass spectrometry. The use of IMS on acetic acid-treated PETS enabled us to identify peptides and obtain their specific localizations in correlation with the undisturbed histological structure of the tissue samples.     

Partial Characterization of a Novel Cardioinhibitory Peptide from the Brain of the Snail Helix aspersa

1. We report the isolation of a peptide from the brain of the snail Helix aspersa by radioimmunoassay using an antisomatostatin.2. The sequencing of an immunopositive fraction showed the presence of a new tridecapeptide, termed Helix cardioinhibitory peptide (HCIP), with the following primary structure : H-Val-Phe-Gln-Asn-Gln-Phe-Lys-Gly-Ile-Gln-Gly-Arg-Phe-NH₂. It is structurally related to the Achatina cardioexcitatory peptide (ACEP-1) and the terminal-ammo acid sequence of HCIP is identical to that of FMRFamide family peptides.3. The synthetic HCIP was tested on heart and neuronal activities and it was found to have inhibitory actions not only on the ventricle but also on visceral neurons of the central nervous system of Helix. Immunocytochemical investigation indicates its presence in visceral and parietal ganglia, in which cells taking part in the regulation of the heartbeat have been previously identified .     

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 regionspecific 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.     

The neurochemical basis of recurrent inhibition in the reflex arch of the defensive reaction]

Mechanisms of habituation in the network of identified neurones were investigated in isolated preparation of central nervous system in the snail Helix. It has been found that intracellularly induced spike discharge in premotor command neurones decreases synaptic responses to repeated nerve stimulation in all recorded command neurones. Application of the neuropeptide FMRFamide elicits similar changes in the network. Taking into account that the investigated command neurones contain FMRFamide, as was shown immunochemically, it is possible to assume the existence of recurrent inhibition in the network underlying avoidance reactions. This recurrent inhibition causes habituation of the network output in the cases when the repeated stimuli do not evoke sensitization via activation of serotonergic cells.     

The actions of FxRFamide related neuropeptides on identified neurones from the snail, Helix aspersa

Intracellular recordings were made from identified neurones in the suboesophageal ganglia of the snail, Helix aspersa. The actions of the eight FxRFamide analogues were investigated on these neurones. These peptides included ones isolated from arthropods and nematodes. All the peptides excited certain neurones while inhibiting others, though their relative potencies varied. Overall on neurones inhibited by these peptides the potency order was: DNFLRFamide > FMRFamide > PDVDHVFLRFamide = KNEFIRFamide > FLRFamide > SDRNFLRFamide = SDPNFLRFamide > KHEYLRFamide. However, if the responses are compared on individual cell types, then the picture becomes more complex. For example, on cell F-2, KNEFIRFamide proved to be potent with an EC-50 value of 0.54 microM. On neurones F-13/16 and E-16, PDVDHVFLRFamide was inhibitory while FMRFamide, FLRFamide, SDRNFLRFamide and SDPNFLRFamide were excitatory. In terms of overall excitatory actions, the data are less complete but an approximate order of potency is: FMRFamide > DNFLRFamide > SDPNFLRFamide > PDVDHVFLRFamide > KNEFIRFamide = KHEYLRFamide = SDRNFLRFamide. However this again varies between specific neurones. These results demonstrate that peptides from insects, crustacea and nematodes are active on Helix neurones and may activate specific receptor subtypes, indicating the possible presence of endogenous analogues of these non-molluscan peptides in the Helix nervous system.     

Immunocytochemical localization of FMRFamide in the central nervous system and the kidney of Helisoma duryi (Mollusca): its possible antidiuretic role

The distribution of FMRFamide-immunoreactive neurons in the central nervous system of the freshwater pulmonate, Helisoma duryi is described. All parts of the central nervous system except the two pleural and the right parietal ganglia, contain immunoreactive neurons. By immunogold techniques, only one kind of neurosecretory FMRFamide-immunoreactive cell (previously identified as the type-3 cell) was localized in the visceral and left parietal ganglia. This cell type has been previously implicated in an antidiuretic role. FMRFamide-immunoreactive material is found in the whole mount of the kidney as well as in kidney sections. Electron microscopic examination shows that the axons innervating either the smooth muscles of the kidney or the kidney itself contain neurosecretory granules morphologically similar to type-3 cells of the visceral and left parietal ganglia. When incubated in saline containing nanogram quantities of FMRFamide, the wet weight of the kidney increased. It is suggested that FMRFamide-like substance may function as an antidiuretic factor and that the kidney is a target organ of this peptide for osmoregulation.     

Imaging mass spectrometry: principle and application

Imaging mass spectrometry (IMS) is two-dimensional mass spectrometry to visualize the spatial distribution of biomolecules, which does not need either separation or purification of target molecules, and enables us to monitor not only the identification of unknown molecules but also the localization of numerous molecules simultaneously. Among the ionization techniques, matrix assisted laser desorption/ionization (MALDI) is one of the most generally used for IMS, which allows the analysis of numerous biomolecules ranging over wide molecular weights. Proper selection and preparation of matrix is essential for successful imaging using IMS. Tandem mass spectrometry, which is referred to MSⁿ, enables the structural analysis of a molecule detected by the first step of IMS. Applications of IMS were initially developed for studying proteins or peptides. At present, however, targets of IMS research have expanded to the imaging of small endogenous metabolites such as lipids, exogenous drug pharmacokinetics, exploring new disease markers, and other new scientific fields. We hope that this new technology will open a new era for biophysics.     

Molecular histoproteomy by MALDI mass spectrometry imaging to uncover markers of the impact of Nosema on Apis mellifera

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful technology used to investigate the spatio-temporal distribution of a huge number of molecules throughout a body/tissue section. In this paper, we report the use of MALDI IMS to follow the molecular impact of an experimental infection of Apis mellifera with the microsporidia Nosema ceranae. We performed representative molecular mass fingerprints of selected tissues obtained by dissection. This was followed by MALDI IMS workflows optimization including specimen embedding and positioning as well as washing and matrix application. We recorded the local distribution of peptides/proteins within different tissues from experimentally infected versus non infected honeybees. As expected, a distinction in these molecular profiles between the two conditions was recorded from different anatomical sections of the gut tissue. More importantly, we observed differences in the molecular profiles in the brain, thoracic ganglia, hypopharyngeal glands, and hemolymph. We introduced MALDI IMS as an effective approach to monitor the impact of N. ceranae infection on A. mellifera. This opens perspectives for the discovery of molecular changes in peptides/proteins markers that could contribute to a better understanding of the impact of stressors and toxicity on different tissues of a bee in a single experiment.     

Imaging mass spectrometry at cellular length scales

Imaging mass spectrometry (IMS) allows the direct investigation of both the identity and the spatial distribution of the molecular content directly in tissue sections, single cells and many other biological surfaces. In this protocol, we present the steps required to retrieve the molecular information from tissue sections using matrix-enhanced (ME) and metal-assisted (MetA) secondary ion mass spectrometry (SIMS) as well as matrix-assisted laser desorption/ionization (MALDI) IMS. These techniques require specific sample preparation steps directed at optimal signal intensity with minimal redistribution or modification of the sample analytes. After careful sample preparation, different IMS methods offer a unique discovery tool in, for example, the investigation of (i) drug transport and uptake, (ii) biological processing steps and (iii) biomarker distributions. To extract the relevant information from the huge datasets produced by IMS, new bioinformatics approaches have been developed. The duration of the protocol is highly dependent on sample size and technique used, but on average takes approximately 5 h.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

ADENYLATE CYCLASE IN HELIX AND APLYSIA GANGLIA: CHARACTERISTICS OF ITS STIMULATION BY A PEPTIDE-CONTAINING NERVOUS SYSTEM EXTRACT

Abstract— A crude particulate fraction, prepared from the central ganglia of Helix or Aplysia , contains levels of adenylate cyclase activity comparable to those in mammalian brain. This activity can be stimulated up to 50-fold by NaF, and 4- to 10-fold by guanyl nucleotides such as GTP and guanylylimidodiphosphate (Gpp(NH)p). A peptide-containing extract from Helix or Aplysia nervous system also stimulates the adenylate cyclase, by 50-400°. In contrast, a number of peptides known to occur in vertebrate and invertebrate nervous system are without effect. The adenylate cylase stimulation by the endogenous molluscan peptide-containing extract may be receptor-mediated, but the effect is not enhanced in the presence of guanyl nucleotides: in this respect it differs from many other hormone-sensitive adenylate cyclases. The endogenous extracts prepared from Helix and Aplysia each stimulate both Helix and Aplysia adenylate cyclases, suggesting that the putative cyclase-linked receptors may be similar in the two species. Furthermore, the active components in the extracts from Helis and Aplysia appear to be similar, since preliminary evidence suggests that they may interact with the same adenylate cyclase-linked receptor in particulate fraction from Helix ganglia.     

Probing neuropeptide signaling at the organ and cellular domains via imaging mass spectrometry

Imaging mass spectrometry (IMS) has evolved to be a promising technology due to its ability to detect a broad mass range of molecular species and create density maps for selected compounds. It is currently one of the most useful techniques to determine the spatial distribution of neuropeptides in cells and tissues. Although IMS is conceptually simple, sample preparation steps, mass analyzers, and software suites are just a few of the factors that contribute to the successful design of a neuropeptide IMS experiment. This review provides a brief overview of IMS sampling protocols, instrumentation, data analysis tools, technological advancements and applications to neuropeptide localization in neurons and endocrine tissues. Future perspectives in this field are also provided, concluding that neuropeptide IMS would greatly facilitate studies of neuronal network and biomarker discovery.     

Immunocytochemical demonstration of neuropeptides in the fish-gill parasite, Diclidophora merlangi (Monogenoidea)

Using the indirect immunofluorescence technique, immunoreactivity (IR) to three mammalian and one invertebrate regulatory peptide has been demonstrated in the nervous system of the monogenean gill parasite Diclidophora merlangi. IR to pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY) and FMRFamide was evident throughout central and peripheral nervous tissues, whereas vasoactive intestinal polypeptide (VIP)-IR was confined to a portion of the longitudinal ventral nerve cords. Staining patterns revealed the orthogonal arrangement of the nervous system consisting of paired cerebral ganglia, connecting post-pharyngeal commissure, three pairs of longitudinal nerve cords and associated neurones. PP-IR, PYY-IR and FMRFamide-IR were intense throughout the central nervous system of the worm. A small plexus of nerve fibres and somata in each peduncle was immunoreactive for FMRFamide and provided innervation to each of the eight posterior clamps. In the peripheral nervous system, PP-IR, PYY-IR and FMRFamide-IR occurred in an extensive nerve-net with fine, possibly sensory nerve endings in the tegument. PP-IR was also present in nerve fibres in the walls of the ootype, seminal vesicle and uterus. PYY- and FMRFamide-IRs, while evident in nerve fibres of the ootype wall, were also present in a distinct population of cells that encircles the ootype, and which are linked to it by fine cytoplasmic connectives. The majority of these somata were bipolar or multipolar. PYY-IR and FMRFamide-IR were also associated with nerve fibres and bipolar cells in the wall of the vitelline reservoir. Regulatory peptides would appear to play an integral role in neuronal functioning and egg development in D. merlangi.     

Dynamics of FMRFamide immunoreactivity in response to physiologically active substances in the central nervous system of the snail, Achatina fulica

1. The changes in FMRFamide (Phe-Met-Arg-Phe-NH2) immunoreactivity in response to incubation in dopamine, serotonin, met-enkephalin, oxytocin, arg-vasopressin and FMRFamide were examined in the central nervous system of the snail, Achatina fulica. 2. When the central nervous system was cultured in medium which contained dopamine and in medium which contained serotonin, the number of immunoreactive neurons increased in the anterior part of the cerebral ganglion and decreased in the sub-esophageal ganglion. 3. When arg-vasopressin was added to the culture medium, the number of immunoreactive neurons increased in the pedal ganglion and decreased in the other sub-esophageal ganglion. 4. By contrast, when the central nervous system was cultured in medium which contained oxytocin, the number of immunoreactive neurons did not increase, but rather decreased, in each ganglion. 5. No changes in immunoreactivity were detected in the central nervous system when it was cultured in medium which contained FMRFamide. 6. It appears, from these results, that the production and release of FMRFamide from different neurons are differentially affected by the physiologically active substances tested.     

Distribution and ontogeny of glial fibrillary acidic protein in the snail Megalobulimus abbreviatus

Glial fibrillary acidic protein (GFAP) is the major component of intermediate glial filaments in the central nervous system of many vertebrates and invertebrates. In vertebrates, this protein is mainly expressed in mature astrocytes and provides structural cell stability. The highly conserved structure and glial specificity of this protein have allowed studies of ontogeny and phylogeny using antibodies. The present study investigated the ontogenetic profile and molecular weight of GFAP in the snail, Megalobulimus abbreviatus, particularly in cerebral ganglia and subesophageal mass, by immunohistochemistry and immunoblotting. Our results confirm and extend previous studies about glial intermediate filaments in snails, showing: (i) a higher GFAP content in cerebral ganglia than in subesophageal mass; (ii) a developmental increase of GFAP immunocontent in cerebral ganglia, as described in Vertebrates; and (iii) an electrophoretic band for GFAP of approximately 55 kDa.