Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I. Our observations support the hypothesis that light yellow cells are involved in maintaining the shape of the animal via the regulation of ion- and waterbalance processes and blood pressure.     

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization

Summary The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material.A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as ectopic CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral gangion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed. The nerves emanating from this ganglion contained many more immunoreactive axons than those from the left pedal ganglion. A polyclonal antibody raised against the synthetic fragment of CDCH stained, in addition to the neurons and axons revealed with the monoclonal antibody and the cDNA probe, three other major groups of neurons. Two are located in the cerebral ganglion, the other in the left pedal ganglion.The present findings suggest the presence of a system of neurons that contain CDCH or CDCH-like peptides. The role this system may play in the control of egg-laying and egg-laying behaviour is discussed.     

The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry

Summary VD₁ and RPD₂ are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD₁/RPD₂ gene. To this end, in situ hybridization with the cDNA probe of the VD₁/RPD₂ gene and immunocytochemistry with antisera specific to VD₁ and RPD₂ (the 1-antiserum, Mab4H5 and ALMA 6) and to R₁₅ (the 1 and 16-mer antisera) were performed on alternate tissue sections. A VD₁/RPD₂ neuronal system comprising three classes of neurons (A1–A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD₁ and RPD₂) immunoreact with the 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1–5 small and 1–5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3–4 medium-sized neurons and a cluster of 4–5 small neurons located in the pedal ganglion) immunoreact with the 1-antiserum only. All neurons of the system are immunonegative to the R₁₅ antisera. The observations suggest that the neurons of the VD₁/RPD₂ system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.     

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.     

Ultrastructural immunocytochemical evidence for peptidergic neurotransmission in the pond snail Lymnaea stagnalis

Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.     

Immunocytochemical demonstration of peptidergic cells in the pond snail Lymnaea stagnalis with an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide

Summary With an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide immunoreactive perikarya and nerve fibers were identified in the central and peripheral nervous system of the pond snail Lymnaea stagnalis. Their localization is described. The same antiserum yielded reactive product in particular cells of the epithelium of the alimentary tract. The use of two different fixatives, glutaraldehyde, and a mixture of glutaraldehyde, picric acid, and acetic acid (GPA) showed that certain nerve cells can be identified only in material fixed with either the one or the other of these two fixatives, a result which indicates that in Lymnaea more than one FMRF-amide-like substance may occur.Positive axon endings were found in the periphery of various nerves, i.e., in places where neurohormones are released into the blood. Other fibers were found to end, probably synaptically, on other neurons, on epithelial cells in the stomach, and between muscle cells in various parts of the body, e.g., in the heart. In these cases the FMRF-amide-like substance may function as a neurotransmitter or a neuromodulator.     

Unusual distribution of tubulin isoforms in the snail Lymnaea stagnalis

Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vitro in the presence of taxol. Various broad-spectrum antibodies raised against -tubulin and -tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome -tubulin, however, labelled -tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total -tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated -tubulin and acetylated -tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.     

Co-existence of immunoreactivity to anti-dopamine,anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis

Summary Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with antivasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.     

Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the shell by way of an adhesive substance containing proteins and mucopolysaccharides.This research was made possible by a grant from the Netherlands Organization for Pure Research (Z.W.O.)     

Immunocytochemical demonstration of a humoral defence factor in blood cells (amoebocytes) of the pond snail,Lymnaea stagnalis

Summary Monolayers of blood cells (amoebocytes) and sections of connective tissue and of amoebocyte pellets of the freshwater pulmonate gastropod Lymnaea stagnalis were stained immunocytochemically with antisera to snail agglutinin/opsonin. The presence of this substance was demonstrated light microscopically both in the cytoplasm and on the cell membrane of amoebocytes. This suggests that amoebocytes synthesize agglutinin/opsonin, and bear it at their surface as receptors for foreign materials.     

Effect of a toxicant on phagocytosis pathways in the freshwater snail Lymnaea stagnalis

The disturbance of plasma membrane carbohydrates and of lipopolysaccharide (LPS) ligands in relation to cytoskeletal transformations of haemocytes has been investigated after chronic exposure of pond snails (Lymnaea stagnalis) to the peroxidizing toxicant fomesafen. Neither of the two lectins used (concanavalin A and wheat germ agglutinin) showed any binding modification after incubation of the snails in the presence of the toxicant. However, after exposure of the snails to fomesafen, a clear and persistent reduction in LPS labelling of haemocytes occurred. The actin cytoskeleton of the same cells also appeared to be sensitive to the toxicant. The reduction in LPS-binding sites was related to actin staining, leading to the hypothesis that LPS ligands and actin could be similarly modulated by the toxicant. Damaged cells showed non-adherent membrane portions with reduced filopodial extrusions, exhibiting a smooth surface free of microvilli. These changes could lower the spreading and adhesion of the cells and could therefore account for the loss in their phagocytic capabilities.     

Haemocyanin production in pore cells of the freshwater snail Lymnaea stagnalis

Summary Electron micrographs of pore cells of Lymnaea stagnalis suggest that these cells produce and store haemocyanin.