Distribution of catecholamines and of immunoreactivity to substances like vertebrate enzymes for the synthesis of catecholamines within the central nervous system of the snail, Lymnaea stagnalis.

Catecholamines (CAs) were detected histochemically within over 185 cell bodies in the central nervous system (CNS) of juvenile and young adult Lymnaea. This distribution of CA-containing cells in all central ganglia except the pleural ganglia is more widespread than previously described but is consistent with other reports suggesting numerous roles for CAs within the nervous system. This study also describes the distribution of substances which are antigenically similar to four bovine enzymes for catecholamine synthesis, but the distribution patterns showed little or no overlap with each other or with CA. These results suggest the need for caution in the interpretation of such immunohistochemical studies.

A comparison of four techniques for mapping the distribution of serotonin and serotonin-containing neurons in fixed and living ganglia of the snail, Lymnaea.

The distribution of serotonin and serotonin-containing neurons was studied in the ganglia of the CNS of the snail Lymnaea stagnalis. Results of the application of three different labelling techniques on wholemount preparations were compared with each other and with the serotonin content of the ganglia, measured by high-performance liquid chromatography. Serotonin immunocytochemistry resulted in the highest number of labelled neurons, but the more recently developed in vivo method of 5,6- or 5,7-dihydroxytryptamine-induced pigmentation also proved to be a reliable technique for the visualization of serotonin-containing cell bodies. In comparison with these two techniques, the glyoxylic acid fluorescence method appeared to be less sensitive. The distribution and number of serotonin-containing neurons and biochemically measured serotonin in specific ganglia showed a close correlation. By combining the results of the three labelling techniques, a detailed map of serotonin-containing neurons was constructed, and this was compared with maps of identified neurons prepared from earlier electrophysiological studies. Previously described serotonergic neurons were consistently found, as well as several new serotonin-containing cell types in the cerebral, visceral and parietal ganglia. A network of serotonin-containing inter- and intraganglionic axon tracts, and thin serotonergic fibres in the perineurium were also demonstrated. This in vivo and in vitro identification of serotonin-containing neurons will facilitate further neurophysiological analysis of serotonergic neural mechanisms in Lymnaea.

Characterization of pre- and postsynaptic dopamine receptors in Lymnaea.

1. The effects of dopamine and several synthetic agonists and antagonists were studied using two identified neurons of the snail Lymnaea stagnalis. 2. In both the buccal-2 (B-2) neurons and the pedal giant (RPeD1) neuron dopamine elicited a hyperpolarizing response at least partly due to potassium efflux. RPeD1 is itself dopaminergic, implicating autoreceptors in its response to dopamine. 3. The following agents were tested: agonists--LY171555, pergolide, SKF38393, (-)-3-PPP, R(-)NPA and dopamine; antagonists--SCH23390, sulpiride, and metaclopramide. Dibutyryl cAMP was applied to determine whether the response is cAMP-mediated. 4. Results indicate that the pharmacological profiles of dopamine receptors on these neurons are inconsistent with those of either D-1, D-2 or autoreceptors in mammals.

Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization: a model study for mRNA detection.

To develop and optimize non-radioactive in situ hybridization techniques for mRNA detection, we used the neuropeptidergic system of the pond snail Lymnaea stagnalis as a biological model system. First, we investigated the in situ hybridization procedure using radioactive-labeled cDNA and synthetic oligonucleotide probes specific for egg-laying hormone (ELH) mRNA and molluscan insulin-like peptide (MIP) mRNA. The results show an intense grain deposit above the caudodorsal cells and light-green cells expressing, respectively, ELH mRNA and MIP mRNA. Good results with relation to signal strength and tissue morphology were obtained with freeze-dry paraformaldehyde vapor fixation. The necessity to perform tissue pre-treatment appeared to be dependent on the cell type of interest. The optimized in situ hybridization protocol proved to be applicable using probes that are either sulfonated/transaminated or labeled with acetylaminofluorene (AAF). In situ hybridization of such haptenized probes led to intense and specific staining of the cytoplasm of the caudodorsal cells. Egg-laying hormone mRNA appeared not to be homogeneously distributed in the cytoplasm but showed a "patch-like" pattern. Nuclear and axoplasmic staining for mRNA was also observed.

Structure of one of the neuropeptides of the egg-laying hormone precursor of Lymnaea.

The neurosecretory caudo-dorsal of the freshwater pulmonate snail Lymnaea stagnalis, a peptidergic system controlling egg laying and egg-laying behavior, produce several neuropeptides. One of these peptides, calfluxin, causes the influx of Ca2+ into the albumen gland, a female sex gland of the snail. In the present study calfluxin was purified and the amino acid composition was determined. The ratio of the amino acids appeared to be very close to that of one of the predicted peptides present on the egg-laying hormone precursor of Lymnaea. This peptide was synthesized and shows a clear biological activity in the bioassay. Furthermore, it shows a chromatographic behavior similar to that of the natural peptide. Based on these evidences it is concluded that the sequence of calfluxin is: Arg-Val-Asp-Ser-Ala-Asp-Glu-Ser-Asn-Asp-Asp-Gly-Phe-Asp. Calfluxin shows a remarkable homology with the sequence of one of the predicted peptides on the egg-laying hormone precursor of the marine opisthobranch Aplysia californica.

[Fasciola hepatica L.: the productivity of a sporocyst as a function of the size of Lymnaea truncatula Müller]. / Fasciola hepatica L.: etude de la productivité d'un sporocyste en fonction de la taille de Lymnaea truncatula Müller.

Experimental studies were undertaken on the rediae of Fasciola hepatica and on their content in two populations of Lymnaea truncatula snails, individually exposed to a single miracidium and fixed for histologic observations just after their death between days 74 and 81 postexposure at 20 degrees C. The numbers of free rediae and of larval stages per snail increased with the shell height. A constant number of free rediae was observed in snails measuring 11 mm and more in size. There was no limit of increase in number for the different types of larval stages, but these increases were lower for the youngest larval types. The number of larval stages per redia also increased with snail size; it was always higher in the first redial generation than in the following generations. Cercariae were produced by 40%-43% of the rediae contained in a snail: 65%-80% of these mature rediae belonged to the first generation and to the first cohort of the second generation.

Crecimiento y alometría de Lymnaea cubensis (Pfeiffer, 1839) y L. columella (Say, 1817) / Growth and allometry of Lymnaea cubensis (Pfeiffer, 1839) y L. columella (Say, 1817)

Mediante el cultivo en el laboratorio de cuatro cohortes de Lymnaea cubensis, proveniente de especímenes silvestres de la localidad de Mendoza Fría, en Los Andes Venezolanos, y seis de L. columella provenientes de especímenes cubanos, se determinó la longitud total de la concha del recién nacido: en promedio 0,63 mm para la primera especie y 0,85 mm para la segunda; la tasa de crecimiento de la longitud de la concha: 0,053 mm/día para L. cubensis (CV: 18,2%, n: 4) y 0,167 mm para L. columella (CV: 15,5%, n: 6); la talla máxima alcanzada: 8,44 y 19,66 mm respectivamente. En condiciones naturales se determinó la tasa de crecimiento de L. cubensis: 0,020 mm/día. Una relación de alometría negativa fue estimada para ambas especies entre la longitud total de la concha y el ancho total y entre el ancho de la abertura y la longitud total. Por el contrario, una relación de isometría se determinó entre la longitud total y el largo de la espira

Equilibrium and kinetic studies of oxygen binding to fragments of Lymnaea stagnalis (freshwater snail) haemocyanin obtained by proteolytic digestion.

Functional fragments of the haemocyanin from the gastropod mollusc Lymnaea stagnalis (freshwater snail) were obtained by partial digestion with trypsin and plasmin. The fragments were purified by ion-exchange chromatography and characterized by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Three types of single-functional unit fragment were isolated from the trypsin digest, and two immunologically distinct three-functional unit fragments and a single-functional unit fragment were isolated from the plasmin digest. The O2-binding behaviour of the fragments was investigated by equilibrium and kinetic methods. Over the pH range 7.0-8.2, in the presence of 10-20 mM-CaCl2, all of the single-functional unit fragments displayed non-co-operative O2 binding and showed no evidence of a Bohr or a salt effect. A Hill coefficient of less than 1.0 was obtained with one of the two three-functional unit fragments studied, whereas both of these fragments displayed a Bohr effect. Functional heterogeneity of the fragments was indicated by the variation in the O2 affinity, the P50 (partial pressure of O2 at half saturation) ranging between 0.26 and 0.77 kPa (approx. 2-6 mmHg). Stopped-flow data reflected the O2 equilibrium behaviour. Thus there was a fall in the value of the O2 dissociation rate constant from approx. 15 to 1s-1 in parallel with the increase in O2 affinity.

The site of action of lithium ions in morphogenesis of Lymnaea stagnalis analyzed by secondary ion mass spectroscopy.

Exogastrulation as a disturbance of development in eggs of Lymnaea stagnalis is caused by the action of LiCl at the second cleavage stage and not at the first or third. The percentage of exogastrulae formed is strongly concentration dependent. To determine the site of action of lithium ions, the cellular contents of Li, C, Na, Mg, P, K, and Ca were analyzed by secondary ion mass spectroscopy (SIMS). The mean elemental concentrations of Na, Mg, K, and Ca are close to those found earlier by electron probe microanalysis and atomic absorption spectroscopy. Lymnaea eggs at the first, second, and third cleavage stage were treated with LiCl in a series of concentrations ranging from 50 to 0.1 mM. In all cases the cells contained a few mM lithium after treatment. After treatment at the insensitive first cleavage stage the lithium content is carried over by the cells through the sensitive second cleavage to the insensitive third cleavage stage. These data allow the conclusion that it is the external lithium concentration which is responsible for the specific effect. This presents direct analytical evidence that the primary action of lithium ions is located at the cell membrane.

Isolation and properties of vitellogenic ferritin from snails.

The iron storage protein ferritin is the principal yolk protein in oocytes of the snails Planorbarius corneus L. and Lymnaea stagnalis L. This report gives an account of the isolation procedure and of some properties of snail ferritins. The isolation procedure includes a heat-denaturation step, gel filtration on Sepharose 6B and two ultracentrifugation steps followed by electrophoresis. Ferritins from both snails are highly reminiscent of vertebrate and plant ferritins in terms of heat stability, absorption spectrum and ultrastructure, and both share common antigen determinants with horse spleen ferritin. In different electrophoresis systems snail ferritins display considerable heterogeneity and microheterogeneity. Electrophoresis in the presence of sodium dodecyl sulphate (SDS) yields two major polypeptides with molecular weights of 19 000 and 24 000, which are interpreted to be authentic subunits of the ferritin molecule. Different organs and tissues of the snails differ in subunit composition. Midgut gland ferritin consists predominantly of the 19 000 Mr polypeptide, while in embryos only the 24 000 Mr band was found. No carbohydrates or lipids could be detected by staining acrylamide gels. Results from SDS/acrylamide electrophoresis, electrophoresis under non-denaturing conditions on gradient gels and from isoelectric focusing indicate that the ferritins of both snails are composed of at least two different types of ferritin that are tissue-specific. One ferritin is typical of somatic tissue (midgut gland) and is most probably a homopolymer of the 19 000 Mr subunit. The other ferritin is typical of oocytes, but since it is an exogenous protein it is also encountered in the midgut gland (the presumed site of yolk synthesis) and the haemolymph. Vitellogenic ferritin is either a homopolymer of the 24 000 Mr subunit or is predominantly composed of it. So far, there is no evidence for a precursor-product relationship between the two subunits.

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

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 substances 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.

Lipid composition in ganglia of Mollusca.

The aim of the present study is to ascertain lipid composition in the ganglia of Mollusca. Nervous ganglia in the periesophageal ring dissected from Helix pomatia, Lymnaea stagnalis, Murex trunculus and Murex brandaris were studied by biochemical and histochemical procedures. Glycosphyngolipids are present mainly as sulpholipid; sialic acid and gangliosides are not present as revealed by Svennerholm's reaction and TLC separation. The phospholipid/cholesterol ratios are: 0.47 (Helix), 0.42 (Lymnaea), 0.86 (Murex brandaris) and 1.01 (Murex trunculus).

Immunocytochemical identification of neural elements in the central nervous systems of a snail, some insects, a fish, and a mammal with an antiserum to the molluscan cardio-excitatory tetrapeptide FMRF-amide.

With an antiserum to the molluscan cardio-excitatory tetrapeptide FMRF-amide neurons and/or nerve fibers were immunocytochemically identified in the central nervous systems of a snail (Lymnaea stagnalis), some insects (Leptinotarsa decemlineata, Periplaneta americana, Locusta migratoria, Pieris brassicae), a fish (Poecilia latipinna) and a mammal (mouse). The fact that immunoreactive material was observed in neurohaemal organs (corpora cardiaca of the insects) as well as in axon terminals ending on other neurons, seems to indicate that this peptide can function as a neurohormone and/or as a neurotransmitter. The results sustain the hypothesis that biologically active peptides have a wide distribution in the animal kingdom.

Characterization of domains obtained from a mollusc haemocyanin by limited proteolytic digestion.

The haemocyanin from the freshwater gastropod Lymnaea stagnalis was digested with proteolytic enzymes under conditions where it existed as whole (native) molecules (mol.wt. approx. 9 X 10(6)), or as one-tenth molecules. Digestion of whole molecules yielded a fragment of mol.wt. approx. 110,000 believed to correspond to the 'collar' of the molecule, and an aggregate some 20--30 times the size of the original native molecule formed by end-to-end polymerization of the molecule after removal of the collar. Digestion of one-tenth molecules yielded a mixture of products that could be separated into three fractions by gel filtration. Analysis of these by sodium dodecylsulphate/polyacrylamide-gel electrophoresis revealed that they typically contained two or three components. The collar fragment was present as a component of the intermediate-molecular-weight fraction, and it dissociated on sodium dodecyl sulphate/polyacrylamide gels to give two bands corresponding to apparent mol.wts. 65,000 and 60,000. The c.d. spectra of the separated fractions were recorded and fitted with Gaussian curves by a computer procedure. The fractions each possessed distinct c.d. spectra, by which they could be identified: the collar-fragment c.d. and absorption spectra showed the most striking differences compared with those of the other fragments. The results were interpreted in terms of the postulated existence, within the haemocyanin molecule, of multi-domain structures, each comprising a single polypeptide chain of mol.wt. 200,000--300,000.