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.     

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.)     

Fixed phagocytes in the freshwater snail Lymnaea stagnalis

Morphological and histochemical examination of the blood and connective tissue of the freshwater snail Lymnaea stagnalis injected with various types of foreign particulate materials has shown the existence of free as well as fixed phagocytic cells. The morphology of the fixed phagocytes is described, and the phagocytic system of the snail is compared with that of other molluscan species.     

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.     

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.     

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.     

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.     

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.     

GABAA- and AMPA-like receptors modulate the activity of an identified neuron within the central pattern generator of the pond snail Lymnaea stagnalis

To examine the neurochemistry underlying the firing of the RPeD1 neuron in the respiratory central pattern generator of the pond snail, Lymnaea stagnalis, we examined electrophysiologically and pharmacologically either “active” or “silent” preparations by intracellular recording and pharmacology. GABA inhibited electrical firing by hyperpolarizing RPeD1, while picrotoxin, an antagonist of GABA_A receptors, excited silent cells and reversed GABA-induced inhibition. Action potential activity was terminated by 1 mM glutamate (Glu) while silent cells were depolarized by the GluR agonists, AMPA, and NMDA. Kainate exerted a complex triphasic effect on membrane potential. However, only bath application of AMPA desensitized the firing. These data indicate that GABA inhibits RPeD1 via activation of GABA_A receptors, while Glu stimulates the neuron by activating AMPA-sensitive GluRs.     

Oral water ingestion in the pulmonate freshwater snail,Lymnaea stagnalis

InLymnaea stagnalis, oral uptake of ambient medium was studied using⁵¹Cr-ethylenediaminetetra-acetic acid. In normal snails Cr-ethylenediaminetetra-acetic acid uptake showed two components: a high uptake rate within the first hour followed by moderate uptake proportional with time. The tracer accumulated mainly in the digestive system. All animals showed initial, transient uptake. Moderate uptake proportional with time did not occur in snails in which the buccal ganglia had been extirpated, in which both the buccal ganglia had been extirpated and the oesophagus was sectioned, or in snails provided with an oesophageal fistula. These snails did not accumulate tracer in the intestinal system. This type of tracer accumulation clearly represented oral ingestion of surrounding water. The oral water ingestion rate ranged from 8 to 12 μl·h⁻¹·g⁻¹. Assuming complete absorption, this accounts for 20–30% of the urine production rate. At low external concentrations the contribution of oral water ingestion to Na⁺ balance is negligible. However, its importance will grow with increasing external concentrations and becomes a major factor at higher concentrations. The ingestion rate increased almost sixfold when starving snails were allowed to feed. It is suggested that oral water ingestion is a consequence of making bite cycles and swallowing.     

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.     

Purification and properties of the carbonic anhydrase of Rhodospirillum rubrum

The carbonic anhydrase (EC 4.2.1.1) of Rhodospirillum rubrum has been purified to apparent homogeneity and some of its properties have been determined. The enzyme was cytoplasmic and was found only in photosynthetically grown cells. It had a molecular weight of about 28,000, and was apparently composed of two equal subunits. The amino acid composition was similar to that of other reported carbonic anhydrases except that the R. rubrum enzyme contained no arginine. The isoelectric point of the enzyme was 6.2 and the pH optimum was 7.5. It required Zn(II) for stability and enzymatic activity. The K _m(CO₂) was 80 mM. Typical carbonic anhydrase inhibition patterns were found with the R. rubrum enzyme. Strong acetazolamide and sulfanilamide inhibition confirmed the importance of Zn(II) for enzymatic activity as did the anionic inhibitors iodide, and azide. Other inhibitors indicated that histidine, sulfhydryl, lysine and serine residues were important for enzymatic activity.Abbreviation CA carbonic anhydrase In memory of R. Y. Stanier     

The osphradium is involved in the control of egg-laying in the pond snail Lymnaea stagnalis

Summary

Ablation of the osphradium (a peripheral putative chemo-sensory organ) in Lymnaea stagnalis resulted in significant increase of egg-laying activity as compared with control animals (observed for 50 days). Cutting the osphradial nerve increased egg-laying to a lesser degree and only for the first 15–20 days. Water changes in the aquaria had a similar stimulating effect on egg-laying activity in both osphradium-deprived and control snails. Permanent aeration of the aquaria abolished this effect. Possible mechanisms of tonic inhibitory action of the osphradium on egg-laying are discussed.