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PubMed1992

Evolution of the vasopressin/oxytocin superfamily: characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis.

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not clear. To investigate this issue, we cloned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressin, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapeptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalian copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

Amino Acid Sequence↗
PubMed1989

Age-related changes in female reproductive activity and growth in the mollusc Lymnaea stagnalis.

Age-related changes in reproduction and growth of the pulmonate snail Lymnaea stagnalis were studied in animals under standard culture conditions. Longitudinal studies as well as cross-sectional studies were done. Egg-laying activity starts in Lymnaea at an age of about 2 months. Up to an age of about 250 days it increases, and in older snails it decreases with age. Cross-sectional studies showed that the increase in egg-laying activity is due to an increase in the number of animals that lay eggs and to an increase in the egg-laying frequency of individuals. The decrease in egg-laying activity is due to a decrease in the egg-laying frequency of individual animals and to an increase in the number of animals that stop egg laying. Growth (shell length and body weight) ceases at about the same age at which egg laying begins to decrease. The results are discussed in relation to the organization of the neuroendocrine system in Lymnaea and to results of earlier studies on age-related changes in peptidergic neurons in the CNS of Lymnaea.

Aging↗
PubMed2006

Comparative study of visuo-vestibular conditioning in Lymnaea stagnalis.

In this review, we compare the current understanding of visuo-vestibular conditioning in Hermissenda crassicornis and Lymnaea stagnalis on the basis of behavioral, electrophysiologic, and morphologic studies. Paired presentation of a photic conditioned stimulus (CS) and an orbital rotation unconditioned stimulus (US) results in conditioned escape behavior in both species. In Hermissenda, changes in excitability of type B photoreceptors and morphologic modifications at the axon terminals follow conditioning. Caudal hair cells, which detect mechanical turbulence, have reciprocal inhibition with type B photoreceptors. In Lymnaea, the interaction between photoreceptors and hair cells is dependent on statocyst location. Furthermore, the organization of the Lymnaea eye is complex, with more than 100 photoreceptors distributed in a uniquely folded retina. Although the optimal conditions to produce long-term memory (memory persistent for >1 week) are almost identical in Hermissenda and Lymnaea, physiologic and morphologic differences suggest that the neuronal mechanisms underlying learning and memory are distinct.

Animals↗
PubMed2000

Modulation in firing pattern and oscillation in nerve cells of Lymnaea during network reconstruction.

The modulation and reconstruction of the cardio-respiratory neural circuit of Lymnaea stagnalis L. was compared to that of Helix ponatia L. where the input variation and signal molecules were found to have primary importance in network reorganization. From the cardio-respiratory circuit only neurons connected by afferent or efferent pathways to the peripheral chemosensory organ, the osphradium, were used. It was shown that, the general principles of the network reorganization is similar in the two species. The firing pattern of the neurons altered in Lymnaea depending on the input activation or presence of signal molecules in the vicinity of the neurons. The responses of the neurons to the same sensory information, originating from osphradium varied depending on their firing patterns. On central neurones the generation of phasic pattern and/or oscillation was an indicator of network disintegration leading to insensibility to the osphradial sensory inputs. Co-application of signal molecules (5HT, DA, GABA with opioid peptides) to the neurons caused a phasic firing pattern and/or oscillation leading to disintegration of one network and activation of another one. The effect of mu-opioid peptides on GABA-induced and voltage activated ion currents were shown to be the cellular target in reconstruction of neural networks in Lymnaea. The neural network reconstruction in vertebrate brain evoked by signal molecules can be compared to that observed in the identified network of Lymnaea stagnalis making this latter a useful model in further studies, too.

Animals↗
PubMed2004

Toxicological effect and biochemical alterations induced by different fractions of Euphorbia royleana latex in freshwater harmful vector snail Lymnaea acuminata.

Laboratory evaluation was made to assess the molluscicidal activity of different fractions of Euphorbia royleana (Family- Euphorbiaceae) latex obtained through sephadex gel column against freshwater snail Lymnaea (Radix) acuminata Lamarack. This snail is the vector of liver fluke, Fasciola hepatica Linnaeus and Fasciola gigantica Cobbold, which causes endemic fascioliasis in cattle and livestock. The toxic effect of the different fractions was time dependent and fifth fraction obtained through benzene: ethyl acetate (5:5) had maximum molluscicidal activity against Lymnaea acuminata. There was a significant negative correlation between LC values and exposure periods thus increase in exposure time, the LC50 value of V fraction of Euphorbia royleana latex was decreased from 14.28 mg/l (24 hr) to 9.28 mg/l (96 hr) against Lymnaea acuminata. After exposure to sub-lethal concentrations of this fraction there were significant time and dose dependent alterations observed in pyruvate, lactate levels, ALAT, AAT, AChE and cytochrome oxidase enzyme activities in different body tissues of Lymnaea acuminata. It is proposed that the fifth fraction of E. royleana latex can be used as a molluscicide for controlling the harmful snail population from aquatic ecosystem without any harm due to their reversible toxic action.

Animals↗
PubMed1995

Degeneration of CDCs and egg laying in the pond snail Lymnaea stagnalis.

In the pond snail Lymnaea stagnalis egg laying is induced by multiple peptides, which are released by the neuroendocrine caudo-dorsal cells (CDCs). Egg laying in Lymnaea starts at about 50 days and increases initially with age to decreases again at an age of about 250 days. From that age on, the total number of animals that stop egg laying increases with age. The maximum age of Lymnaea is about 660 days. Lucifer Yellow fills of individual ventral CDCs showed reduced branching patterns in old animals. Immunocytochemical stainings demonstrated degeneration in ventral, dorsal and lateral CDCs. In animals that had stopped egg laying, CDCs could still be electrically activated. Regeneration studies after crushing the cerebral commissure showed that in old animals CDCs still can regenerate connections in the cerebral commissure. The properties of CDCs in old animals are discussed in relationship with possible mechanisms of cessation of egg laying in Lymnaea.

Aging↗
PubMed1997

Cloning, characterization, and expression of a G-protein-coupled receptor from Lymnaea stagnalis and identification of a leucokinin-like peptide, PSFHSWSamide, as its endogenous ligand.

Neuropeptides are known to be important signaling molecules in several neural systems of the pond snail Lymnaea stagnalis. Although the functions of these peptides have been studied in many neurons, the nature of the postsynaptic signal transduction is mainly unknown. The cloning and characterization of neuropeptide receptors in Lymnaea thus would be very valuable in further elucidating peptidergic pathways. Indirect evidence suggests that these neuropeptides operate via G-protein-coupled mechanisms indicating the presence of G-protein-coupled receptors as the initial postsynaptic targets. Here we describe the cloning of a neuropeptide receptor from Lymnaea and the isolation of an endogenous ligand. This peptide, PSFHSWSamide, belongs to the leucokinin family of peptides, and, thus, this Lymnaea receptor is the first example of a leucokinin-like neuropeptide receptor, representing a new subfamily of G-protein-coupled neuropeptide receptors.

Amino Acid Sequence↗
PubMed1998

Multi-messenger innervation of the male sexual system of Lymnaea stagnalis.

The male copulation behaviour of the hermaphrodite pond snail Lymnaea stagnalis is under the control of five groups of central neurons that produce a variety of neuropeptides and a classical transmitter, 5-hydroxy tryptamine (5HT). In this article, we describe how the male sexual organs of this snail are innervated by axons from these central neurons. We carried out immunocytochemistry with antisera against the tetra peptide Ala-Pro-Gly-TRP-NH2 (APGWamide), the Lymnaea form of neuropeptide tyrosine (LNPY), conopressin, pedal peptide, the FRMFamide copeptide SEEPLY, the GDPFLRFamide co-peptide DEILSR, myomodulin, Lymnaea inhibitory peptide, and 5HT on tissue sections of the following male sexual organs that receive input from the penis nerve: the prostate gland, vas deferens, preputium, and penis. The results demonstrate that the axons of the separate muscle systems contain particular combinations of transmitters. In addition, two networks of peripheral neurons were revealed. In the tip of the everted preputium lies what appears to be a network of conopressin-containing sensory neurons, which is possibly involved in probing; probing is the part of copulation behaviour in which the male searches for the female genital pore. The other network of peripheral neurons surrounds the most proximal part of the vas deferens and is most likely involved in the pacemaker control of vas deferens motility. On the basis of the data obtained, we hypothesize how the preputium and penis are everted during copulation and which transmitters and central neurons might be involved.

Animals↗
PubMed1998

Transmitter identification in neurons involved in male copulation behavior in Lymnaea stagnalis.

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Animals↗
PubMed2004

Lymnaea stagnalis and the development of neuroelectronic technologies.

The recent development of techniques for stimulating and recording from individual neurons grown on semiconductor chips has ushered in a new era in the field of neuroelectronics. Using this approach to construct complex neural circuits on silicon from individual neurons will require improvements at the neuron/semiconductor interface and advances in controlling synaptogenesis. Although devices incorporating vertebrate neurons may be an ultimate goal, initial investigations using neurons from the pond snail Lymnaea stagnalis have distinct advantages. Simple two-cell networks connected by electrical synapses have already been reconstructed on semiconductor chips. Furthermore, considerable progress has been made in controlling the processes that underlie chemical synapse formation in Lymnaea. Studies of Lymnaea neural networks on silicon chips will lead to a deeper understanding of the long-term dynamics of simple neural circuits and may provide the basis for reliable interfaces for new neuroprosthetic devices.

Animals↗
PubMed2000

Appetitive learning using visual conditioned stimuli in the pond snail, lymnaea.

Feeding can be conditioned in the pond snail Lymnaea stagnalis to two different visual stimuli (a black panel or a 5-mm black and white check surround) by pairing the potential conditioned stimulus (CS) with sucrose. Exclusion of chemical cues (associated with differences between the water in home tank and that in training apparatus) that could serve as CS is important for successful visual conditioning. A featureless gray surround, used as an alternative to the check (to which it was matched in luminance) in counterbalanced training designs, was discriminated from the check, showing that resolution (for which the eyes would be necessary) was occurring. The gray surround was largely ineffective as a CS. Single-trial learning was possible with the black panel, but not with the check; it is argued that this may be due to lack of prior experience of stimuli like the check. Conditioning of feeding has now been obtained in Lymnaea to chemical, tactile, and visual cues, opening the way to comparative studies of the neural circuitry underlying appetitive conditioning in different senses, so far explored in Lymnaea only for tactile CS. Such comparative studies are as yet largely lacking in invertebrates.

Animals↗
PubMed1994

Comparison of the migrations of Fasciola hepatica sporocysts in Lymnaea truncatula and other related snail families.

The migration and definitive localization of sporocysts in the snail body were studied in four species (Aplexa hypnorum, Lymnaea glabra, L. truncatula, Physa acuta), each of which was exposed to a single miracidium and killed during a period ranging from the 1st to the 5th day postexposure. When penetration occurred through the mantle, tunnels were found mainly in the mantle or kidney in Lymnaea adults and also extended to the foot in many physidid adults and in newborns of the four species. The sporocysts preferentially localized in the zone surrounding the kidney and heart beginning on the 4th day in Lymnaea spp. and disappeared in Aplexa and Physa snails on the 3rd day. When penetration occurred through the foot and extended inconstantly to the mantle according to the snail species involved. Sporocysts were encountered in the foot as well as the mantle or the zone surrounding the kidney and heart. The significance of these results is discussed in relation to the snail host and the entry site used by the Fasciola hepatica miracidium.

Animals↗
PubMed2004

Effect of herbal molluscicides and their combinations on the reproduction of the snail Lymnaea acuminata.

Effects of sublethal treatment (20 and 60% of LC50/24 h) of the plant-derived molluscicides Annona squamosa Linn. and Lawsonia inermis Linn. and their combinations with other herbal molluscicides, such as Cedrus deodara Roxb, Azadirachta indica A. Juss, bulb powder of Allium sativum Linn. and Polianthes tuberosa Linn., and oleoresin of Zingiber officinale Rosc., and acetogenins extracted from the seeds of A. squamosa Linn., on the reproduction of the snail Lymnaea acuminata have been studied. It was observed that the plant-derived molluscicides singly and in binary combinations with other herbal molluscicides and the extracted acetogenins caused a significant reduction in the fecundity, hatchability, and survival of young snails. Withdrawal of the snails to fresh water after the above treatment caused a significant recovery in the fecundity of the snail Lymnaea acuminata. Twenty-four-hour sublethal treatment with the acetogenins caused a maximum reduction in the protein, amino acid, DNA, and RNA in the ovotestis of treated Lymnaea acuminata.

Amino Acids↗
PubMed2002

Nitric oxide level regulates the embryonic development of the pond snail Lymnaea stagnalis: pharmacological, behavioral, and ultrastructural studies.

On the basis of the distribution of NADPH-diaphorase (NADPH-d) activity, we have previously suggested a role for nitric oxide (NO) in the development of Lymnaea stagnalis. In the present study, the long-term effects of NO donors (sodium nitroprusside, S-nitroso-N-acetyl-penicillamine) and nitric oxide synthase (NOS) inhibitors (nitro-L-arginine methyl-ester [L-NAME], N(G)-nitro-L-arginine [L-NOARG]) were tested on the survival, length of embryonic (intracapsular) life, locomotion (gliding), heartbeat activity and feeding behavior, as well as on the ultrastructure of the developing ganglia in the embryonic Lymnaea. No effect of any of the substances applied can be observed under 10(-5) M concentration, whereas at 10(-3) M concentration both kinds of treatment proved to be toxic. Between 10(-5) M and 10(-3) M concentrations the effects are reversible. At 10(-4) M concentration, NO donors slightly increase the frequency of gliding and heartbeat of E70% embryos, and evoke a more than twofold enhancement of the feeding activity, i.e., the frequency of radula protrusions in the E90% embryonic stage. In contrast, NOS inhibitors at 10(-4) M concentration strongly inhibit the locomotion and heartbeat of E70% embryos, and the feeding of E90% embryos. Under 10(-3) M concentration, L-arginine diminishes the effect of NOARG, whereas the D-isomer of NAME has little or no significant effect. Neither type of treatment alters the course of gangliogenesis, and the light-microscopic appearance of neurons also remains unaffected. Ultrastructural analysis of the central nervous system of E90% embryos treated with 10(-4) M NOS inhibitors revealed a significant reduction of the glycogen granule content and accumulation of lipid droplets in a number of the neuronal perikarya, as well as the occurrence of disintegrated mitochondria in axonal profiles. The effect of 10(-4) M NO donors is mainly characterized by the increased number of lysosomes, disintegrated mitochondria and degenerating axonal profiles. The present findings suggest that NO is involved in the regulation of different behaviors and physiological functions, such as feeding activity, locomotion and heartbeat, during the embryonic development of Lymnaea. Changes observed in neuronal ultrastructure in ganglia seem to indicate NOergic regulatory processes at the central level.

Animals↗
PubMed2004

Expression and distribution of transcription factor CCAAT/enhancer-binding protein in the central nervous system of Lymnaea stagnalis.

The transcription factor, CCAAT/enhancer-binding protein (C/EBP), is involved in important physiological processes, such as cellular proliferation and differentiation, homeostasis, and higher-order functions of the brain. In the present study, we investigated the distribution of mRNA and protein of C/EBP in the central nervous system of the pond snail, Lymnaea stagnalis, by in situ hybridization and immunohistochemistry. Specificity of the anti-mammalian C/EBP antibody against Lymnaea C/EBP (LymC/EBP) was confirmed by combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing and immunoblotting. Cells positive for in situ hybridization were immunoreactive for LymC/EBP in all 11 ganglia. The motoneurons (B1, B2, B4, and B4 clusters) in the buccal ganglia and interneurons (cerebral giant cell, CGC) in the cerebral ganglia were positive for in situ hybridization and were immunopositive. In the pedal ganglion, the right pedal dorsal 1 (RPeD1), pedal A, and pedal C clusters exhibited positive signals of in situ hybridization and immunohistochemistry for LymC/EBP. CGC and RPeD1 are key neurons for associative learning. In addition, the neuropeptidergic cells in the cerebral, pleural, parietal, and visceral ganglia were positive for in situ hybridization and immunoreactive. Interestingly, although the cytoplasm of almost all immunopositive cells was stained, some neuropeptidergic cells located in the light parietal and visceral ganglia exhibited immunoreactivity in nuclei. Our results suggest that LymC/EBP is involved in learning and memory and in the expression and/or secretion of neuropeptides in Lymnaea.

Animals↗
PubMed1999

Pigment-dispersing hormone-like immunoreactive neurons in the central nervous system of the gastropods, Helix pomatia and Lymnaea stagnalis.

By using an antiserum raised against a crustacean beta-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons was investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system was found to be large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons showed immunoreactivity. The immunostained neurons in Helix were characterized by rich arborizations in all central ganglia and revealed massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex were also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea.

Animals↗
PubMed1992

Molecular cloning of G protein alpha subunits from the central nervous system of the mollusc Lymnaea stagnalis.

The central nervous system of the pond snail, Lymnaea stagnalis, contains many large, identified neurons which can be easily manipulated making it an advantageous model system to elucidate in vivo the architecture of neuronal signal transduction pathways. We have isolated three cDNA clones encoding G protein alpha subunits that are expressed in the Lymnaea CNS, i.e. G alpha o, G alpha s and G alpha i. The deduced proteins exhibit a very high degree of sequence identity to their vertebrate and invertebrate counterparts. The strong conservation of G protein alpha subunits suggests that functional insights into G protein-mediated signalling routes obtained through the experimental amenability of the Lymnaea CNS will have relevance for similar pathways in the mammalian brain.

Amino Acid Sequence↗
PubMed1994

Structural characterization of a Lymnaea putative endoprotease related to human furin.

A number of peptides have been identified in the central nervous system of the freshwater snail, Lymnaea stagnalis, that function as hormones and neurotransmitters/neuromodulators. These peptides are typically proteolytically processed from larger prohormones mostly at sites composed of single or multiple basic amino acid residues. Previously we demonstrated a diversity of putative prohormone convertases that may be involved in prohormone processing in the Lymnaea brain. In the present report, we have characterized a cDNA clone encoding a putative endoprotease of 837 amino acids. The primary structure of endoprotease (Lfur2) was comparable to that of human furin and contained a putative catalytic domain, a Cys-rich domain, and a transmembrane region. The catalytic domain of Lfur2 demonstrated about 70% residue identity when compared with human furin, PACE4 and Drosophila Dfur1 and dKLIP-1. The Lfur2 gene was expressed in the central nervous system as well as various peripheral tissues of Lymnaea.

Amino Acid Sequence↗