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PubMed1990

Communication compartments in the post-trochal ectoderm of the mollusc Lymnaea stagnalis.

Cell-to-cell communication via gap junctions provides a pathway for the transfer of small molecules and ions which may be significant for control of metabolic cooperation, cell proliferation, and differentiation. We have assessed the patterns of gap junctional communication in embryos of the mollusc Lymnaea stagnalis during the subdivision of the post-trochal ectoderm into developmental domains. We have microinjected the tracer Lucifer Yellow CH and subsequently analyzed its transfer to other cells. The post-trochal ectoderm of mollucs develops the shell field, the foot, and the stomodeum anlagen. We have found that the cells within the separate anlagen are well dye-coupled but poorly coupled to cells of adjacent anlagen. These results indicate that in Lymnaea embryos the specification of the different developmental domains is associated with the development of corresponding dye-coupling compartments.

Animals↗
PubMed1992

Molluscan putative prohormone convertases: structural diversity in the central nervous system of Lymnaea stagnalis.

In the cerebral ganglia of the central nervous system of the freshwater snail Lymnaea stagnalis many neuropeptides are proteolytically processed from larger prohormones at sites marked by both single and multiple basic amino acids. In the present study we identified cloned cDNA and PCR products corresponding to three putative endoproteases that may be involved in prohormone processing. The cDNA encodes a protein of 653 residues with an overall sequence identity of 60%, 41%, 35%, 40%, and 27% with the recently characterized endoproteases PC2, PC1/3, PC4, furin and Kex2, respectively. The Lymnaea preproconvertase has approximately 80% homology with the catalytic domain, and approximately 40% and approximately 50% with the N- and C-terminal part, respectively, of the vertebrate PC2. Two cloned PCR products, Lfur 1 and Lfur 2, show highest sequence identity to furin. Expression of the LPC2 gene is exclusively in the central nervous system, where two LPC2 transcripts of 3.0 and 4.8 kb were detected.

Amino Acid Sequence↗
PubMed1985

Direct effects of the hyperglycemic factor of the freshwater snail Lymnaea stagnalis on isolated glycogen cells.

In the freshwater pulmonate snail Lymnaea stagnalis special cells, the glycogen cells (GC) are present for the storage of glycogen reserves. These cells occur in large numbers in the anterior mantle region. In a previous paper in vitro experiments with intact anterior mantle tissue indicated that a hyperglycemic factor is released from the central nervous system (CNS) which stimulates glycogen mobilization in mantle tissue (M. A. Hemminga, J. J. Maaskant, W. Koomen, and J. Joosse (1985). Neuroendocrine control of glycogen mobilization in the freshwater snail Lymnaea stagnalis. Gen. Comp. Endocrinol. 57, 117-123). In the present study the question of whether this factor affects glycogen metabolism of GC isolated from mantle tissue was investigated. It is reported that in high-K+ Ringers the CNS is stimulated to release a factor which, in a dose-dependent way, inhibits glycogen synthesis in isolated GC (measured as a decreased incorporation of [14C]glucose into glycogen). Simultaneous with this glycogen synthesis-inhibiting effect, stimulation of glycogen degradation is found (measured as a decreased retention of prelabeled glycogen). It is concluded that (1) the factor released by the CNS having these effects on isolated GC is the same as the hyperglycemic factor which was reported to stimulate glycogen mobilization in intact mantle tissue, and (2) GC after isolation from mantle tissue have retained their ability to respond to this factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A G-protein beta subunit that is expressed in the central nervous system of the mollusc Lymnaea stagnalis identified through cDNA cloning.

We have cloned cDNA encoding a G-protein beta subunit from the central nervous system (CNS) of the mollusc Lymnaea stagnalis. The deduced protein is very homologous to other metazoan beta subunits. Thus, the Lymnaea CNS can be used as a model system to study beta gamma subunits in their native setting since its large neurons can be manipulated and studied relatively easily in vivo.

Amino Acid Sequence↗
PubMed1991

Characterization and evolutionary aspects of a transcript encoding a neuropeptide precursor of Lymnaea neurons, VD1 and RPD2.

We isolated and characterized a cDNA clone encoding the major prohormone of VD1 and RPD2, two electrotonically coupled identified neurons in the central nervous system of the freshwater snail, Lymnaea stagnalis. The VD1/RPD2 prohormone may be cleaved to generate a set of 4 different neuropeptides, called epsilon, delta, alpha 1 and beta peptides, as well as a single aspartate. Since VD1 and RPD2 probably are involved in O2 perception and modulation of cardio-respiratory functions, it is thought that the neuropeptides synthesized and released by these neurons coordinate the adaptive physiological and behavioural processes that occur in response to changes in O2 availability. Comparison of the Lymnaea VD1/RPD2 precursor with two related precursors, prohormones R15-1 and R15-2, identified from neuron R15 in the marine mollusc Aplysia californica revealed a similar pattern of organization of the preprohormones. The overall homology is rather low, however, detailed comparisons show a highly differential pattern of conservation of peptide regions on the precursors.

Amino Acid Sequence↗
PubMed1992

Characterization of a cDNA clone encoding molluscan insulin-related peptide V of Lymnaea stagnalis.

A cDNA clone encoding molluscan insulin-related peptide V (MIP V) was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP II cDNA. The MIP V cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, however, in this case connected by two distinct C peptide, C alpha and C beta, instead of one single C peptide. This phenomenon, which is shared by the MIP II precursor, represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIPs V, I and II, differ remarkably in primary structure; in contrast, the C alpha peptide domains are almost identical. MIP V has only limited sequence similarity with insulins and related peptides. Both MIP V and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I, II and V genes are expressed in a single type of neuron: the growth controlling neuroendocrine light green cells of the Lymnaea CNS.

Amino Acid Sequence↗
PubMed1994

Structural identification, neuronal synthesis, and role in male copulation of myomodulin-A of Lymnaea: a study involving direct peptide profiling of nervous tissue by mass spectrometry.

We used a strategy combining immunodetection, peptide chemistry, and a novel method, direct peptide fingerprinting of neurons and small pieces of nerve by using matrix-assisted laser desorption ionization mass spectrometry, to structurally identify and localize the neuropeptide myomodulin-A in the mollusc, Lymnaea stagnalis. Lymnaea myomodulin appeared to be identical to Aplysia myomodulin-A and is produced by many central neurons, including neurons located in the ventral lobe of the right cerebral ganglion that innervate the penis complex via the penis nerve. Myomodulin-A could also be characterized from the penis complex, and physiological concentrations of the peptide enhanced the relaxation rate of electrically induced contractions of the penis retractor muscle in vitro in a dose-dependent fashion.

Animals↗
PubMed1994

Processing, axonal transport and cardioregulatory functions of peptides derived from two related prohormones generated by alternative splicing of a single gene in identified neurons VD1 and RPD2 of Lymnaea.

The VD1/RPD2 mRNA precursor in identified neurons VD1 and RPD2 of the freshwater snail Lymnaea stagnalis is alternatively spliced to yield two related variants encoding two distinct yet related preprohormones, named the VD1/RPD2-A and -B preprohormones. Here, we report the isolation and structural characterization of alpha 1, alpha 2 and beta peptides from dissected neurons VD1 and RPD2. The alpha 1 and alpha 2 peptides are derived from VD1/RPD2-A and B prohormones, respectively, whereas beta peptide is identical for both prohormones. In addition, we report the isolation and structural characterization of the alpha 2 peptide from the heart, demonstrating that the mature peptides are transported and released in the heart. The pharmacological actions of synthetic alpha 1 and alpha 2 peptides on isolated auricle preparations of the Lymnaea heart were examined. The two alpha peptides have similar excitatory effects on beat rate and beat amplitude, while their potencies differed considerably, indicating that alternative splicing results in structurally and functionally overlapping, through non-identical, sets of peptides.

Amino Acid Sequence↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1991

Survival of snails and characteristic lesions of Fasciola hepatica infection in four European species of Lymnaea.

The survival of snails and characteristic organ lesions were investigated in four different age categories (new-born, 1 and 2 mm shell height, adults) and in four species of Lymnaea (L. glabra, L. palustris, L. peregra ovata, L. truncatula) infected by Fasciola hepatica. Mortalities were higher in infected groups on Days 30 and 45 post-infection (+42% and +45%, respectively) than in controls. Lesions depended on the species and age of the Lymnaea. Epithelial necrosis of the kidney was recorded in L. glabra, L. palustris and L. p. ovata, whereas intralamellar deposits were found in the same organ of L. truncatula. All categories of L. truncatula had evolutive infections (snails with living parthenitae); the young snails of other species became infected, but the adults did not, as estimated at Day 45 post-infection. The lesions were related to the intensity of evolutive infections: necrosis of the digestive gland was found in moderate to low infections and, conversely, gonadal atrophy with epithelial necrosis was found in highly infected snails.

Animals↗
PubMed1995

5-HT receptors on identified Lymnaea neurones in culture: pharmacological characterization of 5-HT3 receptors.

1. The selective agonist, 1-(m-chlorophenyl)-biguanide (m-CPBG) and antagonist, 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) were used to characterize the 5-HT3 receptors in cultured identified neurones; the serotonin-containing cerebral giant cells (CGCs) and some follower neurones in the buccal ganglia of Lymnaea stagnalis. 2. 5-HT and its agonists were pressure ejected, while the 5-HT antagonists were bath applied. 3. Although m-CPBG evoked mostly depolarizing responses, hyperpolarizing responses were sometimes evoked. 4. At 10(-4) M, m-CPBG failed to mimic the responses of 5-HT, but at a concentration higher, 10(-3) M, pressure-ejected m-CPBG mimicked most 5-HT responses. 5. The 5-HT2 antagonist ketanserin failed to block the m-CPBG-evoked responses, whilst partially blocking the 5-HT responses. 6. These results suggest the presence of 5-HT3 receptors similar to those found in mammalian neurones, and that multiple subtypes of these receptors may be present in Lymnaea neurones.

Animals↗
PubMed1994

5-HT receptors on identified Lymnaea neurones in culture: pharmacological characterization of 5-HT2 receptors.

1. Pressure ejection techniques were used to investigate the identify of receptors mediating 5-HT (5-Hydroxytryptamine) effects on the serotonin-containing cerebral giant cells (CGCs) of the cerebral ganglia and some of their follower motorneurones from the buccal ganglia of Lymnaea stagnalis in culture. 2. The vertebrate 5-HT2 receptor agonist alpha-methylserotonin maleate (10(-4) M), inhibited most of the neurones inhibited by 5-HT (10(-3) M). Others were excited by both agonists. In cells where 5-HT failed to evoke any effects, the 5-HT2 agonist also lacked an effect. 3. Bath application of the 5-HT2 receptor antagonists ketanserin and methysergide (10(-4) M), not only blocked spike generation, but also reduced both the excitatory and inhibitory responses to both 5-HT and alpha-methylserotonin maleate, while the 5-HT3 antagonist MDL 72222 (10(-4) M) failed to block alpha-methylserotonin maleate effects. 4. At 10(-3) M, alpha-methylserotonin maleate increased the amplitudes of the hyperpolarizing responses in a dose-dependent manner. These responses were blocked by ketanserin (10(-4) M). 5. The above results suggest that 5-HT2 receptors are involved in the responses of the CGCs and the buccal motorneurones to 5-HT in Lymnaea stagnalis. The pharmacological characterization of these receptors indicates that the compounds that interact with the 5-HT2 receptors in mammals also interact with the 5-HT2 receptors in molluscs.

Animals↗
PubMed1996

Behavioral changes induced by GABA-receptor agonists in Lymnaea stagnalis L.

1. GABA, the GABAA receptor agonist, muscimol, and the GABAB receptor agonist, baclofen, were tested to study the involvement of the GABA neurotransmitter system in control of behaviour in the freshwater pulmonate snail, Lymnaea stagnalis L. Single injections of GABA (1-10 micrograms/ gbw) into the haemocoel of intact snails elicits a sequence of behavioural changes subsequently affecting feeding, locomotion, escape reactions, male mating behaviour and respiration. 2. Both muscimol and baclofen mimic distinct aspects of GABA action implying that the GABA action is mediated by both types of receptors. 3. The modulatory actions of GABA, muscimol, and baclofen on feeding were manifested as opening of the mouth and triggering of specific radular movements (e.g., protraction, retraction, rasping). 4. Baclofen (1-10 micrograms/gbw) evoked the full erection of the penis for a time long enough for natural copulation, while GABA itself caused only partial eversion of the preputium. Muscimol was less effective than GABA. The latency to penis eversion varied in a dose-dependent manner. The data emphasize the participation of baclofen-sensitive receptors in control of reproduction. 5. GABA and baclofen induced withdrawal of the head and anterior part of the foot accompanied by arrest of locomotion and respiration could be considered to be an escape reaction. 6. Muscimol (1-5 micrograms/gbw) failed to elicit withdrawal reactions but caused the loss of normal orientation and longitudinal contractions of the foot. 7. All the agents tested inhibited locomotion in a dose-dependent manner, for a substantial period of time. A decrease in total locomotor activity lead to the failure of animals to attach to the underlying surface, to have foot contractions and also to diminish locomotor velocity. 8. The majority of animals maintained the stereotyped complex reactions of respiration, but with restricted clockwise turning of the shell after drug treatment. 9. All the substances were shown to shift the background behavioural state characterised by high BSSs to a state similar to "rest" which was awarded a low BSS. It is concluded that the GABA neurotransmitter system can play an important role in activation and coordination of neuronal ensembles underlying behaviour and behaviour selection in Lymnaea stagnalis L.

Animals↗
PubMed1996

Characterization of the GABA response on identified dialysed Lymnaea neurons.

1. The effect of gamma-aminobutyric acid has been studied on identified, internally perfused dialysed neurons of Lymnaea stagnalis L. (Pulmonata, Basommatophora). It was shown that: On the majority of neurons GABA (10(-8)-10(-3) M) depolarized the membrane with a decrease in input resistance and activated a Cl- dependent inward current with -20 +/- 4 mV E(GABA). In some cells, the outward current with -67 +/- 8 mV E(GABA) was also recorded. 2. The GABA-induced inward current was fast (1.5 +/- 0.3 sec, n = 4) or slow (3.2 +/- 0.2 sec, n = 3) peaking in a voltage-independent manner. The inactivation phase could be fitted by one or two exponentials characterized with fast (tau = 0.7 sec) and slow (tau = 3.6 sec) time constants. The outward current component was slow and activated at more positive Vh(-30-20 mV). 3. The agonist effects (GABA and muscimol) indicated the involvement of GABA(A) receptors and Cl-permeability changes in activating inward current. Picrotoxin (10(-5)-10(-4) M) and Cd2+ completely inhibited the GABA-activated inward current also affecting E(GABA). Furosemide was without effect on the peak value of GABA-induced inward current, but slightly modified the slope of inactivation. 4. High concentrations of Ca-ions and their substitution with Ba-ions in extracellular saline failed to alter the GABA-induced inward current. However, omission of Cl-ions from extracellular media shifted E(GABA) to the right by 18 +/- 8 mV (n = 4). 5. Omission of Cl-ions from intracellular saline led to inhibition of the fast component of GABA-induced inward current. Full recovery followed readdition of Cl-ions. 6. The results are in agreement with the data obtained on cloned Lymnaea GABA receptors.

Animals↗
PubMed1996

Expression and characterization of molluscan insulin-related peptide VII from the mollusc Lymnaea stagnalis.

A complementary DNA clone encoding molluscan insulin-related peptide VII was identified from a complementary DNA library of the cerebral ganglia of the CNS of the freshwater snail, Lymnaea stagnalis. The novel molluscan insulin-related peptide VII complementary DNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and an A and B chain, and is connected by an unusual long C peptide. The A and B chains, as well as the C peptide of molluscan insulin-related peptide VII, differ remarkably in primary structure with the previously identified molluscan insulin-related peptides. The C peptide of molluscan insulin-related peptide VII shares no significant sequence identity with counterparts in other molluscan insulin-related peptides. Both molluscan insulin-related peptide VII and the other molluscan insulin-related peptides exhibit structural features which make them a unique class of the insulin superfamily. Molluscan insulin-related peptide VII complementary DNA was shown to hybridize in situ with messenger RNA present in the cerebral light green cells, neuroendocrine cells that control growth and that have previously been shown to produce molluscan insulin-related peptides I-III and V. Uniquely, the molluscan insulin-related peptide VII gene is also expressed in neurons that may form part of the feeding circuitry in Lymnaea, indicating that it may function as a neurotransmitter/neuromodulator.

Amino Acid Sequence↗
PubMed1988

The impairment of mobility and development in freshwater snails (Physa fontinalis and Lymnaea stagnalis) caused by herbicides.

1. The pulmonate freshwater snails Physa and Lymnaea and the earthworms Eisenia and Lumbricus can take up and concentrate a carbamate herbicide (chlorpropham). 2. The mobility of freshwater snails was diminished in the presence of the herbicides chlorpropham (carbamate), cycloate (thiocarbamate), pentanochlor (amide) and chloroxurone (urea derivate). 3. The egg-assemblies of Lymnaea stagnalis turned out to be suitable objects for testing the influences of herbicides upon embryonic development. 4. In the presence of chlorpropham, chloroxurone, cycloate, propanil, simazine and terbutryne, all applied in concentrations lower than in practical use, the period of egg-maturing was delayed and the total number of dead embryos increased. 5. Developing snail eggs were very sensitive towards triazine herbicides (simazine, terbutryne) reflecting in an ED50-value lower than 10(-7) M. 6. Similar herbicidal-induced effects suggested that the developing stages of snail embryos may be suitable models for the ecologically more important but experimentally less accessible earthworms.

Animals↗
PubMed1989

The actions of three volatile general anaesthetics on withdrawal responses of the pond-snail Lymnaea stagnalis (L.).

1. The gastropod mollusc Lymnaea stagnalis (L.) is an ideal model system for studies on anaesthesia. It is reversibly anaesthetized by the general anaesthetics halothane, enflurane and isoflurane. 2. Criteria for "anaesthesia" in Lymnaea were established. The reflex used in ED50 trials was the whole animal withdrawal reflex. 3. ED50 values for halothane, enflurane and isoflurane were, 0.83% v.v. (volume for volume), 1.01% v.v. and 1.09% v.v. respectively. 4. Relationships between anaesthetic concentrations, weights of animals and mortality rates are reported.

Animals↗
PubMed2004

Diversity of nicotinic receptors mediating Cl- current in Lymnaea neurons distinguished with specific agonists and antagonist.

Diversity of nicotinic acetylcholine receptors (nAChRs) mediating Cl- current in voltage-clamped identifiable Lymnaea stagnalis neurons was studied using acetylcholine (ACh), three agonists and alpha-conotoxin ImI (ImI). Cytisine, nicotine, and choline, full agonists at alpha7 subunit-containing nAChRs of vertebrates, were found to evoke at saturating concentration 84-92% of the maximal current elicited by ACh. ImI, known to block selectively alpha7 and alpha9 nAChRs, markedly diminished the responses to ACh. The average maximal ImI-induced block was 80%, leaving a residual current which had very slow kinetics. The choline-, cytisine-, and nicotine-induced currents were blocked by ImI almost completely, suggesting that they activate only ImI-sensitive receptors. Two groups of cells which differ in desensitization kinetics and in sensitivity to ImI were revealed. IC50 values for ImI against ACh were 10.3 and 288 nM, respectively, with the rapidly desensitizing current being the more sensitive to ImI. The data obtained suggest the existence of at least three pharmacologically distinct subtypes of nicotinic receptors in Lymnaea neurons. Two of the subtypes are similar to alpha7 nAChRs of vertebrates, but differ from each other in their affinity for ImI and in their desensitization kinetics. The third subtype is quite distinct, in that it is resistant to ImI, is not activated by nicotine, cytisine or choline, and mediates a very slowly developing current.

Animals↗