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.     

The expression pattern of CREB genes in the central nervous system of the pond snail Lymnaea stagnalis

To analyze the expression pattern of genes of cAMP responsive element binding protein (CREB), we performed in situ hybridization for the whole central nervous system (CNS) of the pond snail Lymnaea stagnalis. The CREB1 (activator) and CREB2 (repressor) homologues have already been cloned in L. stagnalis, and they are referred to as LymCREB1 and LymCREB2. Using the frozen sections and the whole mount preparations of the CNS, we mapped the distribution of LymCREB1 and LymCREB2 mRNA containing neurons. The present findings showed that the LymCREB1 mRNA containing neurons are a relatively few, whereas LymCREB2 mRNA is contained ubiquitously in the whole CNS of L. stagnalis.     

Neurocalcin-like immunoreactivity in embryonic stages of the gastropod molluscs Aplysia californica and Lymnaea stagnalis

Abstract. Neurocalcin is a calcium-binding protein that has been localized in neural and non-neural tissues of vertebrates, the arthropod Drosophila melanogaster , and in juveniles and adults of the mollusc Aplysia californica . We examine the distribution of neurocalcin in pre-hatching stages of the molluscs A. californica and Lymnaea stagnalis to elucidate where this calcium-binding protein functions in early development, as well as to localize novel neuronal populations in early stages of ontogeny. Aplysia neurocalcin (ApNc)-like immunoreactivity was localized in shell-secreting cells in embryonic stages of both A. californica and L. stagnalis . In A. californica , central and anterior regions of the embryo were diffusely labeled, as were a few identifiable neurons in veliger stages, On the other hand, in L. stagnalis , ApNc-like immunoreactivity was clearly detected in cells and fibers in the same locations as neuronal elements that have been previously identified very early in development and throughout the embryonic period using techniques to localize specific transmitters and peptides. Furthermore, additional neurons are also identified with anti- ApNc in this species. Establishing the distribution of neurocalcin-like proteins in embryonic stages of these two molluscs provides the first step to understanding the role of such proteins during development.     

Central representation of internal and external sensory information in the CNS of Helix pomatia L. and Lymnaea stagnalis L

The central representation of intero- and exteroreceptors located in visceral organs and the osphradium were compared in the CNS of Helix pomatia L. (Gastropoda, Stylommatophora) and Lymnaea stagnalis L. (Gastropoda, Basommatophora), two pulmonate snail species inhabiting a terrestrial and anaquatic environment, respectively. Semi-intact preparations were used comprising the CNS connected by the corresponding nerves either to the cardio-renal, respiratory and genital systems or to the osphradium. Spike discharges of central neurons and the nerves were recorded simultaneously. The central representation of intero- and exteroreceptors was found to be distributed throughout the CNS and involved about 300 neurons. The majority of the neurons received sensory information from all the studied visceral organs and the osphradium. Among the neurons responding to intero- and exteroreceptors a multimodal reaction to tactile, chemical and osmotic stimuli prevailed while in the osphradium specific reactions also were demonstrated. Central neurons receiving sensory information from visceral organs and the osphradium form overlapping and reorganizing neural circuits using the same neurons in the regulation of heart activity, respiration or reproduction producing the appropriate behaviour. In the selection of sensory information the firing pattern appears to be the main determining factor as bursting neurons do not receive sensory information. The central representation of intero- and exteroreceptors and its variability can be a model system for cellular studies of motivational state and self-perception.     

Hyperpolarization by glucose of feeding-related neurons in snail

In the pond snail Lymnaea stagnalis, D-glucose action was investigated on electrical activity of identified central neurons. In the CNS preparations isolated from specimens that starved for 24-96 h, D-glucose added to a standard or HiDi saline at 500-700 microg/ml effectively hyperpolarized ca. 90% of feeding related neurons B1, SO and CGC. However, not all feeding-related neurons examined were responsive to glucose. Experiments on cells of the serotonergic Pedal A cluster have shown that hyperpolarizing action of D-glucose is retained following complete isolation of "hunger" neurons. Threshold concentration producing 1-3 mV hyperpolarization was ca. 50 microg/ml. The results suggest a direct glucose involvement in the mechanisms that control feeding behavior in Lymnaea.     

Identification of catecholamines in the rat brain and mollusk tissues using semithin slices and retrograde staining

The suggested technique allows revealing the transport-specific dye (primulin) and catecholamine fluorescence simultaneously in the same cell of brain. Intense fluorescence is observed when brain tissue is quickly dehydrated and embedded in the epoxy resin. The same method is suggested for the identification of catecholamines in the embryonal and juvenile tissues of gastropod Lymnaea stagnalis (Mollusca Pulmonata) without using of primulin dye.     

Cryopreserved neurons of a mollusc are capable of morphological differentiation in culture in vitro

It was shown in culture in vitro that neurons isolated from the cryopreserved brain of adult molluscs Lymnaea stagnalis L. retain viability. Isolated brains were frozen in liquid nitrogen vapors at a rate of 400-500 degrees C/min in the presence of 2 M dimethylsulfoxide. The samples were then plunged into liquid nitrogen and stored from 1 month to 2 years. Upon thawing and removing the cryoprotectant, the neurons were isolated from the brain and then introduced into a cellular culture in vitro. It was shown that the thawed neurons were capable of regenerating new nerve processes similar to those formed by unfrozen neurons in the control.     

The effects of mating system and genetic variability on susceptibility to trematode parasites in a freshwater snail, Lymnaea stagnalis

The amount and distribution of genetic variability in host populations can have significant effects on the outcome of host-parasite interactions. We studied the effect of mating system and genetic variability on susceptibility of Lymnaea stagnalis snails to trematode parasites. Mating system of snails from eight populations differing in the amount of genetic variability was manipulated, and self- and cross-fertilized offspring were exposed to naturally occurring trematode parasites in a controlled lake experiment. Susceptibility of snails varied between populations, but mating-system treatment did not have a significant effect. Heterozygosity of snails was negatively correlated with the probability of trematode infection, however, suggesting that parasitic diseases may pose a serious threat to populations lacking genetic variability.     

Effects of temperature on respiration defensive behavior and locomotion of fresh water snail Lymnaea stagnalis

Temperature dependence of lung respiration, defensive behavior and locomotion of Lymnaea stagnalis snail was studied. At the temperature in the range of 4-6 degrees C the rates of locomotion and respiration were reduced (as compared to control temperature of 14-16 degrees C), whereas defensive reactions were much more intense. Vice versa, the temperature rise to 24-26 degrees C activates respiration and locomotion but inhibits defensive behavior. It is suggested that the observed changes in Lymnaea behavior result from temperature-dependent reactions of neurons underlying these activities.     

Distribution of serotonin and FMRF-amide in the brain of Lymnaea stagnalis with respect to the visual system

Despite serotonin’s and FMRF-amide’s wide distribution in the nervous system of invertebrates and their importance as neurotransmitters,the exact roles they play in neuronal networks leaves many questions.We mapped the presence of serotonin and FMRF-amide-immunoreactivity in the central nervous system and eyes of the pond snail Lymnaea stagnalis and interpreted the results in connection with our earlier findings on the central projections of different peripheral nerves.Since the chemical nature of the intercellular connections in the retina of L.stagnalis is still largely unknown,we paid special attention to clarifying the role of serotonin and FMRF-amide in the visual system of this snail and compared our findings with those reported from other species.At least one serotonin-and one FMRF-amidergic fibre were labeled in each optic nerve,and since no cell bodies in the eye showed immunoreactivity to these neurotransmitters,we believe that efferent fibres with somata located in the central ganglia branch at the base of the eye and probably release 5HT and FMRF-amide as neuro-hormones.Double labelling revealed retrograde transport of neurobiotin through the optic nerve,allowing us to conclude that the central pathways and serotonin-and FMRF-amideimmunoreactive cells and fibres have different locations in the CNS in L.stagnalis.The chemical nature of the fibres,which connect the two eyes in L.stagnalis,is neither serotoninergic nor FMRF-amidergic.     

Effect of the endogenous peptide factor and vasopressin on the neurons of mollusks of different ages

Electric reactions of the identified neurons in adult (10-12 months) and old (22-24 months) molluscs Lymnaea stagnalis to endogenous peptide isolated from the water soluble fraction of the Helix Pomatia brain homogenate as well as to vasopressin are studied. The endogenous peptide causes a significant activation of the burst both in adult and old neurons of the molluscs. No significant age-related differences in the pronouncement of the above shifts of the molluscan neurons are found. There is an increased sensitivity to the identified peptide, vasopressin, in the old individuals. A non-uniform change in the sensitivity of mollusk neurons to various neuropeptides may affect the synaptic conduction in old age.     

Multi-linear regression analysis, preliminary biotic ligand modeling, and cross species comparison of the effects of water chemistry on chronic lead toxicity in invertebrates

The current study examined the chronic toxicity of lead (Pb) to three invertebrate species: the cladoceran Ceriodaphnia dubia, the snail Lymnaea stagnalis and the rotifer Philodina rapida. The test media consisted of natural waters from across North America, varying in pertinent water chemistry parameters including dissolved organic carbon (DOC), calcium, pH and total CO(2). Chronic toxicity was assessed using reproductive endpoints for C. dubia and P. rapida while growth was assessed for L. stagnalis, with chronic toxicity varying markedly according to water chemistry. A multi-linear regression (MLR) approach was used to identify the relative importance of individual water chemistry components in predicting chronic Pb toxicity for each species. DOC was an integral component of MLR models for C. dubia and L. stagnalis, but surprisingly had no predictive impact on chronic Pb toxicity for P. rapida. Furthermore, sodium and total CO(2) were also identified as important factors affecting C. dubia toxicity; no other factors were predictive for L. stagnalis. The Pb toxicity of P. rapida was predicted by calcium and pH. The predictive power of the C. dubia and L. stagnalis MLR models was generally similar to that of the current C. dubia BLM, with R(2) values of 0.55 and 0.82 for the respective MLR models, compared to 0.45 and 0.79 for the respective BLMs. In contrast the BLM poorly predicted P. rapida toxicity (R(2)=0.19), as compared to the MLR (R(2)=0.92). The cross species variability in the effects of water chemistry, especially with respect to rotifers, suggests that cross species modeling of invertebrate chronic Pb toxicity using a C. dubia model may not always be appropriate.     

Parasites flicking the NPY gene on the host's switchboard: why NPY?

It was investigated whether up-regulation of the NPY gene by the schistosome Trichobilharzia ocellata in its snail host Lymnaea stagnalis redirects the host's energy flows. We cloned the cDNA encoding Lymnaea NPY (LyNPY), purified and sequenced the peptide, and used synthesized peptide for physiological and morphological studies. Increasing the LyNPY titer in nonparasitized snails (mimicking parasitosis) by 1) implantation of slow-release pellets and 2) injections suppressed reproductive activity and reduced growth in a dose- and time-dependent manner without affecting food intake. When the LyNPY titer was back to normal, reproduction and growth were resumed, coinciding with a transient increase of food intake serving to replenish glycogen stores. Observations on double-immunostained whole mount preparations of brains support these data. A close association was found between LyNPY-positive axons and axons both from ovulation hormone-producing neurons and molluscan insulin-like peptide-producing neurons involved in regulation of growth. As no synaptic(-like) contacts were observed, it is supposed that LyNPY acts nonsynaptically. No morphological interaction was found between LyNPY-positive axons and motoneurons innervating the feeding apparatus. Our data explain why it is an advantageous strategy for endoparasites to up-regulate the highly conserved NPY gene in their host.     

Neuroanatomical and immunocytochemical studies of the head retractor muscle innervation in the pond snail, Lymnaea stagnalis L

Axonal tracing and immunocytochemical techniques were used to study the innervation of the head retractor muscle (HRM) in the pond snail Lymnaea stagnalis L. Fibers of both the superior and inferior cervical nerves which innervate the HRM form endings that comply with the structure of chemical synapses. The somata of neurons with axons in these nerves are located in all except the buccal ganglia of the central nervous system, and this seems to be a special feature of the HRM motor system. By staining the filamentous actin with Oregon-green conjugated phalloidin, we demonstrated that the HRM has a multiterminal innervation and one muscle fiber can contain several synaptic endings which appear to be both morphologically and physiologically different. The morphological diversity of synaptic vesicles suggests a multiplicity of neurotransmitters acting on these nerve-muscle junctions. Immunocytochemical evidence was found for a strong serotonergic and FMRFamidergic innervation of muscle fibers through axons of the inferior cervical nerve. The thin fibers of the inferior cervical nerve possess immunoreactivity to glutamate, gamma-aminobutyric acid (GABA) and choline-acetyltransferase, and form sparser innervation patterns in the muscle. Our results indicate that several neurotransmitters are present in the nerves innervating the Lymnaea HRM and may therefore participate in the control of this muscle. The possible behavioral significance of such different neurotransmitter sets involved in the regulation of contractions is discussed.     

Molecular cloning of a cDNA encoding a member of a novel cytochrome P450 family in the mollusc Lymnaea stagnalis.

We isolated a cDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved home-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.     

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.     

Structure of visual pathways in the nervous system of fresh water pulmonary molluscs

The central nervous system of freshwater pulmonary molluscs Lymnaea stagnalis and Planorbarins corneus was stained by the method of neurobiotin retrograde transport along optic nerve fibers. In the animals of both species, bodies and fibers of stained neurons are found in all ganglia except for the buccal ones. Afferent fibers of the optic nerve form a dense sensor neuropil located in a small volume of cerebral ganglia. Characteristic groups of neurons sending their processes into optic nerves both of ipsi- and of contralateral half of the body are described. Revealed among them are neurons of visceral and parietal ganglia, which simultaneously innervate both eyes as well as give projections into peripheral nerves. It is suggested that these neurons can perform function of integration of sensor signals and, on its base, regulate photosensitivity of retina as well as activity of peripheral organs. There is established the presence of bilateral connections of the mollusc eye with cells of pedal ganglia and statocysts, which seems to be the structural basis of manifestation of the known behavior forms associated with stimulation of visual inputs of the studied gastropod molluscs.     

Analysis and modulation of spike trains and oscillations in identified neural network of Lymnaea stagnalis L

Identified neurons and members of functionally characterized clusters of the central nervous system of Lymnaea stagnalis L. were studied. Long-term spike trains (10-100 min) were collected using current clamp method. Firing patterns were analyzed by several mathematical tools e.g.: spike density function (SDF), interspike interval (ISI), Fourier-transform. Both the spike trains and oscillation of firing were modulated by 5HT (2 x 10(-5) M) and mu-opioid peptides (10(-5) M). Co-application of 5HT (2 x 10(-5) M) and DAGO (10(-5) M) turned the firing of the neurons (RPeD1 and A cells) opposite to the running pattern and eliminated the 0.3 Hz oscillation causing a new slow periodicity (0.1-0.05 Hz).     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.