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The electrical responses of osphradial nerve and central neurons to chemicals stimulation of Lymnaea osphradium.

The osphradial sensory system of the mollusc Lymnaea stagnalis was studied by electrophysiological methods. Bioelectrical activity of the osphradial nerve appeared to be dependent on concentrations of D2, mannit, urea and NaC1 in surrounding media. Studies on impulse activities of axons of the osphradial nerve disclose diverse units. Each of them has its own range of response, often overlapping with the others. Several large neurons and cell clusters, answering to the adequate osphradial stimulation, were identified in the viscero-parietal complex. This response is expressed as excitation, inhibition and redistribution of impulse patterns. The participation of the osphradial sensory system in the regulation of vegetative functions of Lymnaea is discussed.

Animals↗

Inhibition of calcium spikes by gamma-amino-butyric acid in the neurons of Lymnaea stagnalis L.

The modulation of action potentials (APs) and high-threshold calcium currents by gamma-aminobutyric acid (GABA) and GABAB receptor agonist baclofen was investigated in identified internally perfused neurons of Lymnaea stagnalis using current-clamp and whole-cell voltage-clamp techniques. GABA (5 x 10(-5) M) and baclofen (5 x 10(-5) M) reversibly reduced overshoot and afterhyperpolarization phase of the action potentials. Application of GABA or baclofen inhibited inward and outward voltage-gated ionic currents. GABA (10(-8)-10(-4) M) was found to suppress high-threshold calcium current in voltage- and dose-dependent manner. These effects were mimicked by baclofen (5 x 10(-5) M) but not by muscimol (5 x 10(-5) M). Inhibition of Ca(2+)-currents could not be prevented by pentylenetetrazole (5 x 10(-5) M) or with pertussis toxin (1 microgram/ml). These results provide evidences for the presence of GABAB receptors in the identified central neurons of Lymnaea stagnalis L.

Action Potentials↗

Octopamine in the developing nervous system of the pond snail, Lymnaea stagnalis L.

Development of the octopaminergic system in the pond snail, Lymnaea stagnalis, was investigated by means of immunocytochemistry and radioenzymatic assay. The earliest octopamine-immunoreactive neurons appear at a late embryonic stage (E85) of development following metamorphosis. At this moment of development, the adult-like pattern of distribution and projection (arborization) characteristics of octopamine-immunoreactive neurone can already be observed. During hatching and postembryonic (juvenile) development the number of labelled neurons increases only within the ventro-medial cell groups of the cerebral ganglia, whereas the extent of varicose axon arborization of the labelled neurons increases gradually. No peripheral projections of the embryonic and postembryonic octopamine-immunoreactive neurons were observed. The postembryonic increase in number of immunoreactive neurons and development of axonal arborization is accompanied by a rapid, exponential enhancement of octopamine content of Lymnaea CNS, as detected radioenzymatically. A possible role of the octopaminergic neurons in the regulation of certain physiological function(s), active only from a late stage of embryonic development, is suggested.

Animals↗

Unusual transmission of the liver fluke, Fasciola hepatica, by Lymnaea glabra or Planorbis leucostoma in France.

Cases of fasciolosis in ruminants have been recorded in several French farms in the absence of Lymnaea truncatula, which is considered the only snail intermediate host in western Europe. These farms harbored other species of freshwater snails in large numbers (Lymnaea glabra, Physa acuta, or Planorbis leucostoma) and, in many cases, had cattle or sheep infected by another trematode (Paramphistomum daubneyi). These other freshwater snails may serve as intermediate hosts for F. hepatica due to a coexisting infection with P. daubneyi. We have demonstrated that L. glabra, either infected with F. hepatica alone or coinfected by P. daubneyi, was capable of developing a F. hepatica infection. A broader range of L. glabra size classes (up to 10 mm in height) were susceptible to infection if simultaneously infected with P. daubneyi. Planorbis leucostoma can only serve as an intermediate host for F. hepatica, if infected with P. daubneyi. Lastly, P. acuta smaller than 4 mm cannot serve as an intermediate host. These results may explain, in part, the maintenance of low-level F. hepatica infections in the absence of the normal intermediate host, L. truncatula.

Animals↗

The molecular basis of the neuro-endocrine control of egg-laying behaviour in Lymnaea.

The biosynthesis, axonal transport and release of multiple peptides by the egg-laying controlling caudodorsal cells (CDC) of Lymnaea stagnalis were studied. High performance gel permeation chromatography was used to resolve newly synthesized peptides after pulse-chase experiments with radioactive amino acids. The precursor is a 35 kd polypeptide which is produced in the CDC somata. It gives rise to intermediate products (20 kd, 10 kd and 7 kd) and a number of end products which include a approximately 4.5 kd (the ovulation hormone, CDCH), a 1.5 kd peptide (the autotransmitter) and other peptides (6 kd, 3.5 kd and 2 kd). The end products are transported in neurosecretory granules to the CDC axon terminals in the cerebral commissure where they are released into the medium during electrical discharges of the CDC system. A cDNA clone encoding part of the CDCH precursor was isolated from a Lymnaea CNS cDNA library by differential hybridisation and use of synthetic oligonucleotide probes. Examination of the deduced amino acid sequence indicates that the precursor contains additional peptides, besides CDCH.

Animals↗

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.

Animals↗

Learning and memory in Lymnaea are negatively altered by acute low-level concentrations of hydrogen sulphide.

Hydrogen sulphide (H(2)S) is a common industrial pollutant as well as an endogenous neural transmitter/neural modulator. Experiments were performed on the pond snail Lymnaea stagnalis to determine the acute effects of low-level exposure to H(2)S (50-100 micromol l(-1)) on aerial respiratory behaviour, associative learning, and its subsequent consolidation into long-term memory (LTM). A 3-neuron network whose sufficiency and necessity have been demonstrated drives aerial respiratory behaviour in Lymnaea. In the presence of 100 micromol l(-1) H(2)S the number of bouts of aerial respiration and the total breathing time were significantly increased compared to the control hypoxic situation, but were equivalent to those observed in snails that had been subjected to a 'more intense hypoxic challenge'. In addition, at a concentration of 100 micromol l(-1) H(2)S neither associative learning nor long-term memory (LTM) were observed. However, snails subjected to a 'more intense hypoxic challenge' still had the capacity to learn and form LTM. These snails, in fact, showed statistically the best learning and memory performance of any group. While learning and memory were observed at 50 and 75 micromol l(-1) H(2)S, respectively, they were statistically poorer than the learning and memory exhibited by snails in the standard hypoxia condition. Hence the ability to learn and form memory was compromised by H(2)S. Thus an invertebrate model system with a well-defined neural network can be used to study of the effects of H(2)S on the processes of learning and memory.

Analysis of Variance↗

DIFFERENTIAL TRACER COUPLING BETWEEN PAIRS OF IDENTIFIED NEURONES OF THE MOLLUSC LYMNAEA STAGNALIS

Electrical coupling is a common means of cell-to-cell communication in both neuronal and non-neuronal tissues (Lowenstein, 1985). Within the nervous system, many electrically coupled neurones exhibit dye coupling (Bennett, 1973; Stewart, 1978; Glantz and Kirk, 1980; Spencer and Satterlie, 1980; Fraser and Heitler, 1993); however, some electrically coupled cells do not dye-couple (Audesirk et al. 1982; Murphy et al. 1983; Berdan, 1987; Robinson et al. 1993; Veenstra et al. 1993). Electrical coupling and dye coupling, often considered in parallel, are in fact two different parameters that can vary independently (e.g. Audesirk et al. 1982; Perez-Armendariz et al. 1991). The giant identified neurones of pulmonate and opisthobranch molluscs have frequently been used for studies of neuronal communication and its plasticity (Winlow and McCrohan, 1987; Bulloch, 1989). In the present study, we explored the relationship between electrical and tracer coupling in both strongly and weakly coupled pairs of molluscan neurones. Specifically, we examined electrically coupled, identified neurones in a freshwater pond snail, Lymnaea stagnalis L., and tested for tracer coupling with Lucifer Yellow CH and biocytin. The cells examined were the strongly electrically coupled neurones, visceral dorsal 1 (VD1) and right parietal dorsal 2 (RPD2) (Boer et al. 1979; Benjamin and Pilkington, 1986), and the weakly coupled neurones, left buccal 1 (LB1) and right buccal 1 (RB1) (Benjamin and Rose, 1979). The use of these particular neurones made it possible to compare electrical coupling with tracer coupling in the molluscan central nervous system (CNS). All experiments were performed on laboratory-bred Lymnaea stagnalis (Mollusca, Pulmonata), maintained as previously described (Ridgway et al. 1991). The CNS was dissected from mature animals (16­18 mm shell length) and pinned to the silicone rubber (RTV 616 GE) base of a recording dish in normal saline (51.3 mmol l-1 NaCl, 1.7 mmol l-1 KCl, 4.1 mmol l-1 CaCl2, 1.5 mmol l-1 MgCl2 and 5 mmol l-1 Hepes, pH 7.9). Following removal of the outer connective tissue sheath, a small Pronase crystal (Sigma, type XIV, P-5147), held by forceps, was carefully applied to specific ganglia; this treatment softened the inner sheath and facilitated microelectrode penetration. The CNS was then rinsed several times at 5 °C in normal saline.

Journal Article↗

Chemosensitivity of the osphradium of the pond snail Lymnaea stagnalis

The osphradium of the pond snail Lymnaea stagnalis was studied to determine the stimuli to which this organ responds. The following stimuli were tested: hypoxia, hypercapnia, a mixture of amino acids, a mixture of citralva and amyl acetate and a mixture of lyral, lilial and ethylvanillin. The mean nerve activity consistently increased with elevated PCO2, whereas hypoxia produced variable effects. The nerve activity became rhythmic upon application of citralva and amyl acetate, but it increased in a non-rhythmic way upon application of the other two odorant mixtures tested. Whole-cell patch-clamp recordings were made from a group of 15 neurones that lay next to the issuing osphradial nerve, to determine whether ganglion cells were involved in olfactory signal processing. All neurones tested responded to at least one of the three mixtures of odorants. Both excitatory and inhibitory responses occurred. Our results indicate that the osphradium of the pond snail Lymnaea stagnalis is sensitive to elevated PCO2 as well as to three different classes of odorants. In addition, at least some neurones within the osphradium are involved in the processing of olfactory information.

Journal Article↗

Pore properties of Lymnaea stagnalis neuron stretch-activated K+ channels

In many neurons, variations in membrane excitability are determined by a resting K+ conductance whose magnitude is modulated via neurotransmitters. The S-channel in Aplysia californica mechanosensory neurons is such a conductance, but it has also been shown to be a stretch-activated K+ channel. In this, it resembles stretch-activated K+ channels common to all molluscan neurons. Comparable channels are widespread, having been reported in molluscan and insect muscle and various vertebrate cells. The pore properties of the S-channel and similar stretch-activated K+ channels have received only sporadic attention. Here we examine, at the single-channel level, the permeation characteristics of a stretch-activated K+ channel from neurons of the mollusc Lymnaea stagnalis. Michaelis­Menten constants (Km) for the conductance, obtained separately for inward (28 mmol l-1) and outward (91 mmol l-1) K+ currents, suggest that the channel presents to the external medium, where [K+] is lower, a higher-affinity site than it presents to the cytoplasmic medium. This may help to ensure that influx is not diffusion-limited at potentials near the resting potential, i.e. near the K+ equilibrium constant. Anomalous mole fraction behavior, observed when the ratio of permeant ion (K+ and Rb+) was varied, indicated that the stretch-activated K+ channel is a multi-ion pore. The ion selectivity sequence determined using reversal potentials under bi-ionic conditions was Cs+>K+>Rb+>NH4+>Na+>Li+, and using relative conductance in symmetrical solutions, the sequence was Tl+=K+>Rb+>NH4+>>Na+=Li+=Cs+. Extreme variations in extracellular pH from 4.7 to 11.4 had no effect on stretch-activated K+ channel conductance, whereas normal concentrations of extracellular Mg2+ reduced inward K+ current. Intracellular, but not extracellular, Ba2+ produced a slow, open channel block with an IC50 of 140±80 µmol l-1. These pore properties are compared with those of other stretch-activated K+ channels and of K+ channels in general. In spite of a greater than half order of magnitude difference in the cytoplasmic [K+] in marine (Aplysia californica) and freshwater (Lymnaea stagnalis) molluscs, the conductances of stretch-activated K+ channels from the two groups are very similar.

Journal Article↗

Operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis

In this study, we operantly conditioned the aerial respiratory behaviour of the freshwater snail Lymnaea stagnalis. Aerial respiration in Lymnaea stagnalis is accomplished by the spontaneous opening and closing of its respiratory orifice, the pneumostome, at the water surface. Weak tactile stimulation of the pneumostome area, when the pneumostome is open, evoked only the pneumostome closure response, which is one aspect of the escape-withdrawal reflex. Pneumostome stimulation resulted in its closure and the termination of aerial respiratory activity. A contingent tactile stimulation paradigm was used to operantly condition the animals. Stimulation of the pneumostome whenever the animal attempted to breathe resulted in significantly fewer attempts to open the pneumostome as training progressed. The latency of the first breath (subsequent to stimulation), the number of breaths and the total breathing time were measured before and after each training period. Significant, quantifiable changes in these behavioural parameters were observed only in the operant conditioning group animals. Control animals receiving tactile stimulation to their pneumostome not contingent upon pneumostome opening movements (yoked controls) or those that were physically prevented from surfacing to breathe (hypoxic controls), did not exhibit significant changes in these behavioural parameters. Our data provide the first direct evidence for operant conditioning of respiration in any animal.

Journal Article↗

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: II. Postembryonic development of central and peripheral cells.

Catecholamines have long been thought to play important roles in different mollusc neural functions. The present study used glyoxylate- and aldehyde-induced histofluorescence to identify central and peripheral catecholaminergic neurons in the snail Lymnaea stagnalis. The majority of these cells were also found to react to antibodies raised against tyrosine hydroxylase. A minority of the catecholaminergic neurons, however, exhibited no such immunoreactivity. The number of central catecholaminergic neurons nearly doubled (from about 45 to about 80 cells) during the first 2-3 days of postembryonic development. Thereafter, catecholaminergic neurons again doubled in number and generally grew by about 100-200% in soma diameter as the snails grew by 1,000% in overall linear measurements. In contrast to the relatively meager addition of central catecholaminergic neurons, several thousand catecholaminergic somata were added to different peripheral tissues during postembryonic development. These small, centrally projecting neurons were particularly concentrated in the lips, esophagus, anterior margin of the foot, and different regions of the male and female reproductive tracts. Chromatographic analyses indicated that dopamine was the major catecholamine present in the central ganglia, foot, and esophagus, although detectable levels of norepinephrine (approximately 20% of dopamine levels) were also found in the ganglia. The total content but not the concentration of dopamine increased within the tissue samples during postembryonic development. The companion study (Voronezhskaya et al. [1999] J. Comp. Neurol. 404:285-296) and the present study furnish a complete description of central and peripheral catecholaminergic neurons from their first appearance in early embryonic development to adulthood.

Animals↗

Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis: possible involvement in reproduction.

Peptides of the gonadotropin-releasing hormone (GnRH) family are present in neural and nonneural tissues throughout the chordate phylum. Although GnRH peptides have been implicated in nonreproductive functions, their primary function is to control reproduction by regulating sexual behaviors and inducing gonadotropin hormone release from the pituitary. Evidence suggesting the presence of a similar peptide in the central nervous system (CNS) of the gastropod mollusc Helisoma trivolvis has recently been provided. In the present study, we examined the tissue distribution of the peptide and found that it is likely restricted to the nervous system. The neuronal system containing the endogenous GnRH-like peptide is described further and is shown, in part, to innervate the male reproductive tract. Immunostaining in the closely related snail, Lymnaea stagnalis, showed a conservation in the locations of some immunoreactive neurons. Notably, staining occurred in and adjacent to the lateral lobes of both snails. Because these lobes contain neurons involved in the stimulation of egg laying and GnRH staining occurred in additional areas in the Helisoma CNS that are involved in reproduction, we suggest that the endogenous GnRH-like peptide plays a role in regulating reproduction in freshwater snails.

Animals↗

Ciliary neurotrophic factor, unlike nerve growth factor, supports neurite outgrowth but not synapse formation by adult Lymnaea neurons.

The nerve growth factor (NGF) family and ciliary neurotrophic factor (CNTF) support survival and/or neurite outgrowth of many cell types. However, it is not known whether the neurite outgrowth induced by neurotrophic factors results in the formation of synapses. We tested NGF and CNTF for their ability to induce neurite outgrowth and synapse formation in vitro by interneurons from the mollusc Lymnaea. Dopaminergic and peptidergic interneurons survived in the absence of neurotrophic factors but exhibited robust outgrowth in response to both NGF and CNTF. Chemical synapses formed between these interneurons and their target neurons cultured in NGF, but synapses were absent in CNTF. Survival, neurite outgrowth, and synaptogenesis are therefore differentially regulated in these neurons.

Age Factors↗

Target cell contact suppresses neurite outgrowth from soma-soma paired Lymnaea neurons.

Neurite extension from developing and/or regenerating neurons is terminated on contact with their specific synaptic partner cells. However, a direct relationship between the effects of target cell contact on neurite outgrowth suppression and synapse formation has not yet been demonstrated. To determine whether physical/synaptic contacts affect neurite extension from cultured cells, we utilized soma-soma synapses between the identified Lymnaea neurons. A presynaptic cell (right pedal dorsal 1, RPeD1) was paired either with its postsynaptic partner cells (visceral dorsal 4, VD4, and Visceral dorsal 2, VD2) or with a non-target cell (visceral dorsal 1, VD1), and the interactions between their neurite outgrowth patterns and synapse formation were examined. Specifically, when cultured in brain conditioned medium (CM, contains growth-promoting factors), RPeD1, VD4, and VD2 exhibited robust neurite outgrowth within 12-24 h of their isolation. Synapses, similar to those seen in vivo, developed between the neurites of these cells. RPeD1 did not, however, synapse with its non-target cell VD1, despite extensive neuritic overlap between the cells. When placed in a soma-soma configuration (somata juxtaposed against each other), appropriate synapses developed between the somata of RPeD1 and VD4 (inhibitory) and between RPeD1 and VD2 (excitatory). Interestingly, pairing RPeD1 with either of its synaptic partner (VD4 or VD2) resulted in a complete suppression of neurite outgrowth from both pre- and postsynaptic neurons, even though the cells were cultured in CM. A single cell in the same dish, however, extended elaborate neurites. Similarly, a postsynaptic cell (VD4) contact suppressed the rate of neurite extension from a previously sprouted RPeD1. This suppression of the presynaptic growth cone motility was also target cell contact specific. The neurite suppression from soma-soma paired cells was transient, and neuronal sprouting began after a delay of 48-72 h. In contrast, when paired with VD1, both RPeD1 and this non-target cell exhibited robust neurite outgrowth. We demonstrate that this neurite suppression from soma-soma paired cells was target cell contact/synapse specific and Ca(2+) dependent. Specifically, soma-soma pairing in CM containing either lower external Ca(2+) concentration (50% of its control level) or Cd(2+) resulted in robust neurite outgrowth from both cells; however, the incidence of synapse formation between the paired cells was significantly reduced. Taken together, our data show that contact (physical and/or synaptic) between synaptic partners strongly influence neurite outgrowth patterns of both pre- and postsynaptic neurons in a time-dependent and cell-specific manner. Moreover, our data also suggest that neurite outgrowth and synapse formation are differentially regulated by external Ca(2+) concentration.

Animals↗

Effect of molluscicidal components of Abrus precatorius, Argemone mexicana and Nerium indicum on certain biochemical parameters of Lymnaea acuminata.

Exposure to 40% and 80% of the 24 h LC50 of the molluscicidal component of Abrus precatorius (abrin and glycyrrhizin), Argemone mexicana (protopine and sanguinarine) and Nerium indicum (oleandrin) caused a significant decrease in the levels of protein, free amino acid, DNA and RNA in the nervous tissue of Lymnaea acuminata. Except for glycyrrhizin, all the above molluscicides caused a significant reduction in phospholipid levels and a simultaneous increase in the rate of lipid peroxidation in the nervous tissue of treated snails.

Abrin↗

Millimeter waves thermally alter the firing rate of the Lymnaea pacemaker neuron.

The effects of millimeter waves (mm-waves, 75 GHz) and temperature elevation on the firing rate of the BP-4 pacemaker neuron of the pond snail Lymnaea stagnalis were studied by using microelectrode techniques. The open end to a rectangular waveguide covered with a thin Teflon film served as a radiator. Specific absorption rates (SARs), measured in physiological solution at the radiator outlet, ranged from 600 to 4,200 W/kg, causing temperatures rises from 0.3 to 2.2 degrees C, respectively. Irradiation at an SAR of 4200 W/kg caused a biphasic change in the firing rate, i.e., a transient decrease in the firing rate (69 +/- 22% below control) followed by a gradual increase to a new level that was 68 +/- 21% above control. The biphasic changes in the firing rate were reproduced by heating under the condition that the magnitude (2 degrees C) and the rate of temperature rise (0.96 degrees C/s) were equal to those produced by the irradiation (for an SAR of 4,030 W/kg). The addition of 0.05 mM of ouabain caused the disappearance of transient responses of the neuron to the irradiation. It was shown that the rate of temperature rise played an important role in the development of a transient neuronal response. The threshold stimulus for a transient response of the BP-4 neuron found in warming experiments was a temperature rise of 0.0025 degrees C/s.

Animals↗

Gene expression and function of FMRFamide-related neuropeptides in the snail Lymnaea.

FMRFamide and a large family of related peptides (FaRPs) have been identified in every major metazoan phylum examined, including chordates. In the pulmonate snail Lymnaea this family of neuropeptides is encoded by a five-exon locus that is subject to alternative splicing. The two alternative mRNA transcripts are expressed in the CNS in a mutually exclusive manner at the single cell level, resulting in the differential distribution of the distinct sets of FaRPs that they encode in defined neuronal networks. Biochemical peptide purification, single-cell analysis by mass spectroscopy, and immunocytochemistry have led to an understanding of the post-translational processing patterns of the two alternative precursor proteins and identified at least 12 known and novel peptides contained in neuronal networks involved in cardiorespiration, penial control and withdrawal response. The pharmacological actions of single or co-expressed peptides are beginning to emerge for the cardiorespiratory network and its central and peripheral targets. Peptides derived from protein precursor 1 and contained in the heart excitatory central motoneurons E(he) have distinct functions and also act in concert in cardiac regulation, based on their unique effects on heartbeat and their differential stimulatory effects on second messenger pathways. Precursor-2 derived peptides, contained in the Visceral White Interneuron, a key neuron of the cardiorespiratory network, have mostly inhibitory effects on the VWI's central postsynaptic target neurons but with some of the peptides also exhibiting excitatory effects on the same cells.

Animals↗