Snail Helix Pomatia Research Articles

Dopamine inhibits transmission between the interneuron initiating pacemaker activity in a bursting neuron and the bursting neuron on the snail Helix pomatia

1. The effect of external application of dopamine on connection between a peptidergic interneuron initiating bursting pacemaker activity in non-active burster RPal and the bursting neuron itself of the snail Helix pomatia has been investigated under clamp conditions. 2. External application of dopamine in a dose-dependent manner ( K d 15μM) produces a reversible inhibition of slow inward current (SIC) in the RPal neuron evoked by stimulation of peptidergic interneuron. At the same time, an increase of duration and amplitude of interneuronal action potential was observed. 3. Stimulation of the anal nerve evokes in RPal neuron postsynaptic current consisting of at least three components: two fast ones with reversal potentials −50 and−70mV and a long-lasting one with no reversal potential between −50 and −95mV. 4. It is concluded that inhibition of SIC by dopamine is due to the processes occurring in the postsynaptic RPal neuron. 5. Based on the hypothesis that bursting activity of RPal neuron results from the activation of presynaptic unidentified peptidergic interneuron(s) with persistent activity and on data presented it is suggested that inhibition of bursting activity evoked by application of dopamine or anal nerve stimulation is a consequence of decreased efficiency of synaptic transmission between the interneuron initiating bursting activity and the burster.

Ultrastructural, biochemical and electrophysiological changes induced by 5,6-dihydroxytryptamine in the CNS of the snail Helix pomatia L.

The serotonin neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT), was injected into the body cavity of snails. Changes induced in the central nervous system (CNS) by the neurotoxin were studied by morphological, electrophysiological and biochemical techniques for up to 90 days following injection. The neurotoxin induced a variety of ultrastructural alterations during the early phase (1st to 6th days) following treatment. On day 6 after treatment, membranous structures first appeared in the synaptic-like areas and apparently migrated to cell bodies where they were detected by day 14. Their number increased with time. Neurotoxin-induced structural alterations were found in neuronal processes and cell bodies of the serotonergic metacerebral giant cells injected intracellularly with horseradish peroxidase and in serotonin immunoreactive axons. These findings suggest that the toxin-induced alterations are rather selective for the serotonin-containing neuronal elements. The neurotoxin decreased the concentration of 5-HT in and [ 3H]5-HT uptake into cerebral and pedal ganglia, with a maximum effect between the 3rd and 5th day following drug administration. 5-HT levels and 5-HT uptake returned to normal by 19–21 days after treatment. The concentration of dopamine and of [ 3H]DA uptake capacity were reduced between 3–5 days after injection of 5,6-DHT by 6–7 days following treatment. The transmission from identified serotonergic synapse to targets was reduced beyond day 5 after 5,6-DHT administration. By 15 days after treatment, synaptic transmission between the metacerebral giant cell (MGC) and buccal followers was blocked. Transmission recovered by day 21 after 5,6-DHT. Comparison of the time-course of functional and structural recovery indicates that while functional recovery takes place within 21 dyas after treatment, certain structural alterations, e.g. the membranous structures and dense particles, remain in the nerve fibres and cell bodies. These may serve as specific intracellular markers of the serotonin-containing neuronal elements long after functional recovery from the effect of 5,6-DHT.

17α,20β-Dihydroxy-4-pregnen-3-one 20-sulphate: A major new metabolite of the teleost oocyte maturation-inducing steroid

This paper describes the discovery of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate (17,20β-P-sulphate) in urine of male and female plaice Pleuronectes platessa, in female Atlantic salmon Salmo salar and in female Dover sole Solea solea. 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) induces oocyte final maturation in teleosts and, whereas levels of the free steroid in maturing/ovulating female plaice are generally < 1 ng ml −1 and poorly associated with the stage of maturation, the levels of 17,20β-P-sulphate are around 1500 ng ml −1 urine, 11 ng ml −1 blood plasma and six-fold higher in maturing than in nonmaturing fish. There are also high levels in spermiating male plaice (ca. 2300 ng ml −1 urine and 20 ng ml −1 blood plasma). 17,20β-P-sulphate cannot be hydrolysed by snail ( Helix pomatia) sulphatase, but can be completely solvolysed by treatment with trifluoroacetic acid (TFA)/ethyl acetate ( 1 100 , v v ) at 45° for 18 hr. A number of other sulphated steroids have been identified in plaice urine: cortisol, 11-deoxycortisol and 3α,17α,21-trihydroxy-5β-pregnan-20-one (which can all be hydrolysed by snail juice); 17α,20α-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one (which can both be solvolysed by TFA/ethyl acetate).

Electron-cytochemical study of carbohydrate components of the surface membrane of cultured neurons of the edible snail Helix pomatia

An electron-cytochemical study of the structure and topography of carbohydrate determinants of the surface membrane of CNS neurons of the edible snailHelix pomatia cultured in vitro was carried out by means of a set of colloidal goldlabeled lectins. An analysis of binding of lectins having different specificity to carbohydrate determinants shows the inhomogeneity of the carbohydrate pool of the membrane of the body of nerve cells having a different mediator nature. It was established that monoaminergic and peptidergic neurons equally bind lectins PNA, RCA, and WGA and do not demonstrate binding with PVA and LPA. Here HPA, a product of the albumen gland of the edible snailH. pomatia, marks only the membrane of peptidergic neurons. A conclusion is made about the absence of terminal residues of sialic acid on the membrane of many types of the snail neurons, which may be due to characteristics of its molecular structure. It is suggested also that the differences in the carbohydrate composition of the glycocalyx of different types of cells can serve as the basis for the formation of specific connections between different types of neurons of the developing brain.

Extrusion of calcium from a single isolated neuron of the snailHelix pomatia

Simultaneous optical measurements of extra- and intracellular Ca2+ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca2+. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the micro-chamber containing the cell. The velocity of Ca2+ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2-0.5 microM, the velocity of Ca2+ extrusion from the neuron was approximately 0.3-4.6 microM/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca2+ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 microM/sec per cell volume.

Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular-point attachments

A simple micromechanical method has been developed to measure the rupture strength of a molecular-point attachment (focal bond) between two macroscopically smooth membrane capsules. In the procedure, one capsule is prepared with a low density coverage of adhesion molecules, formed as a stiff sphere, and held at fixed position by a micropipette. The second capsule without adhesion molecules is pressurized into a spherical shape with low suction by another pipette. This capsule is maneuvered to initiate point contact at the pole opposite the stiff capsule which leads to formation of a few (or even one) molecular attachments. Then, the deformable capsule is slowly withdrawn by displacement of the pipette. Analysis shows that the end-to-end extension of the capsule provides a direct measure of the force at the point contact and, therefore, the rupture strength when detachment occurs. The range for point forces accessible to this technique depends on the elastic moduli of the membrane, membrane tension, and the size of the capsule. For biological and synthetic vesicle membranes, the range of force lies between 10(-7)-10(-5) dyn (10(-12)-10(-10) N) which is 100-fold less than presently measurable by Atomic Force Microscopy! Here, the approach was used to study the forces required to rupture microscopic attachments between red blood cells formed by a monoclonal antibody to red cell membrane glycophorin, anti-A serum, and a lectin from the snail-helix pomatia. Failure of the attachments appeared to be a stochastic function of the magnitude and duration of the detachment force. We have correlated the statistical behavior observed for rupture with a random process model for failure of small numbers of molecular attachments. The surprising outcome of the measurements and analysis was that the forces deduced for short-time failure of 1-2 molecular attachments were nearly the same for all of the agglutinin, i.e., 1-2 x 10(-6) dyn. Hence, microfluorometric tests were carried out to determine if labeled agglutinins and/or labeled surface molecules were transferred between surfaces after separation of large areas of adhesive contact. The results showed that the attachments failed because receptors were extracted from the membrane.

Caffeine affects Ca uptake and Ca release from intracellular stores: fura-2 measurements in isolated snail neurones

Using the fluorescent probe fura-2, the average cytoplasmic concentration of free Ca 2+ ([Ca] i) was measured in isolated voltage-clamped neurons of the snail Helix pomatia. In normal Ringer solution [Ca] i transients elicited by membrane depolarizations lasting 30–100 s have a voltage dependence similar to that of the calcium current. In the presence of caffeine [Ca] i transients did not depend on the testing voltage, indicating Ca release from intracellular stores. In both cases [Ca] i decayed after the transient increase. The rate of [Ca] i decline was monoexponential and independent of the membrane potential. In caffeine-containing solution the decline was 3 times faster. Steady membrane depolarization in the presence of caffeine induced periodic changes in [Ca] i. A simple model to describe these oscillations on the basis of Ca release from and Ca uptake into intracellular stores predicted that the oscillations could be initiated and modulated by Ca influx into the cytoplasm, which is in line with experimental data.

Diurnal rhythms of heart rate in the snail Helix pomatia L.

1. 1. Under constant temperature and humidity conditions and a 12:12 light-dark cycle heart rates of Helix pomatia show a diurnal rhythm with minimum values at 12.00 hr and maximum values at 24.00 hr. 2. 2. Diurnal rhythm persists during estivation. 3. 3. In hibernating snails periodic fluctuations of heart rate occur which are not synchronized by the prevailing photoperiod.

Experimental epilepsy in vitro: Neuromodulating activity of anti-brain auto antibodies from rats exposed to electroconvulsive shock

Neuromodulating activity of anti-brain autoantibodies obtained from electroshocked (ECS) rats was tested on the neurons of isolated suboesophageal ganglion of the snail Helix pomatia. In 16 out of 18 spontaneously active (pacemaker) neurons, ECS IgG containing anti-brain autoantibodies induced short-lasting epileptiform discharges and membrane depolarization. Membrane input resistance and time constant decreased, while membrane capacitance increased after addition of ECS IgG. Amplitude of evoked action potential (AP) decreased, whereas AP duration, rise time and fall time slightly increased. Thus, anti-neural autoantibody-positive IgG from rats with experimental epilepsy, but not autoantibody-negative IgG from control rats, significantly affected the bioelectrical properties of the isolated snail neurons. These results suggest that anti-neural autoantibodies present in epileptic animals are capable of influencing in vivo the function of the brain neurons.

Composition of lipids and proteins of several species of molluscs, marine and terrestrial, from the Adriatic Sea and Serbia

Results of the analysis of lipids, proteins and amino acids as well as calculation of nutritive value parameters in samples of two sea shellfish ( Venus verucosa and Mytilus galloprovincialis), a marine snail ( Monodonta turbinata) and two terrestrial snails ( Helix pomatia and Helix nemoralis) are presented. Lipid analysis revealed a high relative content of cholesterol among the other identified and unidentified sterols. Of significant nutritional importance is a high content of polyunsaturated fatty acids with more than 20 C atoms. Hydrolysed proteins contain all essential amino acids. Calculated from amino acids, pure proteins represent more than 36% and 54% of dry matter of shellfish and snails, respectively. In the case of snails a large difference between crude proteins (determined by Kjeldahl's method) and pure proteins (calculated from amino acid content) indicates the presence of other nitrogen-containing material in considerable quantities. Nutritive values indicate high biological value of proteins in shellfish, very similar to the biological values of other meat sources. Biological value of the snail's protein is lower and similar to soya protein.

Interneuronal monosynaptic peptidergic contact responsible for the bursting activity generation in the rpal neuron of the snail Helix pomatia L. is axo-axonal

1. 1. The connection between an identified interneuron and RPal bursting neuron from Helix pomatia L. brain was studied electrophysiologically and morphologically. 2. 2. Electrical stimulation of interneuron induced an increase or evoked bursting activity in the RPal neuron. 3. 3. The interneuron and RPal neurons were stained by CoCl 2 and NiCl 2 intracellular injections. 4. 4. The axon from interneuron had two large branches, the first branch was traced in visceral nerve and the second branch was traced in the direction of RPal axon, which in turn went into visceral nerve. 5. 5. According to these and the literature data, it is proposed that contact between interneuron and RPal neuron is axo-axonal.

Free calcium transients and oscillations in nerve cells

Changes in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xanthine-induced release. IP3 and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated.

Influence of caffeine on processes of regulation of the intracellular Ca2+ concentration in isolated neurons of the edible snail

Changes in the intracellular concentration of Ca2+ ([Ca2+]in) that occur during prolonged depolarization of the plasma membrane were studied in isolated neurons of the edible snailHelix pomatia, using the calcium-sensitive probe Fura-2. The dependence of the amplitude of the calcium response on the value of the depolarization in the presence of 5 mM caffeine, in contrast to that observed in a normal solution, practically disappeared. This fact indicates that caffeine promotes calcium-dependent release of Ca2+ from the intracellular depots, which is the determining factor in the increase in [Ca2+]in during depolarization. Processes of reduction of [Ca2+]in to the steady-state levels were described by one exponential function, and in the presence of caffeine they occurred twice as rapidly as in the normal solution. Such an acceleration of the kinetics of the relaxation of [Ca2+]in is evidently associated with an increase in the efficiency of the work of the calcium pump of the intracellular calcium depots, which might lead to a decrease in the steady-state of level of [Ca2+]in even below the level observed for the normal extracellular solution.

The reaction of nitrite with the haemocyanin of the Roman snail (Helix pomatia)

The reaction of nitrite at pH 5.0-7.0 with the deoxyhaemocyanin of a mollusc, the Roman snail (Helix pomatia), yielded nitrosylhaemocyanin (CuIA.NO+ CuIIB), in contrast with the formation of methaemocyanin with the deoxyhaemocyanin of the crustacean Astacus leptodactylus (mud crayfish). With Helix haemocyanin 1 NO was thereby liberated per active site, as shown by m.s., as against 2 NO with Astacus haemocyanin. Helix nitrosylhaemocyanin was characterized in c.d. by the negative extremum at 336 nm (CuIA.NO+) and by the mononuclear e.p.r. signal at g = 2 (CuIIB). Binuclear e.p.r. signals have been observed after the addition of nitrite to methaemocyanins. With Astacus methaemocyanin, no further reaction occurred, whereas with Helix methaemocyanin the mononuclear e.p.r. signal, characteristic for nitrosylhaemocyanin gradually appeared. This formation of Helix nitrosylhaemocyanin implicates the binding, most likely on CuIIA, of a second nitrite besides a bridging nitrite, so that a dismutation into NO and NO2 can occur there. A further dismutation of NO2 yields nitrite and nitrate. The formation of the latter was demonstrated by Raman spectrometry. The reaction rate of Helix methaemocyanin with nitrite decreased with increasing pH according to the Henderson-Hasselbalch equation with a pKa value of 6.77, attributed to a mu-aquo bridging ligand, which can be exchanged for nitrite, in equilibrium with a mu-hydroxo ligand which cannot. These data also favour the formulation of the final reaction product as nitrosylhaemocyanin instead of semi-methaemocyanin, with or without bound nitrite.

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 μm); (ii) large neurons (80–100 μm); (iii) medium-sized neurons (40–70 μm); (iv) small neurons (12–30 μm). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.

Plateau-Generating Nerve Cells in Helix: Morphological and Electrophysiological Characteristics

ABSTRACT We describe the anatomical and electrophysiological characteristics of a group of Helix nerve cells, styled P cells, that generate long-lasting depolarizations in response to repeated stimulations at low frequencies. Four neurones were identified in the perioesophageal ganglia of the snail Helix pomatia. Their structure was determined by intracellular injection of Lucifer Yellow, cobaltlysine or horseradish peroxidase. The soma was found to contain neurosecretory granules. These cells innervated the whole foot muscle and the mantle, but were not involved in muscle movement or locomotion. They may participate in mucus secretion. Upon depolarization they fired Ca2+-dependent spikes; at a critical firing rate (5–6Hz), the spikes were converted into depolarized plateaus (+10 to +20mV) lasting for several seconds. The plateau was Ca2+-dependent and persisted in Na+-free saline. It was sustained by a slowly inactivating Ca2+ current that produced a large intracellular Ca2+ accumulation (monitored with the Ca2+-sensitive dye Arsenazo III). The plateau was restricted to the soma and the proximal axon and may act as a driver potential inducing axon firing and prolonging the release of neurosecretory materials.

Cyclic adenosine monophosphate (cAMP)-induced potentiation of synaptic responses in Helix neurons.

1. The effect of intracellularly injected cAMP on the amplitude of excitatory postsynaptic potentials was studied using identified neurons of the snail Helix pomatia. 2. In 25% of the experiments, postsynaptic cAMP elevation caused a pronounced augmentation of the excitatory postsynaptic potential (EPSP) amplitude, lasting up to 15-30 min. 3. The results suggest that a cAMP increase in the postsynaptic neuron may be involved in the enhancement of synaptic efficiency.

Aluminium, citrate and shell-repair. Effects on the distribution of metals in the snail Helix pomatia L.

1. 1. In shell-repairing snails ( Helix pomatia L.) injected with AlCl 3, Al was found to be significantly more concentrated in the mantle than in the digestive gland. This contrasts with both controls and Na-citrate injected animals, in which no significant differences were found, and also with the reversed relationship recorded in Al-citrate treated snails. 2. 2. The weights of the shell-repair membranes were significantly reduced in the AlCl 3-group only. 3. 3. Ca, Fe and Mg were also affected by the treatments employed, while no consistent effects on Cd, Co, Cr, Cu, Mn, Ni, Pb, V and Zn were recorded. 4. 4. It is concluded that citrate diminishes the deleterious effects of Al on the repair process and that a decreased weight of the repair membrane is associated with a high concentration of Al in the mantle.

Aluminium, phosphatase activities and shell-repair. Studies in the snail Helix pomatia L.

1. Aluminum, injected as A1Cl 3 in the snail Helix pomatia L., affected the activities of both acid and alkaline phosphatase in homogenates prepared from the mantle. 2. In snails with intact as well as damaged shells the activity of acid phosphatase was increased by Al, while that of alkaline phosphatase was decreased. 3. In the shell-damaged snails the activity of the acid phosphatase was approximately 15% lower and the activity of the alkaline phosphatase roughly 20% higher than that of the intact snails receiving the same Al-dose. 4. As the changes in enzyme activity associated with shell-repair are unaffected by Al, it is concluded that reduced weights of the shell-repair membranes and effects on the phosphatases, although both a result of the Al-injections, are independent of each other.

Structural studies on the galactan from the snail Helix pomatia

1. 1. The galactan of the snail Helix pomatia was subjected to two cycles of Smith-degradation and the resulting products were isolated by gel filtration and thin layer chromatography. 2. 2. The structures of the low molecular weight oligosaccharides were elucidated being identical to those obtained from Lymnaea stagnalis galactan. However, the quantities release differed significantly between the two species. 3. 3. The high molecular fractiosn comprising about 66% of the material were not obtained in a similar degradation of the Lymnaea stagnalis galactan. 4. 4. Thus the observed structural differences can explain easily the species-specific reactivity among the two polysaccharides seen earlier with lectins, enzymes and antibodies.