Snail Helix Pomatia Research Articles

Do weak, low pulsed frequency, high-frequency electromagnetic or magnetic fields alter the basic bioelectrical parameters of nerve cells in vineyard snails ( Helix pomatia L.)? I. Electromagnetic fields

Resting potentials (RPs) of the vineyard snail ( Helix pomatia) were investigated to find how they were influenced by weak, low pulsed frequency, high-frequency electromagnetic fields. The flux densities of the fields were chosen such that thermal effects could not occur in the tissue exposed to the field. An experimental method was developed which allowed electrophysiological measurements to be carried out in the high-frequency electromagnetic field without artifacts. Non-thermic effects were achieved with fields with 150 MHz, 8.3 Hz and 124 μT. The RP of the nerve cells being investigated hyperpolarized. A relationship between the resulting value of the strength of the field-induced hyperpolarization and the RP before the start of the exposure was established. A model of the sequence of events for the effect was developed in order to explain the exposure-induced hyperpolarization. Fundamental to the hypothesis which was drawn up is the interaction of a possible field-induced release of calcium from intracellular depots in the cytoplasma of nerve cells and the resultant activation of the calcium-dependent potassium channels.

Conversion of adenosine(5′)oligophospho(5′)adenosines into inosn(5′)oligophospho(5′)inosines by non-specific adenylate deaminase from the snail Helix pomatia

Until now, the catabolism of adenosine(5′)triphospho(5′)adenosine (Ap 3A) and adenosine(5′)tetraphospho(5′)adenosine (Ap 4A) has been thought to commence with either hydrolytic or phosphorolytic cleavage of their oligophosphate chains, depending on the organism. Here, we show that in the extracts from the retractile ‘foot’ of the snail Helix pomatia deamination predominates; the adenosine moieties of these and other adenosine(5′)oligophospho(5′)adenosines (Ap nAs) undergo successive deamination leading, via an inosine(5′)oligophospho(5′)adenosine (Ip nA), to the corresponding inosine(5′)oligophospho(5′)inosine (Ip nI). The reactions are catalyzed by the non-specific adenylate deaminase described earlier (Stankiewicz, A.J. (1983) Biochem. J. 215, 39–44). We describe TLC and HPLC systems which allow the separation of any of the deaminated derivatives from its parent compound; Ap 2A, Ap 3A, Ap 4A or Ap 5A. The K m values for these substrates are 20, 22, 32 and 39 μM, respectively, whereas the K m for 5′-AMP is 12 μM. Relative substrate specificities for these compounds amount to 25, 18, 14, 7 and 100. The enzyme was shown also to deaminate phosphonate and thiophosphate analogues of Ap 3A.

Simulation of bursting activity in theHelix RPa1 neuron: Role of calcium ions

A model of bursting activity in the RPal neuron of the snailHelix pomatia has been developed. In this model, calcium conductances do not play a key role in generation of slow oscillations of membrane potential (MP). The possibility of simulating the maintenance of bursting in the presence of cadmium ions is shown. Inclusion in the model of the calcium-inactivated calcium conductance makes it possible to reproduce both adaptation of the neuron to constant polarizing current, which modifies bursting, and the development of slow inward current when MP is clamped at different phases at the slow wave. In our simulations, the characteristic properties of bursts (such as an increase in the frequency of action potentials and a decrease in spike undershoot at the beginning of a burst) are due to the cumulative inactivation of potassium current. The advantages of the presented mathematical model of bursting compared with other models are discussed.

Purification and Characterization ofα-Glucuronidase from Snail Acetone Powder

We have purified the p-nitrophenyl α-D-glucuronide-hydrolyzing enzyme (PNP-GAase) from snail (Helix pomatia) acetone powder by chromatographies on DEAE-cellulose, CM-Toyopearl, and Toyopearl HW-SSF. The molecular weight of the enzyme was 180,000 by gel filtration chromatography with Superose 6, and 97,000 by SDS–polyacrylamide gel electrophoresis, and the pI was 6.8 by isoelectric focusing. The enzyme showed the maximum activity at pH 3.0 and 50°C, and was stable at pH between 3.0–7.0 and up to 50°C. The enzyme activity was greatly inhibited by Hg2+, p-chloromercuribenzoic acid, sodium dodecylsulfate, and N-bromosuccinimide. The enzyme released D-glucuronic acid not only from PNP-GA but also from 2-, 3-, and 4-O-α-D-glucuronosyl-D-xyloses, O-α-D-glucuronosyl α-D-glucuronide, and benzyl 4-O-α-D-glucuronosyl-β-D-glucoside. The results suggest that the α-glucuronidase from snail acetone powder had a broad substrate specificity comparing with α-glucuronidases from Aspergillus niger and basidiomycetes.

Three-dimensional reconstruction of the α D and β C-hemocyanins of Helix pomatia from frozen-hydrated specimens

The three-dimensional (3D) reconstructions of the di-decameric forms of α Dand β C-hemocyanins of the Roman snail Helix pomatia and of the decameric half molecules of α D-hemocyanin were carried out on frozen-hydrated specimens observed in the electron microscope by using the random conical tilt series method. The three 3D volumes were examined by computing solid-body surface representations and slices through the volume and by eroding the structure progressively through raising of the threshold. The di-decameric molecule of α D and β C-hemocyanins, reconstructed from side views, are very similar and are composed of a cylindrical wall, comprising ten oblique wall units, and of two collar complexes located at both ends of the cylinder, comprising each five arches and an annular collar made up of five collar units. Erosion of the structure reveals that the wall looks like a segment of a five-stranded right-handed helix and that each oblique wall unit resembles a figure 8 inclined to the right. The decameric half molecule of α D-hemocyanin, reconstructed from end-on views, resembles the whole molecule, except that the collar is thinner and appears composed of five independent collar complex units. It is suggested that the difference in structural appearance of the collar complex between the whole and the half α D-hemocyanin may be due to the missing cone artifact, induced by the angular limitations imposed by the goniometer of the electron microscope. The comparison between the α D-hemocyanin and the β C-di-decameric hemocyanin at high thresholds suggests that in the β C-hemocyanin the oblique wall units of each half molecule may be linked by two connections, whereas in α D-hemocyanin there may be only one. This difference in the number of connections may be responsible for the lower stability of the α D molecule at high salt concentration.

Electron cytochemical study of carbogydrate components of surface membrane in cultured glial cells of the snailHelix pomatia

Using a variety of colloidal gold-labelled lectins, the structure and topography of carbohydrate determinants of the surface membrane in different types of cultured glial cells of the snailHelix pomatia have been electron cytochemically investigated. Analysis of lectin binding having different sugar specificities have shown heterogeneity of carbohydrate pools between glial and nerve cells and among different types of glial cells. It was found that satellite glial cells displaying ultrastructural traits of intensive metabolism (type II cells) selectively bindGNA, which is specific for terminal α-D-mannose residues, and do not interact (Con A) or slightly interact (LCA) with other mannose-specific lectins.GNA determinants remain during the whole period of cell growth and are absent in satellite type-I glial cells, fibrous glial cells, microglia, and neurons.LTA, PVA, andLABA do not bind to any glial cells.WGA determinants, which are abundant on the neurons, are completely absent onGNA-binding glial cells and single on other types of glial cells. The density ofPNA determinants on microglial cells is the highest, as compared with other types of glial cells or neurons. It is concluded that some lectin determinants (forRCA-1, PNA, LPA) are present on all types of glial cells, while another determinant (GNA) is specific for a certain type of glial cells only and can serve as a marker of these cells. The role of specific carbohydrate determinants for neuron-glia interaction in mature brain is discussed.

Helix pomatia agglutinin binding is a useful prognostic indicator in colorectal carcinoma

Most deaths from colorectal carcinoma are due to metastases. A relatively reliable prognostic indicator at surgery to date is the Dukes' stage, but this is a morphologic approach that does not elucidate biochemical changes to explain why cells became metastatic. The binding sites for the lectin from the Roman snail Helix pomatia (HPA) were shown to be good prognostic indicators in breast and gastric cancer, and accordingly, this study was performed to evaluate the use of HPA binding sites as prognostic markers in colorectal carcinoma. The histochemically detected expression of HPA binding sites in colorectal carcinomas (n = 130) was increased. The results of the histochemical findings were correlated with patient survival and tumor recurrence. The results indicated that the prognosis for the groups of patients whose colorectal cancer cells binded to HPA in tissue sections was almost as bad as those with Dukes' Stage C disease. Because HPA binds to N-acetylgalactosamine, the authors' results indicate that this sugar residue is at least partly involved in the process of human colorectal carcinoma cells metastasizing to regional lymph nodes and possibly also to distant sites.

Peripheral connections of FMRFamide-like immunoreactive neurons in the snail, helix pomatia: an immunogold electron microscopic study.

Using a postembedding immunogold electron microscopic method, the ultrastructure and synaptic connections of FMRFamide-like immunoreactive varicosities were investigated in different peripheral organs of the snail Helix pomatia, including the heart (auricle), intestine, hepatopancreas, upper tentacle and salivary gland. The FMRFamide-like immunoreactive varicosities contained granules and vesicles as described in a previous study of the CNS of this species, and additionally, based on their granule content, two novel types of varicosities were found in the auricle. A selective accumulation of gold particles over the granules could be demonstrated. The FMRFamide-like immunoreactive varicosities formed unspecialized contacts with postsynaptic target cells in all peripheral organs investigated, with the exception of the tentacle retractor muscle. Both the neuro-muscular and the neuro-glandular contacts were characterized by either unspecialized close apposition of the 'pre- and postsynaptic' membranes or the immunoreactive elements faced the target cell(s) across a relatively wide extracellular space. In the tentacle retractor muscle some of the neuromuscular contacts showed appositions of electron dense material along the presynaptic membrane, clustering of agranular synaptic vesicles and intersynaptic cleft material. The present observations support previous electrophysiological findings and suggest a versatile modulatory role of FMRFamide and related substances in the Helix PNS.

Calibration of gel-permeation columns in the high-molecular-mass range: fixed human thrombocytes for the estimation of interstitial volume and the haemocyanin of the Vineyard snail Helix pomatia as a molecular mass calibration substance

Human thrombocytes, fixed with formaldehyde, are shown to be a universal indicator of the interstitial volume m gel-permeation chromatography (size-exclusion chromatography) for gels of all molecular mass separation ranges. The fixed thrombocytes are simple to prepare and to handle. Furthermore, native or intramolecularly cross-linked haemocyanin of the Roman snail Helix pomatia is shown to be well suited as a molecular mass calibration substance. We propose 8.8 · 10 6 as its molecular mass for calibration. This haemocyanin can therefore be added to the list of globular protein molecular mass calibration substances in the range of very high molecular masses.

Tachykinin-related neuropeptides in the central nervous system of the snail Helix pomatia: an immunocytochemical study

The distribution of neurons reacting with an antibody raised against an insect neuropeptide, locustatachykinin I, was investigated in the CNS of the snail Helix pomatia. The localization of the neurons was compared with that of the substance P-like immunoreactive (SPLI) neurons in the different ganglia. Altogether, there are ∼ 800–1000 locustatachykinin-like immunoreactive (LomTKLI) neurons in the Helix CNS, occurring with an overwhelming dominancy (83.5%) in the cerebral ganglia. Within the cerebral ganglia, the majority of LomTKLI neurons were localized in the procerebrum. The number of SPLI neurons was high; ∼ 2000 SPLI nerve cells were found in the Helix CNS. The majority (44.5%) of SPLI neurons was also found in the cerebral ganglia and they were also concentrated in the procerebrum. The neuropils of all ganglia were densely innervated by both LomTKLI and SPLI fibers except the medullary mass of the procerebrum where only SPLI elements form an extremely dense innervation. In addition to the neuropil processes, LomTKLI neurons sent axon processes to the peripheral nerves. SPLI fibers also formed a dense network of varicose fibers in the connective tissue sheath around the ganglia where they innervated the blood vessel walls too. Immunolabeling on alternating cryostat sections revealed that LomTKLI and SPLI neurons are localized near each other in most cases; co-localization of the two immunoreactive materials could be seen in a very small number of neurons of the pedal and pleural ganglia. The present results show that the Helix CNS possesses distinct neuronal populations using different tachykinin-related peptides. It is suggested that the differential distribution of these neuropeptides also implies a diversity in their central and peripheral functions.

A wide-acting peptidergic interneurone in the snail Helix pomatia : Graded recruitment and local firing of terminal processes

Simultaneous pre- and postsynaptic intracellular recordings were used to study the mechanism of presynaptic terminal recruitment in a multifunction interneurone in the snail Helix pomatia. The interneurone was presynaptic to at least 20 neurones. The synaptic efficiency was correlated with the presence of presynaptic depolarizing after-potentials (DAPs) electrotonically produced by the delayed firing of remote terminal processes. These processes have large swellings filled with neurosecretory vesicles. The terminals were recruited in a graded manner when the interneurone was fired with a prolonged current. The terminal recruitment was enhanced by stimulating various efferent nerves, which presumably activated presynaptic receptors for dopamine. A few animals (three out of 300) had two electrically coupled interneurones. Simultaneous recordings from both cells showed that the terminals could be fired independently of the soma-axon activity. The graded and local firing of the presynaptic terminals was attributed to the electrical load that the large boutons exert on electrotonically spreading presynaptic impulses.

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

The effect of acetylcholine and serotonin on calcium transients and calcium currents in identified Helix pomatia L. neurons.

The results presented demonstrate that in D neurons of the snail Helix pomatia L., acetylcholine (ACh) (10 divided by 100 microM) and serotonin (5-HT) (0.1 divided by 1000 microM) applications reduce both the basal intracellular concentration level ([Ca2+]in) and the amplitudes of calcium transients induced by membrane depolarization. It is likely that the mechanism of [Ca2+]in changes in the suppression of calcium inward currents (ICa). Influences of Ach and 5-HT on ICa were studied. Both effects were dose-dependent (ACh--0.01 divided by 100 microM and 5-HT--0.1 divided by 1000 microM). The half-maximal effects (IC50) were evoked by ACh concentration of 0.15 microM and 5-HT--15 microM. Furthermore we have also shown that in some cells 5-HT could evoke a transient increase in ICa (IC50 = 2 microM). The effects of Ach and 5-HT were nonadditive--the subsequent application of ACh after 5-HT, and vice versa, produced no inhibitory effects. This may indicate that both substances act through a common intermediate (possibly, G-protein).

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia.

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.

Electron cytochemical study of carbohydrate components in different types of cultured glial cells of snail Helix pomatia

Using a variety of colloidal gold-labelled lectins with different sugar specificities, the structure and topography of carbohydrate determinants of the surface membrane of in vitro cultured glial and nerve cells of the snail Helix pomatia have been electron cytochemically studied. Heterogeneity of carbohydrate pools among different types of glial cells and between glial and nerve cells was established. It was found that satellite glial cells having the ultrastructural signs of cells with high metabolic level (type II cells) selectively bind GNA which is specific to terminal α-d-mannose residues and do not bind other mannose-specific lectins. Con A and LCA. GNA determinants are absent in satellite type I glial cells, fibrous glial cells, microglia and neurons. It has been found that glial cells (satellite type I and II glial cells, filamentous glial cells and microglial cells) do not bind PVA and LABA. LTA did not bind to any glial cells and binds weakly to neurons. Con A and WGA determinants which are abundant on the neurons are completely absent on satellite type II glial cells but present on satellite type I glial cells and filamentous glial cells. Microglial cells contain Con A and LCA determinants and the density of PNA determinants on these cells is the highest compared to other types of glial cells or neurons. It is concluded that some lectin determinants (for RCA-1, PNA, LPA) are present on all types of glial cells, while another determinant (GNA) is specific for a definite type of glial cells and can serve as a marker of these cells. The role of specific carbohydrate determinants in the functioning of a neuron-glia complex is discussed.

Cytoplasmic Ca 2+ activity regulation as measured by a calcium-activated current

Calcium-activated non-selective cation (CAN) currents were activated by quantitative injections of Ca 2+ into voltage clamped bursting neurons of the snails Helix aspersa or Helix pomatia. Membrane potential was held at the potassium equillibrium potential and CAN currents were fit with a rising and falling exponential function. Ca 2+ transporters and pumps of the cell membrane, endoplasmic reticulum, and mitochondria were selectively blocked with pharmacological agents. Bath solutions containing 0 Na Ringers, chlorpromazine, Na 3VO 4, or thapsigargin did not significantly change the CAN current decay constants from those measured in Ringers. External 2,4-dinotrophenol or internal ruthenium red, however, significantly lengthened the CAN current decay constant. It is concluded that mitochondria are the most important sink for sub-membrane Ca 2+ activity in the range necessary to effectively activate CAN currents.

Satellite glial cell responses to neuronal firing in the nervous system of Helix pomatia

Patch clamp experiments were conducted on satellite glial cells attached to the cell body of neurons in place within the nervous system of the snail Helix pomatia. The glial cells were studied using cell-attached and whole-cell patch clamp configurations while the underlying neurons were under current or voltage clamp control. The resting potential of the glial cells (-69 mV) was more negative than that of the underlying neurons (-53 mV), due to their high K+ selectivity. Densely packed K+ channels were present, some of which were active at the cell resting potential. Neuronal firing elicited a cumulative depolarization of the glial cells. Large K+ currents flowing from V-clamped neurons depolarized the glial layer by up to 30 mV. The glial depolarization was directly correlated with the size of the neuronal K+ current. The glial cells recovered their resting potential within 2-5 sec. The neuronal depolarization induced a delayed (20-30 sec) and persistent (3-4 min) increase in the glial K+ channel opening probability. Likewise, pulses of K+ (20-50 mM)-rich saline activated the glial channels, unless the underlying neuron was held hyperpolarized. In low Ca(2+)-high Mg2+ saline, neuron depolarization and K(+)-rich saline did not activate the glial K+ channels. These data indicate that a calcium-dependent signal released from the neuronal cell body was involved in glial channel regulation. Neuron-induced channel opening may help eliminate the K+ ions flowing from active neurons.

Excitatory and inhibitory monosynaptic peptidergic transmissions in the Cns of the snail Helix pomatia.

The results of an investigation of the properties of excitatory and inhibitory monosynaptic peptidergic transmission between identified nerve cells of the snail Helix pomatia are presented in this paper. It was demonstrated that stimulation of the presynaptic interneuron activates stationary and potential-and time-dependent ionic channels in the postsynaptic cells.

Structural organization of the sensory systems of the snail.

The author's data on the structural organization of the nervous system of the body wall, the gravitational organs (the statocysts, the eyes, and the chemosensory organs), and the head tentacles of the snails Helix vulgaris and Helix pomatia are correlated in this paper. The localization in the CNS and some structural characteristics of the central divisions of the sensory systems are shown. Information is presented on the structure of the procerebrum, an associative center participating in the processing of chemosensory information. The data were obtained using the Golgi method, the retro- and anterograde transport of CoCl2, horseradish peroxidase, and lucifer yellow, as well as a number of other classical histological methods.

Newly identified nerve cells of the snail, Helix pomatia, associated with the generation of pacemaker activity.

Data on new, previously unidentified nerve cells of the snail Helix pomatia are presented in this paper. The identified neurons described may serve as a convenient model for the investigation of the cellular mechanisms of pacemaker activity, the role of neuropeptides in the generation and regulation of pacemaker activity, peptidergic transmission, and the functional role of the inward calcium current.