Microelectrode investigations of learning phenomena in snail (Helix pomatia) neurones.

We have examined changes of postsynaptic potentials and of pattern activity of the identified silent and oscillatory snail neurones in Helix pomatia during conditioning. Local changes of EPSP or IPSP have been recorded during association following the first stimulus in the silent cells, whereas spike discharges could be observed in response to the 2nd stimulus. In the oscillatory neurones changes of pattern activity have been recorded following the 2nd stimulus, while the first stimulus proved to be ineffective. The formation of temporary connections of snail neurones seemed to be a specific phenomenon, because it was necessary to pair stimuli of different inputs for the development of these modifications. These plastic changes seemed to depend on the interstimulus as well as on the intertrial intervals. Our experimental data underline the probable role of the stimulus parameters and of the electrical properties of neurones during the formation of learned neuronal responses.     

Food-attraction conditioning in the snail,Helix pomatia

Adult pulmonate snails (Helix pomatia) were released equidistant between two types of food, carrot and potato, respectively. Naive snails moved in different directions and did not locate either food above chance, although both foods were readily eaten upon direct contact. After a single carrot feeding episode, 75% of the carrot-fed snails moved directly towards the carrot and ate it. Conversely, potato-fed snails located the potato in 67% of the cases. Snails that were fed apple or lettuce behaved like naive animals, with the majority of animals (75% in both cases) locating neither the carrot nor the potato.The ability of snails to locate this particular food after a single feeding episode was maintained for at least 11 days, provided that the snails were not exposed to other foods in the interim. If the animals were fed a different food (but still tested for food-finding ability to the initially conditioned food) their orientation preference decreased gradually over a period of 5 days.Although the snails' orientation is based upon olfactory cues, exposure to food odor alone is not sufficient to enable food-finding; additional feeding related stimuli are necessary.These findings indicate that Helix do not possess a predisposition for the foods tested, and further suggest that processes underlying food-finding and food selection are strongly influenced by learning experiences. The conditioning phenomenon underlying food-finding behavior has been called Food-Attraction Conditioning, and appears to be a crucial link between the ecologies of learning and foraging behaviors. The accessibility of the snail's nervous system should permit neuronal analysis of the mechanisms underlying such a unique and complex learning phenomenon.     

The influx of tryptamine into snail (Helix pomatia) ganglia: comparison with 5-hydroxytryptamine.

Isolated ganglia possess the ability to concentrate tryptamine from an external medium by a process which is temperature sensitive and independent of sodium and other cations. Kinetic analysis of the accumulation process showed the influx of tryptamine to be a single mechanism with Km and Vmax values of 1.4 X 10(-4)M and 5 X 10(-8) mole/g/min. The influx of tryptamine is an unspecific process and is insensitive to a number of metabolic inhibitors and various analogues. The process of tryptamine influx is thus similar in principle to the low affinity uptake mechanism for 5-HT (see Osborne et al., 1975). The present data, which include some experiments on the release of 5-HT and tryptamine, are discussed from the point of view of a functional role for 5-HT and tryptamine in the snail CNS.     

Growth classes in the adult roman snail (Helix pomatia L.)

Summary a method of dividing adult Helix pomatia into growth classes, which are probably also age classes, is described. Differences in behaviour of individuals previously thought to be associated with their status in a population are probably differences between individuals of different age.     

Intracellular pH regulation in isolated hepatopancreas cells from the Roman snail (Helix pomatia)

The mechanisms of intracellular pH (pHi) regulation were studied in isolated hepatopancreas cells from the Roman snail, Helix pomatia. The relationship between intracellular and extracellular pH indicated that pHi is actively regulated in these cells. At least three pHi-regulatory ion transporters were found to be present in these cells and to be responsible for the maintenance of pHi: an amiloride-sensitive Na+/H+ exchanger, a 4-acetamido-4'-isothiocyanostilbene-2,2'disulfonic acid (SITS)-sensitive, presumably Na(+)-dependent, Cl-/HCO3-exchanger, and a bafilomycin-sensitive H(+)-pump. Inhibition of one of these transporters alone did not affect steady state pHi, whereas incubation with amiloride and SITS in combination resulted in a significant intracellular acidification. Following the induction of intracellular acidosis by addition of the weak acid Na+propionate, the Na+/H+ exchanger was immediately activated leading to a rapid recovery of pHi towards the baseline level. Both the SITS-sensitive mechanism and the H(+)-pump responded more slowly, but were of similar importance for pHi recovery. Measurement of pHi recovery from acidification in the three discernible types of hepatopancreas cells with a video fluorescence image system revealed slightly differing response patterns, the physiological significance of which remains to be determined.     

Activation of snail (Helix pomatia) nervous tissue tyrosine monooxygenase by calcium in vitro.

Addition of calcium chloride to soluble preparations of tyrosine monooxygenase from snail brain appears to produce an activation of the enzyme when assayed with subsaturating concentrations of the pteridine cofactor 6 MPH4 (2-amino-4-hydroxy-6-methyltetrahydropteridine). While some increase in the activity occurs with calcium chloride at a concentration of 0.01 mM, activation is increased by about 100% at 1mM and reaches a maximum at 5mM (144%) where it remains more or less constant up to 10mM. Barium chloride also produces an activating effect although it is much less pronounced while magnesium chloride is without effect. EGTA has no direct effect on the enzyme but antagonises the activation produced by calcium chloride. The activation of tyrosine monooxygenase by calcium is reflected in changes in the kinetic properties of the enzyme, decreasing the Km from 43 muM to 19 muM for tyrosine and from 670muM to 230muM for the pteridine cofactor. No change was observed with V values for either tyrosine or pteridine cofactor. It is suggested that calcium, which enters the nerve terminal during nerve stimulation, regulates the transmitter dopamine by activating the rate-limiting enzyme tyrosine monooxygenase.     

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.     

Acute toxicity of cadmium and copper in hepatopancreas cells from the Roman snail (Helix pomatia)

The toxic effects of cadmium (Cd) and copper (Cu) on cellular metabolism and cell morphology were investigated in isolated hepatopancreas cells from the Roman snail (Helix pomatia). Cell viability was unaffected during 1 h of incubation with 100 microM Cd, but was significantly reduced from 93% in controls to 87% and 85% with 100 microM Cu and 500 microM Cd, respectively. The adverse effect of 500 microM Cd on cell viability was not observed in cells isolated from Cd pretreated snails. Oxygen consumption remained constant in the presence of 100 microM Cu but was inhibited by 38% after 1 h of exposure to 500 microM Cd. Hepatopancreas cells showed enhanced formation of reactive oxygen species when exposed to 100 microM Cu, but not in the presence of Cd. Morphologically, an increase in cell volume of Cd-exposed cells was noted, while cell membrane bleb formation was induced by both metals. The latter may have been induced by metal effects on the actin filamentous network of the cells which showed distinct actin-staining within the blebs at the cell surface. Overall, our data indicate that both Cd and Cu are acutely toxic for hepatopancreas cells form the Roman snail with Cu being more toxic than Cd.     

Structural studies on the galactan from the snail Helix pomatia

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. The structures of the low molecular weight oligosaccharides were elucidated being identical to those obtained from Lymnaea stagnalis galactan. However, the quantities released differed significantly between the two species. The high molecular fractions comprising about 66% of the material were not obtained in a similar degradation of the Lymnaea stagnalis galactan. 4. Thus the observed structural differences can explain easily the species-specific reactivity among the two polysaccharides seen earlier with lectins, enzymes and antibodies.     

The reaction of nitrogen monoxide with the haemocyanins of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The rate of the reaction of Astacus leptodactylus methaemocyanin with NO follows the Henderson-Hasselbalch equation with a pKa of 5.85, suggesting that one imidazole ligand of Cu was exchanged for NO. The reaction is blocked by F- as a bridging ligand. The same imidazole residue might be responsible for the decomposition of nitrosylhaemocyanin, [Cu1NO+CuII], with an unlocated binding site for NO, into methaemocyanin and NO, as the rate increase with pH. NO could react preferentially with CuA of Helix pomatia methaemocyanin, CuA'IICuBII, as it possibly has only two histidine ligands instead of the three of CuA in Astacus haemocyanin. This difference might explain the higher formation rate and the much greater stability of Helix nitrosylhaemocyanin. The fast reaction is governed by a pKa of 6.80, probably of a bridging mu-aquo ligand. With F- or a mu-hydroxo bridging ligand a low reaction rate was still observed, in contrast with Astacus methaemocyanin. Helix nitrosylhaemocyanin was transformed by N3- into methaemocyanin with the liberation of N2 and N2O. This methaemocyanin could almost quantitatively be regenerated with H2O2. Helix nitrosylhaemocyanin was only partially regenerated by a direct treatment with H2O2 and almost quantitatively by HONH2 in a similar fairly fast reaction, followed by a much slower one.     

The possible roles of the monoaminergic system in the feeding of the snail Helix pomatia

The possible role of serotonin and dopamine in the feeding of Helix pomatia was studied applying immunocytochemical, biochemical, and behavioral techniques as well as bioassay experiments. Immunocytochemistry showed that dopamine-containing (thyrosin-hydroxylase-immunoreactive) neuronal elements of the crop and the gizzard belong to the intrinsic part, whereas serotonin-containing (serotonin-immunoreactive) neuronal elements belong to the extrinsic part of the gastrointestinal nervous system. Bioassay studies on the spontaneous contractions of the crop and the gizzard showed that dopamine affected only the longitudinal muscle contractions by increasing both the tonus and contractility, whereas serotonin was effective on both the longitudinal and circular muscle contractions. Serotonin increased the tonus and contractility of longitudinal muscles in the crop but decreased them in the gizzard. Serotonin decreased the tonus and contractility of the circular muscles in the crop but increased them in the gizzard. Serotonin effects on the circular muscle of the gizzard were concentration dependent between a range of 10(-5) M-3 x 10(-5) M. HPLC measurements of monoamines in starved and satiated animals showed that the concentration of both dopamine and serotonin significantly decreased in both the CNS and different parts of the gastrointestinal tract of satiated animals, suggesting a significant monoamine liberation during feeding. The injection of monoamines (10(-3) and 10(-2) M) into the body cavity of starved animals showed that only dopamine was able to induce feeding whereas serotonin increased the general activity of the animals suggesting that the initiation of feeding is rather dopamine than serotonin dependent.