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.     

Spectroscopic characterization of metallothionein from the terrestrial snail, Helix pomatia.

The Cd-sequestering metallothionein (MT) isoform isolated from the midgut gland of Roman snails exposed to Cd supplements in the feed was characterized by compositional and spectroscopic analysis. The preparations contained nearly 5 mol of Cd, small amounts of Cu and about 1 mol of Zn per chain mass of 6620 Da, in numerical agreement with the apoprotein's measured capacity of firmly binding a maximum of 6 equivalents of Cd per molecule. As with other Cd-containing MTs the occurrence of a prominent Cd-mercaptide-specific shoulder at 250 nm in its absorption spectrum showed that Cd is complexed in tetrahedral symmetry by the cysteine residues of the protein, and the multiphasic ellipticity profile in the CD spectrum revealed that these complexes are joined to form one or more oligonuclear Cd-mercapto clusters. Both spectral features vanished with the removal of the metal but were reconstituted to maximum amplitudes by readdition of Cd to the metal-free apoprotein, provided precautions were taken to prevent air oxidation of the latter. Quantitative analysis of snail MT reconstituted with Cd established that the 18 cysteine side chains bind the metal in a 3-to-1 ratio; spectroscopic studies on fractionally restored forms demonstrated that the six Cd ions were bound to the apoprotein molecule in succession in two sets of three Cd ions each. Thus, one can infer from the observed stoichiometry and the coordinating preferences of Cd that this gastropod MT, like the Cd-bearing MTs of marine crustaceans, harboured the metal in two separate cyclically constructed Cd3Cys9 clusters. The snail clusters differed, however, from other MTs in their response to acidification. Their protolytic dissociation proceeded through two separate protonation steps with the manifestation of spectroscopically distinguishable intermediate forms. Thus, this snail isoform displays in its metal composition and its chemical and spectroscopic features both similarities and differences to other animal kingdom MTs. Its properties suggest that it serves an important role in the protection of the terrestrial gastropod from Cd.     

Structural investigations of the galactan of the snail Lymnaea stagnalis

A galactan, isolated from the spawn of the snail Lymnaea stagnalis, contained d-galactose and 0.9% of nitrogen, but neither l-galactose nor phosphate groups. The [α]_D²⁰ values of the galactan and its first Smith-degradation product were +19.5° and +20°, respectively. During each of two consecutive Smith-degradations of the galactan, 1 mol of periodate was consumed and 0.45 mol of formic acid was liberated per mol of “anhydrogalactose” unit. Methylation analyses of the galactan and its first Smith-degradation product yielded equal proportions of 2,3,4,6-tetra-O-methyl- and 2,4-di-O-methyl-galactose. Only small quantities of 2,4,6- (4.9 mol%) and 2,3,4-tri-O-methylgalactose (0.7 mol%) were formed from the galactan, whereas the first Smith-degraded product gave 15.6 and 20.4 mol%, respectively. The product of the second Smith-degradation disintegrated and the following oligosaccharides were identified: β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→6)-d-Gal-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-[β-d-Gal-(1→6)]-β-d-Gal-(1→1)-l-Gro, β-d-Gal-(1→3)-β-d-Gal-(1→6)-β-d-Gal-(1→1)-l-Gro, and β-d-Gal-(1→3)-β-d-Gal-(1→3)-β-d-Gal-(1→1)-l-Gro. Thus, the galactan is highly branched with the backbone containing sequences of either exclusively (1→6)-linked or of more or less regularly alternating (1→3)- and (1→6)-linked units. The side chains vary in length and in the degree of branching. In immunoprecipitin studies, a high degree of species-specificity was seen when various snail galactans were tested with the antiserum to the Lymnaea stagnalis galactan.     

蜗牛纤维素酶提取工艺的初步研究

利用白玉蜗牛的内脏废弃物,以蜗牛纤维素酶为主要提取酶类,采用正交实验的方法初步探讨了蜗牛酶的提取工艺条件。研究结果表明:蜗牛纤维素酶的提取时间约为10h、pH为5.0、提取温度为0℃,盐析饱和度为70%,得到纤维素粗品的最高酶活力为1.402U·ml^-1。     

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.     

Neuronal background of positioning of the posterior tentacles in the snail Helix pomatia

The location of cerebral neurons innervating the three recently described flexor muscles involved in the orientation of the posterior tentacles was investigated by applying parallel retrograde Co- and Ni-lysine tracing via the olfactory and the peritentacular nerves. Their innervation patterns in the flexor muscles were studied by applying anterograde neurobiotin tracings via these nerves. The labeled neurons are clustered in eight groups in the cerebral ganglion. They send both common and distinct innervation pathways to the flexor and the tegumental muscles and to the tentacular retractor muscle. The common pathway reaches the muscles via the olfactory nerve, whereas the distinct pathways innervate via the internal and external peritentacular nerves. The three anchoring points of the three flexor muscles at the base of the tentacle outline the directions of three force vectors generated by the contraction of the muscles and enable the protracted tentacle to bend around a basal pivot. In the light of earlier physiological and the present anatomical findings, we suggest that the common innervation pathway to the muscles is required for tentacle withdrawal and the retractor mechanism, whereas the distinct pathways primarily serve the bending of the protracted posterior tentacles during foraging.     

Mechanisms of membrane potential oscillation in bursting neurons on the snail,Helix pomatia

• 1.1. The mechanism of generation of membrane potential (MP) oscillations was studied in identified bursting neurons from the snail Helix pomatia.
• 2.2. Long-lasting stimulation of an identified peptidergic interneuron produced a persistent bursting activity in a non-active burster.
• 3.3. External application of calcium channel blockers (1 mM Cd²⁺ or 5 mM La²⁺) resulted in a transient increase in the slow-wave amplitude and subsequent prevention of pacemaker activity generation in bursting neurons. Application of these blockers together with endogenous neuropeptide initiating bursting activity generation, increased MP wave amplitude without prevention of bursting activity generation.
• 4.4. Replacement of all NaCl in normal Ringer's solution with isoosmotic CaCl₂, glucose or Tris-HCl produced a reversible block of bursting activity generation. Stationary current-voltage relation (CVR) of bursting neuron membrane has a region of negative resistance (NRR) and does not intersect the potential axis in threshold region for action potential (AP) generation in normal Ringer's solution. In Na-free solution stationary CVR is linear and intersects the potential axis near — 52 mV.
• 5.5. Novel potential- and time-dependent outward (E_rev = − 58 mV) current, I_B, activated by hyperpolarization was found in the bursting neuron membrane. Having achieved a maximal value, this current decayed with a time constant of about 1 sec. Hyperpolarization inactivated maximal conductance, g_B, responsible for I_B, and depolarization abolished inactivation of g_B.
• 6.6. Short-lasting (0.01 sec) hyperpolarization of the bursting neuron membrane by inward current pulse induced the development of prolonged hyperpolarization wave lasting up to 10 sec.
• 7.7. These results suggest that: (a) persistent bursting activity of RPal neuron in the snail Helix pomatia is not endogenous but is due to a constant activation of peptidergic synaptic inputs of these neurons; (b) Ca²⁺ ions do not play a pivotal role in the ionic mechanism of MP oscillations but play a determining role in the process of secretion of a peptide initiating bursting activity by the interneuron presynaptic terminal; (c) depolarizing phase of the MP wave is due to specific properties of stationary CVR and hyperpolarization phase is due to regenerative properties of hyperpolarization-activated outward current I_B. The minimal mathematical version of MP oscillations based on the experimental data is presented.

     

Transmembrane outward hydrogen current in intracellularly perfused neurones of the snail Helix pomatia

The ionic nature and pharmacological properties of the outward current activated by membrane depolarization were studied on isolated neurones of the snail Helix pomatia, placed in Na+- and Ca2+-free extracellular solutions and intracellularly perfused with K+-free solution ("nonspecific outward current"). It was shown that the amplitude and reversal potential of this current (estimated from instantaneous current-voltage characteristics) are determined mainly by the transmembrane gradient for H+ ions. Lowering of pHi induced an increase in the current amplitude and a shift of the reversal potential to more negative values; the shift magnitude was comparable with that predicted for the hydrogen electrode. Raising pHi, as well as lowering pHo, induced a decrease in the current amplitude and a displacement of the current activation curve to more positive potentials. Addition of EGTA (8 mmol/l) to the intracellular perfusate did not affect the current amplitude. Extracellular 4-aminopyridine (10 mmol/l), verapamil (0.25 mmol/l) or Cd2+ (0.5 mmol/l) blocked the current. It is concluded that the current studied is carried mainly by H+ ions. In the same neurones the nature of the fast decay of the calcium inward current was also studied (in the presence of extracellular Ca2+ ions). This decay considerably slowed when pHi was raised or pHo was lowered, and it became less pronounced upon extracellular application of 4-aminopyridine or upon intracellular introduction of phenobarbital (4 mmol/l) and tolbutamide (3 mmol/l). It is suggested that the fast decay of the calcium inward current is due to activation of a Ca-sensitive component of the hydrogen current which depends on accumulation of Ca2+ ions. The possible physiological role of the transmembrane hydrogen currents is discussed.