Immunodetection and localization of nitric oxide synthase in the olfactory center of the terrestrial snail, Helix pomatia

The procerebrum of stylommatophoran snails produces nitric oxide (NO)-modulated oscillatory local field potentials which are considered the basis of olfactory information processing. Although the function of NO is well characterized in the PC, the identification and distribution of NO synthase (NOS) has not known completely. In the present study, applying a mammalian anti-NOS antibody, a 170 kDa molecular weight NOS-like protein was demonstrated in the procerebrum homogenate of Helix pomatia. NOS-like immunolabeling of the globuli cells, the internal and terminal neuropils displayed an identical distribution compared to that of NADPH-diaphorase reactive material, confirming the specificity of immunohistochemistry. The detailed characteristics of the immunostaining (different intensity of the neural perikarya, a gradual appearance in the terminal neuropil and in the axon bundles of the tentacular nerve, as well as an intense, homogeneous distribution of NOS-like immunoreactivity in the internal neuropil) suggest that NOS is expressed constitutively, maintaining a high level of the enzyme in neuropil areas. NOS accumulation in the internal neuropil suggests that NO plays an important role in delivering olfactory signals extrinsic to the procerebrum, and integrating them with other sensory modalities, respectively. Our results are the first, demonstrating unequivocally the presence of NOS and resolving its differential distribution in the Helix procerebrum.     

Electrophoretic detection of protein fractions in extracts of the ocular tentacles of Helix aspersa Müller, 1774 (Gasteropoda, Pulmonata, Helicidae)

The extracts from ocular tentacles of Helix aspersa hare a heterochronic inhibitive action on the development of albumen gland. In view of elucidating the origin of this heterochrony, we make an electrophoretic study of the different extracts from juveniles, adults in activity, in natural sleep and awakning. This study has allowed us to spot 19 proteic fractions common to the four extracts of the tentacles under study, but these fractions show only differences in concentration. Particularly, the proteinic fractions 7 and 8 are more concentrated in the extract of juveniles snails tentacles than in the extract of adults snails. This difference in proteic concentration could explain the heterochronic inhibitive action of ocular tentacles extracts on the development of albumen gland of this snail.     

The 5-HT immunoreactive innervation of the Helix procerebrum

In the procerebrum of terrestrial snails, 5-HT is a key modulatory substance of the generation of synchronous oscillatory activity and odor learning capability. In this study, we have analyzed the characteristics of the 5-HT-immunoreactive (5-HT-IR) innervation of the distinct anatomical regions of the procerebrum of Helix pomatia, applying correlative light- and electron microscopic immunocytochemistry. A dense network of 5-HT-IR innervation was demonstrated in the cell body layer, meanwhile a varicose fiber system of different density occurred in the different neuropil regions. At the ultrastructural level, labeled varicosities were found to contact both procerebral cell bodies, and different unlabeled axon profiles in the neuropils. The labeled structures established mostly close non-specialized membrane contacts with the postsynaptic profiles. The overall dense distribution of 5-HT-IR innervation supports a general modulatory role of 5-HT in processing different olfactory events.     

Twitching and quivering of the tentacles during snail olfactory orientation

In Helix aspersa the posterior tentacles house a sensitive olfactory organ. We studied two types of tentacular movements, twitch and quiver. A twitch is a brief retraction (mean duration, 4.1 s); a quiver is a rapid lateral movement (350 ms) unaccompanied by retraction. We videotaped the tentacles while snails explored an open field. When an attractive odor source, linalool, was present at one side of the arena, the snails consistently moved towards it. By contrast, if only the carrier substance was present the snails moved in random directions. Twitching was 50 times more frequent during linalool trials than during control trials, while quivering was 1.4 times more frequent. Twitching increased steadily and dramatically as snails approached the linalool source and, in the temporal dimension, the maximum rate of twitching occurred when the snails arrived at the odor source. Quivers occurred at a fairly constant rate. Twitching is interpreted as a mechanism to remove odor molecules trapped in the liquid covering of the olfactory epithelium, thus resulting in better temporal resolution for olfactory perception. Quivering may be a mechanism to increase access of odor molecules to receptors by decreasing the boundary layer at the surface of the tentacle. Accepted: 24 May 1997     

Recording of spontaneous oscillations in the procerebrum of terrestrial mollusk Helix in free behavior

Procerebrum is the central part of the olfactory system in terrestrial snails. Spontaneous rhythmic oscillations were described in this structure. The role of these oscillations in the mechanisms of odor perception and discrimination is unknown yet. Electrical activity of the Helix procerebrum was recorded in vivo. Changes in spontaneous rhythmic oscillations in response to olfactory stimulation were observed. Within the first 10 s after odor application (cineole) in low concentration, a statistically significant decrease in the frequency and increase in the amplitude of procerebrum oscillations were revealed in freely behaving animals. Timing of those changes corresponded to the time of defensive reaction realization of the tentacle withdrawal. The increase in the amplitude and a tendency to a decrease in the frequency of oscillations in response to odor application in high concentration were observed in time period 11-20 s, which corresponded to an increased duration of tentacle withdrawal. The results suggest an implicit relation of the amplitude and frequency of oscillations in odor perception and discrimination.     

Neurogenesis in the procerebrum of the snail Helix aspersa: a quantitative analysis

The procerebrum, a specialized structure for olfaction in terrestrial pulmonate molluscs, contains 20,000 to 50,000 small, uniformly sized neurons that increase in number with age. Here I show the likely source of neurons added to the procerebrum of Helix aspersa and that the rate of neuron addition depends on snail weight. After hatching, during the initial exponential growth phase, H. aspersa adds neurons to the procerebral apex by mitosis and from a cerebral tube. In the logistic growth phase beginning 30-40 days post-hatch, neurons also seem to be added to the procerebrum from the peritentacular and olfactory nerves, causing the rate of neuron addition to approximately double; but as in the earlier exponential growth phase, this rate remains a function of snail weight. This neuron addition throughout the life of the snail can be predicted by snail weight. In the two growth phases, the number of neurons in the procerebrum is given by logarithmic functions of snail weight. The results here for H. aspersa provide the basis for experiments to determine the peripheral origin and destination of neuronal precursors that are added to the procerebrum and to determine how neuron addition affects the function of the procerebrum.     

Optical recording of the odor-evoked responses in olfactory structures of the brain of the terrestrial mollusk Helix

Regular oscillations were recorded in olfactory part of the brain (procerebrum) of gastropod mollusk Helix both electrographically and optically. In general, oscillations resembled those in slugs reported earlier. Odor application caused a transient change in the procerebral oscillations followed by appearance of a special pattern. For the first time the evoked potential was recorded in procerebrum and mapped in reference to the area of oscillations. The area of spreading of evoked potential roughly corresponded to the neuropil projection, while the oscillations were recorded in the projection of the cell body layer of procerebrum. The wave of the evoked potential emerged near the place of the olfactory nerve entrance into the procerebrum and propagated via the procerebrum neuropil towards the cell body layer. The evoked potential did not produce a phase-independent wave in rhythmical oscillations.     

Neurophysiological and behavioral responses to olfactory stimuli in the snail Helix pomatia L

Some aspects of olfactory sensitivity in the pulmonate Helix pomatia L. were studied by means of neurophysiological and behavioral methods. Single fiber recordings were carried out in the olfactory nerve of the posterior tentacles. Olfactory stimulations with different odors were performed by means of a continuous air stream. The order of neuronal sensitivity to different odors was as follows: ethanol > or = ethyl acetate > pentanol > hexanol > octanol > or = diethyl malonate > vanillin. Furthermore, the results revealed a relative specificity for some substances. A comparison between neurophysiological and behavioral data shows that those substances, which cause the highest increases in impulse frequency, also evoke a behavioral avoidance reaction.     

Selective involvement of opioids in mechanisms of synapse-specific plasticity in Helix lucorum snail during sensitization acquisition

Effects of met-enkephalin (opioid peptide) and naloxone (opioid antagonist) on nociceptive sensitization were studied in L-RP11 Helix neurons. In control snails sensitizing stimulation produced reversible membrane depolarization and depression of neural responses evoked by sensory stimuli during the short-term stage of sensitization and facilitation of these responses at the long-term stage. Met-enkephalin (10 but not 0.1 microM) suppressed the neural responses evoked by nociceptive stimuli. Sensitizing stimulation during metenkephalin application prevented the facilitation of neural responses evoked by tactile stimulation of snail head, whereas facilitation of neural responses evoked by chemical stimulation of head or tactile stimulation of foot were similar to that in control sensitized snails. Sensitizing stimulation during met-enkephalin and/or naloxone application prevented the facilitation of neural responses evoked by chemical stimulation of snail head, whereas responses evoked by tactile stimulation of snail head or foot were facilitated (as in neurons of control sensitized snails). Opioids are suggested to be involved in regulation of nociceptive mechanisms and selective induction of long-term plasticity in L-RP11 neural inputs activated by tactile of chemical stimulation of snail head.     

Postembryonic neuronogenesis in the procerebrum of the terrestrial snail,Helix lucorum L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70–80%) of the snail procerebrum are produced during the first 1–2 months of posthatching development. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 271–276, 1998     

Consequences of food-attraction conditioning in Helix: a behavioral and electrophysiological study

Food-attraction conditioning is a learning phenomenon by which adult Helix pomatia acquire the ability to locate food through exposure to that particular food. Food-conditioned snails can be distinguished from naive snails during their approach to food. Naive snails keep their tentacles upright — whereas food-conditioned animals bend the tentacles down-ward, in a horizontal orientation, pointed in the direction of the food.Tentacle musculature is innervated by two peritentacular nerves (PTn), each projecting to approximately one hemi-section of the tentacle wall. Stimulating the peritentacular nerves caused the tentacles to bend downward in a manner reflecting the full complement of tentacle movements performed by conditioned snails.The neural correlate of tentacle movements was investigated in isolated ganglion preparations with the posterior tentacles attached. PT nerve activity was recorded while the olfactory epithelia were stimulated with natural food odors. Preparations obtained from conditioned animals responded with a substantial increase in unit activity (mean increase 280%) to stimulation with odor of the conditioned food but not to other odors. Preparations from naive animals did not respond to food odor stimulation. The electrophysiological results demonstrated that plasticity due to conditioning the snails in vivo survived dissection and could be monitored in vitro.Abbreviations ext PTn external peritentacular nerve - int PTn internal peritentacular nerve     

Neural control of olfaction and tentacle movements by serotonin and dopamine in terrestrial snail

We investigated the role of serotonin (5HT) and dopamine (DA) in the regulation of olfactory system function and odor-evoked tentacle movements in the snail Helix. Preparations of the posterior tentacle (including sensory pad, tentacular ganglion and olfactory nerve) or central ganglia with attached posterior tentacles were exposed to cineole odorant and the evoked responses were affected by prior application of 5HT or DA or their precursors 5-hydroxytryptophan (5HTP) and l-DOPA, respectively. 5HT applications decreased cineole-evoked responses recorded in the olfactory nerve and hyperpolarized the identified tentacle retractor muscle motoneuron MtC3, while DA applications led to the opposite changes. 5HTP and l-DOPA modified MtC3 activity comparable to 5HT and DA action. DA was also found to decrease the amplitude of spontaneous local field potential oscillations in the procerebrum, a central olfactory structure. In vivo studies demonstrated that injection of 5HTP in freely moving snails reduced the tentacle withdrawal response to aversive ethyl acetate odorant, whereas the injection of l-DOPA increased responses to “neutral” cineole and aversive ethyl acetate odorants. Our data suggest that 5HT and DA affect the peripheral (sensory epithelium and tentacular ganglion), the central (procerebrum), and the single motor neuron (withdrawal motoneuron MtC3) level of the snail’s nervous system.     

Cerebral parts of chemosensory systems of the snail: Structural bases of intersensory integration

Presented are data on distribution of afferent fibers from tentacular and labial nerves innervating chemosensory tentacular organs, lips, and mouth area in the cerebral ganglia of terrestrial pulmonary snails are in the article. By using Golgi impregnation and infusion of cobalt chloride and nickel, it has been shown that most terminal branches of afferent fibers of all chemosensory organs are located in several symmetric areas of both metacerebrums. A part of fibers both of tentacular and of labial nerves form peculiar tracts and terminate in the interior neuropil of procerebrums. In metacerebrums, various neurons providing connections with afferent fibers of chemosensory organs are revealed. Many of them also innervate interior neuropil and procerebrum cell bodies area. The data obtained allow considering procerebrums as higher integrative centers of chemosensory information.     

Further characterization of passively transferred resistance to Schistosoma mansoni in the snail intermediate host Biomphalaria glabrata.

A heat-labile plasma factor from genetically resistant 10-R2 Biomphalaria glabrata snails confers passively transferred resistance (PTR) to Schistosoma mansoni when injected into susceptible snails within 24-hr of exposure to miracidia. However, no additional details on PTR have emerged since the initial 1984 report, nor has the plasma resistance factor been characterized. In the present study, new information is provided on the occurrence of resistance factor in plasma of additional types of snails, effect of "priming" resistant plasma donors by prior exposure to miracidia, duration of PTR, molecular weight of resistance factor, and fate of sporocysts in snails with PTR. Susceptible NIH albino snails injected 24 hr prior to exposure to miracidia with individual samples of plasma from a different strain (Salvador B. glabrata) or a different species (B. obstructa) of nonsusceptible snail displayed infection prevalences of 49% or 59% of control levels, respectively, whereas injections of homologous plasma had no effect. PTR was not enhanced by prior exposure of resistant Salvador plasma donors to miracidia. Unexpectedly, PTR induced by injections of Salvador plasma persisted for at least 21 days. The molecular weight of the resistance factor(s) was between 10 and 30 kDa, based on results of centrifugal ultrafiltration. A significantly higher proportion of dead sporocysts occurred in histological sections of tentacles from snails injected with Salvador plasma than in tentacles of snails injected with NIH albino plasma at 7 days postexposure to miracidia. Most dead sporocysts in Salvador plasma-injected snails were undergoing gradual degeneration, rather than rapid, hemocyte-mediated destruction, as occurred in Salvador snails.     

Effect of zif268 early gene antisense oligonucleotide on mechanisms of synapse-specific plasticity

It was found that nociceptive sensitization was followed by long-term facilitation of synaptic responses evoked by chemical sensory stimulation of the snail "head", tactile stimulation of the snail "head" and foot in LP11 command neuron of defence behavior in snail Helix lucorum. Sensitizing stimulation during the intracellular injection of antisense olygonucleotide immediate early gene zif268 resulted in a selective suppression of synaptic facilitation in LP11 neuron responses evoked by tactile and chemical stimulation of the snail "head". At the same time, development of synaptic facilitation of responses in the LP11 neuron evoked by tactile stimulation of the foot was the same as in control sensitized snails. The results suggest that immediate early gene zif268 is selectively involved in the mechanisms of specific regulation of plasticity of the synaptic "input" of LP11 neuron from sensory receptors of the snail "head".     

BIOCHEMICAL COMPOSITION OF HELIX SNAILS: INFLUENCE OF GENETIC AND PHYSIOLOGICAL FACTORS

This paper describes the biochemical composition of differentspecies (Helix lucorum, Helix pomatia) and sub-species of snails(Helix aspersa aspersa, Helix aspersa maxima) reared in thesame conditions with a feed (‘Helixal’) speciallydesigned for edible snails. In addition, the composition ofwild H. pomatia and H. lucorum is presented to allow comparisonbetween snails of different origins. Analyses determined thepercentages of proteins, lipids and minerals. They reveal bothsimilarities and differences in composition according to thespecies and the part analysed (whole body, pedal mass, and visceralmass). H. pomatia contains the highest percentage of mineralmatter and the lowest percentage of lipids. Surprisingly, proteincontents are slightly different between artificially rearedH. aspersa maxima of 3 months old and wild H. pomatia. The resultsmake it possible to evaluate nutritional quality of snails withthe composition of the body of four edible snail species. (Received 16 September 1996; accepted 24 May 1997)     

EVOLUTION, DURING DIGESTION, OF THE BACTERIAL FLORA IN THE ALIMENTARY SYSTEM OF HELIX ASPERSA (GASTROPODA: PULMONATA): A SCANNING ELECTRON MICROSCOPE STUDY

The use of scanning electron microscopy (SEM) allowed a studyof the distribution of bacteria in the various digestive organsof the snail Helix aspersa Müller. The bacteria are enclosedby mucous secretions (mucous film or mucous grains) and sometimesattached on the cilia of some of the digestive walls. Accordingto the food that was given to the snails, different morphologicaltypes appeared, two of which dominated. Adult snails were fasted for 4 days, given a dehydrated artificialfood and then sacrificed at different times during digestion.The presence of bacteria may be related to the time of digestion.In fact, bacteria seem to accompany the food mass; they developmostly in the stomach and in the intestine where they may helpdigest the food. Fasting or hibernating snails do not possess bacteria in thealimentary lumen or on the digestive walls. However, the residualfaeces localized in the distal, intestinal lumen, lodge greatquantities of bacteria. From these results, the endogenous or/and exogenous existenceof the bacterial flora in alimentary system of Helix aspersais discussed. (Received 26 June 1989; accepted 16 October 1989)     

Dynamics of elaboration of a conditioned reflex and differentiations in the snail

It is shown that dynamics of percentage of conditioned food refusals by snails Helix pomatia and Helix lucorum is satisfactorily approximated by regression equation of exponential type with included coefficient reflecting the animals state before the beginning of learning. The ability is established of the snails to practically completely differentiate various alimentary conditioned stimuli. The introduction of differentiation always improved the reflex consolidation. Conditioned reactions to differentiation stimulus appeared at the elaboration stage and were absent at the stage of conditioned reflex consolidation.     

Structural organization of procerebrums of terrestrial molluscs: Characteristics of neuronal pattern, plasticity, and age peculiarities

In terrestrial snails and slugs, the presence of five types of neurons has been shown: typical granular, horizontal, stellate, apical, and basal cells. A peculiarity of procerebrum neurons is a loop-like segment of the basal process. Granular cells have been established to spread both as cell columns and as single cells. The number of the columns and the number of cells in each column depend on the age and individual peculiarities of the animal. In the course of ontogenesis, there also occur changes of the number of processes-outgrowths of granular cell bodies as well as of the character of thickenings and the type of endings on their main processes. Basal neurons and single granular cells of the columns form several independent tracts running from the procerebrum cell body areas to metacerebrums. The single granular cells, horizontal cells, and a part of basal neurons are considered as associative elements providing formation in procerebrums of the single system that is a basis for the synchronized activity of the whole neuronal complex. A similarity of organization of procerebrums and of associative-integrative centers of higher animals is discussed.