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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 synapses 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 days 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.

5,6-Dihydroxytryptamine↗

[Comparative analysis of the statistical parameters of synaptic transmission of central neurons in the snail Helix lucorum and in the rabbit].

Comparative analysis of the parameters of synaptic transmission in the central synapses of the snail (parietal ganglia) and in the brain of rabbit (hippocamp and sensorymotor cortex) has been made. Both differences and common features were revealed in synaptic organization of these two objects. Functional re-arrangements may alleviate differences between central synapses of the snail and rabbit. It is concluded that evolution of the central nervous system involved mainly the decrease of the effectiveness of separate synapses at the cost of the decrease of the number of quanta of the released transmitter and the increase of the number of various synaptic connections on each neuron.

Action Potentials↗

The moroccan food snail, Helix aspersa, as a source of Salmonella.

A total of 270 samples, nine lots of 30 samples each, of imported Moroccan food snails was examined for the presence of Salmonella. Eighty-four samples (an overall incidence of 31.11%) and all nine lots contained Salmonella. No significant difference (P greater than 0.25) in the number of positive samples was observed by using either selenite cystine both or tetrathionate broth when the samples had been pre-enriched in lactose broth. When used as direct selective enrichments with samples not pre-enriched in lactose broth, tetrathionate broth was significantly (P less than 0.05) more productive than selenite cystine broth. The overall detection of Salmonella-positive samples by direct enrichment was significantly greater (P less than 0.001) than by pre-enrichment. A variety of uncommon serotypes occurrence and incidence, and the concomitant human health potential, of Salmonella in one species of live, imported food snails.

Culture Media↗

Characterization of myomodulin-related peptides from the pulmonate snail Helix aspersa.

Three myomodulin-related peptides--pQLSMLRLamide, PMSMLRLamide, and SLGMLRLamide--have been purified and sequenced from extracts of whole snails. The level of immunoreactive myomodulin was shown by HPLC and RIA to be widely distributed among 26 different snail tissues, with the highest levels (higher even than those in the central ganglia) occurring in certain male reproductive organs. Synthetic pQLSMLRLamide modified either the spontaneous rhythmic activity or the resting tone of several isolated muscular organs: the aorta, ventricle, upper gut, epiphallus, flagellum, and spermatheca; but the retractor muscles of the pharynx, penis, and tentacle were unaffected.

Animals↗

CO2 chemoreception in the pulmonate snail, Helix aspersa.

We have studied the response of the pneumostome to CO2, O2 and combined CO2 and O2 in intact snails. We found that pneumostomal opening increases in response to both hypercapnia and mild hypoxia. We determined which neural structures were essential for the pneumostomal response to CO2 by eliminating parts of the nervous system: the subesophageal ganglia and an intact anal nerve were necessary and sufficient elements for the CO2 response. Within the subesophageal ganglia, we identified a discrete region on the medial margin of the visceral ganglion that was capable of increasing pneumostomal area when focally stimulated with 6% CO2. Ion substitution experiments indicated that pneumostomal responses to hypercapnia were not mediated by the pneumostomal motor neurons themselves, but rather by interneurons connected polysynaptically to the motor neurons controlling pneumostomal function. In conclusion, intact H. aspersa have a ventilatory response to CO2, and this response is mediated by CO2 sensitive cells located in a small area of the central nervous system.

Animals↗

[Changes in the chemosensitive properties of the central neurons of the snail Helix pomatia and of the mouse as affected by opioid peptides].

In experiments on neurones of the isolated ganglia of the snail and surviving hippocampal slices from mice, studies have been made of the effect of Met- and Leu-enkephalins on neuronal sensitivity to acetylcholine and norepinephrine. It was shown that in some cases the enkephalins do not affect bioelectrical activity of the neurones, but changed their responses to acetylcholine and norepinephrine. It is concluded that during investigation of the effect of enkephalins on the nervous cells, alongside with changes in the bioelectrical activity, special attention should be paid to changes in chemoreceptive properties of the neurones.

Acetylcholine↗

Correlation of axon projections and peptide immunoreactivity in mesocerebral neurons of the snail Helix aspersa.

The purpose of this investigation was to elucidate the organization of efferent neurons in the mesocerebrum of a terrestrial snail. The mesocerebrum is one of three regions, or lobes, that can be identified by gross inspection. Previous studies have indicated a possible function for the mesocerebrum in the control of mating behavior. We used both anterograde and retrograde tracing methods to determine the axon projections of mesocerebral neurons. Virtually all the neurons (96%) send an axon into the cerebropedal connective nerve, and about 25% of these fibers continue into the nervus cutaneous pedalis primus dexter, which innervates the dart sac. Many neurons have additional axon branches in other nerves, especially the penial nerve, which receives projections from about 25% of mesocerebral cells. Neurons that are backfilled from the nervus cutaneus pedalis primus dexter are predominantly immunoreactive for FMRF amide, whereas neurons that are backfilled from the penial nerve are predominately immunoreactive for APGWamide. These results suggest a functional association between FMRFamide and dart shooting on the one hand, and between APGWamide and penial eversion on the other. Some cells contain both APGWamide and FMRFamide; these cells may have dual projections in both the penial nerve and the nervus cutaneous pedalis primus dexter.

Animals↗

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.

Animals↗

Dynamic and static calcium gradients inside large snail (Helix aspersa) neurones detected with calcium-sensitive microelectrodes.

We have used quartz Ca2+-sensitive microelectrodes (CASMs) in large voltage-clamped snail neurones to investigate the inward spread of Ca2+ after a brief depolarisation. Both steady state and [Ca2+]i transients changed with depth of penetration. When the CASM tip was within 20 microm of the far side of the cell the [Ca2+]i transient time to peak was 4.4+/-0.5s, rising to 14.7+/-0.7s at a distance of 80 microm. We estimate that the Ca2+ transients travelled centripetally at an average speed of 6 microm2 s(-1) and decreased in size by half over a distance of about 45 microm. Cyclopiazonic acid had little effect on the size and time to peak of Ca2+ transients but slowed their recovery significantly. This suggests that the endoplasmic reticulum curtails rather than reinforces the transients. Injecting the calcium buffer BAPTA made the Ca2+ transients more uniform in size and increased their times to peak and rates of recovery near the membrane. We have developed a computational model for the transients, which includes diffusion, uptake and Ca2+ extrusion. Good fits were obtained with a rather large apparent diffusion coefficient of about 90+/-20 microm2 s(-1). This may assist fast recovery by extrusion.

Animals↗

Nitric oxide, but not serotonin, is involved in acquisition of food-attraction conditioning in the snail Helix pomatia.

The effects of inhibition of nitric oxide (NO) or serotonin (injection of nitro-L-arginine methyl ester (L-NAME) or 5,6-dihydroxytryptamine (5,6-DHT), respectively) on food-attraction conditioning was investigated in Helix. Blocking NO synthase (NOS) prior to conditioning significantly impaired the food-finding ability of the snails. Food-conditioned snails, after inhibition of NOS, remained able to locate the conditioned food. These results indicate that the acquisition of memory depends on NO, whereas memory recall and olfactory orientation are not dependent. Ablating the serotonergic system did not influence food-attraction conditioning, suggesting that food-attraction conditioning may be at variance with conventional associative conditioning procedures.

5,6-Dihydroxytryptamine↗

Studies on the extra- and intracellular acid-base status and its role on metabolic depression in the land snail Helix lucorum (L.) during estivation.

The aim of the present study was to examine the acid-base status of extra- and intracellular fluids and its possible role on the regulation of the metabolic rate of Helix lucorum during prolonged estivation. For this purpose, the rate of oxygen consumption for active and estivating snails was determined. The acid-base status was also examined in the hemolymph and tissues from active and estivating snails acclimated at 25 degrees C. In addition, the buffer values of hemolymph and tissues were determined in order to examine whether there is a change in the snails during estivation. The rate of oxygen consumption decreased significantly within the 1st 10 days of estivation from 122.51+/-10 microl.g(-1).h(-1) to 25.86+/-5.2 microl.g(-1).h(-1), indicating a marked decrease in metabolic rate. P(CO2)increased within the 1st 20 days of estivation from 13.52+/-0.68 mmHg to 25.09+/-2.05 mmHg, while the pH of hemolymph (pH(e)) decreased from 7.72+/-0.04 to 7.44+/-0.06. The level of bicarbonates decreased in the hemolymph of estivating snails, indicating a metabolic acidosis, which was moderate in extracellular fluids. In contrast to pH(e), the intracellular pH (pH(i)) was maintained in the tissues of estivating H. lucorum, indicating a regulation of pH(i) despite the developed hypercapnia. According to the results presented here, it seems that the timing of pH(e) changes does not correlate with the timing of metabolic rate reduction in estivating H. lucorum.

Acid-Base Equilibrium↗

Influence of photoperiod on the behavioral response to olfactory stimulation in the snail Helix pomatia L.

An ethanol vapor concentration of 1.6 mmol/l was used to test the diurnal variations of the olfactory response in two groups of snails, which were adapted to different light-dark cycles. The results revealed that the olfactory sensitivity to stimulation with ethanol was significantly increased during the day-time, which corresponds to the scotophase of the light-dark cycle, to which the animals had been adapted (chi2-test, P < 0.01).

Animals↗

Lead reduces shell mass in juvenile garden snails (Helix aspersa).

In an earlier paper examining inherited tolerance to Pb, the shell growth of laboratory-bred offspring of Helix aspersa from contaminated sites was compared with that ofjuveniles from naive populations on dosed and undosed diets. Eight-week-old snails were fed either 500 microg g(-1) Pb or a control food in competitive trials between two populations. In the first series of trials, a parental history of exposure to Pb did not confer any advantage to either of two populations (BI and MI) competing with a naïve population (LE). whether Pb was present in the diet or not. However, in the analysis of their metal concentrations reported here, LE are found to retain higher levels of Pb in the soft tissues than either BI or MI. Compared to their siblings on the unleaded diet, dosed LE and BI juveniles had lower soft tissue concentrations of Ca and Mg. Although the growth in shell height is unaffected by diet, LE and BI juveniles build lighter shells on the Pb-dosed diet, achieving around 75% of the shell mass of their controls. In contrast, the shell weights of dosed MI juveniles are depressed by only 15% and show no change in the essential metal concentrations of their soft tissues. A second experiment using five populations fed only the dosed food show that the shell weight/soft tissue weight ratios are comparable to the dosed snails of the previous experiment. Building a lighter shell thus appears to be the common response of all Helix populations to a high Pb diet, at least amongst juveniles. The reduction in its mass means that less Ca and Mg is added to the shell and, along with the lowered soft tissue concentrations observed in some populations. may be a consequence of an increased effort to excrete Pb. The possibility that the MI population shows a genotypic adaptation. perhaps as some form of modification of its Ca metabolism, is briefly discussed.

Adaptation, Physiological↗

[Effect of hydrolases and digitonin on the electrophysiological characteristics of the neurons of the snail, Helix pomatia].

Studies have been made on the effect of trypsin, chymotrypsin, pronase, lipases, hyaluronidase and digitonin on electrophysiological properties of the neurons of the snail H. pomatia under external application. Proteases and lipases gradually depolarize the neuronal membrane, decrease the amplitude and prevent the onset of action potentials, initially increase and then decrease the membrane resistance. The decrease in the membrane resistance coincides with the period of maximum inhibition of resting and action potentials in the neurons. The enzymes studied do not affect the membrane capacitance. Changes in electrophysiological characteristics induced by the enzymes are partially reversible provided the preparation is soaked in Ringer's solution for a sufficient time. Digitonin rapidly and irreversibly depolarizes the membrane, decreases its resistance and blocks action potentials. Hyaluronidase does not significantly affect neuronal electrophysiological properties when applied solely, but facilitates the development of changes during subsequent effect of proteases.

Animals↗

Effects of 5,7-dihydroxytryptamine on an identified 5-hydroxytryptamine-containing neurone in the central nervous sytem of the snail Helix pomatia.

1. The effect of 5,7-dihydroxytryptamine (5,7-DHT) on an identified 5-hydroxytryptamine (5-HT)-containing neurone in the CNS of the snail was studied by histochemical, biochemical and electrophysiological methods. 2. Low concentrations of 5,7-DHT decreased the endogenous 5-HT content of the neurone without affecting the amino acids, while relatively large amounts of the drug proportionately lowered 5-HT and in addition slightly decreased the tryptophan and methionine content of the cell. 3. 5,7-DHT blocked the uptake of [3H]-5-HT into the neurone; the close analogue 5,6-DHT was more potent. 4. As well as slightly influencing the accumulation of [3H]-tryptophan by the neurone 5,7-DHT inhibited the metabolism of this amino acid to form 5-HT, probably by affecting the enzyme tryptophan-hydroxylase. 5. 5,7-DHT produced a postsynaptic blockade of transmission from the neurone by blocking the 5-HT receptors of the follower neurones. This effect appeared to be specific for 5-HT receptors. 6. The data support the idea that 5,7-DHT is neurotoxic for indoleamine-containing neurones.

Action Potentials↗

"Central arousal" and sexual responsiveness in the snail, Helix aspersa.

In molluscs, a "central arousal" system is thought to positively modulate both an animal's level of activity and its behavioral responsiveness. This hypothesis is examined in Helix aspersa by testing the relationships between activity, feeding, and sexual behavior. Activity, feeding, and mating exhibit parallel daily rhythms. Snails are most active, and eat and mate most frequently, during scotophase and the first 3 h of photophase. Handling and injections of serotonin (5 x 10(-7) m/kg body wt) increase general activity. Inducing activity during late photophase increases food consumption, but it does not induce sexual activity. Moreover, serotonin and handling have no effect on sexual arousal; treated snails show no increase in genital eversion stage and require the same length of courtship as controls. If snails are sexually deprived, increasing activity in late photophase does increase copulation frequency, but snails do not copulate more frequently than do unhandled snails tested during early photophase. These results suggest that while manipulations thought to increase central arousal do increase activity and responsiveness to food stimuli, they do not directly affect sexual proclivity or sexual arousal. Moreover, the relationship between feeding and sexual behavior differs between Helix and Aplysia. This difference between the two species may be related to the differences in their reproductive strategies.

Animals↗

The effects of HCl and CaCl(2) injections on intracellular calcium and pH in voltage-clamped snail (Helix aspersa) neurons.

To investigate the mechanisms by which low intracellular pH influences calcium signaling, I have injected HCl, and in some experiments CaCl(2), into snail neurons while recording intracellular pH (pH(i)) and calcium concentration ([Ca(2+)](i)) with ion-sensitive microelectrodes. Unlike fluorescent indicators, these do not increase buffering. Slow injections of HCl (changing pH(i) by 0.1-0.2 pH units min(-1)) first decreased [Ca(2+)](i) while pH(i) was still close to normal, but then increased [Ca(2+)](i) when pH(i) fell below 6.8-7. As pH(i) recovered after such an injection, [Ca(2+)](i) started to fall but then increased transiently before returning to its preinjection level. Both the acid-induced decrease and the recovery-induced increase in [Ca(2+)](i) were abolished by cyclopiazonic acid, which empties calcium stores. Caffeine with or without ryanodine lowered [Ca(2+)](i) and converted the acid-induced fall in [Ca(2+)](i) to an increase. Injection of ortho-vanadate increased steady-state [Ca(2+)](i) and its response to acidification, which was again blocked by CPA. The normal initial response to 10 mM caffeine, a transient increase in [Ca(2+)](i), did not occur with pH(i) below 7.1. When HCl was injected during a series of short CaCl(2) injections, the [Ca(2+)](i) transients (recorded as changes in the potential (V(Ca)) of the Ca(2+)-sensitive microelectrode), were reduced by only 20% for a 1 pH unit acidification, as was the rate of recovery after each injection. Calcium transients induced by brief depolarizations, however, were reduced by 60% by a similar acidification. These results suggest that low pH(i) has little effect on the plasma membrane calcium pump (PMCA) but important effects on the calcium stores, including blocking their response to caffeine. Acidosis inhibits spontaneous calcium release via the RYR, and leads to increased store content which is unloaded when pH(i) returns to normal. Spontaneous release is enhanced by the rise in [Ca(2+)](i) caused by inhibiting the PMCA.

Animals↗

Quantitative changes in the digestive gland cells of the snail Helix aspersa after exposure to the fungicide copper oxychloride.

This study investigated the relationship between accumulated copper in the digestive gland of Helix aspersa, after exposure to the fungicide copper oxychloride, and quantitative changes in the digestive gland cells of this snail. Snails were exposed to 80 and 240 microg g(-1) copper oxychloride for 6 weeks. Histological sections of digestive glands were analyzed by means of computer-assisted image analysis. Results indicate that copper is strongly accumulated in the digestive gland and that this organ is the main site of copper accumulation in the snail body. Also, as a result of copper accumulation in the digestive gland, measurable changes in epithelium cell height and area of this organ occur. In the case of cell height, these changes are dose-related. It is concluded that changes in digestive gland cells, as a result of copper oxychloride exposure, may possibly serve as biomarkers of exposure to this fungicide.

Animals↗