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Fasciola hepatica: characteristics of infection in Lymnaea truncatula in relation to the number of miracidia at exposure.

Experimental infections of Lymnaea truncatula by Fasciola hepatica were carried out in three snail populations to determine whether the number of miracidia used for each snail at exposure (1, 2, 5, 10, or 20 per snail) had any influence on the characteristics of Fasciola infection and metacercarial production. The number of miracidia had a significant influence on snail survival at day 30 postexposure and the frequency of infected L. truncatula that died without shedding (NCS snails). The frequency of NCS snails, the growth of cercaria-shedding snails throughout the experiment, the time between exposure and the first cercarial shedding, the duration of shedding, and the number of metacercariae were independent of the number of miracidia used for each snail. The highest metacercaria productivity for each miracidium was found in single-miracidium infections. Single-miracidium infections were the most effective, as the mean number of cercariae was the same as in other groups, whereas their survival rate was much higher.

Animals↗

The stress of Lymnaea truncatula just before miracidial exposure with Fasciola hepatica increased the prevalence of infection.

Single-miracidium infections of Lymnaea truncatula with Fasciola hepatica were carried out under laboratory conditions to determine whether the stress of snails just before miracidial exposure had any influence on the prevalence of Fasciola infection, redial burden, and cercarial shedding. Three methods, i.e., the fasting of L. truncatula for 3 days in water filtered through a Millipore membrane, the effect of 6-8 degrees C water for 15 min, or the immersion of L. truncatula in a detergent solution at low concentration for 15 min, were used to stress snails. Enhanced susceptibility of snails to F. hepatica infection was noted in stressed groups (93-96% vs 48-50% in controls). The number of free rediae did not show any variation in controls as well as in stressed groups, except for fasted snails in which free rediae were significantly fewer. No differences in cercarial production between controls and the cold group were noted. Fasting, cold shock, or detergent exposure prior to exposure to F. hepatica miracidia might have weakened the snails so that they were not as efficient in avoiding miracidial penetration, thus leading to higher infection rates.

Animals↗

Context extinction and associative learning in Lymnaea.

Aerial respiratory behavior in the pond snail Lymnaea was operantly conditioned so that snails learned not to perform aerial respiration in a hypoxic environment. Snails were trained in either the standard context (no food odorant) or a carrot (food-odorant) context. An operant training procedure of two 45-min training sessions with a 1-h interval between the sessions followed by a third 45-min training session 18 h later was sufficient to produce associative learning and long-term memory (LTM) that persisted for at least 5 days. If, however, following the third operant training session snails received three 45-min extinction training sessions, with each extinction session separated by at least a 1-h interval, LTM was not observed when tested the following day. That is, the memory was extinguished. Extinction, however, did not occur if the context of the extinction training was different from the context of the associative training. That is, in the snails trained in the standard context, extinction did not occur if the extinction training sessions were performed in the food-odorant context and vice versa.

Animals↗

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.

Animals↗

In vivo modulation of vincristine-induced neurotoxicity in Lymnaea stagnalis, by the ACTH(4-9) analogue Org 2766.

The use of the cytostatic agent vincristine (VCR) is limited by the occurrence of peripheral neuropathy. This side-effect is probably caused by interference with axonal microtubules. VCR depolymerizes microtubules and reacts with tubulin to form paracrystals. The potential of a neurotrophic ACTH(4-9) analogue, Org 2766, to counteract peripheral neuropathy caused by cytostatic agents is being investigated. In the present ultrastructural study, modulatory effects of Org 2766 on VCR-induced neurotoxicity were studied in vivo in neurons of the pond snail Lymnaea stagnalis, which has been shown previously to be a suitable test system to investigate neurotoxic side-effects of cytostatic agents. 24 h after treatment with VCR (25 microM), 68.4 +/- 34.7 paracrystals were counted per cross-section of the cerebral commissure and the number of microtubules in the axons had been lowered to 46% of the control level. After a survival period of two weeks all paracrystals had disappeared. By that time, no recovery of the axonal microtubular system could be observed. However, post-treatment with Org 2766 (10(-6) M) on day 6 after VCR treatment had induced a significant increase in the number of microtubules (+55%) on day 7. This beneficial effect lasted for the rest of the experimental period (14 days). These results suggest that post-treatment with Org 2766, i.e. after VCR clearance, can induce long-lasting beneficial effects on VCR-induced neurotoxicity in vivo.

Adrenocorticotropic Hormone↗

Immunocytochemical demonstration of a humoral defense factor in blood cells (Amoebocytes) of the pond snail, Lymnaea stagnalis.

Monolayers of blood cells (amoebocytes) and sections of connective tissue and of amoebocyte pellets of the freshwater pulmonate gastropod Lymnaea stagnalis were stained immunocytochemically with antisera to snail agglutinin/opsonin. The presence of this substance was demonstrated light microscopically both in the cytoplasm and on the cell membrane of amoebocytes. This suggests that amoebocytes synthesize agglutinin/opsonin, and bear it at their surface as receptors for foreign materials.

Agglutinins↗

Sensory input to growth stimulating neuroendocrine cells of Lymnaea stagnalis.

Several environmental factors influence the growth of the basommatophoran freshwater snail Lymnaea stagnalis. Growth is hormonally controlled by 4 cerebral clusters of ca 50-75 peptidergic, neuroendocrine Light Green Cells (LGC). The present light, transmission, and scanning electron microscopic study shows that the LGC are synaptically contacted by a tentacle sensory system (TSS). The TSS consists of 2 types of primary sensory neurone, viz. ca 150 S1-cells and ca 50-100 S2-cells. A S1-cell has a non-ciliated dendrite and an axon branch that synaptically contacts the soma of a S2-cell. A S2-cell has a branching, ciliated dendrite. Probably, S1- and S2-cells have different sensory modalities and can integrate sensory information by intersensory interaction. The S2-axons run through the tentacular nerves, the cerebral ganglia, and the intercerebral commissure. In each ganglion S2-axons branch and form synaptic contacts on the axons and somata of the LGC and on glial cells that surround the LGC. In an LGC-cluster, 1-3 LGC-somata are particularly strongly innervated. Probably, the TSS is involved in the environmental control of growth in L. stagnalis.

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Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization.

The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material. A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as "ectopic" CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral ganglion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Neuronal and non-neuronal control of the neurosecretory caudo-dorsal cells of the freshwater snail Lymnaea stagnalis (L.).

The cerebral ganglia of the freshwater snail Lymnaea stagnalis contain two clusters of neurosecretory Caudo-Dorsal Cells (CDC). These cells produce a neurohormone which stimulates ovulation. Ganglion transplantation and quantitative electron microscopy show that neuronal isolation of the cerebral ganglia complex (CCC) results in an activation of the CDC. It was, therefore, concluded that the CDC are controlled by an inhibitory neuronal input originating outside the cerebral ganglia. Ultrastructural studies on synaptic degeneration in the CCC suggest that this input reaches the CDC via a special type of synapse-like structure, the type C-SLS. Furthermore, transplantation of CCC into acceptor snails leads to a reduced release and an increased intracellular brekdown of neurohormone in the CDC of the nervous system of the acceptors. It is supposed that these phenomena are caused by the release of an (unknown) factor from the transplanted CCC. Special attention was given to the formation and degradation of a peculiar type of neurohormone granule, the large electron dense granule. The physiological significance of the neuronal and non-neuronal control mechanisms which regulate CDC activity is discussed.

Animals↗

An ultrastruct study of the innervation of the musculature of the pond snail Lymnaea stagnalis (L.) with reference to peripheral neurosecretion.

The ultrastructure of nerve endings in the musculature of Lymnaea stagnalis was studied. Seventeen different types of axon ending were distinguished according to the size and morphology of the granules or vesicles they contain. Nine types of axon endings form neuromuscular synapses. Some of these types also form axo-somatic synapses on peripheral neurones. One axon may innervate several muscle cells. Furthermore more than one type of axon may innervate one muscle cell. One type of axon ending forms axo-axonic synapses on the presynaptic part of neuromuscular junctions. In the connective tissue near muscle cells seven types of free axon endings were found. These seem to be peripheral neurosecretory endings. Some of these are probably derived from known types of neurosecretory cells in the central nervous system, whereas others appear to originate from peripheral neuronal perikarya. Body wall muscles appear to be innervated by neuromusculatur synapses, whereas in the visceral musculature both neuromuscular junctions and free axon endings are found.

Animals↗

Regulation of neurosecretory activity in the freshwater pulmonate Lymnaea stagnalis (L.) with particular reference to the role of the eyes: a quantitative electron microscopical study.

The process of neurosecretion in the Caudo-Dorsal Cells (CDC) of the freshwater snail Lymnaea stagnalis, which produce an ovulation hormone, shows a diurnal rhythmicity. Synthesis, transport and release of the neurosecretory material (NSM) is high during the evening and the early night and low during the rest of the day, while storage of NSM mainly occurs during the daytime. In the present study the role of the eyes in the regulation of the CDC-rhythm was investigated. During a 24-hr period, at time intervals of 6 hrs, cerebral ganglia, which contain CDC, of blinded and control snails (5 per group) were fixed and the CDC were studied with quantitative electron microscopical methods. The CDC of the controls showed a distinct diurnal thythmicity. Blinding, on the other hand, clearly affected this rhythmicity. The results indicate that after blinding the circadian CDC-rhythms of individual snails are no longer synchronous with each other ("interanimal desynchronization"). It is suggested that the rhythm of CDC neurosecretory activity is synchronized by the natural light/dark cycle via the eyes. The information from the eyes probably reaches the CDC via a nervous pathway. True snyapses and three types of synapse-like structures were found on the CDC. Their role in the regulation of CDC-activity is discussed. The effect of blinding is specific for the CDC; blinding does not influence the diurnal rhythmicity of another type of cerebral neurosecretory cells, the Light Green Cells (LGC). The CDC within a cluster act synchronously. This synchrony does not depend upon the presence of the eyes. Some structures which may be involved in establishing this synchrony, such as subsurface cisterns, desmosome-like structures and "specific release sites", are described.

Animals↗

An ultrastructural in vitro study on the regulation of neurosecretory activity in the freshwater snail Lymnaea stagnalis (L.) with particular reference to caudo-dorsal cells.

The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.

Animals↗

Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.)..

The neurosecretory Dark Green Cells (DGC) in the pleural and parietal ganglia of the freshwater snail Lymnaea stagnalis seem to be involved in osmoregulation. Previous experiments have indicated that changes of the osmolality of the environment induce activity changes of the DGC. Furthermore, it was shown that information on environmental osmolality reaches the DGC via the blood. In the present study right pleural and parietal ganglion complexes were cultured for 3 days in vitro under different osmotic conditions. Quantitative electron microscopy revealed that, compared with the control osmolality (130mOsm/kg H2O), osmolalities of 160 and 190 mOsm/kg H2O caused a reduced synthesis and an increased storage of neurohormone in the DGC. Apparently, the activity of the DGC depended on the osmotic pressure of the medium. It is proposed that in vivo the osmotic pressure of the blood (which is related to the osmolality of the environment) regulates DGC activity.

Animals↗

Calcium dynamics, exocytosis, and membrane turnover in the ovulation hormone-releasing caudo-dorsal cells of Lymnaea stagnalis.

The axon terminals of the neuroendocrine caudo-dorsal cells (CDC) of the freshwater snail Lymnaea stagnalis release an ovulation-stimulating hormone by exocytosis in a calcium-dependent way. Ultrastructural studies of the terminals, involving the K-pyroantimonate method for the demonstration of calcium and control tests with EGTA, show that calcium occurs in mitochondria and in various types of vesicular structure. Quantitative investigations indicate that mitochondria accumulate calcium during a short period of high neurohormone-release activity ("active state"; ca. 45 min) and release it again into the axoplasm some hours later, during a period of low secretory activity ("resting state"). Probably, in this way mitochondria play an important role in the buffering of the axoplasmic calcium concentration during high hormone-release activity. HRP-incorporation studies strongly suggest that the calcium-containing vesicular structures constitute a mechanism of membrane sequestration by which the CDC axon terminals resorb, transform, and release parts of the axolemma after exocytotic hormone release. The results furthermore indicate that this mechanism also may be involved in the control of the calcium concentration of the axoplasm, by taking up calcium from the axoplasm and releasing it into the extracellular space.

Animals↗

Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the sheel by way of an adhesive substance containing proteins and mucopolysaccharides.

Animals↗

A morphological, enzyme-cytochemical, and physiological study of the blood-gonad barrier in the hermaphroditic snail Lymnaea stagnalis.

In the hermaphroditic pulmonate snail Lymnaea stagnalis a blood-gonad (blood-testis) barrier appears to exist. Septate junctions between Sertoli cells and epithelial cells of the neck areas of the gonadal acini constitute this barrier; they separate the male from the female compartment. Experiments with tracer substances (colloidal gold particles, lanthanum nitrate, tannic acid) showed that the basal lamina around the acini hardly forms a barrier; only the larger colloidal gold particles do not pass this lamina. Physiological, the blood-gonad barrier is apparent in studies on the composition of gonadal fluid, which differs considerably from that of haemolymph. The osmolarity and the concentration of protein and amino acids in gonadal fluid exceed those of haemolymph. As to the major ions, in the gonadal fluid Na+ is partly replaced by K+, and HCO-3 is almost totally replaced by Cl-. Such a distribution of HCO-3 and Cl- is indicative of metabolic acidosis. The cytochemical localization of carbonic anhydrase activity in cells lining the acinar lumen (Sertoli cells, epithelial cells) suggests that these cells are involved in the process of ion exchange. The metabolic acidosis in the gonad might result from the anaerobic production of lactate and succinate by Sertoli cells; these cells lack the enzymes cytochrome oxidase, lactate dehydrogenase, and succinate dehydrogenase. Spermatogenic cells, on the other hand, do possess these enzymes. This probably indicates that these cells metabolize lactate and succinate secreted by Sertoli cells.

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An ultrastructural study of the musculature of the pond snail Lymnaea stagnalis (l.).

The ultrastructure of the musculature of Lymnaea stagnalis was studied. Each of the six muscle systems of the body wall, previously distinguished in an anatomical study, has its own type of smooth muscle, characterised by the size and number of the myofilaments, number of mitochondria and distribution of smooth endoplasmic reticulum. The visceral musculature comprises both smooth and striated muscle. Cross-striated muscle is found in the heart and proximal aorta, obliquely striated muscle in the buccal mass, gizzard and vas deferens. Myofibroblasts and myoendothelial cells were also distinguished. On the basis of the observations it is concluded that striated muscles in L. stagnalis contract and relax more rapidly and have a higher endurance than smooth muscles, but that the latter can contract over a wider range. Among smooth muscles the head retractor muscle contracts most rapidly, the shell muscle is most powerful and the diagonal muscle is slow to contract but has a relatively high endurance. The latter muscle, together with the horizontal foot muscle, plays a major role in maintenance of the hydrostatic skeleton. A model for the organisation of the smooth muscles is deduced from the ultrastructural observations. It implies that the myosin-paramyosin filaments change their actin filament partners during contraction. This agrees with a model deduced for other smooth muscles on the basis of physiological experiments and X-ray diffraction analysis.

Animals↗

Localization of dopamine and its relation to the growth hormone producing cells in the central nervous system of the snail Lymnaea stagnalis.

The distribution of dopamine in the central nervous system of the pond snail Lymnaea stagnalis was investigated by using immunocytochemistry and HPLC measurements. With both methods it was demonstrated that dopamine is predominantly present in the cerebral and pedal ganglia. The dopamine-immunoreactivity was mainly observed in nerve-fibers in the neuropile of the ganglia. Relatively few dopamine-immunopositive cell bodies (diameters 10-30 microns) were found. A large cell in the right pedal ganglion (the so-called RPeD1) stained positively with the dopamine antibody. It has previously been demonstrated that the growth hormone producing cells (GHCs) possess dopamine receptors on their cell bodies. However, dopamine-immunopositive fibers were observed only in the vicinity of the GHC nerve-endings and not close to the GHC cell bodies.

Animals↗