Nerve-granular cell communication in the atrium of the snail Achatina achatina occurs via the cardioexcitatory transmitters serotonin and FMRFamide.

In the present study, the anatomical association and functional interaction between nerve fibres and granular cells in the atrium of the snail Achatina achatina are investigated using a combination of scanning electron microscopy (SEM), pharmacological and immunofluorescence techniques. The SEM studies support a close anatomical association of axons with granular cells and new features of surface morphology are revealed. Pharmacological experiments showed that both serotonin and FMRFamide were able to induce degranulation of granular cells and the release of cysteine-rich atrial secretory protein. Serotonin- and FMRFamide-immunoreactive nerve fibres were observed at variable distances from granular cells, ranging from close contact to distances as far as the diameter of a muscle bundle. These results suggest that serotonin and FMRFamide play a role as paracrine excitatory transmitters in nerve-to-granular cell communication.

Effects of repeated hemolymph withdrawals on the hemocyte populations and hematopoiesis in Pomacea canaliculata.

Pomacea canaliculata is a freshwater gastropod considered an invasive pest by several European, North American and Asiatic countries. This snail presents a considerable resistance to pollutants and may successfully face stressful events. Thanks to the unusual possibility to perform several hemolymph collections without affecting its survival, P. canaliculata is a good model to study the hematopoietic process and the hemocyte turnover in molluscs. Here we have analyzed the effects of repeated hemolymph withdrawals on circulating hemocyte populations and pericardial organs, i.e., the heart, the main vessels entering and leaving the heart and the ampulla, of P. canaliculata. Our experiments revealed that the circulating hemocyte populations were maintained constant after 3 collections performed in 48 h. The tissue organization of the heart and the vessels remained unaltered, whereas the ampulla buffered the effects of hemolymph collections acting as hemocyte reservoir, and its original organization was progressively lost by the repeated hemolymph withdrawals. The hematopoietic tissue of P. canaliculata was evidenced here for the first time. It is positioned within the pericardial cavity, in correspondence of the principle veins. Mitoses within the hematopoietic tissue were not influenced by hemolymph collections, and circulating hemocytes never presented mitotic activity.

Involvement of Na+/K+ pump in fine modulation of bursting activity of the snail Br neuron by 10 mT static magnetic field.

The spontaneously active Br neuron from the brain-subesophageal ganglion complex of the garden snail Helix pomatia rhythmically generates regular bursts of action potentials with quiescent intervals accompanied by slow oscillations of membrane potential. We examined the involvement of the Na(+)/K(+) pump in modulating its bursting activity by applying a static magnetic field. Whole snail brains and Br neuron were exposed to the 10-mT static magnetic field for 15 min. Biochemical data showed that Na(+)/K(+)-ATPase activity increased almost twofold after exposure of snail brains to the static magnetic field. Similarly, (31)P NMR data revealed a trend of increasing ATP consumption and increase in intracellular pH mediated by the Na(+)/H(+) exchanger in snail brains exposed to the static magnetic field. Importantly, current clamp recordings from the Br neuron confirmed the increase in activity of the Na(+)/K(+) pump after exposure to the static magnetic field, as the magnitude of ouabain's effect measured on the membrane resting potential, action potential, and interspike interval duration was higher in neurons exposed to the magnetic field. Metabolic pathways through which the magnetic field influenced the Na(+)/K(+) pump could involve phosphorylation and dephosphorylation, as blocking these processes abolished the effect of the static magnetic field.

Cellular imaging using matrix-enhanced and metal-assisted SIMS.

Imaging mass spectrometry (IMS) allows the direct investigation of both the identity and the spatial distribution of the entire molecular content directly in tissue sections, single cells, and many other biological surfaces. We describe here the steps required to retrieve the molecular information from tissue sections using matrix-enhanced (ME) and metal-assisted (MetA) secondary ion mass spectrometry (SIMS). Surface metallization by plasma coating enhances desorption/ionization of membrane components such as lipids and sterols in imaging time-of-flight (ToF) SIMS of tissues and cells. High-resolution images of cholesterol and other membrane components can be obtained for single neuroblastoma cells and reveal subcellular details. Alternatively, in ME-SIMS, 2,5-dihydroxybenzoic acid electrosprayed on neuroblastoma cells allows intact molecular ion imaging of phosphatidylcholine (PC) and sphingomyelin (SM) at the cellular level.

Development beyond the gastrula stage and digestive organogenesis in the apple-snail Pomacea canaliculata (Architaenioglossa, Ampullariidae)

Development of Pomacea canaliculata from the gastrula stage until the first day after hatching is described. Trochophore embryos are developed after gastrulation, showing the prototroch as a crown of ciliated orange-brownish cells. However, no true veliger embryos are formed, since the prototroch does not fully develop into a velum. Afterward, the connection between the fore- and midgut is permeated and the midgut becomes full of the pink-reddish albumen, which is stored into a central archenteron's lake, from where it is accumulated into the large cells forming the midgut wall ("giant cells"). Electron microscopy of giant cells in late embryos showed that albumen is engulfed by large endocytic vesicles formed between the irregular microvilli at the top of these cells. By the end of intracapsular development, giant cells become gradually replaced by two new epithelial cell types which are similar to those found in the adult midgut gland: the pre-columnar and the pre-pyramidal cells. Pre-columnar cells have inconspicuous basal nuclei and are crowned by stereocilia, between which small endocytic vesicles are formed. Pre-pyramidal cells have large nuclei with 2-3 nucleoli and show a striking development of the rough endoplasmic reticulum. The genesis of the three cell lineages (giant, pre-columnar and pre-pyramidal cells) is hypothetically attributed to epithelial streaks that occur at both sides of the midgut since early stages of development.

Dopamine-induced programmed cell death is associated with cytochrome c release and caspase-3 activation in snail salivary gland cells.

BACKGROUND INFORMATION: PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). RESULTS: SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-beta-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. CONCLUSION: Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

The effects of ferric iron on voltage-gated potassium currents in molluscan neurons.

In isolated neurons of Helix pomatia, a two-microelectrode voltage clamp technique was used to study the effect of Fe3+ on voltage-gated potassium currents: a low-threshold fast-inactivating current (I(A)) and a high threshold slow-inactivating current with calcium-dependent (I(C)) and calcium-independent (I(DR)) components. Extracellular application of FeCl3 rapidly, reversibly and dose-dependently reduced the amplitude of I(A), I(C) and I(DR) with IC50 values of 49, 45 and 70 microM, respectively. Complete inhibition of K+ currents was reached at 100-500 microM Fe3+. The threshold for the total slow-inactivating potassium current shifted in a positive direction by 10-30 mV in the presence of Fe3+ (50-300 microM). Our work is the first demonstration of the strong blocking effect of Fe3+ on potassium currents of neuronal membrane.

Effects of rolipram on induction of action potential bursts in central snail neurons.

Effects of rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of the giant African snail (Achatina fulica Ferussac). Oscillations of membrane potential bursts were elicited by rolipram and forskolin. The bursts of potential elicited by rolipram were not inhibited after administration with (a) calcium-free solution, (b) high-magnesium solution (30 mM) or (c) U73122. However, the bursts of potential elicited by rolipram were inhibited by pretreatment with KT-5720 (10 microM). Voltage-clamp studies revealed that rolipram decreased the total inward current and steady-state outward currents of the RP4 neuron. The negative slope resistance (NSR) was not detectable in control or rolipram treated RP4 neurons. TEA elicited action potential bursts and an NSR at membrane potential between -50 mV and -30 mV. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters; (2) NSR of steady-state I-V curve; (3) phospholipase activity of the neuron. The rolipram-elicited bursts of potential were dependent on the phosphodiesterases inhibitory activity and the cAMP signaling pathway in the neuron.

The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials in the central neuron of snail, Achatina fulica Ferussac.

The modulation effects of d-amphetamine and procaine on the spontaneously generated action potentials were studied on the RP1 central neuron of giant African snails (Achatina fulica Ferussac). Extra-cellular application of d-amphetamine or procaine reversibly elicited bursts of potential (BoP). Prazosin, propranolol, atropine or d-tubocurarine did not alter the BoP elicited by either d-amphetamine or procaine. KT-5720 or H89 (protein kinase A inhibitors) blocked d-amphetamine-elicited BoP, whereas they did not block the procaine-elicited BoP. U73122, neomycin (phospholipase C inhibitors) blocked the procaine-elicited BoP, whereas they did not block the d-amphetamine-elicited BoP in the same neuron. These results suggest that BoP elicited by d-amphetamine or procaine were associated with protein kinase A and phospholipase C activity in the neuron.

Acid phosphatase complex from the freshwater snail Viviparus viviparus L. under standard conditions and intoxication by cadmium ions.

Acid phosphatases differing in both subcellular localization and substrate specificity were isolated for the first time from the liver of the freshwater snail Viviparus viviparus L. by preparative isoelectrofocusing. One of five characterized phosphatases is highly specific to ADP and the others can hydrolyze (at variable rate) a series of natural substrates. A scheme is proposed for the involvement of the studied phosphatases in carbohydrate metabolism. We have also studied some peculiarities of the effect of Cd2+ in vitro and in vivo on the activities of individual components of the acid phosphatase complex and corresponding changes in metabolism of the freshwater snail as a new test-object allowing the estimation of toxicity in water.

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation.

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.

[Morphofunctional parameters of nucleoli during development of the albumen gland polyploid cells in the snail Succinea lauta]. / Morfofunktsional'nye parametry iadryshek v razvitii poliploidnykh kletok belkovoi zhelezy ulitki iantarki Succinea lauta.

Ag-protein content, the total area and the number of nucleoli were estimated on AgNO3-methyl green stained squashed preparations of the albumen gland of Succinea lauta during polyploidization, differentiation and functioning of secretory cells. The investigation was made by means of morphometry and cytophotometry using the computer videoimage system. During the gland growth, the mean number of nucleoli increased from 1-2 in diploid (2c) nuclei to 3 in polyploid (16c-32c) nuclei, but commonly only one large nucleolus was formed in mature nuclei. As a whole, the area and Ag-protein content of nucleoli was changed proportionally to the gene dosage, but this dependence was not linear during several endocycles. Before 8c-level the coefficients of increase in each endocycle were more than 2 (2.7-4.0), and these decreased to 1.5-1.0 during the following endocycles (8c-16c-32c). The area of nucleoli increased more quickly than Ag-protein content in the course of endocycles of growing cells. On the contrary, a relative area of nucleoli decreased with the increase in ploidy levels in mature cells. This irregular dynamics reflects the influence of several factors of the nucleolar activity: endomitotic polyploidy (gene dosage effect), cell differentiation, inhibition of transcription, and its reactivation in mature cells. In addition, individual variations of nucleolar parameters in mature snails are associated with the rhythmical functioning of sexual system, and aging of animals.

[Somatic polyploidy in neurons of the gastropod molluscs. II. Dynamics of DNA synthesis in the process of postnatal growth of CNS neurons in Succineid snail]. / Somaticheskaia poliploidiia v neironakh briukhonogikh molliuskov. II. Dinamika sinteza DNK v protsesse postnatal'nogo rosta neironov TsNS ulitki Iantarki.

A study was made of the age dynamics of polyploidization and dynamics of DNA synthesis in neuron cell nuclei during the postnatal growth of the gastropod pulmonate snail Succinea lauta. According to cytophotometrical results, the degree of polyploidization in neuron nuclei increases from young to adult individuals, varying from 2c to 16,384c. In the visceral complex, the maximum and medium ploidy values of the neuron nuclei are higher by almost 4-8 times than those in cerebral and pedal ganglia. The medium level of ploidy in adult snails increases by 5.7 times in the visceral complex of ganglia and by 4.1-4.2 times in the pedal and cerebral ganglia. According to 3H-thymidine autoradiography, DNA synthesis in neuron nuclei occurs during the whole life of the snail. In young individuals the neurons have the highest activity of DNA synthesis--the index of labeled nuclei of neurons making in total 50.2%. In older age, a steady decrease in the index of labeled nuclei is observed--in total to 35.8% and 7.0% in small and large adult snails, respectively. The state of summer hibernation completely stops DNA syntheses in neurons, but emergency from hibernation is accompanied by restoration of DNA syntheses.

[Somatic polyploidy in neurons from gastropod mollusks. I. Morphological characteristics of ganglia and neurons in the CNS of the snail Succinea lauta]. / Somaticheskaia poliploidiia v neironakh briukhonogikh molliuskov. I. Morfologicheskaia kharakteristika gangliev i neironov TsNS ulitki iantarki.

A study was made of general anatomy and histological organization of the central nervous system in Succinea lauta, a gastropod pulmonate snail. Neurons grow by means of polyploidy during the postnatal succineid ontogeny. In the adult individuals diameters of perikaria of large neurons increase 2.5 to 5 times, in comparison with young individuals. As a whole, in adult snails sizes of ganglian cells vary from 3 to 380 mkm. Most of giant neurons are in parietal, abdominal and pallial ganglia. The chromatin structure has been described in neurons of young and adult snails. The endomitotic mechanism of polyploidization in the succineid ganglioneurons is proposed.

[Genome multiplication mechanisms in the development of albumen gland polyploid cells of Succinea lauta (Gastropoda:Pulmonata). VI. Ultrastructural research of endomitotic structure]. / Izuchenie mekhanizmov umnozheniia genoma v razvitii poliploidnykh kletok belkovol zhelezy ulitki iantarki. VI. Ultrastrukturiaia kharakteristika endomitoticheskogo tsikla.

This ultrastructural study of endomitosis in polyploid albumen gland cells of succineid snail. S. lauta was made to extend the previous research (Anisimov, 1997). The investigation was prosecuted on ultrathin sections by means of transmission electron microscopy. The insertion of 3H-thymidine and 3H-uridine into the nuclei and the light microscopy control of nuclear ploidy allowed to identify glands on early and intensive polyploidization stages and to ignore pseudoendomitotic terminally differentiated cells. Considering ultrastructural qualities and information about the cell population kinetics enabled us to discriminate between predifferentiation (2-4c), protodifferentiation (4-8c) and promoted differentiation (8-16c) cell stages. The endomitotic cycle was observed synchronously with the development of cytoplasmatic structures and secretion production in differentiating cells of ploidy levels 4-8-16c. According to morphological condition of the chromatin and chromosomes, G1-, S- and G2-interphase periods can be determined, such as successive stages of endomitosis (endopro-, meta-, ana-, and telophase). The following main features of endomitosis are accentuated: the normal (mitotic) chromosome cycle with total compaction in endometaphase and differential decompaction in interphase; the absence of the mitotic spindle; integrity of the nuclear envelope, nucleolus and contacts between them and chromosomes; part of chromosomes split nonsimultaneously and incompletely in the endoanaphase, or endomitosis with diplochromosomes rarely occurs. Chromosome nondisjunction is a temporary process that does not lead to a permanent chromosome endoreduplication (polyteny). It is emphasized that the normal chromosome cycle is combined with a complete block of cytoplasmic structures. Possible reasons of mitotic cycle reduction during the transition of differentiating cells to endomitotic polyploidy are discussed.

[Genome multiplication in the development of albumen gland polyploid cells of Succinea lauta (Gastropoda:Pulmonata).VII. Transcriptive activity of the nuclei in the endomitotic cycle]. / Izuchenie mekhanizmov umnozheniia genoma v razvitii poliploidnykh kletok belkovol zhelezy ulitki iantarki. VII. Transkriptsionnaia aktivnost iader v endomitoticheskom tsikle.

Using 3H-uridine autoradiography and electron microscopy a study was made of the activity of RNA synthesis in the interphasic nuclei, mitoses and endomitoses of different ploidy classes (4c-32c) in the albumen gland cells of Succinea lauta. The incorporation of 3H-uridine into mitotic and endomitotic condensed chromosomes was temporarily stopped or kept at the lowest level: 0.7% in normal and abnormal mitoses and about 3-4% in endomitoses, as calculated from the highest possible level in the resting nuclei of the respective ploidy classes. In the interphasic nuclei, chromatid and chromonemal structures are revealed at the ultrastructural level, in addition to thin euchromatinous fibres with perichromatia granules, which makes the brushy appearance of chromonemes and chromocentres. During endomitosis the chromosomes become compacted and free from granules, which well compares with autoradiographical data on transcription stopping. The results obtained corroborate once again that endomitosis is a real endoreproductive mechanism, and allow to make distinctions between real endomitosis and pseudoendomitotic nuclei.

[Genome multiplication mechanisms in the development of albumen gland polyploid cell of Succinea lauta (Gastropoda:Pulmonata). VIII. Pseudomitosis in the terminally differentiated cells]. / Izuchenie mekhanizmov umnozheniia genoma v razvitii poliploidnykh kletok belkovol zhelezy ulitki iantarki. VIII. Psevdoéndomitoz terminalno differentsirovannykh kletok.

Using a mature albumen gland of Succinea lauta, a study was made of the chromatin structure and RNA synthesis activity in polyploid nuclei (16c-32c-64c) of terminally differentiated cells, by means of electron microscopy and 3H-uridine radioautography of squash preparations. No 3H-thymidine incorporation in these cells was observed, which suggested the absence of DNA synthesis and endomitotic cycles at the examined functional stage of gland development. Some different stages of secretory cells could be distinguished: a resting period of the terminally differentiated cells, with the least 3H-uridine incorporation; a phase of intense heterosynthetic transcription, with the highest 3H-uridine incorporation; intervals in daily cycles; nuclear degeneration in ageing cells. At all these states the nuclei demonstrated some morphological features of endomitosis. This may be possible, because in the course of terminal cell differentiation the main chromatin mass is compacting like chromatids or chromocentres with thin euchromatinous filaments, which can be covered with perichromatinous granules. Since the true endomitosis is deliberately excluded at this stage, the above described morphological state of polyploid nuclei is regarded as postendomitotic and is designated as pseudoendomitosis. The view of the existence of "two fundamentally different types of endomitosis", one of which is "endomitosis in general", and the other "shows up as stationary" (Therman et al., 1983), is accepted as groundless.

Microglia in invertebrate ganglia.

The results of this study lend strong support to the concept of the existence in insects and molluscs of a distinctive class of neuroglial cells comparable to vertebrate microglia. The evidence presented is as valid as that used in reference to the separate status of vertebrate microglia--i.e., the demonstration of a close structural and functional relationship of these cells with cells of the immune system. As in vertebrates, the excision of ganglia from three invertebrate species (the molluscs Planorbarius corneus and Mytilus edulis and the insect Leucophaea maderae) and their maintenance in incubation media led to an exodus of small cells and their accumulation in the culture dish. During this process, they underwent conformational changes from stellate to rounded, and then to more or less ameboid, comparable to those indicative of the process of activation in the animals' immunocytes. Functional characteristics which these translocated microglia-like cells share with immunocytes are motility, phagocytotic activity, and adherence to the culture dish. Furthermore, the two cells have certain biochemical features in common--e.g., the presence of certain cytokines and (at least in Planorbarius) that of corticotropin. An additional phenomenon of particular interest for the classification of microglial elements is their response to morphine. At 10(-6) M, this drug decreases not only the number of cells emerging from the excised ganglia but also the degree of their transformation to the "active" ameboid form. This dose-dependent and naloxone-sensitive effect of morphine on microglial cells parallels that on activated immunocytes of the same species. Corresponding results demonstrating an inhibitory effect of morphine on mobilized microglial cells of the frog Rana pipiens indicate that this relationship between the two cell types under consideration also exists in vertebrates. Binding and displacement experiments with membrane homogenates of microglial cells as well as immunocytes of Mytilus have shown that the effects of morphine on both cell types are mediated by the same special opiate receptor (mu 3).