Changes in Membrane and Threshold Potentials of Command Neurons in Terrestrial Snail during Development of a Conditioned Situational Defensive Reflex.

Changes of the electrical characteristics of command neurons of defensive behavior caused by the development of a conditioned situational defensive reflex were studied experimentally under in vitro conditions on preparations of the nervous system of snails. After learning, the membrane and threshold potentials of command neurons LPa3 and RPa3 significantly decreased and excitability of the studied neurons increased.

Relationship in Pacemaker Neurons Between the Long-Term Correlations of Membrane Voltage Fluctuations and the Corresponding Duration of the Inter-Spike Interval.

Several studies of the behavior in the voltage and frequency fluctuations of the neural electrical activity have been performed. Here, we explored the particular association between behavior of the voltage fluctuations in the inter-spike segment (VFIS) and the inter-spike intervals (ISI) of F1 pacemaker neurons from H. aspersa, by disturbing the intracellular calcium handling with cadmium and caffeine. The scaling exponent α of the VFIS, as provided by detrended fluctuations analysis, in conjunction with the corresponding duration of ISI to estimate the determination coefficient R 2 (48-50 intervals per neuron, N = 5) were all evaluated. The time-varying scaling exponent α(t) of VFIS was also studied (20 segments per neuron, N = 11). The R 2 obtained in control conditions was 0.683 ([0.647 0.776] lower and upper quartiles), 0.405 [0.381 0.495] by using cadmium, and 0.151 [0.118 0.222] with caffeine (P < 0.05). A non-uniform scaling exponent α(t) showing a profile throughout the duration of the VFIS was further identified. A significant reduction of long-term correlations by cadmium was confirmed in the first part of this profile (P = 0.0001), but no significant reductions were detected by using caffeine. Our findings endorse that the behavior of the VFIS appears associated to the activation of different populations of ionic channels, which establish the neural membrane potential and are mediated by the intracellular calcium handling. Thus, we provide evidence to consider that the behavior of the VFIS, as determined by the scaling exponent α, conveys insights into mechanisms regulating the excitability of pacemaker neurons.

[ULTRASTRUCTURAL ORGANIZATION OF CELLULAR ELEMENTS AND INTERCELLULAR CONNECTIONS IN STATOCYSTS OF TERRESTRIAL PULMONARY SNAIL H. LUCORUM].

The organ of mollusc equilibrium - statocyst appears to be the analogue of acoustic-vestibular system in vertebrate animals. In terrestrial pulmonary snail the epithelial lining of statocyst cavity is created by two types of the cells - a small amount of large cells, provided with kinocilia of sensitive cells and considerably a large number of small supporting or inserted cells, covered with the microvilli. By means of transmission and scanning electron microscopy the ultrastructure and intercellular connections of these cells were studied. The sensitive cells have in a certain way structured cytoplasm, which consists of three layers: ectoplasm, granular layer and hyaloplasm. Myelin-like bodies having the granular, vesicular and drop-like formations in the centre appear to be the special structure of the cytoplasm. In the cytoplasm there are areas, saturated with electron dense glycogen granules. On the electronograms sometimes it is observed how the pinocytic vesicles in the supporting cells are created from the diverticulum of plasmatic membrane of sensitive cells. The boundary areas of plasmatic membrane of adjacent cells (sensitive cells with supporting or supporting cells with the support) are also characterized by the presence of specialized contacts, which are analogous to desmosomes in the epithelial tissues, as well as by the existence of cellular desmosomes, interdigitations. Numerous lacunas have been revealed in the intercellular space, which are connected by the thin tubules and ducts resulting in the formation of a complicated configuration of extensive system of communicating with each other lacunae, which have the exit in statocyst cavity.

[Spontaneous EPSPs in Command Helix Lucorum Neurons during Geterosynaptic Potentiation].

To develop the presynaptic mechanism of the short-term plasticity of synaptic transmission the influence of the rhythmical orthodromic stimulation of intestinal nerve on the spontaneous excitatory postsynaptic potentials (EPSPs) have been analyzed in the command neurons of the defensive behavior of land snail Helix lucorum. It was shown early that the tetanic stimulation induced the short-term potentiation of evoked EPSPs. The rhythmical stimulation did not influence the amplitude of spontaneous EPSPs, but considerably increased their number. Discovered augmentation of the frequency of spontaneous EPSPs makes it possible to assume the participation of presynaptic mechanism in the short-term potentiation of synaptic transmission. The presynaptic mechanism can include reduction in the thresh- old of action potentials (APs) generation in the presynaptic neurons, which will lead to an increase in the fraction of spontaneous active neurons and, correspondingly, it will increase the number of spontaneous presynaptic APs.

Fractal-like correlations of the fluctuating inter-spike membrane potential of a Helix aspersa pacemaker neuron.

We analyzed the voltage fluctuations of the membrane potential manifested along the inter-spike segment of a pacemaker neuron. Time series of intracellular inter-spike voltage fluctuations were obtained in the current-clamp configuration from the F1 neuron of 12 Helix aspersa specimens. To assess the dynamic or stochastic nature of the voltage fluctuations these series were analyzed by Detrended Fluctuation Analysis (DFA), providing the scaling exponent α. The median α result obtained for the inter-spike segments was 0.971 ([0.963, 0.995] lower and upper quartiles). Our results indicate a critical-like dynamic behavior in the inter-spike membrane potential that, far from being random, shows long-term correlations probably linked to the dynamics of the mechanisms involved in the regulation of the membrane potential, thereby endorsing the occurrence of critical-like phenomena at a single-neuron level.

Voltage-gated membrane currents in neurons involved in odor information processing in snail procerebrum.

The procerebrum (PC) of the snail brain is a critical region for odor discrimination and odor learning. The morphological organization and physiological function of the PC has been intensively investigated in several gastropod species; however, the presence and distribution of ion channels in bursting and non-bursting cells has not yet been described. Therefore, the aim of our study was to identify the different ion channels present in PC neurons. Based on whole cell patch-clamp and immunohistochemical experiments, we show that Na(+)-, Ca(2+)-, and K(+)-dependent voltage-gated channels are differentially localized and expressed in the cells of the PC. Different Na-channel subtypes are present in large (10-15 µm) and small (5-8 µm) diameter neurons, which are thought to correspond to the bursting and non-bursting cells, respectively. Here, we show that the bursting neurons possess fast sodium current (I NaT) and NaV1.9-like channels and the non-bursting neurons possess slow sodium current (I NaT) and NaV1.8-like channels in addition to the L-type Ca(-), KV4.3 (A-type K-channel) and KV2.1 channels. We suggest that the bursting and/or non-bursting character of the PC neurons is at least partly determined by the battery of ion-channels present and their cellular and subcellular compartmentalization.

Selective modulation of chemical and electrical synapses of Helix neuronal networks during in vitro development.

BACKGROUND: A large number of invertebrate models, including the snail Helix, emerged as particularly suitable tools for investigating the formation of synapses and the specificity of neuronal connectivity. Helix neurons can be individually identified and isolated in cell culture, showing well-conserved size, position, biophysical properties, synaptic connections, and physiological functions. Although we previously showed the potential usefulness of Helix polysynaptic circuits, a full characterization of synaptic connectivity and its dynamics during network development has not been performed. RESULTS: In this paper, we systematically investigated the in vitro formation of polysynaptic circuits, among Helix B2 and the serotonergic C1 neurons, from a morphological and functional point of view. Since these cells are generally silent in culture, networks were chemically stimulated with either high extracellular potassium concentrations or, alternatively, serotonin. Potassium induced a transient depolarization of all neurons. On the other hand, we found prolonged firing activity, selectively maintained following the first serotonin application. Statistical analysis revealed no significant changes in neuronal dynamics during network development. Moreover, we demonstrated that the cell-selective effect of serotonin was also responsible for short-lasting alterations in C1 excitability, without long-term rebounds.Estimation of the functional connections by means of cross-correlation analysis revealed that networks under elevated KCl concentrations exhibited strongly correlated signals with short latencies (about 5 ms), typical of electrically coupled cells. Conversely, neurons treated with serotonin were weakly connected with longer latencies (exceeding 20 ms) between the interacting neurons. Finally, we clearly demonstrated that these two types of correlations (in terms of strength/latency) were effectively related to the presence of electrical or chemical connections, by comparing Micro-Electrode Array (MEA) signal traces with intracellularly recorded cell pairs. CONCLUSIONS: Networks treated with either potassium or serotonin were predominantly interconnected through electrical or chemical connections, respectively. Furthermore, B2 response and short-term increase in C1 excitability induced by serotonin is sufficient to trigger spontaneous activity with chemical connections, an important requisite for long-term maintenance of firing activity.

Organization of the procerebrum in terrestrial pulmonates (Helix, Limax) reconsidered: cell mass layer synaptology and its serotonergic input system.

The synaptology of the cell body layer of the olfactory center, procerebrum, was investigated in two prominent terrestrial pulmonate gastropod species, Helix pomatia and Limax valentianus. In addition, the analysis of the 5-HT-immunoreactive innervation, including ultrastructural level, was performed at high resolution in H. pomatia. A highly complex system of synaptic and non-synaptic connections was found in the procerebrum of both species connected to local neuropil areas of different size. The procerebral (globuli) cell perikarya were richly innervated by varicosities meanwhile the axon profiles also established contacts with each other. Synaptic configurations including convergence, divergence and presynaptic modulation were also revealed. The frequent occurrence of unspecialized but close axo-somatic and axo-axonic membrane contacts referring to the modulatory forms of transmitter release were also accompanied by membrane configurations indicative of active exocytosis. In H. pomatia, the cell mass layer was shown to receive a rich 5-HT-immunoreactive innervation, forming a dense network around the cell bodies. At ultrastructural level, 5-HT-immunoreactive varicosities contacted both cell bodies and different unlabeled axon profiles. Our results suggest that the local neuropil regions in the cell body layer are site of local circuits, which may play a decisive role in olfactory integrative processes bound to the procerebrum. The pattern and form of the 5-HT-immunoreactive innervation of extrinsic origin suggest an overall modulatory role in the cell body layer. The results may serve a basis for considering the role of local intercellular events, connected to microcircuits, within the procerebrum cell body layer involved in oscillation activities.

[Light microscopy of statocyst cell elements from Helix lucorum (space experiment aboard the orbital station "MIR")].

Statocyst epithelial lining of terrestrial pulmonary snail Helix lucorum is a spatially arranged structure consisting of 13 cell ensembles. Each ensemble has a sensory cell surrounded by companion cells. The sensory cell on the anterior statocyst pole is star-shaped due to multiple protoplasmatic protrusions on its body. The remaining 12 polygon-shaped cells form 3 tires along the statocyst internal perimeter: anterior, middle or equatorial and posterior. There are 4 cells in each tire. Topography of every sensory cell on the statocyst internal surface was described as well as cell nuclei size and form, nucleoli number and patterns of cytoplasm vacuolization. Space free of sensory cells is occupied by supporting or intercalary cells. Exposure to space microgravity over 40, 43, 102 and 135 days aboard the orbital station MIR affected morphology of the sensory cells. Specifically, this appeared as reductions in cell height and, consequently, extension of the statocyst cavity internal diameter and volume in the space-flown snails.

Neurotoxic effects evoked by cyanobacterial extracts suggest multiple receptors involved in electrophysiological responses of molluscan (CNS, heart) models.

The responses of the snail central neurons (Helix pomatia, Lymnaea stagnalis) and the isolated Helix heart were characterized evoked by cyanobacterial extracts (Cylindrospermopsis raciborskii ACT strains) isolated from Lake Balaton (Hungary). The nicotinergic acetylcholine (ACh) receptors in the CNS (both excitatory and inhibitory) were blocked by the extracts of ACT 9502 and ACT 9505 strains and the anatoxin- a (homoanatoxin-a) producing reference strain of Oscillatoria sp. (PCC 6506), similar to the inhibitory effects of the pure anatoxin-a. The enhancement of the ACh responses by the ACT 9504 extract suggests additional, probably acetylcholine esterase inhibitory mechanisms. On the isolated Helix heart the crude ACT 9505 and PCC 6506 extracts evoked frequency increase and transient twitch contraction, opposite to the ACh evoked heart relaxation. Anatoxin-a similarly contracted the heart but did not increase its contration frequency. These data suggest the involvement of some non-cholinergic mechanisms, acting very likely by direct modulation of the electrical or contractile system of the isolated heart. Diversity of the effects evoked by the cyanobacterial extracts in the CNS and heart suggest pharmacologically different neuroactive components among the secondary metabolites of the cyanobacteria acting on both (anatoxin-a like) cholinergic and (unidentified) non-cholinergic receptors.

Immunohistochemical localization of hepatopancreatic phospholipase A2 in Hexaplex trunculus digestive cells.

BACKGROUND: Mammalian sPLA2-IB localization cell are well characterized. In contrast, much less is known about aquatic primitive ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes and the mode of digestion of lipid food. RESULTS: The marine snail digestive phospholipase A2 (mSDPLA2) has been previously purified from snail hepatopancreas. The specific polyclonal antibodies were prepared and used for immunohistochimical and immunofluorescence analysis in order to determine the cellular location of mSDPLA2. Our results showed essentially that mSDPLA2 was detected inside in specific vesicles tentatively named (mSDPLA2+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion in the snail might occur in specific granules inside the digestive cells. CONCLUSION: The cellular location of mSDPLA2 suggests that intracellular phospholipids digestion, like other food components digestion of snail diet, occurs in these digestive cells. The hepatopancreas of H. trunculus has been pointed out as the main region for digestion, absorption and storage of lipids.

Morphological peculiarities statoconia in statocysts of terrestrial pulmonary snail Helix lucorum.

Vast majority of statoconia in statocysts of Helix lucorum are of oval shape and have smooth surface. Each statoconium in its central part has a nucleus, a spherical mass of 1.5 µ in diameter, surrounded by concentric structures. Minority of statoconia are of subcircular, elongated, rectangular, triangular, irregular, and sometimes fanciful shape and are structured around several nuclei or around small statoconia consolidated by shared growth layers. Apart from statoconia, spherical formations lacking mineral composition of 0.3-2.5 µ in diameter were also found in the statocyst cavity. Similar formations were found in vacuoles of sensory cells of statocyst epithelial lining. It is hypothesized that statoconium nuclei and growth layers around them are of different origin: the nuclei are formed by statocyst sensory cells, while the mineral component is a result of activity of supporting cells.

Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland.

In terrestrial pulmonate snails, cadmium (Cd) uptake leads to the induction of a Cd-specific metallothionein isoform (Cd-MT) that protects against adverse interactions of this toxic metal ion. Increasing concentrations of Cd cause increased individual mortality possibly linked to pathological alterations in the snail midgut gland. Histological, immuno-histochemical, and electron-microscopic methods in combination with tissue metal analyses and quantification of MT induction parameters were applied to the midgut gland of Cd-exposed Roman snails (Helix pomatia). Conspicuous concentration-dependent alterations occurred in this organ, including the metal-induced increase of Cd-MT concentration and manifestation of Cd-MT mRNA precipitations in all midgut gland cell types. The most evident alteration was an increase of cellular turnover reflected by enhanced cell proliferation. Intensified vesiculation of endoplasmic reticulum was noted in basophilic cells and an increasing formation of lipofuscin granules in excretory cells. At the highest Cd concentrations, mitochondrial membranes were disrupted in basophilic cells, and lipofuscin granules were released from excretory cells into the midgut gland tubular system. Some of these alterations (e.g., increased cell proliferation rate, vesiculation of endoplasmic reticulum) detected at low Cd concentrations were interpreted as adaptive response processes enhancing the tolerance of exposed individuals to metal stress. Cellular alterations at higher Cd concentrations (e.g., mitochondrial structural damage) clearly represented ongoing irreversible cellular disruption. Combined evaluation of cellular biomarkers and MT saturation levels indicated that the transition from stress resistance to depletion of resistance capacity occurred above a threshold of 0.8 micromol Cd/g dry weight in the midgut gland of H. pomatia. At these Cd concentrations, Cd-MT was saturated with Cd(2+) ions, whereas at the cellular level, structural alterations turned into pathological deterioration.

Helix neuronal ensembles with controlled cell type composition and placement develop functional polysynaptic circuits on Micro-Electrode Arrays.

Neuronal cell cultures on Micro-Electrode Arrays (MEAs) provide an essential experimental tool for studying the connectivity and long-term activity of complex neuronal networks. MEA studies are generally based on the analysis of mixed neuronal populations constituted by a large number of cultured cells with cell type composition and connectivity patterns which are quite unpredictable a priori. In this work, we propose a different approach which consists of assembling on MEAs neuronal circuits formed by individually identifiable C1, C3, and B2 Helix neurons. Cells were plated under conditions of controlled number and position to form neuronal networks of defined composition. We performed multi-site electrophysiological recordings, and we characterized the firing dynamics. By means of cross-correlation analysis, we studied the electrophysiological properties of MEA-coupled microcircuits and characterized their activity patterns. We showed how the synaptic connectivity, actually observed in polysynaptic circuits of C1, C3 and B2 neurons, correlates well with the expected connectivity of C1-B2, B2-B2 and B2-C3 cell pairs as previously reported in conventional electrophysiological studies in culture.

Quantitative changes in digestive gland cells and oocytes of Helix aspersa, as biomarkers of copper oxychloride exposure under field conditions.

This study investigated the effects of accumulated copper, on digestive epithelium height and percentage area, and on oocyte numbers of the snail Helix aspersa, in a vineyard where copper oxychloride is sprayed. The ultimate aim was to determine the usefulness of these cellular responses as biomarkers. Results showed that snails collected 2 months after fungicide application, had a significantly lower mean digestive epithelium height and percentage area, as well as significantly fewer oocytes per 1 mm(2) of ovotestis, compared to snails collected only 1 week after fungicide application and those from a control vineyard. It was concluded that these cellular responses are clear, measurable responses to copper oxychloride exposure and copper accumulation. However, they do not provide an early warning of copper exposure, which impacts on their usefulness as biomarkers.

Serotonin precursor 5-hydroxytryptophan disturbs the protective effect of low doses of antibodies to S100B protein during the formation of long-term sensitization.

We studied the effect of ultralow doses of antibodies to calcium-binding protein S-100B and 5-hydroxytryptophan, a metabolic precursor of serotonin, on the formation of long-term sensitization as a neurobiological model of anxiety and depression. Daily administration of antibodies to S-100B to edible snail before the formation of long-term sensitization prevents its development. 5-Hydroxytryptophan administered before the formation of long-term sensitization abolished the protective effect of antibodies to S-100B protein.

Intravital investigation of the effects of serotonin and glutamate on the dynamics of DNA activity in L-RPl1 neurons of edible snail.

Microloci of varying size with increased fluorescence were observed in L-RPl1 neuronal nuclei of snails using DNA-selective dye SYTO16. Application of serotonin and glutamate increased the number of small (<1 micro in diameter) microloci, while the number of medium and large (1-3 micro) loci decreased. Combined application of neurotransmitters produced more pronounced changes in the number of microloci compared to individual administration. RNA synthesis inhibitors abolished the effects of the transmitters. We hypothesized that the revealed small microloci of fluorescence are "active" DNA zones, where transcription of new genes are initiated.

Changes in electrical properties of command neurons during protective effect of low doses of antibodies to S100 protein on the development of long-term sensitization in Helix lucorum.

We studied the effect of antibodies to Ca(2+)-binding protein S100 in a dilution of 10(-12) (LAT-S100) on the development of long-term sensitization in Helix lucorum, a neurobiological model of anxious and depressive states. After administration of LAT-S100 preventing the development of long-term sensitization before training, the membrane and threshold potentials in command neurons regulating defense behavior decreased less markedly than during long-term sensitization. It is assumed that the "protective" effect is associated with mechanisms of long-term potential maintenance and changes in intra- and extracellular balance of Ca(2+)-binding protein S100.

Effects of preliminary administration of haloperidol in low doses on the effects of haloperidol on behavioral reactions and command neuron membrane potential in edible snail.

We studied the effects of preliminary administration of haloperidol in low doses on changes in motor activity of edible snail and in electrical properties of defensive behavior command neurons induced by chronic administration of haloperidol. The rate of locomotion decreased after injections of haloperidol preparations (C6, C12, C30, C200 and a mixture C12+C30+C200) for 3 days. Similar changes were observed after 3 days of haloperidol administration. Haloperidol preparations in low doses produced a modulating effect on the decrease in locomotion rate and hyperpolarization of command neurons in edible snails caused by chronic exposure to haloperidol: the decrease in locomotion rate caused by chronic haloperidol treatment was prevented by preliminary injection of haloperidol in low doses C6, C12 and C30; the depolarizing shift of command neuron membrane potential was also abolished after consecutive injection of the same haloperidol preparations C6, C12 and C30.