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Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP., Summary: PACAP has a neuroprotective effect in different toxin-induced rat and snail parkinsonian models, acting partially through the same mechanisms.

In this study, we investigate the potentiating effect of PACAP27 on cholinergic neuromuscular transmission in the recently discovered flexor muscles of the land snail, Helix pomatia. Using immunohistochemistry, we show that PACAP and PAC1 receptors are present in nerve fibers innervating the flexor muscles but not in the muscle itself. We also observed that PACAP27 exerts both pre- and postsynaptic effects on the cholinergic synapse and performed tests using a broad spectrum of chemicals in order to explore the possible intracellular pathways through which PACAP mediates its stimulatory effect. Our pharmacological data demonstrate that PACAP27 presynaptically enhances the release of acetylcholine by activating the adenylate cyclase-cAMP-PKA pathway. Postsynaptically, PACAP27 was found to enhance muscle contractility by PKC-mediated signaling pathway resulting in an increased Ca(2+) release from intracellular stores. These findings suggest that regulation of Ca(2+) release may contribute to the stimulatory effect of PACAP. Our data are the first demonstration of the potentiating effect of PACAP27 at the molluscan excitatory neuromuscular contact.

Having been investigated for over 40 years, some aspects of the biology of terrestrial gastropod's olfactory system have been challenging and highly contentious, while others still remain unresolved. For example, a number of terrestrial gastropod species can track the odor of food, while others have no strong preferences toward food odor; rather they find it by random encounter. Here, while assessing the most recent findings and comparing them with earlier studies, the aspects of the food selection based on olfactory cues are examined critically to highlight the speculations and controversies that have arisen. We analyzed and compared the potential role of airborne odors in the feeding behavior of several terrestrial gastropod species. The available results indicate that in the foraging of most of the terrestrial gastropod species odor cues contribute substantially to food finding and selection. The results also suggest, however, that what they will actually consume largely depends on where they live and the species of gastropod that they are. Due to the voluminous literature relevant to this object, this review is not intended to be exhaustive. Instead, I selected what I consider to be the most important or critical in studies regarding the role of the olfaction in feeding of terrestrial gastropods.

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis, exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry. The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species.

The distribution and functional presence of three voltage-dependent potassium channels, K v 2.1, K v 3.4, K v 4.3, respectively, were studied in the central nervous system of the snail Helix pomatia by immunohistochemical and electrophysiological methods. Cell clusters displaying immunoreactivity for the different channels were observed in all parts of the CNS, although their localization and number partly varied. Differences were also found in their intracellular, perikaryonal and axonal localization, as well as in their presence in non-neuronal tissues nearby the CNS, such as the perineurium and the aorta wall. At ultrastructural level K v 4.3 channel immunolabeling was observed in axon profiles containing large 80–100 nm granular vesicles. Blotting analyses revealed specific signals for the K v 2.1, K v 3.4 and K v 4.3 channels, confirming the presence of the channels in the Helix CNS. Voltage-clamp recordings proved that outward currents obtained from neurons displaying K v 3.4 or K v 4.3 immunoreactivity contained transient components while K v 2.1 immunoreactive cells were characterized by delayed currents. The distribution of the K + -channels containing neurons suggests specific roles in intercellular signaling processes in the Helix CNS, most probably related to well-defined, partly local events. The cellular localization of the K + -channels studied supports their involvement in both pre- and postsynaptic events at perikaryonal and axonal levels.

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.

Programmed cell death leading to apoptosis is essential for normal development and homeostasis in plants and throughout the animal kingdom. Although there are differences in apoptotic mechanisms between lower animals and vertebrates, crucial biochemical components of the programmed cell death pathways remained remarkably conserved throughout evolution. Despite decades of studies on the neurobiology and development of mollusks, comparatively little is known about the mechanisms of apoptosis in this phylum. In this review, an attempt is made to summarize data obtained on mollusks so far, and to discuss the molecular mechanisms, the functional and ecological significance of apoptosis and the advantages of snail preparations as tools for programmed cell death research. A definitive comparison of the data obtained on mollusks with those obtained on the more widely studied vertebrates, will contribute to the better understanding of the apoptotic process in general and of its evolutionary development.

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-β-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.

Pituitary adenylate cyclase activating polypeptide (PACAP) shows a remarkable sequence similarity among species and several studies provide evidence that the functions of PACAP have also been conserved among vertebrate species. Relatively little is known about its presence and functions in invertebrates. The aim of the present study was to investigate whether the well-known anti-apoptotic effect of PACAP can also be demonstrated in invertebrates. This effect was studied in the salivary gland of a molluscan species, Helix pomatia. In this work, we first showed the presence of PACAP-like immunoreactivity in the Helix salivary gland by means of immunohistochemistry. Radioimmunoassay measurements showed that PACAP38-like immunoreactivity dominated in the salivary gland of both active and inactive snails and its concentration was higher in active than in inactive animals in contrast to PACAP27-like immunoreactivity, which did not show activity-dependent changes. PACAP induced a significant elevation of cAMP level in salivary gland extracts. Application of apoptosis-inducing agents, dopamine and colchicine, led to a marked increase in the number of terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the salivary gland, which was significantly attenuated by PACAP treatment. In a similar manner, the number of caspase-positive cells was reduced after co-application of dopamine and PACAP. Taken together, the data indicate that PACAP activates cAMP in a molluscan species and we show, for the first time, that PACAP is anti-apoptotic in the invertebrate Helix pomatia.

GPRCs are regulated via phosphorylation by different protein kinases including GRKs and PKA and PKC. The purpose of this study was to determine the presence and physiological role of GRKs in the tissues of the snail, Helix pomatia . Here we report that immunoblotting of brain homogenate with anti-GRK2/3 antibody prepared from mammalian tissue can be detected in snail GRK-like immunoreactivity. The GRK2/3 immunoreactivity was found at ∼ 80 kDa in a variety of cells, including salivary duct, salivary gland and eye. Intracellular injection of the anti-GRK2/3 prevented the neuron from desensitization and agonist-induced activation augmented the phosphorylated GRKs in the membrane fraction suggesting that GRKs may have a functional role in the neuropeptide receptor desensitization in snail.

The aim of the present study was to assess the cellular mechanism of secretion in the salivary gland of the snail, Helix pomatia, using electrophysiological, electron microscopic and immunohistochemical techniques. A homogeneously distributed membrane potential (-56.6 +/- 9.8 mV) was determined mainly by a K+ -electrochemical gradient and partly by the contribution of the electrogenic Na+ -pump and Cl- conductance. Low resistance electrical coupling sites were identified physiologically. Transmission electron microscopy and innexin 2 antibody revealed the presence of gap-junction-like membrane structures between gland cells. It is suggested that gap-junctions are sites of electrotonic intercellular communication, which integrate the gland cells into a synchronized functional unit in the acinus. Stimulation of the salivary nerve elicited secretory potentials (depolarization) which could be mimicked by local application of acetylcholine, dopamine or serotonin. In voltage-clamp experiments four major conductances were identified: a delayed rectifier (IK), a transient (IA) and a Ca2+ -activated outward K+ current (IK(Ca)) and Ca2+ -inward currents (ICa). It is suggested that one or more of these conductances may give rise to a stimulus activated secretory potential leading to excitation-secretion coupling and subsequent the release of the mucus from the gland cells.

Members of the mytilus inhibitory peptide (MIP) family play an important role in the modulation of many physiological processes in molluscs. The signal transduction pathways affected by the MIP effect have not, however, been elucidated. Application of guanosine 5'-[gamma-thio]triphosphate tetralithium salt (GTPgammaS), guanosine 5'-[beta-thio]diphosphate trilithium salt (GDPbetaS), the G-protein inhibitor suramin and pertussis toxin (PTX) demonstrated the involvement of the PTX-insensitive G-protein in the signal transduction pathway mediating MIP effects. Both G-protein alpha(i) and betagamma subunits were identified in D-neurons of Helix pomatia by immunoblotting. Their role in signal transduction was shown in electrophysiological experiments, which supported the notion that, in addition to the Galpha subunit, the betagamma dimer also participates in the neuropeptide-induced activation of K-channels in snail neurons. Finally, neuropeptide-activated responses were inhibited by the activation of adenylyl cyclase and by blockers of the phospholipase pathway. We suggest that bifurcation of the signal transduction takes place at the level of G-protein subunits. The alpha subunit may have a direct effect on adenylyl cyclase, while the betagamma subunit may have a direct effect on phospholipase enzymes.

Blocking of the GABA-activated chloride current by cadmium was investigated in identified nerve cells (RPeD1, RPaD1) of the pond snail (Lymnaea stagnalis L.), using a two-microelectrode voltage-clamp technique. Cd2+, at 50 μM extracellular concentration, inhibited GABA-activated chloride currents, both in normal and Ca2+-free solution. Intracellular injection of Ca2+ or the application of caffeine mimicked the inhibitory effect of Cd2+ on GABA-elicited currents. Cd2+-block was eliminated, or it was substantially decreased, when neurons were intracellularly loaded with EGTA, or when the Ca2+-release was blocked by ruthenium red. The blocking effect of Cd2+ was also eliminated by applying G-protein inhibitors: pertussis toxin, suramin or GTP-γ-S. Finally, intracellularly injected Cd2+ was ineffective at eliciting an inward current on GABA-activated currents, suggesting that the Cd2+-binding site resides extracellularly. These results suggest that cadmium inhibited GABA-activated chloride currents by increasing the intracellular Ca2+ level, by releasing it from intracellular stores and by interacting with a putative G-protein-coupled cell-surface “metal-receptor”.

Distribution, chemical-neuroanatomy, concentration, and uptake-release properties of histamine (HA)-containing neurons and the possible physiological effects of HA in the central and peripheral nervous system of the pulmonate snails, Helix pomatia and Lymnaea stagnalis, are described. In the CNS of both species, the distribution pattern of HA-immunoreactive (HA-IR) neurons was similar. In both species the majority were located in the buccal, cerebral, and pedal ganglia. In Helix, approximately 400 HA-IR neurons were seen, whereas in Lymnaea approximately 130 labeled cells were visualized. The neuropils, connectives, commissures, several peripheral nerves, and a part of the peripheral tissues (lip and foot of both species and the upper tentacles of Helix) were innervated by HA-IR elements. Numerous sensory cells were found in the tentacles, lip, and statocysts. The HA concentration values assayed by HPLC ranged from 4.8 to 47.4 pmol/mg in the different central ganglia of Helix, and from 4.3 to 18.6 pmol/mg in Lymnaea CNS, whereas the peripheral tissues contained 0.33-1 pmol/mg HA in Helix and 0.26-0.46 pmol/mg in Lymnaea. In the Lymnaea CNS, a high-affinity (37.6 microM), single component 3H-HA uptake system was demonstrated. 3H-HA release evoked by either electrical stimulation or 100 mM K+ could be prevented in Ca2+-free physiological solution. Voltage-clamp experiments indicated specific changes caused by HA in the membrane conductance of identified central neurons of Helix and Lymnaea. Exogenously applied 10(-5) M HA resulted in the acceleration of locomotion (gliding by foot cilia) of Lymnaea. The findings suggest an important signaling role of HA, described here for the first time, in the nervous system of higher-order, pulmonate, gastropods, involving efferent, integrative, and sensory functions. The data can also be applied as a background for further specification of HA in the regulation of different behaviors in these species.

The block of the transient outward K-current, I(K(A)) by 4-aminopyridine (4-AP) and blood-depressing substances (BDS) was investigated in identified Helix pomatia neurons (LPa3) using the two microelectrode voltage-clamp technique. The present study shows that 4-AP inhibits I(K(A)) in snail neurons in a voltage- and concentration-dependent manner. The 4-AP block of I(K(A)) involves the block of both open and closed states of the channel, however binding to open channels is preferred. It is suggested that 4-AP have two binding sites on the identified Helix neuron. One site causes an open channel block, which affects the N-type inactivation, and binding to the second site induces closed channel block, which affects C-type inactivation. In control solution the inactivating phase of the current is biexponential, suggesting simultaneous presence of two types of inactivation. The counterplay of these mechanisms results in the crossover of the current traces recorded from control and 4-AP blocked channels. It is assumed that use-dependence does not occur through blocker 'trapping', but rather by a different mechanism. BDS had no effect on Helix I(K(A)), suggesting that transient potassium channels in LPa3 neuron are not Kv3.4 type channels.

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15–35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5–7 µm diameter) and light neurons with 12–18 (T1 type) and 18–30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus–response pathway, consisting of olfactory stimulus—local motor neuron—motor response components, to generate local lateral movements of the tentacle tip (“quiver”). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus—distal segments of central motor axons—motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.

Hibernation, as behavior, is an evolutionary mode of adaptation of animal species to unfavorable environmental conditions. It is generally characterized by suppressed metabolism, which also includes down regulation of the energy consuming ion-channel functioning. Experimental data regarding decreased ion-channel function are scarce. Therefore, our goal was to study the possible down regulation of voltage-gated sodium channel (NaV) subtypes in the neurons of hibernating snails. Our immunohistochemical experiments revealed that the expression of NaV1.8-like channels in the central nervous system was substantially down regulated in hibernating animals. In contrast to NaV1.8-like, the NaV1.9-like channels were present in neurons independently from hibernating and non-hibernating states. Our western blot data supported the immunohistochemical results according to which the band of the NaV1.8-like channel protein was less intensively labeled in the homogenate of the hibernating snails. The NaV1.9-like immunoreactivity was equally present both in hibernating and active snails. Micro-electrophysiological experiments show that in hibernating snails both NaV1.8- and NaV1.9-like currents are substantially decreased compared to that of the active snails. The contradictory electrophysiological and immunohistochemical or western blot data suggest that the molecular mechanisms of the "channel arrest" could be different in diverse NaV channel subtypes. Climate changes will affect temperature extremes and a question is how different species beyond their physiological tolerance will or able to adapt to changing environment. Hibernation is an important mode of adaptation to extreme climatic variations, and pursuant to this the present results may contribute to the study of the behavioral ecology.

Effects of seven, pressure applied MIP (Mytilus inhibitory peptides) had been studied on D-neurons of the CNS of Helix pomatia in voltage-clamp experiments. In physiological saline, the peptides produced a hyperpolarization usually coupled with the cessation of any spontaneous spiking activity. Clamped at the resting potential ( approximately -60 mV), peptide applications elicited an outward current, which increased its amplitude by shifting the holding potential towards depolarisation. The response was concentration-dependent and accompanied by an increased membrane conductance. Reversal potentials obtained at different [K+]o were plotted with a slope of 52 mV per ten-fold change in [K+]o showing that the peptide-elicited current was mainly due to the increased K+-conductance(s). The peptide-induced outward current could partially be blocked by Ba2+ (5 mM), CdCl2 (1 mM), TEACl (10 mM) or apamin (2.5x10(-5) M) or furosemide (10 mg/ml) and decreased either in Na+-free or Cl--free solutions. 4-Aminopyridine at 5 mM concentration completely blocked the peptide-induced current. In the presence of high [K+]o, the peptide(s) was still found to induce an outward current at membrane potentials beyond K+-reversal potential. This component was not present in Cl--free saline, suggesting that the current was due to the inward flow of Cl- ions. Our results show that the MIPs have at least two (three) independent actions, each associated with different voltage-, concentration-dependence and ionic mechanisms. It is suggested, that the peptide-induced currents are carried by K+, and Cl- ions. According to our present finding, the observed effects are mediated by the same receptor, activating different second messenger systems, inducing multiple conductance changes in the membrane of neurons of the snail ganglia.

Recently, three novel flexor muscles (M1, M2 and M3) in the posterior tentacles of the snail have been described, which are responsible for the patterned movements of the tentacles of the snail, Helix pomatia. In this study, we have demonstrated that the muscles received a complex innervation pattern via the peritentacular and olfactory nerves originating from different clusters of motoneurons of the cerebral ganglia. The innervating axons displayed a number of varicosities and established neuromuscular contacts of different ultrastructural forms. Contractions evoked by nerve stimulation could be mimicked by external acetylcholine (ACh) and glutamate (Glu), suggesting that ACh and Glu are excitatory transmitters at the neuromuscular contacts. Choline acetyltransferase and vesicular glutamate transporter immunolabeled axons innervating flexor muscles were demonstrated by immunohistochemistry and in Western blot experiments. Nerve- and transmitter-evoked contractions were similarly attenuated by cholinergic and glutamatergic antagonists supporting the dual excitatory innervation. Dopamine (DA, 10−5 M) oppositely modulated thin (M1/M2) and thick (M3) muscle responses evoked by stimulation of the olfactory nerve, decreasing the contractions of the M1/M2 and increasing those of M3. In both cases, the modulation site was presynaptic. Serotonin (5-HT) at high concentration (10−5 M) increased the amplitude of both the nerve- and the ACh-evoked contractions in all muscles. The relaxation rate was facilitated suggesting pre- and postsynaptic site of action. Our data provided evidence for a DAergic and 5-HTergic modulation of cholinergic nerves innervating flexor muscles of the tentacles as well as the muscles itself. These effects of DA and 5-HT may contribute to the regulation of sophisticated movements of tentacle muscles lacking inhibitory innervation.

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+-, Ca2+-, 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.

1. The paper summarizes the effects of the metal ions Cu2+, Pb2+, Ag+, Hg2+, Zn2+, and Cd2+ applied externally or internally to the surface membrane of different excitable cells. 2. Conductance changes induced by metal ions, and metal ion-activated current, are compared with respect to their ion and voltage dependence. 3. It is suggested that metal ion-induced effects can be realized through special structures of the cell membrane, the metal ion "receptors," although other mechanisms, as, for example, competition for Ca-binding sites in the channel forming proteins, cannot be excluded.

The purpose of this review is to highlight the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in a range of physiological and behavioral processes of gastropod molluscs, Helix and Lymnaea. Since its discovery in 1989 PACAP has become increasingly recognized for its important and diversified roles in the central and peripheral nervous system and in several peripheral organs of a variety of vertebrate and invertebrate species. Twenty-two years after its discovery, PACAP is now one of the most extensively studied of the neuropeptides. This review surveys the importance of PACAP and PACAP-like peptides in invertebrates, focusing mainly on the gastropod molluscs. The relevance of studies on lower vertebrates and invertebrates, which do not have a pituitary gland, is to contribute to the unraveling of fundamental effects of PACAP or PACAP-like peptides and to provide a comparative view.

Neuropeptides, the most diverse group of signaling molecules, are responsible for regulating a variety of cellular and behavioral processes in all vertebrate and invertebrate animals. The role played by peptide signals in information processing is fundamentally different from that of conventional neurotransmitters. Neuropeptides may act as neurotransmitters or neuromodulators and are released at either synaptic or non-synaptic sites. Peptide signals control developmental processes, drive specific behaviors or contribute to the mechanisms of learning and memory storage. Co-transmission within or across peptide families, and between peptide and non-peptide signaling molecules, is common; this ensures the great versatility of their action. How these tasks are fulfilled when multiple neuropeptides are released has become an important topic for peptide research. Although our knowledge concerning the physiological and behavioral roles of most of the neuropeptides isolated from molluscs is incomplete, this article provides examples to address the complexity of peptide signaling.

In this study we examined changes in a persistent sodium current (I(NaP)) after behavioral aversive classical conditioning in the snail Helix pomatia. We trained animals by pairing food with a mild electric shock that triggered the whole-body withdrawal reflex. This aversive training resulted in transcription dependent long-term associative memory. Isolated central nervous system preparations were set up from trained, random control and naive animals and using two-electrode voltage clamp methods, I(NaP) was activated and measured in identified body withdrawal interneurons RPa3 and LPa3. We show here that in preparations from conditioned animals I(NaP) is increased, suggesting that modifications in intrinsic cellular properties contribute to the formation of the memory trace. Blocking RNA synthesis by systemic injection of actinomycin D (0.12microM) suppressed both memory consolidation in intact animals and the learning-induced increase of I(NaP) in withdrawal interneurons, suggesting that aversive classical conditioning affects sodium channel expression at the transcriptional level.

Neurones RPa3 and LPa3 were identified as 'command' neurones in the Helix parietal ganglia. The physiological role of these cells is the integration of sensory information before triggering withdrawal behaviour. Properties of the Na-channels are poorly understood in these neurones which produce Na(+)-dependent action potentials in Ca(2+)-free solution. Our aim was to describe the kinetic properties and TTX-sensitivity of the Na-channels of these cells, and to provide evidence for the existence of a persistent inward sodium current (I(NaP)) in them. Two-microelectrode voltage- and patch-clamp techniques were used on isolated or semi-isolated neurones. The kinetics and potential dependence of the transient inward sodium current (I(NaT)) agreed well with those obtained on other molluscan neurones. We concluded that I(NaT) present in these neurones is slow and TTX-resistant (k(D)=8 microM of TTX) and has two components with different rates of inactivation. In addition, the presence of an I(NaP) component was revealed. We showed that I(NaP) is neither an artifact nor the contribution of a Ca-channel or a 'window' current. With slow voltage ramp pulses I(NaP) could be activated and separated from I(NaT). Like I(NaT) it appeared to be TTX-resistant and Na-dependent. I(NaP) was upregulated by increased pH (8.0) and decreased by elevated extracellular Mg(2+) concentration parallel with the I(NaT). Our results suggest that I(NaP) originates from the same set of sodium channels that underlie I(NaT).

The effect of anatoxin (ANTX), the crude extract (AlgTX) and purified fraction (F1) isolated from cyanobacterium C. raciborskii was studied on the neurones of two snail species. ANTX and AlgTX exerted excitatory, inhibitory and biphasic effects on the spontaneous activity of identified neurones. Both ANTX and AlgTX elicited an inward current, which could be decreased by curare or amiloride. On the contrary, F1 had no direct effect on the spontaneous activity; it was not able to induce conductance changes of the neuronal membrane, but it did antagonise the acetylcholine (ACh)-induced inward current. We concluded that ANTX affects the neuronal membrane of neurones acting on ACh receptors. The AlgTX had similar effects, and therefore the extract of C . raciborskii may contain an ANTX-like component. The purified fraction prolonged and decreased the ACh-elicited response, but had no direct membrane effect. We suggest, therefore, that both AlgTX and the purified fraction F1 interact with the ACh receptor, but they have different binding sites on the neuronal ACh receptor–ion channel complex. The possible neurotoxic effects of the C. raciborskii extract and F1 are demonstrated for the first time; the molecular mechanism of their action, however, remains to be elucidated.

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.

1. The effect of Mytilus inhibitory peptide-related peptide RAPYFVamide, isolated from Helix pomatia brain, was studied on 21 different identified Helix neurons. 2. It was found that the neuropeptide hyperpolarized 11 of the 21 neurons studied, inducing a K-dependent current. Not all neurons responded by hyperpolarization to the peptide application, though, in all cases the voltage-dependent outward K and inward Ca currents were depressed. 3. This may show that, in the peptide effect, more than one type of K conductance is involved. 4. It is proposed that RAPYFVamide may have a functional role in the modulation of the feeding behavior in H. pomatia.

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10(-3), 10(-4), 10(-5) M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.

The results presented demonstrate that in D neurons of the snail Helix pomatia L., acetylcholine (ACh) (10 divided by 100 microM) and serotonin (5-HT) (0.1 divided by 1000 microM) applications reduce both the basal intracellular concentration level ([Ca2+]in) and the amplitudes of calcium transients induced by membrane depolarization. It is likely that the mechanism of [Ca2+]in changes in the suppression of calcium inward currents (ICa). Influences of Ach and 5-HT on ICa were studied. Both effects were dose-dependent (ACh--0.01 divided by 100 microM and 5-HT--0.1 divided by 1000 microM). The half-maximal effects (IC50) were evoked by ACh concentration of 0.15 microM and 5-HT--15 microM. Furthermore we have also shown that in some cells 5-HT could evoke a transient increase in ICa (IC50 = 2 microM). The effects of Ach and 5-HT were nonadditive--the subsequent application of ACh after 5-HT, and vice versa, produced no inhibitory effects. This may indicate that both substances act through a common intermediate (possibly, G-protein).

Single sodium channel currents were studied in mouse neuroblastoma cells. Channel conductance (gamma) was 11.6 +/- 3.4 pS at 6-8 degrees C. Unitary current amplitudes and channel activity increased with increasing temperature. The Arrhenius plot of the conductance was linear between 5 and 35 degrees C with an activation enthalpy of 27.1 kJ/mole (Q10 = 1.28). Amplitude distributions were fitted by the sum of two Gaussian functions indicating the presence of two different single channel amplitudes: smaller i1 and larger i2. The relative probability of appearance of i1, which has a shorter mean open time, was higher during the early phase of depolarization (t less than 16 ms). The open time histograms, inactivating phase of macroscopic currents and delay time histograms were fitted by the sum of two exponentials. The distinct kinetic and steady-state parameters reflect two open states of sodium channels.

1. 1. The effect of a pyrethroid insecticide deltamethrin was investigated on transient outward potassium currents of identified snail (Helix pomatia) neurones LPa1 and RPa3. 2. 2. In 5 × 10−5 M concentration the deltamethrin decreased the IA amplitude and the slope of I–V curve. The activation variable was shifted left along the voltage axis by 10–20 mV, while the inactivation variable remained unchanged. 3. 3. Time constant of inactivation decreased, and the relaxation of IA described by one exponential. “Modified” ionic channel fraction was not observed. 4. 4. It is suggested that deltamethrin acts on IA channels through a different molecular mechanism to INa channels, since not only the gating machinery but the permeability of the channels were influenced.

The effect of Ca-free solution upon the ultrastructure and electrical activity of the heart muscle cells of Helix pomatia was investigated. The ultrastructure of the heart of Helix pomatia differs from that of vertebrate species but is similar to the heart of other Mollusca and shows characteristic alterations to Ca-lack. Short incubation or perfusion in Ca-free solution for 30 min or 1 h, caused the swelling of the sub-sarcolemmal cisterns. In the experiments of longer duration (4 h), the ultrastructural damage to the mitochondria was significant. In a Ca-free solution the contractile activity disappeared and the generation of action potentials ceased. It seems that Ca plays an important role in Helix heart both in the excitation-contraction coupling and in generation of the action potentials.