Ageing is associated in many organisms with a reduction in motor movements. We have previously shown that the rate of feeding movements of the pond snail, Lymnaea, decreased with age but the underlying cause is not fully understood. Here, we show that dopamine in the cerebro‐buccal complex is an important signalling molecule regulating feeding frequency in Lymnaea and that ageing is associated with a decrease in CNS dopamine. A proteomic screen of young and old CNSs highlighted a group of proteins that regulate stress responses. One of the proteins identified was 14‐3‐3, which can enhance the synthesis of dopamine. We show that the Lymnaea 14‐3‐3 family exists as three distinct isoforms. The expression of the 29 kDa isoform (14‐3‐3Lym3) in the cerebro‐buccal complex decreased with age and correlated with feeding rate. Using a 14‐3‐3 antagonist (R18) we were able to reduce the synthesis of L‐DOPA and dopamine in ex vivo cerebro‐buccal complexes. Together these data suggest that an age‐related reduction in 14‐3‐3 can decrease CNS dopamine leading to a consequential reduction in feeding rate.

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 Ca2+ release from intracellular stores. These findings suggest that regulation of Ca2+ 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.

The involvement of serotonin in mediating hunger-related changes in behavioral state has been described in many invertebrates. However, the mechanisms by which hunger signals to serotonergic cells remain unknown. We tested the hypothesis that serotonergic neurons can directly sense the concentration of glucose, a metabolic indicator of nutritional state. In the snail Lymnaea stagnalis, we demonstrate that completely isolated pedal serotonergic neurons that control locomotion changed their biophysical characteristics in response to glucose application by lowering membrane potential and decreasing the firing rate. Additionally, the excitatory response of the isolated serotonergic neurons to the neuroactive microenvironment of the pedal ganglia was significantly lowered by glucose application. Because hunger has been reported to increase the activity of select neurons and their responses to the pedal ganglia microenvironment, these responses to glucose are in accordance with the hypothesis that direct glucose signaling is involved in the mediation of the hunger-related behavioral state.

Behavioural/motivational state is known to influence nearly all aspects of physiology and behaviour. The cellular basis of behavioural state control is only partially understood. Our investigation, performed on the pond snail Lymnaea stagnalis whose nervous system is useful for work on completely isolated neurons, provided several results related to this problem. First, we demonstrated that the behavioural state can produce long-term changes in individual neurons that persist even after neuron isolation from the nervous system. Specifically, we found that pedal serotonergic neurons that control locomotion show higher activity and lower membrane potential after being isolated from the nervous systems of hungry animals. Second, we showed that the modulatory state (the chemical neuroactive microenvironment of the central ganglia) changes in accordance with the nutritional state of an animal and produces predicted changes in single isolated locomotor neurons. Third, we report that observed hunger-induced effects can be explained by the increased synthesis of serotonin in pedal serotonergic neurons, which has an impact on the electrical activity of isolated serotonergic neurons and the intensity of extrasynaptic serotonin release from the pedal ganglia.

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.

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.

Purpose : To study the effect of single, 30-min long, whole-body, homogeneous static magnetic field (SMF)-exposure of magnetic induction 147 +/- 3 mT on the response latency of the snail Helix pomatia.Materials and methods : The response was investigated using the hot plate test.Results : The effect caused by exposure to SMF was compared to sham-exposure and resulted in significant differences (up to 47.1%, p < 0.001). The response latency depended on the day-night cycle; response latency was higher by 51.2% (p < 0.001) during the night. This trend also held for SMF-exposure (28.6%, p < 0.001). Serotonin alone increased response latency (55.7%, p < 0.001), whereas serotonin antagonist tryptamine decreased it (-97.8%, p < 0.001). Using naloxone, response latency decreased (-52.5%, p < 0.001); however both SMF-exposure and serotonin in combination with naloxone rose it back to above the control level (116.9%, p < 0.001 or 150.2%, p < 0.001, respectively).Conclusions : This study provides evidence that SMF-exposure mediates peripheral thermal nociceptive threshold by affecting the serotonerg as well as the opioiderg system.

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 location of cerebral neurons innervating the three recently described flexor muscles involved in the orientation of the posterior tentacles was investigated by applying parallel retrograde Co- and Ni-lysine tracing via the olfactory and the peritentacular nerves. Their innervation patterns in the flexor muscles were studied by applying anterograde neurobiotin tracings via these nerves. The labeled neurons are clustered in eight groups in the cerebral ganglion. They send both common and distinct innervation pathways to the flexor and the tegumental muscles and to the tentacular retractor muscle. The common pathway reaches the muscles via the olfactory nerve, whereas the distinct pathways innervate via the internal and external peritentacular nerves. The three anchoring points of the three flexor muscles at the base of the tentacle outline the directions of three force vectors generated by the contraction of the muscles and enable the protracted tentacle to bend around a basal pivot. In the light of earlier physiological and the present anatomical findings, we suggest that the common innervation pathway to the muscles is required for tentacle withdrawal and the retractor mechanism, whereas the distinct pathways primarily serve the bending of the protracted posterior tentacles during foraging.

We investigated the effect of homogeneous and inhomogeneous static magnetic field (SMF) exposure on the thermal nociceptive threshold of snail in the hot plate test (43 °C). Both homogeneous (hSMF) and inhomogeneous (iSMF) SMF increased the thermo-nociceptive threshold: 40.2%, 29.2%, or 41.7% after an exposure of 20, 30, or 40 min hSMF by p < 0.001, p < 0.0001, or p < 0.001, and 32.7% or 46.2% after an exposure of 20 or 40 min iSMF by p < 0.05 or p < 0.0001. These results suggest that SMF has an antinociceptive effect in snail. On the other hand, naloxone as an atypical opioid antagonist in an amount of 1 μg/g was found to significantly decrease the thermo-nociceptive threshold (41.9% by p < 0.002), which could be antagonized by hSMF exposure implying that hSMF exerts its antinociceptive effect partly via opioid receptors.

The anatomy of three novel flexor muscles in the posterior tentacles of Helix pomatia is described. The muscles originate from the ventral side of the sensory pad and are anchored at different sites in the base of the tentacle stem. The muscles span the tentacle and always take the length of the stem which depends on the rate of tentacle protrusion indicating that the muscles are both contractile and extremely stretchable. The three anchoring points at the base of the stem determine three space axes along which the contraction of a muscle or the synchronous contraction of the muscles can move the tentacle in space.

We investigated the presence and distribution of the D1 dopamine receptor in the CNS of Lymnaea stagnalis applying immunobloting and immunocytochemistry. We also investigated the effect of dopamine as well as the specific D1 receptor blocker, SCH23390, on the firing activity of the feeding modulator serotonergic neuron, CGC, which displayed D1 immunoreactivity. Immunoblot experiments showed one specifically labeled band with 62 kDa mw which is close to that of the mammalian D1 receptor. Neurons displaying D1-like immunoreactivity can be observed in each ganglion of the CNS but particularly in the pedal ganglia which are the center for locomotion. Dopamine regularly evokes burst activity in the serotonergic CGC at 1 mM and this effect could be antagonized by SCH23390. These observations suggest that a D1-like receptor molecule is present in the CNS of Lymnaea.

Termeszetes fitoplankton tomegprodukcio soran gyűjtott mintak es hazai cianobakterium torzsek in vivo toxikologiai elemzese (THAMNOTOX-F, ArTox, DAPHTOX-FTM, ToxAlert 100) ill. in vitro biomarker meresei bizonyitjak, hogy sporadikusan mind a Balatonban mind a Kis-Balaton teruleten megjelenhetnek potencialisan toxikus algatorzsek. A Balatonbol izolalt Cylindrosprmopsis raciborskii torzsek toxikus mechanizmusai a mert biokemiai parameterek (LDH, GST szintek, AchE gatlas) alapjan elternek a cilindrospermopszint termelő ausztral C. raciborskii (AQS) torzs hatasatol, es jobban hasonlitanak az anatoxinokat termelő Oscillatoria (PCC 6505) torzsre. Puhatestű neuronokon kapott elektrofiziologiai eredmenyek (az algakivonatok kolinerg receptorblokkolo, esetenkent AchE gatlo hatasa) is a PCC torzs hatasahoz merhető. In vitro, CHO-K1 sejttenyeszeten vegzett eletkepessegi tesztek es a sejtvaz szerkezeteben kialakulo degenerativ valtozasok rovid-es hosszutavu (feltehetően elterő bioaktiv komponensek vagy mechanizmusok reven) kialakulo citotoxikus hatasokat is kimutattak. Molekularis genetikai vizsgalatok szerint a balatoni C. raciborskii torzsek nem termelnek cilindrospermopszint, es analitikai vizsgalatok sem igazoltak eddig ismert cianotoxinok (anatoxin-a, anatoxin-a(S) vagy szaxitoxinok) jelenletet. Az in vivo es in vitro tesztek szerint okozott toxicitas igy feltehetően egyeb, eddig nem azonositott toxikus metabolitok jelenletenek, illetve ezek szinergista hatasanak koszonhetőek. | Cyanobacterial bloom samples and endogenous cyanobacterial isolates were assessed for environmental risk by a battery of in vivo toxicity tests (THAMNOTOX-F, ArTox, DAPHTOX-FTM, ToxAlert 100) and in vitro biochemical assays of test animals. The results revealed the sporadic appearance of toxic strains of cyanobacteria in the lake Balaton and Kis-Balaton area. Biochemical data (LDH, GST and AchE levels) suggested different toxic mechanisms of the extracts of Cylindrospermopsis raciborskii isolates from the Lake Balaton compared with the cylindrospermopsin producing Australian C. raciborskii strains. The electrophysiological results on identified gastropod neurons showed similar features (cholinegic blocking and AchE inhibiting effects) with the extracts made from the anatoxin-a producing Oscillatoria (PCC 6505) strains. In vitro viability tests and morphological studies on CHO-K1 cell culture separated the short term and long term cytotoxic effects of the C. raciborskii strains, probably due to their different toxic components and target mechanisms. Molecular genetic studies did not prove the presence of cylindrospermopsin, neither analitical analysis showed any of the recently known cyanotoxins (anatoxin-a, anatoxin-a(s), saxitoxin) in the extracts of C. raciborskii strains from Balaton, suggesting that their in vivo and in vitro toxicity is the product of other, recently unidentified toxic substances and their likely synergistic interactions.

In the present study, the ability of a range of endogenous neuropeptides to modulate neuromuscular transmission was examined in the salivary duct neuromuscular preparation of the terrestrial snail, Helix pomatia. Immunohistochemical and physiological techniques were used to localize the neuropeptides (GSPYFVamide, CARP, FMRFamide and APGWamide) and to investigate whether contractions elicited by the stimulation of the salivary nerve or by exogenously applied 5-HT are subject to peptidergic modulation. All of the neuropeptides studied decreased the tonus by a direct action on the muscle fibers in a concentration dependent manner in a range of 10−9 to 10−6 M. Neuropeptides distinctly affected the 5-HT evoked contraction or relaxation and GSPYFVa and APGWa decreased also the amplitude of contractions elicited by the stimulation of the salivary nerve. All four neuropeptides facilitated the relaxation phase providing further evidence for the postsynaptic action of neuropeptides. Low Ca2+/high Mg2+ saline abolished the nerve-elicited contractions, however the denervated muscle retained the ability to contract due to the mobilization of the Ca2+ from intracellular stores. It was concluded, that peptides belonging to different peptide families exerted their effects through pre- and postsynaptic mechanisms. The modulatory effect of neuropeptides can be assigned to the partial co-localization of 5-HT and neuropeptides in the nerves innervating muscles of the salivary duct, as it was demonstrated by double-labeling immunohistochemistry. A double origin of the 5-HTergic innervation was demonstrated, including efferents originating from both the cerebral and visceral ganglia.

PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

The osmotic stimulation which is able to change the behavioral state of the animal are most effective during rainy weather while they are less effective during dry weather conditions. In isolated CNS preparations from aestivated animals the highest firing activity and serotonin sensitivity of the serotonergic (RPas) heart modulator neurons are recorded during rainy weather and the lowest parameters are observed in dry conditions. In aestivated animals the serotonin (5HT) content in both the CNS and the foot is higher than the dopamine (DA) content during rainy weather, while in dry weather the DA level is higher than the 5HT. The inactivation-reactivation process is accompanied by decreasing both the DA and 5HT levels in the CNS and increasing them in the peripheral organs. Our results suggest that aestivated animals adapt to (favorable and unfavorable) environmental conditions by modulating their central arousal state through changing the levels and distribution of monoamines (5HT, DA) in their body.

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia. PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an approximately 14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.