Novel triplet of flexor muscles in the posterior tentacles of the snail, Helix pomatia.

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.

Whole body static magnetic field exposure increases thermal nociceptive threshold in the snail, Helix pomatia.

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.

Immunological and pharmacological identification of the dopamine D1 receptor in the CNS of the pond snail, Lymnaea stagnalis.

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.

The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia.

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.

Humoral serotonin and dopamine modulate the feeding in the snail, Helix pomatia.

We investigated the effect of elevated levels of humoral 5HT and DA on the feeding latency of Helix pomatia, 1 day, 3 days and 10 days following satiation, by injecting monoamines into the haemocoel. HPLC assay of monoamines showed that both 5HT and DA are present in pmol/ml concentrations in the haemolymph of both starved and non-starved animals. Elevated levels of 5HT and DA were most effective at decreasing the feeding latency 10 days following satiation when DA decreased the feeding latency in a concentration dependent manner between 10(-7) and 10(-5) M whereas 5HT levels decreased the feeding latency only at 10(-6) M but increased it at 10(-5) M. Immunocytochemistry revealed that both 5HT3 and D1 receptor-like immuno-reactivity are present in cell bodies located in the same areas of the buccal ganglia. Our observations suggest that both humoral DA and 5HT mutually modulate the activity of the feeding CPG through neurons which have these receptors.

The terrestrial snail, Helix pomatia, adapts to environmental conditions by the modulation of central arousal.

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.

Topographic organization of serotonergic and dopaminergic neurons in the cerebral ganglia and their peripheral projection patterns in the head areas of the snail Helix pomatia.

The distribution of monoaminergic neurons within the cerebral ganglia was investigated in the pulmonate snail Helix pomatia. Simultaneous serotonin and tyrosine hydroxylase double immunostaining revealed that the immunoreactive cell groups are concentrated in a putative monoaminergic center on the ventral surface of the cerebral ganglia. Simultaneous cobalt (Co)- and nickel (Ni)-lysine backfills of cerebral nerves were combined with 5, 6-dihydroxytryptamine pigment-labelling of serotonergic neurons, or with fluorescence immunocytochemistry of dopaminergic neurons. This showed that the serotonergic and dopaminergic cell groups can be divided into smaller subgroups on the basis of their axonal projections into different cerebral nerves. These subgroups show a topographic organization within the serotonergic and dopaminergic neuronal clusters. In the serotonergic system, the different regions of the head are represented in a rostrocaudal direction, whereas a caudorostral organization is characteristic for the dopaminergic system. No serotonin- or dopamine-immunoreative cell bodies but numerous fibers were observed in the head areas, indicating that these are innervated by cerebral monoaminergic neurons and show different innervation patterns. Serotonin-immunoreactive fibers mostly innervate muscle fibers, whereas dopamine-immunoreactive processes do not innervate effector cells, but terminate within the nerve branches of the head areas. On the basis of their innervation pattern, we suggest that dopaminergic neurons may take part in en route modulation of sensory afferent and efferent processes in an as yet unknown manner. The serotonergic neurons, on the other hand, may play a direct role in the modulation of muscle function.

Peptidergic neurons in the snail Helix pomatia: distribution of neurons in the central and peripheral nervous systems that react with an antibody raised to the insect neuropeptide, leucokinin I.

In this study, an antiserum raised against an insect myotropic peptide, leucokinin I (DPAFNSWGamide), was used for mapping leucokinin-like immunoreactive (LK-LI) neurons in the gastropod mollusc, Helix pomatia. Immunocytochemistry performed on both whole-mounts and cryostat sections demonstrated LK-LI neurons in all ganglia of the central nervous system (CNS), except the visceral ganglion. Altogether about 700 immunolabelled neurons have been found, with nearly one-half (46%) in the cerebral ganglia. A large proportion of the LK-LI neurons have small cell bodies and are likely to be interneurons. The most prominent LK-LI cell group is represented by the entire neuron population of the mesocerebri, which is the major source of a thick fiber bundle system, encircling and innervating the whole CNS. One single LK-LI giant neuron was found, which is located in the left pedal ganglion and is termed GLPdLKC (giant left pedal leucokinin immunoreactive cell). This cell has not been identified previously. The ganglion neuropils are heavily innervated by varicose LK-LI fiber arborizations. Some integrative centers, such as the medullary neuropil of the procerebri, reveal an extreme density of LK-LI innervation. All major peripheral nerves contain a large number of LK-LI axons, and LK-LI innervation is found in the musculature of different peripheral organs (buccal mass, lip, tentacles, oviduct, intestine). Among the peripheral organs investigated, the intestine contains a rich varicose LK-LI network, composed of both intrinsic and extrinsic elements. Radioimmunoassay (RIA) demonstrates a very high content of LK-LI material in Helix ganglion extracts (about 50 pmol/CNS). This is the first report on the occurrence of a substance resembling the myotropic neuropeptide leucokinin I in a phylum outside arthropods. Based on our immunocytochemical observations, a role for leucokinin-like peptides in both central and peripheral regulatory processes in Helix is suggested. According to double-labelling experiments, only a small number of the LK-LI neurons are labelled with an antibody to the vertebrate tachykinin substance P.

Topographic organization of efferent neurons with different neurochemical characters in the cerebral ganglia of the snail Helix pomatia.

This study provides a description of the organization of neurons efferent to different head areas in the cerebral ganglia of Helix pomatia, revealed by simultaneous Ni-lysine and Co-lysine back-filling of different pairs of cerebral nerves. The backfills show that labeled cerebral neurons that innervate the head areas are concentrated in seven representation foci distributed in different parts of the cerebral ganglia. Almost each head area is represented in each focus. At a gross level, the representation of the different head areas in the representation foci shows a topographic arrangement. Each focus is constituted by neurochemically different groups of neurons. All head areas are innervated by serotonin-containing fibers from a single focus (Focus 2) and by dopamine-containing fibers from Foci 1, 2, and 4. However, they are innervated by CARP and FMRFamide-containing fibers from all of the foci. The combination of retrograde labeling with 5, 6-dihydroxytriptamine induced pigment labeling of serotonin-containing neurons or with fluorescence tyrosinehydroxylase immunocytochemistry to detect dopamine-containing neurons showed that the different head areas are topographycally represented in the clusters of both the serotonin- and dopamine-containing cells. The combination of Ni-lysine backfillings from different cerebral nerves with fluorescence CARP and FMRFamide immunocytochemistry revealed that the head areas are represented also in both the CARP and FMRFamide immunoreactive groups of neurons in the different foci.

Ultrastructural, biochemical and electrophysiological changes induced by 5,6-dihydroxytryptamine in the CNS of the snail Helix pomatia L.

The serotonin neurotoxin, 5,6-dihydroxytryptamine (5,6-DHT), was injected into the body cavity of snails. Changes induced in the central nervous system (CNS) by the neurotoxin were studied by morphological, electrophysiological and biochemical techniques for up to 90 days following injection. The neurotoxin induced a variety of ultrastructural alterations during the early phase (1st to 6th days) following treatment. On day 6 after treatment, membranous structures first appeared in the synaptic-like areas and apparently migrated to cell bodies where they were detected by day 14. Their number increased with time. Neurotoxin-induced structural alterations were found in neuronal processes and cell bodies of the serotonergic metacerebral giant cells injected intracellularly with horseradish peroxidase and in serotonin immunoreactive axons. These findings suggest that the toxin-induced alterations are rather selective for the serotonin-containing neuronal elements. The neurotoxin decreased the concentration of 5-HT in and [3H]5-HT uptake into cerebral and pedal ganglia, with a maximum effect between the 3rd and 5th day following drug administration. 5-HT levels and 5-HT uptake returned to normal by 19-21 days after treatment. The concentration of dopamine and of [3H]DA uptake capacity were reduced between 3-5 days after injection of 5,6-DHT by 6-7 days following treatment. The transmission from identified serotonergic synapses to targets was reduced beyond day 5 after 5,6-DHT administration. By 15 days after treatment, synaptic transmission between the metacerebral giant cell (MGC) and buccal followers was blocked. Transmission recovered by day 21 after 5,6-DHT. Comparison of the time-course of functional and structural recovery indicates that while functional recovery takes place within 21 days after treatment, certain structural alterations, e.g. the membranous structures and dense particles, remain in the nerve fibres and cell bodies. These may serve as specific intracellular markers of the serotonin-containing neuronal elements long after functional recovery from the effect of 5,6-DHT.

A new double labelling technique of the serotonergic neurons in wholemount CNS preparations of the snail Helix pomatia.

5,6-dihydroxytryptamine-induced pigmentation of serotonergic neurons was combined with retrograde cobalt- or nickel-lysine labelling in order to identify serotonergic cell bodies among cobalt- or nickel-labelled ones. Cobalt- or nickel-labelled serotonergic cell bodies could easily be distinguished from labelled, nonserotonergic neurons. The nonserotonergic cobalt-labelled neurons have only orange, whereas the nickel-labelled neurons have only blue colorization in their somata, while the labelled serotonergic neurons have dark pigment granules in their somata besides the blue or orange colorization. By this double labelling technique the central serotonergic neurons which send processes to different body regions through CNS nerves, can be identified in wholemount preparations.

Somatotopic representation of the head areas in the cerebral ganglion of the snail Helix pomatia.

This study gives a detailed description of the central representation of the head regions in the cerebral ganglion after parallel nickel-lysine and cobaltic-lysine backfilling of different pairs of cerebral nerves. The backfilling of the cerebral nerves demonstrate different groups of labelled neurons in the cerebral ganglion as in the procerebrum, mesocerebrum, metacerebrum, as well as in the pleural, pedal and commissural lobes of the postcerebrum. Each head area except the anterior and posterior tentacle is represented in each labelled cell group. The tentacles are not represented in the mesocerebrum and the pedal lobe of the post-cerebrum. The different head areas in the procerebrum, metacerebrum and the pleural lobe are represented in fronto-caudal a somatotopic order.

Peptidergic and aminergic centers in the Helix cerebral ganglia: somatotopy and immunocytochemistry.

In Helix cerebral ganglia the relationship between location of serotonin, dopamine, CARP, FMRFamide, Met-enkephalin, and substance P immunoreactive neurons and the different representation foci of the head areas was compared. The majority of immunoreactive neurons was located in representation foci, however, the extension of immunoreactive centers are larger than the representation foci. Therefore, the real extension of a representation focus with the additional non-efferent neurons might be larger than revealed by retrograde labelling.

Neurons with different immunoreactivity form clusters in the CNS of Helix pomatia.

The distribution of serotonin (5HT-ir), FMRF amide (FMRF a-ir), catch-relaxing-peptide (CARP-ir), dopamine (DA-ir), gamma-amino-butyric-acid (GABA-ir), and leucokinin (LK-ir) immunoreactive neurons were compared in the ganglia of Helix CNS. These neurons are not distributed randomly, but their location outlines distinct groups in the ganglia. In a few groups only DA-ir, GABA-ir and LK-ir neurons can be seen, whereas in the majority of groups FMRFa-ir, CARP-ir and 5HT-ir neurons are localized together. In the latter groups of immunoreactive neurons either FMRFa- and CARP- or 5HT- and FMRFa-immunoreactivities coexist in numerous neurons. Immunoreactive groups composed of DA-ir, GABA-ir and LK-ir neurons are localized exclusively in the areas of the origin of skin nerves, suggesting that these neurons are related to the processing of cutaneous afferent information. Other groups constituted by 5HT-ir, FMRFa-ir and CARP-ir neurons are localized first of all in ganglia the neurons of which innervate large masses of muscle fibers. The primary role of these neurons in motor/efferent processes is assumed.

Mercury and cadmium induced structural alterations in the taste buds of the fish Alburnus alburnus.

The ultrastructural damages of the taste buds of the fish, Alburnus alburnus were studied after applying 0.05 microM and 0.5 microM mercury chloride as well as 0.1 microM and 1 microM cadmium chloride. The most conspicuous alterations were induced during the first week of heavy metal exposition. The main structural alterations are: 1) the swelling of sensory microvilli and cilia; 2) the extreme dilation of the rER tubules and nuclear membranes, which is most expressed after cadmium exposition; 3) the increase in the number of lysosomes and dens bodies, which is more expressed after mercury exposition; 4) the swelling of the innervating nerve fibres at the synaptic areas of the taste buds, especially after mercury exposition. The damaging processes induced by the applied dose of heavy metals did not increase after the first week of exposition. The taste buds showed regenerated structural appearance after two weeks of exposition to 1 microM CdCl2, while the evoked structural alterations could be detected even after two weeks of exposition to 0.5 microM HgCl2.

Input and output organization of cerebral neurones responding to tactile and taste stimuli applied to the lip of Helix pomatia L.

Mechano- and chemoafferent responsiveness as well as outputs of identified cerebral neurones were investigated by electrophysiological methods in Helix pomatia L. the axonal projections of the identified cells were studied by intracellular staining. The studied neurones proved to be unipolar, their main axon branches were found in ipsilateral lip nerves. They could be divided into several groups according to their spontaneous activity, input and output organization and the selectivity of their responses to different tactile and taste stimuli applied to the lip. The activity of most of the neurones could be influenced by both ipsi- and contralateral inputs. They receive afferent input mostly through the medial lip nerves and their efferent information is transferred to the periphery mainly through the pair of inner lip nerves. There were seven neurones among the identified cells which responded selectively to taste stimuli identified in previous behavioural tests as phagostimulants. They can be considered as elements of the cerebral system regulating taste discrimination and feeding.

Central representation and functional connections of afferent and efferent pathways of Helix pomatia L. lip nerves.

Localization and distribution of cerebral neurones sending axons into the three pairs of Helix pomatia lip nerves were investigated by the method of retrograde axonal NiCl2 transport. Using electrophysiological technics (extracellular recordings) the dependence of lip nerve's activity on inputs of other lip nerves was studied after application of various types of stimuli to the lip of semi-intact preparations. All lip nerves have neuronal representation in each lobe of the cerebral ganglia but in different proportions. Labelled neurones were located mainly on the ventral surface of the cerebral ganglia, most of them projecting to the medial, the least to the inner lip nerve. Lip nerves differ from each other according to the proportions of neurones of various size. They share in the axons of large (55-70 microns) and medium sized (30-40 microns) neurones in the order inner greater than outer greater than medial and medial greater than outer greater than inner lip nerve, respectively. Most neurones projecting to different nerves are located in discrete groups. According to the electrophysiological results the medial lip nerve has the most prominent afferent, while the inner one has the strongest efferent activity. Both the afferent and efferent activities of the outer lip nerve proved to be the least significant compared to the other lip nerves. Contralateral cerebral connections play an important role in the sensory information processing. The sensory input of a given nerve usually activates the contralateral member of another pair of lip nerves. Mechano- and chemo-afferent pathways have almost the same properties but there are some differences in latencies and other parameters.

The presence and specificity of crustacean cardioactive peptide (CCAP)-immunoreactivity in gastropod neurons.

CCAP-like immunoreactivity was detected in central neurons with small and medium diameters in both Helix and Lymnaea CNS. The intensity of immunoreactivity showed seasonal changes with a maximum intensity during spring. The overwhelming majority of nerve cell bodies exhibiting CCAP immunoreactivity is located in the cerebral and parietal ganglia of both Helix and Lymnaea. The neurons of pleural and buccal ganglia were devoid of CCAP-immunoreactivity. Following preabsorbtion of CCAP antibody in 1:15000 dilution with 10(-3) M CCAP or CCAP-related peptide (Helix -CCAP), immunoreactivity could not be observed in neurons, demonstrating the specificity of the antibody to CCAP-related molecules in both Helix and Lymnaea.

Characterization of catch-relaxing peptide (CARP) immunoreactive neurons in the Helix nervous system.

Both small and large diameter of CARP immunoreactive neurons could be observed in the different ganglia of CNS of Helix but not in the foot musculature. The immunoreactivity is the strongest in the varicose segments of immunoreactive fibers. The present findings suggest a transmitter or modulatory role of CARP in both central and peripheral regulatory processes.

Serotonergic input on identified command neurons in Helix.

In Helix, serotonin evokes long-lasting alteration of activity of withdrawal triggering neurons. Cell bodies of these neurones are surrounded by a dense network of serotonin-containing fibres without any synaptic membrane specializations, which confirm the suggestion on the non-synaptic, modulatory action of serotonin on the withdrawal command elements.