An octopaminergic system in the CNS of the snails,Lymnaea stagnalis and Helix pomatia

Octopamine (OA) levels in each ganglion of the terrestrial snail, Helix pomatia, and the pond snail, Lymnaea stagnalis, were measured by using the HPLC technique. In both species an inhomogeneous distribution of OA was found in the central nervous system. The buccal ganglia contained a concentration of OA (12.6 pmol mg^-1 and 18.8 pmol mg^-1) that was two to three times higher than the pedal (4.93 pmol mg^-1 and 9.2 pmol mg^-1) or cerebral (4.46 pmol mg^-1 and 4.9 pmol mg^-1) ganglia of Helix and Lymnaea, respectively, whereas no detectable amount of OA could be assayed in the visceroparietal complex. In Lymnaea ganglia, the OA uptake into the synaptosomal fraction had a high (K_m1 = 4.07 ± 0.51 μM, V_max1 = 0.56 ± 0.11 pmol mg^-1 per 20 min), and a low (K_m2 = 47.6 ± 5.2 μM, V_max2 = 4.2 ± 0.27 pmol mg^-1 per 20 min), affinity component. A specific and dissociable ³H-OA binding to the membrane pellet prepared from the CNS of both Helix and Lymnaea was demonstrated. The Scatchard analysis of the ligand binding data showed a one-binding site, representing a single receptor site. The K_d and B_max values were found to be 33.7 ± 5.95 nM and 1678 ± 179 fmol g^-1 tissue in Helix and 84.9 ± 17.4 nM and 3803 ± 515 fmol g^-1 tissue in Lymnaea preparation. The pharmacological properties of the putative molluscan OA receptor were characterized in both species and it was demonstrated that the receptor resembled the insect OA₂ rather than to the cloned Lymnaea OA receptor. Immunocytochemical labelling demonstrated the presence of OA-immunoreactive neurons and fibres in the buccal, cerebral and pedal ganglia in the central nervous system of both species investigated. Electrophysiological experiments also suggested that the Lymnaea brain possessed specific receptors for OA. Local application of OA onto the identified buccal B2 neuron evoked a hyperpolarization which could selectively be inhibited by the OAergic agents phentolamine, demethylchlordimeform and 2-chloro-4-methyl-2-(phenylimino)-imidazolidine. Among the dopamine antagonists, ergotamine reversibly inhibited the OA response, whereas sulpiride had no effect. Based on our findings, a neurotransmitter-modulator role of OA is suggested in the gastropod CNS.     

A histoenzymatic investigation of galactogen synthesis in the albumen gland of Helix pomatia and Lymnaea stagnalis

Summary A histoenzymatic investigation of Galaotogen synthesis has been attempted in the albumen gland of Helix pomatia and Lymnaea stagnalis. Using histoenzymatic methods the authors succeeded in finding only two enzymes (E.C. 2.7.7.9.=uridylyltransferase and E. C. 5.1.3.2.=epimerase) demonstrated by biochemical assays and isolated. They therefore assume that there may be other metabolic pathways in addition to those demonstrated by biochemical methods.     

Pigment-dispersing hormone-like immunoreactive neurons in the central nervous system of the gastropods, Helix pomatia and Lymnaea stagnalis

By using an antiserum raised against a crustacean #-pigment-dispersing hormone (PDH), the distribution and chemical neuroanatomy of PDH-like immunoreactive neurons was investigated in the central nervous system of the gastropod snails, Helix pomatia and Lymnaea stagnalis. The number of immunoreactive cells in the Helix central nervous system was found to be large (700-900), whereas in Lymnaea, only a limited number (50-60) of neurons showed immunoreactivity. The immunostained neurons in Helix were characterized by rich arborizations in all central ganglia and revealed massive innervation of all peripheral nerves and the neural (connective tissue) sheath around the ganglia and peripheral nerve trunks. A small number of Helix nerve cell bodies in the viscero-parietal ganglion complex were also found to be innervated by PDH-like immunoreactive processes. Hence, a complex central and peripheral regulatory role, including neurohormonal actions, is suggested for a PDH-like substance in Helix, whereas the sites of action may be more limited in Lymnaea.     

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 fine structural organization of sensory nerve endings in the lip of Helix pomatia L.

The fine structural and cytochemical characteristics of sensory nerve cells have been studied in the lip of Helix pomatia. A ruthenium red positive cuticular layer was found on the surface of the sensory epithelium. Among the undifferentiated epithelial cells two types of sensory dendrites were observed, namely ciliated and non-ciliated ones. A large amount of smooth surfaced endoplasmic reticulum, microtubes and ribosomes were present in the neuroplasm of the sensory dendrites. However, rough surfaced endoplasmic reticulum, mitochondria, and electrodense bundles of long filaments were characteristic in the simple epithelial cells. The cell bodies of the sensory dendrites lie subepithelially among the muscle cells and they generally contain empty or dense core vesicles.     

The ultrastructure of the sensory cells in the chemoreceptor of the ommatophore of Helix pomatia L.

Most of the sensory cells found in the chemoreceptor of the ommatophore of Helix pomatia are typical bipolar cells. The chemoreceptor is deveded by a furrow into two parts; within the ventral subdivision the layer of sensory cell bodiesis thicker than in the dorsal part. According to the differentiations of the apical surface of the dendrites, it is possible to distinguish six different classes: a) dendrites with one cilium and 75 nm thick cytofila (sometimes dendrites of identical appearance posses more than one cilium); b)dendrites with several cilial and 150 nm thick cytofila; c) dendrites with several cilia, 50 nm thick cytofila, and long, striated rootlets; d) dendrites with several cilia bur without cytofila; e) dendrites with 130 nm thick cytofila but without cilia; and f) dendrites with 65 nm thick cytofila but without cilia; dendrites of this class are the only ones with a cytoplasm more electron dense than that of the surrounding supporting cells. All these dendrites are connected to the surrounding supporting cells by terminal bars, each consisting of zonula adhaerens, aonula intermedia and zonula septata. The perikarya of the sensory cells measure approximately 15 mum by 8 mum and enclose 10 mum by 6 mum large nuclei. Axons, originating from these perikarya, extend to the branches of the digital ganglion. In the distal part of this gangloin the axons come into synaptic contact with interneurons, but in our electron micrography it was not possible to coordinate processes and synapses with the corresponding neurons.     

Analysis of the Neural Network Regulating the Cardio-Renal System in the Central Nervous System of Helix pomatia L.

In the central nervous system of Helix pomatia 22 cells takingpart in the regulation of the cardio-renal system have beenidentified. These cells are scattered throughout the visceraland right parietal ganglia. Among identified cells sensory,motor, and interneurons were found and the hierarchical characterof the network was stated. The network regulating heart activitywas found to be of the over-guaranteed, convergent type, wherethe inputs predominate and the outputs form independent, parallelpathways, being coordinated by interneurons. This neural networkcan be divided into two levels: collectors and coordinators,which are responsible for the conduction or analysis of theafferent inputs, respectively. The localization of identifiedcells was studied by intracellular and retrograde injectionof CoCl₂. Primary sensory cells were found to be bipolar, motoneuronesand interneurones were unipolar or pseudo-unipolar with richarborization within the ganglia. Interneuron V21 showed a phasic or tonic pattern of firing.The phasic activity of the neuron V21 appeared as a burst correlatedwith the individual heartbeats. The tonic pattern of firingwas caused by activation of various inputs of the cardio-renalsystem and led always to the stopping of the heartbeat. Thenetwork regulating the heartbeat was organized around the interneurones.In the activity pattern of the interneurones the origin of theinput was not distinguishable; however, on the middle levelof the system it can be verified. These cells play a role inthe storage of information originating Irom various receptorareas. According to this the regulatory network can be dividedinto subsystems.     

Processing of mechano- and chemosensory information in the lip nerve and cerebral ganglia of the snail Helix pomatia L.

Neurophysiologists have long been seeking out simple model systems in which to analyse the neuronal mechanisms underlying the organisation of behaviour. The feeding behaviour of molluscs has proved to be one of the most useful simple systems for the analysis of cyclical motor patterns, the interactions of central pattern generating interneurones and the role of sensory inputs in the initiation and maintenance of the behaviour. Considerable progress has been made in one or both of the first two aspects of this research in Lymnaea, Helisoma, Limax, Planorbarius, Pleurobranchaea and Tritonia (for reviews see [3, 7, 8, 15]) and more recently, in Aplysia [39] and Planorbis [1]. The role of mechano- and chemosensory inputs in the organisation of the feeding behaviour was studied in at least twenty molluscan species (for a review see [3]). However, in only less than half of them was the analysis extended to the effect of tactile and chemical inputs on identified neurones in the buccal and cerebral ganglia which contain the feeding circuitry (Aplysia: [12, 22, 36, 41]; Pleurobranchaea: [9, 16, 17]; Tritonia: [2]; Helisona: [21]; Limax: [11, 14, 35]; Helix: [6, 19, 24-26, 32, 38]). In present chapter I would like to review our earlier findings on the processing of mechano- and chemosensory information in the lip nerves and cerebral ganglia of Helix pomatia L. These findings were published in a series of papers between 1982 and 1987 [19, 20, 24-26]. The results reviewed here prepared the way for the development of new lines of research in our laboratory on the plasticity and serotonergic modulation of feeding in this widely used experimental animal [27, 40].     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

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.     

Functional characteristics of an identified pair of neurones in the CNS of the pond snail (Lymnaea stagnalis L.).

The properties of 2 giant electrically coupled neurones (A10 and P1) identified in the visceral and right parietal ganglia of Lymnaea stagnalis were examined. The active and passive electrical parameters of the neurones, as well as the junction between them were measured. The main peripheral and interneuronal connections of the neurones were demonstrated using both electrophysiological and morphological methods. It is shown that the coupled cells are not neurones of the same function, but they are asymmetrical ones. This finding is supported by the following results: (1) the axonal pathways of neurones A10 and P1 are different; (2) there are significant differences in their afferent and efferent connections; (3) though the electrical junction between them is bidirectional, the junctional electrical characteristics prefer P1-A10 transmission. According to the electron microscopic results both neurones are possible neurosecretory cells. The differences demonstrated between the 2 giant neurones may have significance concerning their role in a special neuronal network.     

Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the shell by way of an adhesive substance containing proteins and mucopolysaccharides.This research was made possible by a grant from the Netherlands Organization for Pure Research (Z.W.O.)     

Environmental osmolarity and neurosecretory neurones in Lymnaea stagnalis (L.).

Cell body volume and Alcian blue-Alcian Yellow staining properties of neurosecretory neurones in the brain of Lymnaea stagnalis were compared for snails kept in de-ionised water and standard tapwater. In the same experiment, the ionic content of the blood, blood volume and body weight and environmental ionic composition were measured. Five days of immersion in de-ionised water resulted in significant decreases in body weight, blood volume and blood, Na+ and Cl- concentrations but no change in blood Ca2+, K+,and HCO3- concentrations, compared with controls. No consistently significant differences across the 5 day period were found in cell body volumes for Dark Green Cells, Yellow Cells or Light Green Cells (used as a control) when these volumes were compared for large numbers of cells from snails kept in de-ionised water and standard tapwater. However, the number of Yellow Cells which could be counted in snails kept in de-ionised water was lower than the number from standard tapwater by day 2 of the experiment and lower for Yellow-green Cells by day 5. We interpret this lower number to be the result of depletion of Alcian blue-Alcian Yellow stained neurosecretory material in these cells which made them impossible to distinguish. This was confirmed by examination of visceral Yellow Cells which could be identified on the basis of known location close to the visceral-right parietal connective.     

Tales of two snails: sexual selection and sexual conflict in Lymnaea stagnalis and Helix aspersa

Sexual selection and sexual conflict have been shown to playkey roles in the evolution of species with separate sexes. Experimentalevidence is accumulating that this is also true for simultaneoushermaphrodites. For example, many species of land snails forcefullystab their mating partners with love darts. In the brown gardensnail (Helix aspersa, now called Cantareus asperses), this dartincreases sperm storage and paternity, probably via the transferof an allohormone that inhibits sperm digestion. A recent interspeciescomparison of dart-possessing land snails revealed coevolutionbetween darts and spermatophore-receiving organs that is consistentwith counteradaptation against an allohormonal manipulation.The great pond snail (Lymnaea stagnalis) seems to use a seminalproduct to manipulate its partner and mates in the male rolewhen enough seminal fluid is available in the prostate gland.Receipt of semen not only initiates egg laying in virgin animals,but also feminizes the mating partner later in life. These increasesin the female function have been shown to go at the expenseof growth and seminal fluid production of the sperm recipient.Although in Helix, and probably also Lymnaea, the sperm donorbenefits from the induced changes through increased fertilizationsuccess, the sperm recipient may experience injury, imposedreallocation of resources, and altered sperm storage. Thesefindings support the existence of sexual conflict in simultaneouslyhermaphroditic snails, and its importance for the evolutionof mating behaviors and reproductive morphologies is discussed.     

Formation of basal bodies in the ciliary epithelium of molluscs Buccinum undatum L. and Lymnaea stagnalis L

Electron microscopic studies of the leg ciliary epithelium was carried out in two mollusks. In the epithelium of the leg of adult animals, the centrioles were mostly formed de novo with participation of deuterosomes during the formation of basal bodies. Transformation of the centriolar cylinder in a mature basal body is accompanied by the cylinder elongation and appearance of pericentriolar structures, such as rootlet system, basal legs, and basal plate. Centriolegenesis proceeds in both ciliate and nonciliate (with microvilli) cells of the epithelium. It has been proposed that the cell with microvilli represent a transitional stage in differentiation of the ciliary cells.     

Formation of Basal Bodies in Ciliary Epithelium of Molluscs Buccinum undatum L. and Lymnaea stagnalis L.

Electron microscopic studies of the leg ciliary epithelium was carried out in two mollusks. In the epithelium of the leg of adult animals, the centrioles were mostly formed de novo with participation of deuterosomes during the formation of basal bodies. Transformation of the centriolar cylinder in a mature basal body is accompanied by the cylinder elongation and appearance of pericentriolar structures, such as rootlet system, basal legs, and basal plate. Centriolegenesis proceeds in both ciliate and nonciliate (with microvilli) cells of the epithelium. It has been proposed that the cell with microvilli represent a transitional stage in differentiation of the ciliary cells.     

The dependence of thermopreferendum in Helix pomatia L. on air temperature

Behavioural tests with 192 specimen of the roman garden snail Helix pomatia L. were performed in order to clarify whether the thermopreferendum of this pulmonate is influenced not only by the temperature of the substratum but also by air temperature. For this purpose, the snails were exposed to a horizontal temperature gradient of the substratum at different air temperatures between 10 and 30 degrees C. The results show that the selection of substratum temperature depends on the air temperature: the lower the air temperature, the lower is the temperature of the substratum preferred by the snails. Obviously, the animals tend to keep the difference between air temperature and temperature of the substratum as small as possible. This behaviour might be a mechanism to reduce the temperature gradient in the snail's body and to adjust the metabolic rate to cardiac output.