Structural and functional characterization of neuropeptides involved in the control of male mating behavior of Lymnaea stagnalis.

Mating as a male in the simultaneous hermaphrodite freshwater snail, Lymnaea stagnalis, comprises a series of complex behaviors that are a prelude to copulation. Copulatory behavior itself is assumed to be controlled by various types of peptidergic neurons as well as serotonergic cells. Here we report the primary structure of two peptides that were extracted from a cluster of neurons that innervates the penial complex and that is located in the anterior lobe of the right cerebral ganglion. The sequences of the peptides were determined as: Ala-Pro-Gly-Trp-amide and Ser-Gly-Ser-Asp-Tyr-Cys-Glu-Thr-Leu-Lys-Glu-Val-Ala-Asp-Glu-Tyr-Ile-Leu- Leu- Ser-Tyr-Lys-Ile-Glu-Glu-Gln-Arg-Ala-Ala-Asp-Cys-Gly-Gly-Glu-Pro-Pro-Asn- Ser- Gln(amide), respectively. The longer peptide is a homodimer. Both peptides are processed from the recently identified Ala-Pro-Gly-Trp-amide prohormone, which is expressed in the neurons of the anterior lobe of the right cerebral ganglion. Ala-Pro-Gly-Trp-amide could also be recovered from the penial complex. This peptide, when applied in vitro, inhibits the contractions of the penis retractor muscles evoked by serotonin in a dose-dependent fashion.     

Immuno-electron microscopy of sorting and release of neuropeptides in Lymnaea stagnalis

Summary The cerebral caudodorsal cells of the pulmonate snail Lymnaea stagnalis control egg laying and egglaying behavior by releasing various peptides derived from two precursors. The biosynthesis, storage, intracellular breakdown and release of three caudodorsal cell peptides were studied by means of immuno-electron microscopy using antisera raised to fragments of these peptides: (1) Caudodorsal Cell Hormone-I (CDCH-I; derived from precursor I), (2) Caudodorsal Cell Hormone-II (CDCH-II; from precursor II), and (3) -Caudodorsal Cell Peptide ( CDCP; from both precursors). After affinity purification of the antisera, the specificity of the sera was confirmed with dotting immunobinding assays. From the ultrastructural immunocytochemical data it has been concluded that the precursor molecules are cleaved at the level of the Golgi apparatus after which the C-terminal parts (containing CDCP) and N-terminal parts (containing CDCH-I or CDCH-II) are sorted and preferentially packaged into large electron-dense granules (MD 150 nm), respectively. Very probably, the content of the large electron-dense granules is degraded within the cell body. The immunoreactivity of the secretory granules increases during discharge from the Golgi apparatus, indicating further processing. At least a portion of the secretory granules contains all three peptides, as shown by double and triple immunopositive stainings whereas other granules appear to contain only one or two of these peptides. The caudodorsal cells release multiple peptides via exocytosis from neurohemal axon terminals into the hemolymph and from blindly ending axon collaterals into the intercellular space of the cerebral commissure (nonsynaptic release).     

Respiratory behavior in the pond snail Lymnaea stagnalis

Summary This study describes the neural basis of respiratory behavior in a pulmonate mollusc, Lymnaea stagnalis. We describe and identify muscles of the respiratory orifice (pneumostome) and mantle cavity as well as relevant motor neurons innervating these muscles. All of these identified motor neurons are active during spontaneously occurring respiratory behavior and a sporadically occurring synaptic input, termed Input 3, controls the activities of these motor neurons. This spontaneous input can also be recorded from isolated brain preparations, suggesting that the respiratory motor program is generated centrally. However, evidence is also presented that in semi-intact preparations the role of peripheral feedback is important for the initiation and termination of respiratory behavior in Lymnaea.     

The effect of hydroxyurea on ciliogenesis in ciliary epithelium of the mollusk Lymnaea stagnalis

Electron-microscopy study of the ciliary epithelium structure of the mollusk Lymnaea stagnalis was carried out under the action of hydroxyurea. By the method of radioautography, a high proliferative activity of the ciliary epithelium was established as the norm; a cluster distribution of cells, including the label, was noted. The presence of hydroxyurea in the mollusk organism was shown to inhibit proliferation. Scanning electron microscopy of the molluskan foot surface revealed clusters of nonciliated cells and of cells with short villi in control epithelial folds. Under hydroxyurea treatment for 24 h, such sites disappeared completely and ciliary epithelium looked uniform and was composed of cells with long cilia. By transmission electron microscopy, it was established that hydroxyurea did not affect the formation of the basal body and course of ciliogenesis. It has been suggested that hydroxyurea not only inhibits proliferative activity of epithelial cells, but also induces differentiation of unciliated into the ciliated cells.     

Peptidergic modulation of male sexual behavior in Lymnaea stagnalis: structural and functional characterization of -FVamide neuropeptides

In the simultaneous hermaphrodite snail Lymnaea stagnalis, copulation as a male is controlled by neurons that send axons to the male copulatory organs via a single penis nerve. Using direct mass spectrometry of a penis nerve sample, we show that one of the molecular ions has a mass corresponding to GAPRFVamide, previously identified from the buccal ganglia, and named Lymnaea inhibitory peptide (LIP). The identity of this peptide is confirmed by partial peptide purification from the penis nerve, followed by post source decay mass spectrometry. We cloned the LIP-encoding cDNA, which predicts a prohormone that gives rise to five copies of LIP (now re-named LIP A), two other -FVamide peptides (LIPs B and C), and five structurally unrelated peptides. The LIP gene is expressed in neurons of the right cerebral ventral lobe that send their axons into the penis nerve. We show that the LIP A peptide is present in these neurons and in the penis nerve, and confirmed the presence of LIP B and C in the penis nerve by post source decay mass spectrometry. Finally, we demonstrate that LIP A, B and C inhibit the contractions of the penis retractor muscle, thereby implicating their role in male copulation behavior.     

Detection of biogenic monoamines in the developing nervous system of Lymnaea stagnalis mollusk embryos]

Formaldehyde-induced fluorescence of intraneuronal monoamines can be demonstrated in the Lymnaea embryos from the "late veliger" stage on. Green specific fluorescence indicating the presence of a primary catecholamine occurs in two paired formations which contain a mass of fibres and varicosities. The formations are supposed to correspond to cerebral and pedal ganglia. Single fibres of the same type can be seen in the foot and other organs of the embryo.     

Seasonal variation in the content of free reducing sugars in body fluids of freshwater mollusk Lymnaea stagnalis

Accumulation of certain mono-and disaccharides in body fluids of ectotherms in autumn and winter suggests their protective role in adaptation to near-zero temperatures. In this context, the effect of seasonal temperature variation on the content of free reducing sugars was studied in freshwater snail Lymnaea stagnalis. The summer and autumn carbohydrate spectrum essentially featured high concentrations of fructose and glucose. After 2.5-month hypobiosis (4°C), the concentration of fructose and glucose decreased 35 and 12 times, respectively. Maltose and other disaccharides (presumably cellobiose) undetectable in summer became detectable in autumn before hypobiosis started. The subsequent level of maltose increased to almost reach the sharply decreased glucose level and to exceed the fructose level after 2.5-month hypobiosis. The involvement of maltose in the mechanisms of L. stagnalis adaptation to near-zero temperatures as a membrane protectant is proposed.     

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.     

Ultrastructural changes in the MP3 neuron of the mollusk Lymnaea stagnalis after cryopreservation of the isolated brain

Investigation of a possibility of long-term storage of frozen (-196 degrees C) viable neurons and nervous tissue is one of the central present day problems. In this study ultrastructural changes in neurons of frozen-thawed snail brain were examined as a function of time. We studied the influence of cryopreservation, cryoprotectant (Me2SO), cooling to 4-6 degrees C, and a prolonged incubation in physiological solution at 4-6 degrees C on dictyosomes of Golgi apparatus, endoplasmic reticulum (ER) cisternae and mitochondria. It has been found that responses of these intracellular structures of cryopreserved neurons to the above influences are similar: dissociation of Golgi dictyosomes, swelling of endoplasmic reticulum cisternae and mitochondrial cristae. Both freezing-thawing and cryoprotectant were seen to cause an increase in the number of lysosomes, liposomes, myelin-like structures, and to form large vacuoles. The structural changes in molluscan neurons caused by cryopreservation with Me2SO (2 M) were reversible.     

The osphradium is involved in the control of egg-laying in the pond snail Lymnaea stagnalis

Summary

Ablation of the osphradium (a peripheral putative chemo-sensory organ) in Lymnaea stagnalis resulted in significant increase of egg-laying activity as compared with control animals (observed for 50 days). Cutting the osphradial nerve increased egg-laying to a lesser degree and only for the first 15–20 days. Water changes in the aquaria had a similar stimulating effect on egg-laying activity in both osphradium-deprived and control snails. Permanent aeration of the aquaria abolished this effect. Possible mechanisms of tonic inhibitory action of the osphradium on egg-laying are discussed.     

Cyclic nucleotide-gated channels are involved in phototransduction of dermal photoreceptors in Lymnaea stagnalis

Dermal photoreceptors in the pond snail Lymnaea stagnalis mediate the whole-body withdrawal response, including pneumostome closure, elicited by a shadow passing over the pneumostome area. The pneumostome closure response is part of the defense reaction in Lymnaea. The shadow or ‘light-off’ stimulus elicits activity in a higher order interneuron, RPeD11, which has a major role in mediating defensive withdrawal behavior elicited by noxious or threatening stimuli. Here, we tested our hypothesis that cyclic nucleotide-gated (CNG) channels are involved in the dermal photoreceptor-mediated transduction of the shadow stimulus. The response to the shadow stimulus recorded in RPeD11 was abolished by 500 μM cis-diltiazem, which blocks cGMP-activated conductance of CNG channels. On the other hand, the shadow response elicited in RPeD11 was not blocked by 2-amino ethyldiphenyl borate (2-APB), a transient receptor potential (TRP) channel blocker. Consistent with the electrophysiologic data, cis-diltiazem blocked the shadow-evoked withdrawal response, whereas 2-APB did not block the withdrawal response evoked by the shadow stimulus in intact freely behaving Lymnaea. Together, these findings support the hypothesis that the second messenger in dermal photoreceptors involves CNG and not TRP channels.     

Variability of morphologic parameters of individual neurons and neuron clusters in the visceral ganglion of the mollusk Lymnaea stagnalis L

Quantitative investigations on variability of visual morphological parameters in neurons BB1, BB3-5 and clusters BBb have been performed on the ventral side of the visceral ganglion in 20 molluscs Lymnaea stagnalis L. (height of the shell -45.5-1.3 mm, variation coefficient -2.9%) cought in the same pond and on the same day. Statistical analysis of the data obtained is performed by means of the methods applied for analysing small independent samples. A probability character of visual identification and an essential variability are demonstrated for the parameters of the neuron BB1 (diameter, position, colour), of the number of giant and/or large neurons BB3-5 and the cluster BBb neurons. The variation character of the parameters mentioned is asymmetric. It is possible to speak on a greater determinancy of a preferable gradation of the parameters. The interconnection of the variability and the determinancy of the morphological signs with the identificity and functioning of the neuronal elements are discussed. A suggestion is made that identification of the neurons according to both their morphological and functional criteria, as a whole, can have a probability character. The data considered in the work make doubt the invariance of the neurons studied in the mollusc Lymnaea stagnalis L.     

LFRFamides: a novel family of parasitation-induced -RFamide neuropeptides that inhibit the activity of neuroendocrine cells in Lymnaea stagnalis

We report the characterization of a cDNA encoding a novel -RFamide neuropeptide precursor that is up-regulated during parasitation in the snail Lymnaea stagnalis. Processing of this precursor yields five structurally related neuropeptides, all but one ending with the C-terminal sequence -LFRFamide, as was confirmed by direct mass spectrometry of brain tissue. The LFRFamide gene is expressed in a small cluster of neurons in each buccal ganglion, three small clusters in each cerebral ganglion, and one cluster in each lateral lobe of the cerebral ganglia. Application of two of the LFRFamide peptides to neuroendocrine cells that control either growth and metabolism or reproduction induced similar hyperpolarizing K+-currents, and inhibited electrical activity. We conclude that up-regulation of inhibitory LFRFamide neuropeptides during parasitation probably reflects an evolutionary adaptation that allows endoparasites to suppress host metabolism and reproduction in order to fully exploit host energy recourses.     

A behavioural and neuronal analysis of the locomotory system of Lymnaea stagnalis

• 1.1. During locomotion the pond snail Lymnaea stagnalis (L.) exhibits recurrent cyclical movements; in water this behaviour consists of anteriorly and posteriorly directed shell movements and on land these same movements are coupled with longitudinal contractions and extensions of the foot.
• 2.2. Motoneurones innervating the foot, body wall and column have been identified. The activity of some of these neurones is correlated with shell movements during spontaneous locomotor discharges in reduced preparations.
• 3.3. The structure of these motoneurones has been determined by intracellular injection of the fluorescent dye Lucifer Yellow CH.