Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

Identification of two novel genes specifically expressed in the D-group neurons of the terrestrial snail CNS

A search for genes specifically expressed in the giant interneurons of parietal ganglia of the snailHelix lucorum yielded, among others, two genes named HDS1 and HDS2. According to data obtained by Northern hybridization and whole-mountin situ hybridization, both genes are neurospecific and expressed almost exclusively in the peptidergic D-group neurons (Sakharov, 1974) located in the right parietal ganglion.In situ hybridization of the HDS1 and HDS2 probes with CNS of several related species of the Helicoidea superfamily identified in all cases similarly located homologous groups of neurons. Sequencing of the near full-length cDNA copies of the HDS1 and HDS2 genes revealed open reading frames 107 and 102 amino acids long for HDS1 and HDS2, respectively. Both putative proteins contain a hydrophobic leader peptide and putative recognition sites for furin-like and PC-like endopeptidases. Predicted amino acid sequences of the HDS1 and HDS2 proteins were found to be moderately homologous to each other, as well as to the LYCP preprohormone expressed by the light yellow cells of the freshwater snailLymnaea stagnalis. These results confirm an earlier hypothesis that the D-group of theHelix family and the light yellow cells ofLymnaea stagnalis represent homologous neuronal groups. Our data suggest that the HDS1 and HDS2 genes encode precursors of secreted molecules, most likely neuropeptides or neurohormones.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

Distribution of tyrosine-hydroxylase-immunoreactive and dopamine-immunoreactive neurons in the central nervous system of the snail Helix pomatia

The distribution and characterization of dopamine-containing neurons are described in the different ganglia of the central nervous system of Helix on the basis of the distribution of tyrosine hydroxylase immunoreactive (TH-ir) and dopamine immunoreactive (DA-ir) neurons. Both TH-ir and DA-ir cell bodies of small diameter (10–25 m) can be observed in the buccal, cerebral and pedal ganglia, dominantly on their ventral surface, and concentrated in small groups close to the origin of the peripheral nerves. The viscero-parietal-pleural ganglion complex is free of immunoreactive cell bodies but contains a dense fiber system. The largest number of TH-ir and DA-ir neurons can be detected in the pedal, and cerebral ganglia. The average number of TH-ir and DA-ir neurons significantly differs but all the identifiable groups of TH-ir neurons also show DA-immunoreactivity. Therefore, we consider the TH-ir neurons in those groups as being DA-containing neurons. The amounts of DA in the different ganglia assayed by high performance liquid chromatography correspond to the distribution and number of TH-ir and DA-ir neurons in the different ganglia. The axon processes of the labeled small-diameter neurons send thin proximal branches toward the cell body layer but only rarely surround cell bodics, whereas distally they give off numerous branches in the neuropil and then leave the ganglion through the peripheral nerves. In the cerebral ganglia, the analysis of the TH-ir pathways indicates that the largest groups of labeled neurons send their processes through the peripheral nerves in a topographic order. These results furnish morphological evidence that DA-containing neurons of Helix pomatia have both central and peripheral roles in neuronal regulation.     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.     

Detection of FMRFamide-like immunoreactivities in the sea scallopPlacopecten magellanicus by immunohistochemistry and Western blot analysis

FMRFamide-like immunoreactivity was detected histochemically in the sea scallopPlacopecten magellanicus. Most immunoreactivity was concentrated in the cerebral, pedal, and parietovisceral ganglia, particularly in the cortical cell bodies and in their fibers which extend into the central neuropile. Whole-mount immunofluorescence studies were used to localize concentrations of immunoreactive cells on the dorsal and ventral surfaces of each ganglion. Immunoreactivity was also detected in nerves emanating from the ganglia. Strong immunoreactivity was localized in peripheral organs, including the gut and gills of juvenile and adult scallops. Weak immunoreactivity was detected in the gonads, heart, and adductor muscle of the adults. A broad FMRFamide-like immunoreactive band of 2.5–8.2 kDa was detected by Western blotting of acetone extracts of the parietovisceral ganglia. In the presence of protease inhibitors, two FMRFamide-like immunoreactive bands (7.2–8.2 kDa and >17 kDa) were obtained. Neither of these bands comigrated with the FMRFamide standard. It is concluded that peptides of the FMRFamide family are probably regulators of numerous central and peripheral functions inP. magellanicus.     

Spermatogenesis of a marine snail,Littorina sitkana

Summary The fine structure of the spermatogonium, spermatocyte and spermatid of a marine snail, Littorina sitkana is described. The ring centriole (annulus) is formed from the distal centriole and it migrates to the base of the mitochondrial region where it lies in a joint-like structure which is formed by an area of invaginated plasma membrane. The distal and proximal centrioles are at first perpendicular to each other but the proximal centriole rotates to a position coaxial with the distal centriole and fuses with it. The peripheral doublet fibers are continuous between the two centrioles but the central fibers originate only in the distal centriole. The acrosome differentiates from the proacrosomal granule which is derived from a Golgi body. Microtubules, present at this stage, may assist acrosomal formation. Chromatin condensation begins with the formation of fibrous strands, then to lamellar plates which become folded and later twisted around the flagellar shaft. In the final stages the lamellae appear in cross section as concentric rings which eventually fuse to form a homogeneously dense nuclear tube.     

Distribution of oxytocin-like and vasopressin-like immunoreactivities within the central nervous system of the cuttlefish, <Emphasis Type="Italic">Sepia officinalis</Emphasis>

We have investigated the distribution of oxytocin/vasopressin (OT/VP) superfamily peptides in the central nervous system (CNS) of the cuttlefish, Sepia officinalis, by using antibodies raised against mammalian OT and VP. Several populations of OT-like and VP-like immunoreactive cell bodies and fibers were widely distributed in cerebral structures involved in learning processes (vertical lobe complex, optic lobes), behavioral communication (peduncle, lateral basal and chromatophore lobes), feeding behavior (inferior frontal, brachial and buccal lobes), sexual activity (dorsal basal, subpedunculate, olfactory lobes), and metabolism (visceral lobes). The two most remarkable findings of this study were the occurrence of OT-like immunoreactivity in many amacrine cells of the vertical lobe and the dense accumulation of VP-like immunoreactive cell bodies in the subpedunculate 1 lobe. No double-immunolabeled cell bodies or fibers were found in any lobes of the CNS, indicating, for the first time in a decapod cephalopod mollusc, the existence of distinct oxytocinergic-like and vasopressinergic-like systems. The widespread distribution of the immunoreactive neurons suggests that these OT-like and VP-like peptides act as neurotransmitters or neuromodulators. This research was supported by grants from the “Région Basse-Normandie” (FRANCE) and the LARC-Neurosciences network (FRANCE).     

Neural structures in the receptive field of pleural ganglion mechanosensory neurons of Aplysia californica

The peripheral processes of the mechanoafferents that, when stimulated, initiate the much-studied tail withdrawal reflex of Aplysia californica have not been characterized. We show that immunofluorescence staining for class III -tubulin highlights neurons and reveals nerve tracts and fine neuronal processes in Aplysia tissue. Coupled with transmission and scanning electron microscopy, class III -tubulin immunofluorescence is consistent with the possibility that mechanoafferents in the receptive field of pleural ganglion mechanosensory neurons penetrate the tail epidermis and terminate as ciliated endings. This view is reinforced by comparisons among neuronal processes in several mechanosensory epidermal regions and in a chemosensory epidermis.     

Distribution of serotonin-like immunoreactive neurons in the slug<Emphasis Type="Italic"> Limax valentianus</Emphasis>

Immunohistochemical techniques were used to study the distribution of serotonin-containing neurons in the nervous system of the slug Limax valentianus. Approximately 350 serotonin-like immunoreactive cell bodies were found in the central nervous system. These were located in the cerebral, pedal, visceral and right parietal ganglia. Most serotonin-like immunoreactive neurons had small cell bodies, which were aggregated into discrete clusters. A pair of previously identified metacerebral giant cells were found on the anterior side of the cerebral ganglion, and two additional pairs of uniquely identifiable, serotonin-like immunoreactive cells were found on the posterior side of the cerebral ganglion. The whole-mount maps of these stained neurons will be useful in further physiological and biochemical studies of olfactory learning at the cellular level in Limax valentianus.This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology, Japan (nos. 12307053 and 13771353)     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix 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.     

Innervation of the gastrointestinal canal of the toad Bufo marinus by neurons containing 5-hydroxytryptamine-like immunoreactivity

Summary The gut of the toad, Bufo marinus, was examined for evidence of enteric neurons containing 5-hydroxytryptamine-like immunoreactivity. Such neurons were absent from the stomach. They were present in the small intestine, with processes confined to the myenteric plexus. Immunoreactive nerve cell bodies lay on branches of the pelvic nerves supplying the large intestine; fibres were found in the submucosa of the posterior large intestine and in the muscularis externa of the anterior large intestine. It is concluded, on morphological grounds, that the neurons in the small intestine are interneurons, whereas those in the large intestine are postganglionic parasympathetic motoneurons.     

Studies on Ca2+-accumulating vesicles in oocytes of the snail Helix aspersa

Summary Ultrastructural, cytochemical and autoradiographic techniques were used to study the functional significance of Ca²⁺-accumulating vesicles in Helix aspersa oocytes. The organic material of these vesicles is capable of adsorbing calcium ions, thus removing them from the cytosol and forming osmotically inactive complexes that are concentrated in the core. Ca²⁺-accumulating vesicles likely act as subcellular detoxifying compartments that sequestrate excess cytosolic Ca²⁺ (and plausibly other metal cations) to maintain physiologically adequate ionic concentrations.Part of this paper was presented at the VII Congreso Nacional de Malacología (Sevilla, November 1988)     

In vitro capsaicin-induced cytological changes and alteration in calcium distribution in giant serotonergic neurons of the snail Helix pomatia: a light- and electron-microscopic study

Morphological changes induced by capsaicin were studied in the serotonergic metacerebral giant neurons of the cerebral ganglia of Helix pomatia under in vitro conditions. Capsaicin at a concentration of 10^-4 M caused characteristic structural alterations in the giant serotonergic neurons but did not significantly influence serotonin immunoreactivity in the neurons. At the lightmicroscopic level, the most conspiciuous structural alterations were swelling of the cell bodies, which contained a swollen pale nucleus. Under the electron microscope, the nuclei,mitochondria and the cisternae of the endoplasmic reticulum were swollen in the capsaicin-affected metacerebral giant neurons. Electron-microscopic cytochemical techniques for calcium demonstration revealed electron-dense deposits in the swollen mitochondria and in the cisternae of the endoplasmic reticulum, suggesting an increased Ca²⁺ influx. The serotonergic metacerebral giant neurons could be labelled by cobalt (1 mM) in the presence of capsaicin (10^-4 M) suggesting that capsaicin opens the cation chanels of the capsaicin-sensitive neuronal membrane. The morphological and cytochemical alterations induced by capsaicin in the serotonergic metacerebral giant neurons of Helix pomatia closely resemble those induced in sensory neurons of mammalian dorsal root ganglion.This work was supported by OTKA grants No.: 2477, T016861, T017127 and ETT 587/93     

Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail,Lymnaea stagnalis,studied by in situ hybridization and immunocytochemistry

Summary The functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8–17 and 64–73, respectively. SIS-peptide sequences 10–20 and 67–76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle. The morphology of the system is discussed in relation to the process of sodium ion uptake from the ambient medium and from pro-urine, and to that of regulating blood pressure. In the central nervous system and other organs, neurons and axons not labeled with the DNA probes, but immunoreactive to one or two of the antibodies, were observed. It seems unlikely that these elements are functionally related to the SIS-peptide system.     

Effect of a toxicant on phagocytosis pathways in the freshwater snail Lymnaea stagnalis

The disturbance of plasma membrane carbohydrates and of lipopolysaccharide (LPS) ligands in relation to cytoskeletal transformations of haemocytes has been investigated after chronic exposure of pond snails (Lymnaea stagnalis) to the peroxidizing toxicant fomesafen. Neither of the two lectins used (concanavalin A and wheat germ agglutinin) showed any binding modification after incubation of the snails in the presence of the toxicant. However, after exposure of the snails to fomesafen, a clear and persistent reduction in LPS labelling of haemocytes occurred. The actin cytoskeleton of the same cells also appeared to be sensitive to the toxicant. The reduction in LPS-binding sites was related to actin staining, leading to the hypothesis that LPS ligands and actin could be similarly modulated by the toxicant. Damaged cells showed non-adherent membrane portions with reduced filopodial extrusions, exhibiting a smooth surface free of microvilli. These changes could lower the spreading and adhesion of the cells and could therefore account for the loss in their phagocytic capabilities.