Functional morphology of the neuropeptidergic light-yellow-cell system in pulmonate snails

The light yellow neuropeptidergic cell system of the basommatophoran snail Lymnaea stagnalis is homologous to the R3-R14 system of the opisthobranch Aplysia californica, and produces three different neuropeptides. Systems homologous to the light yellow cells of Lymnaea stagnalis have been investigated morphologically in two Basommatophora (Lymnaea ovata, Bulinus truncatus) and three Stylommatophora (Helix aspersa, Cepaea nemoralis, Deroceras reticulatum). To this end, an antibody to synthetic light-yellow-cell peptide-II and oligonucleotides to mRNAs encoding parts of peptide-I and peptide-III, were used. The in situ hybridization probes gave negative results. On the other hand, neuronal cell clusters were observed in the central nervous system of all specias studied by immunocytochemistry. These clusters were located in the ganglia of the visceral complex. The neurons project axons into all nerves of these ganglia, especially into the pallial nerves, into the connective tissue of the central nervous system, and into the neuropile of various ganglia. The morphology of the systems is similar to that of the light-yellow-cell system of Lymnaea stagnalis. In all species, the wall of the aorta was innervated by immunoreactive axons. Peripheral innervation by the light-yellow-cell system was investigated in Helix aspersa and Deroceras reticulatum. Serial and alternate sections of whole snails were studied. Reconstructions were made of the heart-kidney-lung complex of these animals. In both species, the muscular vessels of the pulmonary system at the right side of the body were strongly innervated by immunoreactive axons. Furthermore, immunopositive innervation was observed to muscles in the secondary ureter-pneumostome area. The light-yellow-cell system of pulmonates is thus probably involved in the regulation of blood pressure and urine release.     

Light- and electron-microscopic immunocytochemistry of a molluscan insulin-related peptide in the central nervous system of Planorbarius corneus

Summary Two groups of cerebral dorsal cells of the pulmonate snail Planorbarius corneus stain positively with antisera raised against synthetic fragments of the B- and C-chain of the molluscan pro-insulin-related prohormone, proMIP-I, of another pulmonate snail, Lymnaea stagnalis. At the light-microscopic level the somata of the dorsal cells and their axons and neurohemal axon terminals in the periphery of the paired median lip nerves are immunoreactive with both antisera. Furthermore, the canopy cells in the lateral lobes of the cerebral ganglia are positive. In addition, MIP_B-immunoreactive neurons are found in most other ganglia of the central nervous system. At the ultrastructural level, pale and dark secretory granules are found in somata and axon terminals of the dorsal cells. Dark granules are about 4 times as immunoreactive to both antisera as pale granules. Release of anti-MIP_B- and anti-MIP_C-immunopositive contents of the secretory granules by exocytosis is apparent in material treated according to the tannic acid method. It is concluded that the dorsal and canopy cells synthesize a molluscan insulin-related peptide that is packed in the cell body into secretory granules and that is subsequently transported to the neurohemal axon terminals and released into the hemolymph by exocytosis. Thus, MIP seems to act as a neurohormone on peripheral targets. On the basis of the analogy between the dorsal cells and the MIP-producing cells in L. stagnalis, it is proposed that the dorsal cells of P. corneus are involved in the control of body growth and associated processes.     

The central nervous system of Bulla gouldiana: Peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

Sensorin-A immunocytochemistry reveals putative mechanosensory neurons inLymnaea CNS

The pond snailLymnaea stagnalis is a useful model system for studying the neural basis of behaviour but the mechanosensory inputs that impact on behaviours such as respiration, locomotion, reproduction and feeding are not known. InAplysia, the peptide sensorin-A appears to be specific to a class of central mechanosensory neurons. We show that in theLymnaea central nervous system sensorin-A immunocytochemistry reveals a discrete pattern of staining involving well over 100 neurons. Identifiable sensorin positive clusters of neurons are located in the buccal and cerebral ganglia, and a single large neuron is immunopositive in each pedal ganglion. These putative mechanosensory neurons are not in the same locations as previously identified motoneurons, interneurons or neurosecretory cells. As would be expected for a mechanoafferent, sensorin positive fibres were found in nerve tracts innervating the body wall. This study lays the foundation for future electrophysiological and behavioural analysis of these putative mechanosensory neurons.     

Co-existence of immunoreactivity to anti-dopamine,anti-serotonin and anti-vasotocin in the cerebral giant neuron of the pond snail Lymnaea stagnalis

Summary Consecutive sections of certain neurons in the central ganglia of the pond snail Lymnaea stagnalis appear to be immunoreactive to anti-dopamine and anti-serotonin. The Cerebral Giant Neurons stain in addition with antivasotocin. The observations indicate the presence of two biogenic amines within the same neuron and in addition their co-existence with a biologically active peptide.     

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 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.     

Unusual distribution of tubulin isoforms in the snail Lymnaea stagnalis

Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vitro in the presence of taxol. Various broad-spectrum antibodies raised against -tubulin and -tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome -tubulin, however, labelled -tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total -tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated -tubulin and acetylated -tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Neurons in a variety of molluscs react to antibodies raised against the VD1/RPD2 α-neuropeptide of the pond snail Lymnaea stagnalis

The VD₁ and RPD₂ neurons of Lymnaea stagnalis innervate other central neurons, certain skin areas, the pneumostome area, and the auricle of the heart. Recently, a set of four (, , , ) neuropeptides produced by these giant neurons and by certain other central neurons has been characterized. Although alternative splicing of the preprohormone of these neurons yields at least 10 different neuropeptides, an affinity-purified antiserum directed against a domain common to all neuropeptides has previously been shown to be highly selective in staining VD₁, RPD₂ and other neurons that produce the preprohormone. Since the gene encoding the neuropeptides is structurally similar to that expressed in R15 of the marine opisthobranch Aplysia californica, we have used the affinity purified antiserum as a marker for VD₁/RPD₂-related systems in other molluscs. Immunopositive neurons and fibers are observed in the central nervous systems of all species studied (Achatina fulica, Anodonta sp., Aplysia brasiliana, A. californica, Bulinus truncatus, Cepea sp., Eobania vermiculata, Helix aspersa, H. pomatia, Limax maximus, Mytilus edulis, Nassarius reticulatus, Viviparus viviparus). Several medium-sized and small neurons and 1–4 giant neurons are found in the pulmonates and opisthobranchs. The giant neurons in pulmonates have locations in the subesophageal ganglion, axonal branching patterns, and terminal arborizations in the auricle of the heart; all these characteristics are similar to those of VD₁ and RPD₂. Double-labelling (Lucifer yellow injection, immunocytochemistry) confirms that the two giant neurons in Helix pomatia are Br and Br. The immunoreactive cells in A. fulica appear to include the VIN and PON neurons. The antiserum also stains cells that appear to be the R15 neurons in two Aplysia species. The small and medium-sized neurons are distributed widely over the central ganglia of opisthobranchs and pulmonates. In prosobranchs and bivalves, small neurons are found in the cerebral and abdominal ganglia. No evidence has been found for innervation of the heart in these latter groups but in M. edulis, immunoreactive terminals can be observed in the gill. The results suggest the evolutionary conservation of immunoreactive peptides and the neurons that produce them, and thus support and extend previous hypotheses regarding the homology of certain giant neurons across molluscan species.     

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.     

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.     

Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I. Our observations support the hypothesis that light yellow cells are involved in maintaining the shape of the animal via the regulation of ion- and waterbalance processes and blood pressure.     

The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry

Summary VD₁ and RPD₂ are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD₁/RPD₂ gene. To this end, in situ hybridization with the cDNA probe of the VD₁/RPD₂ gene and immunocytochemistry with antisera specific to VD₁ and RPD₂ (the 1-antiserum, Mab4H5 and ALMA 6) and to R₁₅ (the 1 and 16-mer antisera) were performed on alternate tissue sections. A VD₁/RPD₂ neuronal system comprising three classes of neurons (A1–A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD₁ and RPD₂) immunoreact with the 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1–5 small and 1–5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3–4 medium-sized neurons and a cluster of 4–5 small neurons located in the pedal ganglion) immunoreact with the 1-antiserum only. All neurons of the system are immunonegative to the R₁₅ antisera. The observations suggest that the neurons of the VD₁/RPD₂ system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.     

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.     

Localization of Lom-AG-myotropin I-like substances in the male reproductive and nervous tissue of the locust,Locusta migratoria

Summary Lom-AG myotropin I (Lom-AG-MTI) was the first peptide to be isolated from the male accessory reproductive glands of the locust, Locust migratoria. It shows no sequence similarity to any of the peptides identified from vertebrate or invertebrate tissues. A polyclonal antiserum was used to localize Lom-AG-MTI-like material in the male reproductive system and nervous system of the locust. Immunoreactivity was found in two of the hyaline gland tubules. In the brain, cell bodies were detected in the proto- and deuterocerebrum as well as the frontal ganglion. Nerve fibers were stained in the neuropils of the brain and throughout the labial nerves into the recurrent nerve. Thoracic and last abdominal ganglia contained neurons which could be stained with Lom-AG-MTI antiserum. The pronounced reactivity in the central nervous system suggests a possible neuroregulatory function of the peptide.     

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.     

Structure of visual pathways in the nervous system of freshwater pulmonate molluscs

Central nervous system of freshwater pulmonate molluscs Lymnaea stagnalis and Planorbarius corneus was stained using retrograde transport of neurobiotin in the optic tract fibers. In both species, perikarya and fibers of the stained neurons are found in all ganglia except the buccal ones. Afferent fibers of the optic nerve form dense sensory neuropil located in relatively small volume of cerebral ganglia. Typical neuronal groups sending their processes into the optic nerves of ipsilateral and contralateral body halves are described. Among them, neurons of visceral and parietal ganglia innervating both eyes concurrently as well as sending projections into peripheral nerves are revealed. These neurons, supposedly, have a function to integrate sensory signals, which may be a basis for regulation of light sensitivity of retina and functioning of peripheral organs. Bilateral links of the molluscan eye with the pedal ganglia cells and statocysts are found, which is, likely, a structural basis of certain known behavioral patterns related to stimulation of visual inputs in the studied gastropod molluscs.