Neuronal and non-neuronal control of the neurosecretory Caudo-Dorsal cells of the freshwater snail Lymnaea stagnalis (L.)

The cerebral ganglia of the freshwater snail Lymnaea stagnalis contain two clusters of neurosecretory Caudo-Dorsal Cells (CDC). These cells produce a neurohormone which stimulates ovulation. Ganglion transplantation and quantitative electron microscopy show that neuronal isolation of the cerebral ganglia complex (CCC) results in an activation of the CDC. It was, therefore, concluded that the CDC are controlled by an inhibitory neuronal input originating outside the cerebral ganglia. Ultrastructural studies on synaptic degeneration in the CCC suggest that this input reaches the CDC via a special type of synapse-like structure, the type C-SLS.Furthermore, transplantation of CCC into acceptor snails leads to a reduced release and an increased intracellular breakdown of neurohormone in the CDC of the nervous system of the acceptors. It is supposed that these phenomena are caused by the release of an (unknown) factor from the transplanted CCC. Special attention was given to the formation and degradation of a peculiar type of neurohormone granule, the large electron dense granule.     

Membrane mechanism of neuroendocrine caudo-dorsal cell inhibition by the ring neuron in the pond snail Lymnaea stagnalis

Ovulation in the pond snail Lymnaea stagnalis is controlled by the neuroendocrine caudo-dorsal cells (CDCs) in the cerebral ganglia, which release an ovulation hormone during a period of impulse activity. Firing of the single RN in the right cerebral ganglion hyperpolarizes the CDCs. This hyperpolarization is caused by the opening of potassium channels in the axons that connect both the CDC clusters. By this action, that presumably is mediated by axonal branches of the RN in the intercerebral commissure closely associated with these CDC axons, the RN decouples both the CDC clusters. Although the RN has negative feedback on the CDC, it does not control afterdischarge characteristics. The authors suggest that the RN, next to the egg-laying behavior, is involved in another behavior, not compatible with ovulation. Male reproductive activity is presented as a possible candidate for such behavior.     

Regulation of neurosecretory activity in the freshwater pulmonate Lymnaea stagnalis (L.) with particular reference to the role of the eyes

The process of neurosecretion in the Caudo-Dorsal Cells (CDC) of the freshwater snail Lymnaea stagnalis, which produce an ovulation hormone, shows a diurnal rhythmicity. Synthesis, transport and release of the neurosecretory material (NSM) is high during the evening and the early night and low during the rest of the day, while storage of NSM mainly occurs during the daytime. In the present study the role of the eyes in the regulation of the CDC-rhythm was investigated. During a 24-hr period, at time intervals of 6 hrs, cerebral ganglia, which contain CDC, of blinded and control snails (5 per group) were fixed and the CDC were studied with quantitative electron microscopical methods. The CDC of the controls showed a distinct diurnal thythmicity. Blinding, on the other hand, clearly affected this rhythmicity. The results indicate that after blinding the circadian CDC-rhythms of individual snails are no longer synchronous with each other ("interanimal desynchronization"). It is suggested that the rhythm of CDC neurosecretory activity is synchronized by the natural light/dark cycle via the eyes. The information from the eyes probably reaches the CDC via a nervous pathway. True snyapses and three types of synapse-like structures were found on the CDC. Their role in the regulation of CDC-activity is discussed. The effect of blinding is specific for the CDC; blinding does not influence the diurnal rhythmicity of another type of cerebral neurosecretory cells, the Light Green Cells (LGC). The CDC within a cluster act synchronously. This synchrony does not depend upon the presence of the eyes. Some structures which may be involved in establishing this synchrony, such as subsurface cisterns, desmosome-like structures and "specific release sites", are described.     

Monoamines in the nervous system of the tube-wormChaetopterus variopedatus (Polychaeta): Biochemical detection and serotonin immunoreactivity

Summary The presence and distribution of biogenic monoamines in the tube-wormChaetopterus variopedatus were investigated by a radioenzymatic method and HPLC with electrochemical detection, and the cellular localization of serotonin by peroxidase-antiperoxidase (PAP) immunohistochemistry with an antibody against serotonin-formaldehyde-protein conjugate. Dopamine, norepinephrine, epinephrine, serotonin (5-HT) and some of their metabolites were detectable, dopamine and norepinephrine being present in substantially larger amounts than 5-HT and epinephrine. With few exceptions, the largest amounts of amines were localized in the most nerve-rich tissues such as tentacles, and those containing cerebral ganglia and the ventral nerve cord. Serotonin-immunoreactive unipolar neurons were widely distributed in the dorso-lateral cerebral ganglia, the neurosecretory pharyngeal ganglion and the segmental ganglia of the anterior (dorsolateral) and posterior (medioventral) nerve cords. Some nerve-fiber tracts stained in the cerebral ganglia, but the neuropiles of segmental ganglia were the most intensely reactive CNS structures. Numerous reactive fibers were also present in connectives, commissures and segmental nerves. All peripheral sensory structures included serotonin-immunoreactive cells and neurites, especially the parapodial cirri and the bristle receptors of the setae. Trunk and parapodial muscles contained reactive varicose fibers and neuronal somata. These results suggest that monoamines are abundant and widespread in these worms and that 5-HT appears to have a key sensory role.     

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.     

Localization of ovulation hormone-like neuropeptide in the central nervous system of the snail Lymnaea stagnalis by means of immunocytochemistry and in situ hybridization

Summary The caudo-dorsal cells (CDC) in the cerebral ganglia of the pond snail Lymnaea stagnalis synthesize the 36-amino acid ovulation hormone (CDCH). We have used immuno-cytochemistry and in situ hybridization to reveal the localization of neurons and axons containing CDCH-like material.A monoclonal antibody to a fragment of CDCH and a cDNA probe encoding CDCH reacted with the CDC-system, with specific cell groups in the cerebral and pleural ganglia, and with individually occurring neurons throughout the central nervous system. The cells in the pleural ganglia, which were found in about 50% of the preparations studied, are considered as ectopic CDC. They are morphologically similar to CDC in their somal dimensions and axonal organization. By means of immuno-electron microscopy it was shown that these neurons contain secretory vesicles that are similar to those of the CDC. The neurons of the bilateral groups occurring in the cerebral ganglia in addition to the CDC are smaller and more intensely stained than the CDC. Axons of these small neurons probably have varicosities located on the CDC axons in the neuropil of the cerebral ganglion, indicating synaptic contacts. Two major axon tracts could be followed from (or toward) the neuropil of the cerebral ganglion. One tract runs from the cerebral gangion via the pleural and parietal ganglia to the visceral ganglion, giving off branches to most nerves emanating from these ganglia. The other tract could be traced through the cerebro-pedal connective to the pedal ganglia. Only in the right pedal ganglion was extensive axonal branching observed. The nerves emanating from this ganglion contained many more immunoreactive axons than those from the left pedal ganglion. A polyclonal antibody raised against the synthetic fragment of CDCH stained, in addition to the neurons and axons revealed with the monoclonal antibody and the cDNA probe, three other major groups of neurons. Two are located in the cerebral ganglion, the other in the left pedal ganglion.The present findings suggest the presence of a system of neurons that contain CDCH or CDCH-like peptides. The role this system may play in the control of egg-laying and egg-laying behaviour is discussed.     

Structure of visual pathways in the nervous system of fresh water pulmonary molluscs

The central nervous system of freshwater pulmonary molluscs Lymnaea stagnalis and Planorbarins corneus was stained by the method of neurobiotin retrograde transport along optic nerve fibers. In the animals of both species, bodies and fibers of stained neurons are found in all ganglia except for the buccal ones. Afferent fibers of the optic nerve form a dense sensor neuropil located in a small volume of cerebral ganglia. Characteristic groups of neurons sending their processes into optic nerves both of ipsi- and of contralateral half of the body are described. Revealed among them are neurons of visceral and parietal ganglia, which simultaneously innervate both eyes as well as give projections into peripheral nerves. It is suggested that these neurons can perform function of integration of sensor signals and, on its base, regulate photosensitivity of retina as well as activity of peripheral organs. There is established the presence of bilateral connections of the mollusc eye with cells of pedal ganglia and statocysts, which seems to be the structural basis of manifestation of the known behavior forms associated with stimulation of visual inputs of the studied gastropod molluscs.     

Purification and chemical characterization of caudodorsal cell hormone-II from the egg-laying controlling caudodorsal cells of Lymnaea stagnalis.

Caudodorsal cell hormone-I (CDCH-I), a 36 amino acid peptide synthesized by the caudodorsal cells (CDCs), is one of the major neuropeptides present in the cerebral ganglia of the freshwater snail, Lymnaea stagnalis. This peptide induces ovulation and egg-mass formation, and, in addition, acts on neuronal circuits involved in the control of overt egg-laying behavior. cDNA cloning studies revealed that there is a CDCH gene family comprising two genes coding for CDCH-I and the related yet distinct CDCH-II, respectively. In the present study, using sequential high performance gel permeation and reversed phase high performance liquid chromatography, we have isolated and sequenced the peptide CDCH-II. This neuropeptide has the following sequence: Ser-Ile-Thr-Asn-Asp-Leu-Arg-Ala-Ile-Ala-Asp-Ser-Tyr-Leu-Tyr-Asp-Gln-His- Lys-Leu - Arg-Glu-Gln-Gln-Glu-Glu-Asn-Leu-Arg-Arg-Arg-Phe-Tyr-Glu-Leu-Ser-Leu-Arg- Pro-Tyr - Pro-Asp-Asn-Leu. The sequence differs from those predicted by the cDNA sequence encoding preproCDCH-II at two positions, suggesting the polymorphism of CDCH-II exists in L. stagnalis.     

Differential localization of vasopressin- and oxytocin-immunoreactive cells and conventional neurosecretory cells in the ganglia of the earthworm Pheretima hilgendorfi

Cells containing arginine vasopressin (AVP)- and oxytocin (OXT)-like substances were immunohistochemically visualized in the cerebral, subesophageal, and ventral nerve cord ganglia of the earthworm Pheretima hilgendorfi. Whether these anti-AVP– and anti-OXT–reactive cells are identical with classical aldehyde fuchsin (AF)-positive neurosecretory cells was tested in serial sections. In all ganglia, groups of scattered neuronal cell bodies and axons strongly reactive to AVP and OXT antisera were observed, but AF-positive cells consisting of type a (dark blue) and type b (purple) cells were predominantly present in the cerebral and subesophageal ganglia. In the cerebral and subesophageal ganglia anti-AVP– and anti-OXT–reactive cells were generally larger than AF-positive cells. Some AF-positive cells were reactive either to anti-AVP or anti-OXT serum, but some failed to react to either serum. Anti-AVP– and anti-OXT–reactive cells were not immunoreactive to OXT and AVP antisera, respectively. Electron microscopic observations showed that the granules of type a cells were larger and less electron dense than those of type b cells and anti-AVP–reactive cells. The present cytological observations clearly showed that AVP- and OXT-like substances were widely present in the ganglionic cells of the earthworm     

On the neurosecretory system of Dendrobaena atheca Cernosvitov

Four kinds of neurosecretory cells A, B, U and C are distinguished in the central nervous system of Dendrobaena atheca Cernosvitov. A cells, which show different morphological characteristics under different physiological states and during their cyclic changes, are the most active neurosecretory cells. They form the outer layer of the cortical cell zone in the cerebral ganglion. B cells are large and medium sized and are distributed in all parts of the central nervous system. U cells are found only in the sub-pharyngeal ganglion while C cells are distributed in the sub-pharyngeal as well as in the ventral nerve cord ganglion. The number and secretory activity of C cells decrease in caudal direction. Further, Gomori-positive cells are also observed in the ganglia of the vegetative nervous system. A rudimentary neurohaemal organ, the storage zone, has been observed in the cerebral ganglion and there appears to be another neurohaemal area in the ventral nerve cord ganglion. The storage zone is formed by the terminal ends of the axons of A cells. The chrome alum haematoxylin phloxin (CHP) and aldehyde fuchsin (AF) positive substances in the form of granules are found in this area. The cerebral ganglion is richly supplied by blood capillaries. The distal end of the axons of B cells are swollen like a bulb while in some cases the axons are united to form an axonal tract. Extra-cellular material is abundant in different parts of the nervous system. In all cell types, the perinuclear zone is the first to show activity in the secretory cycle. It appears that the nucleus may be involved in the elaboration of the neurosecretory material in the cells.     

Electron microscopical and histochemical observations on the relation between medio-dorsal bodies and neurosecretory cells in the basommatophoran snails Lymnaea stagnalis,Ancylus fluviatilis,Australorbis glabratus and Planorbarius corneus

Summary In Basommatophora medio-dorsal bodies (MDB) are closely attached to the cerebral ganglia, in which, just underneath the bodies, groups of Gomori-positive neurosecretory cells (MDC) occur. It has been suggested that the MDB-cerebral ganglion complex should be regarded as a neuro-endocrine association.In the present study the morphological relation between MDB and the ganglion is histochemically and ultrastructurally investigated in Lymnaea stagnalis, Ancylus fluviatilis, Australorbis glabratus and Planorbarius corneus.Histochemical tests showed the paraldehyde-fuchsin positive material of fibers in the MDB to be different from the neurosecretory material (NSM) in the MDC. At the ultrastructural level no penetration of nerve cell processes through the perineurium, separating the MDB from the ganglion, into the medulla of the MDB was observed. However, excepting for Lymnaea, the perineurium at these places shows particular differentiations. In the medulla of the MDB granule laden profiles (granule ø 700–900 Å) occur. They appeared to be processes of MDB cells.From these results it is concluded that the medulla of the MDB should not be regarded as a neurosecretory neuropile. Apparently, the MDB-cerebral ganglion complex is no neuroendocrine association. Probably the MDB is an endocrine organ. The small electron dense granules of the profiles in the medulla were also found in the MDB cell bodies. They are thought to represent a secretion product. The close morphological relation between MDB and cerebral ganglion may be connected with the origin of the MDB cells from perineural elements.     

Distribution of alpha CDCP-immunoreactive neurons in the central nervous system of the snail Helix aspersa.

alpha CDCP is a neuropeptide produced by the caudodorsal cells of Lymnaea stagnalis and encoded by the genes of the egg-laying hormone (ELH). The use of a polyclonal antiserum raised against alpha CDCP resulted in the detection of about 800 immunoreactive neurons in the parietal ganglia and a small population (60 cells) in the cerebral ganglia of Helix aspersa. As the genes of ELH are well conserved among the gastropod species, these data designate the parietal ganglia as a putative source for the egg-laying hormone in Helix aspersa.     

The Fine-Structural Localization of Phosphatases in Neurosecretory Cells Within the Ganglia of Certain Gastropod Snails

The neurosecretory cells in the cerebral ganglia of the snail,Planorbis trivolvis, have been examined for structural detailsand distribution of phosphatase activities. A more preliminarystudy on the localization of phosphatases in the neurosecretorycells of Helix aspersa has also been made. These neurons inboth species contain typical elementary neurosecretory granuleswhich appear to be elaborated or condensed in the Golgi saccules.Immature elementary granules are identifiable as "primary lysosomes"in that they contain acid phosphatase activity as well as nucleosidephosphatases. The mature elementary granules display no phosphataseactivity. Some saccules of the Golgi are also reactive for severalnucleoside phosphatases, including thiamine pyrophosphatase(TPPase). In Planorbis, TPPase is also present in the cisternaeof the endoplasmic reticulum. Larger, membrane bound, electron-densebodies (lipochondria) are found in close spatial associationwith the Golgi region. These also possess acid phosphatase;in addition, they contain nucleoside diphosphatases and (inPlanorbis) adenosine triphosphatase. Their content of acid phosphataseand the features of their fine structure indicate that theyare lysosomes akin to the dense bodies of vertebrate neurones. The significance and implications of these results are considered,as are other details of the neuronal and glial structure inPlanorbis.     

Antioxidative protection in the central nervous ganglia of a mollusc Lymnaea stagnalis at modulation of activity of the NO-ergic system

In experiments on molluscs Lymnnaea stagnalis the state of antioxidative protection is studied in central nervous ganglia during a long-term activation (inhibition) of synthesis of nitric monoxide (NO) in the body. Effect of the blocker of NO-synthase N(G)-nitro-L-arginine (L-NNA) at the background of enhancement of pulmonary respiration has been established to be associated with a rise of levels of reduced glutathione and TBK-active products in the nervous tissue at preservation of a relatively high superoxide dismutase activity and a low glutathione peroxidase activity as compared with control group and the animals treated with the metabolic precursor of NO synthesis L-arginine. In spite of the revealed disturbances of balance of the body pro- and antioxidative system, DNA electrophoresis detected no products of its degradation, which can indicate the absence of massive programmed death of the nervous tissue cells in Lymnaea stagnalis during modulation of activity of the NO-ergic system.     

New vistas on the initiation and maintenance of insect motor behaviors revealed by specific lesions of the head ganglia

In insects, thoracic pattern generators are modulated by the two head ganglia, the supraesophageal ganglion (brain) and the subesophageal ganglion, which act as higher-order neuronal centers. To explore the contribution of each head ganglion to the initiation and maintenance of specific motor behaviors in cockroaches (Periplaneta americana), we performed specific lesions to remove descending inputs from either the brain or the subesophageal ganglion or both, and quantified the behavioral outcome with a battery of motor tasks. We show that ‘emergency’ behaviors, such as escape, flight, swimming or righting, are initiated at the thoracic level independently of descending inputs from the head ganglia. Yet, the head ganglia play a major role in maintaining these reflexively initiated behaviors. By separately removing each of the two head ganglia, we show that the brain excites flight behavior and inhibits walking-related behaviors, whereas the subesophageal ganglion exerts the opposite effects. Thus, control over specific motor behaviors in cockroaches is anatomically and functionally compartmentalized. We propose a comprehensive model in which the relative permissive versus inhibitory inputs descending from the two head ganglia, combined with thoracic afferent sensory inputs, select a specific thoracic motor pattern while preventing the others.     

Morphology and physiology of pleural-to-buccal neurons coordinating defensive retraction with feeding arrest in the pond snail Lymnaea stagnalis

1. We have studied morphology, physiology and chemistry of a bilateral pair of pleural-to-buccal projecting neurons (PlB cells) of the pond snail Lymnaea stagnalis. Intracellular dye fills revealed axon arborization within neuropiles of ipsilateral pedal and cerebral ganglia, as well as in both buccal ganglia. Terminal axons of the left and right PlBs showed close proximity within the buccal commissure. 2. The left and right PlB neurons have been found electrotonically coupled and, sometimes, generating synchronous spikes. 3. The results show that two PlB cells operate as a single unit, and that paired buccal networks responsible for feeding rhythm are treated by the PlBs as a single target.     

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.     

NADPH-diaphorase activity in the nervous system of the embryonic and juvenile pond snail, Lymnaea stagnalis

Nicotinamide-adenine-dinucleotide-phosphate-diaphorase (NADPH-d) histochemistry has been applied in the present study to determine the distribution of putative nitric oxide (nitric oxide synthase)-producing cells during embryonic and early postembryonic development in the pond snail, Lymnaea stagnalis L., with special reference to the nervous system. The first NADPH-d-positive structures appear as early as 18% of development (E18, trochophore stage) and correspond to the pair of protonephridia. These structures later show disintegration, although after metamorphosis (E26=75%) staining of their individually spreading cells can be observed until hatching. Peripheral sensory neurons in the foot, mantle edge and lips, and their afferents projecting to the central nervous system reveal NADPH-d activity in the postmetamorphosis period (E25–E27=E60%–E80%) of embryogenesis. After hatching (P1–P3), a number of stained sensory cells appear in the pharynx and esophagus. Some NADPH-d positive neuronal perikarya occur in the pedal and pleural ganglia, and a few weakly stained cells in the cerebral and buccal ganglia of juvenile snails. At the same time, a continuous bundle of reactive fibers is formed in the neuropil both through and through around the circumesophageal ganglion ring. The localization of NADPH-d activity in the developing nervous system of Lymnaea suggests that nitric oxide participates mainly in sensory processes. However, its role in specific intraganglionic integrative events cannot be excluded following embryonic metamorphosis.     

Egg-laying-hormone immunoreactivity in the neural ganglia and ovary of Haliotis asinina Linnaeus

Immunoreactivity against the abalone egg-laying hormone (aELH) was detected in the fine granules of type 1 and 2 neurosecretory (NS) cells, neurites in the neuropil, and blood sinuses in the connective tissue sheath of the cerebral, pleuropedal, and visceral ganglia of the tropical abalone, Haliotis asinina Linnaeus. The number of positive NS cells, and the intensity of staining in the ganglia, varied and might be related to the stage of ovarian cycle. At any stage, positive cells were most numerous in the pleuropedal, and least numerous in the visceral ganglion. In addition, several cells of the statocyst and associated nerves also exhibited the immunoreactivity. In the ovary, the most intense reactivity was detected in the follicular and granular cells adjacent to mature oocytes, in the trabeculae and the ovarian capsule. The cytoplasm of mature oocytes was also moderately stained. The results indicate that the cerebral, pleuropedal, and visceral ganglia are the main sites of aELH-producing cells. The ovary may also produce aELH locally.