The organization of the nervous system in Plathelminthes. The neuropeptide F-immunoreactive pattern in Catenulida,Macrostomida, Proseriata

The organization of the nervous system of Archilopsis unipunctata Promonotus schultzei and Paramonotus hamatus (Monocelididae, Proseriata) and Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) was studied by immunocytochemistry, using antibodies to the authentic flatworm neuropeptide F (NPF) (Moniezia expansa). The organization of the nervous system of the Monocelididae was compared to that of the nervous system of Bothriomolus balticus (Otoplanidae), a previously studied species of another family of the Proseriata. The results show that the main nerve cords (MCs), independent of lateral or ventral position in the Monocelididae and the Otoplanidae, correspond to each other. The study also confirms the status of the lateral cords as main cords (MCs) in S. leucops and M. lineare. Common for MCs in the members of the investigated taxa are the following features: MCs consist of many fibres, originate from the brain and are adjoined to 5-HT-positive neurons. In Monocelididae and Otoplanidae, the MCs additionally have the same type of contact to the pharyngeal nervous system. Also common for both proseriate families is the organization of the two lateral nerve cords, with weaker connections to the brain, and the pair of dorsal cords running above the brain. The organization of the minor cords differs. The Monocelididae have a pair of thin ventral cords forming a mirror image of the dorsal pair. Furthermore, an unpaired ventral medial cord connecting medial commissural cells was observed in P. schultzei. Marginal nerve cords, observed in Otoplanidae, are absent in Monocelididae. All minor nerve cords are closely connected to the peripheral nerve plexus. The postulated trends of condensation of plexal fibres to cords and/or the flexibility of the peripheral nerve plexus are discussed. In addition, the immunoreactivity (IR) pattern of NPF was compared to the IR patterns of the neuropeptide RFamide and the indoleamine, 5-HT (serotonin). Significant differences between the distribution of IR to NPF and to 5-HT occur. 5-HT-IR dominates in the submuscular and subepidermal plexuses. In the stomatogastric plexus of M. lineare, only peptidergic IR is observed in the intestinal nerve net. The distribution of NPF-IR in fibres and cells of the intestinal wall in M. lineare indicates a regulatory function for this peptide in the gut, while a relationship with ciliary and muscular locomotion is suggested for the 5-HT-IR occurring in the subepidermal and submuscular nerve, plexuses. In M. lineare, the study revealed an NPF- and RFamide-positive cell pair, marking the finished development of new zooids. This finding indicates that constancy of these cells is maintained in this asexually reproducing and regenerating species.     

Organization of the orthogon – main and minor nerve cords

The central nervous system of flatworms has been regarded as comprised of the bilobed brain, the longitudinal cords and the connecting transverse commissures forming a so called orthogon. The peripheral nervous system comprises the submuscular and subepidermal plexuses. As a confusion in the terminology of the longitudinal nerve cords has prevailed, two concepts have been introduced, the main nerve cords (MCs) and the minor cords. The MCs have been defined as the pair of longitudinal nerve cords that (1) start with strongest roots in the brain, (2) consist of wide fibre bundles and (3) are associated with more neurons (particularly amninergic marker neurons) than the other cords. Longitudinal nerve cords in other positions are thinner and have less pronounced contact with the brain. They have collectively been named minor cords. Support for the special status of the MCs has been obtained from studies of the neuroanatomy of Catenulida, Macrostomida, Proseriata, Tricladida and Lecithoepitheliata and of parasitic flatworms. Using the above mentioned criteria for the MCs and the results of recent studies, we present the following hypothese: The MCs in all flatworms correspond to each other and have a common origin. This revised version was published online in July 2006 with corrections to the Cover Date.     

The nervous system of Bothriomolus balticus (Proseriata) —a contribution to the knowledge of the orthogon in the Plathelminthes

Summary The nervous system (NS) of Bothriomolus balticus (Proseriata) was studied by the immunocytochemical (ICC) method with antisera to RFamide, SALMFamide and serotonin and with the histochemical GAIF method. The use of the ICC technique provided a much more precise morphological account of the nervous system than had previously been possible. The obtained data are discussed in connection with the comparative morphology of the nerve cords of the Plathelminthes. A similar position does not grant direct correspondence between nerve cords in the taxon Seriata. Marginal cords had probably an independent origin in the Monocelididae and Otoplanidae. The ventral (main) cords of B. balticus seem to correspond to the lateral (main) cords of the Monocelididae. It can be hypothesized that both: (1) a shift of the main cords accompanied by formation of new cords from the plexus and fusion of other cords and (2) a redistribution of nerve processes and perikarya between the cords, take part in the evolution of cords in the Plathelminthes. The first hypothesis seems to explain the difference in the position of main cords in proseriates, though, the second hypothesis might dominate, for example, in the Neorhabdocoela and the Neodermata. The correctness of the evolutionary analysis of the nerve cords in plathelminths can only be provided by neurons or neuron groups marking these structures.     

Hydra size and budding rate: influence of feeding

Summary

In the present study of Dugesia tigrina, the development of the nervous system is followed and compared during regeneration after fission and after decapitation. Immunocytochemistry was used, with antisera raised against the biogenic mine, 5-hydroxyhyptamine (5-HT) and the two neuropeptides, neuropeptide F (NPF), and FMRFamide. The results indicate that two processes are involved in the formation of the new cerebral ganglion. First, new processes sprouting from the original main longitudinal nerve cords bend transversely, indicating the position of the developing horseshoe-shaped anterior cerebral commissure. Then new nerve cells in front of the commissure differentiate from neoblasts and their growth cones fasciculate with the fibres from the old main longitudinal nerve cords. In the cerebral ganglion, 5-HT-IR cells appear before NPF-IR cells, in contrast to the pharynx where NPF-IR cells differentiate before the 5-HT-IR cells. In the peripheral nervous system, NPF-IR fibres and cells appear at a very early stage and dominate the whole regeneration process. A role for the PNS in early pattern formation is suggested.     

The nervous system of the tube-worm Chaetopterus variopedatus (Polychaeta)

Silver impregnation of serial histological sections of the tubeworm Chaetopterus variopedatus revealed the presence of a subepidermal nervous system. The anterior nervous system is delimited by the first 11 segments and comprises (1) two dorsolateral cerebral ganglia and lateral instead of ventral nerve cords which are widely separated and thus connected by unusually long commissures, (2) a pharyngeal ganglion in the fourth segment which is connected to the cerebral ganglia by pharyngeal nerves and constitutes along with the pharyngeal plexus a stomatogastric or enteric nervous system, and (3) small, presumably segmental ganglionic swellings along the lateral nerve cords from which emerge commissures and parapodial nerves. No subesophageal ganglion or periesophageal connective could be identified. The lateral nerve cords converge toward the midline in the 12th segment to form the posterior nervous system comprising a pair of ventromedian nerve cords with their repetitive segmental ganglia from which emerge numerous short commissures and three segmental nerves coursing toward the dorsal and ventral regions of parapods and toward the neuropod. Light and electron microscopic investigations of cerebral and segmental ganglia showed an arrangement of inner neuropile and of unipolar neuron somata at the periphery. The neuropile comprises numerous neurites ranging in diameter from 0.5 to 10 μm and making polarized or symmetrical synaptic junctions with each other. The pharyngeal ganglion consists of a similar neuropile and of a large mass of cell bodies which is traversed by an elaborate network of sinuses and harbors three types of neurosecretory cells in addition to the conventional neuron somata. These findings are interpreted in the framework of the highly specialized morphological features and habits of Chaetopterus, and the welldeveloped stomatogastric system is considered to be related to control of the feeding activities.     

The nervous system of Tricladida. I. Neuroanatomy ofProcerodes littoralis (Maricola,Procerodidae): An immunocytochemical study

The organization of the nervous system ofProcerodes littoralis (Tricladida, Maricola, Procerodidae) was studied by immunocytochemistry, using antibodies to authentic flatworm neuropeptide F (NPF) (Moniezia expansa). Compared to earlier investigations of the neuroanatomy of tricladid flatworms, the pattern of NPF immunoreactivity inProcerodes littoralis reveals differences in the following respects: 1. Shape and structure of the brain. 2. Number and composition of longitudinal nerve cords. 3. Shape of branches of, and transverse connections between, main ventral nerve cords. 4. Composition of the pharyngeal nervous system. The rich innervation by NPF immunoreactive (IR) fibres and cells of the subepithelial muscle layer, the pharynx musculature and the musculature of the male copulatory apparatus indicates a neurotransmitter or neuromodulatory influence on muscular activity.     

Localisation of GYIRFamide immunoreactivity inMacrostomum hystricinum marinum (Plathelminthes,Macrostomida)

The distribution of GYIRFamide immunoreactivity in the nervous system ofMacrostomum hystricinum marinum has been demonstrated by an indirect fluorescence technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was extensive in both the central (CNS) and peripheral (PNS) nervous systems, revealing detailed information on the microanatomy of the peptidergic nervous system of this free-living plathelminth. In the CNS, immunoreactive nerve cell bodies and nerve fibres occurred in the brain and along two pairs of longitudinal nerve cords: the main nerve cords and the ventral nerve cords. In the PNS, immunostaining was prevalent in nerve cells and fibres innervating the pharynx and the gut. The employed antibody is directed against a recently characterised FMRF-amide-related peptide (FaRP), GYIRFamide, isolated from two species of the Tricladida,Dugesia tigrina andBdelloura candida. Phylogenetically, GYIRFamide represents the most ancient neuropeptide thus far identified within the Bilateria     

The nervous system of Tricladida. IV. Neuroanatomy of Planaria torva (Paludicola,Planaridae): an immunocytochemical study

Characters of the nervous system have scarcely been used in consideration of the phylogenetic relationships of the Tricladida. In order to contribute characters toward this end, we studied the nervous system of Planaria torva with immunocytochemical methods and we compare the results with what is known of nervous system structure in other triclads. Antisera against four neuroactive substances were used: neuropeptide F (NPF), RFamide, GYIRFamide, and serotonin (5-HT). All four substances showed positive immunoreactions throughout the nervous system; and the patterns of distribution of the first three were similar, while 5-HT showed a distinct distribution. The organization of these nervous system elements in Planariidae and Dendrocoelidae resembles that of the Maricola but differs from that of the Dugesiidae. The results support the hypotheses that the Paludicola is a more advanced taxon than the Maricola and that the Planariidae and Dendrocoelidae together are a sister group of the Dugesiidae. This revised version was published online in July 2006 with corrections to the Cover Date.     

The structure of Archiannelid Dinophilus gyrociliatus ventral nerve cords

Dinophilus gyrociliatus is a typical representative of the synthetic group Archiannelida. Here we describe the structure of Dinophilus young adults ventral nerve cords. Five nerve cords, irregular commissures and intersegmental nerves are revealed by tubulin and FMRFamide immunostaining. 5-HT-stainning marks the complex varicose nerve plexus with numerous irregularly distributed small perikarya. Four nerve cords and solitary cell bodies occur after clyoxylic-induced reaction for catecholamines visualization. Altogether, our results indicated that morphology of Dinophilus ventral nerve cords differs from the Polychaetes archetype in many respects.     

Antisera to the sequence Arg-Phe-amide visualize neuronal centralization in hydroid polyps

Summary Antisera to the sequence Arg-Phe-amide (RF-amide) have a high affinity to the nervous system of fixed hydroid polyps. Whole-mount incubations of several Hydra species with RFamide antisera visualize the three-dimensional structure of an ectodermal nervous system in the hypostome, tentacles, gastric region and peduncle. In the hypostome of Hydra attenuata a ganglion-like structure occurs, consisting of numerous sensory cells located in a region around the mouth opening and a dense plexus of processes which project mostly radially towards the bases of the tentacles. In Hydra oligactis an ectodermal nerve ring was observed lying at the border of hypostome and tentacle bases. This nerve ring consists of a few large ganglion cells with thick processes forming a circle around the hypostome. This is the first direct demonstration of a nerve ring in a hydroid polyp.Incubation of Hydractinia echinata gastrozooids with RFamide antisera visualizes an extremly dense plexus of neuronal processes in body and head regions. A ring of sensory cells around the mouth opening is the first group of neurons to show RFamide immunoreactivity during the development of a primary polyp. In gonozooids the oocytes and spermatophores are covered with strongly immunoreactive neurons.All examples of whole-mount incubations with RF-amide antisera clearly show that hydroid polyps have by no means a diffuse nerve net, as is often believed, and that neuronal centralization and plexus formation are common in these animals. The examples also show that treatment of intact fixed animals with RFamide antisera is a useful technique to study the anatomy or development of a principal portion of the hydroid nervous system.     

A robust molecular phylogeny of the Tricladida (Platyhelminthes: Seriata) with a discussion on morphological synapomorphies.

The suborder Tricladida (Platyhelminthes: Turbellaria, Seriata) comprises most well-known species of free-living flatworms. Four infraorders are recognized: (i) the Maricola (marine planarians); (ii) the Cavernicola (a group of primarily cavernicolan planarians); (iii) the Paludicola (freshwater planarians); and (iv) the Terricola (land planarians). The phylogenetic relationships among these infraorders have been analysed using morphological characters, but they remain uncertain. Here we analyse the phylogeny and classification of the Tricladida, with additional, independent, molecular data from complete sequences of 18S rDNA and 18S rRNA. We use maximum parsimony and neighbour-joining methods and the characterization of a unique gene duplication event involving the Terricola and the dugesiids to reconstruct the phylogeny. The results show that the Maricola is monophyletic and is the primitive sister group to the rest of the Tricladida (the Paludicola plus the Terricola). The Paludicola are paraphyletic since the Terricola and one paludicolan family, the Dugesiidae, share a more recent common ancestor than the dugesiids with other paludicolans (dendrocoelids and planariids). A reassessment of morphological evidence may confirm the apparent redundancy of the existing infraorders Paludicola and Terricola. In the meantime, we suggest replacing the Paludicola and Terricola with a new clade, the Continenticola, which comprises the families Dugesiidae, Planariidae, Dendrocoelidae and the Terricola.     

Primitive nervous system: Electrophysiology of inhibitory events in flatworm nerve cords

Evoked potentials from the major longitudinal nerve cords of Notoplana acticola are potentiated if the preparation is decerebrated or if certain nerves in the ventral submuscular plexus are severed. Concomitant with an increase in amplitude of the response (in some preparations over fourfold) is a decrease in latency of the response and a decrease in the threshold stimulus intensity needed to evoke activity. Evoked activity can also be depressed with moderate increases in stimulus intensity and is induced some distance from the recording site. The depressant effects can be lifted if the brain is bisected, if the contralateral nerve VI is severed close to the brain, and if the commissures between the two nerves VI are cut. The depressant effects of nerves V and VI are additive. Depression and the subsequent lifting of the inhibition occur in Ringer baths containing either normal sea water or equal mixtures of sea water and isotonic MgCl₂. The possibility of inhibitory synapses immune to high concentrations of magnesium ions is discussed.     

The pattern of neuropeptide F and RF-amide in two tapeworms

Two years ago the first platyhelminth regulatory peptide, neuropeptide F (NPF), was isolated from the tapeworm Moniezia expansa by Maule et al. (1991). NPF is a 39 amino acid peptide with a C terminal phenylalaninamide. NPF is the first platyhelminth neuropeptide to be sequenced fully. Preabsorption with NPF quenches the immunostaining with anti-FMRF-amide and anti-bovine PP (Halton et al. 1992). As the first authentic flatworm neuropeptide, the occurence and distribution of NPF along the whole flatworm line are under investigation. Both free-living and parasitic flatworms are being studied. So far NPF-immunoreactivity has been reported from three free- living flatworms (see Grahn et al., 1995) and from four parasitic flatworms (Marks et al., 1993).FMRF- and RF-amide immunoreactive (IR) nerve cells and fibres are common in the gull-tapeworm Diphyllobothrium dendriticum. In order to test whether the patterns for NPF- and RF-immunoreactivity co- localize in the gull-tapeworm, immunostaining with anti-NPF and anti-RF were performed. To broaden the study, adult Proteochepalus exiguus from the intestine of whitefish were included in the experiment.The study was performed on whole mounts of skinned worms (Gustafsson, 1991). Anti-NPF was used in concentrations 1:500 and 1:1000. Controls included liquid phase absorption with the homologous antigen (1000 ng ml^–1).In D. dendriticum NPF-immunoreactivity occurs in nerve cells and varicose nerve fibres of larval and adult worms. The NPF-IR cell bodies are more common in the peripheral nerve cords than along the main nerve cords, which contain nerve fibres with large varicosities. The cell bodies in the PNS are often triangular in shape. Immediately beneath the tegumental surface a thin NPF-IR nerve fibre is observed. As to the co-localization of NPF and RF nothing definite can be said but the general pattern seems tobe the same. In the brain commissure of D. dendriticum one large ganglion cell stains with both antisera, indicating coexistence.In P. exiguus NPF- and RF-immunoreactivity was observed in the two main nerve cords situated laterally and in the pairs of thin dorsal and ventral longitudinal nerve cords. Numerous transverse commissures connect the longitudinal cords forming an orthogonal pattern. The cell bodies along the nerve cords are multipolar. Thin projections extend from the main nerve cords to the surface of the worm. The main nerve cords are lined with NPF-and RF-IR cell bodies. The general staining patterns of NPF and RF are very similar.     

The nervous system of Microstomum lineare (Turbellaria,Macrostomida)

Summary The nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida) was studied by electron and light microscopy, combined with fluorescence histochemistry (Falck-Hillarp method for biogenic monoamines). The NS is primitively organized, with a bilobed brain, two lateral nerve cords lacking commissures, and peripheral nerve cells scattered along the nerve cords. The stomatogastric NS, with a pharyngeal nerve ring, is joined to the central NS by a pair of connective ganglia. A green fluorescence in all parts of the NS indicates catecholaminergic neurons as the dominant neuron type.Ultrastructurally, two types of neurons were identified on the basis of their vesicle content: 1. Aminergic (catecholaminergic) neurons containing densecore vesicles of varying electron-density and size, i.e., small dense-core vesicles (diameter 50–100 nm), vesicles with a highly electron-dense core (60–140 nm), and vesicles with an eccentric dense-core. 2. Presumed peptidergic neuro-secretory neurons containing large granular vesicles (diameter about 200 nm) in the stomatogastric NS and peripheral parts of the central NS. In light microscopy, paraldehyde-thionin stained neurons were observed in the same areas.     

Comparative analysis of the nervous systems in presumptive progenetic dinophilid and dorvilleid polychaetes (Annelida) by immunohistochemistry and cLSM

Entire nervous systems of the dinophilids Dinophilus (two species) and Trilobodrilus (three species) and the dorvilleids Parapodrilus psammophilus and Ophryotrocha gracilis (larva) were stained with antisera directed against serotonin, Phe‐Met‐Arg‐Phe‐NH₂ (FMRFamide) and acetylated α‐tubulin and analysed by confocal laser scanning microscopy (cLSM). Adult dinophilids and the dorvilleid larva exhibit the same structure of the ventral nerve cord, with two main nerves spaced far apart, one median and two paramedian nerves. A serotonergic plexus is situated between the paramedian nerve pair, above the ventral locomotory ciliary band. These similarities between adults and larva corroborate the presumed progenetic origin of dinophilids. However, since larval nervous systems of other polychaete taxa also seem to be organized in this way, this result cannot support the view that dinophilids originate from dorvilleids. In P.psammophilus the main nerve cords are widely separated only in the last segment, indicating that this pattern may be correlated with the absence of parapodia. The unpaired median nerve of dinophilids, P. psammophilus and many other polychaetes, is considered to be part of the basic annelid body plan. The ground pattern of the ventral paired dinophilid ganglia is represented by three (anterior, main, posterior) commissures, conserved in most of the ganglia in the Dinophilus species and mostly reduced to a main commissure in the Trilobodrilus species. The dinophilid species and P. psammophilus possess six pairs of ganglia indicating six trunk segments – in contrast to former views. The two rings behind the prostomium in both the dinophilid and the dorvilleid species contain one pair of ganglia only, corroborating the presumed homology of this peristomial region in the two taxa. The Dinophilus nervous system with 12 longitudinal nerves and three perpendicular nerve rings per segment resembles orthogonal nervous structures characteristic of platyhelminths.     

The catecholaminergic nerve plexus of Holothuroidea

Catecholamines have been extensively reported to be present in most animal groups, including members of Echinodermata. In this study, we investigated the presence and distribution of catecholaminergic nerves in two members of the Holothuroidea, Holothuria glaberrima (Selenka, 1867) (Aspidochirotida, Holothuroidea) and Holothuria mexicana (Ludwig, 1875) (Aspidochirotida, Holothuroidea), by using induced fluorescence for catecholamines on tissue sections and immunohistochemistry with an antibody that recognizes tyrosine hydroxylase. The presence of a catecholaminergic nerve plexus similar in distribution and extension to those previously reported in other members of Echinodermata was observed. This plexus, composed of cells and fibers, is found in the ectoneural component of the echinoderm nervous system and is continuous with the circumoral nerve ring and the radial nerves, tentacular nerves, and esophageal plexus. In addition, fluorescent nerves in the tube feet are continuous with the catecholaminergic components of the radial nerve cords. This is the first comprehensive report on the presence and distribution of catecholamines in the nervous system of Holothuroidea. The continuity and distribution of the catecholaminergic plexus strengthen the notion that the catecholaminergic cells are interneurons, since these do not form part of the known sensory or motor circuits and the fluorescence is confined to organized nervous tissue.     

Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis

The neuroendocrine system of the starfish Marthasterias glacialis was investigated immunocytochemically using antisera specific for rat neuronal, bovine aortic endothelial, and mouse macrophage, nitric oxide (NO) synthases. Immunoreactivity was detected only with the antibodies specific for the neural enzyme, in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract. The pyloric stomach showed more immunoreactive structures than the other digestive organs, with the rectal caeca showing the least activity. Immunoreactive endocrine cells were located in the cardiac and pyloric stomachs and in the pyloric caeca. Co-localization of the enzyme immunoreactivity, and the staining for NADPH-diaphorase, demonstrate the presence of NO synthase in echinoderms. These results provide further evidence that NO is a neuronal messenger of early phylogenetic origin which has been conserved throughout evolution.     

The pineal complex of the three-spined stickleback,Gasterosteus aculeatus L.

Summary The pineal complex of the three-spined stickleback (Gasterosteus aculeatus L.) was investigated by light and electron microscopy, as well as fluorescence histochemistry for demonstration of catecholamines and indolamines. The pineal complex of the stickleback consists of a pineal organ and a small parapineal organ situated on the left side of the pineal stalk. The pineal organ, including the entire stalk, is comprised mainly of ependymal-type interstitial cells and photoreceptor cells with well-developed outer segments. Both unmyelinated and myelinated nerve fibres are present in the pineal organ. Nerve tracts from the stalk enter the habenular and posterior commissures. A small bundle of nerve fibres connects the parapineal organ and the left habenular body. The presence of indolamines (5-HTP, 5-HT) was demonstrated in cell bodies of both the pineal body and the pineal stalk, and catecholaminergic nerve fibres surround the pineal complex.