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.     

Growth factors in asexually reproducing catenulida and macrostomida (Plathelminthes)?

Summary The occurrence and localization of immuno-reactivity (IR) to epidermal growth factor receptor (EGF-r), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), revealed by the present study, indicate growth-factor-like substances in the asexually reproducing flatworms,Stenostomum leucops andMicrostomum lineare. IR to all three antibodies occurs in the nervous system. Differences in the number of positive cells and intensity of IR during asexual development of new individuals were observed. By confocal scanning laser microscopy, immunopositive growth cones of nerve fibres were seen in developing zooids, and weakly or unstained perikarya were observed in close contact with the positive nerve fibres. Antibodies to the growth factors EGF-r, EGF and bFGF as well as to the neuroactive substances 5-HT and RF-amide had a negative influence on the growth and asexual reproduction of culturedS. leucops. No significant differences in the influence of antibodies to growth factors and antibodies to the neuroactive substances were observed.     

Development and differentiation of neuronal subsets in asexually reproducing Microstomum lineare

Summary The development of immunoreactivity (IR)_in the nervous system of asexually reproducing Microstomum lineare has been studied by a combination of simultaneous and double immunostaining with antisera to 5-HT and RF-amide, as well as with monoclonal antibodies to SCP_B (molluscan small cardiactive peptide). Immunoreactivity appears in a distinct sequential order. 5-HT antigenicity in the postpharyngeal commissure indicates the initiation of the development of a new zooid. The development of a new brain and pharyngeal plexus always starts in connection to the parental nerve cords. Significantly different developmental patterns are observed for the IR to 5-HT and RF-amide, whereas IR to SCP_B has the same localization as that to RF-amide, but appears both weaker and later during the development. Influences of the immunoreactive substances on the asexual reproduction and the feeding behaviour are discussed.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

Immunocytochemical demonstration of 5-HT-like and FMRF-amide-like substances in whole mounts of Microstomum lineare (Turbellaria)

Summary A whole mount immunofluorescence method is used for an investigation of immunoreactivity (IR) to anti-(a-) 5-HT and anti-(a-)FMRF-amide in the nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida, Platyhelminthes). New details of the organization of the NS are demonstrated, differences in 5-HT and FMRF amide IR are revealed, and new information on the development of the NS in zooids is obtained. In contrast to previous reports of a reduction (one pair of nerve cords without transverse processes) of the basic turbellarian plan, IR to both antisera reveals three pairs of longitudinal nerve cords, and features of the orthogonal organization, characterized by transverse commissures. The lateral pair of nerve cords is the most prominent. The following differences in the patterns of 5-HT and FMRF-amide IR are observed: 1. Perikarya positive for a-5-HT and a-FMRF-amide in the brain show different localizations. 2. Perikarya positive for a-5HT occur along the main lateral nerve cords, while the cords visualized by FMRF-amide IR look double-stranded and lack associated perikarya. 3. 5-HT IR is observed in a postpharyngeal commissure, which is absent in the a FMRF-amide-stained preparations. 4. In developing zooids 5-HT IR is first observed in the postpharyngeal commissure and later on in an increasing number of perikarya and in the neuropile. The first FMRF-amide IR in developing zooids appears in the cerebral commissure and in two perikarya in front of this commissure.     

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.     

Stem cells in microturbellarians

Summary A combination of microscopical, immunocytochemical, and autoradiographic techniques were employed to study stem cells and their fates during asexual reproduction and regeneration in two microturbellarians,Microstomum lineare (Macrostomida) andStenostomum leucops (Catenulida). Special attention was paid to the development of the immunoreactivity (IR) to FMRF/RF-amide and 5-HT in differentiating nerve cells.Asexual reproduction inM. lineare andS. leucops occurs by paratomy, i.e., fragmentation after completed differentiation of the new organs. Regeneration, on the other hand, involves a combination of morphallactic and epimorphic processes without the formation of a regeneration blastema. The only cells incorporating tritiated thymidine ([³H]T) were the mesenchymal and gastrodermal neoblasts, which proliferate continuously replenishing the population of stem cells available for growth, asexual reproduction and regeneration. These proliferative cells occurred in two ultrastructurally different forms, differing from each other only by the presence or absence of ciliar basal bodies in the cytoplasm. Few differentiated cells were labeled in the head piece after completed regeneration. A greater amount of labeled differentiated cells were, however, observed postpharyngeally in the first zooid as well as in zooids having developed during the same time (i.e., 20–45 h after the treatment with [³H]T). Furthermore, many labeled cells were still undifferentiated at that time or just in the beginning of the differentiation process. It can therefore be concluded that neoblasts function both as reserve cells and as functional stem cells for all differentiated cell types in these worms. IR to FMRF/RF-amide neuropeptides was not observed in nerve cells differentiating from neoblasts until the occurrence of dense-core vesicles in their cytoplasm. Due to methodological difficulties only weak or no IR to 5-HT could be traced in the nervous system of the asexual and regenerating worms.Abbreviations ICC Immunocytochemical - IR immunoreactivity - [³H]T tritiated thymidine     

Catecholamines demonstrated by glyoxylic-acid-induced fluorescence and HPLC in some microturbellarians

The microturbellarians Stenostomum leucops (Catenulida), Microstomum lineare (Macrostomida), Promonotus schultzei (Proseriata) and Gyratrix hermaphroditus (Rhabdocoela) were subjected to the glyoxylic-acid-induced-fluorescence method to reveal catecholaminergic parts of their nervous systems. Histofluorescence indicative of catecholamines was evident in nerve cells and fibers of all investigated species. The patterns of distribution were compared to those of other neuroactive substances. The catecholaminergic fluorescence was at least partly confined to separate neuronal subsets in S. leucops and M. lineare and probably also in P. schultzei. In S. leucops, the presence of dopa and dopamine was demonstrated by high-pressure liquid chromatography. The occurence of catecholamines in these representatives of four orders of microturbellarians suggests that catecholamines arose early in the evolution of flatworms and underscores the importance of dopaminergic substances for the function of early nervous systems.     

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.     

Ciliary and nervous structures in juvenile females of the annelid Dinophilus gyrociliatus (O. Schmidt, 1848) (Annelida: Polychaeta)

Ciliary and nerve structures were described in juvenile female Dinophilus gyrociliatus (O. Schmidt, 1848) after immunochemical staining with tubulin, serotonin, and FMRFamide antibodies. Anti-tubulin antibodies revealed the following external structures: two head and seven body ciliary bands, a ventral ciliary band, and head ciliary fields. Gut cilia and five pairs of protonephridia were detected inside the body. The nervous system consists of an oval headed neuropile with anterior and posterior nerves extending from it, seven longitudinal nerve cords, commissures, and circular nerves. Anti-serotonin antibodies revealed the head neuropile, neurons at the base of the ventral ciliary band, an oesophageal ring, and seven longitudinal ventral cords. Anti-FMRFamide antibodies revealed approximately ten neurons in the cerebral ganglion, five longitudinal cords, and the oesophageal and caudal-nerve rings. The presented data suggest the simplification of the nervous system structure in D. gyrociliatus, which probably reflects pedomorphosis.     

Immunocytochemical localization of histamine in flatworms

Summary Specific antibodies against histamine were used to demonstrate the occurrence and cellular distribution of histamine-like immunoreactivity in three species of flatworms (phylum Platyhelminthes). In the parasitic cestode Diphyllobothrium dendriticum, histamine-reactivity was found in neurons of the main nerve cords, and in cells lining the central and peripheral excretory ducts. In the free-living microturbellarian Microstomum lineare and in the planarian Polycelis nigra, histamine-immuno-reactivity was restricted to cells and fibres of the nervous system. The occurrence of histamine or a related substance in the nervous system of flatworms, which represent primary bilateria, indicates the importance of this neuroactive substance in the animal kingdom.     

Immunoreactivity to a specific echinoderm neuropeptide in the nervous system of the flatworm Macrostomum hystricinum marinum (Turbellaria,Macrostomida)

Immunocytochemical characterization of the neuropeptides FMRF-amide and serotonin (5-HT) is a well-known method successfully applied to demonstrate nervous-system morphology in several platyhelminths (see Wikgren & Reuter, 1985, and Reuter, 1988, for review). We have immunolabeled whole-mount preparations of Macrostomum hystricinum marinum Rieger from cultures (see Rieger et al., 1988) with anti-SALMF-amide, an antibody specific for the C-terminal pentapeptide sequence of the neuropeptide GNFSALMF-amide recently isolated from echinoderms (source M. Thorndyke, England). Immunoreactivity to SALMF-amide gave a more detailed picture of the nervous system of M. hystricinum than FMRF-amide. Conventional light microscopy (Luther, 1905) shows this nervous system to consist of a bilobed brain, a pharyngeal nerve-ring system, a posterior commissure, and two main ventrolateral nerve cords. Immunostaining reveals, in addition, two thin paired longitudinal nerve strands and fine subepithelial and submuscular nerve nets. Anti-SALMF-amide labels a distinct class of neurons, causing the main lateral longitudinal cords and pharyngeal nerve-ring system to appear more filamentous than with other techniques. Recent fine-structural investigations on the nervous system of Macrostomum hystricinum marinum revealed several axon types with characteristic vesicles and neurotubules (D. R. pers. obs.). Partly supported by FWF grant P7816.     

Antigenicity to neuropeptide F (NPF) in Stenostomum leucops and Microstomum lineare

The first native flatworm regulatory peptide, neuropeptide F (NPF) has recently been isolated and sequenced from the cestode Moniezia expansa (see Maule et al., 1991) and the turbellarian Artioposthia triangulata, (see Curry et al., 1992). NPF belongs to the neuropeptide Y (NPY) superfamily and the antiserum is known to show cross-reactivity to the vertebrate neuropeptides of the NPY superfamily. It terminates in RFamide, like the invertebrate neuropeptides FMRFamide and RFamide, and may cross-react with neuropeptides of the FMRFamide family. Strong immunoreactivity (IR) to FMRF- and RF-amide has been demonstrated in members of most flatworm groups. In the present study, IR to NPF (diluted 1:1000) is demonstrated in Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida). The controls included: omitting primary antibody, using non-immune serum and liquid-phase absorption with the homologous antigen (1000 ng ml^–1). The NPF IR pattern was compared to the FMRF and RF-amide IR patterns in order to reveal differences or co-localization. In addition, the sequential appearance of NPF-positive cells in developing zooids was followed and double staining with a-5-HT made to complete the study.     

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 release sites and targets of nerve cells immunoreactive to RFamide — an ultrastructural study ofMicrostomum lineare andDiphyllobothrium dendriticum (Plathelminthes)

Summary A detailed study of the ultrastructure and RFamide immunoreactivity (RF-IR) in the flatwormsMicrostomum lineare andDiphyllobothrium dendriticum has been made with the immunogold technique. The present ultrastructural study confirms the localization of RF-IR cells observed by light microscopic immunocytochemistry. The RF-IR is demonstrated in small, electron-dense vesicles in neuronal cell bodies and processes. RF-IR is not detected in the rough endoplasmatic reticulum or the Golgi system of the nerve cells. The targets of RF positive fibres are nerve fibres, muscles and glands. Gold labelling occurs in classic synapses, which points to a role in neurotransmission. RF-IR is also observed in nerve terminals lacking the characteristics of synapses. These release sites occur close to muscle fibres in the intestine and body musculature ofM. lineare. Thus, an additional, paracrine action of the neuropeptide is suggested.     

The nervous system of Tricladida. III. Neuroanatomy ofDendrocoelum lacteum andPolycelis tenuis (Plathelminthes,Paludicola): an immunocytochemical study

The organization of the nervous system ofDendrocoelum lacteum (Tricladida, Paludicola, Dendrocelidae) andPolycelis tenuis (Tricladida, Paludicola, Planariidae) was studied by immunocytochemical double staining, using neuropeptide RFamide and serotonin (5-HT) antisera on cryosections. The study confirmed the status of the main nerve cords (MCs) as the most important and stable of the longitudinal cords and supported the hypothesis of a common phylogenetic origin of the MCs in flatworms. The ganglion-like structures along the MCs at the beginning of transverse commissures and laterla branches showed a close contact with ventral fibres of the submuscular nerve plexus indicating an origin from crossing points of insunken ring commissures. The distributional pattern and morphology of the RFamide-IR cell bodies inD. lacteum corresponded to that of neurosecretory cells. Most RFamide-IR cells were unipolar and rounded while 5-HT-IR cells were uni- bi- and multipolar. The neutropile consisted of a dense RFamide-IR and a loose 5-HT-IR network. RFamide dominated in all parts of the genital plexus.     

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release. Work in this study was funded by the National Health and Medical Research Council (grant numbers: 114215 and 216704; Senior Research Fellowship to B.S.), a Melbourne University Research Scholarship and the Murdoch Children’s Research Institute.     

On the serotonergic nervous system of two planktonic rotifers, Conochilus coenobasis and C. dossuarius (Monogononta, Flosculariacea, Conochilidae)

The serotonergic nervous systems of two non-colonial species of Conochilus were examined to obtain the first immunohistochemical insights into the neuroanatomy of species of Flosculariacea (Rotifera, Monogononta). Species of Conochilus, subgenus Conochiloides, were examined using serotonin (5-HT) immunohistochemistry, epifluorescence and confocal laser scanning microscopy, and 3D computer imaging software. In specimens of C. coenobasis and C. dossuarius, the serotonergic nervous system is defined by a dorsal cerebral ganglion, apically directed cerebral neurites, and paired nerve cords. The cerebral ganglion contains approximately four pairs of small 5-HT-immunoreactive perikarya; one pair innervates the posterior nerve cords and three pairs innervate the apical field. The most dorsal pair innervates a coronal nerve ring that encircles the apical field. Within the apical field is a second nerve ring that outlines the inner border of the coronal cilia. Together, both the inner and outer nerve rings may function to modulate ciliary activity of the corona. The other two pairs of perikarya innervate a region around the mouth. Specific differences in the distribution of serotonergic neurons between species of Conochilus and previously examined ploimate rotifers include the following: (a) a lack of immunoreactivity in the mastax; (b) a greater number of apically directed serotonergic neurites; and (c) a complete innervation of the corona in both species of Conochilus. These differences in nervous system immunohistochemistry are discussed in reference to the phylogeny of the Monogononta.     

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.