Nadezhda B. Terenina

NO nerves and their targets in a tapeworm: an immunocytochemical study of cGMP in Hymenolepis diminuta

We studied the pattern of cGMP immunostaining (IS) after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase in adult Hymenolepis diminuta. cGMP-IS was detected in the peripheral nervous system, especially in nerve fibres close to the body muscle fibres. cGMP-IS also occurred in terminals beneath the basal lamina of the tegument and between the muscle fibres of the suckers. The pattern of cGMP-IS was compared to that of 5-HT-IS and GYIRFamide-IS. TRITC-conjugated phalloidin was used to stain the musculature.

NO nerves in a tapeworm. NADPH-diaphorase histochemistry in adult Hymenolepis diminuta.

The free radical nitric oxide (NO), which is synthesized by nitric oxide synthase (NOS), has recently been discovered to function as a neuronal messenger. The presence of NOS was detected in the nervous system of adult Hymenolepis diminuta with NADPH-diaphorase (NADPH-d) histochemistry. The NADPH-d histochemical reaction is regarded as a selective marker for NOS in neuronal tissue. NADPH-d staining was observed in nerve fibres in the main and minor nerve cords and the transverse ring commissures, and in cell bodies in the brain commissure, along the main nerve cords, in the suckers and the rostellar sac. NADPH-d staining was also observed in the wall of the internal seminal vesicle and the genital atrium. The pattern of NADPH-d staining was compared with that of the 5-HT immunoreactive nervous elements. The NADPH-d staining reaction and the 5-HT immunoreactivity occur in separate sets of neurons. This is the first time the NADPH-d reaction has been demonstrated in the nervous system of a flatworm, indicating that NOS is present and that NO can be produced at this level of evolution.

The nervous system of Tricladida. II. Neuroanatomy ofDugesia tigrina (Paludicola, Dugesiidae): An immunocytochemical study

The nervous system (NS) ofDugesia tigrina has been studied by immunocytochemical double-staining, using the authentic flatworm neuropeptide, neuropeptide F (NPF), and serotonin (5-HT) on cryosections. This technique provides a precise morphological (descriptive) account of the NS. The results show that the central nervous system is shaped like a horseshoe. The brain is composed of two lateral lobes connected by three commissures, one antero-dorsal in front of the cerebral eyes and two, more ventral, behind the eyes. The pair of main nerve cords extend from the lateral lobes of the brain to the tail end of the worm. Cross sections reveal a very close contact between lateral branches from the main cords and the submuscular plexus. Thin cord-like lateral nerves are formed by longitudinal plexal fibres. No dorsal cords were observed. The patterns of immunoreactivity to NPF and 5-HT differ from each other in several respects. In the walls of gut diverticula only NPF immunoreactive (IR) cells and fibres were observed. Only NPF-immunoreactive cells occur in the parenchyma along dorso-ventral nerve fibres connecting the dorsal and ventral parts of the submuscular plexus. The number of 5-HT-immunoreactive cells associated with the main nerve cords (MCs) is greater than that of the NPF-immunoreactive cells, and the spongy structure of the MCs is more apparent following immunostaining for 5-HT. Thin 5-HT-immunoreactive fibres were observed in the subepithelial plexus, penetrating the basal lamina and innervating a rhabdite-free ventro-lateral sensory area along the body periphery. The correspondence between MCs in the lower flatworms (Catenulida and Macrostomida) and the Seriata (Tricladida and Proseriata) confirms the status of the MCs in flatworms as the most important and stable neuronal characteristic, and constitutes support for the hypothesized common origin of the MCs in flatworms.

Neuropeptides in flatworms.

The use of well-characterized antibodies raised to neuronal signal substances and their application through immunocytochemistry and confocal scanning laser microscopy has revolutionized studies of the flatworm nervous system (NS). Data about flatworm neuropeptides and the spatial relationship between neuropeptides and other neuronal signal substances and muscle fibers are presented. Neuropeptides form a large part of the flatworm NS. Neuropeptides are especially important as myoexcitatory transmitters or modulators, controlling the musculature of the attachment organs, the stomatogastric and the reproductive systems.

An experimental, NADPH-diaphorase histochemical and immunocytochemical study of Mesocestoides vogae tetrathyridia.

In order to test the role of nitric oxide in flatworms, Mesocestoides vogae tetrathyridia were incubated together with L-arginine, which is the substrate for nitric oxide synthesis, or with NG-nitro-L-arginine, which is an irreversible inhibitor of nitric oxide synthase. Normally, tetrathyridia attach to each other with the aid of their suckers, forming clusters. The rate of cluster formation was followed during the incubations. L-Arginine stimulated, and NG-nitro-L-arginine clearly inhibited, the cluster formation. This is the first time that an effect of nitric oxide has been observed in a flatworm. In addition, the pattern of the NADPH-diaphorase histochemical reaction in the nervous system and the pattern of F-actin filaments in the musculature stained with TRITC-labelled phalloidin were studied. NADPH-d staining occurred in the brain and the main nerve cords but also followed the muscle fibres stained with phalloidin. The pattern of the NADPH-d reaction was compared with that of 5-HT immunoreactivity. The implications of the results are discussed in relation to the background of data on neuronal signal substances in M. vogae.

Comparative study of the spatial relationship between nicotinamide adenine dinucleotide phosphate-diaphorase activity, serotonin immunoreactivity, and GYIRFamide immunoreactivity and the musculature of the adult liver fluke, Fasciola hepatica (Digenea, Fasciolidae)

This is the first detailed description of the nitrergic nervous system in a fluke. In this study, the authors analysed the distribution of the nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) reactivity in neuronal and nonneuronal tissues of the adult fluke Fasciola hepatica and compared this with the distribution of the musculature using tetramethylrhodamine isothiocyanate‐phalloidin. To assess the correlation between the number of muscle cells in different parts of the fluke and the NADPH‐d‐stained cells, the nuclei were stained with Hoechst 333 42, which is specific for chromatin. The spatial relation between the NADPH‐d‐positive nerves and the 5‐hydroxytryptamine (serotonin; 5‐HT)‐immunoreactive (‐IR) and GYIRFamide‐IR nervous elements was also examined. The methods complement each other. NADPH‐d‐positive staining occurs in both in neuronal tissue and nonneuronal tissue. Large, NADPH‐d‐stained neurones were localised in the nervous system. The oral and ventral suckers are innervated with many large NADPH‐d‐stained neurones. In addition, the NADPH‐d staining reaction follows closely the muscle fibres in both the suckers, in the body, and in the ducts of the reproductive organs. The presence of NADPH‐d activity along muscle fibres in F. hepatica and in other flatworms supports a possible myoinhibitory role for nitric oxide. Neuronal nitric oxide synthase in flatworms may form a novel drug target, which would facilitate the development of a novel anthelminthic. J. Comp. Neurol. 429:71–79, 2001.

A radiometric analysis of nitric oxide synthase activity in Hymenolepis diminuta

The free radical nitric oxide (NO) is a neuronal messenger which is synthesized from L-arginine and O2 by nitric oxide synthase (NOS). In the synthesis NO and L-citrulline are produced in a stoichiometric 1:1 relation. The activity of NOS was analysed in homogenates of the rat tapeworm Hymenolepis diminuta by measuring the formation of L-[3H]citrulline after incubation with L-[3H]arginine. The nature of NOS in H. diminuta was determined by studying the effect of 3 types of NOS inhibitors: (1) L-NAME, (2) EGTA, (3) 7-nitro-indazole. All inhibitors caused a significant but not complete reduction in the formation of L-[3H]citrulline. The results are discussed against the background of nerve cells and fibres positive for NADPH-diaphorase staining in H. diminuta.

Nitric oxide and its target cells in cercaria of Diplostomum chromatophorum: a histochemical and immunocytochemical study

Abstract. In order to advance our knowledge of the nitrergic nervous system in flatworms, the patterns of the NADPH-diaphorase (NADPH-d) reaction and cGMP immunoreactivity, after stimulation with a nitric oxide donor in the presence of an inhibitor of phosphodiesterase, were investigated in cercaria of Diplostomumchromatophorum. This is the first time the presence of NADPH-d activity has been detected in a larval fluke, and the first time the presence of cGMP immunoreactivity has been detected in a flatworm. The NADPH-d reaction occurs in the ventral sucker, the hind body and the tail. cGMP immunoreactivity was detected in the muscle cells of the body and in two pairs of sensory cells at the anterior end of the body and in the middle of the furca. The sensory cells also showed 5-HT immunoreactivity. The spatial relationship between the cGMP and the 5-HT immunoreactivities was studied. By staining with TRITC-labelled phalloidin, the pattern of the muscle fibres was revealed.

The Nervous System of the Pike-Tapeworm Triaenophorus nodulosus (Cestoda: Pseudophyllidea): Ultrastructure and Immunocytochemical Mapping of Aminergic and Peptidergic Elements

The nervous system of the adult pike-tapeworm Triaenophorus nodulosus was studied to identify nerve cells and fibers immunoreactive to serotonin (5-HT) and RFamide (RF) on whole-mount preparations and frozen sections. Neurons immunoreactive to 5-HT were seen solely in the central nervous system, while those immunoreactive to RF occurred in the peripheral nervous system as well as in the central nervous system. In the scolex, both types of nerve fibers were found. While the gonads were not innervated by either fiber type, the reproductive tract showed RF-immunoreactive nerves. On the ultrastructural level, five types of neurons and three types of release sites and a neuromuscular junction could be distinguished. Levels of 5-HT, measured spectrofluorimetrically, were found to be lower in the tapeworm than in the tissues where it resides in its host, indicating a possibility that the parasite absorbs this bioamine from its environment. Additional key words: 5-HT, RFamide, neuron types, neurotransmitter release sites, spectrofluorimetry During the last two decades, knowledge of the neuroanatomy of flatworms has grown (for review, see Reuter & Gustafsson 1995). For the parasitic flatworms, however, ultrastructure of neuronal elements is still rather poorly known, and information on the nature and number of neuronal mediators in flatworms in general is sparse. To fill part of this gap, we have studied the nervous system (NS) of the pike-tapeworm Triaenophorus nodulosus (PALLAS 1781) with immunocytochemical, ultrastructural, and spectrofluorimetric methods. T. nodulosus parasitizes the intestine of pike as an adult and parasitizes other economically important fishes, such as burbot, as a plerocercoid, the last larval stage. The first parasitic larval stage, the procercoid, develops in fresh-water crustaceans. Apart from the studies of Michajlow (1932, 1933, 1934) little information is available on the histology, especially the neuroanatomy, of the Triaenophoridae. Gustafsson (1973), investigating cellular composition in the neck region of plerocercoids of T. nodulosus, a To whom correspondence should be addressed. Phone: 358 21 2654603; FAX: 358 21 2654748; E-mail: magustaf@ra.abo.fi noted that the cells surrounding the main nerve cords constitute as much as 19% of the total cell population. In light microscopical studies of the general neuroanatomy of T. nodulosus, Kotikova & Kuperman (1977) monitored acetylcholinesterase activity during ontogenesis and documented formation of nerve plexuses and cholinergic nervous elements in the scolex and an increase in the number of longitudinal nerve cords at each successive stage of development; Biserova et al. (1991) used the glyoxylic-acid method to study the aminergic NS of adults and showed evidence of serotonin in ganglia of the brain, connecting commissure, and main nerve cords. Ultrastructure of sensory structures in the scolex was investigated by Biserova et al. (1991); and ultrastructure of the integument, frontal glands, and excretory system was examined by Kuperman (1973, 1988) and Kuperman & Davydov (1982a,b). We have extended the study of adults of T. nodulosus by comparing patterns of peptidergic and aminergic nerve elements, distinguishing types of nerve cells and neurotransmitter-release sites, and measuring serotonin (5-HT) concentrations. The nervous system of flatworms can be subdivided into central and peripheral elements (see Reuter & This content downloaded from 157.55.39.35 on Thu, 01 Sep 2016 04:58:52 UTC All use subject to http://about.jstor.org/terms Biserova, Gustatsson, Reuter, & Terenina Gustafsson 1995 and Reuter et al., in press, for discussion of terminology). The central nervous system of T. nodulosus consists of the bilobed brain and the two main nerve cords (MCs). The peripheral nervous system comprises all the other longitudinal cords (the minor cords), ring commissures in the cortical parenchyma, and nervous structures associated with the genital organs.

Serotonin, reserpine, and motility inMesocestoides tetrathyridia

The relationship between serotonin (5-HT) and motility in tetrathyridia of Mesocestoides vogae (syn. M. corti) was studied with the aid of reserpine. Reserpine decreases the content of 5-HT as measured spectrofluorometrically and immunocytochemically and, furthermore, inhibits the motility, thus indicating a connection between the two. The results support the hypothesis about 5-HT being an excitatory neurotransmitter of motor activity in M. vogue. New neuroanatomical details were revealed by immunocytochemical staining.The relationship between serotonin (5-HT) and motility in tetrathyridia ofMesocestoides vogae (syn.M. corti) was studied with the aid of reserpine. Reserpine decreases the content of 5-HT as measured spectrofluorometrically and immunocytochemically and, furthermore, inhibits the motility, thus indicating a connection between the two. The results support the hypothesis about 5-HT being an excitatory neurotransmitter of motor activity inM. vogae. New neuroanatomical details were revealed by immunocytochemical staining.