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 pharmacological actions of 5-hydroxytryptamine,FMRF-amide and substance P and their possible occurrence in the heart of the snail Helix aspersa L.

The actions of 5-hydroxytryptamine (5-HT), FMRF-amide and substance P were tested on the isolated heart of Helix aspersa. All three compounds were found to produce positive inotropic and chronotropic effects, the order of potency being 5-HT > substance P > FMRF-amide. However, whereas the responses to 5-HT and FMRF-amide were maximal within a few seconds, the response to substance P had a longer latency. Two other similar undecapeptides, eledoisin and physalaemin, were also tested and were found to have very similar actions to those of substance P. The effects of 5-HT and FMRF-amide could be separated using the 5-HT blockers methysergide and ketanserin, which had relatively little effect on the response to FMRF-amide.Immunohistochemical staining was carried out on sections through the Helix auricle and ventricle for 5-HT, FMRF-amide and substance P. Substantial 5-HT-like and substance P-like immunoreactivity was observed, apparently concentrated in nerve endings, but the level of FMRF-amide-like immunoreactivity was considerably lower. The fluorescence produced by all three compounds was evenly distributed throughout the heart tissue. HPLC analysis of heart tissue extract demonstrated a high level of 5-HT (about 8 μg/g wet weight) but a negligible catecholamine content.     

Immunocytochemical Demonstration of Serotonin and Neuropeptides in the Nervous System of Gyrodactylus salaris (Monogenea)

A whole mount immunocytochemical (ICC) method has been used for the investigation of immunoreactivity (IR) to the molluscan cardioactive peptide FMRF-amide, to 9 vertebrate neuropeptides—leu-enkephalin, growth hormone-releasing factor (GRF), urotensin I, urotensin II, bovine pancreatic peptide (BPP), β-endorphin, substance P, secretin and insulin—and to the bioamine 5-HT in the nervous system (NS) of the trematode Gyrodactylus salaris belonging to the taxon Monogenea. Positive results were obtained using antisera to FMRF-amide, leu-enkephalin, urotensin I, GRF and to 5-HT. The present results are the first documentation of the presence of neuroregulatory peptides and a bioamine in the nervous system (NS) of Monogenea. Differences in the distribution pattern of the IR to the different antisera indicate that different subsets of neurons are revealed. In addition, details of the basic neuroanatomical pattern in monogeneans are confirmed by the whole mount ICC method used in this study. Negative results were obtained with antisera to urotensin II, substance P, β-endorphin, secretin, insulin and bovine pancreatic polypeptide (BPP).     

Immunohistochemical localization of tryptophan hydroxylase and serotonin transporter in the carotid body of the rat

It has been proposed that serotonin (5-HT) facilitates the chemosensory activity of the carotid body (CB). In the present study, we investigated mRNA expression and immunohistochemical localization of the 5-HT synthetic enzyme isoforms, tryptophan hydroxylase 1 (TPH1) and TPH2, and the 5-HT plasma membrane transport protein, 5-HT transporter (SERT), in the CB of the rat. RT-PCR analysis detected the expression of mRNA for TPH1 and SERT in extracts of the CB. Using immunohistochemistry, 5-HT immunoreactivity was observed in a few glomus cells. TPH1 and SERT immunoreactivities were observed in almost all glomus cells. SERT immunoreactivity was seen on nerve fibers with TPH1 immunoreactivity. SERT immunoreactivity was also observed in varicose nerve fibers immunoreactive for dopamine beta-hydroxylase, but not in nerve fibers immunoreactive for vesicular acetylcholine transporters or nerve terminals immunoreactive for P2X₃ purinoreceptors. These results suggest that 5-HT is synthesized and released from glomus cells and sympathetic nerve fibers in the CB of the rat, and that the chemosensory activity of the CB is regulated by 5-HT from glomus cells and sympathetic nerve fibers.     

Substance P immunoreactivity in sensory structures and the central and pharyngeal nervous system of Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida)

Immunoreactivity (IR) obtained by monoclonal antibodies to substance P (SP) was studied in the asexually reproducing microturbellarians Stenostomum leucops and Microstomum lineare. The IR pattern was studied by confocal and ordinary fluorescence microscopy. In both species, IR occurs in the brain in peripheral cells, neuropilar fibres, in longitudinal cords and in the pharyngeal nervous system. The IR patterns reveal neuroanatomical details not observed with other neuroactive substances. In both species, immunopositive cells send fibers to the ciliary pits. In M. lineare, additional fibres run to more frontally located sensory structures. In S. leucops, two pharyngeal nerve rings are visualized. The pharyngeal nerve ring close to the surface associated with symmetrical immunopositive cell pairs is demonstrated for the first time, while the deeper-lying pharyngeal nerve ring has been previously demonstrated by antibodies to the molluscan cardioactive peptide FMRF-amide. Two cells with strong IR are connected by short fibres to the pharyngeal nerve ring in M. lineare. In the developing new individuals, i.e., the zooids of M. lineare, IR to SP is first revealed in nerve fibres growing out from parental lateral nerve cords towards the centre of the worm where the new brain commissure will appear. Immunopositive cells in the brain periphery and close to the developing ciliary pits appear later. Simultaneous staining by antibodies to SP and 5-HT shows that IR to SP appears later than IR to 5-HT.     

Actions of the molluscan neuropeptide FMRF-amide on neurones in the suboesophageal ganglia of the snail Helix aspersa

The effects of the molluscan neuropeptide FMRF-amide were tested on several neurones in the suboesophageal ganglia of the snail Helix aspersa. Almost all neurones tested responded to the peptide, some being hyperpolarized (H response) and others depolarized (D response). The H response is due primarily to an inward potassium current and may be blocked in 20 microM 4-aminopyridine. The hyperpolarizing actions of FMRF-amide and dopamine may be separated by ergometrine which blocks the response to dopamine but not to FMRF-amide. The D response is due mainly to an inward sodium current but this is not blocked by d-tubocurarine, morphine or TTX. It appears to be mediated by a distinct receptor/ionophore as excitation by ACh and 5-HT are both antagonized by d-tubocurarine. The Leu2-substituted analogue FLRF-amide was found to produce similar H responses to FMRF-amide, but was much less potent at producing D responses. It did, however, produce cross-desensitization of the D response to FMRF-amide, suggesting that it does bind to the FMRF-amide receptor.     

The physiological role of peripheral serotoninergic neurones. A review

1. Serotoninergic neurones and neurones which takes up 5-hydroxytryptamine (5-HT) have been observed in sensory ganglia, but their physiological role remains unknown. 2. Serotoninergic neurones participating in the neural control of gut motility are present within the enteric intramural nervous system. 3. 5-HT applied to the serosa inhibits the peristaltic reflex in the small intestine. In contrast, peristalsis is stimulated by 5-HT applied to the serosa. 4. Intracellular microelectrode investigations indicate that some neurones of the enteric nervous system are depolarized, whereas others are hyperpolarized by 5-HT. In addition, 5-HT can also decrease the release of acetylcholine by acting on presynaptic receptors located on cholinergic nerve endings. The release of 5-HT from serotoninergic enteric neurones is very probably under the control of a noradrenergic mechanism. 5. Electromyographic studies on the rabbit colon indicate that a nerve-mediated descending inhibition is modified by drugs interacting with the synthesis of 5-HT or its reuptake.     

Organisation of the nervous system in the Acoela: an immunocytochemical study

In order to broaden the information about the organisation of the nervous system in taxon Acoela, an immunocytochemical study of an undetermined Acoela from Cape Kartesh, Faerlea glomerata, Avagina incola and Paraphanostoma crassum has been performed. Antibodies to 5-HT and the native flatworm neuropeptide GYIRFamide were used. As in earlier studies, the pattern of 5-HT immunoreactivity revealed an anterior structure composed mainly of commissures, a so-called commissural brain. Three types of brain shapes were observed. No regular orthogon was visualised. GYIRFamide immunoreactive cell clusters were observed peripherally to the 5-HT immunoreactive commissural brain. Staining with anti-GYIRFamide revealed more nerve processes than did staining with anti-FMRFamide. As no synapomorphies were found in the organisation of the nervous system of the Acoela and that of the Platyhelminthes, the results support the view that the Acoela is not a member of the Platyhelminthes.     

An immunocytochemical and ultrastructural study of the nervous and muscular systems of Xenoturbella westbladi (Bilateria inc. sed.)

The phylogenetic position of the Xenoturbellida is highly disputed. Are they primitive flatworms? Are they related to Deuterostomia? Do they form a sister taxon to other Bilateria? Are they bivalve molluscs? In order to provide more data for this discussion, a study of the nervous system of Xenoturbella westbladi and its relation to the musculature was performed, using 5-HT and FMRFamide immunocytochemistry, TRITC-conjugated phalloidin fluorescence for staining of F-actin filaments, confocal scanning laser microscopy and transmission electron microscopy. The nervous system comprises solely an intraepidermal net of nerve cells and processes. No ganglia or any other internal nervous structures could be detected. No evidence of 5-HT- or FMRFamide-immunoreactive innervation below the subepidermal membrane complex was obtained. The 5-HT and FMRFamide immunoreactivity occurs in separate sets of neurones. On the ultrastructural level, three types of neurones were observed: (1) the predominating ”light” neurones, (2) the smaller ”dark” neurones and (3) the bipolar sensory neurones bearing a single cilium with a long bipartite rootlet. Non-synaptic, paracrine, release sites are common and synapses are inconspicuous. In the layer of epidermal cells, close to the lateral furrow, F-actin filaments were observed. They reach from the basal membrane to the surface. The organisation of the nervous system appears very simple. Our results are compatible with the hypothesis of Xenoturbellida forming a sister taxon to Bilateria. No evidence was obtained for inclusion of X. westbladi in either the Mollusca or Plathelminthes.     

Development and differentiation of neuronal subsets in asexually reproducing Microstomum lineare. Immunocytochemistry of 5-HT, RF-amide and SCPB

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 SCPB (molluscan small cardioactive 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 SCPB 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.     

The appearance of neural and glial cell markers during early development of the nervous system in the amphibian embryo

Cell-type-specific antibodies have been used to follow the appearance of neurones and glia in the developing nervous system of the amphibian embryo. Differentiated neurones were recognized with antibodies against neurofilament protein while glial cells were identified with antibodies against glial fibrillary acidic protein (GFAP). The appearance of neurones containing the neurotransmitters 5-hydroxytryptamine and dopamine has been charted also. In Xenopus, neurofilament protein in developing neurones was observed occasionally at NF stage 21 and was present reliably in the neural tube and in caudal regions of the brain at stage 23. Antibodies to the low molecular weight fragment of the neurofilament triplet recognized early neurones most reliably. Radial glial cells, identified with GFAP antibody, were identified from stage 23 onwards in the neural tube and caudal regions of the brain. In the developing spinal cord, GFAP staining was apparent throughout the cytoplasm of each radial glial cell. In the brain, the peripheral region only of each glial cell contained GFAP. By stage 36, immunohistochemically recognizable neurones and glia were present throughout the nervous system. In the axolotl, by stage 36 the pattern of neural and glial staining was identical to that observed in Xenopus. GFAP staining of glial cells was obvious at stage 23, although neuronal staining was clearly absent. This implies that glial cells differentiate before neurones. 5-HT-containing cell bodies were first observed in caudal regions of the developing brain on either side of the midline at stage 26. An extensive network of 5-HT neurones appeared gradually, with a substantial subset crossing to the opposite side of the brain through the developing optic chiasma. 5,7-dihydroxytryptamine prevented the appearance of 5-HT. Depletion of 5-HT had little effect on development or swimming behaviour. Dopamine-containing neurones in the brain first differentiated at stage 35-36 and gradually increased in number up to stage 45-47, the latest stage examined. The functional role of 5-HT- or dopamine-containing neurones remains to be elucidated. We conclude that cell-type-specific antibodies can be used to identify neurones and glial cells at early times during neural development and may be useful tools in circumstances where functional identification is difficult.     

Immunohistochemical localisation of FMRF-amide-like peptide in the brain of icefish and red-blooded Antarctic Teleosts

The distribution of Phe-Met-Arg-Phe-NH₂ (FMRF-amide) -like immunoreactivity was investigated by indirect immunofluorescence technique using the molluscan FMRF-amide antibody in the brain of icefish (Pagetopsis macropterus and Chionodraco hamatus used as positive control) and red blooded (Trematomus bernacchii, Gymnodraco acuticeps, Histiodraco velifer, Cygnodraco mawsoni) Antarctic Teleosts. Immunoreactive perikarya were localised in the ventral thalamus, in the hypothalamus (preoptic and periventricular regions) and in the intermedioventral rhombencephalon (vagal motor nucleus) as well as in the telencephalon and in the mesencephalon. Positive nerve fibres were seen to project towards the caudal brainstem to reach the rhombencephalon. No differences were observed in the immunopositivity of FMRF-amide new distribution in the Antarctic Teleosts examined. In the icefishes the immunoreaction was stronger than in the hemoglobin-rich Teleosts. The distribution patterns of the FMRF-amide immunostaining suggest that this peptide may play a pivotal role in the cardiovascular regulation in the Antarctic Teleosts.     

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.     

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.     

The brain of the Nemertodermatida (Platyhelminthes) as revealed by anti-5HT and anti-FMRFamide immunostainings

The taxa Nemertodermatida and Acoela have traditionally been considered closely related and classified as sister groups within the Acoelomorpha Ehlers 1984 (Platyhelminthes). Recent molecular investigations have questioned their respective position. In this study, the 5-HT and FMRFamide immunoreactivity (IR) in the nervous system of two nemertodermatids, Nemertoderma westbladi and Meara stichopi, is described. The 5-HT immunoreactive pattern differs in the two nemertodermatids studied. In M. stichopi, two loose longitudinal bundles of 5-HT-immunoreactive fibres and an basi-epidermal nerve net were observed. In N. westbladi the 5-HT-IR shows a ring-shaped commissural structure, different from the commissural brain of acoels. In both nemertodermatids, FMRFamide immunoreactive nerve fibres followed the 5-HT-immunoreactive fibres. It is demonstrated that the Nemertodermatida have neither a 'commissural brain' structure similar to that of the Acoela, nor a 'true', ganglionic brain and orthogon, typical for other Platyhelminthes. The question of the plesiomorphic or apomorphic nature of the nervous system in Nemertodermatida cannot yet be answered. The neuroanatomy of the studied worms provides no synapomorphy supporting the taxon Acoelomorpha.     

Serotonin augments gut pacemaker activity via 5-HT3 receptors

Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT's actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca(2+) and electric activities in ICC were performed by employing fluorescent Ca(2+) imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca(2+) antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca(2+) oscillations that are considered to underlie electric pacemaker activity in ICC. LY-278584, a 5-HT(3) receptor antagonist suppressed spontaneous Ca(2+) activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5-HT(3) receptor agonist, restored it. GR113808, a selective antagonist for 5-HT(4), and O-methyl-5-HT (O-Me-5-HT), a non-selective 5-HT receptor agonist lacking affinity for 5-HT(3) receptors, had little effect on ICC Ca(2+) activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT(3) receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT(3) receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome.     

Distribution of serotonin and FMRF-amide in the nervous system of different zooidal types of cheilostome bryozoa: A case study of <Emphasis Type="Italic">Arctonula arctica</Emphasis>

In the White Sea bryozoans Arctonula arctica, the structure of the nervous system and distribution of 5-hydroxytryptamine (5-HT) and FMRF-amide were studied for the first time using immunohistochemical methods and confocal scanning microscopy. The neurotransmitters studied have been actively involved into the integrative processes, gut functioning, and regulation of motion activity. In avicularia, 5-HT and FMRF-amide receptors are capable of performing the same functions, except for participation in the gut functioning, because they have no digestive system.     

Cholinergic, serotoninergic and peptidergic components of the nervous system of Discocotyle sagittata (Monogenea: Polyopisthocotylea)

Cholinergic, serotoninergic (5-HT) and peptidergic neuronal pathways have been demonstrated in both central and peripheral nervous systems of adult Discocotyle sagittata, using enzyme histochemistry and indirect immunocytochemistry in conjunction with confocal scanning laser microscopy. Antisera to 2 native flatworm neuropeptides, neuropeptide F and the FMRFamide-related peptide (FaRP), GNFFRFamide, were employed to detect peptide immunoreactivity. The CNS is composed of paired cerebral ganglia and connecting dorsal commissure, together with several paired longitudinal nerve cords. The main longitudinal nerve cords (Iateral, ventral and dorsal) are interconnected at intervals by a series of annular cross-connectives, producing a ladder-like arrangement typical of the platyhelminth nervous system. At the level of the haptor, the ventral cords provide nerve roots which innervate each of the 8 clamps. Cholinergic and peptidergic neuronal organisation was similar, but distinct from that of the serotoninergic components. The PNS and reproductive system are predominantly innervated by peptidergic neurones.     

A commissural brain! The pattern of 5-HT immunoreactivity in Acoela (Plathelminthes)

 In order to solve the question whether Plathelminthes belonging to the taxon Acoela have a brain and an orthogon of the common flatworm type, an immunocytochemical study of the pattern of serotonin (5-HT) in four species was performed. In all species the 5-HT immunoreactivity revealed no ganglionic cell mass typical for other Plathelminthes, only a symmetrical brain-like structure composed of commissural fibres associated with a few cell bodies. 5-HT immunoreactivity was detected in three to five pairs of longitudinal nerve cords, connected by an irregular network of immunoreactive transverse fibres. No regular orthogon was visualised. All the surface sensilla were strongly immunoreactive. The unique commissural brain and cordal nervous system found in Acoela support the view of a deep gap lying between Acoela and other Plathelminthes. Accepted: 15 December 1997