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.     

Plathelminthes and Plathelminthomorpha — paraphyletic taxa

The concept of monophyly of Plathelminthes and Plathelminthomorpha (Plathelminthes and Gnathostomulida) is critically re-analysed. Based on new morphological and molecular evidence, it is concluded that both taxa are paraphyletic assemblages. The Acoelomorpha (Xenoturbellida?, Nemertodermatida and Acoela) are considered as the earliest offshoot of the bilaterian stem line, primarily lacking paired cerebral ganglia, an orthogonal nervous system and ultrafiltration nephridia. They are followed by Plathelminthes sensu stricto (i.e. the rhabditophoran turbellarians and Neodermata), Catenulida, and Gnathostomulida, the latter group is probably the sister group of the Syndermata (Rotifera and Acanthocephala). The respective characters, as well as the implication for the proposed stem species of the Bilateria are outlined and discussed.     

Arthropod interrelationships – the phylogenetic-systematic approach1

The Arthropda are a monolphyletic taxon sharing many synapomorphies. The alternative models of polyphyletic origins of the arthropods are shown to be the result of 1. wrong interpretation of data, e. g. interpretation of the tracheate mandible as a “whole limb mandible”, 2. typological thinking, defining taxa on the basis of their overall similarity, and 3. unscientific reasoning, postulation unkown palaeozoic taxa instead of known ones as closest relatives. The main controversy concerns the position of the Tracheata. Are they Mandibulata and the sister group of the Crustacea or are they “Uniramia” and the sister group of the Onychophora? Whereas the Tracheata and Onychophora share only symplesiomorphies, the Tracheata share convincing synapomorphies with the Crustacea. The most important of these are the transformation of the third head appendage to the principal part of the mouth apparatus – this would even be true if the tracheate mandible were a “whole lim mandible”–, and the identical composition of the compound eye ommatidia. The Chelicerata are used as an example, following Lauterbach 'S considerations, to demonstrate how fossils enhance our understanling of the evolutionary history of arthropods.     

Immunohistochemical investigations of Myzostoma cirriferum and Mesomyzostoma cf. katoi (Myzostomida,Annelida) with implications for the evolution of the myzostomid body plan

Although part of the annelid radiation, Myzostomida exhibit a highly specialized body plan that lacks many typical annelid characters. Their annelid ancestry is evident from their trochophora-like larvae, adult myoanatomy and parts of the nervous system, whereas segmentation is considered at best to be incomplete in myzostomids. We analyzed the morphology of two myzostomid species, the ectocommensal Myzostoma cirriferum and the endoparasitic Mesomyzostoma cf. katoi using a broad set of fluorescent markers to reveal the degree of segmentation in myzostomids. We used immunocytochemical and classical fluorescent staining methods combined with confocal laser-scanning microscopy to visualize tissues labeled with antibodies directed against classical invertebrate neurotransmitters (serotonin, dopamine, FMRFamide), synapsin, which labels nerve cell terminals, and the marker phalloidin–rhodamine which binds F-actin in muscle. Our data provide a broad body of additional evidence for the segmented origin of Myzostomida. It becomes apparent that the adult nervous system of M. cirriferum exhibits signs of pseudoradial symmetry with repetitive patterns of putative FMRFamide, serotonin and dopamine-like immunoreactivity. An analysis of the staining patterns in juvenile M. cirriferum yielded evidence for positional changes, as well as additions and reductions of neuronal structures during development. Interestingly, the neuroanatomy and myoanatomy of Mesomyzostoma cf. katoi indicate further reductions of neuronal and myoanatomical patterns in this species. Notably this taxon shows a presumably secondarily evolved cylindrical and strictly bilateral morphology, which is supposed to have evolved from a flat, disk-shaped Myzostoma-like ancestor with an underlying pseudoradial symmetry.     

An analysis of the 5-hydroxytryptamine (serotonin) receptor subtypes of central neurones of Helix aspersa

1. Intracellular recordings were made from identified neurones in the central nervous system of Helix aspersa. Two types of cell were used, those excited by 5-hydroxytryptamine (5-HT) and acetylcholine and those inhibited by 5-HT and dopamine. The actions of a range of 5-HT agonists and antagonists were tested for their ability to interact with 5-HT receptors.2. 5-Carboxyamidotryptamine, α-methyl-5-HT and N-methyl-5-HT were active on cells excited by 5-HT, with similar potencies to 5-HT. Only 5-carboxyamidotryptamine and 5-methoxytryptamine were equiactive with 5-HT on cells inhibited by 5-HT. Most of the non-indole analogues were inactive or very weak agonists on both receptors.3. MDL 72222 was the most active antagonist tested against 5-HT excitation, showing some selectivity for 5-HT over acetylcholine. Cinanserin and ketanserin also showed selectivity for 5-HT over acetylcholine.4. Tryptamine was inhibitory on both cell types and was a potent antagonist of 5-HT excitation, showing selectivity for 5-HT over acetylcholine.5. It is concluded that the 5-HT excitatory receptor recognizes the indole nucleus with substitution on position 5, save for 5-fluorotryptamine which was inhibitory. It does not appear that these 5-HT receptors can be classified in terms of the vertebrate subtypes of 5-HT receptor. However, it should be noted that only two receptor subtypes located on a small number of neurones were studied in these experiments and other 5-HT receptor subtypes may be located on other groups of neurones and peripheral tissues. These receptors may recognize other 5-HT receptor ligands including non-indoles.     

Histochemical localisation of FMRFamide-gated Na+ channels in Helisoma trivolvis and Helix aspersa neurones

FMRFamide-gated Na⁺ channels of molluscan neurones belong to the ENa/Deg family of channels which have diverse functions. FMRFamide (Phe-Met-Arg-Phe-NH₂) Na⁺ channels were detected electrophysiologically in specified neurones of Helix (Helix aspersa) and Helisoma (Helisoma trivolvis), and clones (FaNaCs) subsequently identified. We have now made a study to determine the distribution of mRNA for the clones HaFaNaC (Helix) and HtFaNaC (Helisoma) in the nervous systems of these species using standard in situ hybridization techniques. Immunohistochemical experiments were also made using an HtFaNaC antibody to detect the channel protein in Helisoma neurones. Many neurones in the central ganglia, including those which exhibit the FMRFamide Na⁺ current, stained for FaNaC-mRNA, suggesting a much wider distribution of the channel than was indicated by the earlier work. An immunoreactive response to the channel antibody was also observed in some Helisoma neurones, such as the giant dopamine neurone of the left pedal ganglion, also shown to possess HtFaNaC-mRNA and to exhibit the FMRFamide Na⁺ current. Taken together, these experiments suggest that the clones HaFaNaC and HtFaNaC are major, if not the only, subunits of the FMRFamide-gated Na⁺ channel detected electrophysiologically in the identified neurones of these species. However, fewer neurones in Helisoma reacted with the HtFaNaC-antibody than those which exhibited message for the channel. This discrepancy may be due to a difference in sensitivity of the two techniques, or because not all of the channel mRNA is normally expressed as a membrane protein.     

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.     

The nervous and muscular systems in the free-living flatworm Castrella truncata (Rhabdocoela): an immunocytochemical and phalloidin fluorescence study

In order to broaden the information about the organisation of the nervous system in the Plathelminthes, an immunocytochemical (ICC) study of the free-living flatworm Castrella truncata (Dalyellioida) has been performed. This is the first time a representative of the taxon Rhabdocoela has been studied with the ICC technique. Antibodies to 5-HT and FMRF-amide and confocal scanning laser microscope were used. 5-HT and FMRF-amide immunoreactivity was observed in the neuropile of the brain, in the cerebral neurones, in the three pairs of longitudinal nerve cords and the adjoining neurones, in the pharyngeal nerve ring and in twelve neurones associated with the pharynx. 5-HT and FMRF-amide immunoreactivity occurs in separate sets of neurones. Only FMRF-amide immunoreactivity was observed in a peculiar ellipse-shaped structure in the brain, and in the genital plexus. The type of orthogon is discussed. Staining of the muscular system with TRITC conjugated phalloidin revealed muscle patterns that have not been described previously.     

The actions of FxRFamide related neuropeptides on identified neurones from the snail, Helix aspersa

Intracellular recordings were made from identified neurones in the suboesophageal ganglia of the snail, Helix aspersa. The actions of the eight FxRFamide analogues were investigated on these neurones. These peptides included ones isolated from arthropods and nematodes. All the peptides excited certain neurones while inhibiting others, though their relative potencies varied. Overall on neurones inhibited by these peptides the potency order was: DNFLRFamide > FMRFamide > PDVDHVFLRFamide = KNEFIRFamide > FLRFamide > SDRNFLRFamide = SDPNFLRFamide > KHEYLRFamide. However, if the responses are compared on individual cell types, then the picture becomes more complex. For example, on cell F-2, KNEFIRFamide proved to be potent with an EC-50 value of 0.54 microM. On neurones F-13/16 and E-16, PDVDHVFLRFamide was inhibitory while FMRFamide, FLRFamide, SDRNFLRFamide and SDPNFLRFamide were excitatory. In terms of overall excitatory actions, the data are less complete but an approximate order of potency is: FMRFamide > DNFLRFamide > SDPNFLRFamide > PDVDHVFLRFamide > KNEFIRFamide = KHEYLRFamide = SDRNFLRFamide. However this again varies between specific neurones. These results demonstrate that peptides from insects, crustacea and nematodes are active on Helix neurones and may activate specific receptor subtypes, indicating the possible presence of endogenous analogues of these non-molluscan peptides in the Helix nervous system.     

5-HT receptors on identified Lymnaea neurones in culture. Pharmacological characterization of 5-HT1A receptors

The ability of the selective 5-HT_1A receptor agonist R(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) to bind with 5-HT receptor(s) on cultured, identified neurones in Lymnaea stagnalis was examined. The identified neurones studied were from the buccal ganglia and the serotonin-containing cerebral giant cells (CGCs). 5-HT and its agonists were applied from puffer pipettes, whilst 5-HT antagonists were applied in the bathing medium. At 10⁻³ M, the 5-HT_1A agonist, always produced paroxysmal depolarizing shifts (PDS) while at a lower concentration (10⁻⁴ M), it always mimicked the effects of 10⁻³ M 5-HT. 8-OH-DPAT (10⁻⁴ M) and 5-HT 10⁻³ M produced dose-dependent increases in the responses they evoked. At 10⁻⁴ M, the 5-HT₃ receptor agonist 1-(m-chlorophenyl)-biguanide hydrochloride (m-CPBG), failed to hyperpolarize most of the neurones hyperpolarized by 5-HT. At 10⁻⁴ M, the antagonists ketanserin (5-HT₂), MDL 72222 (5-HT₃), and pindobind-5-HT_1A (5-HT_1A) consistently abolished spike generation ii spontaneously active neurones. Both ketanserin and MDL 72222 failed to block the actions of 8-OH-DPAT and only partially blocked those of 5-HT, but pindobind-5-HT_1A completely, but reversibly,blocked the 8-OH-DPAT effects while greatly reducing those of 5-HT. These results suggest that 5-HT_1A receptor subtypes might be involved in the hyperpolarizing responses of the CGCs and their follower motor neurones in the buccal ganglia of Lymnaea stagnalis to 5-HT. The presence of 5-HT_1A receptors on these neurones can be considered to correspond with those found in mammals because their pharmacological responses resemble those of mammalian 5-HT_1A receptors.     

Innervation of bivalve larval catch muscles by serotonergic and FMRFamidergic neurons

Bivalve larvae use catch muscles for rapid shell closure and maintenance of the closed condition. We used specific antibodies against the muscle proteins together with phalloidin and neuronal markers, FMRFamide and serotonin (5-HT), to analyze mutual distribution of muscle and neuronal elements in larvae of the mussel, Mytilus trossulus, and the oyster, Crassostrea gigas. At trochophore and early veliger stages no anatomical connections between muscular and nervous system were detected. By the pediveliger stage the 5-HT innervation of the anterior adductor developed in oyster only, while rich FMRFa innervation of the adductor muscles developed in both species. Possible roles and mechanisms of FMRFamide and serotonin in the regulation of the catch state are discussed.     

Tryptamine and 5-hydroxytryptamine: actions and interactions of cortical neurones in the rat

The actions of iontophoretically applied tryptamine (T) and 5-hydroxytryptamine (5-HT) were compared on single neurones in the rat somatosensory cortex. The firing rate of the vast majority of neurones tested was depressed by T. However, 5-HT excited and depressed approximately equal numbers of neurones. Depressant effects of 5-HT could be profoundly enhanced by a very weak concurrent application of T (0–10 nA) which itself did not alter the baseline cell firing rate. Excitatory responses to 5-HT were consistently reversed into depressant responses during weak applications of T. These observations could support a modulatory role for endogenous T in 5-HT-mediated transmission in the central nervous system (CNS).     

The neurochemical basis of recurrent inhibition in the reflex arch of the defensive reaction]

Mechanisms of habituation in the network of identified neurones were investigated in isolated preparation of central nervous system in the snail Helix. It has been found that intracellularly induced spike discharge in premotor command neurones decreases synaptic responses to repeated nerve stimulation in all recorded command neurones. Application of the neuropeptide FMRFamide elicits similar changes in the network. Taking into account that the investigated command neurones contain FMRFamide, as was shown immunochemically, it is possible to assume the existence of recurrent inhibition in the network underlying avoidance reactions. This recurrent inhibition causes habituation of the network output in the cases when the repeated stimuli do not evoke sensitization via activation of serotonergic cells.     

Mesodermal Gene Expression in the Acoel Isodiametra pulchra Indicates a Low Number of Mesodermal Cell Types and the Endomesodermal Origin of the Gonads

Acoelomorphs are bilaterally symmetric small marine worms that lack a coelom and possess a digestive system with a single opening. Two alternative phylogenetic positions of this group within the animal tree are currently debated. In one view, Acoelomorpha is the sister group to all remaining Bilateria and as such, is a morphologically simple stepping stone in bilaterian evolution. In the other, the group is a lineage within the Deuterostomia, and therefore, has derived a simple morphology from a more complex ancestor. Acoels and the closely related Nemertodermatida and Xenoturbellida, which together form the Acoelomorpha, possess a very limited number of cell types. To further investigate the diversity and origin of mesodermal cell types we describe the expression pattern of 12 orthologs of bilaterian mesodermal markers including Six1/2, Twist, FoxC, GATA4/5/6, in the acoel Isodiametra pulchra. All the genes are expressed in stem cells (neoblasts), gonads, and at least subsets of the acoel musculature. Most are expressed in endomesodermal compartments of I. pulchra developing embryos similar to what has been described in cnidarians. Our molecular evidence indicates a very limited number of mesodermal cell types and suggests an endomesodermal origin of the gonads and the stem cell system. We discuss our results in light of the two prevailing phylogenetic positions of Acoelomorpha.     

Ultrastructure of the subepidermal musculature of Xenoturbella bocki, the adelphotaxon of the Bilateria

 Xenoturbella bocki is the only species of the high-ranked taxon Xenoturbellida. The species lives on marine mud bottoms at a depth of 20–120 m and moves extremely slowly by ciliary gliding. Nevertheless it possesses a well-developed body wall musculature with outer circular muscles, a prominent layer of inner longitudinal muscles and radial muscles that extend from the outer circular myocytes to the musculature surrounding the gastrodermis. The longitudinal myocytes are not compact cells, but form fascicles of fibrils running parallel to each other. Fine cytoplasmic cords connect the fibres of a cell to each other and with its nuclear region. The muscles are embedded within a sometimes expansive extracellular matrix (ECM) that lacks any fibrillar components. All muscle cells display conspicuous and numerous cytoplasmic extensions that are intermingled with each other. Tight coupling between adjacent cell membranes is not found, but zonula adhaerens-like junctions exist. Fibrils belonging to different myocytes, but also fibrils of the same cell, are coupled by such cytoplasmic extensions. Circular, radial and at least the peripheral longitudinal myocytes display cell-matrix connections with the internal lamina, a component of the subepidermal ECM. This internal lamina projects down into the centres of the fascicles with longitudinal muscle fibrils and forms extensive attachment zones with the muscle cells, reminiscent of focal contacts. For the ingestion of food, X. bocki opens the simple mouth pore and protrudes the aciliated gastrodermis. The body wall musculature is responsible for this protrusion and also for the withdrawal of the gastrodermis. In the past, possible phylogenetic kinships with the Acoelomorpha (Plathelminthes) or the Enteropneusta and Holothuroidea were discussed, but, on the basis of all information available, X. bocki is hypothesized to be the sister taxon of the Bilateria. Accepted: 2 April 1997     

An initial screen of a series of neuroactive peptides for activity on identified central neurones of Helix aspersa

1. Intracellular recordings were made from identified neurones in the suboesophageal ganglia of Helix aspersa. Seven neuropeptides were tested for activity and their actions compared with acetylcholine and FMRFamide.2. Three peptides isolated from nematodes, AF-1, AF-2 and PAN-1 had mainly inhibitory effects with thresholds of around 1 nM. This inhibition was due to an increase in potassium conductance.3. The molluscan neuropeptides LSSFVRIamide, CARP and ACEP-1 were all active on certain neurones; the first two showed only inhibitory effects while ACEP-1 was mainly excitatory. The thresholds in each case were 0.1–10 μM. When norleucine replaced methionine in CARP, the potency was reduced by at least 100 times.4. The echinoderm peptide, SALMF-1, only excited neurones but with a very low threshold, around 1.0 fM.5. There was no obvious correlation between the action of these peptides and either acetylcholine or FMRFamide.     

New names inPhanerogamae 6

94 new nomenclatural combinations are proposed, resulting mostly from the study of taxonomic problems on the generic level. The reasons for the acceptance of the generic rank are given in the case ofAcetosa, Pulegium and of some sections ofOrobanche s. l. andSilene s. l. The taxonomic rank ofGentiana blepharophora Bordzi?. from the Caucasus has been revised and the taxon is classified as a subspecies ofGentianopsis ciliata. Nomenclatural problems of not numbered “species” inHoffmann's “Flora Deutschlands” from 1800 are demonstrated on the example ofArmeria elongata.     

FMRFamide-related peptides and serotonin regulate Drosophila melanogaster heart rate: mechanisms and structure requirements

Drosophila melanogaster FMRFamide-related peptides (FaRPs) include SDNFMRFamide, PDNFMRFamide, and TDVDHVFLRFamide (dromyosuppressin, DMS); each peptide contains a C-terminal FMRFamide but a different N-terminal extension. FaRPs and serotonin (5-HT) each affect the frequency of D. melanogaster heart contractions in vivo. We examined the cellular expression of FaRPs and 5-HT, and the activities of FMRFamide, SDNFMRFamide, PDNFMRFamide, or DMS and 5-HT on heart rate. FaRPs and 5-HT were not co-localized; FaRP-and 5-HT-immunoreactive fibers extended from different brain cells and innervated the anterior D. melanogaster dorsal vessel. However, no neuron expressed both a FaRP and 5-HT. The effect of FMRFamide and 5-HT was not different from the effect of 5-HT alone on heart rate. The effect of PDNFMRFamide and 5-HT showed an additive effect on heart rate. SDNFMRFamide and 5-HT or DMS and 5-HT resulted in non-additive effects on heart rate. Our data provide evidence for the complexity of FaRP and 5-HT interactions to regulate frequency of heart contractions in vivo. Our results also confirm the biological importance of FaRP N-terminal amino acid extensions.     

The muscular system of Nemertoderma westbladi and Meara stichopi (Nemertodermatida, Acoelomorpha)

Nemertodermatida is a small taxon of marine worm-like animals; its position in the tree of life is highly contested. The musculature of Nemertoderma westbladi and Meara stichopi is studied here in detail using fluorescent phalloidin and confocal microscopy. In both species, the musculature is composed of an outer layer of circular and an inner layer of longitudinal musculature, diagonal muscles form a distinct layer in N. westbladi, but in M. stichopi these fibres connect to both other layers. The supraterminally opening male pore and antrum are formed by invagination of the whole body-wall in both species, and the seminal vesicle is lined by a thin net of musculature only in full male maturity. Modifications of the ventral body-wall adjacent to the mouth are small and transient in N. westbladi including no extra musculature, whereas it consists of additional strong U-shaped musculature in M. stichopi. Myogenesis in N. westbladi is not finished in hatchlings and will be completed dorsally in juvenile specimens and ventrally in male mature ones, after the loss of the mouth. Musculature between the two species differs considerably and might give insights into the internal relationships of Nemertodermatida and might prove to be useful in studies investigating their phylogenetic position. More data of other species and developmental changes are needed.