Different extrinsic trophic factors regulate neurite outgrowth and synapse formation between identified Lymnaea neurons

The requirement for trophic factors in neurite outgrowth is well established, though their role in synapse formation is yet to be determined. Moreover, the issue of whether the trophic factors mediating neurite outgrowth are also responsible for synapse specification has not yet been resolved. To test whether trophic factors mediating neurite outgrowth and synapse formation between identified neurons are conserved in two molluscan species and whether these developmental processes are differentially regulated by different trophic factors, we used soma-soma and neurite-neurite synapses between identified Lymnaea neurons. We demonstrate here that the trophic factors present in Aplysia hemolymph, although sufficient to induce neurite outgrowth from Lymnaea neurons, do not promote specific synapse formation between excitatory partners. Specifically, the identified presynaptic neuron visceral dorsal 4 (VD4) and postsynaptic neuron left pedal dorsal 1 (LPeD1) were either paired in a soma-soma configuration or plated individually to allow neuritic contacts. Cells were cultured in either Lymnaea brain-conditioned medium (CM) or on poly-L-lysine dishes that were pretreated with Aplysia hemolymph (ApHM), but contained only Lymnaea defined medium (DM; does not promote neurite outgrowth). In ApHM-coated dishes containing DM, Lymnaea neurons exhibited extensive neurite outgrowth, but appropriate excitatory synapses failed to develop between the cells. Instead, inappropriate reciprocal inhibitory synapses formed between VD4 and LPeD1. Similar inappropriate inhibitory synapses were observed in Aplysia hemolymph-pretreated dishes that contained dialyzed Aplysia hemolymph. These inhibitory synapses were novel and inappropriate, because they do not exist in vivo. A receptor tyrosine kinase inhibitor (Lavendustin A) blocked neurite outgrowth induced by both Lymnaea CM and ApHM. However, it did not affect inappropriate inhibitory synapse formation between the neurons. These data demonstrate that neurite outgrowth but not inappropriate inhibitory synapse formation involves receptor tyrosine kinases. Together, our data provide direct evidence that trophic factors required for neurite outgrowth are conserved among two different molluscan species, and that neurite extension and synapse specification between excitatory partners are likely mediated by different trophic factors.     

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.     

Characterization of a novel molluskan tyrosine kinase receptor that inhibits neurite regeneration

Receptor tyrosine kinases play many important roles in neuronal signaling including regulating neurite outgrowth. We have identified a novel receptor tyrosine kinase, neurite outgrowth regulating kinase (nork) from Aplysia californica. A fragment of this kinase was also identified in another mollusk, Lymnaea. The kinase domain is equally homologous to the Ret (rearranged during transformation) and fibroblast growth factor receptor families, but the extracellular domain is entirely novel, suggesting that it binds a nonconserved ligand. Overexpression of neurite outgrowth regulating kinase, but not a kinase dead form, causes a reduction in neurite outgrowth of Aplysia sensory neurons. Thus, we have identified a novel receptor tyrosine kinase implicated in regulating neurite outgrowth.     

Fertilization in inhibited in five diverse animal phyla by erythrosin B

Erythrosin B inhibited fertilization in species from the following phyla: Echiuroida, Annelida, Mollusca, Echinodermata, and Chordata. This effect was not due to diminished gamete viability but may reflect a general interference of sperm-egg membrane fusion by Erythrosin B.     

Structure and Function of Metazoan Ciliary Bands and Their Phylogenetic Significance

Ciliated epithelia, especially the ciliary bands used in swimming and filter feeding, of representatives of the following phyla have been investigated: Porifera, Cnidaria, Annelida, Mollusca, Sipuncula, Nemertini, Platyhelminthes, Entoprocta, Ectoprocta, Rotifera, Pterobranchia, Phoronida, Brachiopoda, Echinodermata and Enteropneusta. The trochaea theory predicts that Porifera and Cnidaria have only monociliate cells and lack ciliary bands used in filter-feeding, that the gastroneuralian larvae have downstream-collecting ciliary bands with prototroch and metatroch of compound cilia on multiciliate cells, and that notoneuralian larvae have an upstream-collecting neotroch on monociliate cells. The observations generally fit these predictions and the exceptions are discussed. In all the ciliated epithelia, except that of the sponge larva, each ciliated cell has an accessory centriole perpendicular to the basal body of the cilium and situated on its downstream side.     

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.     

Phylogenetic position of Sipuncula derived from multi‐gene and phylogenomic data and its implication for the evolution of segmentation

The phylogenetic position of Sipuncula, a group of unsegmented marine worms, has been controversial for several decades: Especially based on morphological data, closer relationships to Mollusca or Annelida were among the most favoured hypotheses. Increasing amounts of molecular data in recent years have consistently placed Sipuncula either in close affinity to or even within Annelida, the segmented worms, and rejected a close relationship to Mollusca. Yet, it remained uncertain whether Sipuncula is the sister group of Annelida or an annelid subtaxon. Therefore, herein we gathered data for five nuclear genes, which have been rarely used regarding Annelida and Sipuncula, and combined these with data for six previously used genes to further elucidate the phylogenetic position of Sipuncula. We also compiled a data set for 78 ribosomal proteins from publicly available genomic data sets. These are the two largest data sets for annelids with more than 10 taxa to date. All analyses placed Sipuncula within Annelida. For the first time, topology tests significantly rejected the possibility that Sipuncula is sister to Annelida. Thus, our analyses revealed that Sipuncula had secondarily lost segmentation. Given that unsegmented Echiura is also an annelid subtaxon, segmentation, a key character of Annelida, is much more variable than previously thought. Yet, this conclusion does not support the hypothesis that the last common ancestor of Annelida, Arthropoda and Chordata was segmented, assuming several losses along the branches leading to them. As yet no traces of segmentation could be shown in taxa exhibiting serially organized organ systems such as certain Mollusca, while in Sipuncula and Echiura such traces could be demonstrated. An independent origin of segmentation in Annelida, Arthropoda and Chordata thus appears to be more plausible and parsimonious.     

Single-molecule force spectroscopy of the aplysia cell adhesion molecule reveals two homophilic bonds

Aplysia californica neurons comprise a powerful model system for quantitative analysis of cellular and biophysical properties that are essential for neuronal development and function. The Aplysia cell adhesion molecule (apCAM), a member of the immunoglobulin superfamily of cell adhesion molecules, is present in the growth cone plasma membrane and involved in neurite growth, synapse formation, and synaptic plasticity. apCAM has been considered to be the Aplysia homolog of the vertebrate neural cell adhesion molecule (NCAM); however, whether apCAM exhibits similar binding properties and neuronal functions has not been fully established because of the lack of detailed binding data for the extracellular portion of apCAM. In this work, we used the atomic force microscope to perform single-molecule force spectroscopy of the extracellular region of apCAM and show for the first time (to our knowledge) that apCAM, like NCAM, is indeed a homophilic cell adhesion molecule. Furthermore, like NCAM, apCAM exhibits two distinct bonds in the trans configuration, although the kinetic and structural parameters of the apCAM bonds are quite different from those of NCAM. In summary, these single-molecule analyses further indicate that apCAM and NCAM are species homologs likely performing similar functions.     

The effect of brain-derived neurotrophic factor on neuritogenesis and synaptic plasticity in Aplysia neurons and the hippocampal cell line HiB5

Brain-derived neurotrophic factor (BDNF) plays a key role in the differentiation and neuritogenesis of developing neurons, and in the synaptic plasticity of mature neurons, in the mammalian nervous system. BDNF binds to the receptor tyrosine kinase TrkB and transmits neurotrophic signals by activating neuron-specific tyrosine phosphorylation pathways. However, the neurotrophic function of BDNF in Aplysia neurons is poorly understood. We examined the specific effect of BDNF on neurite outgrowth and synaptic plasticity in cultured Aplysia neurons and a multipotent rat hippocampal stem cell line (HiB5). Our study indicates that mammalian BDNF has no significant effect on the neuritogenesis, neurotransmitter release, excitability, and synaptic plasticity of cultured Aplysia neurons in our experimental conditions. In contrast, BDNF in combination with platelet-derived growth factor (PDGF) increases the length of the neurites and the number of spine-like structures in cells of HiB5.     

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.     

Molluscan neurons in culture: shedding light on synapse formation and plasticity

From genes to behaviour, the simple model system approach has played many pivotal roles in deciphering nervous system function in both invertebrates and vertebrates. However, with the advent of sophisticated imaging and recording techniques enabling the direct investigation of single vertebrate neurons, the utility of simple invertebrate organisms as model systems has been put to question. To address this subject meaningfully and comprehensively, we first review the contributions made by invertebrates in the field of neuroscience over the years, paving the way for similar breakthroughs in higher animals. In particular, we focus on molluscan (Lymnaea, Aplysia, and Helisoma) and leech (Hirudo) models and the pivotal roles they have played in elucidating mechanisms of synapse formation and plasticity. While the ultimate goal in neuroscience is to understand the workings of the human brain in both its normal and diseased states, the sheer complexity of most vertebrate models still makes it difficult to define the underlying principles of nervous system function. Investigators have thus turned to invertebrate models, which are unique with respect to their simple nervous systems that are endowed with a finite number of large, individually identifiable neurons of known function. We start off by discussing in vivo and semi-intact preparations, regarding their amenability to simple circuit analysis. Despite the 'simplicity' of invertebrate nervous systems however, it is still difficult to study individual synaptic connections in detail. We therefore emphasize in the next section, the utility of studying identified invertebrate neurons in vitro, to directly examine the development, specificity, and plasticity of synaptic connections in a well-defined environment, at a resolution that it is still unapproachable in the intact brain. We conclude with a discussion of the future of invertebrates in neuroscience in elucidating mechanisms of neurological disease and developing neuron-silicon interfaces.     

Processing of mechano- and chemosensory information in the lip nerve and cerebral ganglia of the snail Helix pomatia L.

Neurophysiologists have long been seeking out simple model systems in which to analyse the neuronal mechanisms underlying the organisation of behaviour. The feeding behaviour of molluscs has proved to be one of the most useful simple systems for the analysis of cyclical motor patterns, the interactions of central pattern generating interneurones and the role of sensory inputs in the initiation and maintenance of the behaviour. Considerable progress has been made in one or both of the first two aspects of this research in Lymnaea, Helisoma, Limax, Planorbarius, Pleurobranchaea and Tritonia (for reviews see [3, 7, 8, 15]) and more recently, in Aplysia [39] and Planorbis [1]. The role of mechano- and chemosensory inputs in the organisation of the feeding behaviour was studied in at least twenty molluscan species (for a review see [3]). However, in only less than half of them was the analysis extended to the effect of tactile and chemical inputs on identified neurones in the buccal and cerebral ganglia which contain the feeding circuitry (Aplysia: [12, 22, 36, 41]; Pleurobranchaea: [9, 16, 17]; Tritonia: [2]; Helisona: [21]; Limax: [11, 14, 35]; Helix: [6, 19, 24-26, 32, 38]). In present chapter I would like to review our earlier findings on the processing of mechano- and chemosensory information in the lip nerves and cerebral ganglia of Helix pomatia L. These findings were published in a series of papers between 1982 and 1987 [19, 20, 24-26]. The results reviewed here prepared the way for the development of new lines of research in our laboratory on the plasticity and serotonergic modulation of feeding in this widely used experimental animal [27, 40].     

岳麓山枫树群落大型土壤动物种类组成及生态研究

2004年3月至2004年4月,在岳麓山选取3个样地作枫树群落土壤大型无脊椎动物调查.共采获大型土壤无脊椎动物317头和18窝蚂蚁;分别隶属于扁形动物门、环节动物门、软体动物门、节肢动物门4门;涡虫纲、寡毛纲、腹足纲、蛛形纲、多足纲、昆虫纲、软甲纲7纲,共21目.其中优势种类为钻螺、红蚁、黑蚁、鼠妇,优势种类所在的纲为腹足纲、昆虫纲、软甲纲.同时根据结果及分析得出:土壤动物的最适pH值为6～7;土壤动物活动的起点温度为10℃,最适温度为17℃左右;降雨可以减少土壤动物的活动;土壤板结不利于土壤动物的生存;植物凋落物处于碎裂阶段时,土壤动物种类和个体数量多;空间异质性程度高,土壤动物种类及个体数量均较多;人类的活动既可以给土壤动物的生存造成有利条件,也可以造成不利环境从而影响土壤动物的数量和种类组成;枫树与其他树木交错分布的地方土壤动物的种类和个体数量均较高,具明显的边缘效应.     

Feeding repellence of Antarctic and sub-Antarctic benthic invertebrates against the omnivorous sea star Odontaster validus

Antarctic and sub-Antarctic benthic invertebrates are subjected to intense predation by mobile macroinvertebrates. Accordingly, chemical protection as well as other defensive mechanisms are expected to be common in organisms inhabiting these ecosystems. In order to evaluate anti-predation activities and allocation of chemical defenses within the anatomy of marine benthic Antarctic and sub-Antarctic invertebrates, 55 species were tested for feeding repellence against the sea star Odontaster validus, a common eurybathic sympatric predator. The invertebrates tested were collected from the deep waters of two poorly surveyed areas in terms of chemical ecology studies: the eastern Weddell Sea (Antarctica) and the vicinities of Bouvet Island (sub-Antarctica). Experiments were conducted at the Spanish Antarctic Base in Deception Island. In the feeding deterrence experiments, shrimp pieces were treated with crude lipophilic fractions obtained from each species, and were offered to the sea stars. A total of 29 species (53 %) from 7 different phyla (Porifera, Cnidaria, Chordata, Bryozoa, Echinodermata, Mollusca, and Annelida) showed feeding repellence against O. validus, and are therefore chemically protected against this keystone predator. Furthermore, 25 species were dissected into parts to investigate the possible allocation of defensive compounds. Some of the results obtained from these analyses support the prediction that the most exposed/vulnerable tissues concentrate chemical defenses to avoid predation against the sea stars. In summary, the results obtained in our survey support the hypothesis that deep-water Antarctic and sub-Antarctic benthic invertebrates are well protected chemically against sympatric predators, similarly to what has been reported in previous studies investigating shallow-water Antarctic species.     

Fine structure of the pharyngeal apparatus of the pelagosphera larva in Phascolosoma agassizii (Sipuncula) and its phylogenetic significance

Sipuncula is a small taxon of worm-like marine organisms of still uncertain phylogenetic position. Sipunculans are characterized by an unsegmented body composed of a trunk into which the anterior part, the introvert, can be withdrawn. The group has been placed at various positions within Metazoa; currently, it is either seen as sister group of a clade comprising Mollusca and Annelida or as sister to each of these. An in-group position in either Mollusca or Annelida has usually been precluded till now due to the lack of so-called annelid or molluscan “key-characters” such as segmentation and chaetae or the radula. In the development of certain taxa the trochophore stage is followed by a planktonic larva, the pelagosphera, which might exhibit phylogenetically important structures. Among these is the buccal organ, which has been considered homologous either to the ventral pharyngeal organ present in many sedentary polychaetes or to the radular apparatus of molluscs. In the present paper, the ventral pharynx of the pelagosphera larva of Phascolosoma agassizii is investigated by transmission electron microscopy. The pharynx comprises dorsolateral ciliary folds, a muscle bulb formed by transverse muscle fibres with large intercellular spaces, and an investing muscle. A tongue-like organ is lacking. These results show great structural correspondences to the ventral pharynx of polychaetes, especially to that of the flabelligerid Diplocirrus longisetosus. In contrast, there are no signs of structural similarities to the corresponding structures of molluscs. Thus evidence increases that Sipuncula are closely related to annelids; moreover, an in-group position of Sipuncula within Annelida, as suggested by recent molecular studies, is not precluded by the present data. Instead these studies find additional support. Hence the lack of segmentation and chitinous chaetae in Sipuncula would be a secondary rather than a primary situation, as has recently been shown for Echiura and Pogonophora.     

Distribution of sulfated mucopolysaccharides in invertebrates.

The sulfated mucopolysaccharide composition of 22 species of invertebrates belonging to the phyla Arthropoda, Mollusca, Annelida, Tunicata, Echinodermata, Coelenterata, and Porifera was analyzed. It is shown that all the species contain variable amounts of one or more types of sulfated mocopolysaccharides, most of which similar to the ones found in vertebrates. It is shown also that each species has a characteristic composition, differing from each other regarding the relative amount and type of chondroitin sulfates A, B, and C, heparitin sulfate, and heparin. The possible biological role of the sulfated mucopolysaccharides in cell recognition or aggregation or both is discussed in view of the present findings.