Search

The regulation of the settlement process in barnacles has attracted research interest due to their role as fouling organisms. The involvement of neurotransmitters in the regulation of settlement of marine invertebrate larvae has been described in several species. In this work, we reported the effects of the neurotransmitters dopamine and serotonin on the settlement of cyprids of the barnacle Balanus improvisus and described differences among cyprids of different ages. Also, we tested the effects of dopaminergic and serotonergic agonists and antagonists on settlement of cyprid larvae. We found that dopamine significantly stimulated settlement of 2- and 4-day-old cyprids, while serotonin exerted an inhibitory effect, regardless of cyprid age. The agonists and antagonists to the two neurotransmitters were not able to stimulate settlement but in general had either an inhibitory or no effect. We compared our results to those previously reported of the roles of dopamine and serotonin in the settlement of Balanus amphitrite. There appeared to be striking differences in the effects of these neurotransmitters between the two species because it has been reported that serotonin induces settlement in B. amphitrite and that dopamine inhibits it. This suggested that dopamine and serotonin play pivotal roles in settlement of barnacle but may act in different ways in the two species. [ABSTRACT FROM AUTHOR]

Most ascidian larvae settle and begin adhesion by means of three mucus secreting and sensory organs, the adhesive papillae or palps. However, the adhesive papillae of Botrylloides genus larvae, despite their name, have only a sensory function. By immunohistochemical localization of serotonin and β-tubulin, we demonstrated that the adhesive papillae of Botrylloides leachi contain two distinct types of neurons with different localization and possibly a different function. The central neurons emerge from the tunic at the apex of papillae and probably have a mechanosensory function. The lateral neurons contain serotonin and may play a role in the mechanism of metamorphosis triggering. Moreover, by histological analysis we found numerous secreting cells, clustered in the centre of the area among the three papillae, forming a glandular organ. This organ could perform the attachment of the larva to the substrate, which is traditionally considered to be operated by the anterior epidermis of the larva, with a sucker-like mechanism. [ABSTRACT FROM AUTHOR]

The organization of the cnidarian nervous system has been widely documented in polyps and medusae, but little is known about the nervous system of planula larvae, which give rise to adult forms after settling and metamorphosis. We describe histological and cytological features of the nervous system in planulae of the hydrozoan Clava multicornis. These planulae do not swim freely in the water column but rather crawl on the substrate by means of directional, coordinated ciliary movement coupled to lateral muscular bending movements associated with positive phototaxis. Histological analysis shows pronounced anteroposterior regionalization of the planula's nervous system, with different neural cell types highly concentrated at the anterior pole. Transmission electron microscopy of planulae shows the nervous system to be unusually complex, with a large, orderly array of sensory cells at the anterior pole. In the anterior half of the planula, the basiectodermal plexus of neurites forms an extensive orthogonal network, whereas more posteriorly neurites extend longitudinally along the body axis. Additional levels of nervous system complexity are uncovered by neuropeptide-specific immunocytochemistry, which reveals distinct neural subsets having specific molecular phenotypes. Together these observations imply that the nervous system of the planula of Clava multicornis manifests a remarkable level of histological, cytological, and functional organization, the features of which may be reminiscent of those present in early bilaterian animals., (Copyright © 2011 Wiley-Liss, Inc.)

Paraquat is an herbicide widely used in agriculture, that proved to have toxic effect on many animal models. Moreover, it is considered a potential etiologic factor of Parkinson's disease. Ascidians are invertebrate chordates, whose larval central nervous system shares basic structural homologies with the vertebrate one. Ascidian larvae exposed to paraquat developed specific alterations of the CNS, that were characterized by histological and immunohistochemical analysis. Tyrosine hydroxylase (TH) expression was examined by "in situ" hybridization. A decrease of dopamine content in anterior CNS of treated larvae was observed. In combined treatments with paraquat and l-ascorbic acid, a common anti-oxidant, the severity of the malformations was significantly reduced, confirming that the oxidative stress is involved in the toxicity mechanism of paraquat on ascidians. For its sensitivity to paraquat and its simple chordate body plan, ascidian larva is a promising animal model to further investigate the molecular mechanism of paraquat neurotoxicity., (Copyright © 2009 Elsevier B.V. All rights reserved.)

Imazalil (IMA) is a fungicide that is used extensively in fruit plantations and post-harvest treatments, but has teratogenic effects on vertebrate development, possibly due to the perturbation of retinoic acid (RA) levels in the embryo. Ascidians are sessile marine invertebrate chordates that develop through a tadpole larva, with a body plan that shares basic homologies with vertebrates. In this work, we tested the effects of IMA on the development of the solitary ascidian Ciona intestinalis by treating two-cell stage embryos with a range of concentrations (0.1, 0.5, 1, 2.5, 5, 10, 20 and 50microThe fungicide significantly altered ascidian development even at low concentrations and its effects were dose-dependent. Probit analysis revealed that the median lethal concentration, LC(50), was 4.87microM and the median teratogenic concentration, TC(50), was 0.73microM. Larvae developing from embryos exposed to IMA showed malformations of the anterior structures, which became more severe as IMA concentration increased. In particular, the anterior nervous system and the sensory vesicle were reduced, and the pigmented organs (the ocellus and the otolith) progressively lost their pigmentation. The larval phenotype induced by 5microM IMA exposure was further characterized by means of molecular analysis, through whole mount in situ hybridization with probes for genes related to the nervous system: Ci-Otp, Ci-GAD, Ci-POU IV, which are markers of the anterior neuro-ectoderm, the central nervous system and the peripheral nervous system respectively, and Ci-Hox-1, a gene specifically activated by RA, and Ci-Aldh2, a gene for aldehyde dehydrogenase, which is involved in RA synthesis. The altered expression of Ci-Otp, Ci-GAD, Ci-POU IV in 5microM IMA-exposed larvae compared to control larvae showed that this fungicide could affect the differentiation of the anterior nervous system, particularly of the sensory vesicle neurons. Recent studies suggest a similarity between IMA- and RA-induced phenotypes in tunicates, indicating that triazoles may also alter RA metabolism in ascidians. The observed Ci-Hox-1 and Ci-Aldh2 expression in control and treated larvae did not allow a direct link between IMA teratogenic potential and RA-dependent morphogenesis to be identified. It is likely that the fungicidal teratogenic mechanism involved RA signalling but that its effects on ascidian development depend on a more complex mechanism.

We describe Ciona intestinalis gamma-aminobutyric acid (GABA)-ergic neurons during development, studying the expression pattern of Ci-GAD (glutamic acid decarboxylase: GABA synthesizing enzyme) by in situ hybridization. Moreover, we cloned two GABA(B) receptor subunits (Ci-GABA(B)Rs), and a phylogenetic analysis (neighbor-joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci-GAD and Ci-GABA(B)Rs expression patterns in C. intestinalis embryos and larvae. At the tailbud stage, Ci-GAD expression was widely detected in central and peripheral nervous system (CNS/PNS) precursors, whereas Ci-GABA(B)Rs expression was evident at the level of the precursors of the visceral ganglion. GABA was localized by immunohistochemistry at the same developmental stage. In the larva, Ci-GAD transcripts and GABA immunofluorescence were also detected throughout the CNS and in some neurons of the PNS, whereas transcripts of both GABA(B) receptor subunits were found mainly in the CNS. The expression pattern of Ci-GABA(B)Rs appeared restricted to Ci-GAD-positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci-GABA(B)Rs transcripts were found in ventral motoneurons that did not express Ci-GAD. Insofar as GABAergic neurons are widely distributed also in the CNS and PNS of vertebrates and other invertebrate chordates, it seems likely that GABA signaling was extensively present in the protochordate nervous system. Results from this work show that GABA is the most widespread inhibitory neurotransmitter in C. intestinalis nervous system and that it can signal through GABA(B) receptors both pre- and postsynaptically to modulate different sensory inputs and subsequent swimming activity.

Fluconazole (FLUCO) is an azole derivative used to treat fungal and yeast infections. Embryotoxicity tests on the ascidian Phallusia mammillata were performed to evaluate the effects of this drug. FLUCO proved to have strong consequences on P. mammillata development. Incidence of malformations and of lethality increased in a dose dependent way. Probit analysis showed that FLUCO had a high TI value (Teratogenic Index, LC(50)/TC(50)), thus this substance could be classified as a teratogenic compound for ascidians. Larvae exposed to FLUCO showed a typical phenotype characterized by malformations restricted to the trunk region: the trunk appeared round in shape with flat palps, the sensory vesicle cavity was absent or reduced and the anterior central nervous system failed to correctly differentiate. These anomalies resulted similar to those induced by retinoic acid (RA) treatment. Thus, it could be hypothesized that FLUCO and RA may act with a similar pathogenic mechanism in ascidian larvae, as it has been proposed for mammals., (Copyright © 2006 Elsevier B.V. All rights reserved.)

To describe the serotonergic system in a tunicate larva, we cloned a gene encoding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, in the ascidian Ciona intestinalis and studied its expression pattern during development. Ci-TPH expression was found from tailbud stage in the precursor cells of the visceral ganglion and in the tail. In the larva, TPH-expressing neurons formed two clusters in the anterior central nervous system at the level of the visceral ganglion. Moreover, we found Ci-TPH expression at the level of the muscle cells of the tail and suggested that this localisation might be at the level of neuro-muscular junctions. Moreover, we discussed the involvement of serotonin in the control of larval locomotory activity.

Hydroid planulae metamorphose in response to an inducing external stimulus, usually a bacterial cue. There is evidence that neurotransmitters participate in the signal transduction pathway of hydroid metamorphosis. Eudendrium racemosum is a colonial hydroid common in the Mediterranean Sea. It lacks the medusa stage and the planulae develop on female colonies during the fertile season. In this work, serotonin (5-HT) was localized in some planula ectodermal cells. Co-localization of serotonin and beta-tubulin suggested that 5-HT was present in sensory nervous cells and in different ectodermal cells. To investigate the role of neurotransmitters in metamorphosis, E. racemosum planulae were treated with serotonin and dopamine and with agonists and antagonists of the corresponding receptors. Serotonin and a serotonin receptor agonist induced metamorphosis, while a 5-HT receptor antagonist inhibited it. Dopamine and all dopaminergic drugs used did not show any significant effect on the onset of metamorphosis. Results from this work showed that 5-HT could stimulate metamorphosis in E. racemosum planulae in the presence of a natural inducer. A mechanism by which this neurotransmitter could act in this phase is proposed.

Azole compounds are fungicides used in agriculture and in clinical area and are suspected to produce craniofacial malformations in vertebrates. Toxicity tests on sperm viability, fertilization and embryogenesis of the solitary ascidian Phallusia mammillata were performed to evaluate the effects of two azole derivatives, Imazalil and Triadimefon. Ascidian (Chordata, Ascidiacea) embryos and larvae could provide biological criteria for seawater quality standards because the larvae are lecitotrophic and have a short pelagic period, allowing to run the larval toxicity tests over a short period of time. Imazalil and Triadimefon proved to have strong consequences on P. mammillata. They could influence the reproductive rate of the animal exerting their effects at different levels: acting as spermiotoxic agents, inhibiting fertilization and impairing embryological development. Fertilization rate significantly decreased after 30min exposure of sperm to 25microM Imazalil (P<0.0001) and after exposure of both gametes to 50microM Imazalil (P<0.05) and 1mM Triadimefon (P<0.0001) as compared to controls. Malformations caused by exposure of embryos to both substances were dose dependent. Imazalil median teratogenic concentration (TC50 concentration, the concentration that resulted in 50% malformed larvae) value was 0.67microM and median lethal concentration (LC50, the concentration that resulted in 50% embryos dead before completing the development) value was 10.23microM while for Triadimefon TC50 value was 29.56 and LC50 value was 173.7microM. Larvae developed from embryos treated with Imazalil and Triadimefon showed alterations of the anterior structures of the trunk: papillary nerves and the anterior central nervous system failed to correctly differentiate, as showed by immunostaining with anti-beta-tubulin antibody. Comparing the anomalies caused by retinoic acid, reported in a previous study, it was possible to hypothesize that malformations induced by Imazalil and Triadimefon could be due to a perturbation of the endogenous retinoid content, as it has been proposed for mammals. Ascidians proved to be good models to study the toxic effects of pesticides since they offered both the convenience of working with an invertebrate species and the tissue sensitivity to chemical compound comparable to vertebrates.

The aim of this study was to characterize the swimming behaviour of C. intestinalis larvae during the first 6 h after hatching by measuring tail muscle field potentials. This recording method allowed a quantitative description of the responses of the larva under light and dark conditions. Three different larval movements were distinguished by their specific frequencies: tail flicks, 'spontaneous' swimming, and shadow response, or dark induced activity, with respective mean frequencies of about 10, 22 and 32 Hz. The shadow response develops at about 1.5 h post hatching (h.p.h.). The frequency of muscle potentials associated with this behaviour became higher than those of spontaneous swimming activity, shifting from 20 to 30 Hz, but only from about 2 h.p.h. onwards. Swimming rate was influenced positively for about 25 s after the beginning of the shadow response. Comparison of swimming activity at three different larval ages (0-2, 2-4 and 4-6 h.p.h.) showed that Ciona larvae swim for longer periods and more frequently during the first hours after hatching. Our results provide a starting point for future studies that aim to characterize the nervous control of ascidian locomotion, in wild-type or mutant larvae.

Neurotransmitters play an important role in larval metamorphosis in different groups of marine invertebrates. In this work, the role of dopamine and serotonin during metamorphosis of the ascidian Phallusia mammillata larvae was examined. By immunofluorescence experiments, dopamine was localized in some neurons of the central nervous system and in the adhesive papillae of the larvae. Dopamine and serotonin signaling was inhibited by means of antagonists of these neurotransmitters receptors (R(+)-SCH-23390, a D(1) antagonist; clozapine, a D(4) antagonist; WAY-100635, a 5-HT(1A) antagonist) and by sequestering the neurotransmitters with specific antibodies. Moreover, dopamine synthesis was inhibited by exposing 2-cell embryos to alpha-methyl-l-tyrosine. Dopamine depletion, obtained by these different approaches, caused early metamorphosis, while serotonin depletion delayed the onset of metamorphosis. The opposite effects were obtained using agonists of the neurotransmitters: lisuride, a D(2) agonist, inhibited metamorphosis, while DOI hydrochloride and 8-OH-DPAT HBr, two serotonin agonists, promoted it. So, it is possible to suppose that dopamine signaling delayed metamorphosis while serotonin signaling triggers it. We propose a mechanism by which these neurotransmitters may modulate the timing of metamorphosis in larvae.

Serotonin (5-HT) is a neurotransmitter which is supposed to play a key role during development. In the last few years 5-HT receptors have been cloned in many animal species, and there is evidence that different 5-HT receptors are also present in ascidians. Ascidians and vertebrates are both members of the phylum Chordata and both have a dorsal tubular central nervous system. Embryos of the ascidian Phallusia mammillata have been treated with WAY-100635, a potent and selective 5-HT(1A) receptor antagonist. The larvae developed from treated embryos showed a dramatic reduction of their anterior sensory vesicles and the pigment of two sensory organs, the ocellus and the otolith. Immunofluorescence experiments with an anti beta-tubulin monoclonal antibody specific for the neural system showed that the anterior neural system of treated animals was radically altered by the action of the drug in a dose-dependent way. These results suggest that 5-HT plays a role in the development of the neural system in ascidians and its action is mediated by receptors similar to the members of the 5-HT(1A) receptor subtype of mammals.

It is well known that deep hypothermia used in open-heart surgery is usually associated with a marked reduction in carbohydrate tolerance, especially dangerous in the diabetic patient, since it might result in severe metabolic complications, namely ketosis and hyperosmolar coma. In order to prevent the occurrence of such complications we treated an insulin-dependent diabetic patient undergoing cardiovascular surgery, with a feed-back insulin infusion operated by an artificial pancreas (GCIIS, Biostator). The Biostator was then used to perform a continuous (minute by minute) blood glucose monitoring in 2 more patients, a type II (non insulin-dependent) diabetic and a non diabetic. Blood samples were drawn sequentially in order to determine plasma free insulin concentration. The glycemic profile observed in the insulin-dependent diabetic under artificial pancreas treatment was similar to that in the non-diabetic. Plasma free insulin levels dropped near to zero during by-pass cooling, returning toward basal level during the rewarming phase. Such results are then discussed by the Authors and given a pathogenetic interpretation.

The glucose clamp technique has been used to evaluate the metabolic activity of NPH biosynthetic insulin in diabetic subjects free from anti-insulin antibodies. After overnight blood glucose normalization with a glucose-controlled insulin infusion system (Biostator), an s.c. injection of NPH insulin was given in the abdominal region. The insulin dose (0.236 +/- 0.05 U/kg body wt) was related to the usual intermediate-acting insulin requirement in the morning. Glucose was clamped at 100 mg/dl by a feedback i.v. glucose infusion. The end of the action of s.c. injected insulin considered conventionally to be the time of the spontaneous rise of blood glucose to 110 mg/dl. Free insulin levels were higher and the length of action was longer after NPH porcine than after NPH biosynthetic human insulin (BHI) (area under the free insulin curve: porcine 1423 +/- 556 mU/L/h; BHI 1045 +/- 338 mU/L/h, P less than 0.05; length of action: porcine 16.0 +/- 3.2 h; BHI 13.7 +/- 0.9 h, P less than 0.05); the glucose requirement was higher after porcine (76.8 +/- 13.5 g) than after BHI (58.5 +/- 14.6 g) without reaching statistical significance. However, the metabolic activity of the bioavailable insulin (index of plasma free insulin activity) was similar for the two insulins (porcine 381 +/- 77.4, BHI 342.8 +/- 54.2 mU/L/g of glucose/h). We conclude that a difference in pharmacokinetics exists between NPH BHI and porcine NPH insulin, which makes the latter metabolically more active. The different behavior does not seem to be related to the insulin molecule itself but could be a consequence of the unequal content of protamine in the two pharmacologic preparations.