Michel Anctil

Chemical transmission in the sea anemone Nematostella vectensis: A genomic perspective

The sequencing of the starlet sea anemone (Nematostella vectensis) genome provides opportunities to investigate the function and evolution of genes associated with chemical neurotransmission and hormonal signaling. This is of particular interest because sea anemones are anthozoans, the phylogenetically basal cnidarians least changed from the common ancestors of cnidarians and bilaterian animals, and because cnidarians are considered the most basal metazoans possessing a nervous system. This analysis of the genome has yielded 20 orthologues of enzymes and nicotinic receptors associated with cholinergic function, an even larger number of genes encoding enzymes, receptors and transporters for glutamatergic (28) and GABAergic (34) transmission, and two orthologues of purinergic receptors. Numerous genes encoding enzymes (14), receptors (60) and transporters (5) for aminergic transmission were identified, along with four adenosine-like receptors and one nitric oxide synthase. Diverse neuropeptide and hormone families are also represented, mostly with genes encoding prepropeptides and receptors related to varying closeness to RFamide (17) and tachykinin (14), but also galanin (8), gonadotropin-releasing hormones and vasopressin/oxytocin (5), melanocortins (11), insulin-like peptides (5), glycoprotein hormones (7), and uniquely cnidarian peptide families (44). Surprisingly, no muscarinic acetylcholine receptors were identified and a large number of melatonin-related, but not serotonin, orthologues were found. Phylogenetic tree construction and inspection of multiple sequence alignments reveal how evolutionarily and functionally distant chemical transmitter-related proteins are from those of higher metazoans.

Modulation of a rhythmic activity by serotonin via cyclic AMP in the coelenterateRenilla kllikeri

Summary1.Factors influencing peristalsis-like rhythmic contractions were investigated in the rachidial tissue of a colonial anthozoan, the sea pansyRenilla köllikeri.2.Allowing water to flow slowly over turgescent preparations resulted in transient increases in the amplitude of rhythmic contractions. Suprathreshold electrical stimulation of the rachidial tissue during relaxed periods of the cycle had a similar effect in addition to causing a rise in tonus.3.Serotonin (5-HT) and tryptamine induced increases in the amplitude of rhythmic contractions that were similar to those obtained by mechanical (water flow) or electrical stimulation. The threshold concentration for this indolaminergic response was 1 μmol·l−1. Washing off the drugs usually further enhanced the rise in amplitude of the rhythmic contractions. The 5-HT-induced response was reduced or abolished by the 5-HT blockers naphthyl piperazine and methysergide, but not mianserin.4.Dibutyryl or 8-bromo cyclic AMP mimicked the 5-HT-induced response. In addition, the levels of cyclic AMP, measured in rachidial tissue by radioimmunoassay, increased significantly in tissues exposed to 5-HT.5.Incubation of rachidial tissues in the serotonergic neurotoxin 5,7-dihydroxytryptamine and the serotonin synthesis inhibitor p-chlorophenylalanine resulted in a sharp reduction of the amplitude of rhythmic and electrically stimulated contractions concomitent with potentiation of the 5-HT-induced response. The monoamine storage depletor reserpine had similar effects except for the 5-HT response.6.This and other evidences suggest that a specific cyclic AMP-mediated serotonergic mechanism is involved in the modulation of rhythmic contractions and peristalsis in the sea pansy.

Melatonin in a primitive metazoan: Seasonal changes of levels and immunohistochemical visualization in neurons

Monthly day/night melatonin activity profiles were determined by radioimmunoassay over a 13‐month period in the colonial anthozoan Renilla köllikeri, and no daily rhythmic oscillation was found. Averaging those monthly values yielded a seasonal quantitative rhythm in both colonial and non‐colonial tissues of this cnidarian, with spring and summer levels found to be four to five times higher than autumn and winter ones. The annual rise, which occurred in two successive Aprils, coincided with the first stages of sexual maturation in R. köllikeri. Immunohistochemistry with a melatonin antibody raised in sheep revealed an exclusively neuronal distribution of melatonin‐immunoreactivity (MEL‐IR) in the endodermal septal filaments wrapped around gametophores, in endodermal walls of the rachis, and in the ectoderm of polyps. The MEL‐IR ectodermal neurons shared many morphological features with serotonin‐immunoreactive (5‐HT‐IR) neurons previously described in this animal but showed either weak or absent 5‐HT‐IR in double‐labelling experiments. In contrast, MEL‐IR and 5‐HT‐IR were strongly colocalized in endodermal neurons. These results indicate that melatonin is not a daily photoperiodic messenger but may instead act as a seasonal marker for reproduction in this cnidarian. We also provide the first evidence of a neuronal localisation of melatonin in an invertebrate, which suggests that melatonin may act as a neurotransmitter or neurohormone in the least evolved animals endowed with a nervous system. J. Comp. Neurol. 387:243–254, 1997.

Evidence for Intercellular Coupling and Connexin-like Protein in the Luminescent Endoderm of Renilla koellikeri (Cnidaria, Anthozoa)

Gap junction plaques are abundant in Hydrozoa, where they play an important role in signal propagation through epithelia and nerve nets, but they have not been found in the two other classes of Cnidaria, the Scyphozoa and the Anthozoa. Here several lines of evidence are presented that point to the existence of intercellular coupling in tissues of the anthozoan Renilla koellikeri, especially in the luminescent endoderm. Dye-exchange experiments show that calcein vital stains spread between cultured cells after their reassociation. Polyp luminescence evoked by KCI depolarization, electrical stimulation, or {beta}-adrenergic agonists was largely and reversibly suppressed in the presence of the gap junction uncouplers octanol, heptanol, and low pH sodium acetate. A connexin43-like protein was isolated on Western blots of R. koellikeri membrane extracts by using a monoclonal connexin-43 antibody. Loading this antibody in R. koellikeri tissues resulted in the suppression of luminescence evoked by electrical stimulation. Immunohistochemical investigations using this antibody revealed mostly punctate immunostaining associated with endodermal cells of the luminescent tissue and with the mesogleal nerve net. Electron microscopic observations confirmed the absence of conventional gap junction plaques in these tissues, but revealed the presence of tiny zones of close membrane apposition between light-emitting and other endodermal cells, with gaps of 2-4 nm. Taken together, these results are consistent with the notion of the existence in R. koellikeri of intercellular coupling (1) involved in local transmission of luminescence signals, and (2) mediated by connexin43-based connexons that are not assembled into typical gap junction plaques.

Annual variations and sex-related differences of estradiol-17β levels in the anthozoan Renilla koellikeri

In the sea pansy Renilla koellikeri, estradiol-17beta (E(2)) levels exhibited an annual pattern of secretion that correlated with the reproductive cycle, and displayed sex-specific and tissue-specific differences. The E(2) levels were low during the non-reproductive period extending from autumn to winter. A first rise of E(2) concentrations occurred in March when gonad maturation resumed, as indicated by an increase in lipid storage. This suggests that E(2) may influence the beginning of gonad maturation in the sea pansy. Estradiol-17beta returned to basal levels in April and May when lipids rose sharply. A second, more significant surge of E(2) levels occurred in June when spawning was initiated and it was more marked in female than in male colonies. This suggests that E(2) may participate in synchronising of oocyte maturation around spawning time to optimise the probability of fertilisation. The higher E(2) levels in somatic tissues (peduncle and polyps) than in eggs during the March peak revealed a non-reproductive origin for E(2) and the need for transport of E(2) through the gastrovascular cavity to reach reproductive tissues. Further analyses are required to determine the relative contribution of E(2) to reproductive events and its pathway of synthesis in this colonial anthozoan.

Nitric oxide modulates peristaltic muscle activity associated with fluid circulation in the sea pansy Renilla koellikeri

SUMMARY Nitric oxide (NO) is a well-known regulator of vascular activities in vertebrates and it has also been implicated as a vasodilatatory agent in a cephalopod. In the sea pansy Renilla koellikeri, an octocorallian representative of the most basal animals with a nervous system, we investigated the role of NO in peristalsis, an activity that moves body fluids through the coelenteron (gastrovascular cavity) of the polyps across the colony. NO donors increased the amplitude of peristaltic contractions and increased tonic contractions in relaxed preparations, but caused a relaxation of basal tension in contracted preparations. The NO synthase (NOS) inhibitors L-NAME (N(ω)-nitro-L-arginine methyl ester) and 7-nitroindazole reduced the amplitude of peristaltic contractions and lowered basal tension. In contrast, aminoguanidine, a specific inhibitor of inducible NOS, increased the amplitude but reduced the rate of peristalsis. Zaprinast, a cGMP-specific phosphodiesterase inhibitor, decreased the amplitude of peristaltic contractions, a decrease that was amplified by dibutyryl cGMP. In contrast, the inhibitor of soluble guanylyl cyclase ODQ (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one) enhanced peristalsis. Putative NOS-containing neurons, revealed by NADPH-diaphorase activity and citrulline immunohistochemistry, were observed in the basiectoderm at the base of the autozooid polyp tentacles and in a nerve-net around the oral disc. Their neurites ran up the tentacles and down to the polyp body wall, crossing from the ectoderm through the mesoglea and into the endoderm musculature where musculo-epithelial cells were also reactive. These data suggest that two distinct nitrergic pathways, one of which is mediated by cGMP, regulate peristalsis and muscle tone in the sea pansy and that these pathways may involve NOS-containing ectodermal neurons and musculo-epithelial cells.

A new G protein-coupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells

Biogenic amine receptors mediate wide‐ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic‐like receptors were previously characterized pharmacologically and found to engender control of the animals bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT‐PCR strategy, we obtained a full‐length cDNA encoding a polypeptide with typical G protein‐coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK‐ and COS‐7 cells, based on both cAMP and Ca2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11‐fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.

Bioactivity of FMRFamide and related peptides on a contractile system of the coelenterateRenilla kllikeri

Summary1.The pharmacological activity of peptides terminating with RFamide was investigated in the rachidial tissue of the colonial anthozoanRenilla köllikeri.2.FMRFamide and RFamide induced tonic rachidial contractions in a dose-dependent maner, the threshold concentration being 0.1 μM. This response was elicited to a lesser extent by FLRFamide and abolished in the presence of an antibody to FMRFamide.3.Colonial activities coordinated by the through-conducting nerve-net ofRenilla such as stimulation-induced luminescence and rachidial contractions were enhanced in the presence of FMRFamide.4.FMRFamide-induced tonic contractions were independent of external calcium or sodium, were not influenced by adding lanthanum or cobalt to the bathing medium, but were blocked by D600 and verapamil.5.Contrary to the peptide-elicited tonic contraction, rhythmic and electrically stimulated rachidial contractions were dependent on external calcium, thus demonstrating that the oinic mechanisms associated with these agents differ from those involved with the FMRFamide response.6.This and other (immunohistochemical) evidence suggest that a FMRFamide-related peptide plays a role in neuromuscular transmission inRenilla.

Antho-RFamide-Containing Neurons in the Primitive Nervous System of the Anthozoan Renilla koellikeri

The neuropeptide Antho‐RFamide is extremely abundant in Renilla koellikeri (sea pansy), a representative of the cnidarians (octocorallians) considered to be closest to the stem ancestors of metazoans with nervous systems. Therefore, a knowledge of the distribution of Antho‐RFamide‐containing neurons in this species would contribute to our understanding of the early evolution of nervous systems. Using antisera raised against RFamide and FMRFamide, we detected immunostaining in numerous neurons throughout the nervous system of the sea pansy. The antisera revealed ectodermal nerve‐nets on the upper and lower sides of the colony and on the oral side of tentacles, in the oral disk, and in the pharynx of feeding polyps. Neurons were immunostained also in the mesogleal nerve‐net of feeding polyps and in the through‐conducting mesogleal nerve‐net of the colonial mass. Varying densities of stained neurons were observed in the different compartments of the endoderm: muscular walls of the feeding and water circulation polyps, mesenteric filaments and their derived follicles containing either ovocytes or spermatophores, in the endodermal channels connecting the different compartments of the colony, and in circular muscle of the peduncle. The distribution of immunostained neurons suggests that they play important roles in feeding, reproduction, neuromuscular transmission, and in neuro‐neuronal transmission coordinating the different parts of the colony. J. Comp. Neurol. 472:208–220, 2004.

Evidence for biosynthesis and catabolism of monoamines in the sea pansy Renilla koellikeri (CNIDARIA)

The biosynthesis of catecholamines and indoleamines was investigated in the sea pansy Renilla koellikeri by radiochemical screening of tissue samples exposed in vivo to labelled amino acid precursors and analysed by high-performance liquid chromatography coupled with electrochemical detection. Incubation of sea pansy tissues in [3H]tyrosine resulted in substantial accumulation of radioactivity recovered in chromatograms coeluting with tyrosine and 4-hydroxy-3-methoxy mandelic acid and, to a lesser extent, with 3,4-dihydroxy-phenylalanine, dopamine, norepinephrine, epinephrine, normetanephrine and 3,4-dihydroxyphenyl acetic acid. The catecholamine synthesis inhibitor alpha-methyl-p-tyrosine effectively reduced several of these [3H]tyrosine by-products formed as well as endogenous stores of these amines. Incubations in [3H]tryptophan resulted in large amounts of radioactivity associated with liquid chromatographic peaks coeluting with tryptophan and 5-hydroxytryptophan and lesser amounts with 5-hydroxytryptamine, N-acetyl-5-hydroxytryptamine and 5-hydroxy-3-indole acetic acid. The indoleamine synthesis inhibitor p-chlorophenylalanine reduced the amounts of products formed and depleted stores of the endogenous indoleamines. Enzyme activities which appear to involve tyrosine hydroxylase (EC 1. 12. 16. 2), tryptophan hydroxylase (EC 1. 14. 16. 4) and phenylethanolamine N-methyltransferase (EC 2. 1. 1. 28) were also detected in rachidial tissues by HPLC analysis of reaction products (hydroxylases) and by a radioenzymatic assay (methyltransferase). The sea pansy being a representative of the earliest invertebrates possessing a nervous system, these results support the hypothesis that vertebrate-like enzymatic pathways for the biosynthesis and degradation of monoamine neurotransmitters were conserved throughout evolution.