Immunohistochemical localization of a retinoic acid-like receptor in nerve cells of two colonial anthozoans (Cnidaria).

Retinoic acid is known to induce vertebrate stem cells to differentiate into a variety of cell types, including neurons. Although retinoic acid was reported to affect morphogenetic pattern specification in the hydrozoan Hydractinia (Müller, W.A., 1984. Retinoids and pattern formation in a hydroid. J. Embryol. Exp. Morph. 81, 253-271) and a retinoid RXR receptor was cloned in the jellyfish Tripedalia (Kostrouch, Z., Kostrouchova, M., Love, W., Jannini, E., Piatigorsky, J., Rall, J.E., 1998. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc. Natl. Acad. Sci. U.S.A. 95, 13442-13447), the cellular targets of retinoids were not investigated. We used Western immunoblotting and immunohistochemistry to investigate the presence and cellular distribution of a RXR-like receptor in the sea pansy Renilla koellikeri and in the staghorn coral Acropora millepora (Cnidaria, Anthozoa). Western blots revealed a 64 kDa protein from a sea pansy extract in a band that co-migrated with a RXR protein from the rat brain. Using antibodies raised against an epitope of human alpha RXR, we visualized putative ectodermal sensory cells in the polyp column of the adult sea pansy. Immunoreactivity was absent in staghorn coral larvae but present in the polyp column of adult colonies in the form of clusters of neuron-like cells in the basiectoderm near the ectoderm-mesoglea interface. These observations suggest that a RXR-like receptor is involved in epithelial nerve cell specification in adult anthozoans and that this role is conserved throughout evolution.

Variations of lipid and fatty acid contents during the reproductive cycle of the anthozoan Renilla koellikeri.

Lipid and fatty acid contents were monitored in the sea pansy Renilla koellikeri during its reproductive cycle. The fluctuations of lipids and fatty acids were closely related to sexual development and gonads represented the principal site of lipid storage in the colony. Between April and May, fatty acids and lipids greatly accumulated just prior to spawning. The subsequent decrease of lipid and fatty acid contents in June probably resulted from loss due to spawning. Fatty acid composition was assessed for the whole animal and was compared between male and female gonads, and between gonads and somatic tissues. Arachidonic acid (AA) was the principal fatty acid in the whole colony followed by palmitic acid and eicosapentaenoic acid (EPA). The examination of fatty acid variations in gonads showed that EPA and AA were highly incorporated in eggs during the month preceding spawning, whereas in the same period, EPA was the only fatty acid to accumulate in spermatophores. The differences of fatty acid levels observed in gonads reflected sex differences observed in colonies. The seasonal variations of the omega6/omega3 ratio of the colonies were largely influenced by EPA and AA, and appeared as a reliable indicator of the sexual maturity of the sea pansy.

Monoamine release by neurons of a primitive nervous system: an amperometric study.

We measured monoamine release from dissociated neurons of the sea pansy Renilla koellikeri, a representative of the most evolutionarily ancient animals with nervous systems, by real-time monitoring of exocytosis using the amperometric method with carbon-fiber microelectrodes. Depolarization-induced, as well as spontaneously active, neurons exhibited calcium-dependent exocytotic events at both the soma and the terminal bulb of neuritic processes. All spontaneously active neurons exhibited a bursting activity pattern in which amplitudes of exocytotic events appeared to be distributed in a quantal-like fashion. Fast Fourier transform analysis of bursting activity in 20 such neurons revealed burst harmonics with a major frequency of 8 Hz and a dominant rate of 95 Hz for individual exocytotic events within bursts. The results suggest that exocytotic transmitter release is as ancient as neurons and that endogenously bursting neurons in the sea pansy are as complex as those of higher animals. In addition, the observation that both soma and neuritic terminals of the same neuron can release transmitter suggests that local release sites in these cnidarian neurons are not critical for nerve net function.

Evidence for gonadotropin-releasing hormone-like peptides in a cnidarian nervous system.

There is increasing evidence that peptides of the gonadotropin-releasing hormone (GnRH) family, long considered a vertebrate preserve, are also present in invertebrate (molluscan) nervous systems. The possibility was examined that GnRHs are present and bioactive in cnidarians, considered to be representatives of the most primitive animals possessing a nervous system. Immunoreactive GnRH was detected in endodermal neurons of two anthozoans, the sea pansy Renilla koellikeri and the sea anemone Nematostella vectensis. In the sea pansy, immunoreactivity was detected throughout the autozooid polyps, including gamete-producing endoderm. High-performance liquid chromatography and radioimmunoassays of extracts from whole sea pansy colonies yielded two elution peaks exhibiting GnRH immunoreactivity with antisera raised against shark or mammalian GnRH. Vertebrate GnRHs as well as the two sea pansy GnRH-like factors inhibited the amplitude and frequency of peristaltic contractions in the sea pansy, and these actions were blocked by the LHRH analog [D-pGlu(1),D-Phe(2),D-Trp(3,6)]-LHRH. These results suggest that the GnRH family of neuropeptides is more widespread in metazoans than previously thought. Although our physiological data are preliminary, they point to a role for GnRHs as inhibitory modulators of neuromuscular transmission in the sea pansy.

Distribution of serotonin uptake and binding sites in the cnidarian Renilla koellikeri: an autoradiographic study.

Using [(3)H]-serotonin ([(3)H]-5-HT) as radiolabel and autoradiography, we have mapped the distribution of 5-HT uptake and binding sites in the sea pansy Renilla koellikeri in order to identify potential cellular pathways of 5-HT inactivation and to identify cellular substrates for the previously characterized 5-HT receptor involved in the modulation of peristaltic behavior. Uptake measured in fresh polyp tissues occurred via two processes: a high affinity (uptake(1)), clomipramine-sensitive process with a K(m) of 0.45 microM, and another of lower affinity (uptake(2)) with a Km of 11.6 microM. Autoradiograms of high affinity uptake sites revealed a diffuse distribution of label with higher density in the ectoderm and endoderm, and lower density in the mesoglea. No subsets of cells, including serotonergic neurons, appeared to retain label preferentially, thus suggesting that removal of 5-HT and its chemical conversion is a general property of sea pansy tissues. Under incubation conditions identical to those used in a previous radiobinding analysis, autoradiograms of binding sites were generated on sections from lightly fixed and cryosectioned polyps. In contrast to uptake sites, binding sites appeared as aggregations of label around neurons of the subectodermal, mesogleal and endodermal nerve nets. In the endoderm where the myoepithelia subtend peristalsis, myoepithelial cells appeared unlabeled, suggesting that 5-HT exerts its modulatory effect on peristalsis principally via neurons. Taken together, these results indicate that 5-HT is released as a neurohormone in the sea pansy and that it may act as a broad-range neuromodulator.

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.

Characterization of a serotonin receptor in the cnidarian Renilla koellikeri: a radiobinding analysis.

Serotonin (5-HT) binding sites in membrane preparations from the sea pansy Renilla koellikeri were identified using [3H]5-HT as radioligand and unlabelled 5-HT as displacer of specific binding. Saturation and kinetic studies revealed a mixed population of [3H]5-HT binding sites which as a whole displayed slow association kinetics, saturability, negligible dissociation and low affinity. The dopaminergic antagonist trifluoperazine specifically displaced the non-dissociated binding sites, allowing the characterization of a homogenous population of saturable sites exhibiting faster association and full dissociation. Scatchard analysis of this population revealed a KD of 23-34 nM and binding site density (Bmax) of 8.5-20.6 pmol/mg protein depending on body region. The pharmacological profile of the dissociable [3H]5-HT binding sites could not be categorized with that of any of the existing vertebrate and invertebrate 5-HT receptor subtypes in the current nomenclature. Especially noteworthy was the absolute inability of LSD, a ligand of choice for all hitherto identified invertebrate 5-HT receptors, to compete for the binding sites in sea pansy membrane preparations. It is proposed that these binding sites represent an evolutionary forerunner of the primordial 5-HT receptor that diversified as multiple subtypes in higher invertebrates and vertebrates.

Neuronal and nonneuronal taurine-like immunoreactivity in the sea pansy, Renilla koellikeri (Cnidaria, Anthozoa).

A quantitative evaluation of putative amino acid neurotransmitters in sea pansy polyps by high-performance liquid chromatography indicates that the taurine content exceeds that of other amino acids by a 100-fold. The cellular source of this taurine was investigated by immunohistochemistry with two polyclonal antisera raised in rabbit, one against a glutaraldehyde-polylysine-taurine conjugate and the other against a succinylated ovalbumin-carbodiimide-taurine conjugate. Taurine-immunoreactive neurons were localized in a perioral subectodermal nerve net and in the zooid nerve net of the endodermal retractor muscle of the polyp mesenteries. Double labeling experiments revealed that taurine immunostaining does not colocalize with Phe-Mat-Ang-Phe -NH2 FMRFamide immunoreactivity. In addition, strong taurine immunoreactivity was found in nematocytes and other ectodermal cells, in myoepithelial cell bodies of the endoderm, and in calcareous spicule-producing cells of the colonial tissue mass. The limited distribution of neuronal taurine immunostaining to nerve nets associated with muscle systems subtending autozooid polyp retraction supports a role for taurine as a neuromuscular transmitter for this protective reflex. In contrast, the widespread distribution of taurine immunoreactivity in nematocytes and in other nonneuronal cells points to additional cellular functions of taurine, one of which may be to mediate responses to osmotic or metabolic stress.

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.

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.

Melatonin activity rhythms in eyes and cerebral ganglia of Aplysia californica.

In this study, we used a sensitive and specific radioimmunoassay to detect a substance which appears to be melatonin, an acetylation and methylation product of serotonin, in the eyes and central nervous system of the opisthobranch mollusc Aplysia californica. This identification was confirmed in the eyes by HPLC with fluorimetric detection. Melatonin activity was high in the eyes during the day and in the cerebral ganglia during the night. Only small amounts of melatonin were present at midday or midnight in the pedal ganglia. A single 1-hr exposure to light in the middle of the dark phase resulted in a sharp increase of melatonin in the eyes, whereas no such activity was detectable in cerebral and pedal ganglia. Eyes maintained in culture exhibited a diurnal rhythm of released melatonin activity over a 3-day period. These results suggest that melatonin is produced in A. californica in a rhythmic pattern different from that associated with pineal melatonin production in vertebrates.

Distribution of beta 2-like adrenergic receptors in the cnidarian Renilla koellikeri as revealed by autoradiography and in situ hybridization.

Autoradiography and in situ hybridization were used to examine the histological distribution of the previously characterized beta 2-like adrenergic receptors involved in the bioluminescent activity of the sea pansy Renilla koellikeri. The use of [3H]-(+/-)CGP12177 as radioligand revealed autoradiographic labelling of the refringent granule-filled endoderm at the base of autozooid tentacles and autozooid columns, and in the corresponding endoderm of siphonozooid polyps, all areas where photocytes are concentrated. The presence of excess (10 microM) unlabelled (+/-)CGP12177 or atenolol in the incubation mixture substantially reduced total [3H]-(+/-)CGP12177 labelling. Under low stringency hybridization washing, human beta 2-adrenoceptor oligonucleotide probe signals were detected in granular cells located in those areas of polyp endoderm that were labelled by [3H]-(+/-)CGP12177. These cells were previously shown to be distinct from, but in close proximity to photocytes. No other cell or tissue type was labelled in polyps or throughout colonial tissues. The results suggest that a conserved form of beta 2-adrenergic receptors is present and synthesized in a unique type of endodermal cell indirectly involved in sea pansy bioluminescence control.

Glutamate Immunoreactivity in Non-neuronal Cells of the Sea Anemone Metridium senile.

The distribution of glutamate in the tentacles and oral disk of the sea anemone Metridium senile was investigated by wholemount immunohistochemistry with the use of a monoclonal antibody raised against the derivatization product {gamma}-L-glutamyl-L-glutamic acid. Immunoreactivity was localized in one class of tentacle nematocysts and on their associated threads. These nematocysts were concentrated at the distal end of tentacles, none being found at the base of tentacles or in the oral disk. Muscle end-feet of epitheliomuscular cells also stained in the longitudinal muscle of tentacle ectoderm. In contrast, immunostaining in the oral disk was confined to ectodermal granule-containing cells overlying the radial muscle. These results support a role for glutamate as an osmolyte precursor in nematocysts but provide little clue as to the functional significance of this amino acid in muscle and gland cells.

Positive coupling of beta-like adrenergic receptors with adenylate cyclase in the cnidarian Renilla koellikeri.

Coupling of the previously characterized beta1- and beta2-like adrenoceptors in the sea pansy Renilla koellikeri with adenylate cyclase was examined in membrane preparations from this cnidarian. Adenylate cyclase activity was stimulated by several guanine nucleotides, such as GTP, Gpp(NH)p and GTPgammaS. Fluoride ions and cholera toxin greatly enhanced the enzyme activity, whereas forskolin had no effect on basal or isoproterenol-induced stimulation of the enzyme. The stimulation of adenylate cyclase activity by several beta-adrenergic agonists in different parts of the animal reflected a positive coupling with the beta2- and beta1-like adrenoceptors in autozooid and peduncle tissues, respectively. In addition, isoproterenol-induced stimulation of adenylate cyclase activity was dependent on guanine nucleotides, suggesting coupling mediated by a G protein. The pharmacological profile of various antagonists on isoproterenol-sensitive adenylate cyclase in autozooid and peduncle tissues matched that of previous radioligand binding studies. Isoproterenol-induced stimulation of adenylate cyclase activity in rachidial tissues was partially inhibited by trifluoperazine of (+/-)CGP12177 and was completely blocked in the presence of both antagonists. This suggests that coupling of the enzyme occurs with beta1- and beta2-like adrenoceptors, both being present in the rachis. Serotonin and dopamine were also found to stimulate adenylate cyclase activity. Their stimulatory effect was additive to isoproterenol-induced activation, suggesting the presence of dopaminergic and serotonergic receptors in the tissues of the sea pansy. Along with the data presented previously on beta-adrenergic binding, this study suggests that elements of receptor-dependent G protein signal transduction originated early in invertebrate evolution.

Biogenic amines in coelenterates.

1. This mini review highlights the most important findings during three decades of research on biogenic amines in coelenterates. 2. Histochemical, analytic chemical and physiological evidences clearly indicate that dopamine is used as an intercellular messenger in hydrozoans. 3. The colonian anthozoan Renilla, has beta-adrenergic mechanisms in monitoring bioluminescence and serotoninergic mechanisms in rhythmic contractions.

Melatonin secretion in vitro from the pineal complex of the lamprey Petromyzon marinus.

Melatonin secretion from cultured pineal complexes of the lamprey, Petromyzon marinus, was measured by radioimmunoassay (RIA) for 6 days under either a 12:12 light:dark cycle (L:D) or continuous darkness (D:D) at either 10 degrees or 20 degrees. Under the L:D cycle, melatonin release was completely suppressed during daytime and high during night-time, with larger amplitude at 20 degrees than at 10 degrees. Under D:D conditions, no significant circadian rhythmicity in secretion could be revealed by periodogram analysis at either 10 degrees or 20 degrees. This is the first direct evidence of melatonin secretion in a lamprey, suggesting that both light and temperature can affect in vitro melatonin secretion.

Serotonin-immunoreactive neurons in the cnidarian Renilla koellikeri.

The cellular localization of 5-hydroxytryptamine (5-HT) was investigated in the pennatulid anthozoan Renilla koellikeri by means of peroxidase-antiperoxidase-immunohistochemistry with an antiserum against 5-HT-formaldehyde-protein conjugate. In many colonies, strong 5-HT-immunoreactivity was displayed by the cell bodies and beaded processes of relatively small neuronlike elements predominating in the outer ectoderm or scattered in the underlying mesoglea. The immunostained neurons of the mesoglea were generally bipolar and their relatively short processes extended toward myoepithelial cells. In the ectoderm, most immunostained neurons appeared pseudounipolar. These cell bodies were endowed with a small, superficially directed, conical appendage reaching the outer surface of the epithelium. Their neurites emerged from the inner pole of the perikarya and branched toward other immunopositive ectodermal and mesogleal nerve cells, or nematocytes in the tentacles. The networklike distribution of the presumed 5-HT ectodermal neurons varied between the different regions of colonies and along the autozooid column. In the context of earlier observations in cnidarians, these cytological features suggest a sensory as well as a modulatory function for 5-HT in Renilla koellikeri.

Excitatory action of the native neuropeptide antho-rfamide on muscles in the pennatulid Renilla köllikeri.

1. Antho-RFamide (pGlu-Gly-Arg-Phe-amide), a neuropeptide recently isolated from the sea pansy Renilla köllikeri induced sustained (tonic) contractions in the rachis and peduncle of the colony, and in the individual autozooid polyps. 2. The threshold concentration for this effect was 5 nM in summer colonies and 1 microM in autumn or winter colonies. 3. The peptide-induced tonic contractions were unaffected in sodium-free sea water. There was a 30% reduction of the contraction amplitude in sea water lacking calcium. 4. Peptides related to Antho-RFamide and other peptides were also examined for activity on rachidial muscles. Only peptides containing the carboxyterminal sequence Arg-Phe-amide were active. 5. It is concluded that Antho-RFamide acts on Renilla muscles via a specific receptor and that it is a candidate for neurotransmitter or modulator in this pennatulid.

Serotoninlike immunoreactivity in the central and peripheral nervous system of the scale worm Harmothoe imbricata (Polychaeta).

The distribution of serotoninergic neurons in the nervous system of the scale worm Harmothoe imbricata was visualized in the anterior half of the body by the peroxidase-antiperoxidase (PAP) immunohistochemical method with a specific antiserotonin antibody. Immunoreactive neuronal somata were localized in discrete ganglion cell masses of the dorsally situated cerebral ganglion and in segmental ganglia of the ventral nerve cord. They also make up the majority of neurons present in the parapodial ganglia. Large and small varicose fibers stained in the neuropile of all the above-mentioned ganglia but also in interganglionic connectives and segmental nerves. On the basis of soma size and location and of fiber distribution, the reactive neurons were identified as primarily interneuronal with a few motoneurons and presumptive afferent neurons. The presence of a motor component was substantiated by observations of several reactive varicose fibers spread over longitudinal muscle layers of the trunk. In addition, neurites of the subepidermal nerve plexus and enterochromaffinlike cells of the gut epithelium reacted with the serotonin antibody. It is concluded that serotoninergic pathways are ubiquitous elements in the organization of the central and peripheral nervous system of this polychaete. The significance of these findings in relation to other annelid groups and to the physiological role of serotonin is discussed.

Luminescent activity and ultrastructural characterization of photocytes dissociated from the coelenterate Renilla köllikeri.

Dissociation and Percoll sedimentation techniques were used to separate and pool the autofluorescent luminescent cells (photocytes) of the pennatulid anthozoan Renilla köllikeri. Photometric recordings of luminescent activity of photocyte suspensions show that activation of flashing and glowing by KCl depolarization is suppressed in calcium-free sea water and by cobalt but enhanced by trifluoperazine, thus suggesting that luminescence excitation is dependent on extracellular calcium and calmodulin-mediated mechanisms. Of several neuroactive substances tested, adrenaline, dopamine, N-methyl-N-phenylethanolamine, serotonin and the native neuropeptide Antho-RFamide all induced photocyte responses at high concentrations (0.1-1 mM) only, whereas lower concentrations of adrenaline and Antho-RFamide are known to activate or enhance luminescence or muscular contractions in intact Renilla tissues (Anctil et al., 1982; Anctil, 1987). Hence, none of these substances is a likely neurotransmitter candidate for direct photocyte activation. Ultrastructural observations of dissociated photocytes reveal that they are musculo-epithelial cells containing numerous 0.2-mum vesicles resembling previously extracted and light-emitting lumisomes (Anderson and Cormier, 1973). Similar cells were traced ultrastructurally in situ in the endodermal luminescent zones, but not in non-luminescent endoderm.