Catecholamines and dihydroxyphenylalanine in metamorphosing larvae of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

The content of catecholamines and dihydroxyphenylalanine in larvae of the nudibranch Phestilla sibogae was analyzed by high-performance liquid chromatography with electrochemical detection. Dihydroxyphenylalanine, norepinephrine and dopamine were identified in larvae of all ages examined (5 through 12 days post-fertilization). Dihydroxyphenylalanine could be accurately quantified only in larvae of ages 8 through 12 days, when its average concentration increased from 0.62 to 6.71 × 10⁻² pmol μg protein⁻¹. Between ages 5 and 12 days dopamine rose from 0.081 to 0.616 pmol μg protein⁻¹, and norepinephrine from 0.45 to 2.17 × 10⁻² pmol μg protein⁻¹. Dihydroxyphenylalanine, dopamine and norepinephrine were also measured at different stages of metamorphic progress in 10- to 12-day larvae. Dihydroxyphenylalanine increased by a factor of 2.4 between the onset and completion of metamorphosis, but levels of dopamine and norepinephrine remained stable. One millimolar alpha-methyl-dl-m-tyrosine, an inhibitor of catecholamine synthesis, inhibited natural metamorphosis and depleted endogenous norepinephrine and especially dopamine, respectively, to 75% and 35% of control values. The existence of unexpectedly high levels of catecholamines in metamorphically competent larvae, and the association of catecholamine depletion with inhibition of metamorphosis, indicate that these compounds may participate in the control of gastropod development. Accepted: 18 April 1997     

The apical sensory organ of a gastropod veliger is a receptor for settlement cues

On the basis of anatomy and larval behavior, the apical sensory organ (ASO) of gastropod veliger larvae has been implicated as the site of perception of cues for settlement and metamorphosis. Until now, there have been no experimental data to support this hypothesis. In this study, cells in the ASO of veliger larvae of the tropical nudibranch Phestilla sibogae were stained with the styryl vital dye DASPEI and then irradiated with a narrow excitatory light beam on a fluorescence microscope. When its ASO cells were bleached by irradiation for 20 min or longer, an otherwise healthy larva was no longer able to respond to the usual metamorphic cue, a soluble metabolite from a coral prey of the adult nudibranch. The irradiated cells absorbed the dye acridine orange, suggesting that they were dying. When larvae not stained with DASPEI were similarly irradiated, or when stained larvae were irradiated with the light beam focused on other parts of the body, there was no loss of ability to metamorphose. Together these data provide strong support for the hypothesis. Potassium and cesium ions, known to induce metamorphosis in larvae of many marine-invertebrate phyla, continue to induce metamorphosis in larvae that have lost the ability to respond to the coral inducer due to staining and irradiation. These results demonstrate that (1) the ASO-ablated larvae have not lost the ability to metamorphose and (2) the ions do not act only on the metamorphic-signal receptor cells, but at other sites downstream in the metamorphic signal transduction pathway.     

Catecholamine release and uptake in the mouse prefrontal cortex

Monitoring the release and uptake of catecholamines from terminals in weakly innervated brain regions is an important step in understanding their importance in normal brain function. To that end, we have labeled brain slices from transgenic mice that synthesize placental alkaline phosphatase (PLAP) on neurons containing tyrosine hydroxylase with antibody-fluorochrome conjugate, PLAP-Cy5. Excitation of the fluorochrome enables catecholamine neurons to be visualized in living tissue. Immunohistochemical fluorescence with antibodies to tyrosine hydroxylase and dopamine beta-hydroxylase revealed that the PLAP labeling was specific to catecholamine neurons. In the prefrontal cortex (PFC), immunohistochemical fluorescence of the PLAP along with staining for dopamine transporter (DAT) and norepinephrine transporter (NET) revealed that all three exhibit remarkable spatial overlap. Fluorescence from the PLAP antibody was used to position carbon-fiber microelectrodes adjacent to catecholamine neurons in the PFC. Following incubation with L-DOPA, catecholamine release and subsequent uptake was measured and the effect of uptake inhibitors examined. Release and uptake in NET and DAT knockout mice were also monitored. Uptake rates in the cingulate and prelimbic cortex are so slow that catecholamines can exist in the extracellular fluid for sufficient time to travel approximately 100 microm. The results support heterologous uptake of catecholamines and volume transmission in the PFC of mice.     

Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia)

SUMMARY The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae . Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.     

Catecholamines induce metamorphosis in the hydrozoan Halocordyle disticha but not in Hydractinia echinata

Summary Planula larvae of the marine hydroids Halocordyle disticha and Hydractinia echinata were treated with the catecholamines epinephrine, norepinephrine and dopamine, as well as with certain of their precursors and agonists. Norepinephrine, l-dopa, dopamine and the dopamine agonist ADTN at concentrations ranging from 0.1 to 0.001 mM induced metamorphosis within 24 h in Halocordyle disticha, with no observable morphogenetic abnormalities. Epinephrine, the adrenergic agonists phenylephrine, isoproterenol and methoxyamine, and the catecholamine precursors phenylalanine and tyrosine were found not to induce metamorphosis at the concentrations employed. None of the compounds was effective in inducing metamorphosis in Hydractinia echinata. A model is presented for neural control of metamorphosis in Halocordyle disticha     

The effect of intrastriatal injection of liposome-entrapped tyrosinase on the dopamine levels in the rat brain.

Parkinson's disease is a neurodegenerative disorder which is mainly characterized by degeneration of the dopaminergic cells in the nigro-striatal system. Due to a lowered L-tyrosine 3-monooxygenase activity, L-tyrosine is not sufficiently transformed to L-DOPA. To date the most common therapy is the administration of the dopamine precursor L-DOPA, with severe collateral effects. Therefore, the substitution of the lacking tyrosine hydroxylase with tyrosinase might be a novel therapeutical approach that would generate specifically L-DOPA from L-tyrosine. We present here evidence that stereotaxic injection of liposome-entrapped tyrosinase is able to significatively increase the levels of dopamine in the rat brain. The catecholamines L-DOPA, dopamine, L-epinephrine, L-norepinephrine were extracted by acid treatment from the brains and detected by HPLC.     

Glucocorticoids Increase Catecholamine Synthesis and Storage in PC 12 Pheochromocytoma Cell Cultures

Abstract: Glucocorticoids, cholera toxin and high plating density all increase the activity of tyrosine 3-monooxygenase (TH) in cultured PC12 pheochromocytoma cells. Glucocorticoids increase enzyme activity in cells treated with cholera toxin and in cells grown at high plating density. Glucocorticoids also increase the content of stored catecholamines in the cells. In cells cultured under routine conditions, glucocorticoids primarily increase the stores of dopamine. The addition of ascorbate to the culture medium increases the storage of norepinephrine in both steroid-treated and untreated cells. Incubation of the cells in media containing 56 n M K⁺ causes the release of the same percentage of stored dopamine from steroid-treated as from untreated cells. Steroid-treated cells contain more dopamine than do untreated cells and therefore, in response to high K⁺, the steroid-treated cells secrete more dopamine than do untreated cells. We conclude that the activity of tyrosine 3-monooxygenase in PC12 cells can be regulated by several distinct mechanisms; that glucocorticoids cause a coordinate increase in TH activity and in catecholamine storage; that steroids increase the storage of catecholamines in a releasable pool; and that the steroid-induced increase in catecholamine storage may result in increased secretion of catecholamines from steroid-treated cells.     

Timing is everything: the effects of putative dopamine antagonists on metamorphosis vary with larval age and experimental duration in the prosobranch gastropod Crepidula fornicata

The signal transduction pathway through which excess potassium ion stimulates the larvae of many marine invertebrates to metamorphose is incompletely understood. Recent evidence suggests that dopamine plays important roles in the metamorphic pathway of Crepidula fornicata. Therefore, we asked whether blocking dopamine receptors might prevent excess potassium ion from stimulating metamorphosis in this species. Surprisingly, the effects of the three putative dopamine antagonists tested (all at 10 microM) varied with exposure duration and the age of competent larvae. Chlorpromazine, a nonspecific dopamine antagonist known to have a number of other pharmacological effects, blocked the inductive action of excess potassium ion during the initial 5-8-h exposure periods in most assays, particularly for younger or smaller competent larvae. However, chlorpromazine in the absence of excess potassium ion also stimulated metamorphosis, particularly over the next 18 h, and worked faster on older competent larvae than on younger competent larvae. The specific D(1) antagonist R(+)-Sch-23309 had similar effects, blocking potassium-stimulated metamorphosis in short-term exposures and stimulating metamorphosis in longer exposures, particularly for older competent larvae. Although the specific D(2) antagonist spiperone (SPIP) blocked the inductive effects of excess potassium ion in only 1 of 6 assays during the first 6 h of exposure, it blocked metamorphosis in 2 of the assays during 24-h exposures. Our results indicate that dopamine receptors are involved in the pathway through which excess potassium ion stimulates metamorphosis in C. fornicata. In addition, the largely latent inductive effects of chlorpromazine, an inhibitor of nitric oxide synthase, suggest that endogenous nitric oxide may play a natural role in inhibiting metamorphosis in this species. Overall, our results would then suggest that exposing larvae of C. fornicata to excess K(+) leads to a shutdown of nitric oxide synthesis via a dopaminergic pathway, a pathway that can be blocked by some dopamine antagonists. Alternatively, chlorpromazine might eventually be stimulating metamorphosis by elevating endogenous cyclic nucleotide (e.g., cAMP) concentrations, again acting downstream from the steps acted on directly by excess K(+).     

Neural correlates of settlement in veliger larvae of the gastropod,Crepidula fornicata

Settlement behavior of molluscan veliger larvae prior to metamorphosis requires cessation of swimming, accomplished by arrest of prototrochal cilia on the margin of the velum (the larval swimming organ). Ciliary arrest in larvae of gastropods is mediated by an action potential that occurs synchronously across the velum as a consequence of electrical coupling between the prototrochal ciliated cells. We developed a preparation for extracellular recording of such ciliary arrest spikes from intact swimming and crawling veliger larvae of the caenogastropod Crepidula fornicata, using a fine wire electrode. Ciliary arrest spike rates during bouts of substrate crawling were significantly higher than those recorded during preceding swimming periods in larvae that were competent for metamorphosis, but not in precompetent larvae. Spike rates were similar on clean polystyrene substrates, and on substrates that had been coated with a natural cue for metamorphosis (mucus from conspecific adults). We used immunohistochemical methods to localize neuromodulators that might regulate the function of velar cilia. Labeled terminals for serotonin, FMRFamide, and tyrosine hydroxylase (an enzyme for catecholamine synthesis) were located in positions consistent with modulatory effects on the prototrochal ciliated cells. Prototrochal ciliary arrest spike rates and beat frequencies were measured in isolated velar lobes from competent larvae, which were exposed to serotonin, FMRFamide, and dopamine (10^?5 mol L^?1). Serotonin abolished arrest spiking and increased beat frequency; dopamine also increased beat frequency, and FMRFamide depressed it. Competent larvae tested in a small static water column swam to the top of the column when exposed to serotonin, but occupied lower positions than controls when in the presence of dopamine and FMRFamide. The larval nervous system appears to regulate velar functions that are critical for settlement behavior, and is likely to do so by integrating different sensory modalities in an age‐dependent manner.     

Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.     

Characterization of a natural inducer of coral larval metamorphosis

External chemical signals used by scleractinian corals to recognize suitable substrata for larval settlement and metamorphosis were identified from crustose coralline red algae (CCA). A fragment of coral rubble with CCA induced larval metamorphosis of the scleractinian coral Pseudosiderastrea tayamai. A natural inducer and compounds that enhanced its effect in larval metamorphosis were isolated from the methanol extracts of coral rubble with CCA. A bromotyrosine derivative, 11-deoxyfistularin-3 (10^− 7 M) isolated from the CCA, induced the metamorphosis of P. tayamai larvae (27.5 ± 24.0%). In the presence of fucoxanthinol (10^− 9 M) and fucoxanthin (10^− 9 M), the percentage of metamorphosis induced by the bromotyrosine derivative was further enhanced to 87.8 ± 13.0 and 88.4 ± 17.8%, respectively. Both carotenoids are also found in the coral rubble with CCA. These results suggest that bromotyrosine derivative and carotenoids have a synergistic effect in the metamorphosis of P. tayamai larvae. The synergistic effect provides a higher selectivity for recruitment than a single-component natural inducer for recognizing suitable substrata for larval metamorphosis. Thus, the effect might offer a survival advantage for benthic marine invertebrates.     

The Low Affinity Dopamine Binding Site on Tyrosine Hydroxylase: The Role of the N-Terminus and In Situ Regulation of Enzyme Activity

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is inhibited in vitro by catecholamines binding to two distinct sites on the enzyme. The N-terminal regulatory domain of TH contributes to dopamine binding to the high affinity site of the enzyme. We prepared an N-terminal deletion mutant of TH to examine the role of the N-terminal domain in dopamine binding to the low affinity site. Deletion of the N-terminus of TH removes the high affinity dopamine binding site, but does not affect dopamine binding to the low affinity site. The role of the low affinity site in situ was examined by incubating PC12 cells with L-DOPA to increase the cytosolic catecholamine concentration. This resulted in an inhibition of TH activity in situ under both basal conditions and conditions that promoted the phosphorylation of Ser40. Therefore the low affinity site is active in situ regardless of the phosphorylation status of Ser40.     

A study on melanogenesis and catecholamine biosynthesis during Bufo bufo development

Tyrosinase and L-DOPA decarboxylase activities have been investigated during Bufo bufo development since catecholamines and melanin are formed from common substrates in homologous cells. Catecholamines first appear at stage 13 (neural plate), but tyrosinase, at a very low level, and L-DOPA decarboxylase are present throughout all of prior development. Hence, L-DOPA decarboxylase activity is not likely to be correlated with the control of catecholamine synthesis, although at stage 17 it is mainly localized in the nonneural part of the embryo. The distribution of young melanosomes and L-DOPA decarboxylase suggest a separation between melanogenesis and catecholamine synthesis.     

Natural inducers for coral larval metamorphosis

 Coral gametes from Acropora millepora (Ehrenberg, 1834) and from multi-species spawning slicks provided larvae for use in metamorphosis assays with a selection of naturally occurring inducer chemicals. Four species of crustose coralline algae, one non-coralline crustose alga and two branching coralline algae induced larval metamorphosis. However, one additional species of branching coralline algae did not produce a larval response. Metamorphosis was also observed when larvae were exposed to skeleton from the massive coral Goniastrea retiformis (Lamarck, 1816) and to calcified reef rubble, demonstrating metamorphosis is possible in the absence of encrusting algae. Chemical extracts from these algae and the coral skeleton, obtained using either decalcification or simple methanol extraction procedures, also contained active inducers. These results extend the number of crustose algal species known to induce coral metamorphosis, suggest that some inducers may not necessarily be strongly associated with the calcified algal cell walls, and indicate that inducer sources in reef habitats may be more diverse than previously reported. Accepted: 21 May 1999     

The biology of coral metamorphosis: molecular responses of larvae to inducers of settlement and metamorphosis

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring > 8000 Acropora unigenes were used to follow gene expression changes during the 12 h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common—an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.     

Loss of sensory elements in the apical sensory organ during metamorphosis in the nudibranch Phestilla sibogae

Larvae of the nudibranch Phestilla sibogae are induced to metamorphose by a water-borne chemical cue released by the adult nudibranch's prey, the coral Porites compressa. In competent larvae, the apical sensory organ (ASO) includes five serotonergic parampullary neurons; five ampullary neurons, the ampullae of which are filled with sensory cilia; and a basal neuropil. After sensing the coral cue, the ASO undergoes radical morphological changes: a deterioration of sensory elements in the ASO and serotonergic axons originating from them to innervate the velum. Three hours after metamorphic induction, the velar lobes are lost, the serotonergic axons begin to break apart, the five parampullary neurons begin to degenerate, and the five ampullary neurons retract away from the epidermal surface. The extent of deterioration evident by this time suggests that the parampullary and ampullary components of the ASO are no longer functional. By 10 h after metamorphic induction, labeling of the ciliary bundles in the ampullary neurons has disappeared, and it is likely that these cells have degenerated. The results presented here provide evidence that the sensory neurons of the ASO and probably the entire organ are solely larval structures that do not persist into the adult sensory-nervous system in P. sibogae.     

Are G-protein-coupled receptors involved in mediating larval settlement and metamorphosis of coral planulae?

Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae.     

An inducer of molluscan metamorphosis transforms activity patterns in a larval nervous system

Larvae of the nudibranch mollusc Phestilla sibogae metamorphose in response to a small organic compound released into seawater by their adult prey, the scleractinian coral Porites compressa. The transformations that occur during metamorphosis, including loss of the ciliated velum (swimming organ), evacuation of the shell, and bodily elongation, are thought to be controlled by a combination of neuronal and neuroendocrine activities. Activation of peripheral chemosensory neurons by the metamorphosis-inducing compound should therefore elicit changes within the central nervous system. We used extracellular recording techniques in an attempt to detect responses of neurons within the larval central ganglia to seawater conditioned by P. compressa, to seawater conditioned by the weakly inductive coral Pocillopora damicornis, and to non-inductive seawater controls. The activity patterns within the nervous systems of semi-intact larvae changed in response to both types of coral exudates. Changes took place in two size classes of action potentials, one of which is known to be associated with velar ciliary arrests.     

5-s-Cysteinyl-conjugates of catecholamines induce cell damage,extensive DNA base modification and increases in caspase-3 activity in neurons

A decrease in reduced glutathione levels in dopamine containing nigral cells in Parkinson's disease may result from the formation of cysteinyl-adducts of catecholamines, which in turn exert toxicity on nigral cells. We show that exposure of neurons (CSM 14.1) to 5-S-cysteinyl conjugates of dopamine, L-DOPA, DOPAC or DHMA causes neuronal damage, increases in oxidative DNA base modification and an elevation of caspase-3 activity in cells. Damage to neurons was apparent 12-48 h of post-exposure and there were increases in caspase-3 activity in neurons after 6 h. These changes were paralleled by large increases in pyrimidine and purine base oxidation products, such as 8-OH-guanine suggesting that 5-S-cysteinyl conjugates of catecholamines are capable of diffusing into cells and stimulating the formation of reactive oxygen species (ROS), which may then lead to a mechanism of cell damage involving caspase-3. Indeed, intracellular ROS were observed to rise sharply on exposure to the conjugates. These results suggest one mechanism by which oxidative stress may occur in the substantia nigra in Parkinson's disease.     

Immunohistochemistry of endogenous L-DOPA in the rat posterior hypothalamus

The aim of this work was to study L-DOPA-containing neuronal structures of the rat posterior and dorsal hypothalamus by means of immunohistochemistry using antiserum against glutaraldehyde conjugated L-DOPA. Aspects and distribution of L-DOPA immunoreaction among cells of the supramammillary nucleus and the A11, A13c and A13 cell groups are described and compared to dopamine immunoreactivity, mainly through a double colored labelling procedure employing a color modification of the DAB reaction by metallic ions. Differences between L-DOPA and dopamine stainings within cell groups as the presence of cells with predominant or exclusive L-DOPA coloration are tentatively explained under the light of previous findings using immunohistochemistry of catecholamines synthesizing enzymes and catecholamines histofluorescence.