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SummaryThe presence of serotonergic system during early pre-neural development is enigmatic and conserved amongst all studied invertebrate and vertebrate animals. We took advantage of zebrafish model system to address what is the role of early serotonin before first neurons form. Unexpectedly, we experimentally revealed the existence of delayed developmental neurogenic and behavioral effects resulting from the manipulations of pre-neural (zygote, blastula and gastrula) serotonergic system. In particular, the delayed effects included differences in the synthesis of serotonin in early serotonergic neurons in the central nervous system as well as in behavioral alterations after habituation in zebrafish larvae. These effects appeared as highly specific and did not coincide with any major abnormalities. The same manipulations of the serotonergic system at neural developmental stages did not show such effects, which confirms that early effects of serotonergic system manipulation are not based on retained serotonin in embryonic cells. Accordingly, gene expression analysis demonstrated specific changes only in response to the elevation of early pre-neural serotonin, which included the delayed and pre-mature onsets of different gene expression programs. Taken together, our results introduce a novel function of early pre-neural serotonergic system in a vertebrate embryo – tuning and fine control of specific mechanisms at later neural developmental stages that result in a mild variation of a behavioral adaptive spectrum.

Serotonin (5-HT) is a key player in many physiological processes in both the adult organism and developing embryo. One of the mechanisms for 5-HT-mediated effects is covalent binding of 5-HT to the target proteins catalyzed by transglutaminases (serotonylation). Despite the implication in a variety of physiological processes, the involvement of serotonylation in embryonic development remains unclear. Here we tested the hypothesis that 5-HT serves as a substrate for transglutaminase-mediated transamidation of the nuclear proteins in the early embryos of both vertebrates and invertebrates. For this, we demonstrated that the level of serotonin immunoreactivity (5-HT-ir) in cell nuclei increases upon the elevation of 5-HT concentration in embryos of sea urchins, mollusks, and teleost fish. Consistently, pharmacological inhibition of transglutaminase activity resulted in the reduction of both brightness and nuclear localization of anti-5-HT staining. We identified specific and bright 5-HT-ir within nuclei attributed to a subset of different cell types: ectodermal and endodermal, macro- and micromeres, and blastoderm. Western blot and dot blot confirmed the presence of 5-HT-ir epitopes in the normal embryos of all the species examined. The experimental elevation of 5-HT level led to the enhancement of 5-HT-ir-related signal on blots in a species-specific manner. The obtained results demonstrate that 5-HT is involved in transglutaminase-dependent monoaminylation of nuclear proteins and suggest nuclear serotonylation as a possible regulatory mechanism during early embryonic development. The results reveal that this pathway is conserved in the development of both vertebrates and invertebrates.

Hatching is an important phase of the development of pulmonate gastropods followed by the adult-like extracapsular foraging life. Right before hatching the juveniles start to display a rhythmic radula movement, executed by the buccal complex, consisting of the buccal musculature (mass) and a pair of the buccal ganglia. In order to have a detailed insight into this process, we investigated the serotonergic regulation of the buccal (feeding) rhythm in 100% stage embryos of the pond snail, Lymnaea stagnalis, applying quantitative immunohistochemistry combined with the pharmacological manipulation of the serotonin (5-HT) synthesis, by either stimulating (by the 5-HT precursor 5-hydroxytryptophan, 5-HTP) or inhibiting (by the 5-HT synthesis blocker para-chlorophenylalanine, pCPA) it. Corresponding to the direction of the drug effect, significant changes of the fluorescence intensity could be detected both in the cerebral ganglia and the buccal complex. HPLC-MS assay demonstrated that 5-HTP increased meanwhile pCPA decreased the 5-HT content both of the central ganglia and the buccal complex. As to the feeding activity, 5-HTP induced only a slight (20%) increase, whereas the pCPA resulted in a 20% decrease of the radula protrusion frequency. Inhibition of 5-HT re-uptake by clomipramine reduced the frequency by 75%. The results prove the role of both central and peripheral 5-HTergic processes in the regulation of feeding activity. Application of specific receptor agonists and antagonists revealed that activation of a 5-HT1-like receptor depressed the feeding activity, meanwhile activation of a 5-HT6,7-like receptor enhanced it. Saturation binding plot of [3H]-5-HT to receptor and binding experiments performed on membrane pellets prepared from the buccal mass indicated the presence of a 5-HT6-like receptor positively coupled to cAMP. The results suggest that 5-HT influences the buccal (feeding) rhythmic activity in two ways: an inhibitory action is probably exerted via 5-HT1-like receptors, while an excitatory action is realized through 5-HT6,7-like receptors.

Serotonin (5.HT) is known to be functionally active during early development in both vertebrates and invertebrates. However, the presence of 5-HT and its synthesis and transport system has not yet been demonstrated in bivalve early development. The presence of 5-HT was immunochemically demonstrated at the cleavage stage of bivalve Mytilus trossulus. 5-HT level dramatically increased within all embryonic cells after incubation with 5-HTP but not after incubation with tryptophan and 5-HT. The first 5-HT uptake by specific transporter was detected at 13 hpf blastula stage only and it was restricted to one distinct cell.

We investigated involvement of different 5-HT receptors in regulation of ciliary rotation, gliding locomotion and heartbeat of Helisoma embryo at pre- and post-metamorphic stages. Pharmacological analysis suggested that activation of 5-HT1 receptor enhance ciliary rotation but do not affect gliding locomotion. Activation of 5-HT4 receptor depresses both types of locomotion. Before metamorphosis heart contraction is depressed by activation of 5-HT4 and enhanced by activation of 5-HT7 receptor. However, the heart became insensitive to all agonists by hatching. We hypothesized that alterations in affinity or expression of particular 5-HT receptors can underlie the well-coordinated character of serotonin-dependent larval behavior.

Serotonin (5-HT) is known to induce a wide range of short-term and long-term (or delayed) effects. In the present paper we demonstrated that short time-window application of the 5-HT precursor 5-hydroxytryptophan during early cleavage stages results in both irreversible morphological malformation (exogastrulation) and distinct changes in behavior of young animals of the freshwater snail, Lymnaea stagnalis (Mollusca: Gastropoda). Pharmacological and immunocytochemical analysis confirmed that both the increase of intracellular 5-HT level within the cleaved blastomers and activation of membrane 5-HT2-like type receptors are required for the appearence of these phenomena.

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.

Freshwater pond snails Helisoma trivolvis and Lymnaea stagnalis undergo larval development and metamorphosis inside egg capsules. We report that their development is permanently under slight tonic inhibitory influence of the anterior sensory monoaminergic neurones, which are the remnants of the apical sensory organ. Conspecific juvenile snails, when reared under conditions of starvation and crowding, release chemical signals that are detected by these neurones in encapsulated larvae and reversibly suppress larval development, thus providing a link between environmental signals and developmental regulation. Induced retardation starts from the trochophore stage and results in up to twofold prolongation of the larval lifespan. Upon stimulation with the signal, the neurones increase synthesis and release of monoamines [serotonin (5-HT) in Helisoma and dopamine in Lymnaea] that inhibit larval development acting via ergometrine-sensitive internal receptors. Thus, the novel regulatory mechanism in larval development of molluscs is suggested and compared with the phenomenon of dauer larvae formation in the nematode Caenorhabditis elegans.

SummaryMany organisms survive in constantly changing environments, including cycling seasons. Developing embryos show remarkable instant adaptations to the variable environmental challenges they encounter during their adult life, despite having no direct contact with the changing environment until after birth or hatching. The mechanisms by which such non-genetic information is transferred to the developing embryos are largely unknown. Here, we address this question by using a freshwater pond snail (Lymnaea stagnalis) as a model system. This snail normally lives in a seasonal climate, and the seasons define its locomotion, feeding, and reproductive behavior. We discovered that the serotonergic system plays a crucial role in transmitting a non-genetic instructive signal from mother to progeny. This maternal serotonin-based signal functions in embryos during a short time window at exclusively early pre-neural developmental stages and modulates the dynamics of embryonic and juvenile growth, feeding behavior, and locomotion.

BACKGROUND: Serotonin (5-HT) is well known as widely distributed modulator of developmental processes in both vertebrates and invertebrates. It is also the earliest neurotransmitter to appear during neuronal development. In aquatic invertebrates, which have larvae in their life cycle, 5-HT is involved in regulation of stages transition including larval metamorphosis and settlement. However, molecular and cellular mechanisms underlying developmental transition in aquatic invertebrate species are yet poorly understood. Earlier we demonstrated that in larvae of freshwater molluscs and marine polychaetes, endogenous 5-HT released from the neurons of the apical sensory organ (ASO) in response to external stimuli retarded larval development at premetamorphic stages, and accelerated it at metamorphic stages. Here we used a freshwater snail Helisoma trivolvis to study molecular mechanisms underlying these dual developmental effects of 5-HT. RESULTS: Larval development of H. trivolvis includes transition from premetamorphic to metamorphic stages and shares the main features of metamorphosis with free-swimming aquatic larvae. Three types of 5-HT receptors (5-HT1-, 5-HT4- and 5-HT7-like) are functionally active at premetamorphic (trochophore, veliger) and metamorphic (veliconcha) stages, and expression patterns of these receptors and respective G proteins undergo coordinated changes during development. Stimulation of these receptors modulated cAMP-dependent regulation of cell divisions. Expression of 5-HT4- and 5-HT7-like receptors and their downstream Gs protein was down-regulated during the transition of pre- to metamorphic stage, while expression of 5-HT1 -like receptor and its downstream Gi protein was upregulated. In accordance with relative amount of these receptors, stimulation of 5-HTRs at premetamorphic stages induces developmental retardation, while their stimulation at metamorphic stages induces developmental acceleration. CONCLUSIONS: We present a novel molecular mechanism that underlies stage-specific changes in developmental tempo of H. trivolvis larvae in response to endogenous 5-HT produced by the neurons of the ASO. We suggest that consecutive changes in expression patterns of different receptors and their downstream partners in the course of larval development represent the molecular base of larval transition from premetamorphic (non-competent) to metamorphic (competent) state. peerReviewed

1. 1. Prolonged exposure of the pond snail Lymnaea stagnalis to micromolar concentrations of chlorpromazine (CPZ) results in marked changes in the serotonin (5-HT) content of the central nervous system. 2. 2. High-performance liquid chromatography with electrochemical detection indicates that levels of 5-HT, but not those of dihydroxyphenyl-alanine (DOPA), dopamine or norepinephrine, were significantly decreased (e.g., to less than 40% of normal after 30 days of exposure to 1 μM CPZ in the bathing water). 3. 3. Glyoxylate-induced fluorescence was depressed to undetectable levels in central, serotonergic neurons. 4. 4. Performance of 5-HT-dependent motor behaviors was impaired. 5. 5. The present results, in accord with earlier studies on the effects of chronic exposure to haloperidol, suggest that previously overlooked mechanisms of monoamine downregulation may contribute to long-term effects of antipsychotic drugs.

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.

Serotonin (5.HT) is known to be functionally active during early development in both vertebrates and invertebrates. However, the presence of 5-HT and its synthesis and transport system has not yet been demonstrated in bivalve early development. The presence of 5-HT was immunochemically demonstrated at the cleavage stage of bivalve Mytilus trossulus. 5-HT level dramatically increased within all embryonic cells after incubation with 5-HTP but not after incubation with tryptophan and 5-HT. The first 5-HT uptake by specific transporter was detected at 13 hpf blastula stage only and it was restricted to one distinct cell.

We investigated involvement of different 5-HT receptors in regulation of ciliary rotation, gliding locomotion and heartbeat of Helisoma embryo at pre- and post-metamorphic stages. Pharmacological analysis suggested that activation of 5-HT1 receptor enhance ciliary rotation but do not affect gliding locomotion. Activation of 5-HT4 receptor depresses both types of locomotion. Before metamorphosis heart contraction is depressed by activation of 5-HT4 and enhanced by activation of 5-HT7 receptor. However, the heart became insensitive to all agonists by hatching. We hypothesized that alterations in affinity or expression of particular 5-HT receptors can underlie the well-coordinated character of serotonin-dependent larval behavior.

The regulation of larval development by starved adults occurs in both freshwater snails, Helisoma trivolvis and marine polychaetes, Platynereis dumerilii. Serotonin (5-HT) links this environmental signal which is detected by larval apical sensory neurons to changes in larval development. A profile of the stage-dependent expression of 5-HT receptors and coupled G proteins is essential in this regulatory mechanism. The final effect on development depends on the modulation of the activity of the larval digestive system.