Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster,Homarus americanus

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A ∼90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 357–373, 1997     

Aspects of the embryology and neural development of the American lobster.

It is feasible to study the anatomical, physiological, and biochemical properties of identifiable neurons in lobster embryos. To exploit fully the advantages of this preparation and to lay the foundation for single-cell studies, our recent goals have been to 1) establish a quantitative staging system for embryos, 2) document in detail the lobster's embryonic development, 3) determine when uniquely identifiable neurons first acquire their transmitter phenotypes, and 4) identify particular neurons that may serve developmental functions. Behavioral, anatomical, morphometric, and immunocytochemical studies have led to a detailed characterization of the growth and maturation of lobster embryos and to the adoption of a percent-staging system based upon the eye index of Perkins (Fish. Bull., 70:95-99, 1972). It is clear from these studies that the lobster nauplius molts at approximately 12% embryonic development (E12%) into a metanauplius, which subsequently undergoes a complete molt cycle within the egg. This molt cycle climaxes with the emergence of the first-stage larva shortly after hatching. Serotonin and proctolin, neurohormones widely distributed in the lobster nervous system, appear at different times in development. Serotonin immunoreactive neurons begin to appear at approximately E10%, with the adult complement being established by E50%. In contrast, proctolin immunoreactive neurons appear later and attain their full complement over a protracted period including larval and juvenile stages. The development of serotonergic deutocerebral neurons and their targets, the olfactory and accessory lobes in the brain, are also examined. The olfactory lobes are forming by E10% and have acquired their glomerular organization by E50%, whereas the formation of the accessory lobes is delayed; the early rudiments of the accessory lobes are seen by E50%, and glomeruli do not form until the second larval stage.     

Serotonin localization in Phallusia mammillata larvae and effects of 5-HT antagonists during larval development

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an important role in a wide range of non-neural processes. Using immunofluorescence with an antiserotonin antibody, 5-HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5-HT on development, we treated embryos with two different 5-HT receptor subtype antagonists. Treatment at the gastrula stage with 10 microM ondansetron, an antagonist of the 5-HT(3) receptor, induced anterior truncation and a short tail. At 10 microM, ritanserin, a 5-HT(2B) receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5-HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5-HT is suggested.     

Some effects of proctolin on the cardiac ganglion of the Maine Lobster, Homarus americanus (Milne Edwards)

The neuropeptide proctolin has excitatory effects on the isolated lobster cardiac ganglion. Selective application to the anterior cell body region produces a dose-dependent (10(-8)--10(-5) M) prolonged depolarization of large anterior cells as well as marked increases in burst frequency and/or duration. In ganglia which have been silenced with tetrodotoxin, proctolin application to anterior cells elicits long-lasting depolarizing responses which are accompanied by a 10-30% increase of the apparent membrane input resistance. Higher proctolin concentrations produce high-frequency trains of driver potentials. It is proposed that a proctolin like peptide may serve a neurohumoral role in the lobster cardiac ganglion and that the anterior motor neurons exhibit endogenous rhythmicity in its presence.     

Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na(+) saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a "borrowed transmitter". Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron-evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron-evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave-form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult.     

The Pre-nervous Serotonergic System of Developing Sea Urchin Embryos and Larvae: Pharmacologic and Immunocytochemical Evidence

Forty serotonin-related neurochemicals were tested on embryos and larvae of Lytechinus variegatus and other sea urchin species. Some of these substances (agonists of 5-HT₁ receptors, antagonists of 5-HT₂, 5-HT₃ or 5-HT₄ receptors, and inhibitors of the serotonin transporter, SERT) perturbed post-blastulation development, eliciting changes in embryonic/larval phenotypes typical for each class of receptor ligand. These developmental malformations were prevented completely or partially by serotonin (5-HT) or 5-HT analogs (5-HTQ, AA-5-HT), providing evidence for the putative localization of cellular targets. Immunoreactive 5-HT, 5-HT receptors and SERT were found in pre-nervous embryos and larvae of both L. variegatus and Strongylocentrotus droebachiensis. During gastrulation, these components of the serotonergic system were localized to the archenteron (primary gut), mesenchyme-like cells, and often the apical ectoderm. These results provide evidence that pre-nervous 5-HT may regulate early events of sea urchin embryogenesis, mediated by 5-HT receptors or the 5-HT transporter.     

Serotonin in the developing stomatogastric system of the lobster,Homarus americanus

We studied the development of the serotonergic modulation of the stomatogastric nervous system of the lobster, Homarus americanus. Although the stomatogastric ganglion (STG) is present early in embryonic development, serotonin immunoreactivity is not visible in the STG until the second larval stage. However, incubation of the STG with exogenous serotonin showed that a serotonin transporter is present in embryonic and early larval stages. Serotonin uptake was blocked by paroxetine and 0% Na⁺ saline. The presence of a serotonin transporter in the embryonic STG suggests that hormonally liberated serotonin could be taken up by the STG, and potentially released as a “borrowed transmitter”. Consistent with a potential hormonal role, serotonin is found in the pericardial organs, a major neurosecretory structure, by midembryonic development. The rhythmic motor patterns produced by embryonic and larval STGs were decreased in frequency by serotonin. Lateral Pyloric (LP) neuron‐evoked excitatory junctional potentials (EJPs) in the embryos and the first larval stage (LI) were larger, slower, and more variable than those in the adult. The amplitude of adult LP neuron‐evoked EJPs was increased more than twofold in serotonin, but in embryos and LI preparations this effect was negligible. In embryos and LI preparations, serotonin increased the occurrence of muscle fiber action potentials and altered the EJP wave‐form. These data demonstrate that serotonin receptors are present in the stomatogastric nervous system early in development, and suggest that the role of serotonin changes from modulation of muscle fiber excitability early in development to enhancement of neurally evoked EJPs in the adult. © 2002 Wiley Periodicals, Inc. J Neurobiol 54: 380–392, 2003     

Immunocytochemical Distribution of 5-HT (Serotonin) in the Nervous System of the Gastrotrich Turbanella cornuta

The 5-HT (serotonin) distribution in the nervous system of the macrodasyoid gastrotrich Turbanella cornuta was studied using immunocytochemical methods. Positive immunoreaction was found in two pairs of neurons. The neurons of one pair had processes which extended peripherally to the surface of the body. Central processes of both pairs entered the brain commissure and proceeded into the longitudinal cords. Unlike other acoelomate worms studied so far, the Platyhelminthes and the Nematoda, in this gastrotrich no 5-HT positive perikarya or processes were present in the pharynx innervation, and no positive neurons sent processes directly to the nerve cords     

Effects of serotonin depletion on local interneurons in the developing olfactory pathway of lobsters

During embryonic life, the growth of the olfactory and accessory lobes of the lobster brain is retarded by serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) (Benton et al., 1997). The local and projection interneurons that synapse with chemosensory cells in the olfactory lobes are potential targets of this depletion. This study documents proliferation and survival in the local interneuron cell clusters, and examines the differentiation of a prominent local interneuron, the serotonergic dorsal giant neuron (DGN), following serotonin depletion. An increase in dye coupling between the DGN and nearby cells is seen after serotonin depletion. However, morphometric analyses of individual DGNs in normal, sham-injected, and 5,7-DHT-treated embryos show that the general morphology and size of the DGNs are not significantly altered by serotonin depletion. Thus, the DGN axonal arbor occupies a greater proportion of the reduced olfactory lobes in the 5,7-DHT-treated embryos than in normal and sham-injected groups. The paired olfactory globular tract neutrophils (OGTNs), where olfactory interneurons synapse onto the DGNs, are 75% smaller in volume than the comparable region in either sham-injected or normal embryos. In vivo experiments using bromodeoxyuridine (BrdU) show that proliferation in the local interneuron soma clusters is reduced by 5,7-DHT treatment and that survival of newly proliferated local interneurons is also compromised. Our data suggest that alterations in the growth of the DGNs do not contribute to the dramatic reduction in size of the olfactory neutrophils following serotonin depletion, but that cell proliferation and survival among the local interneurons are regulated by serotonin during development. Reduced numbers of local interneurons are therefore one likely reason for the growth reduction observed after serotonin depletion.     

Immunohistological localization of serotonin in the CNS and feeding system of the stable fly Stomoxys calcitrans L. (Diptera: Muscidae)

Serotonin, or 5-hydroxytryptamine (5-HT), plays critical roles as a neurotransmitter and neuromodulator that control or modulate many behaviors in insects, such as feeding. Neurons immunoreactive (IR) to 5-HT were detected in the central nervous system (CNS) of the larval and adult stages of the stable fly, Stomoxys calcitrans, using an immunohistological technique. The location and pattern of the 5-HT IR neurons are described and compared for these two different developmental stages. Anatomical features of the fly feeding system were analyzed in third instar larvae and adult flies using a combination of histological and immunohistological techniques. In third instar larvae, the cibarial dilator muscles were observed within the cibarial pump skeleton and innervated by 5-HT IR neurons in nerves arising from the brain. There were four pairs of nerves arising from the frontal surface of the larval brain that innervate the cibarial pump muscles, pharynx, and muscles controlling the mouth hooks. A strong serotoninergic innervation of the anterior stomatogastric system was observed, which suggests 5-HT may play a role in the coordination of different phases of food ingestion by larvae. Similarly, many 5-HT IR neurons were found in both the brain and the thoracico-abdominal ganglia in the adult, some of which innervate the cibarial pump dilator muscles and the stomatogastric muscles. This is tnhe first report describing neuromuscular structures of the stable fly feeding system. The results reported here suggest 5-HT may play a critical role in feeding behaviors of stable fly larvae and adults.     

Dopamine and serotonin modulate the onset of metamorphosis in the ascidian Phallusia mammillata

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 and gastrin/cholecystokinin-like immunorcactive neurons in the larval retrocerebral complex of the blowfly Calliphora erythrocephala

Summary The presence and distribution of neurons immunoreactive against antibodies to serotonin (5-HT) and gastrin/cholecystokinin (gastrin/CCK) has been studied in the larval retrocerebral complex of the blowfly Calliphora erythrocephala, a composite structure which consists of the corpus cardiacum, the corpus allatum, the thoracic gland and a portion of the cephalic aorta. Immunoreactive material was found in all these elements except in the corpus allatum. Six to eight cell bodies in the corpus cardiacum and four to eight cell bodies in the thoracic gland were 5-HT immunoreactive (5-HTi). These 5-HTi cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the cephalic aorta, corpus cardiacum and ventral part of the thoracic gland. Six to eight cell bodies in the corpus cardiacum and four to six cell bodies in the thoracic gland reacted with antibodies against gastrin/CCK. These cell bodies send processes to the neuropil of the corpus cardiacum and to neurohemal sites in the corpus cardiacum and the cephalic aorta in a pattern resembling that of the 5-HTi fibers. Additional gastrin/CCK-like immunoreactive fibers were shown to come from the central nervous system via the two nervi corporis cardiaci. An electron-microscopical analysis was performed to analyze further the morphological features revealed by the light-microscopic immunocytochemical technique. This confirmed the existence of neurosecretory-like terminals among the gland cells of the thoracic glands and the existence of neurohemal sites in several regions of the larval retrocerebral complex. Some functional aspects of the retrocerebral complex are discussed on the basis of the presented data.     

The serotonin 5-HT7Dro receptor is expressed in the brain of Drosophila, and is essential for normal courtship and mating

The 5-HT(7) receptor remains one of the less well characterized serotonin receptors. Although it has been demonstrated to be involved in the regulation of mood, sleep, and circadian rhythms, as well as relaxation of vascular smooth muscles in mammals, the precise mechanisms underlying these functions remain largely unknown. The fruit fly, Drosophila melanogaster, is an attractive model organism to study neuropharmacological, molecular, and behavioral processes that are largely conserved with mammals. Drosophila express a homolog of the mammalian 5-HT(7) receptor, as well as homologs for the mammalian 5-HT(1A), and 5-HT(2), receptors. Each fly receptor couples to the same effector pathway as their mammalian counterpart and have been demonstrated to mediate similar behavioral responses. Here, we report on the expression and function of the 5-HT(7)Dro receptor in Drosophila. In the larval central nervous system, expression is detected postsynaptically in discreet cells and neuronal circuits. In the adult brain there is strong expression in all large-field R neurons that innervate the ellipsoid body, as well as in a small group of cells that cluster with the PDF-positive LNvs neurons that mediate circadian activity. Following both pharmacological and genetic approaches, we have found that 5-HT(7)Dro activity is essential for normal courtship and mating behaviors in the fly, where it appears to mediate levels of interest in both males and females. This is the first reported evidence of direct involvement of a particular serotonin receptor subtype in courtship and mating in the fly.     

Primary afferent responses of a crustacean mechanoreceptor are modulated by proctolin, octopamine, and serotonin

Modulation of sensory responses recorded intracellularly in primary sensory afferents of a crustacean proprioceptor is described. The neuropeptide proctolin enhances the sensory response, whereas the bioamines octopamine and serotonin depress it. The lobster oval organ of the second maxilla, a simple stretch receptor lacking centrifugal control, provides a useful model for studies on nonsynaptic modulation at peripheral sensory loci. Its three large afferents, X, Y, and Z, were prepared for intracellular recording and tested under five experimental conditions: (1) when fully rested, (2) when adapted to maintained stretch and firing tonically, (3) when showing reduced responses after habituation to repetitive stimulation, (4) not stretched but depolarized with current injections, (5) after TTX blockade. The results, taken together, indicate that conductances contributing to the overall amplitude of the receptor potential are major targets for modulators. Thus proctolin increased receptor potential amplitudes with consequent augmentation of spiking, whereas serotonin and octopamine depressed the receptor potentials, often to subthreshold levels with loss of spiking. Octopamine was a less potent agent than serotonin and failed to act upon fibers under TTX blockade. Fibers Y and Z consistently showed sensitivity to the modulators tested. The largest fiber, X, typically was resistant to proctolin, octopamine, and serotonin. Threshold concentrations of 10(-10)-10(-11) M determined in vitro are well below the circulating levels for serotonin and octopamine found in vivo. Proctolin, however, is usually not detectable in the hemolymph, and it is suggested that a significant site of proctolin release may be the oval organ itself.     

Local serotonergic signaling in mammalian follicles, oocytes and early embryos

The involvement of neurotransmitters in mammalian female reproductive tissues has been the object of several studies in past decades. This review focuses on new evidence that serotonin (or 5-hydroxytryptamine, 5-HT) may be an important key player, acting locally in mammalian ovaries and female genital tracts where it may influence granulosa and cumulus cells as well as oocytes and early embryos. Pioneering studies reporting 5-HT in ovaries and other female reproductive tissues and cells are now complemented by the identification of specific 5-HT receptor subtypes (5-HT(1D), 5-HT(2A-B) and 5-HT(7)) in granulosa or cumulus cells, oocytes and early embryos. Additional serotonergic players, including the 5-HT transporter (SERT or Slc6A4) expressed in oocytes and embryos, and the 5-HT-producing enzyme tryptophan hydroxylase-1 (TPH1) expressed in cumulus cells, now make up a complete and autonomous local serotonergic network. Direct demonstrations of intracellular Ca(2+) and cAMP signaling by 5-HT in cumulus cells and its capacity to regulate progesterone secretion by granulosa cells further illustrate some of its potential functions in ovarian physiology. Recent evidence shows that mouse mothers with knocked-out TPH1 have embryos with impaired early development, establishing that maternal 5-HT is required for normal embryonic development. This local regulation of reproductive processes by 5-HT in mammals might have derived from better-known, and possibly ancestral, serotonergic networks similarly at play in several primitive animals, and potential implications for human reproduction may also be foreseen. Specific roles played by 5-HT in mammalian reproduction remain to be further investigated, and now span from steroidogenesis and oocyte maturation to early embryonic development.     

A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns.

Biogenic amines such as serotonin elicit or modulate a wide range of behaviours by interacting with multiple receptor subtypes. We have isolated cDNA clones encoding three distinct Drosophila serotonin receptors which belong to the G protein-coupled receptor family. When expressed in mammalian cells, these receptors activate different intracellular effector systems. The 5HT-dro1 receptor stimulates adenylate cyclase while the 5HT-dro2A and the 5HT-dro2B receptors inhibit adenylate cyclase and activate phospholipase C. Expression of all three receptors starts in late embryos and is restricted to distinct populations of cells in the central nervous system. The 5HT-dro2A receptor is predominantly expressed in midline motor neurons (VUM neurons) that innervate larval muscles thus suggesting a role for this receptor in motor control.     

Serotonin localization and its functional significance during mouse preimplantation embryo development

Serotonin is a neurotransmitter functioning also as a hormone and growth factor. To further investigate the biological role of serotonin during embryo development, we analysed serotonin localization as well as the expression of specific serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos. The functional significance of serotonin during the preimplantation period was examined by studying the effects of serotonin on mouse embryo development. Embryo exposure to serotonin (1 microM) highly significantly reduced the mean cell number, whereas lower concentrations of serotonin (0.1 microM and 0.01 microM) had no significant effects on embryo cell numbers. In all serotonin-treated groups a significant increase in the number of embryos with apoptotic and secondary necrotic nuclei was observed. Expression of serotonin 5-HT1D receptor mRNA in mouse oocytes and preimplantation embryos was confirmed by in situ hybridization showing a clearly distinct punctate signal. Immunocytochemistry results revealed the localization of serotonin in oocytes and embryos to the blastocyst stage as diffuse punctate cytoplasmic labelling. It appears that endogenous and/or exogenous serotonin in preimplantation embryos could be involved in complex autocrine/paracrine regulations of embryo development and embryo-maternal interactions.