Expression and function of the neurotransmitter serotonin during development of the Helisoma nervous system

Exogenous serotonin has been shown to evoke a neuron-selective inhibition of neurite outgrowth and synaptogenesis in identified Helisoma neurons in vitro. We demonstrate here that serotonin is present in the embryonic nervous system of Helisoma and can act as a regulator of neuronal development in vivo. Serotonin-like immunoreactivity was first observed in neurons at an early stage of nervous system development (E20). Throughout embryogenesis, the number of serotonin-immunoreactive neurons increased in a stereotypic pattern that was unique for each type of ganglion. Strikingly, the number of serotonin-immunoreactive neurons continued to increase throughout adult life. Transient perturbation of endogenous serotonin levels during embryogenesis had profound effects on the development of specific identified neurons. Embryos treated with 5,7-dihydroxytryptamine and raised to maturity showed aberrations in neuronal morphology, neuronal dye coupling, and strength of electrical synaptic connections. These effects were restricted to neurons known to be sensitive to the growth-inhibitory effects of serotonin in vitro. These results support the hypothesis that neurotransmitters are an important class of regulatory factors during normal development of the nervous system.     

Motor effects of serotonin in the central nervous system

Serotoneric pathways in the CNS affect posture and movement, as well as behavioral responses to arousing stimuli. Pharmacologic analysis of these effects has led to an increasingly complex and confusing literature. Increased availability of serotonin (5-HT) by administration of precursors, or by its direct intracranial infusion can induce inhibitory or excitatory behavioral effects depending on the conditions of the experiment, although generally motor inhibition has been found. Availability of 5-HT has been decreased by electrolytic lesions of the raphe nuclei, inhibition of tryptophan hydroxylase, or use of selective neurotoxins. These treatments have generally increased motor activity, especially spontaneous locomotion in a familiar environment, as well as sexual or aggressive behaviors; other behaviors, such as responses in a novel environment, presumably associated with curiosity or fear, have been paradoxically decreased after loss of 5-HT. This differentiation may occur to an important extent through 5-HT projections to hippocampus and limbic structures, notably from the median raphe. Increases or decreases of brain 5-HT, respectively, generally tend to decrease and increase responses to catecholamine agonists such as amphetamines, and some effects of 5-HT may be mediated through catecholaminergic systems. Increased availability of 5-HT in the presence of MAO inhibitors, or challenge with 5-HT-agonists after selective 5-HT-denervation in the CNS has led to a behavioral syndrome marked by hyperactivity, autonomic arousal and myoclonic seizures. The mechanisms underlying this complex response may be mediated by descending 5-HT systems that result in motor excitation at the spinal cord level, in contrast to rostral projections to forebrain that may mediate many behaviorally inhibitory responses.     

Neurosecretion of the mediodorsal cells of the central nervous system of the snail Helisoma duryi

Summary The neurosecretory mediodorsal cells that produce a putative growth hormone of the snail Helisoma duryi were studied in fast-growing virgin snails and in slow-growing reproducing snails. There are about 60 mediodorsal cells in clusters on each side of the cerebral commissure of the central nervous system, and they contain dense-cored granules which are 100–200 nm in diameter. The cells of virgin snails have dense Golgi bodies, scattered ER cisternae, and few granules, while those of reproducing snails have pale Golgi bodies, stacked ER cisternae, and numerous granules. Thus the mediodorsal cells of the virgin snails appear to be more active synthetically than those of the reproducing snails. The cells near the endocrine dorsal bodies contain many dorsal body precesses in their membrane interdigitations. There are junction-like interactions between some of the interdigitations. Gap junction-like contacts are seen between mediodorsal cells and glial cells. The axon endings of the mediodorsal cells at the neurohemal area in the labial nerve show more release profiles in virgin snails than in reproducing snails. A daily pattern of release has been observed in reproducing snails, and rates of release are higher in the evening than in the morning.     

Characterization and development of rotational behavior in Helisoma embryos: Role of endogenous serotonin

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Effect of serotonin and acetylcholine on neurons in the central nervous system of snails

Differences in the distribution of neurons distinguished by their responses to serotonin and acetylcholine were found in the central nervous system ofHelix pomatia. When applied to the body of the neuron acetylcholine hyperpolarizes the cell more often than it depolarizes it, but depolarization predominates in some regions, e.g., on the dorsal surface of the visceral ganglion. In most cases serotonin stimulates activity and induces depolarization or the appearance of pacemaker oscillations of membrane potential. The oscillogenic effect of serotonin is characteristic, in particular, of white (peptidergic) neurons and the depolarization effect is characteristic of other neurons, including the paired giant metacerebral neurons which contain serotonin in their cytoplasm. Both effects failed to appear in sodium-free solution. A group of neurons in which hyperpolarization was observed in response to serotonin application was found in the visceral ganglion of hibernating snails. The same cells in active snails were stimulated by serotonin. A giant neuron with two variously located cholinergic structures is present on the ventral surface of the ganglion among this group of cells: acetylcholine hyperpolarized it when applied to the cell body but depolarized it when applied to the axon.     

Behavioral correlates of serotonin depletion.

Depletion of telencephalic serotonin (5-HT) content by medical forebrain bundle lesions, which interrupt the ascending serotonergic pathways or by DL-p-chlorophenylalanine produces an increased sensitivity to pain as measured by the flinch-jump, stabilimetric, or hot-plate methods. Examination of the effects of a number of other lesions and drugs indicated that dopamine, norepinephrine and acetylcholine are not involved in pain sensitivity. Dosages of 75 mg/kg DL-5-hydroxytryptophan(5-HTP), 37.5 mg/kg L-5-HTP or 50 mg/kg Ro 4-4602 (NI-(DL-seryl)-N2-(2,3,4-trihydroxybenzyl)hydrazine) plus 37.5 mg/kg L-5-HTP administered to medical forebrain bundle lesioned rats returned both the telencephalic content of 5-HT and the pain threshold to normal values. Injection of 37.5 mg/kg of D-5-HTP or an equimolar dose of L-dopa had no effect on pain threshold. Normal animals display increased sensitivity to pain and decreased 5-HT contents in frontal pole, hippocampus, and amygdala during dark as compared to light hours. All three of these telencephalic areas are innervated by the ascending serotonergic pathways, and cells in these areas show inhibition of firing following the iontophoretic application of 5-HT. Taken together these data suggest that the serotonergic system normally acts to inhibit the effects of painful stimuli. A review of a variety of behavioral effects of 5-HT depletion including an enhanced response to lysergic acid diethylamide and amphetamine suggests that the ascending serotonergic system may have a general role in the inhibition of arousal, rather than a specific role with respect to various categories of behavior.     

Transient expression of NOS-II during development of the murine enteric nervous system

In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca²⁺ -dependent and Ca²⁺ -independent NOS isoforms.     

Pharmacological manipulation of serotonin levels in the nervous system of the opisthobranch mollusc Tritonia diomedea

Serotonin-related disorders can be treated by manipulating serotonin synthesis with the serotonin precursor 5-hydroxytryptophan (5-HTP) or other pharmacological agents. The mollusc Tritonia diomedea is a model for investigating the effects of altering serotonin content on the functions of identified neurons. We used high-performance liquid chromatography and immunohistochemistry to examine the amount and localization of 5-HTP, serotonin, and the serotonin breakdown product 5-hydroxyindolacetic acid (5-HIAA) in the Tritonia brain after various pharmacological treatments. Exposure to 5-HTP (2 mM for 30 min-1 h) caused an immediate and massive increase in total 5-HTP content, which lasted more than 20 h, and the widespread appearance of 5-HTP immunoreactivity in neurons. Serotonin levels rose gradually, but only a restricted number of additional neurons displayed serotonin immunoreactivity. 5-HTP treatment also caused an increase in the total amount of 5-HIAA and the appearance of 5-HIAA immunoreactivity throughout the brain. Treatment with the synthesis cofactor tetrahydrobiopterin, the initial precursor tryptophan, or serotonin itself had no persistent effect on total serotonin content. The amino acid decarboxylase inhibitor hydroxybenzylhydrazine (NSD-1015) also had no effect on the total serotonin content, although it caused an accumulation of 5-HTP. Thus, serotonin levels in the brain of T. diomedea appear to be maintained by a homeostatic mechanism that can be disrupted by 5-HTP.     

Development and sensitivity to serotonin of Drosophila serotonergic varicosities in the central nervous system

Serotonin is a classical small-molecule neurotransmitter with known effects on developmental processes. Previous studies have shown a developmental role for serotonin in the fly peripheral nervous system. In this study, we show that serotonin can modulate the development of serotonergic varicosities within the fly central nervous system. We have developed a system to examine the development of serotonergic varicosities in the larval CNS. We use this method to describe the normal serotonergic development in the A7 abdominal ganglion. From first to third instar larvae, the volume of the neuropil and number of serotonergic varicosities increase substantially while the varicosity density remains relatively constant. We hypothesize that serotonin is an autoregulator for serotonergic varicosity density. We tested the sensitivity of serotonergic varicosities to serotonin by adding neurotransmitter at various stages to isolated larval ventral nerve cords. Addition of excess exogenous serotonin decreases native varicosity density in older larvae, and these acute effects are reversible. The effects of serotonin appear to be selective for serotonergic varicosities, as dopaminergic and corazonergic varicosities remain qualitatively intact following serotonin application.     

Acute tyrosine depletion reduces tyrosine hydroxylation rate in rat central nervous system

An amino acid cocktail was devised that would rapidly reduce central nervous system (CNS) tyrosine levels in rats following gastric intubation. The effect of this treatment on in vivo tyrosine hydroxylation rate was examined. Serum tyrosine (TYR) levels, the serum ratio of TYR to the sum of its transport competitors, and CNS TYR concentrations fell substantially within 60 minutes of intubation and remained low for at least 3 hr. In vivo tyrosine hydroxylation rate, evaluated in hypothalamus and retina 2 hr after amino acid intubation, also declined significantly. The results suggest that an amino acid mixture can be devised that will cause an acute reduction in TYR levels and hydroxylation rate in rat CNS. This procedure may ultimately prove applicable to humans to examine functional consequences in particular CNS regions of reducing neuronal catecholamine synthesis.     

Effect of carbidine on the dopamine and serotonin receptors of the central nervous system

It has been shown in experiments on mice and rats that unlike haloperidol and sulpiride, carbidine does not influence the intensity and even increases the duration of apomorphine stereotypy. However, similarly to haloperidol carbidine decreases head twitches in mice, induced by administration of 5-hydroxytryptophan. In radioligand-binding experiments in vitro and in vivo carbidine displaced 3H-spiperone but in the brain cortex without having any effect on the binding in the striatum. Carbidine did not affect the dopamine-sensitive adenylate cyclase in rat striatum. Based on these data it is suggested that in contrast to haloperidol and sulpiride, carbidine selectively inhibits serotonin receptors of the brain.     

Immunocytochemical localization of FMRFamide in the central nervous system and the kidney of Helisoma duryi (Mollusca): its possible antidiuretic role

The distribution of FMRFamide-immunoreactive neurons in the central nervous system of the freshwater pulmonate, Helisoma duryi is described. All parts of the central nervous system except the two pleural and the right parietal ganglia, contain immunoreactive neurons. By immunogold techniques, only one kind of neurosecretory FMRFamide-immunoreactive cell (previously identified as the type-3 cell) was localized in the visceral and left parietal ganglia. This cell type has been previously implicated in an antidiuretic role. FMRFamide-immunoreactive material is found in the whole mount of the kidney as well as in kidney sections. Electron microscopic examination shows that the axons innervating either the smooth muscles of the kidney or the kidney itself contain neurosecretory granules morphologically similar to type-3 cells of the visceral and left parietal ganglia. When incubated in saline containing nanogram quantities of FMRFamide, the wet weight of the kidney increased. It is suggested that FMRFamide-like substance may function as an antidiuretic factor and that the kidney is a target organ of this peptide for osmoregulation.     

Alterations of the serotonin level in the central nervous system of Locusta migratoria during larval-adult transformation

Serotonin level was measured in the cerebral, suboesophageal, thoracic and abdominal ganglia of Locusta migratoria during larval-adult transformation. It has been established that the serotonin content shows a progressive increase in the period between the last larval stage and the first 7 days of adult life, being of the highest value in the abdominal ganglia (146%), followed by the thoracic (110%), and then by the cerebral and suboesophageal (75%) ganglia. No significant changes are manifested in the serotonin level in the course of moulting. Considerable increase is also detectable in the protein content of the ganglia during the studied development phase. No correlation is present between the protein and serotonin contents of the ganglia, the changes of the protein content was found to be faster as compared to that of serotonin.     

Novel effects of serotonin on neurite outgrowth in neurons cultured from embryos of Helisoma trivolvis

The neurotransmitter serotonin has been shown to inhibit neurite outgrowth in specific identified neurons isolated from adult Helisoma. While in vivo experiments on Helisoma embryos have supported the hypothesis that endogenous serotonin regulates neurite outgrowth during embryonic development, direct effects of serotonin on embryonic neurons have not been measured. In the present study, cultures of dissociated embryonic neurons were used to test the direct actions of serotonin on developing embryonic neurons. Serotonin arrested neurite outgrowth in a significant percentage of elongating neurites in a dose-dependent manner. Furthermore, analysis of neurons with stable, nonelongating neurites revealed a novel response. Serotonin caused the reinitiation of neurite outgrowth in a significant percentage of nonelongating neurites. The arrestment of outgrowth and reinitiation of outgrowth occurred in similar percentages of elongating and nonelongating neurites, respectively. Parallel experiments on cultures of dissociated adult neurons were carried out to determine whether serotonin could also induce both inhibitory and stimulatory responses in adult cells. Serotonin arrested neurite outgrowth in a similar percentage of neurites to that observed in cultures of embryonic neurons. In contrast, serotonin did not reinitiate neurite outgrowth in a significant percentage of adult neurites. These data support the hypothesis that serotonin regulates neurite outgrowth in developing embryonic neurons. Furthermore, only some of these regulatory effects appear to be conserved from embryonic to adult neurons.     

SALMFamide2 and serotonin immunoreactivity in the nervous system of some acoels (Xenacoelomorpha)

Acoel worms are simple, often microscopic animals with direct development, a multiciliated epidermis, a statocyst, and a digestive parenchyma instead of a gut epithelium. Morphological characters of acoels have been notoriously difficult to interpret due to their relative scarcity. The nervous system is one of the most accessible and widely used comparative features in acoels, which have a so‐called commissural brain without capsule and several major longitudinal neurite bundles. Here, we use the selective binding properties of a neuropeptide antibody raised in echinoderms (SALMFamide2, or S2), and a commercial antibody against serotonin (5‐HT) to provide additional characters of the acoel nervous system. We have prepared whole‐mount immunofluorescent stainings of three acoel species: Symsagittifera psammophila (Convolutidae), Aphanostoma pisae, and the model acoel Isodiametra pulchra (both Isodiametridae). The commissural brain of all three acoels is delimited anteriorly by the ventral anterior commissure, and posteriorly by the dorsal posterior commissure. The dorsal anterior commissure is situated between the ventral anterior commissure and the dorsal posterior commissure, while the statocyst lies between dorsal anterior and dorsal posterior commissure. S2 and serotonin do not co‐localise, and they follow similar patterns to each other within an animal. In particular, S2, but not 5‐HT, stains a prominent commissure posterior to the main (dorsal) posterior commissure. We have for the first time observed a closed posterior loop of the main neurite bundles in S. psammophila for both the amidergic and the serotonergic nervous system. In I. pulchra, the lateral neurite bundles also form a posterior loop in our serotonergic nervous system stainings.     

Regulation of serotonin receptor function in the nervous system by lipid rafts and adaptor proteins

5-HT is a phylogenetically conserved monoaminergic neurotransmitter which is crucial for a number of physiological processes and is dysregulated in several disease states including depression, anxiety and schizophrenia. 5-HT neurons in the central nervous system are localized in the raphe nuclei and project to a wide range of target areas. 5-HT exerts its functions through 14 subtypes of 5-HT receptors. The tertiary structures of seven transmembrane 5-HT receptors contain several important features, including cholesterol consensus motifs, prominent intracellular loops and free C-termini. Alterations of cholesterol levels affect binding of ligands to 5-HT receptors and cholesterol-enriched microdomains in the cell membrane, termed lipid rafts, regulate 5-HT receptor internalization and signaling. The intracellular loops and the C-termini of 5-HT receptors provide binding sites for interacting adaptor proteins. Adaptor proteins affect internalization, desensitization as well as G-protein dependent and independent signaling via 5-HT receptors. We will here briefly review recent progress on the role of lipid rafts and adaptor proteins in the regulation of localization, trafficking, signaling and ligand bias of 5-HT receptors.     

Pharmacological characterization of a serotonin receptor involved in an early embryonic behavior of Helisoma trivolvis

In contrast to the abundance of information on the many physiological and developmental actions of serotonin in molluscan nervous systems, comparatively little is known about the serotonin receptors involved in these responses. Embryos of the pulmonate gastropod, Helisoma trivolvis, display a cilia-driven rotational behavior that is regulated by endogenous serotonin. In the present study, two functional assays were used to determine some of the pharmacological properties of the receptors that mediate the cilio-excitatory action of serotonin. Timelaspe video microscopy was used to measure whole embryo rotation rat and cilia beat frequency in isolated cells. In dose-response experiments, serotonin was approximately 10 times more potent in stimulating cilia beat frequency over embryo rotation. In rotation experiments, 5-carboxyamidotryptamine and methysergide had effective agonist activity in dose ranges similar to that of serotonin (1 to 100 μM). In contrast, 8-hydroxydiproylaminotetralin HBr (8-OH-DPAT) displayed agonist activity of lower potency and effectiveness. Several compounds displayed antagonist activity in the 1 to 100 μM dose range, including mianserin, spiperone, ritanserin, 1-(1-naphthyl) piperazine, and Propranolol. α-Methylserotonin had mixed agonist–antagonist activity, and metoclopramide, MDL-72222, and ketanserin were inactive. Experiments on isolated cells suggested that the extremely effective antagonism displayed by mianserin in the embryo rotation assay was due to its specific activity at ciliary serotonin receptors. These results implicate the presence of a novel serotonin receptor on embryonic ciliated cells that is pharmacologically distinct from those previously characterized in vertebrate or invertebrate systems. 1994 John Wiley & Sons, Inc.     

Characterization and development of rotational behavior in Helisoma embryos: role of endogenous serotonin.

Cilia-driven rotational behavior displayed by embryos of the pond snail Helisoma trivolvis was characterized in terms of its behavioral subcomponents, developmental changes, and response to exogenous serotonin. Rotation was found to be a complex behavior characterized by four parameters; rotational direction, rotation rate, rotational surges, and periods of inactivity. These parameters all exhibited characteristic developmental changes from embryonic stage E15 through stage E30. Notably, both rotation rate and frequency of rotational surges increased from stage E15 to E25 and declined to an intermediate level by stage E30. It appeared that the developmental increase in overall rotation rate was caused primarily by an increase in surge frequency, rather than an increase in the rate of nonsurge rotation. Immersion of embryos inserotonin-containing pond water resulted in a dose-dependent, reversible increase in rotation rate as well as a dose-dependent, reversible decrease in surge frequency. The serotonin antagonist, mianserin, abolished the excitatory effect of exogenous serotonin. Furthermore, application of mianserin alone reduced rotation rate and virtually abolished rotational surges. Taken together, these pharmacological results suggest that endogenous serotonin is responsible for generating rotational surges. Given that early embryos contain only a single pair of serotonergic neurons (Goldberg and Kater, 1989) during the stages when rotational surges are expressed, these results also prompt the hypothesis that these neurons, embryonic neurons C1, act as cilioexcitatory motor neurons during embryonic development.     

Effect on the plasma renin-aldosterone system of lesions to suprachiasmatic nuclei and central serotonin depletion in rats

Renin activity, angiotensin-converting enzyme activity and aldosterone concentration were measured in the plasma of 8 experimental groups of rats: I--sham operated non-treated rats, II--suprachiasmatic nuclei (SCN) lesioned non-treated: III--sham operated + furosemide (4 mg/kg i.p.), IV--SCN lesioned + furosemide, V--shams + 24-hour water deprivation: VI--SCN + 24-hour water deprivation, VII--intact rats + saline: and VIII--intact rats + p-chlorophenylalanine (pCPA, 300 mg/kg, i.p.). No significant changes in basal levels of the three parameters were found after SCN, lesions in comparison with sham operated controls. Furosemide caused a similar increase in all three parameters of both sham and SCN lesioned rats. Similar changes were observed in SCN rats 24 hours after water deprivation and in intact rats 48 hours after serotonin depletion by pCPA: suppressed renin activity together with increased aldosterone concentration. It is concluded that the central serotonergic system and SCN play a similar role in control of the renin-aldosterone system in rats under conditions of negative water-salt balance.