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.     

Failure to demonstrate serotonergic neurons in the myenteric plexus of the rat

Summary Several recent studies suggested that serotonergic neuron-like elements are present in the guinea pig ileum. The present paper reports an extensive study of the digestive tract of the rat with the use of a histofluorescence technique. Administration of the serotonin precursor, tryptophan, associated with a monoamine oxidase inhibitor, did not allow histochemical demonstration of rapidly fading, yellow fluorescent, 6-hydroxydopamine-resistant neurons; conversely such neurons were readily detected in the brain. It is concluded that serotonergic neuron-like elements cannot be detected histochemically in the rat myenteric plexus area after chemical sympathectomy.     

A developmental study of serotonin-immunoreactive neurons in the embryonic brain of the Marbled Crayfish and the Migratory Locust: Evidence for a homologous protocerebral group of neurons

It is well established that the brains of adult malacostracan crustaceans and winged insects display distinct homologies down to the level of single neuropils such as the central complex and the optic neuropils. We wanted to know if developing insect and crustacean brains also share similarities and therefore have explored how neurotransmitter systems arise during arthropod embryogenesis. Previously, Sintoni et al. (2007) had already reported a homology of an individually identified cluster of neurons in the embryonic crayfish and insect brain, the secondary head spot cells that express the Engrailed protein. In the present study, we have documented the ontogeny of the serotonergic system in embryonic brains of the Marbled Crayfish in comparison to Migratory Locust embryos using immunohistochemical methods combined with confocal laser-scan microscopy. In both species, we found a cluster of early emerging serotonin-immunoreactive neurons in the protocerebrum with neurites that cross to the contralateral brain hemisphere in a characteristic commissure suggesting a homology of this cell cluster. Our study is a first step towards a phylogenetic analysis of neurotransmitter system development and shows that, as for the ventral nerve cord, traits related to neurogenesis in the brain can provide valuable hints for resolving the much debated question of arthropod phylogeny.     

Plexin-A3 and plexin-A4 restrict the migration of sympathetic neurons but not their neural crest precursors

During development, the semaphorin family of guidance molecules is required for proper formation of the sympathetic nervous system. Plexins are receptors that mediate semaphorin signaling, but how plexins function during sympathetic development is not fully understood. Using phenotypic analyses of mutant mice in vivo, expression pattern studies, and in vitro assays, we show that plexin-A3 and plexin-A4 are essential for normal sympathetic development. This study confirms our previous in vitro findings that the two plexins differentially regulate the guidance of sympathetic axons. In addition, we find that semaphorin signaling through plexin-A3 and plexin-A4 restricts the migration of sympathetic neurons, but these two plexins function redundantly since migration defects are only observed in plexin-A3/-A4 double mutants. Surprisingly, our analysis also indicates that plexin-A3 and plexin-A4 are not required for guiding neural crest precursors prior to reaching the sympathetic anlagen. Immunoprecipitation studies suggest that these two plexins independently mediate secreted semaphorin signaling. Thus, plexin-A3 and plexin-A4 are expressed in newly-differentiated sympathetic neurons, but not their neural crest precursors. They function cooperatively to regulate the migration of sympathetic neurons and then differentially to guide the sympathetic axons.     

Differential effects of serotonergic and peptidergic cardioexcitatory neurons on the heart activity in the pteropod mollusc, Clione limacina

A group of four cardioexcitatory neurons has been identified in the intestinal ganglia of the mollusc Clione limacina. Relatively weak stimulation of the intestinal neurons induced auricle contractions only, while strong stimulation produced initial auricle contractions followed by full-cycle auricle-ventricle contractions. Intestinal cardioexcitatory neurons probably utilized as their transmitter a peptide similar to Tritonia pedal peptide – they showed pedal peptide-like immunoreactivity, and their effects were mimicked by application of the exogenous pedal peptide. The pedal cardioexcitatory neuron was found to produce strong excitatory effects only on the ventricle contractions. Its stimulation induced ventricle contractions in the quiescent heart or significantly accelerated the rate of ventricle contractions in the rhythmically active heart. The pedal cardioexcitatory neuron apparently utilized serotonin as a neurotransmitter, based upon serotonin immunoreactivity, blocking effect of serotonin antagonists mianserin and methysergide, and the observation that exogenous serotonin mimicked its effect. A dense network of pedal peptide-like immunoreactivity was found both in the auricle and ventricle tissue. Serotonin immunoreactivity was densely present in the ventricle, while the auricle contained only a separate serotonin-immunoreactive unbranched axon. Thus, there are two separate groups of central cardioexcitatory neurons with different effects on heart activity, which together might provide a complex cardio-regulatory function in Clione. Accepted: 14 August 1999     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

Postembryonic development of serotonin-immunoreactive neurons in the central nervous system of the blowfly,Calliphora erythrocephala

Summary The postembryonic development of serotonin-immunoreactive (5-HTi) neurons was studied in the optic lobe of the blowfly. In the adult fly there are 24 5-HTi neurons invading each optic lobe. The perikarya of two of these neurons are situated in the dorso-caudal part of the protocerebrum (LBO-5HT neurons; large bilateral optic lobe 5-HTi neurons). The cell bodies of the remaining 22 neurons are located anteriorly at the medial base of the medulla (2 innervating the lobula, LO-5HT neurons; and 20 neurons innervating the medulla, ME-5HT neurons). The two central neurons (LBO-5HT neurons) are derived from metamorphosing larval neurons, while the ME- and LO-5HT neurons are imaginai optic lobe neurons differentiating during pupal development.The 5-HTi neurons of the optic lobe seem to have different ancestors. The LBO-5HT neurons are probably derived from segmental protocerebral neuroblasts, whereas the ME-and LO-5HT neurons are most likely derived from the inner optic anlage. The first 5-HTi fibers to reach the imaginal optic lobes are seen in the late third instar larva and are derived from the LBO-5HT neurons. The first ME- and LO-5HT neurons become immunoreactive at 24 h (10%) pupal development. At about 96 h (40%) of pupal development all the 5-HTi neurons of the optic lobes have differentiated and attained their basic adult morphology. The further development mainly entails increase in volume of arborizations and number of finer processes. The differentiation and outgrowth of 5-HTi processes follows that of, e.g., columnar neurons in the optic lobe neuropils. Hence, 5-HTi processes invade neuropil relatively late in the differentiation of the optic lobe.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: Potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I)

The synapse number and the related dendritic spine number in the cerebral cortex of primates shows a rapid increase after birth. Depending on the brain region and species, the number of synapses reaches a peak before adulthood, and pruning takes place after this peak (overshoot-type synaptic formation). Human mental disorders, such as autism and schizophrenia, are hypothesized to be a result of either too weak or excessive pruning after the peak is reached. Thus, it is important to study the molecular mechanisms underlying overshoot-type synaptic formation, particularly the pruning phase.     

The Drosophila RNA-binding protein ELAV is required for commissural axon midline crossing via control of commissureless mRNA expression in neurons

Drosophila ELAV is the founding member of an evolutionarily conserved family of RNA-binding proteins considered as key inducers of neuronal differentiation. Although several ELAV-specific targets have been identified, little is known about the role of elav during neural development. Here, we report a detailed characterization of the elav mutant commissural phenotype. The reduced number of commissures in elav mutant embryos is not due to loss or misspecification of neural cells but results from defects in commissural axon projections across the midline. We establish a causal relationship between the elav mutant commissural phenotype and a reduction in the expression of commissureless, a key component of the Robo/Slit growth cone repulsive signalling pathway. In the nerve cord of elav mutant embryos, comm mRNA expression is strongly reduced in neurons, but not in midline glial cells. Furthermore, specific expression of an elav transgene in posterior neurons of each segment of an elav mutant nerve cord restores comm mRNA expression in these cells, as well as the formation of posterior commissures. Finally, forced expression of comm in specific commissural neuron subsets rescues the midline crossing defects of these neurons in elav mutant embryos, further indicating that elav acts cell autonomously on comm expression.     

Migration of neurons between ganglia in the metamorphosing insect nervous system

Migration of neurons over long distances occurs during the development of the adult central nervous system of the sphinx moth Manduca sexta, and the turnip moth Agrotis segetum. From each of the suboesophageal and three thoracic ganglia, bilaterally-paired clusters of immature neurons and associated glial cells migrate posteriorly along the interganglionic connectives, to enter the next posterior ganglion. The first sign of migration is observed at the onset of metamorphosis, when posterio-lateral cell clusters gradually separate from the cortex of neuronal cell bodies and enter the connectives. Cell clusters migrate posteriorly along the connective to reach the next ganglion over the first three days (approximately 15%) of pupal development. During migration, each cell cluster is completely enveloped by a single giant glial cell spanning the entire length of the connective between two adjacent ganglia. Intracellular cobalt staining reveals that each migrating neuron has an ovoid cell body and an extremely long leading process which extends as far as the next posterior ganglion; this is not a common morphology for migrating neurons that have been described in vertebrates. Once the cells arrive at the anterior cortex of the next ganglion, they rapidly intermingle with the surrounding neurons and so we were unable to determine the fate of the migrating neurons at their final location.     

Calcium-induced synergistic inhibition of a translational factor eEF2 in nerve growth cones

Local protein synthesis in nerve growth cones has been suggested, but how it is controlled remains largely unknown. We found eukaryotic elongation factor-2 (eEF2), a key component of mRNA translation, in growth cones by immunocytochemistry. While phosphorylated eEF2 was weakly distributed in advancing growth cones, eEF2 phosphorylation was increased by high potassium-evoked calcium influx. In the growth cone, calcium elevation increased eEF2 kinase (EF2K), a calcim-calmodulin-dependent enzyme. Calcium also decreased the level of phosphorylated p70-S6 kinase (S6K), a kinase known to inhibit EF2K. Moreover, calcium elevation decreased total eEF2 in growth cones. Since phosphorylated eEF2 inhibits mRNA translation, calcium elevation appears to inhibit mRNA translation in growth cones by a synergistic mechanism involving regulation of EF2K, S6K, and eEF2 itself. Time-lapse imaging showed that calcium elevation induced growth arrest of neurites. The inhibitory effect on mRNA translation may thus be involved in the regulation of neurite outgrowth.     

A role of local signalling in the establishment and maintenance of the asymmetrical architecture of a neuron

Significant progress has been made in the identification of intrinsic and extrinsic factors involved in the development of nervous system. It is remarkable that the establishment and maintenance of the asymmetrical architecture of a neuron is coordinated by a limited repertoire of signalling machineries. However, the details of signalling mechanisms responsible for creating specificity and diversity required for proper development of the nervous system remain largely to be investigated. An emerging body of evidence suggests that specificity and diversity can be achieved by differential regulation of signalling components at distinct subcellular localizations. Many aspects of neuronal polarization and morphogenesis are attributed to localized signalling. Further diversity and specificity of receptor signalling can be achieved by the regulation of molecules outside the cell. Recent evidence suggests that extracellular matrix molecules are essential extrinsic cues that function to foster the growth of neurons. Therefore, it is important to understand where the signalling machineries are activated and how they are combined with other factors in order to understand the molecular mechanism underlying neuronal development.     

The effects of 5-HT on sensory, central and motor neurons driving the abdominal superficial flexor muscles in the crayfish

Serotonin (5-HT) induces a variety of physiological and behavioral effects in crustaceans. However, the mechanisms employed by 5-HT to effect behavorial changes are not fully understood. Among the mechanisms by which these changes might occur are alterations in synaptic drive and efficacy of sensory, interneurons and motor neurons, as well as direct effects on muscles. We investigated these aspects with the use of a defined sensory-motor system, which is entirely contained within a single abdominal segment and consists of a ‘cuticular sensory neurons–segmental ganglia–abdominal superficial flexor motor neurons–muscles’ circuit. Our studies address the role of 5-HT in altering (1) the activity of motor neurons induced by sensory stimulation; (2) the inherent excitability of superficial flexor motor neurons; (3) transmitter release properties of the motor nerve terminal and (4) input resistance of the muscle. Using en passant recordings from the motor nerve, with and without sensory stimulation, and intracellular recordings from the muscle, we show that 5-HT enhances sensory drive and output from the ventral nerve cord resulting in an increase in the firing frequency of the motor neurons. Also, 5-HT increases transmitter release at the neuromuscular junction, and alters input resistance of the muscle fibers     

异季条件下稻穗枝梗和颖花分化、退化及其形成的差异性研究

以19个水稻杂交组合为试验材料,通过大田条件下的分期播种,于抽穗期调查了稻穗枝梗、颖花分化、退化及形成有关性状,分析了各性状不同季节的变异及其相互关系。结果表明,单穗一次枝梗分化数、二次枝梗分化数、颖花分化数、一次枝梗退化数、二次枝梗退化数、颖花退化数、一次枝梗退化率、二次枝梗退化率、颖花退化率、一次枝梗形成数、二次枝梗形成数、一次枝梗颖花形成数、二次枝梗颖花形成数和单穗颖花总形成数等性状均存在较大的组合间差异和季节间变异,其中19个组合单穗颖花总形成数异季差异率变幅为5.12%~93.59%,平均变异率为34.19%;总体上,不同季节下二次枝梗各性状的变异性强,颖花性状其次,一次枝梗各性状变异性最小;根据异季条件下稻穗枝梗和颖花分化、退化及其形成性状的变异性,从大穗角度讨论了杂交稻新组合育种资源选择与利用问题。     

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.     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.