Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

Expression and functional analysis of flotillins in Dugesia japonica

FLOTILLIN-1 and FLOTILLIN-2 are membrane rafts associated proteins that have been implicated in insulin and growth factor signaling, endocytosis, cell migration, proliferation, differentiation, cytoskeleton remodeling and membrane trafficking. Furthermore, FLOTILLINs also play important roles in the progression of cancer and neurodegenerative diseases. In this study, the roles of flotillins are investigated in planarian Dugesia japonica. The results show that Djflotillin-1 and Djflotillin-2 play a key role in homeostasis maintenance and regeneration process by regulating the proliferation of the neoblast cells, they are not involved in the maintenance and regeneration of the central nervous system in planarians.     

Regeneration of photoreceptor organs in freshwater planarians at different levels of accumulation of natural methylmercury compounds

The effects of natural methylmercury compounds on regeneration of photoreceptor organs were studied in three freshwater planarians: Polycelis tenuis, Dugesia lugubris, and D. tigrina. Accumulation of methyl mercury in the planarian body suppressed regeneration of P. tenuis with numerous photoreceptor organs to a greater extent than in two other planarians that have only two eyes. High methyl mercury concentrations inhibited the restoration of photoreceptor organs in asexual and sexual D. tigrina races     

Control of Neuronal Network in Caenorhabditis elegans

Caenorhabditis elegans, a soil dwelling nematode, is evolutionarily rudimentary and contains only ∼ 300 neurons which are connected to each other via chemical synapses and gap junctions. This structural connectivity can be perceived as nodes and edges of a graph. Controlling complex networked systems (such as nervous system) has been an area of excitement for mankind. Various methods have been developed to identify specific brain regions, which when controlled by external input can lead to achievement of control over the state of the system. But in case of neuronal connectivity network the properties of neurons identified as driver nodes is of much importance because nervous system can produce a variety of states (behaviour of the animal). Hence to gain insight on the type of control achieved in nervous system we implemented the notion of structural control from graph theory to C. elegans neuronal network. We identified ‘driver neurons’ which can provide full control over the network. We studied phenotypic properties of these neurons which are referred to as ‘phenoframe’ as well as the ‘genoframe’ which represents their genetic correlates. We find that the driver neurons are primarily motor neurons located in the ventral nerve cord and contribute to biological reproduction of the animal. Identification of driver neurons and its characterization adds a new dimension in controllability of C. elegans neuronal network. This study suggests the importance of driver neurons and their utility to control the behaviour of the organism.     

Planarian GSK3s are involved in neural regeneration

Glycogen synthase kinase-3 (GSK3) is a key element in several signaling cascades that is known to be involved in both patterning and neuronal organization. It is, therefore, a good candidate to play a role in neural regeneration in planarians. We report the characterization of three GSK3 genes in Schmidtea mediterranea. Phylogenetic analysis shows that Smed-GSK3.1 is highly conserved compared to GSK3 sequences from other species, whereas Smed-GSK3.2 and Smed-GSK3.3 are more divergent. Treatment of regenerating planarians with 1-azakenpaullone, a synthetic GSK3 inhibitor, suggests that planarian GSK3s are essential for normal differentiation and morphogenesis of the nervous system. Cephalic ganglia appear smaller and disconnected in 1-azakenpaullone-treated animals, whereas visual axons are ectopically projected, and the pharynx does not regenerate properly. This phenotype is consistent with a role for Smed-GSK3s in neuronal polarization and axonal growth. Teresa Adell and Maria Marsal contributed equally to this work. An erratum to this article can be found at     

Protective effect of leptin-mediated caveolin-1 expression on neurons after spinal cord injury

Spinal cord injury (SCI) causes long-term disability and has no effective clinical treatment. After SCI, extracellular adenosine triphosphate (ATP) leads to an influx of extracellular Ca²⁺, and this Ca²⁺ overload causes neuronal toxicosis and apoptosis. The biological functions of leptin have been widely investigated in the central nervous system. In this study, we discovered that the administration of leptin could improve locomotor recovery following SCI. The aim of this study was to determine the neuroprotective mechanism of leptin in vivo and in vitro. The neuronal apoptosis and Ca²⁺ imaging signal induced by ATP were suppressed by leptin, due to elevated caveolin-1 expression. In vivo two-photon observations revealed that leptin reduced the neuronal Ca²⁺ imaging signal in the exposed spinal cords of live Thy1-YFP mice. In conclusion, leptin promotes locomotor functional recovery and suppresses neuronal impairment after SCI, suggesting that leptin has a promising clinical therapeutic value for treatment of SCI.     

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.     

Immunocytochemical identification of serotoninergic neurons in planaria <Emphasis Type="Italic">Girardia tigrina</Emphasis>

Flatworms occupy an important position among simple organisms, which were first in the evolution having bilateral symmetry and centralized nervous system. This paper provides evidence of the presence of a biogenic amine serotonin in free-living flatworms planarians Girardia tigrina (Turbellaria, Platyhelminthes). Using immunohistochemical method, fluorescence and confocal laser scanning microscopy, we have identified serotonin neurons and their fibers using planarian whole-mount preparations and got important information about distribution of serotoninergic components in their body. Information on the number and size of serotonin-immunopositive neurons in the brain ganglion of G. tigrina and on the distribution density of serotoninergic neurons in the central nervous system of worms is presented for the first time. The published data concerning the serotoninergic signalization in flatworms are briefly overviewed.     

The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.     

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

A lot of evidence testifies that aromatase is expressed in the central nervous system where it has been detected not only in hypothalamic and limbic regions but also in the cerebral cortex and spinal cord. In physiological conditions, aromatase is expressed exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than those from males, suggesting a protective role of estradiol. The aim of this study was to evaluate changes in aromatase expression in response to 3-nitro-L-tyrosine, a marker of oxidative stress, in primary neuronal cell cultures from brains of 60-day old sheep fetuses. Cells were identified as neurons by using class III β-tubulin, a marker of neuronal cells. Two morphological types were consistently recognizable: i) bipolar cells with an oval cell body; ii) multipolar cells whose processes formed a wide net with those of adjacent cells. In situ hybridization technique performed on 60-day old fetal neurons revealed that in baseline conditions aromatase gene expression occurs. Importantly, cells exposed to 360 µM 3-nitro-L-tyrosine were fewer and showed more globular shape and shorter cytoplasmic processes in comparison to control cells. The immunocytochemical study with anti-aromatase antibody revealed that cells exposed to 360 µM 3-nitro-L-tyrosine were significantly more immunoreactive than control cells. Thus, it can be postulated that the oxidant effects of the amino acid analogue 3-nitro-L-tyrosine could be counterbalanced by an increase in aromatase expression that in turn can lead to the formation of neuroprotective estradiol via aromatization of testosterone.Key words: 3-nitro-L-tyrosine, aromatase, oxidative injury, neuroprotection, neuronal cell cultures, sheep.The brain is an important site of steroid synthesis in vertebrates (Baulieu, 1997). Neuroendocrine tissue is capable of converting androgens into estrogens by the enzyme P450 aromatase (Naftolin et al., 1971). Estradiol, through its specific receptors, promotes many crucial regulatory effects on various processes, such as viability and survival of neurons in rat primary cultures (Chowen et al., 1992), neural differentiation and plasticity as well as sexual behavior. Aromatase modulates synaptic plasticity in the hippocampus and other brain regions related to cognition.The enzyme may also influence synaptic development and plasticity in other non-reproductive regions of the central nervous system. For instance, Purkinje cells in aromatase-knockout mice show decreased dendritic growth and impairment of formation of dendritic spines and synapses (Sasahara et al., 2007). In addition, numerous studies have shown expression, activity and distribution of aromatase in the central nervous system of rats (Shinoda et al., 1994) and humans (Yague et al., 2006). In the brain, aromatase is predominantly expressed in hypothalamic and limbic regions, but also other structures such as the cerebral cortex, midbrain and spinal cord reveal aromatase activity and immunoreactivity.It has been demonstrated that aromatase is neuroprotective in the central nervous system. For instance, treatment with the neurotoxin kainic acid resulted in significant neuronal loss in the hippocampus of rats treated with the aromatase inhibitor fadrozole (Azcoitia et al., 2001). Under baseline conditions, aromatase is expressed in the central nervous system of mammals exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals (Naftolin et al., 1996). Since aromatase is expressed in several cellular compartments, it can be supposed that it leads to the formation of estrogen that acts not only through the classical receptors but also by direct and rapid effects at neuronal membranes (Roselli, 2007).Aromatase-expressing astrocytes have been observed in rats after stressful conditions such as serum deprivation or ischemia (Azcoitia et al., 2003, Roselli, 2007). The increased expression of aromatase in injured brain areas suggests that the enzyme may be involved in the protection of nervous tissue by increasing levels of local estrogens. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than males, suggesting estradiol has a protective role (Liu et al., 2007). In particular, these Authors demonstrated that astrocytes isolated from neonatal cortex exhibit marked sex differences in the sensitivity to oxygen-glucose deprivation and oxidant cell death since female cells exhibited enhanced aromatization and estradiol formation.The present investigation describes for the first time changes in aromatase expression in response to 3-nitro-L-tyrosine - a marker of oxidative stress - in primary neuronal cultures from fetal sheep brain.     

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudière et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.Download video file.^{(52M, mov)}     

TINP1 homolog is required for planarian regeneration

The planarian flatworm is an ideal system for the study of regeneration in vivo. In this study, we focus on TINP1, which is one of the most conserved proteins in eukaryotic organisms. We found that TINP1 was expressed in parenchymal region through whole body as well as central nervous system (CNS) during the course of regeneration. RNA interference targeting DjTINP1 caused lysis defects in regenerating tissues and a decreased in cell division and expression levels of DjpiwiA and Djpcna. Furthermore, the expression levels of DjTINP1 were decreased when we inhibited the TGF-β signal by knockdown of smad4, which is the sole co-smad and has been proved to control the blastema patterning and central nervous system (CNS) regeneration in planarians. These findings suggest that DjTINP1 participate in the maintenance of neoblasts and be required for proper cell proliferation in planarians as a downstream gene of the TGF-β signal pathway.     

Functional insight into LOAD-associated microglial response genes

Alzheimer''s disease (AD) is characterized by the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs), neuronal and synaptic loss and inflammation of the central nervous system (CNS). The majority of AD research has been dedicated to the understanding of two major AD hallmarks (i.e. Aβ and NFTs); however, recent genome-wide association studies (GWAS) data indicate neuroinflammation as having a critical role in late-onset AD (LOAD) development, thus unveiling a novel avenue for AD therapeutics. Recent evidence has provided much support to the innate immune system''s involvement with AD progression; however, much remains to be uncovered regarding the role of glial cells, specifically microglia, in AD. Moreover, numerous variants in immune and/or microglia-related genes have been identified in whole-genome sequencing and GWAS analyses, including such genes as TREM2, CD33, APOE, API1, MS4A, ABCA7, BIN1, CLU, CR1, INPP5D, PICALM and PLCG2. In this review, we aim to provide an insight into the function of the major LOAD-associated microglia response genes.     

Drosophila Neurotrophins Reveal a Common Mechanism for Nervous System Formation

Neurotrophic interactions occur in Drosophila, but to date, no neurotrophic factor had been found. Neurotrophins are the main vertebrate secreted signalling molecules that link nervous system structure and function: they regulate neuronal survival, targeting, synaptic plasticity, memory and cognition. We have identified a neurotrophic factor in flies, Drosophila Neurotrophin (DNT1), structurally related to all known neurotrophins and highly conserved in insects. By investigating with genetics the consequences of removing DNT1 or adding it in excess, we show that DNT1 maintains neuronal survival, as more neurons die in DNT1 mutants and expression of DNT1 rescues naturally occurring cell death, and it enables targeting by motor neurons. We show that Spätzle and a further fly neurotrophin superfamily member, DNT2, also have neurotrophic functions in flies. Our findings imply that most likely a neurotrophin was present in the common ancestor of all bilateral organisms, giving rise to invertebrate and vertebrate neurotrophins through gene or whole-genome duplications. This work provides a missing link between aspects of neuronal function in flies and vertebrates, and it opens the opportunity to use Drosophila to investigate further aspects of neurotrophin function and to model related diseases.     

Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling

The special glycerophospholipids plasmalogens (Pls) are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor) proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh)-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.     

Emergence of Motor Circuit Activity

In the developing nervous system, ordered neuronal activity patterns can occur even in the absence of sensory input and to investigate how these arise, we have used the model system of the embryonic chicken spinal motor circuit, focusing on motor neurons of the lateral motor column (LMC). At the earliest stages of their molecular differentiation, we can detect differences between medial and lateral LMC neurons in terms of expression of neurotransmitter receptor subunits, including CHRNA5, CHRNA7, GRIN2A, GRIK1, HTR1A and HTR1B, as well as the KCC2 transporter. Using patch-clamp recordings we also demonstrate that medial and lateral LMC motor neurons have subtly different activity patterns that reflect the differential expression of neurotransmitter receptor subunits. Using a combination of patch-clamp recordings in single neurons and calcium-imaging of motor neuron populations, we demonstrate that inhibition of nicotinic, muscarinic or GABA-ergic activity, has profound effects of motor circuit activity during the initial stages of neuromuscular junction formation. Finally, by analysing the activity of large populations of motor neurons at different developmental stages, we show that the asynchronous, disordered neuronal activity that occurs at early stages of circuit formation develops into organised, synchronous activity evident at the stage of LMC neuron muscle innervation. In light of the considerable diversity of neurotransmitter receptor expression, activity patterns in the LMC are surprisingly similar between neuronal types, however the emergence of patterned activity, in conjunction with the differential expression of transmitter systems likely leads to the development of near-mature patterns of locomotor activity by perinatal ages.