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Sample records for neuronal cell cultures

  1. General overview of neuronal cell culture.

    Science.gov (United States)

    Gordon, Jennifer; Amini, Shohreh; White, Martyn K

    2013-01-01

    In this introductory chapter, we provide a general overview of neuronal cell culture. This is a rapidly evolving area of research and we provide an outline and contextual framework for the different chapters of this book. These chapters were all contributed by scientists actively working in the field who are currently using state-of-the-art techniques to advance our understanding of the molecular and cellular biology of the central nervous system. Each chapter provides detailed descriptions and experimental protocols for a variety of techniques ranging in scope from basic neuronal cell line culturing to advanced and specialized methods.

  2. Dissociated neurons of the pupal blowfly antenna in cell culture.

    Science.gov (United States)

    Nakagawa, A; Iwama, A

    1995-12-01

    Primary cell cultures are useful for studying the function of neurons in a simplified and controlled environment. We established a primary culture of antennal cells from pupal blowflies in order to investigate olfactory receptor neurons. In cultures, neuron-like cells were identified on the basis of morphology and immunocytochemical characterization with anti-HRP staining. Neuron-like cells showed variety in the extension pattern of neurites. Many neuron-like cells extended a single prominent long process, which reached about 200 microm after four days, and several short ones. However, some neuron-like cells differentiated in other ways; some exhibited bipolar or multipolar processes, distinct from intact olfactory receptor neurons. The size of cell bodies of neuron-like cells as divisible into two groups; approx. 7 microm diameter and 10-15 microm diameter. Neuron-like cells in culture will provide a good model for electrophysiological analysis and for developmental studies of olfactory receptor neurons.

  3. Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

    Science.gov (United States)

    Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

    2008-01-01

    Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

  4. Rapid method for culturing embryonic neuron-glial cell cocultures

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Shan, Wei-Song; Colman, David R;

    2003-01-01

    A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces...... for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added...... to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric...

  5. [Electrophysiological properties of inhibitory neurones in cultured dissociated hippocampal cells].

    Science.gov (United States)

    Moskaliuk, A O; Kolodin, Iu O; Kravchenko, M O; Fedulova, S A; Veselovs'kyĭ, M S

    2004-01-01

    Electrophysiological properties of inhibitory (GABAergic) neurones were studied in dissociated hippocampal culture using simultaneous whole cell recordings from pairs of monosynaptically coupled neurons. Reliable identification of GABAergic neuron was performed by presence of monosynaptic inhibitory currents at postsynaptic cell in response to action potentials at stimulated cell. It was shown that GABAergic neurons in hippocampal culture are divided in two groups by their firing characteristics: first type generates action potentials at high frequency in response to injection of current (duration 0.5 s)--fast-spiking neurons (FS), cells from second type has no ability for high-frequency action potential generation--regular spiking neurons (RS). These two groups were distinguished by kinetic characteristics of action potentials, adaptation characteristics during continuous generation of action potentials and inhibitory effect making on postsynaptic cell. Application of potassium channel blocker 4-AP to somas of FS neurons in concentration, which selectively inhibits Kv3 potassium channels evoked reversible changes in kinetic of action potentials, frequency and adaptation characteristics during continuous generation of action potentials. It was concluded that there is hight level of expression of Kv3 potassium channels in the first group of neurons.

  6. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

    Svendsen, Winnie Edith; Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya

    2015-01-01

    In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been te...... tested successfully with brain slices and PC12 cells. The culture substrate can be modified using metal electrodes and/or nanostructures for conducting electrical measurements while culturing and for better mimicking the in vivo conditions.......In this paper we demonstrate a novel culturing system for brain slices and neuronal cells, which can control the concentration of nutrients and the waste removal from the culture by adjusting the fluid flow within the device. The entire system can be placed in an incubator. The system has been...

  7. Neuronal stimulation of (3H)thymidine incorporation by primary cultures of highly purified non-neuronal cells.

    Science.gov (United States)

    McCarthy, K D; Partlow, L M

    1976-09-24

    A specific intercellular interaction has been demonstrated between neuronal and non-neuronal cells that appears to increase the rate of non-neuronal cell proliferation. Isolated and recombined primary cultures of both cell types were prepared from 11-day embryonic chick sympathetic ganglia by a method recently developed in this laboratory. When non-dividing neurons were added to an equal number of proliferating non-neuronal cells, the amount of [methyl-3H]thymidine incorporated by these mixed cultures was 230% greater than that incorporated by 99% pure non-neuronal cultures. Removal of all neurons from such non-neuronal cultures by a 48-h preincubation without nerve growth factor resulted in an even greater increase in [3H]thymidine incorporation upon addition of neurons (370%). When increasing numbers of isolated neurons were added to non-neuronal cell cultures, the amount of [3H]thymidine incorporation initially increased in a dose-dependent fashion until it reached a plateau. In contrast, the addition of increasing numbers of non-neuronal cells to a constant number of neurons resulted in a linear increase in [3H]thymidine incorporation. In some cases neurons and non-neuronal cells were not grown in direct physical contact but were only allowed to communicate with one another through the culture medium. Such indirect communication never resulted in a stimulation of [3H]thymidine incorporation. When neurons were added to cultures of embryonic chick fibroblasts, the neurons grew well but did not stimulate [3H]thymidine incorporation by the fibroblasts. These results suggest that embryonic sympathetic neurons selectively stimulate the proliferation of non-neuronal cells derived from the same source.

  8. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    Science.gov (United States)

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  9. Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    . Tests show that the function of neurons cultured on PNWs lies closer to neurons in vivo than neurons cultured on conventional plastic substrates. The second part of the thesis describes a fluidic system for culturing of brain slices. It describes the fabrication and use of the system as well as results...... for culturing of brain tissue. The second goal was to develop a sensor system with the potential for incorporation into both conventional culture systems and fluidic culturing systems. The third and final goal of this project was to develop a system for culturing of neuronal cells with the possibility...... neuronal cells on a Peptide Nano Wires (PNW) modified substrate aiming to bring conventional neuronal cultures closer to mimic the in vivo situation. The work describes both the fabrication of the culture substrates and results comparing the performance of PNWcultured neurons and conventional cultures...

  10. The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study

    National Research Council Canada - National Science Library

    Biffi, Emilia; Regalia, Giulia; Menegon, Andrea; Ferrigno, Giancarlo; Pedrocchi, Alessandra

    2013-01-01

    .... Neuronal cultures plated with different cell densities differ in number of synapses per neuron and thus in single neuron synaptic transmission, which results in a density-dependent neuronal network activity...

  11. Exogenous gangliosides may affect methylation mechanisms in neuronal cell cultures

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    Ferret, B.; Hubsch, A.; Dreyfus, H.; Massarelli, R. (Centre de Neurochimie du CNRS, Strasbourg (France))

    1991-02-01

    Primary neurons in culture from chick embryo cerebral hemispheres were treated with a mixture of gangliosides added to the growth medium (final concentration: 10(-5)M and 10(-8)M) from the 3rd to the 6th day in vitro. Under these conditions methylation processes measured with (3H) and (35S) methionine and (3H)ethanolamine as precursors showed an increased methylation of (3H)ethanolamine containing phospholipids, a correspondent increased conversion of these compounds to (3H)choline containing phospholipids, and a general increased methylation of trichloroacetic acid precipitable macromolecules containing labeled methionine. A small increase in protein synthesis was observed after incubation of neurons with (3H)- and (35S)methionine. This was confirmed after electrophoretic separation of a protein extract with increased 3H- and 35S-labeling in protein bands with moecular weights between 50 and 60 KDaltons. A protein band of about 55 KDaltons appeared to be preferentially labelled when (3H) methionine was the precursor. The treatment with gangliosides increased the incorporation of (methyl-3H) label after incubation of neurons with (3H) methionine, into total DNA and decreased that of total RNA. The treatment of neurons in culture with exogenous gangliosides hence affects differently methylation processes, a finding which may confirm the involvement of gangliosides on the intracellular mediation of neuronal information mechanisms.

  12. Glucose Levels in Culture Medium Determine Cell Death Mode in MPP(+)-treated Dopaminergic Neuronal Cells.

    Science.gov (United States)

    Yoon, So-Young; Oh, Young J

    2015-09-01

    We previously demonstrated that 1-methyl-4-phenylpyridinium (MPP(+)) causes caspase-independent, non-apoptotic death of dopaminergic (DA) neuronal cells. Here, we specifically examined whether change of glucose concentration in culture medium may play a role for determining cell death modes of DA neurons following MPP(+) treatment. By incubating MN9D cells in medium containing varying concentrations of glucose (5~35 mM), we found that cells underwent a distinct cell death as determined by morphological and biochemical criteria. At 5~10 mM glucose concentration (low glucose levels), MPP(+) induced typical of the apoptotic dell death accompanied with caspase activation and DNA fragmentation as well as cell shrinkage. In contrast, MN9D cells cultivated in medium containing more than 17.5 mM (high glucose levels) did not demonstrate any of these changes. Subsequently, we observed that MPP(+) at low glucose levels but not high glucose levels led to ROS generation and subsequent JNK activation. Therefore, MPP(+)-induced cell death only at low glucose levels was significantly ameliorated following co-treatment with ROS scavenger, caspase inhibitor or JNK inhibitor. We basically confirmed the quite similar pattern of cell death in primary cultures of DA neurons. Taken together, our results suggest that a biochemically distinct cell death mode is recruited by MPP(+) depending on extracellular glucose levels.

  13. Non-Neuronal Cells Are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System.

    Directory of Open Access Journals (Sweden)

    Chin Wai Hui

    Full Text Available Chronic inflammation is associated with activated microglia and reactive astrocytes and plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's. Both in vivo and in vitro studies have demonstrated that inflammatory cytokine responses to immune challenges contribute to neuronal death during neurodegeneration. In order to investigate the role of glial cells in this phenomenon, we developed a modified method to remove the non-neuronal cells in primary cultures of E16.5 mouse cortex. We modified previously reported methods as we found that a brief treatment with the thymidine analog, 5-fluorodeoxyuridine (FdU, is sufficient to substantially deplete dividing non-neuronal cells in primary cultures. Cell cycle and glial markers confirm the loss of ~99% of all microglia, astrocytes and oligodendrocyte precursor cells (OPCs. More importantly, under this milder treatment, the neurons suffered neither cell loss nor any morphological defects up to 2.5 weeks later; both pre- and post-synaptic markers were retained. Further, neurons in FdU-treated cultures remained responsive to excitotoxicity induced by glutamate application. The immunobiology of the FdU culture, however, was significantly changed. Compared with mixed culture, the protein levels of NFκB p65 and the gene expression of several cytokine receptors were altered. Individual cytokines or conditioned medium from β-amyloid-stimulated THP-1 cells that were, potent neurotoxins in normal, mixed cultures, were virtually inactive in the absence of glial cells. The results highlight the importance of our glial-depleted culture system and identifies and offer unexpected insights into the complexity of -brain neuroinflammation.

  14. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    Science.gov (United States)

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  15. Neural cell co-culture induced differentiation of bone marrow mesenchymal stem cells into neuronal-like cells

    Institute of Scientific and Technical Information of China (English)

    Nailong Yang; Lili Xu; Fen Yang

    2008-01-01

    BACKGROUND: It has been previously demonstrated that the neural cell microenvironment has the ability to induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into the neural cells.OBJECTIVE: To establish a co-culture system of human BMSCs and neural cells, and to observe effects of this co-culture system on differentiation of human BMSCs into neural cells.DESIGN, TIME AND SETTING: A comparative observation experiment, performed at the Center Laboratory of the Affiliated Hospital of Medical College Qingdao University from October 2006 to December 2007.MATERIALS: Neural cells were obtained from human fetal brain tissue. BMSCs were harvested from female patients that underwent autonomous stem cell transplantation.METHODS: BMSCs in the co-culture group consisted of BMSCs and third passage neural cells. BMSCs in the control group were solely cultured in vitro.MAIN OUTCOME MEASURES: Morphological changes of BMSCs were observed, and expression of the neuronal specific marker, neuron-specific enolase (NSE), was analyzed by immunofluorescence staining after4-5-day co-culture.RESULTS: The number of neural cells in the co-culture group increased and the cells spread on the culture bottle surface. Radial dendrite formed and connected with each other. NSE-immunoreactive cells were also detected. The positive ratio of NSE-positive cells reached (32.7±11.5)%, with morphological characteristics similar to neuronal cells. Human BMSCs did not express NSE in the control group.CONCLUSION: The microenvironment provided by neurons induced differentiation of BMSCs into neuronal-like cells.

  16. Reduced Neurite Density in Neuronal Cell Cultures Exposed to Serum of Patients with Bipolar Disorder

    Science.gov (United States)

    Wollenhaupt-Aguiar, Bianca; Pfaffenseller, Bianca; Chagas, Vinicius de Saraiva; Castro, Mauro A A; Passos, Ives Cavalcante; Kauer-Sant’Anna, Márcia; Kapczinski, Flavio

    2016-01-01

    Background: Increased inflammatory markers and oxidative stress have been reported in serum among patients with bipolar disorder (BD). The aim of this study is to assess whether biochemical changes in the serum of patients induces neurotoxicity in neuronal cell cultures. Methods: We challenged the retinoic acid-differentiated human neuroblastoma SH-SY5Y cells with the serum of BD patients at early and late stages of illness and assessed neurite density and cell viability as neurotoxic endpoints. Results: Decreased neurite density was found in neurons treated with the serum of patients, mostly patients at late stages of illness. Also, neurons challenged with the serum of late-stage patients showed a significant decrease in cell viability. Conclusions: Our findings showed that the serum of patients with bipolar disorder induced a decrease in neurite density and cell viability in neuronal cultures. PMID:27207915

  17. Neurotoxicity of isoniazid and its metabolites in cultures of mouse dorsal root ganglion neurons and hybrid neuronal cell line.

    Science.gov (United States)

    Sanfeliu, C; Wright, J M; Kim, S U

    1999-12-01

    Isoniazid (INH) is one of the anti-tuberculosis drugs widely prescribed for patients since the early 1950s. It is relatively nontoxic but some patients develop peripheral neuropathy attributed to a disturbance of vitamin B6 metabolism. Some isoniazid metabolites are hepatotoxic but little is known about their neurotoxic property. Isoniazid and its metabolites including acetylisoniazid, acetylhydrazine, diacetylhydrazine, isonicotinic acid and hydrazine were examined for their potential neurotoxic effects in cultured mouse dorsal root ganglion (DRG) neurons and mouse neuroblastoma x DRG neuron hybrid cell line N18D3. Isoniazid did not cause neurotoxicity at exposures up to 7 days. Hydrazine was found to be the most toxic metabolite with LC50 values of 2.7 mM and 0.3 mM after 7 days of exposure in DRG neurons and N18D3 hybrid neurons, respectively. Other metabolites including acetylisoniazid, acetylhydrazine, diacetylhydrazine and isonicotinic acid had moderate to minor neurotoxic effects on N18D3 hybrid neurons. Pyridoxine, which is used in clinical practice to prevent or ameliorate the isoniazid-induced neuropathy, did not consistently reverse the neurotoxicity of any of the metabolites in the cell cultures, but some interaction with hydrazine cannot be ruled out. Pyridoxine itself was found to be neurotoxic both in DRG neurons and N18D3 hybrid neurons, in agreement with human peripheral sensory neuropathy caused by prolonged overdosage. The enzymes catalase and superoxide dismutase and the antioxidant agent selenium showed some protection against hydrazine neurotoxicity, suggesting an involvement of the generation of reactive oxygen species in the pathogenesis of isoniazid neuropathy. Both mouse DRG neurons and N18D3 mouse hybrid neurons were shown to be useful culture systems for elucidating the neurotoxicity mechanisms of agents causing sensory neuropathies and general neurotoxic effects in the nervous system.

  18. Visually guided whole cell patch clamp of mouse supraoptic nucleus neurons in cultured and acute conditions.

    Science.gov (United States)

    Stachniak, Tevye J E; Bourque, Charles W

    2006-07-01

    Recent advances in neuronal culturing techniques have supplied a new set of tools for studying neural tissue, providing effective means to study molecular aspects of regulatory elements in the supraoptic nucleus of the hypothalamus (SON). To combine molecular biology techniques with electrophysiological recording, we modified an organotypic culture protocol to permit transfection and whole cell patch-clamp recordings from SON cells. Neonatal mouse brain coronal sections containing the SON were dissected out, placed on a filter insert in culture medium, and incubated for at least 4 days to allow attachment to the insert. The SON was identifiable using gross anatomical landmarks, which remained intact throughout the culturing period. Immunohistochemical staining identified both vasopressinergic and oxytocinergic cells present in the cultures, typically appearing in well-defined clusters. Whole cell recordings from these cultures demonstrated that certain properties of the neonatal mouse SON were comparable to adult mouse magnocellular neurons. SON neurons in both neonatal cultures and acute adult slices showed similar sustained outward rectification above -60 mV and action potential broadening during evoked activity. Membrane potential, input resistance, and rapidly inactivating potassium current density (IA) were reduced in the cultures, whereas whole cell capacitance and spontaneous synaptic excitation were increased, perhaps reflecting developmental changes in cell physiology that warrant further study. The use of the outlined organotypic culturing procedures will allow the study of such electrophysiological properties of mouse SON using whole cell patch-clamp, in addition to various molecular, techniques that require longer incubation times.

  19. Direct Signaling from Astrocytes to Neurons in Cultures of Mammalian Brain Cells

    Science.gov (United States)

    Nedergaard, Maiken

    1994-03-01

    Although astrocytes have been considered to be supportive, rather than transmissive, in the adult nervous system, recent studies have challenged this assumption by demonstrating that astrocytes possess functional neurotransmitter receptors. Astrocytes are now shown to directly modulate the free cytosolic calcium, and hence transmission characteristics, of neighboring neurons. When a focal electric field potential was applied to single astrocytes in mixed cultures of rat forebrain astrocytes and neurons, a prompt elevation of calcium occurred in the target cell. This in turn triggered a wave of calcium increase, which propagated from astrocyte to astrocyte. Neurons resting on these astrocytes responded with large increases in their concentration of cytosolic calcium. The gap junction blocker octanol attenuated the neuronal response, which suggests that the astrocytic-neuronal signaling is mediated through intercellular connections rather than synaptically. This neuronal response to local astrocytic stimulation may mediate local intercellular communication within the brain.

  20. Optimization of single-cell electroporation protocol for forced gene expression in primary neuronal cultures.

    Science.gov (United States)

    Nishikawa, Shin; Hirashima, Naohide; Tanaka, Masahiko

    2014-09-01

    The development and function of the central nervous system (CNS) are realized through interactions between many neurons. To investigate cellular and molecular mechanisms of the development and function of the CNS, it is thus crucial to be able to manipulate the gene expression of single neurons in a complex cell population. We recently developed a technique for gene silencing by introducing small interfering RNA into single neurons in primary CNS cultures using single-cell electroporation. However, we had not succeeded in forced gene expression by introducing expression plasmids using single-cell electroporation. In the present study, we optimized the experimental conditions to enable the forced expression of green fluorescent protein (GFP) in cultured cerebellar Purkinje neurons using single-cell electroporation. We succeeded in strong GFP expression in Purkinje neurons by increasing the inside diameter of micropipettes or by making the size of the original plasmid smaller by digestion and cyclizing it by ligation. Strong GFP expression in Purkinje neurons electroporated under the optimal conditions continued to be observed for more than 25 days after electroporation. Thus, this technique could be used for forced gene expression in single neurons to investigate cellular and molecular mechanisms of the development, function, and disease of the CNS.

  1. In vitro differentiation of bone marrow stromal cells into neurons and glial cells and differential protein expression in a two-compartment bone marrow stromal cell/neuron co-culture system.

    Science.gov (United States)

    Qi, Xu; Shao, Ming; Peng, Haisheng; Bi, Zhenggang; Su, Zhiqiang; Li, Hulun

    2010-07-01

    This study was performed to establish a bone marrow stromal cell (BMSC)/neuron two-compartment co-culture model in which differentiation of BMSCs into neurons could occur without direct contact between the two cell types, and to investigate protein expression changes during differentiation of this entirely BMSC-derived population. Cultured BMSCs isolated from Wistar rats were divided into three groups: BMSC culture, BMSC/neuron co-culture and BMSC/neuron two-compartment co-culture. Cells were examined for neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) expression. The electrophysiological behavior of the BMSCs was examined using patch clamping. Proteins that had significantly different expression levels in BMSCs cultured alone and co-cultured with neurons were studied using a protein chip-mass spectroscopy technique. Expression of NSE and GFAP were significantly higher in co-culture cells than in two-compartment co-culture cells, and significantly higher in both co-culture groups than in BMSCs cultured alone. Five proteins showed significant changes in expression during differentiation: TIP39_RAT and CALC_RAT underwent increases, and INSL6_RAT, PNOC_RAT and PCSK1_RAT underwent decreases in expression. We conclude that BMSCs can differentiate into neurons during both contact co-culture with neurons and two-compartment co-culture with neurons. The rate at which BMSCs differentiated into neurons was higher in contact co-culture than in non-contact co-culture.

  2. Omega-3 Polyunsaturated Fatty Acids Enhance Neuronal Differentiation in Cultured Rat Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Masanori Katakura

    2013-01-01

    Full Text Available Polyunsaturated fatty acids (PUFAs can induce neurogenesis and recovery from brain diseases. However, the exact mechanisms of the beneficial effects of PUFAs have not been conclusively described. We recently reported that docosahexaenoic acid (DHA induced neuronal differentiation by decreasing Hes1 expression and increasing p27kip1 expression, which causes cell cycle arrest in neural stem cells (NSCs. In the present study, we examined the effect of eicosapentaenoic acid (EPA and arachidonic acid (AA on differentiation, expression of basic helix-loop-helix transcription factors (Hes1, Hes6, and NeuroD, and the cell cycle of cultured NSCs. EPA also increased mRNA levels of Hes1, an inhibitor of neuronal differentiation, Hes6, an inhibitor of Hes1, NeuroD, and Map2 mRNA and Tuj-1-positive cells (a neuronal marker, indicating that EPA induced neuronal differentiation. EPA increased the mRNA levels of p21cip1 and p27kip1, a cyclin-dependent kinase inhibitor, which indicated that EPA induced cell cycle arrest. Treatment with AA decreased Hes1 mRNA but did not affect NeuroD and Map2 mRNA levels. Furthermore, AA did not affect the number of Tuj-1-positive cells or cell cycle progression. These results indicated that EPA could be involved in neuronal differentiation by mechanisms alternative to those of DHA, whereas AA did not affect neuronal differentiation in NSCs.

  3. Neural stem cells improve neuronal survival in cultured postmortem brain tissue from aged and Alzheimer patients.

    Science.gov (United States)

    Wu, L; Sluiter, A A; Guo, Ho-Fu; Balesar, R A; Swaab, D F; Zhou, Jiang-Ning; Verwer, R W H

    2008-01-01

    Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cell can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co-cultured postmortem slices from Alzheimer patients and control participants with rat embryonic day 14 (E14) neural stem cells. Viability staining based on the exclusion of ethidium bromide by intact plasma membranes showed that there were strikingly more viable cells and fewer dead cells in slices co-cultured with neural stem cells than in untreated slices. The presence of Alzheimer pathology in the brain slices did not influence this effect, although the slices from Alzheimer patients, in general, contained fewer viable cells. Co-culturing with rat E14 fibroblasts did not improve the viability of neurons in the human brain slices. Since the human slices and neural stem cells were separated by a membrane during co-culturing our data show for the first time that neural stem cells release diffusible factors that may improve the survival of aged and degenerating neurons in human brains.

  4. Neural stem cells improve neuronal survival in cultured postmortem brain tissue from aged and Alzheimer patients

    NARCIS (Netherlands)

    Wu, L.; Sluiter, A.A.; Guo, Ho Fu; Balesar, R. A.; Swaab, D. F.; Zhou, Jiang Ning; Verwer, R. W H

    Neurodegenerative diseases are progressive and incurable and are becoming ever more prevalent. To study whether neural stem cell can reactivate or rescue functions of impaired neurons in the human aging and neurodegenerating brain, we co-cultured postmortem slices from Alzheimer patients and control

  5. Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture.

    Science.gov (United States)

    Vianney, John-Mary; Spitsbergen, John M

    2011-05-16

    Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma×neuroblastoma hybrid cells (NG108-15) were used to create nerve-muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve-myotube co-cultures were blocked with alpha-bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve-muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Culturing rat hippocampal neurons.

    Science.gov (United States)

    Audesirk, G; Audesirk, T; Ferguson, C

    2001-01-01

    Cultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.

  7. Rhinacanthus nasutus protects cultured neuronal cells against hypoxia induced cell death.

    Science.gov (United States)

    Brimson, James M; Tencomnao, Tewin

    2011-07-26

    Rhinacanthus nasutus (L.) Kurz (Acanthaceae) is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington's disease, Parkinson's disease and Alzeheimer's disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL⁻¹ root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL⁻¹ had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL⁻¹ reduced HT-22 cell proliferation. We also used H₂DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  8. Rhinacanthus nasutus Protects Cultured Neuronal Cells against Hypoxia Induced Cell Death

    Directory of Open Access Journals (Sweden)

    James M. Brimson

    2011-07-01

    Full Text Available Rhinacanthus nasutus (L. Kurz (Acanthaceae is an herb native to Thailand and Southeast Asia, known for its antioxidant properties. Hypoxia leads to an increase in reactive oxygen species in cells and is a leading cause of neuronal damage. Cell death caused by hypoxia has been linked with a number of neurodegenerative diseases including some forms of dementia and stroke, as well as the build up of reactive oxygen species which can lead to diseases such as Huntington’s disease, Parkinson’s disease and Alzeheimer’s disease. In this study we used an airtight culture container and the Mitsubishi Gas Company anaeropack along with the MTT assay, LDH assay and the trypan blue exlusion assay to show that 1 and 10 µg mL−1 root extract of R. nasutus is able to significantly prevent the death of HT-22 cells subjected to hypoxic conditions, and 0.1 to 10 µg mL−1 had no toxic effect on HT-22 under normal conditions, whereas 100 µg mL−1 reduced HT-22 cell proliferation. We also used H2DCFDA staining to show R. nasutus can reduce reactive oxygen species production in HT-22 cells.

  9. Cyclooxygenase-2 contributes to VX-induced cell death in cultured cortical neurons.

    Science.gov (United States)

    Tenn, Catherine C; Weiss, M Tracy; Beaup, Claire; Peinnequin, Andre; Wang, Yushan; Dorandeu, Frederic

    2012-04-05

    The link between cell death and increased cyclooxygenases-2 (COX-2) activity has not been clearly established. In this study, we examined whether COX-2 activation contributed to the mechanism of neurotoxicity produced by an organophosphorous nerve agent in cultured rat cortical neurons. Exposure of neuronal cells to the nerve agent, VX resulted in an increase in COX enzyme activity in the culture media. A concentration dependent increase in the activity levels of COX-2 enzyme was observed while there was little to no effect on COX-1. In addition, COX-2 mRNA and protein levels increased several hours post-VX exposure. Pre-treatment of the cortical cells with the COX-2 selective inhibitor, NS 398 resulted in a decrease in both the enzyme activity and prostaglandin (PGE(2) and PGF(2α)) release, as well as in a reduction in cell death. These findings indicate that the increase in COX-2 activity may contribute to the mechanism of VX-induced neurotoxicity in cultured rat cortical neuron.

  10. Mesenchymal stem cells enhance GABAergic transmission in co-cultured hippocampal neurons.

    Science.gov (United States)

    Mauri, Mario; Lentini, Daniela; Gravati, Marta; Foudah, Dana; Biella, Gerardo; Costa, Barbara; Toselli, Mauro; Parenti, Marco; Coco, Silvia

    2012-04-01

    Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent stem cells endowed with neurotrophic potential combined with immunological properties, making them a promising therapeutic tool for neurodegenerative disorders. However, the mechanisms through which MSCs promote the neurological recovery following injury or inflammation are still largely unknown, although cell replacement and paracrine mechanisms have been hypothesized. In order to find out what are the mechanisms of the trophic action of MSCs, as compared to glial cells, on CNS neurons, we set up a co-culture system where rat MSCs (or cortical astrocytes) were used as a feeding layer for hippocampal neurons without any direct contact between the two cell types. The analysis of hippocampal synaptogenesis, synaptic vesicle recycling and electrical activity show that MSCs were capable to support morphological and functional neuronal differentiation. The proliferation of hippocampal glial cells induced by the release of bioactive substance(s) from MSCs was necessary for neuronal survival. Furthermore, MSCs selectively increased hippocampal GABAergic pre-synapses. This effect was paralleled with a higher expression of the potassium/chloride KCC2 co-transporter and increased frequency and amplitude of mIPSCs and sIPSCs. The enhancement of GABA synapses was impaired by the treatment with K252a, a Trk/neurotrophin receptor blocker, and by TrkB receptor bodies hence suggesting the involvement of BDNF as a mediator of such effects. The results obtained here indicate that MSC-secreted factors induce glial-dependent neuronal survival and trigger an augmented GABAergic transmission in hippocampal cultures, highlighting a new effect by which MSCs could promote CNS repair. Our results suggest that MSCs may be useful in those neurological disorders characterized by an impairment of excitation versus inhibition balance.

  11. Dual compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture

    Directory of Open Access Journals (Sweden)

    Thirukumaran T eKanagasabapathi

    2011-10-01

    Full Text Available We developed a dual compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode array (MEA.The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. Cross-correlation analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.

  12. Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Johnsson, Anna-Karin; Karlsson, Roger, E-mail: roger.karlsson@wgi.su.se

    2012-01-15

    Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

  13. Magnetic-activated cell sorting (MACS) can be used as a large-scale method for establishing zebrafish neuronal cell cultures

    OpenAIRE

    Georg Welzel; Daniel Seitz; Stefan Schuster

    2015-01-01

    Neuronal cell cultures offer a crucial tool to mechanistically analyse regeneration in the nervous system. Despite the increasing importance of zebrafish (Danio rerio) as an in vivo model in neurobiological and biomedical research, in vitro approaches to the nervous system are lagging far behind and no method is currently available for establishing enriched neuronal cell cultures. Here we show that magnetic-activated cell sorting (MACS) can be used for the large-scale generation of neuronal-r...

  14. BDNF production by olfactory ensheathing cells contributes to axonal regeneration of cultured adult CNS neurons.

    Science.gov (United States)

    Pastrana, Erika; Moreno-Flores, Maria Teresa; Avila, Jesus; Wandosell, Francisco; Minichiello, Liliana; Diaz-Nido, Javier

    2007-02-01

    Olfactory ensheathing cells (OECs) are the main glial cell type that populates mammalian olfactory nerves. These cells have a great capacity to promote the regeneration of axons when transplanted into the injured adult mammalian CNS. However, little is still known about the molecular mechanisms they employ in mediating such a task. Brain-derived neurotrophic factor (BDNF) was identified as a candidate molecule in a genomic study that compared three functionally different OEC populations: Early passage OECs (OEC Ep), Late passage OECs (OEC Lp) and the OEC cell line TEG3 [Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., Diaz-Nido, J., 2006. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J. Neurosci. 26, 5347-5359]. We have here set out to determine the role played by BDNF in the stimulation of axon outgrowth by OECs. We compared the extracellular BDNF levels in the three OEC populations and show that it is produced in significant amounts by the OECs that can stimulate axon regeneration in adult retinal neurons (OEC Ep and TEG3) but it is absent from the extracellular medium of OEC Lp cells which lack this capacity. Blocking BDNF signalling impaired axonal regeneration of adult retinal neurons co-cultured with TEG3 cells and adding BDNF increased the proportion of adult neurons that regenerate their axons on OEC Lp monolayers. Combining BDNF with other extracellular proteins such as Matrix Metalloproteinase 2 (MMP2) further augmented this effect. This study shows that BDNF production by OECs plays a direct role in the promotion of axon regeneration of adult CNS neurons.

  15. Dual-compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture

    NARCIS (Netherlands)

    Kanagasabapathi, T.T.; Ciliberti, D.; Martinoia, S.; Wadman, W.J.; Decré, M.M.J.

    2011-01-01

    We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for

  16. Cell surface area regulation in neurons in hippocampal slice cultures is resistant to oxygen-glucose deprivation

    Directory of Open Access Journals (Sweden)

    Natalya Shulyakova

    2010-09-01

    Full Text Available Natalya Shulyakova1,2, Jamie Fong2, Diana Diec2, Adrian Nahirny1,2, Linda R Mills1,21Department of Physiology, University of Toronto, Toronto, ON, Canada, M5T 2S8; 2Toronto Western Hospital Research Institute, University Health Network, 11-430, 399 Bathurst St, Toronto, ON, Canada, M5T 2S8Background: Neurons swell in response to a variety of insults. The capacity to recover, ie, to shrink, is critical for neuronal function and survival. Studies on dissociated neurons have shown that during swelling and shrinking, neurons reorganize their plasma membrane; as neurons swell, in response to hypo-osmotic media, the bilayer area increases. Upon restoration of normo-osmotic media, neurons shrink, forming transient invaginations of the plasma membrane known as vacuole-like dilations (VLDs, to accommodate the decrease in the bilayer.Methods: Here we used confocal microscopy to monitor neuronal swelling and shrinking in the three-dimensional (3D environment of post-natal rat hippocampal slice cultures. To label neurons, we used biolistic transfection, to introduce enhanced green fluorescent protein (eGFP targeted to the cytoplasm; and a membrane targeted GFP (lckGFP, targeted to the plasma membrane.Results: Neurons in slice cultures swelled and shrank in response to hypo-osmotic to normo-osmotic media changes. Oxygen-glucose deprivation (OGD caused sustained neuronal swelling; after reperfusion, some neurons recovered but in others, VLD recovery was stalled. OGD did not impair neuronal capacity to recover from a subsequent osmotic challenge.Conclusion: These results suggest cell surface area regulation (SAR is an intrinsic property of neurons, and that neuronal capacity for SAR may play an important role in the brain’s response to ischemic insults.Keywords: neurons, swelling, ischemia, cell surface area, hippocampal slice culture

  17. Isolation and Culture of Pig Spermatogonial Stem Cells and Their in Vitro Differentiation into Neuron-Like Cells and Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiaoyan Wang

    2015-11-01

    Full Text Available Spermatogonial stem cells (SSCs renew themselves throughout the life of an organism and also differentiate into sperm in the adult. They are multipopent and therefore, can be induced to differentiate into many cells types in vitro. SSCs from pigs, considered an ideal animal model, are used in studies of male infertility, regenerative medicine, and preparation of transgenic animals. Here, we report on a culture system for porcine SSCs and the differentiation of these cells into neuron-like cells and adipocytes. SSCs and Sertoli cells were isolated from neonatal piglet testis by differential adhesion and SSCs were cultured on a feeder layer of Sertoli cells. Third-generation SSCs were induced to differentiate into neuron-like cells by addition of retinoic acid, β-mercaptoethanol, and 3-isobutyl-1-methylxanthine (IBMX to the induction media and into adipocytes by the addition of hexadecadrol, insulin, and IBMX to the induction media. The differentiated cells were characterized by biochemical staining, qRT-PCR, and immunocytochemistry. The cells were positive for SSC markers, including alkaline phosphatase and SSC-specific genes, consistent with the cells being undifferentiated. The isolated SSCs survived on the Sertoli cells for 15 generations. Karyotyping confirmed that the chromosomal number of the SSCs were normal for pig (2n = 38, n = 19. Pig SSCs were successfully induced into neuron-like cells eight days after induction and into adipocytes 22 days after induction as determined by biochemical and immunocytochemical staining. qPCR results also support this conclusion. The nervous tissue markers genes, Nestin and β-tubulin, were expressed in the neuron-like cells and the adipocyte marker genes, PPARγ and C/EBPα, were expressed in the adipocytes.

  18. A non-destructive culturing and cell sorting method for cardiomyocytes and neurons using a double alginate layer.

    Directory of Open Access Journals (Sweden)

    Hideyuki Terazono

    Full Text Available A non-destructive method of collecting cultured cells after identifying their in situ functional characteristics is proposed. In this method, cells are cultivated on an alginate layer in a culture dish and released by spot application of a calcium chelate buffer that locally melts the alginate layer and enables the collection of cultured cells at the single-cell level. Primary hippocampal neurons, beating human embryonic stem (hES cell-derived cardiomyocytes, and beating hES cell-derived cardiomyocyte clusters cultivated on an alginate layer were successfully released and collected with a micropipette. The collected cells were recultured while maintaining their physiological function, including beating, and elongated neurites. These results suggest that the proposed method may eventually facilitate the transplantation of ES- or iPS-derived cardiomyocytes and neurons differentiated in culture.

  19. Poly(2-oxazoline)-Based Microgel Particles for Neuronal Cell Culture.

    Science.gov (United States)

    Platen, Mitja; Mathieu, Evelien; Lück, Steffen; Schubel, René; Jordan, Rainer; Pautot, Sophie

    2015-05-11

    An increasing number of in vivo and in vitro neuro-engineering applications are making use of colloidal particles as neuronal cell carriers. Recent studies highlight the shortcomings of commercial glass particles and stress the benefit of using soft microgel particles (MGPs) instead. This study describes first the fabrication of MGPs from telechelic poly(2-methyl-2-oxazoline)s (PMeOx) cross-linkers and hydrophilic neutral (hydroxyethyl)methacrylate (HEMA) or charged 2-methacryloxyethyltrimethylammonium (METAC) monomers by emulsion polymerization, and it discusses their ability to support cell growth. It establishes that uncharged copolymers lead to MGPs with nonfouling properties inappropriate for cell culture, and it provides a protocol to amend their surface properties to enable cell adhesion. Finally, it demonstrates that the introduction of positive charges by METAC is necessary to obtain surface properties suitable for neuronal cell development. Through the optimization of the PMeOx30 MGP properties, this work provides general guidelines to evaluate and tune MGP chemistry to obtain microcarriers for neuro-engineering applications.

  20. Three dimensional neuronal cell cultures more accurately model voltage gated calcium channel functionality in freshly dissected nerve tissue.

    Directory of Open Access Journals (Sweden)

    Yinzhi Lai

    Full Text Available It has been demonstrated that neuronal cells cultured on traditional flat surfaces may exhibit exaggerated voltage gated calcium channel (VGCC functionality. To gain a better understanding of this phenomenon, primary neuronal cells harvested from mice superior cervical ganglion (SCG were cultured on two dimensional (2D flat surfaces and in three dimensional (3D synthetic poly-L-lactic acid (PLLA and polystyrene (PS polymer scaffolds. These 2D- and 3D-cultured cells were compared to cells in freshly dissected SCG tissues, with respect to intracellular calcium increase in response to high K(+ depolarization. The calcium increases were identical for 3D-cultured and freshly dissected, but significantly higher for 2D-cultured cells. This finding established the physiological relevance of 3D-cultured cells. To shed light on the mechanism behind the exaggerated 2D-cultured cells' functionality, transcriptase expression and related membrane protein distributions (caveolin-1 were obtained. Our results support the view that exaggerated VGCC functionality from 2D cultured SCG cells is possibly due to differences in membrane architecture, characterized by uniquely organized caveolar lipid rafts. The practical implication of use of 3D-cultured cells in preclinical drug discovery studies is that such platforms would be more effective in eliminating false positive hits and as such improve the overall yield from screening campaigns.

  1. Progesterone promotes neuronal differentiation of human umbilical cord mesenchymal stem cells in culture conditions that mimic the brain microenvironment

    Institute of Scientific and Technical Information of China (English)

    Xianying Wang; Honghai Wu; Gai Xue; Yanning Hou

    2012-01-01

    In this study, human umbilical cord mesenchymal stem cells from full-term neonates born by vaginal delivery were cultured in medium containing 150 mg/mL of brain tissue extracts from Sprague-Dawley rats (to mimic the brain microenvironment). Immunocytochemical analysis demonstrated that the cells differentiated into neuron-like cells. To evaluate the effects of progesterone as a neurosteroid on the neuronal differentiation of human umbilical cord mesenchymal stem cells, we cultured the cells in medium containing progesterone (0.1, 1, 10 μM) in addition to brain tissue extracts. Reverse transcription-PCR and flow cytometric analysis of neuron specific enolase-positive cells revealed that the percentages of these cells increased significantly following progesterone treatment, with the optimal progesterone concentration for neuron-like differentiation being 1 μM. These results suggest that progesterone can enhance the neuronal differentiation of human umbilical cord mesenchymal stem cells in culture medium containing brain tissue extracts to mimic the brain microenvironment.

  2. Protective effects of N-methyl-D-aspartate receptor antagonism on VX-induced neuronal cell death in cultured rat cortical neurons.

    Science.gov (United States)

    Wang, Yushan; Weiss, M Tracy; Yin, Junfei; Tenn, Catherine C; Nelson, Peggy D; Mikler, John R

    2008-01-01

    Exposure of the central nervous system to organophosphorus (OP) nerve agents induces seizures and neuronal cell death. Here we report that the OP nerve agent, VX, induces apoptotic-like cell death in cultured rat cortical neurons. The VX effects on neurons were concentration-dependent, with an IC(50) of approximately 30 microM. Blockade of N-methyl-D-aspartate receptors (NMDAR) with 50 microM. D-2-amino-5-phosphonovalerate (APV) diminished 30 microM VX-induced total cell death, as assessed by alamarBlue assay and Hoechst staining. In contrast, neither antagonists of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) nor metabotropic glutamate receptors (mGluRs) had any effect on VX-induced neurotoxicity. VX-induced neuronal cell death could not be solely attributed to acetylcholinesterase (AChE) inhibition, since neither the reversible pharmacological cholinesterase inhibitor, physostigmine, nor the muscarinic receptor antagonist, atropine, affected VX-induced cell death. Importantly, APV was found to be therapeutically effective against VX-induced cell death up to 2 h post VX exposure. These results suggest that NMDARs, but not AMPARs or mGluRs, play important roles in VX-induced cell death in cultured rat cortical neurons. Based on their therapeutic effects, NMDAR antagonists may be beneficial in the treatment of VX-induced neurotoxicities.

  3. AlGaN/GaN-based HEMTs for electrical stimulation of neuronal cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Witte, H; Warnke, C; Krost, A [Institute of Experimental Physics, Otto-von-Guericke-University-Magdeburg, Magdeburg (Germany); Voigt, T; De Lima, A [Institute for Physiology, Otto-von-Guericke-University-Magdeburg, Magdeburg (Germany); Ivanov, I; Vidakovic-Koch, T R; Sundmacher, K, E-mail: hartmut.witte@physik.uni-magdeburg.de [Process Systems Engineering, Otto-von-Guericke-University-Magdeburg, Magdeburg (Germany)

    2011-09-07

    Unipolar source-drain voltage pulses of GaN/AlGaN-high electron mobility transistors (HEMTs) were used for stimulation of cultured neuronal networks obtained from embryonic rat cerebral cortex. The HEMT sensor was grown by metal organic vapour phase epitaxy on a 2 inch sapphire substrate consisting of 10 single HEMTs concentrically arranged around the wafer centre. Electrolytic reactions between the HEMT sensor surface and the culture medium were not detected using cyclic voltammetry. During voltage pulses and resulting neuronal excitation, capacitances were recharged giving indications of the contributions of the AlGaN and AlO{sub x} isolation layers between the two-dimensional electron gas channel and the neuron culture. The resulting threshold current for stimulation of neuron activity strongly depended on the culture and HEMT position on the sensor surface under consideration which was caused by different impedances of each neuron culture and position within the culture. The differences of culture impedances could be explained by variations of composition, thickness and conductivity of the culture areas.

  4. Orexins protect neuronal cell cultures against hypoxic stress: an involvement of Akt signaling.

    Science.gov (United States)

    Sokołowska, Paulina; Urbańska, Anna; Biegańska, Kaja; Wagner, Waldemar; Ciszewski, Wojciech; Namiecińska, Magdalena; Zawilska, Jolanta B

    2014-01-01

    Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

  5. Exposure to cell phone radiation up-regulates apoptosis genes in primary cultures of neurons and astrocytes.

    Science.gov (United States)

    Zhao, Tian-Yong; Zou, Shi-Ping; Knapp, Pamela E

    2007-01-22

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working Global System for Mobile Communication (GSM) cell phone rated at a frequency of 1900MHz. Primary cultures were exposed to cell phone emissions for 2h. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 and Asc (apoptosis associated speck-like protein containing a card) gene expression in neurons and astrocytes. Up-regulation occurred in both "on" and "stand-by" modes in neurons, but only in "on" mode in astrocytes. Additionally, astrocytes showed up-regulation of the Bax gene. The effects are specific since up-regulation was not seen for other genes associated with apoptosis, such as caspase-9 in either neurons or astrocytes, or Bax in neurons. The results show that even relatively short-term exposure to cell phone radiofrequency emissions can up-regulate elements of apoptotic pathways in cells derived from the brain, and that neurons appear to be more sensitive to this effect than astrocytes.

  6. Biosynthesis of the D2 cell adhesion molecule: pulse-chase studies in cultured fetal rat neuronal cells

    DEFF Research Database (Denmark)

    Lyles, J M; Norrild, B; Bock, E

    1984-01-01

    D2 is a membrane glycoprotein that is believed to function as a cell adhesion molecule (CAM) in neural cells. We have examined its biosynthesis in cultured fetal rat brain neurones. We found D2-CAM to be synthesized initially as two polypeptides: Mr 186,000 (A) and Mr 136,000 (B). With increasing...... chase times the Mr of both molecules increased to 187,000-201,000 (A) and 137,000-158,000 (B). These were similar to the sizes of D2-CAM labeled with [14C]glucosamine, [3H]fucose and [14C]mannosamine, indicating that the higher Mr species are glycoproteins. In the presence of tunicamycin, which...... pulse or chase times, showing that these molecules are not interconverted. Thus, our data indicate that the neuronal D2-CAM glycoproteins are derived from two mRNAs....

  7. Cell-type-specific and differentiation-status-dependent variations in cytotoxicity of tributyltin in cultured rat cerebral neurons and astrocytes.

    Science.gov (United States)

    Oyanagi, Koshi; Tashiro, Tomoko; Negishi, Takayuki

    2015-08-01

    Tributyltin (TBT) is an organotin used as an anti-fouling agent for fishing nets and ships and it is a widespread environmental contaminant at present. There is an increasing concern about imperceptible but serious adverse effect(s) of exposure to chemicals existing in the environment on various organs and their physiological functions, e.g. brain and mental function. Here, so as to contribute to improvement of and/or advances in in vitro cell-based assay systems for evaluating brain-targeted adverse effect of chemicals, we tried to evaluate cell-type-specific and differentiation-status-dependent variations in the cytotoxicity of TBT towards neurons and astrocytes using the four culture systems differing in the relative abundance of these two types of cells; primary neuron culture (> 95% neurons), primary neuron-astrocyte (2 : 1) mix culture, primary astrocyte culture (> 95% astrocytes), and passaged astrocyte culture (100% proliferative astrocytes). Cell viability was measured at 48 hr after exposure to TBT in serum-free medium. IC50's of TBT were 198 nM in primary neuron culture, 288 nM in primary neuron-astrocyte mix culture, 2001 nM in primary astrocyte culture, and 1989 nM in passaged astrocyte culture. Furthermore, in primary neuron-astrocyte mix culture, vulnerability of neurons cultured along with astrocytes to TBT toxicity was lower than that of neurons cultured purely in primary neuron culture. On the other hand, astrocytes in primary neuron-astrocyte mix culture were considered to be more vulnerable to TBT than those in primary or passaged astrocyte culture. The present study demonstrated variable cytotoxicity of TBT in neural cells depending on the culture condition.

  8. Stiff substrates enhance cultured neuronal network activity.

    Science.gov (United States)

    Zhang, Quan-You; Zhang, Yan-Yan; Xie, Jing; Li, Chen-Xu; Chen, Wei-Yi; Liu, Bai-Lin; Wu, Xiao-an; Li, Shu-Na; Huo, Bo; Jiang, Lin-Hua; Zhao, Hu-Cheng

    2014-08-28

    The mechanical property of extracellular matrix and cell-supporting substrates is known to modulate neuronal growth, differentiation, extension and branching. Here we show that substrate stiffness is an important microenvironmental cue, to which mouse hippocampal neurons respond and integrate into synapse formation and transmission in cultured neuronal network. Hippocampal neurons were cultured on polydimethylsiloxane substrates fabricated to have similar surface properties but a 10-fold difference in Young's modulus. Voltage-gated Ca(2+) channel currents determined by patch-clamp recording were greater in neurons on stiff substrates than on soft substrates. Ca(2+) oscillations in cultured neuronal network monitored using time-lapse single cell imaging increased in both amplitude and frequency among neurons on stiff substrates. Consistently, synaptic connectivity recorded by paired recording was enhanced between neurons on stiff substrates. Furthermore, spontaneous excitatory postsynaptic activity became greater and more frequent in neurons on stiff substrates. Evoked excitatory transmitter release and excitatory postsynaptic currents also were heightened at synapses between neurons on stiff substrates. Taken together, our results provide compelling evidence to show that substrate stiffness is an important biophysical factor modulating synapse connectivity and transmission in cultured hippocampal neuronal network. Such information is useful in designing instructive scaffolds or supporting substrates for neural tissue engineering.

  9. Neurogenic and neurotrophic effects of BDNF peptides in mouse hippocampal primary neuronal cell cultures.

    Directory of Open Access Journals (Sweden)

    Maria del Carmen Cardenas-Aguayo

    Full Text Available The level of brain-derived neurotrophic factor (BDNF, a member of the neurotrophin family, is down regulated in Alzheimer's disease (AD, Parkinson's disease (PD, depression, stress, and anxiety; conversely the level of this neurotrophin is increased in autism spectrum disorders. Thus, modulating the level of BDNF can be a potential therapeutic approach for nervous system pathologies. In the present study, we designed five different tetra peptides (peptides B-1 to B-5 corresponding to different active regions of BDNF. These tetra peptides were found to be non-toxic, and they induced the expression of neuronal markers in mouse embryonic day 18 (E18 primary hippocampal neuronal cultures. Additionally, peptide B-5 induced the expression of BDNF and its receptor, TrkB, suggesting a positive feedback mechanism. The BDNF peptides induced only a moderate activation (phosphorylation at Tyr 706 of the TrkB receptor, which could be blocked by the Trk's inhibitor, K252a. Peptide B-3, when combined with BDNF, potentiated the survival effect of this neurotrophin on H(2O(2-treated E18 hippocampal cells. Peptides B-3 and B-5 were found to work as partial agonists and as partial antagonists competing with BDNF to activate the TrkB receptor in a dose-dependent manner. Taken together, these results suggest that the described BDNF tetra peptides are neurotrophic, can modulate BDNF signaling in a partial agonist/antagonist way, and offer a novel therapeutic approach to neural pathologies where BDNF levels are dysregulated.

  10. Exposure to Cell Phone Radiation Up-Regulates Apoptosis Genes in Primary Cultures of Neurons and Astrocytes

    OpenAIRE

    Zhao, Tian-Yong; Zou, Shi-Ping; Pamela E Knapp

    2006-01-01

    The health effects of cell phone radiation exposure are a growing public concern. This study investigated whether expression of genes related to cell death pathways are dysregulated in primary cultured neurons and astrocytes by exposure to a working GSM (Global System for Mobile Communication) cell phone rated at a frequency of 1900 MHz. Primary cultures were exposed to cell phone emissions for 2 hrs. We used array analysis and real-time RT-PCR to show up-regulation of caspase-2, caspase-6 an...

  11. Long-term stability of grafted polyethylene glycol surfaces for use with microstamped substrates in neuronal cell culture.

    Science.gov (United States)

    Branch, D W; Wheeler, B C; Brewer, G J; Leckband, D E

    2001-05-01

    Crucial to long-term stability of neuronal micropatterns is functional retention of the underlying substratum while exposed to cell culture conditions. We report on the ability of covalently bound PEG films in long-term cell culture to continually retard protein adhesion and cell growth. PDMS microstamps were used to create poly-d-lysine (PDL) substrates permissive to cell attachment and growth, and polyethylene glycol (PEG) substrates were used to minimize protein and cell adhesion. Film thickness was measured using null ellipsometry and atomic force microscopy (AFM). Organosilane film structure was examined using Fourier transform infrared (FT-IR) spectroscopy. Long-term film stability in cell culture conditions was tested by immersion in 0.1 M sodium phosphate buffer pH 7.4 for up to one month. Null ellipsometry and water contact measurements indicated that organosilane films were stable up to one month, whereas the PEG film thickness declined rapidly after day 25. Hippocampal cells plated at 200 cells/mm2 on uniform PEG substrates gave a steady increase in biofilm thickness on PEG films throughout the culture, possibly from proteins of neuronal origin. We found that all the layers in the cross-linking procedure were stable in cell culture conditions, with the exception of PEG, which degraded after day 25.

  12. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    The subcellular localization of neuronal membrane signaling molecules such as receptors and ion channels depends on intracellular trafficking mechanisms. Essentially, vesicular trafficking mechanisms ensure that a large number of membrane proteins are correctly targeted to different subcellular...... compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish...... the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify...

  13. Culturing conditions determine neuronal and glial excitability.

    Science.gov (United States)

    Stoppelkamp, Sandra; Riedel, Gernot; Platt, Bettina

    2010-12-15

    The cultivation of pure neuronal cultures is considered advantageous for the investigation of cell-type specific responses (such as transmitter release and also pharmacological agents), however, divergent results are a likely consequence of media modifications and culture composition. Using Fura-2 based imaging techniques, we here set out to compare calcium responses of rat hippocampal neurones and glia to excitatory stimulation with l-glutamate in different culture types and media. Neurones in neurone-enriched cultures had increased responses to 10 μM and 100 μM l-glutamate (+43 and 45%, respectively; p's< 0.001) and a slower recovery compared to mixed cultures, indicating heightened excitability. In matured (15-20 days in vitro) mixed cultures, neuronal responder rates were suppressed in a neurone-supportive medium (Neurobasal-A, NB: 65%) compared to a general-purpose medium (supplemented minimal essential medium, MEM: 96%). Glial response size in contrast did not differ greatly in isolated or mixed cultures maintained in MEM, but responder rates were suppressed in both culture types in NB (e.g. 10 μM l-glutamate responders in mixed cultures: 29% in NB, 71% in MEM). This indicates that medium composition is more important for glial excitability than the presence of neurones, whereas the presence of glia has an important impact on neuronal excitability. Therefore, careful consideration of culturing conditions is crucial for interpretation and comparison of experimental results. Especially for investigations of toxicity and neuroprotection mixed cultures may be more physiologically relevant over isolated cultures as they comprise aspects of mutual influences between glia and neurones.

  14. Potentiation by histamine of synaptically mediated excitotoxicity in cultured hippocampal neurones: a possible role for mast cells.

    Science.gov (United States)

    Skaper, S D; Facci, L; Kee, W J; Strijbos, P J

    2001-01-01

    Excessive glutamatergic neurotransmission, particularly when mediated by the N:-methyl-D-aspartate (NMDA) subtype of glutamate receptor, is thought to underlie neuronal death in a number of neurological disorders. Histamine has been reported to potentiate NMDA receptor-mediated events under a variety of conditions. In the present study we have utilized primary hippocampal neurone cultures to investigate the effect of mast cell-derived, as well as exogenously applied, histamine on neurotoxicity evoked by excessive synaptic activity. Exposure of mature cultures for 15 min to an Mg(2+)-free/glycine-containing buffer to trigger synaptic transmission through NMDA receptors, caused a 30-35% neuronal loss over 24 h. When co-cultured with hippocampal neurones, activated mast cells increased excitotoxic injury to 60%, an effect that was abolished in the presence of histaminase. Similarly, addition of histamine during magnesium deprivation produced a concentration-dependent potentiation (+ 60%; EC(50) : 5 microM) of neuronal death which was inhibited by sodium channel blockers and NMDA receptor antagonists, although this effect did not involve known histamine receptors. The histamine effect was further potentiated by acidification of the culture medium. Cultures 'preconditioned' by sublethal (5 min) Mg(2+) deprivation exhibited less neuronal death than controls when exposed to a more severe insult. NMDA receptor activation and the extracellular regulated kinase cascade were required for preconditioning neuroprotection. The finding that histamine potentiates NMDA receptor-mediated excitotoxicity may have important implications for our understanding of conditions where enhanced glutamatergic neurotransmission is observed in conjunction with tissue acidification, such as cerebral ischaemia and epilepsy.

  15. Toxic effects of apomorphine on rat cultured neurons and glial C6 cells, and protection with antioxidants.

    Science.gov (United States)

    dos Santos El-Bachá, R; Daval, J; Koziel, V; Netter, P; Minn, A

    2001-01-01

    Many catechol derivatives are currently used as drugs, even if they produce reactive oxygen species that may cause tissue damage. Among them, apomorphine, a potent dopamine agonist, displays efficient anti-parkinsonian properties, but the consequences of its oxidant and toxic properties have been poorly investigated on in vitro models. In the present work, we investigated apomorphine cytotoxicity by incubating cultures of rat glioma C6 cells and primary cultures of neurons with different concentrations of the drug. Apomorphine-promoted cell death was proportional to its concentration and was time-dependent. The ED(50) of apomorphine on C6 cell death after 48 hr was about 200 microM. The cytotoxic effects induced by apomorphine were correlated to its autoxidation, which leads to the formation of reactive oxygen species, semiquinones, quinones, and a melanin-like pigment. C6 cells that underwent treatment with 400 microM apomorphine for 6 hr displayed features of necrosis, including loss of membrane integrity, degeneration of mitochondria, and DNA fragmentation. Thiols, such as cysteine, N-acetyl-L-cysteine, and glutathione, significantly protected cultured neurons and C6 cells against apomorphine-induced cytotoxicity. Thiols also inhibited apomorphine autoxidation. These data strongly suggest that apomorphine cytotoxicity towards neurons and C6 cells results from an intracellular oxidative stress.

  16. Prototypical antipsychotic drugs protect hippocampal neuronal cultures against cell death induced by growth medium deprivation

    Directory of Open Access Journals (Sweden)

    Williams Sylvain

    2006-03-01

    Full Text Available Abstract Background Several clinical studies suggested that antipsychotic-based medications could ameliorate cognitive functions impaired in certain schizophrenic patients. Accordingly, we investigated the effects of various dopaminergic receptor antagonists – including atypical antipsychotics that are prescribed for the treatment of schizophrenia – in a model of toxicity using cultured hippocampal neurons, the hippocampus being a region of particular relevance to cognition. Results Hippocampal cell death induced by deprivation of growth medium constituents was strongly blocked by drugs including antipsychotics (10-10-10-6 M that display nM affinities for D2 and/or D4 receptors (clozapine, haloperidol, (±-sulpiride, domperidone, clozapine, risperidone, chlorpromazine, (+-butaclamol and L-741,742. These effects were shared by some caspases inhibitors and were not accompanied by inhibition of reactive oxygen species. In contrast, (--raclopride and remoxipride, two drugs that preferentially bind D2 over D4 receptors were ineffective, as well as the selective D3 receptor antagonist U 99194. Interestingly, (--raclopride (10-6 M was able to block the neuroprotective effect of the atypical antipsychotic clozapine (10-6 M. Conclusion Taken together, these data suggest that D2-like receptors, particularly the D4 subtype, mediate the neuroprotective effects of antipsychotic drugs possibly through a ROS-independent, caspase-dependent mechanism.

  17. Embryonic Cell Grafts in a Culture Model of Spinal Cord Lesion: Neuronal Relay Formation is Essential for Functional Regeneration

    Directory of Open Access Journals (Sweden)

    Anne Tscherter

    2016-09-01

    Full Text Available Presently there exists no cure for spinal cord injury. However, transplantation of embryonic tissue into spinal cord lesions resulted in axon outgrowth across the lesion site and some functional recovery, fostering hope for future stem cell therapies. Although in vivo evidence for functional recovery is given, the exact cellular mechanism of the graft support remains elusive: either the grafted cells provide a permissive environment for the host tissue to regenerate itself or the grafts actually integrate functionally into the host neuronal network reconnecting the separated spinal cord circuits. We tested the two hypotheses in an in vitro spinal cord lesion model that is based on propagation of activity between two rat organotypic spinal cord slices in culture. Transplantation of dissociated cells from E14 rat spinal cord or forebrain re-established the relay of activity over the lesion site and, thus, provoked functional regeneration. Combining patch-clamp recordings from transplanted cells with network activity measurements from the host tissue on multi-electrode arrays we here show that neurons differentiate from the grafted cells and integrate into the host circuits. Optogenetic silencing of neurons developed from transplanted embryonic mouse forebrain cells provides clear evidence that they replace the lost neuronal connections to relay and synchronize activity between the separated spinal cord circuits. In contrast, transplantation of neurospheres induced neither the differentiation of mature neurons from the grafts nor an improvement of functional regeneration. Together these findings suggest, that the formation of neuronal relays from grafted embryonic cells is essential to re-connect segregated spinal cord circuits.

  18. Dual-compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture.

    Science.gov (United States)

    Kanagasabapathi, Thirukumaran T; Ciliberti, Davide; Martinoia, Sergio; Wadman, Wytse J; Decré, Michel M J

    2011-01-01

    We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures, and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. CC analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.

  19. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts.

    Science.gov (United States)

    Baskin, David S; Ngo, Hop; Didenko, Vladimir V

    2003-08-01

    Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

  20. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

    Science.gov (United States)

    Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

  1. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain

    DEFF Research Database (Denmark)

    Schou-Pedersen, Anne Marie Voigt; Hansen, Stine Normann; Tveden-Nyborg, Pernille

    2016-01-01

    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical d...... and is broadly applicable to monoamine measurements in cell cultures as well as brain biopsies from animal models used in preclinical neurochemistry....

  2. Cannabinoids increase type 1 cannabinoid receptor expression in a cell culture model of striatal neurons: implications for Huntington's disease.

    Science.gov (United States)

    Laprairie, Robert B; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2013-09-01

    The type 1 cannabinoid receptor (CB1) is a G protein-coupled receptor that is expressed at high levels in the striatum. Activation of CB1 increases expression of neuronal trophic factors and inhibits neurotransmitter release from GABA-ergic striatal neurons. CB1 mRNA levels can be elevated by treatment with cannabinoids in non-neuronal cells. We wanted to determine whether cannabinoid treatment could induce CB1 expression in a cell culture model of striatal neurons and, if possible, determine the molecular mechanism by which this occurred. We found that treatment of STHdh(7/7) cells with the cannabinoids ACEA, mAEA, and AEA produced a CB1receptor-dependent increase in CB1 promoter activity, mRNA, and protein expression. This response was Akt- and NF-κB-dependent. Because decreased CB1 expression is thought to contribute to the pathogenesis of Huntington's disease (HD), we wanted to determine whether cannabinoids could increase CB1 expression in STHdh(7/111) and (111/111) cells expressing the mutant huntingtin protein. We observed that cannabinoid treatment increased CB1 mRNA levels approximately 10-fold in STHdh(7/111) and (111/111) cells, compared to vehicle treatment. Importantly, cannabinoid treatment improved ATP production, increased the expression of the trophic factor BDNF-2, and the mitochondrial regulator PGC1α, and reduced spontaneous GABA release, in HD cells. Therefore, cannabinoid-mediated increases in CB1 levels could reduce the severity of some molecular pathologies observed in HD.

  3. Dopaminergic neuronal differentiation from the forebrain-derived human neural stem cells induced in cultures by using a combination of BMP-7 and pramipexole with growth factors

    Directory of Open Access Journals (Sweden)

    Hongna eYang

    2016-04-01

    Full Text Available Transplantation of dopaminergic (DA neurons is considered to be the most promising therapeutic strategy for replacing degenerated dopamine cells in the midbrain of Parkinson’s disease (PD, thereby restoring normal neural circuit function and slow clinical progression of the disease. Human neural stem cells (hNSCs derived from fetal forebrain are thought to be the important cell sources for producing DA neurons because of their multipotency for differentiation and long-term expansion property in cultures. However, low DA differentiation of the forebrain-derived hNSCs limited their therapeutic potential in PD. In the current study, we explored a combined application of Pramipexole (PRX, bone morphogenetic proteins 7 (BMP-7, and growth factors, including acidic fibroblast factor (aFGF, forskolin, and phorbol-12-myristae-13-acetate (TPA, to induce differentiation of forebrain-derived hNSCs towards DA neurons in cultures. We found that DA neuron-associated genes, including Nurr1, Neurogenin2 (Ngn2, and tyrosine hydroxylase (TH were significantly increased after 24h of differentiation by RT-PCR analysis (p<0.01. Fluorescent examination showed that about 25% of cells became TH-positive neurons at 24h, about 5% of cells became VMAT2 (vascular monoamine transporter 2-positive neurons, and less than 5% of cells became DAT (dopamine transporter-positive neurons at 72h following differentiation in cultures. Importantly, these TH-, VMAT2- and DAT-expressing neurons were able to release dopamine into cultures under both of the basal and evoked conditions. Dopamine levels released by DA neurons produced using our protocol were significantly higher compared to the control groups (P<0.01, as examined by ELISA. Our results demonstrated that the combination of PRX, BMP-7, and growth factors was able to greatly promote differentiation of the forebrain-derived hNSCs into DA-releasing neurons.

  4. Human induced pluripotent stem cell (hiPSC)-derived neurons respond to convulsant drugs when co-cultured with hiPSC-derived astrocytes.

    Science.gov (United States)

    Ishii, Misawa Niki; Yamamoto, Koji; Shoji, Masanobu; Asami, Asano; Kawamata, Yuji

    2017-08-15

    Accurate risk assessment for drug-induced seizure is expected to be performed before entering clinical studies because of its severity and fatal damage to drug development. Induced pluripotent stem cell (iPSC) technology has allowed the use of human neurons and glial cells in toxicology studies. Recently, several studies showed the advantage of co-culture system of human iPSC (hiPSC)-derived neurons with rodent/human primary astrocytes regarding neuronal functions. However, the application of hiPSC-derived neurons for seizure risk assessment has not yet been fully addressed, and not at all when co-cultured with hiPSC-derived astrocytes. Here, we characterized hiPSC-derived neurons co-cultured with hiPSC-derived astrocytes to discuss how hiPSC-derived neurons are useful to assess seizure risk of drugs. First, we detected the frequency of spikes and synchronized bursts hiPSC-derived neurons when co-cultured with hiPSC-derived astrocytes for 8 weeks. This synchronized burst was suppressed by the treatment with 6-cyano-7-nitroquinoxaline-2,3-dione, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor antagonist, and D-(-)-2-amino-5-phosphonopentanoic acid, an N-Methyl-d-aspartate (NMDA) receptor antagonist. These data suggested that co-cultured hiPSC-derived neurons formed synaptic connections mediated by AMPA and NMDA receptors. We also demonstrated that co-cultured hiPSC-derived neurons showed epileptiform activity upon treatment with gabazine or kaliotoxin. Finally, we performed single-cell transcriptome analysis in hiPSC-derived neurons and found that hiPSC-derived astrocytes activated the pathways involved in the activities of AMPA and NMDA receptor functions, neuronal polarity, and axon guidance in hiPSC-derived neurons. These data suggested that hiPSC-derived astrocytes promoted the development of action potential, synaptic functions, and neuronal networks in hiPSC-derived neurons, and then these functional alterations result in the epileptiform

  5. Dorsal root ganglia neurons and differentiated adipose-derived stem cells: an in vitro co-culture model to study peripheral nerve regeneration.

    Science.gov (United States)

    de Luca, Alba C; Faroni, Alessandro; Reid, Adam J

    2015-02-26

    Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

  6. Inhibitory synapse formation in a co-culture model incorporating GABAergic medium spiny neurons and HEK293 cells stably expressing GABAA receptors.

    Science.gov (United States)

    Brown, Laura E; Fuchs, Celine; Nicholson, Martin W; Stephenson, F Anne; Thomson, Alex M; Jovanovic, Jasmina N

    2014-11-14

    Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed

  7. Production of compartmented cultures of rat sympathetic neurons.

    Science.gov (United States)

    Campenot, Robert B; Lund, Karen; Mok, Sue-Ann

    2009-01-01

    The compartmented culture, in which primary neurons plated in a proximal compartment send their axons under silicone grease barriers and into left and right distal compartments, has enhanced the experimental capabilities of neuronal cultures. Treatments can be applied separately to cell bodies/proximal axons or distal axons, and cell bodies/proximal axons and distal axons can be separately harvested and analyzed. Distal axons can be axotomized, and the neurons can be studied while their axons regenerate. Construction of the culture dishes requires 3 h for 48 cultures, and preparing the neurons also requires 3 h. Compartmented cultures provide enough cellular material for biochemical analyses such as immunoblotting. The uses of compartmented cultures have included studies of neurotrophic factor retrograde signaling, axonal transport, and axonal protein and lipid biosynthesis. Here we focus on sympathetic neurons cultured from neonatal rats and provide protocols for the production and some of the uses of compartmented cultures.

  8. High-content analysis of factors affecting gold nanoparticle uptake by neuronal and microglial cells in culture.

    Science.gov (United States)

    Stojiljković, A; Kuehni-Boghenbor, K; Gaschen, V; Schüpbach, G; Mevissen, M; Kinnear, C; Möller, A-M; Stoffel, M H

    2016-09-22

    Owing to their ubiquitous distribution, expected beneficial effects and suspected adverse effects, nanoparticles are viewed as a double-edged sword, necessitating a better understanding of their interactions with tissues and organisms. Thus, the goals of the present study were to develop and present a method to generate quantitative data on nanoparticle entry into cells in culture and to exemplarily demonstrate the usefulness of this approach by analyzing the impact of size, charge and various proteinaceous coatings on particle internalization. N9 microglial cells and both undifferentiated and differentiated SH-SY5Y neuroblastoma cells were exposed to customized gold nanoparticles. After silver enhancement, the particles were visualized by epipolarization microscopy and analysed by high-content analysis. The value of this approach was substantiated by assessing the impact of various parameters on nanoparticle uptake. Uptake was higher in microglial cells than in neuronal cells. Only microglial cells showed a distinct size preference, preferring particles with a diameter of 80 nm. Positive surface charge had the greatest impact on particle uptake. Coating with bovine serum albumin, fetuin or protein G significantly increased particle internalization in microglial cells but not in neuronal cells. Coating with wheat germ agglutinin increased particle uptake in both N9 and differentiated SH-SY5Y cells but not in undifferentiated SH-SY5Y cells. Furthermore, internalization was shown to be an active process and indicators of caspase-dependent apoptosis revealed that gold nanoparticles did not have any cytotoxic effects. The present study thus demonstrates the suitability of gold nanoparticles and high-content analysis for assessing numerous variables in a stringently quantitative and statistically significant manner. Furthermore, the results presented herein showcase the feasibility of specifically targeting nanoparticles to distinct cell types.

  9. Culturing of PC12 Cells, Neuronal Cells, Astrocytes Cultures and Brain Slices in an Open Microfluidic System

    DEFF Research Database (Denmark)

    Al Atraktchi, Fatima Al-Zahraa; Bakmand, Tanya; Rømer Sørensen, Ane

    The brain is the center of the nervous system, where serious neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Huntington’s are products of functional loss in the neural cells (1). Typical techniques used to investigate these diseases lack precise control of the cellular surroundings...

  10. The development and characterisation of complex ovine neuron cultures from fresh and frozen foetal neurons.

    Science.gov (United States)

    Kay, Graham W; Oswald, Manfred J; Palmer, David N

    2006-07-15

    Cultures of ovine cerebral and cerebellar neurons from mid-term sheep foetal brains, 9-15 weeks old, have been established for the first time. These foetal brains are relatively mature, being at similar stages of development as peri and post-natal rodent brains. Cultures were routinely maintained for 3-4 weeks, and longer. Nearly all the cells from the younger foetuses adhered as neurons. The proportion of glial cells increased with age, as did the risk of cultures being overtaken by glial cells. Cultured neurons were bipolar, tripolar and multipolar, similar to the morphologies of neurons in vivo. Older foetuses also yield more complex neurons, notably giant cells. Other properties of the cultured neurons also mimic in vivo observations, including neurite beading, complexity in neurotransmitter class (GABAergic and glutamatergic) and calcium binding protein (calbindin and calretinin) content. Single cell divisions of neurons were observed in younger cultures by time-lapse photography and the occurrence of telophase nuclei. The advantage of the high yield of genetically identical cells obtained from a single sheep foetus, 150 million, was extended by cryopreservation of neurons after snap freezing, and later culture. These cultures showed the same characteristics as cultures from the freshly plated cells.

  11. Anion-selective channelrhodopsin expressed in neuronal cell culture and in vivo in murine brain: Light-induced inhibition of generation of action potentials.

    Science.gov (United States)

    Dolgikh, D A; Malyshev, A Yu; Salozhin, S V; Nekrasova, O V; Petrovskaya, L E; Roshchin, M V; Borodinova, A A; Feldman, T B; Balaban, P M; Kirpichnikov, M P; Ostrovsky, M A

    2015-01-01

    Anionic channelrhodopsin slow ChloC was expressed in the culture of nerve cells and in vivo in mouse brain. We demonstrated ability of slow ChloC to suppress effectively the activity of the neuron in response to the illumination with the visible light. It has been shown for a first time that slow ChloC works equally efficiently in both neuronal culture and in the whole brain being expressed in vivo. Thus, slow ChloC could be considered as an effective optogenetic tool capable in response to light stimulation to inhibit the generation of action potentials in the neuron.

  12. Caspase 3 siRNA decreases apoptosis in cultured neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Chunting Ye; Yaoxiong Huang; Xiaohong Yang; Honghui Chen

    2009-01-01

    BACKGROUND: Lentiviral vectors, a type of retroviral vector, are able to infect cells at all phases of cell cycle. They are able to express exogenous target genes in vivo over long periods of time with limited immunological reaction. OBJECTIVE: To inhibit neuronal apoptosis by blocking the apoptotic cascade reaction, gene silencing of Caspase 3, and transfection of Caspase 3 short hairpin ribonucleic acid (shRNA) into Neuro 2a cells using a lentiviral vector. DESIGN, TIME AND SETTING: An observational, genetic engineering cellular biology experiment was performed in Guangzhou Red Cross Hospital and Guangzhou institute of Traumatic Surgery between March 2007 and June 2008.MATERIALS: PLL3.7, PCMV-VSV-G, and PH'8.9△PR plasmids were provided by the CBR institute for Biomedical Research, Harvard Medical School, USA. Staurosporine was purchased from Sigma, USA. METHODS: Caspase 3 siRNA was synthesized and cloned into the PLL3.7 plasmid. The Caspase 3 shRNA-PLL3.7 lentivirus was generated in 293T cells using a calcium phosphate transfection kit. After the lentivirus was transfected into Neuro 2a cells, apoptosis was induced in both the transfected and untransfected cells by staurosporine. Cell apoptosis was assessed by flow cytometry. MAIN OUTCOME MEASURES: Caspase 3 mRNA expression was measured by RT-PCR and Caspase protein expression was assessed by Western blot. Cellular apoptosis was determined by flow cytometry using Annevin V-PE/7aad-Cy7.RESULTS: The transfection rate of caspase 3 shRNA was>98% using the ientiviral vector. RT-PCR and Western blot results demonstrated that significantly reduced Caspase 3 mRNA and protein expression in the transfected Neuro 2a. The control group exhibited 38.7% Annexin V/7aad-positive cells, which suggested apoptotic anaphase, while only 5.0% cells in the Caspase 3 gene silencing group entered apoptotic anaphase. CONCLUSION: Caspase 3 shRNA inhibited Caspase 3 expression in Neuro 2a cells and decreased drug-induced apoptosis of

  13. Ginkgolides protects cultured cortical neurons against excitotoxic and oxidative insults

    Institute of Scientific and Technical Information of China (English)

    ZHANGYu-Yang; YUQing-Hai; YOUSong; SHENGLi

    2004-01-01

    AIM: The neurotoxicity of glutamate is associated with neurological disorders including hypoxic-ischaemic brain injury. Studies using cultured cortical neurons have demonstrated that exposure to glutamate produced delayed degeneration of mature neurons. Oxygen free radicals generated during injury have been postulated to be a major cause of neuronal cell

  14. Successful elimination of non-neural cells and unachievable elimination of glial cells by means of commonly used cell culture manipulations during differentiation of GFAP and SOX2 positive neural progenitors (NHA to neuronal cells

    Directory of Open Access Journals (Sweden)

    Krynska Barbara

    2008-07-01

    Full Text Available Abstract Background Although extensive research has been performed to control differentiation of neural stem cells – still, the response of those cells to diverse cell culture conditions often appears to be random and difficult to predict. To this end, we strived to obtain stabilized protocol of NHA cells differentiation – allowing for an increase in percentage yield of neuronal cells. Results Uncommitted GFAP and SOX2 positive neural progenitors – so-called, Normal Human Astrocytes (NHA were differentiated in different environmental conditions to: only neural cells consisted of neuronal [MAP2+, GFAP-] and glial [GFAP+, MAP2-] population, non-neural cells [CD44+, VIMENTIN+, FIBRONECTIN+, MAP2-, GFAP-, S100β-, SOX2-], or mixture of neural and non-neural cells. In spite of successfully increasing the percentage yield of glial and neuronal vs. non-neural cells by means of environmental changes, we were not able to increase significantly the percentage of neuronal (GABA-ergic and catecholaminergic over glial cells under several different cell culture testing conditions. Supplementing serum-free medium with several growth factors (SHH, bFGF, GDNF did not radically change the ratio between neuronal and glial cells – i.e., 1,1:1 in medium without growth factors and 1,4:1 in medium with GDNF, respectively. Conclusion We suggest that biotechnologists attempting to enrich in vitro neural cell cultures in one type of cells – such as that required for transplantology purposes, should consider the strong limiting influence of intrinsic factors upon extracellular factors commonly tested in cell culture conditions.

  15. Three-dimensional culture of single embryonic stem-derived neural/stem progenitor cells in fibrin hydrogels: neuronal network formation and matrix remodelling.

    Science.gov (United States)

    Bento, Ana R; Quelhas, Pedro; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2016-12-29

    In an attempt to improve the efficacy of neural stem/progenitor cell (NSPC) based therapies, fibrin hydrogels are being explored to provide a favourable microenvironment for cell survival and differentiation following transplantation. In the present work, the ability of fibrin to support the survival, proliferation, and neuronal differentiation of NSPCs derived from embryonic stem (ES) cells under monolayer culture was explored. Single mouse ES-NSPCs were cultured within fibrin (fibrinogen concentration: 6 mg/ml) under neuronal differentiation conditions up to 14 days. The ES-NSPCs retained high cell viability and proliferated within small-sized spheroids. Neuronal differentiation was confirmed by an increase in the levels of βIII-tubulin and NF200 over time. At day 14, cell-matrix constructs mainly comprised NSPCs and neurons (46.5% βIII-tubulin(+) cells). Gamma-aminobutyric acid (GABA)ergic and dopaminergic/noradrenergic neurons were also observed, along with a network of synaptic proteins. The ES-NSPCs expressed matriptase and secreted MMP-2/9, with MMP-2 activity increasing along time. Fibronectin, laminin and collagen type IV deposition was also detected. Fibrin gels prepared with higher fibrinogen concentrations (8/10 mg/ml) were less permissive to neurite extension and neuronal differentiation, possibly owing to their smaller pore area and higher rigidity. Overall, it is shown that ES-NSPCs within fibrin are able to establish neuronal networks and to remodel fibrin through MMP secretion and extracellular matrix (ECM) deposition. This three-dimensional (3D) culture system was also shown to support cell viability, neuronal differentiation and ECM deposition of human ES-NSPCs. The settled 3D platform is expected to constitute a valuable tool to develop fibrin-based hydrogels for ES-NSPC delivery into the injured central nervous system. Copyright © 2016 John Wiley & Sons, Ltd.

  16. MDMA (Ecstasy) Decreases the Number of Neurons and Stem Cells in Embryonic Cortical Cultures

    DEFF Research Database (Denmark)

    Kindlundh-Högberg, Anna M S; Pickering, Chris; Wicher, Grzegorz

    2010-01-01

    Ecstasy, 3,4-methylenedioxymetamphetamine (MDMA), is a recreational drug used among adolescents, including young pregnant women. MDMA passes the placental barrier and may therefore influence fetal development. The aim was to investigate the direct effect of MDMA on cortical cells using dissociated...... CNS cortex of rat embryos, E17. The primary culture was exposed to a single dose of MDMA and collected 5 days later. MDMA caused a dramatic, dose-dependent (100 and 400 muM) decrease in nestin-positive stem cell density, as well as a significant reduction (400 muM) in NeuN-positive cells. By q......PCR, MDMA (200 muM) caused a significant decrease in mRNA expression of the 5HT3 receptor, dopamine D(1) receptor, and glutamate transporter EAAT2-1, as well as an increase in mRNA levels of the NMDA NR1 receptor subunit and the 5HT(1A) receptor. In conclusion, MDMA caused a marked reduction in stem cells...

  17. Pedicularioside A from Buddleia lindleyana inhibits cell death induced by 1-methyl-4-phenylpyridinium ions (MPP+) in primary cultures of rat mesencephalic neurons.

    Science.gov (United States)

    Li, Yan-Yun; Lu, Jiang-Hai; Li, Quan; Zhao, Yu-Ying; Pu, Xiao-Ping

    2008-01-28

    Parkinson's disease is characterized by the progressive degeneration of midbrain dopaminergic neurons. Buddleia lindleyana is a traditional Chinese herb, commonly called Zui Yu Cao. The purification and identification of pedicularioside A and other phenylethanoid glycosides from this plant have been reported. However, their neuroprotective effects on the 1-methyl-4-phenylpyridinium ion (MPP(+))-induced death of rat mesencephalic neuron primary cultures and the precise mechanism of this protection remains unclear. We used the 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay for cellular growth to examine the effects of five phenylethanoid glycosides isolated from B. lindleyana, including pedicularioside A, leucosceptoside A, isoacteoside, acteoside, and arenariside, on the viability of mesencephalic neurons treated with MPP(+). Of the compounds tested, pedicularioside A exhibited the greatest degree of protection from MPP(+)-induced cell death. We also observed a marked increase in the number of tyrosine hydroxylase immunoreactive neurons. Pedicularioside A inhibited expression of the caspase-3 gene and cleavage of poly (ADP-ribose) polymerase (PARP) in cultures exposed to MPP(+). Our results suggest that pedicularioside A has a neuroprotective effect to improve the survival of mesencephalic neurons (dopaminergic neurons and non-dopaminergic neurons). The mode of action appears to be the inhibition of caspase-3 gene expression, thereby protecting mesencephalic neurons from MPP(+)-induced cell death.

  18. Molluscan cells in culture: primary cell cultures and cell lines

    OpenAIRE

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as bi...

  19. Effect of NGF, BDNF, bFGF, aFGF and cell density on NPY expression in cultured rat dorsal root ganglion neurones.

    Science.gov (United States)

    Kerekes, N; Landry, M; Lundmark, K; Hökfelt, T

    2000-07-01

    The effect of neurotrophic factors on neuropeptide Y (NPY) expression was studied in adult rat dispersed dorsal root ganglion (DRG) cultures. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), acidic fibroblast growth factor (aFGF) or basic FGF was included in the culture medium during incubation for 72 h. In untreated cultures, around 18% of all neurones (visualized by antibodies to PGP 9.5) expressed NPY-like immunoreactivity (LI). In contrast, in vivo uninjured neurones do not contain detectable levels of NPY-LI. In the immunohistochemical analysis aFGF increased the percentage of NPY-immunoreactive (-IR) neurones 1.8-fold, while NGF, BDNF or bFGF had no significant effect on NPY expression. When the effect of these growth factors was monitored with non-radioactive in situ hybridization, both aFGF and bFGF caused a significant increase (2.25- and 1.8-fold, respectively), whereas, again, NGF and BDNF had no effect. The results also showed an effect of cell density on NPY expression, whereby fewer neurones expressed NPY in high than in low density cultures. This difference was seen in untreated as well as growth factor-treated cultures. The present results support the hypothesis that DRG neurones in culture are in an axotomized state, since they express NPY to about the same extent as axotomized DRG neurones in vivo. Surprisingly, two growth factors of the FGF family enhance NPY expression in DRG neurones, which is in apparent contrast to a published in vivo study [Ji, R.-R., Zhang, Q., Pettersson, R.F., Hökfelt, T., 1996. aFGF, bFGF and NGF differentially regulate neuropeptide expression in dorsal root ganglia after axotomy and induce autotomy. Reg. Pept. 66, 179-189.]. Finally, NPY expression was also influenced by cell density.

  20. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21 and Nsg-2 (P19.

    Directory of Open Access Journals (Sweden)

    Laura Digilio

    Full Text Available The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65 were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21 and Nsg-2 (P19 are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.

  1. Role of ERK1/2, Akt, and PLCy pathways in proliferation and neuronal differentiation in the adult rat spinal cord neural stem/progenitor cell culture

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    Wai Si eChan

    2013-08-01

    Full Text Available Proliferation of endogenous neural stem/progenitor cells (NSPCs has been identified in both normal and injured adult mammalian spinal cord. Yet the signaling mechanisms underlying the regulation of adult spinal cord NSPCs proliferation and commitment toward a neuronal lineage remain undefined. In this study, the role of three growth factor-mediated signaling pathways in proliferation and neuronal differentiation was examined. Adult spinal cord NSPCs were enriched in the presence of fibroblast growth factor 2 (FGF2. We observed an increase in the number of cells expressing the microtubule-associated protein 2 (MAP2 over time, indicating neuronal differentiation in the culture. Inhibition of the mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK kinase 1 and 2/ERK 1 and 2 (MEK/ERK1/2 or the phosphoinositide 3-kinase (PI3K/Akt pathways suppressed active proliferation in adult spinal cord NSPC cultures; whereas neuronal differentiation was negatively affected only when the ERK1/2 pathway was inhibited. Inhibition of the phospholipase C gamma (PLCy pathway did not affect proliferation or neuronal differentiation. Finally, we demonstrated that the blockade of either the ERK1/2 or PLCy signaling pathways reduced neurite branching of MAP2+ cells derived from the NSPC cultures. Many of the MAP2+ cells expressed synaptophysin and had a glutamatergic phenotype, indicating that over time adult spinal cord NSPCs had differentiated into mostly glutamatergic neurons. Our work provides new information regarding the contribution of these pathways to the proliferation and neuronal differentiation of NSPCs derived from adult spinal cord cultures, and emphasizes that the contribution of these pathways is dependent on the origin of the NSPCs.

  2. Synergy by secretory phospholipase A2 and glutamate on inducing cell death and sustained arachidonic acid metabolic changes in primary cortical neuronal cultures

    DEFF Research Database (Denmark)

    Kolko, M; DeCoster, M A; de Turco, E B

    1996-01-01

    Secretory and cytosolic phospholipases A2 (sPLA2 and cPLA2) may contribute to the release of arachidonic acid and other bioactive lipids, which are modulators of synaptic function. In primary cortical neuron cultures, neurotoxic cell death and [3H]arachidonate metabolism was studied after adding ...

  3. Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism

    DEFF Research Database (Denmark)

    Jayakumar, A R; Bak, L K; Rama Rao, K V

    2016-01-01

    contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH...... dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of (13)C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4-24 h), and whether such events...

  4. Novel Insights into the Distribution and Functional Aspects of the Calcium Binding Protein Secretagogin from Studies on Rat Brain and Primary Neuronal Cell Culture

    Science.gov (United States)

    Maj, Magdalena; Milenkovic, Ivan; Bauer, Jan; Berggård, Tord; Veit, Martina; Ilhan-Mutlu, Aysegül; Wagner, Ludwig; Tretter, Verena

    2012-01-01

    Secretagogin is a calcium binding protein (CBP) highly expressed in neuroendocrine cells. It has been shown to be involved in insulin secretion from pancreatic beta cells and is a strong candidate as a biomarker for endocrine tumors, stroke, and eventually psychiatric conditions. Secretagogin has been hypothesized to exert a neuroprotective role in neurodegenerative diseases like Alzheimer’s disease. The expression pattern of Secretagogin is not conserved from rodents to humans. We used brain tissue and primary neuronal cell cultures from rat to further characterize this CBP in rodents and to perform a few functional assays in vitro. Immunohistochemistry on rat brain slices revealed a high density of Secretagogin-positive cells in distinct brain regions. Secretagogin was found in the cytosol or associated with subcellular compartments. We tested primary neuronal cultures for their suitability as model systems to further investigate functional properties of Secretagogin. These cultures can easily be manipulated by treatment with drugs or by transfection with test constructs interfering with signaling cascades that might be linked to the cellular function of Secretagogin. We show that, like in pancreatic beta cells and insulinoma cell lines, also in neurons the expression level of Secretagogin is dependent on extracellular insulin and glucose. Further, we show also for rat brain neuronal tissue that Secretagogin interacts with the microtubule-associated protein Tau and that this interaction is dependent on Ca2+. Future studies should aim to study in further detail the molecular properties and function of Secretagogin in individual neuronal cell types, in particular the subcellular localization and trafficking of this protein and a possible active secretion by neurons. PMID:22888312

  5. A voltage-sensitive dye-based assay for the identification of differentiated neurons derived from embryonic neural stem cell cultures.

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    Richardson N Leão

    Full Text Available BACKGROUND: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC and embryonic neural stem cell (NSC cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca(2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. METHODOLOGY/PRINCIPAL FINDINGS: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD. We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. CONCLUSIONS/SIGNIFICANCE: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.

  6. Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures.

    Science.gov (United States)

    Pringle, A K; Sundstrom, L E; Wilde, G J; Williams, L R; Iannotti, F

    1996-06-28

    We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not neuroprotective. These data suggest that BDNF may form part of an endogenous neuroprotective mechanism.

  7. Differentiation of human bone marrow precursor cells into neuronal-like cells after transplantation into canine spinal cord organotypic slice cultures

    Institute of Scientific and Technical Information of China (English)

    FEI Zhi-qiang; XIONG Jian-yi; CHEN Lei; SHEN Hui-yong; Ngo Stephanie; Wang Jeffrey; WANG Da-ping

    2012-01-01

    Background Treatments to regenerate different tissue involving the transplantation of bone marrow derived mesenchymal precursor cells are anticipated.Using an alternative methods,in vitro organotypic slice culture method,would be useful to transplant cells and assessing the effects.This study was to determine the possibility of differentiating human bone marrow precursor cells into cells of the neuronal lineage by transplanting into canine spinal cord organotypic slice cultures.Methods Bone marrow aspirates were obtained from posterior superior iliac spine(PSIS)of patients that had undergone spinal fusion due to a degenerative spinal disorder.For cell imaging,mesenchymal precursor cells(MPCs)were pre-stained with PKH-26 just before transplantation to canine spinal cord slices.Canine spinal cord tissues were obtained from three adult beagle dogs.Spinal cords were cut into transverse slices of 1 mm using tissue chopper.Two slices were transferred into 6-well plate containing 3 ml DMEM with antibiotics.Prepared MPCs(1×104)were transplanted into spinal cord slices.On days 0,3,7,14,MPCs were observed for morphological changes and expression of neuronal markers through immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR).Results The morphological study showed:spherical cells in the control and experiment groups on day 0;and on day 3,cells in the control group had one or two thick,short processes and ones in the experiment group had three or four thin,long processes.On day 7,these variously-sized processes contacted each other in the experiment group,but showed typical spindle-shaped cells in the control group.Immunofluorescence showed that PKH-26(+)MPCs stained positive for NeuN(+)and GFAP(+)in experimental group only.Also RT-PCR showed weak expression of β-tubulinⅢ?and GFAP.Conclusions Human bone marrow mesenchymal precursor cells(hMPCs)have the potential to differentiate into the neuronal like cells in this canine spinal cord organotypic

  8. Neuronal Cell Death Induced by Mechanical Percussion Trauma in Cultured Neurons is not Preceded by Alterations in Glucose, Lactate and Glutamine Metabolism

    Science.gov (United States)

    Jayakumar, A. R.; Bak, L. K.; Rama Rao, K. V.; Waagepetersen, H.S.; Schousboe, A.; Norenberg, M.D.

    2016-01-01

    Traumatic brain injury (TBI) is a devastating neurological disorder that usually presents in acute and chronic forms. Brain edema and associated increased intracranial pressure in the early phase following TBI are major consequences of acute trauma. On the other hand, neuronal injury, leading to neurobehavioral and cognitive impairments, that usually develop months to years after single or repetitive episodes of head trauma, are major consequences of chronic TBI. The molecular mechanisms responsible for TBI-induced injury, however, are unclear. Recent studies have suggested that early mitochondrial dysfunction and subsequent energy failure play a role in the pathogenesis of TBI. We therefore examined whether oxidative metabolism of 13C-labeled glucose, lactate or glutamine is altered early following in vitro mechanical percussion-induced trauma (5 atm) to neurons (4–24 h), and whether such events contribute to the development of neuronal injury. Cell viability was assayed using the release of the cytoplasmic enzyme lactate dehydrogenase (LDH), together with fluorescence-based cell staining (calcein and ethidium homodimer-1 for live and dead cells, respectively). Trauma had no effect on the LDH release in neurons from 1 h to 18 h. However, a significant increase in LDH release was detected at 24 h after trauma. Similar findings were identified when traumatized neurons were stained with fluorescent markers. Additionally 13C-labeling of glutamate showed a small, but statistically significant decrease at 14 h after trauma. However, trauma had no effect on the cycling ratio of the TCA cycle at any time-period examined. These findings indicate that trauma does not cause a disturbance in oxidative metabolism of any of the substrates used for neurons. Accordingly, such metabolic disturbance does not appear to contribute to the neuronal death in the early stages following trauma. PMID:26729365

  9. Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases

    Science.gov (United States)

    Engstrom, Anna; Wang, Hao; Xia, Zhengui

    2015-01-01

    Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. PMID:25967738

  10. Evaluation of silicon nitride as a substrate for culture of PC12 cells: an interfacial model for functional studies in neurons.

    Directory of Open Access Journals (Sweden)

    Johan Jaime Medina Benavente

    Full Text Available Silicon nitride is a biocompatible material that is currently used as an interfacial surface between cells and large-scale integration devices incorporating ion-sensitive field-effect transistor technology. Here, we investigated whether a poly-L-lysine coated silicon nitride surface is suitable for the culture of PC12 cells, which are widely used as a model for neural differentiation, and we characterized their interaction based on cell behavior when seeded on the tested material. The coated surface was first examined in terms of wettability and topography using contact angle measurements and atomic force microscopy and then, conditioned silicon nitride surface was used as the substrate for the study of PC12 cell culture properties. We found that coating silicon nitride with poly-L-lysine increased surface hydrophilicity and that exposing this coated surface to an extracellular aqueous environment gradually decreased its roughness. When PC12 cells were cultured on a coated silicon nitride surface, adhesion and spreading were facilitated, and the cells showed enhanced morphological differentiation compared to those cultured on a plastic culture dish. A bromodeoxyuridine assay demonstrated that, on the coated silicon nitride surface, higher proportions of cells left the cell cycle, remained in a quiescent state and had longer survival times. Therefore, our study of the interaction of the silicon nitride surface with PC12 cells provides important information for the production of devices that need to have optimal cell culture-supporting properties in order to be used in the study of neuronal functions.

  11. Developmental exposure to ethanol increases the neuronal vulnerability to oxygen-glucose deprivation in cerebellar granule cell cultures.

    Science.gov (United States)

    Le Duc, Diana; Spataru, Ana; Ceanga, Mihai; Zagrean, Leon; Schöneberg, Torsten; Toescu, Emil C; Zagrean, Ana-Maria

    2015-07-21

    Prenatal alcohol exposure is associated with microencephaly, cognitive and behavioral deficits, and growth retardation. Some of the mechanisms of ethanol-induced injury, such as high level oxidative stress and overexpression of pro-apoptotic genes, can increase the sensitivity of fetal neurons towards hypoxic/ischemic stress associated with normal labor. Thus, alcohol-induced sequelae may be the cumulative result of direct ethanol toxicity and increased neuronal vulnerability towards metabolic stressors, including hypoxia. We examined the effects of ethanol exposure on the fetal cerebellar granular neurons' susceptibility to hypoxic/hypoglycemic damage. A chronic ethanol exposure covered the entire prenatal period and 5 days postpartum through breastfeeding, a time interval partially extending into the third-trimester equivalent in humans. After a binge-like alcohol exposure at postnatal day 5, glutamatergic cerebellar granule neurons were cultured and grown for 7 days in vitro, then exposed to a 3-h oxygen-glucose deprivation to mimic a hypoxic/ischemic condition. Cellular viability was monitored by dynamic recording of propidium iodide fluorescence over 20 h reoxygenation. We explored differentially expressed genes on microarray data from a mouse embryonic ethanol-exposure model and validated these by real-time PCR on the present model. In the ethanol-treated cerebellar granule neurons we find an increased expression of genes related to apoptosis (Mapk8 and Bax), but also of genes previously described as neuroprotective (Dhcr24 and Bdnf), which might suggest an actively maintained viability. Our data suggest that neurons exposed to ethanol during development are more vulnerable to in vitro hypoxia/hypoglycemia and have higher intrinsic death susceptibility than unexposed neurons.

  12. Group IIA secretory phospholipase A2 stimulates exocytosis and neurotransmitter release in pheochromocytoma-12 cells and cultured rat hippocampal neurons.

    Science.gov (United States)

    Wei, S; Ong, W Y; Thwin, M M; Fong, C W; Farooqui, A A; Gopalakrishnakone, P; Hong, W

    2003-01-01

    Recent evidence shows that secretory phospholipase A2 (sPLA2) may play a role in membrane fusion and fission, and may thus affect neurotransmission. The present study therefore aimed to elucidate the effects of sPLA2 on vesicle exocytosis. External application of group IIA sPLA2 (purified crotoxin subunit B or purified human synovial sPLA2) caused an immediate increase in exocytosis and neurotransmitter release in pheochromocytoma-12 (PC12) cells, detected by carbon fiber electrodes placed near the cells, or by changes in membrane capacitance of the cells. EGTA and a specific inhibitor of sPLA2 activity, 12-epi-scalaradial, abolished the increase in neurotransmitter release, indicating that the effect of sPLA2 was dependent on calcium and sPLA2 enzymatic activity. A similar increase in neurotransmitter release was also observed in hippocampal neurons after external application of sPLA2, as detected by changes in membrane capacitance of the neurons. In contrast to external application, internal application of sPLA2 to PC12 cells and neurons produced blockade of neurotransmitter release. Our recent studies showed high levels of sPLA2 activity in the normal rat hippocampus, medulla oblongata and cerebral neocortex. The sPLA2 activity in the hippocampus was significantly increased, after kainate-induced neuronal injury. The observed effects of sPLA2 on neurotransmitter release in this study may therefore have a physiological, as well as a pathological role.

  13. Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype

    Directory of Open Access Journals (Sweden)

    Kobeissy Firas H

    2010-02-01

    Full Text Available Abstract Background Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA. Results NMDA exposure induced light chain-3 (LC-3-immunopositive and monodansylcadaverine (MDC fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment. Conclusions Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.

  14. Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture

    DEFF Research Database (Denmark)

    Sandgren, Katarina; Lin, Zhong; Svenningsen, Åsa Fex;

    2003-01-01

    of VIP, NO donor, VIP antiserum, or NOS inhibitor. A marked loss of neurons was noted during culturing. VIP and NO significantly promoted neuronal survival. Corroborating this was the finding of an enhanced neuronal cell loss when cultures were grown in the presence of VIP antiserum or NOS inhibitor....... adaptation. The aim of this study was to evaluate whether VIP and nitric oxide (NO) influence survival of cultured, dissociated myenteric neurons. Neuronal survival was evaluated after 0, 4, and 8 days in culture. Influence of VIP and NO on neuronal survival was examined after culturing in the presence...

  15. Type 1 cannabinoid receptor ligands display functional selectivity in a cell culture model of striatal medium spiny projection neurons.

    Science.gov (United States)

    Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Dupré, Denis J; Denovan-Wright, Eileen M

    2014-09-05

    Modulation of type 1 cannabinoid receptor (CB1) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB1 are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC)) on arrestin2-, Gα(i/o)-, Gβγ-, Gα(s)-, and Gα(q)-mediated intracellular signaling in the mouse STHdh(Q7/Q7) cell culture model of striatal medium spiny projection neurons that endogenously express CB1. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα(i/o) and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB1 levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα(q)-dependent pathways. CP55,940 and CBD both signaled through Gα(s). CP55,940, but not CBD, activated downstream Gα(s) pathways via CB1 targets. THC and CP55,940 promoted CB1 internalization and decreased CB1 protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB1 leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.

  16. A Modified Technique for Culturing Primary Fetal Rat Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Sui-Yi Xu

    2012-01-01

    Full Text Available The study explored a modified primary culture system for fetal rat cortical neurons. Day E18 embryos from pregnant Sprague Dawley rats were microdissected under a stereoscope. To minimize enzymatic damage to the cultured neurons, we applied a sequential digestion protocol using papain and Dnase I. The resulting sifted cell suspension was seeded at a density of 50,000 cells per cm2 onto 0.1 mg/mL L-PLL-covered vessels. After a four-hour incubation in high-glucose Dulbecco’s Modified Eagle’s Medium (HG-DMEM to allow the neurons to adhere, the media was changed to neurobasal medium that was refreshed by changing half of the volume after three days followed by a complete medium change every week. The cells displayed progressively robust neurite extension, and nonneuronal-like cells could barely be detected by five days in vitro (DIV; cell growth was still substantial at 14 DIV. Neurons were identified by β-tubulin III immunofluorescence, and neuronal purity within the cultures was assessed at over 95% by both flow cytometry and by dark-field counting of β-tubulin III-positive cells. These results suggest that the protocol was successful and that the high purity of neurons in this system could be used as the basis for generating various cell models of neurological disease.

  17. Interactions between neuronal and non-neuronal cells in adult rat isolated dorsal root ganglion cells

    Institute of Scientific and Technical Information of China (English)

    NG K Y; WONG Y H; WISE H

    2008-01-01

    Objective The glial cells of the central nervous system are involved in tripartite signaling, therefore we have been investigating the relationship between sensory neurons and non-neuronal cells in isolated preparations of dorsal root ganglia (DRG). Methods The mixed cell cultures of dissociated DRG cells were separated to yield enriched fractions of IB4-positive cells (small diameter, non-peptidergic cells), IB4-negative cells (small diameter, peptidergic cells, and large diameter cells), and non-neuronal cells (principally satellite glial cells, Schwarm cells and fibroblasts). Adenylyl cyclase activity was assayed by measuring production of [3H]cAMP from cells preloaded with [3H]adenine. Results PGE2 and the PGI2 mimetic eicaprost stimulated adenylyl cyclase activity which was inhibited by ONO-AE3-208 (EP4 antagonist) or CAY10441 (IP antagonist) with estimated pA2 values of 8.9 and 8.2, respectively. Surprisingly, both PGE2 and cicaprost-stimulated [3H] cAMP production was greatest in the non-neuronal cell preparation. Furthermore, when the number of non-neuronal cells was kept constant and the number of neuronal cells was increased, we observed a progressive decrease in prostanoid-stimulated activity. Conclusions Sensory neurons appear to regulate prostanoid receptor-mediated cell signaling in non-neuronal cells within the DRG.

  18. The experimental study of the damage of environmental neurotoxins on the cultured rat dopaminergic neurons

    Institute of Scientific and Technical Information of China (English)

    WANG Jian; LU Chuanzhen; JIANG Yuping

    2000-01-01

    Objective To establish the culture system of rat dopaminergic neurons. and to determine whether Paraquat and Dieldrin selectively destroy cultured rat dopaminergic neurons respectively. Methods The cultured rat dopaminergic neurons were treated for 24h with Paraquat and Dieldrin(0.001 to 100 μ mol/L) respectively, Data were expressed as percentage of surviving TH-positive(TH+) cells and other cells per culture dish. Results Paraquat was not effective in selectively destroying TH+ neurons. Dieldrin (1 μ mol/L) selectively decreased the number of TH+ neurons without affecting other cells. The EC50 of Dieldrin on TH+ neurons was 27.6 l mol/L. Conclusion: Paraquat can not selectively destroy dopaminergic neurons in culture. Dieldrin (1 μ mol/L) can selectively destroy the dopaminergic neurons in culture, which make it a potential etiological agent for PD. The possible parkinsonogenic effect of Dieldrin is deserved for further investigation.

  19. Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies.

    Science.gov (United States)

    Kanski, Jaroslaw; Aksenova, Marina; Stoyanova, Antonia; Butterfield, D Allan

    2002-05-01

    In this study, free radical scavenging abilities of ferulic acid in relation to its structural characteristics were evaluated in solution, cultured neurons, and synaptosomal systems exposed to hydroxyl and peroxyl radicals. Cultured neuronal cells exposed to the peroxyl radical initiator AAPH die in a dose-response manner and show elevated levels of protein carbonyls. The presence of ferulic acid or similar phenolic compounds, however, greatly reduces free radical damage in neuronal cell systems without causing cell death by themselves. In addition, synaptosomal membrane systems exposed to oxidative stress by hydroxyl and peroxyl radical generators show elevated levels of oxidation as indexed by protein oxidation, lipid peroxidation, and ROS measurement. Ferulic acid greatly attenuates these changes, and its effects are far more potent than those obtained for vanillic, coumaric, and cinnamic acid treatments. Moreover, ferulic acid protects against free radical mediated changes in conformation of synaptosomal membrane proteins as monitored by EPR spin labeling techniques. The results presented in this study suggest the importance of naturally occurring antioxidants such as ferulic acid in therapeutic intervention methodology against neurodegenerative disorders such as Alzheimer's disease in which oxidative stress is implicated.

  20. Clivorine, an otonecine pyrrolizidine alkaloid from Ligularia species, impairs neuronal differentiation via NGF-induced signaling pathway in cultured PC12 cells.

    Science.gov (United States)

    Xiong, Aizhen; Yan, Artemis Lu; Bi, Cathy W C; Lam, Kelly Y C; Chan, Gallant K L; Lau, Kitty K M; Dong, Tina T X; Lin, Huangquan; Yang, Li; Wang, Zhengtao; Tsim, Karl W K

    2016-08-15

    Pyrrolizidine alkaloids (PAs) are commonly found in many plants including those used in medical therapeutics. The hepatotoxicities of PAs have been demonstrated both in vivo and in vitro; however, the neurotoxicities of PAs are rarely mentioned. In this study, we aimed to investigate in vitro neurotoxicities of clivorine, one of the PAs found in various Ligularia species, in cultured PC12 cells. PC12 cell line was employed to first elucidate the neurotoxicity and the underlying mechanism of clivorine, including cell viability and morphology change, neuronal differentiation marker and signaling pathway. PC12 cells were challenged with series concentrations of clivorine and/or nerve growth factor (NGF). The cell lysates were collected for MTT assay, trypan blue staining, immunocytofluorescent staining, qRT-PCR and western blotting. Clivorine inhibited cell proliferation and neuronal differentiation evidenced by MTT assay and dose-dependently reducing neurite outgrowth, respectively. In addition, clivorine decreased the level of mRNAs encoding for neuronal differentiation markers, e.g. neurofilaments and TrkA (NGF receptor). Furthermore, clivorine reduced the NGF-induced the phosphorylations of TrkA, protein kinase B and cAMP response element-binding protein in cultured PC12 cells. Taken together, our results suggest that clivorine might possess neurotoxicities in PC12 cells via down-regulating the NGF/TrkA/Akt signaling pathway. PAs not only damage the liver, but also possess neurotoxicities, which could possibly result in brain disorders, such as depression. Copyright © 2016 Elsevier GmbH. All rights reserved.

  1. Physiological maturation and drug responses of human induced pluripotent stem cell-derived cortical neuronal networks in long-term culture.

    Science.gov (United States)

    Odawara, A; Katoh, H; Matsuda, N; Suzuki, I

    2016-05-18

    The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20-30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

  2. Protective effect of keishi-bukuryo-gan and its constituent medicinal plants against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells.

    Science.gov (United States)

    Shimada, Y; Yokoyama, K; Goto, H; Sekiya, N; Mantani, N; Tahara, E; Hikiami, H; Terasawa, K

    2004-07-01

    Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medicinal plants against NO donors, sodium nitroprusside (SNP) and 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC18)-induced neuronal death in cultured rat cerebellar granule cells (CGCs). MTT assay showed cell viability to be significantly increased by the addition of KBG extract (KBGE) (100 microg/ml), Cinnamomi Cortex extract (CCE) (3, 10 and 30 microg/ml), Paeoniae Radix extract (PRE) (100 microg/ml) and Moutan Cortex extract (MCE) (10 and 30 microg/ml) compared with exposure to SNP (30 microM, 24 h) only. Also, cell viability was significantly increased by the addition of KBGE (100 and 300 microg/ml), CCE (30 and 100 microg/ml), PRE (100 and 300 microg/ml) and MCE (30 and 100 microg/ml) compared with exposure to NOC 18 (100 microM, 48 h) only. Persicae Semen extract and Hoelen extract did not protect against NO donor-induced neuronal death. These results suggest that KBG has protective effect against NO-mediated neuronal death in cultured CGCs and that it is derived from Cinnamomi Cortex, Paeoniae Radix and Moutan Cortex.

  3. Inhibitory effects of multiwall carbon nanotubes with high iron impurity on viability and neuronal differentiation in cultured PC12 cells.

    Science.gov (United States)

    Meng, Li; Jiang, Aihua; Chen, Rui; Li, Chen-zhong; Wang, Liming; Qu, Ying; Wang, Peng; Zhao, Yuliang; Chen, Chunying

    2013-11-08

    The increasing use of carbon nanotubes (CNTs) in biomedical applications has garnered a great concern on their potential negative effects to human health. CNTs have been reported to potentially disrupt normal neuronal function and they were speculated to accumulate and cause brain damage, although a lot of distinct and exceptional properties and potential wide applications have been associated with this material in neurobiology. Fe impurities strapped inside the CNTs may be partially responsible for neurotoxicity generation. In the present study, we selected rat pheochromocytoma (PC12) cells to investigate and compare the effects of two kinds of multiwall carbon nanotubes (MWCNTs) with different concentrations of Fe impurities which usually come from the massive production of CNTs by chemical vapor deposition. Exposure to Fe-high MWCNTs can reduce cell viability and increase cytoskeletal disruption of undifferentiated PC12 cells, diminish the ability to form mature neurites, and then adversely influence the neuronal dopaminergic phenotype in NGF-treated PC-12 cells. The present results highlight the critical role of iron residue in the adverse response to MWCNTs exposure in neural cells. These findings provide useful information for understanding the toxicity and safe application of carbon nanotubes.

  4. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    Science.gov (United States)

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  5. Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

    Science.gov (United States)

    Sheikh, Muhammad Abid; Malik, Yousra Saeed; Xing, Zhenkai; Guo, Zhaopei; Tian, Huayu; Zhu, Xiaojuan; Chen, Xuesi

    2017-05-01

    Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor deficits which result from the progressive loss of dopaminergic neurons. Gene therapy using growth factors such as VEGF seems to be a viable approach for potential therapeutic treatment of PD. In this study, we utilized a novel non-viral gene carrier designated as PEI-PLL synthesized by our laboratory to deliver VEGF gene to study its effect by using both cell culture as well as animal models of PD. For cell culture experiments, we utilized 6-hydroxydopamine (6-OHDA) mediated cell death model of MN9D cells following transfection with either a control plasmid or VEGF expressing plasmid. As compared to control transfected cells, PEI-PLL mediated VEGF gene delivery to MN9D cells resulted in increased cell viability, increase in the number of Tyrosine hydroxylase (TH) positive cells and decreased apoptosis following 6-OHDA insult. Next, we studied the therapeutic potential of PEI-PLL mediated VEGF gene delivery in SNPc by using unilateral 6-OHDA Medial forebrain bundle (MFB) lesion model of PD in rats. VEGF administration prevented the loss of motor functions induced by 6-OHDA as determined by behavior analysis. Similarly, VEGF inhibited the 6-OHDA mediated loss of DA neurons in Substantia Nigra Pars Compacta (SNPc) as well as DA nerve fibers in striatum as determined by TH immunostaining. In addition, PEI-PLL mediated VEGF gene delivery also prevented apoptosis and microglial activation in PD rat models. Together, these results clearly demonstrated the beneficial effects of PEI-PLL mediated VEGF gene delivery on dopaminergic system in both cell culture and animal models of PD. In this report, we exploited the potential of PEI-PLL to deliver VEGF gene for the potential therapeutic treatment of PD by using both cell culture and animal models of PD. To the best of our knowledge, this is the first report describing the use of novel polymeric gene carriers for the delivery of VEGF gene

  6. Stearic acid protects primary cultured cortical neurons against oxidative stress

    Institute of Scientific and Technical Information of China (English)

    Ze-jian WANG; Cui-ling LIANG; Guang-mei LI; Cai-yi YU; Ming YIN

    2007-01-01

    Aim: To observe the effects of stearic acid against oxidative stress in primary cultured cortical neurons. Methods: Cortical neurons were exposed to glutamate,hydrogen peroxide (H202), or NaN3 insult in the presence or absence of stearic acid. Cell viability of cortical neurons was determined by MTT assay and LDH release. Endogenous antioxidant enzymes activity[superoxide dismutases (SOD),glutathione peroxidase (GSH-Px), and catalase (CAT)] and lipid peroxidation in cultured cortical neurons were evaluated using commercial kits. {3-[1(p-chloro-benzyl)-5-(isopropyl)-3-t-butylthiondol-2-yl]-2,2-dimethylpropanoic acid, Na}[MK886; 5 pmol/L; a noncompetitive inhibitor of proliferator-activated receptor(PPAR)α], bisphenol A diglycidyl ether (BADGE; 100 μmol/L; an antagonist of PPARγ), and cycloheximide (CHX; 30 μmol/L, an inhibitor of protein synthesis)were tested for their effects on the neuroprotection afforded by stearic acid.Western blotting was used to determine the PPARγ protein level in cortical neurons.Results: Stearic acid dose-dependently protected cortical neurons against glutamate or H202 injury and increased glutamate uptake in cultured neurons.This protection was concomitant to the inhibition of lipid peroxidation and to the promotion activity of Cu/Zn SOD and CAT in cultured cortical neurons. Its neuroprotective effects were completely blocked by BADGE and CHX. After incubation with H2O2 for 24 h, the expression of the PPARγ protein decreased significantly (P<0.05), and the inhibitory effect of H2O2 on the expression of PPARγ can be attenuated by stearic acid. Conclusion: Stearic acid can protect cortical neurons against oxidative stress by boosting the internal antioxidant enzymes.Its neuroprotective effect may be mainly mediated by the activation of PPARγ and new protein synthesis in cortical neurons.

  7. Optophysiological approach to resolve neuronal action potentials with high spatial and temporal resolution in cultured neurons

    Directory of Open Access Journals (Sweden)

    Stephane ePages

    2011-10-01

    Full Text Available Cell to cell communication in the central nervous system is encoded into transient and local membrane potential changes (ΔVm. Deciphering the rules that govern synaptic transmission and plasticity entails to be able to perform Vm recordings throughout the entire neuronal arborization. Classical electrophysiology is, in most cases, not able to do so within small and fragile neuronal subcompartments. Thus, optical techniques based on the use of fluorescent voltage-sensitive dyes (VSDs have been developed. However, reporting spontaneous or small ΔVm from neuronal ramifications has been challenging, in part due to the limited sensitivity and phototoxicity of VSD-based optical measurements. Here we demonstrate the use of water soluble VSD, ANNINE-6plus, with laser scanning microscopy to optically record ΔVm in cultured neurons. We show that the sensitivity (> 10 % of fluorescence change for 100 mV depolarization and time response (submillisecond of the dye allows the robust detection of action potentials (APs even without averaging, allowing the measurement of spontaneous neuronal firing patterns. In addition, we show that back-propagating APs can be recorded, along distinct dendritic sites and within dendritic spines. Importantly, our approach does not induce any detectable phototoxic effect on cultured neurons. This optophysiological approach provides a simple, minimally invasive and versatile optical method to measure electrical activity in cultured neurons with high temporal (ms resolution and high spatial (µm resolution.

  8. Simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell cultures and in sub-regions of guinea pig brain.

    Science.gov (United States)

    Schou-Pedersen, Anne Marie V; Hansen, Stine N; Tveden-Nyborg, Pernille; Lykkesfeldt, Jens

    2016-08-15

    In the present paper, we describe a validated chromatographic method for the simultaneous quantification of monoamine neurotransmitters and their biogenic metabolites intracellularly and extracellularly in primary neuronal cell culture and in sub-regions of the guinea pig brain. Electrochemical detection provided limits of quantifications (LOQs) between 3.6 and 12nM. Within the linear range, obtained recoveries were from 90.9±9.9 to 120±14% and intra-day and inter-day precisions found to be less than 5.5% and 12%, respectively. The analytical method was applicable for quantification of intracellular and extracellular amounts of monoamine neurotransmitters and their metabolites in guinea pig frontal cortex and hippocampal primary neuronal cell cultures. Noradrenaline, dopamine and serotonin were found to be in a range from 0.31 to 1.7pmol per 2 million cells intracellularly, but only the biogenic metabolites could be detected extracellularly. Distinct differences in monoamine concentrations were observed when comparing concentrations in guinea pig frontal cortex and cerebellum tissue with higher amounts of dopamine and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid in frontal cortex, as compared to cerebellum. The chemical turnover in frontal cortex tissue of guinea pig was for serotonin successfully predicted from the turnover observed in the frontal cortex cell culture. In conclusion, the present analytical method shows high precision, accuracy and sensitivity and is broadly applicable to monoamine measurements in cell cultures as well as brain biopsies from animal models used in preclinical neurochemistry.

  9. Cell biology of neuronal endocytosis.

    Science.gov (United States)

    Parton, R G; Dotti, C G

    1993-09-01

    Endocytosis is the process by which cells take in fluid and components of the plasma membrane. In this way cells obtain nutrients and trophic factors, retrieve membrane proteins for degradation, and sample their environment. In neuronal cells endocytosis is essential for the recycling of membrane after neurotransmitter release and plays a critical role during early developmental stages. Moreover, alterations of the endocytic pathway have been attributed a crucial role in the pathophysiology of certain neurological diseases. Although well characterized at the ultrastructural level, little is known of the dynamics and molecular organization of the neuronal endocytic pathways. In this respect most of our knowledge comes from studies of non-neuronal cells. In this review we will examine the endocytic pathways in neurons from a cell biological viewpoint by making comparisons with non-neuronal cells and in particular with another polarized cell, the epithelial cell.

  10. GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived hippocampal slice cultures: a possible effect of glutamate transporter up-regulation.

    Science.gov (United States)

    Bonde, C; Sarup, A; Schousboe, A; Gegelashvili, G; Noraberg, J; Zimmer, J

    2003-01-01

    Besides its neurotrophic and neuroprotective effects on dopaminergic neurons and spinal motoneurons, glial cell line-derived neurotrophic factor (GDNF) has potent neuroprotective effects in cerebral ischemia. The protective effect has so far been related to reduced activation of N-methyl-D-aspartate receptors (NMDAr). This study tested the effects of GDNF on glutamate transporter expression, with the hypothesis that modulation of glutamate transporter activity would affect the outcome of cerebral ischemia. Organotypic hippocampal slice cultures, derived from 1-week-old rats, were treated with 100 ng/ml GDNF for either 2 or 5 days, followed by Western blot analysis of NMDAr subunit 1 (NR1) and two glutamate transporter subtypes, GLAST and GLT-1. After 5-day exposure to GDNF, expression of GLAST and GLT-1 was up-regulated to 169 and 181% of control values, respectively, whereas NR1 was down-regulated to 64% of control. However, despite these changes that potentially would support neuronal resistance to excitotoxicity, the long-term treatment with GDNF was found to aggravate the neuronal damage induced by oxygen-glucose deprivation (OGD). The increased cell death, assessed by propidium iodide (PI) uptake, occurred not only among the most susceptible CA1 pyramidal cells, but also in CA3 and fascia dentata. Given that glutamate transporters are able to release glutamate by reversed action during energy failure, it is suggested that the observed increase in OGD-induced cell death in the GDNF-pretreated cultures was caused by the build-up of excitotoxic concentrations of extracellular glutamate released through the glutamate transporters, which were up-regulated by GDNF. Although the extent and consequences of glutamate release via reversal of GLAST and GLT-1 transporters seem to vary in different energy failure models, the present findings should be taken into account in clinical trials of GDNF.

  11. Molluscan cells in culture: primary cell cultures and cell lines.

    Science.gov (United States)

    Yoshino, T P; Bickham, U; Bayne, C J

    2013-06-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

  12. Effects of glutamate and nimodipine on survival rate of embryonic rat neuronal stem cells cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiaohong Lü; Hailong Fu; Qiang Sun; Li Cui; Dihui Ma; Weihong Lin

    2008-01-01

    BACKGROUND: At least three types of calcium ion channel (T, N, and L) have been recognized in nerve cells, but only the L type of channel is sensitive to drugs. Theoretically, nimodipine can lead to L-type channel inactivation and prevent calcium ion inflow, thereby exhibiting protective effects on nerve cells.OBJECTIVE: To observe the protective effects of nimodipine on glutamate-induced injury to embryonic rat neural stem cells, and to make a comparison with MK-801, a nonselective glutamate receptor antagonist.DESIGN, TIME AND SETTING: The present in vitro experiment pertaining to neural stem cells was performed at the Department of Neurology, First Hospital, Jilin University between January 2005 and December 2006.MATERIALS: Glutamate was sourced from the Shanghai Biological Research Institute of the Chinese Academy of Sciences. Nimodipine was provided by Bayer Company, Germany. Brain tissue was taken from Wistar rats on day 15 of gestation for isolation and culture of neural stem cells.METHODS: Passage 2 neural stem cell spheres were taken for preparation of single cell suspension. The prepared single cell suspension was divided into 4 groups: (1) Normal control, normally cultured. (2)Glutamate, cultured with 50, 100, 200, 500, and 1000 μ mol/L glutamate. (3) Nimodipine, received a 30-minute nimodipine [1 ×(10-8-10-2) g/L] culture followed by a glutamate (200 μ mol/L) treatment step. (4)MK-801, given as 30- minute MK-801 (100 μ mol/L) culture, followed by a glutamate (200 μ mol/L)treatment step.MAIN OUTCOME MEASURES: Determination of glutamate-induced cell death by methyl thiazolyl tetrazolium (MTT) assay; calculation of neural stem cell survival rate following addition of nimodipine.RESULTS: The survival rate of neural stem cells was approximately 25.26% following 24 hour 50 μ mol/L glutamate culture and gradually decreased as the glutamate dose increased (P < 0.05 0.01). Only 9.27% of neural stem cells survived when the glutamate dose was 1 000 μ mol

  13. Biased Type 1 Cannabinoid Receptor Signaling Influences Neuronal Viability in a Cell Culture Model of Huntington Disease.

    Science.gov (United States)

    Laprairie, Robert B; Bagher, Amina M; Kelly, Melanie E M; Denovan-Wright, Eileen M

    2016-03-01

    Huntington disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder with limited treatment options. Prior to motor symptom onset or neuronal cell loss in HD, levels of the type 1 cannabinoid receptor (CB1) decrease in the basal ganglia. Decreasing CB1 levels are strongly correlated with chorea and cognitive deficit. CB1 agonists are functionally selective (biased) for divergent signaling pathways. In this study, six cannabinoids were tested for signaling bias in in vitro models of medium spiny projection neurons expressing wild-type (STHdh(Q7/Q7)) or mutant huntingtin protein (STHdh(Q111/Q111)). Signaling bias was assessed using the Black and Leff operational model. Relative activity [ΔlogR (τ/KA)] and system bias (ΔΔlogR) were calculated relative to the reference compound WIN55,212-2 for Gαi/o, Gαs, Gαq, Gβγ, and β-arrestin1 signaling following treatment with 2-arachidonoylglycerol (2-AG), anandamide (AEA), CP55,940, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD), and THC+CBD (1:1), and compared between wild-type and HD cells. The Emax of Gαi/o-dependent extracellular signal-regulated kinase (ERK) signaling was 50% lower in HD cells compared with wild-type cells. 2-AG and AEA displayed Gαi/o/Gβγ bias and normalized CB1 protein levels and improved cell viability, whereas CP55,940 and THC displayed β-arrestin1 bias and reduced CB1 protein levels and cell viability in HD cells. CBD was not a CB1 agonist but inhibited THC-dependent signaling (THC+CBD). Therefore, enhancing Gαi/o-biased endocannabinoid signaling may be therapeutically beneficial in HD. In contrast, cannabinoids that are β-arrestin-biased--such as THC found at high levels in modern varieties of marijuana--may be detrimental to CB1 signaling, particularly in HD where CB1 levels are already reduced.

  14. Bilaminar co-culture of primary rat cortical neurons and glia.

    Science.gov (United States)

    Shimizu, Saori; Abt, Anna; Meucci, Olimpia

    2011-11-12

    This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons. Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection. A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions(1). At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods(1-3). Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology(4), cellular and molecular biology(5-8), biochemistry(5), imaging and microscopy(4,6,7,9,10). The primary neurons extend axons and dendrites to form functional synapses(11), a process which is not observed in neuronal cell lines, although some

  15. Minimum neuron density for synchronized bursts in a rat cortical culture on multi-electrode arrays.

    Science.gov (United States)

    Ito, D; Tamate, H; Nagayama, M; Uchida, T; Kudoh, S N; Gohara, K

    2010-11-24

    To investigate the minimum neuron and neurite densities required for synchronized bursts, we cultured rat cortical neurons on planar multi-electrode arrays (MEAs) at five plating densities (2500, 1000, 500, 250, and 100 cells/mm(2)) using two culture media: Neuron Culture Medium and Dulbecco's Modified Eagle Medium supplemented with serum (DMEM/serum). Long-term recording of spontaneous electrical activity clarified that the cultures exhibiting synchronized bursts required an initial plating density of at least 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum. Immediately after electrical recording, immunocytochemistry of microtubule-associated protein 2 (MAP2) and Neurofilament 200 kD (NF200) was performed directly on MEAs to investigate the actual densities of neurons and neurites forming the networks. Immunofluorescence observation revealed that the construction of complicated neuronal networks required the same initial plating density as for synchronized bursts, and that overly sparse cultures showed significant decreases of neurons and neurites. We also found that the final densities of surviving neurons at 1 month decreased greatly compared with the initial plating densities and became saturated in denser cultures. In addition, the area of neurites and the number of nuclei were saturated in denser cultures. By comparing both the results of electrophysiological recording and immunocytochemical observation, we revealed that there is a minimum threshold of neuron densities that must be met for the exhibition of synchronized bursts. Interestingly, these minimum densities of MAP2-positive final neurons did not differ between the two culture media; the density was approximately 50 neurons/mm(2). This value was obtained in the cultures with the initial plating densities of 250 cells/mm(2) for Neuron Culture Medium and 500 cells/mm(2) for DMEM/serum.

  16. A novel method for three-dimensional culture of central nervous system neurons.

    Science.gov (United States)

    Puschmann, Till B; de Pablo, Yolanda; Zandén, Carl; Liu, Johan; Pekny, Milos

    2014-06-01

    Neuronal signal transduction and communication in vivo is based on highly complex and dynamic networks among neurons expanding in a three-dimensional (3D) manner. Studies of cell-cell communication, synaptogenesis, and neural network plasticity constitute major research areas for understanding the involvement of neurons in neurodegenerative diseases, such as Huntington's, Alzheimer's, and Parkinson's disease, and in regenerative neural plasticity responses in situations, such as neurotrauma or stroke. Various cell culture systems constitute important experimental platforms to study neuronal functions in health and disease. A major downside of the existing cell culture systems is that the alienating planar cell environment leads to aberrant cell-cell contacts and network formation and increased reactivity of cell culture-contaminating glial cells. To mimic a suitable 3D environment for the growth and investigation of neuronal networks in vitro has posed an insurmountable challenge. Here, we report the development of a novel electrospun, polyurethane nanofiber-based 3D cell culture system for the in vitro support of neuronal networks, in which neurons can grow freely in all directions and form network structures more complex than any culture system has so far been able to support. In this 3D system, neurons extend processes from their cell bodies as a function of the nanofiber diameter. The nanofiber scaffold also minimizes the reactive state of contaminating glial cells.

  17. Human embryonic stem cells for neuronal repair.

    Science.gov (United States)

    Ben-Hur, Tamir

    2006-02-01

    Human embryonic stem cells may serve as a potentially endeless source of transplantable cells to treat various neurologic disorders. Accumulating data have shown the therapeutic value of various neural precursor cell types in experimental models of neurologic diseases. Tailoring cell therapy for specific disorders requires the generation of cells that are committed to specific neural lineages. To this end, protocols were recently developed for the derivation of dopaminergic neurons, spinal motor neurons and oligodendrocytes from hESC. These protocols recapitulate normal development in culture conditions. However, a novel concept emerging from these studies is that the beneficial effect of transplanted stem cells is not only via cell replacement in damaged host tissue, but also by trophic and protective effects, as well as by an immunomodulatory effect that down-regulates detrimental brain inflammation.

  18. An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

    Directory of Open Access Journals (Sweden)

    Ying Huang

    2013-11-01

    Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

  19. Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System.

    Science.gov (United States)

    Hu, Wentao; He, Yongpei; Xiong, Yongjie; Lu, Hong; Chen, Hong; Hou, Limin; Qiu, Zhandong; Fang, Yu; Zhang, Suming

    2016-04-01

    Induced pluripotent stem cells (iPSCs) generated from patient-derived somatic cells provides the opportunity for model development in order to study patient-specific disease states with the potential for drug discovery. However, use of lentivirus and exposure of iPSCs to animal-derived products limit their therapeutic utility and affect lineage differentiation and subsequent downstream functionality of iPSC derivatives. Within the context of this study, we describe a simple and practical protocol enabling the efficient reprogramming of terminally differentiated adult fibroblasts into integration-free human iPSCs (hiPSCs) using a combination of episomal plasmids with small molecules (SMs). Using this approach, there was a 10-fold increase in reprogramming efficiency over single plasmid vector-based methods. We obtained approximately 100 iPSCs colonies from 1 × 10(5) human adult dermal fibroblasts (HADFs) and achieved approximately 0.1% reprogramming efficiencies. Concurrently, we developed a highly conducive culture system using xeno-free media and human vitronectin. The resulting hiPSCs were free of DNA integration and had completely lost episomal vectors, maintained long-term self-renewal, featured a normal karyotype, expressed pluripotent stem cell markers, and possessed the capability of differentiating into components of all three germ layers in vivo. Finally, we demonstrate that the integration-free hiPSCs could be differentiated into motor neurons under xeno-free culture conditions. This induction method will promote the derivation of patient-specific integration-free and xeno-free iPSCs and improve the strategy for motor neuron derivation. Our approach provides a useful tool for human disease models, drug screen, and clinical applications.

  20. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    Science.gov (United States)

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  1. Bis(7)-Tacrine, a Promising Anti-Alzheimer's Agent ,Attenuates Glutamate-Induced Cell Injury in Primary Cultured Cerebrocortical Neurons of Rats

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The effects of bis(7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on gluta mate-induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0. 5 mmol/L glutamate for 30 min resulted in significant cell damage. Pre treatment with bis (7)-tacrine (0. 03-1.0 μmol/L) reduced the glutamate-induced neurotoxicity in a concentra tion dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection.A receptor binding assay showed that bis(7)-tacrine can completely displace MK-801 binding to rat cortical membrane with an IC50 of 0. 57 μmol/L. These findings suggest that bis(7)-tacrine can directly interact with N-methyl-D-aspartate receptor channel complex, which may contribute to the inhibitor's protective effects a gainst glutamate-induced excitotoxicity. Thus, it is possible that anti-glutamate/anti-AChE synergism is re sponsible for potentially better Alzheimer's therapy of bis(7)-tacrine relative to tacrine.

  2. L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells.

    Science.gov (United States)

    Audesirk, G; Audesirk, T; Ferguson, C; Lomme, M; Shugarts, D; Rosack, J; Caracciolo, P; Gisi, T; Nichols, P

    1990-08-01

    The intracellular free Ca2+ concentration, [Ca2+]i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca2+]i is partly a function of Ca2+ influx through voltage-sensitive calcium channels (VSCC), Ca2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd2+, Ni2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd2+ (less than 50 microM), which have been reported to inhibit L-type channels, reduced neurite initiation, but not mean neurite length. Ni2+ (less than 100 microM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (less than 300 microM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (great than 300 microM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 microM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 microM), Cd2+ (5 microM), and streptomycin (200 microM) reduced neurite initiation, while nickel (50 microM) and neomycin (100 microM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd2

  3. [A protocol for primary dissociated astrocyte and neuron co-culture].

    Science.gov (United States)

    Shi, Ying; Zhou, Mi; Jiang, Min

    2013-02-25

    Cultured primary hippocampal neurons are ideal tool for investigating the subcellular localization and trafficking of neuronal proteins. The aim of the present study was to establish a method to co-culture hippocampal neurons and cortical astrocytes, which would guarantee well conditions of neurons. Newborn Sprague-Dawley (SD) rats were sacrificed by decapitation. The cortex of cerebrum was cut into pieces, and the cortical tissue was digested with trypsin. The liquid suspension of single cells was planted onto a 25 cm² culture flask. On the fourth day of culture, the tissue cells except astrocytes were removed by intensive agitation of culture flask. Purified astrocytes were allowed to grow continuously until they reached most area of flask. At this time point, we replaced the culture media with neuronal cell media containing cytarabine, and planted the primary culture of rat hippocampal neurons onto the feed layer of cortical astrocytes. The microscopic observation results showed that, the astrocytes evenly grew without obvious boundaries between each other, and exhibited good purity. The co-cultured hippocampal neurons were in good condition, developed intertwined network of axons and dendrites, lived for a long time, and could tolerate gene transfection. Above all, this method is relatively simple from a technical point of view, yet provides healthy and reliable neuronal culture.

  4. Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19)

    National Research Council Canada - National Science Library

    Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    ...) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19...

  5. Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen-glucose deprivation of hippocampal slice cultures.

    Science.gov (United States)

    Bonde, C; Noraberg, J; Noer, H; Zimmer, J

    2005-01-01

    Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen-glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen-glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen-glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen-glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen-glucose deprivation induced necrotic cell death only. Addition of 10 microM of the N-methyl-D-aspartate glutamate receptor antagonist MK-801, and 20 microM of the non-N-methyl-D-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl

  6. Selective regulation of current densities underlies spontaneous changes in the activity of cultured neurons.

    Science.gov (United States)

    Turrigiano, G; LeMasson, G; Marder, E

    1995-05-01

    We study the electrical activity patterns and the expression of conductances in adult stomatogastric ganglion (STG) neurons as a function of time in primary cell culture. When first plated in culture, these neurons had few active properties. After 1 d in culture they produced small action potentials that rapidly inactivated during maintained depolarization. After 2 d in culture they fired large action potentials tonically when depolarized, and their properties resembled very closely the properties of STG neurons pharmacologically isolated in the ganglion. After 3-4 d in culture, however, their electrical properties changed and they fired in bursts when depolarized. We characterized the currents expressed by these neurons in culture. They included two TTX-sensitive sodium currents, a calcium current, a delayed-rectifier-like current, a calcium-dependent potassium current, and two A-type currents. The changes in firing properties with time in culture were accompanied by an increase in inward and decrease in outward current densities. A single-compartment conductance-based model of an STG neuron was constructed by fitting the currents measured in the biological neurons. When the current densities in the model neuron were matched to those measured for the biological neurons in each activity state, the model neuron closely reproduced each state, indicating that the changes in current densities are sufficient to account for the changes in intrinsic properties. These data indicate that STG neurons isolated in culture change their intrinsic electrical properties by selectively adjusting the magnitudes of their ionic conductances.

  7. Electrical signals polarize neuronal organelles, direct neuron migration, and orient cell division.

    Science.gov (United States)

    Yao, Li; McCaig, Colin D; Zhao, Min

    2009-09-01

    During early brain development, the axis of division of neuronal precursor cells is regulated tightly and can determine whether neurons remain in the germinal layers or migrate away. Directed neuronal migration depends on the establishment of cell polarity, and cells are polarized dynamically in response to extracellular signals. Endogenous electric fields (EFs) orient cell division and direct migration of a variety of cell types. Here, we show that cell division of cultured hippocampal cells (neuron-like cells and glial-like cells) is oriented strikingly by an applied EF, which also directs neuronal migration. Directed migration involves polarization of the leading neurite, of the microtubule-associated protein MAP-2 and of the Golgi apparatus and the centrosome, all of which reposition asymmetrically to face the cathode. Pharmacological inhibition of Rho-associated coiled-coil forming protein kinases (ROCK) and phosphoinositide 3-kinase decreased, leading neurite orientation and Golgi polarization in the neurons in response to an EF and in parallel decreased the directedness of EF-guided neuronal migration. This work demonstrates that the axis of hippocampal cell division, the establishment of neuronal polarity, the polarization of intracellular structures, and the direction of neuronal migration are all regulated by an extracellular electrical cue.

  8. An Optimized Culture Method of Rat Dorsal Root Ganglion Neurons

    Institute of Scientific and Technical Information of China (English)

    LIUYin; CHENJing-Hong; GONGZe-Hui

    2004-01-01

    AIM: To establish a primary culture technique of acutely isolated dorsal root ganglion (DRG) neurons, and provide a simple & useful in vitro model for study of analgesia. Methods: Acutely isolated dorsal root ganglion (DRG) neurons were planted and cultured; the configuration and growth characters of DRG neurons were observed through inverted microscope.

  9. [Induced-division of neurons derived from neural stem cells].

    Science.gov (United States)

    Lin, Qiu-Xia; Que, Hai-Ping; Lu, Shuang-Hong; Liu, Shao-Jun

    2004-04-25

    In order to explore if mature neurons derived from neural stem cells have the potentiality to divide, we utilized the chemical digestion method to disperse the adult rat brain tissue into single cells, and culture them in serum-free medium. After being cultured for about eight days in vitro, the neural stem cells were induced to differentiate into neurons. The neurons were further induced to divide. Utilizing the method of serial photograph and NF-160 immunocytochemistry, the processes of division of some neurons were recorded. At the same time, PCNA+NF-160 (or Chat, GABA, GAD) double label were used to investigate if the dividing-neurons were mature ones. After the neural stem cells were induced to differentiate in vitro for eight days, they possessed the shape and character of mature neurons. The differentiated neuron had a big nucleus and one or two distinct nucleolus in the nuclear. Within the perikaryon,there were a large amount of dense and Nissl body-like structure. Several long processes emerged from various locations of the cell body. Then, EGF and bFGF were added into the medium to induce division. After two days of induced-division, neuron-like cells were observed to divide; moreover, the number of neuron-like cells in the region increased continually. Immunocytochemistry demonstrated these cells were NF-160-positive. Serial photographs of dividing-process of neuron-like cells were obtained and their daughter cells were also NF-160-positive. After PCNA+NF-160 (or Chat, GABA, GAD) double label, some cells showed brown cell plasma and black nucleus. The above-mentioned results indicate that neurons, which were previously thought to be end-differentiated, can be re-called into cell cycle under appropriate conditions. Mature neurons still have the potential to divide, proliferate and self-renew.

  10. Ibuprofen augments bilirubin toxicity in rat cortical neuronal culture.

    Science.gov (United States)

    Berns, Monika; Toennessen, Margit; Koehne, Petra; Altmann, Rodica; Obladen, Michael

    2009-04-01

    Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 microg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 microM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 microg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 microM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

  11. Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture.

    Directory of Open Access Journals (Sweden)

    Sun Hee Kim

    Full Text Available BACKGROUND: Sirtuins (Sirt, a family of nicotinamide adenine nucleotide (NAD dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7, Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: To induce excitotoxic injury, we exposed primary cultured mouse cortical neurons to NMDA (30 µM. NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD in neurons through poly (ADP-ribose polymerase-1 (PARP-1 activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity. CONCLUSIONS: This study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.

  12. Spike Code Flow in Cultured Neuronal Networks.

    Science.gov (United States)

    Tamura, Shinichi; Nishitani, Yoshi; Hosokawa, Chie; Miyoshi, Tomomitsu; Sawai, Hajime; Kamimura, Takuya; Yagi, Yasushi; Mizuno-Matsumoto, Yuko; Chen, Yen-Wei

    2016-01-01

    We observed spike trains produced by one-shot electrical stimulation with 8 × 8 multielectrodes in cultured neuronal networks. Each electrode accepted spikes from several neurons. We extracted the short codes from spike trains and obtained a code spectrum with a nominal time accuracy of 1%. We then constructed code flow maps as movies of the electrode array to observe the code flow of "1101" and "1011," which are typical pseudorandom sequence such as that we often encountered in a literature and our experiments. They seemed to flow from one electrode to the neighboring one and maintained their shape to some extent. To quantify the flow, we calculated the "maximum cross-correlations" among neighboring electrodes, to find the direction of maximum flow of the codes with lengths less than 8. Normalized maximum cross-correlations were almost constant irrespective of code. Furthermore, if the spike trains were shuffled in interval orders or in electrodes, they became significantly small. Thus, the analysis suggested that local codes of approximately constant shape propagated and conveyed information across the network. Hence, the codes can serve as visible and trackable marks of propagating spike waves as well as evaluating information flow in the neuronal network.

  13. Correction: Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    Science.gov (United States)

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-09-01

    Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.

  14. Co-culture of astrocytes with neurons from injured brain A time-dependent dichotomy

    Institute of Scientific and Technical Information of China (English)

    Xiaojing Xu; Min Wang; Jing Liu; Jingya Lv; Yanan Hu; Huanxiang Zhang

    2011-01-01

    As supportive cells for neuronal growth and development, much effort has been devoted to the role of astrocytes in the normal state. However, the effect of the astrocytes after injury remains elusive. In the present study, neurons isolated from the subventricular zone of injured neonatal rat brains were co-cultured with astrocytes. After 6 days, these astrocytes showed a mature neuron-like appearance and the number of survivingneurons, primary dendrites and total branches was significantly higher than those at 3 days. The neurons began to shrink at 9 days after co-culture with shorter and thinner processes and the number of primary dendrites and total branches was significantly reduced. These experimental findings indicate that astrocytes in the injured brain promote the development of neurons in the early stages of co-culture while these cells reversely inhibit neuronal growth and development at the later states.

  15. Effects of amyotrophic lateral sclerosis sera on cultured cholinergic neurons

    Energy Technology Data Exchange (ETDEWEB)

    Touzeau, G.; Kato, A.C.

    1983-03-01

    Dissociated monolayer cultures of chick ciliary ganglion neurons have been used to study the effects of control and ALS sera. The cultured neurons survive and extend neurites for a minimum of 2 weeks in a standard tissue culture medium that contains 10% heat-inactivated human serum. Three parameters of the neurons have been examined when cultured in control and ALS sera for 8 to 12 days: (1) neuronal survival, (2) activity of the enzyme choline acetyltransferase, and (3) synthesis of /sup 3/H-acetylcholine using /sup 3/H-choline as precursor. ALS sera cause a small decrease in these three parameters, but this difference is not significant.

  16. A simplified micropatterning method for straight-line neurite extension of cultured hippocampal neurons.

    Science.gov (United States)

    Suzuki, Ikuro; Nakamura, Kosuke; Odawara, Aoi; Alhebshi, Amani; Gotoh, Masao

    2013-01-01

    We report a simplified micropatterning method for the straight-line extension of the neurites of cultured neurons. We prepared a poly-D-lysine (PDL)-patterned surface using a polydimethylsiloxane microfluidic stamp. Hippocampal neurons were cultured on the PDL-bound substrate with the stamp removed, allowing for conventional cell seeding and detailed optical observation without fluorescent label. Cultured neurons elongated neurites along straight lines at the single-cell level and displayed spontaneous firing as detected by time-lapse imaging and Ca(2+) imaging.

  17. A Highly Efficient Human Pluripotent Stem Cell Microglia Model Displays a Neuronal-Co-culture-Specific Expression Profile and Inflammatory Response

    Directory of Open Access Journals (Sweden)

    Walther Haenseler

    2017-06-01

    Full Text Available Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology.

  18. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models

    Directory of Open Access Journals (Sweden)

    Kung PJ

    2014-10-01

    Full Text Available Pin-Jui Kung,1,* Yu-Chen Tao,1,* Ho-Chiang Hsu,1 Wan-Ling Chen,1 Te-Hsien Lin,1 Donala Janreddy,2 Ching-Fa Yao,2 Kuo-Hsuan Chang,3 Jung-Yaw Lin,1 Ming-Tsan Su,1 Chung-Hsin Wu,1 Guey-Jen Lee-Chen,1 Hsiu-Mei Hsieh-Li1 1Department of Life Science, 2Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan; 3Department of Neurology, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taipei, Taiwan *These authors contributed equally to this work Abstract: In spinocerebellar ataxia type 17 (SCA17, the expansion of a translated CAG repeat in the TATA box binding protein (TBP gene results in a long polyglutamine (polyQ tract in the TBP protein, leading to intracellular accumulation of aggregated TBP and cell death. The molecular chaperones act in preventing protein aggregation to ameliorate downstream harmful events. In this study, we used Tet-On SH-SY5Y cells with inducible SCA17 TBP/Q79-green fluorescent protein (GFP expression to test indole and synthetic derivative NC001-8 for neuroprotection. We found that indole and NC001-8 up-regulated chaperone expression to reduce polyQ aggregation in neuronal differentiated TBP/Q79 cells. The effects on promoting neurite outgrowth and on reduction of aggregation on Purkinje cells were also confirmed with cerebellar primary and slice cultures of SCA17 transgenic mice. Our results demonstrate how indole and derivative NC001-8 reduce polyQ aggregation to support their therapeutic potentials in SCA17 treatment. Keywords: spinocerebellar ataxia type 17, TATA box binding protein, polyQ aggregation, indole and derivative, therapeutics

  19. Cultured cells of the nervous system, including human neurones, in the study of the neuro-degenerative disorder, Alzheimer's disease: an overview.

    Science.gov (United States)

    De Boni, U

    1985-01-01

    Human nervous-system cells in culture are a suitable model for the study of the degenerative changes associated with Alzheimer's disease. Alzheimer-diseased brain contains a factor which induces the formation of paired helical filaments (PHF) in cultured cells, similar to that seen in Alzheimer's disease. The excitotoxic amino acids, glutamate and aspartate, induce similar PHE formation in cultured cells. The neurotoxic element aluminium is present in high concentrations in the brain in several human neurological disorders, including Alzheimer's disease. In cultured-cell systems, aluminium interacts with acidic nuclear proteins, decreases steroid binding, produces a form of neurofibrillary degeneration and alters nucleoside metabolism.

  20. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidoptera

  1. Insect Cell Culture

    NARCIS (Netherlands)

    Oers, van M.M.; Lynn, D.E.

    2010-01-01

    Insect cell cultures are widely used in studies on insect cell physiology, developmental biology and microbial pathology. In particular, insect cell culture is an indispensable tool for the study of insect viruses. The first continuously growing insect cell cultures were established from lepidoptera

  2. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine

    DEFF Research Database (Denmark)

    Leke, Renata; Bak, Lasse Kristoffer; Anker, Malene

    2011-01-01

    in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure...... enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important......Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme...

  3. Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity.

    Science.gov (United States)

    Xu, Jin-Chong; Fan, Jing; Wang, Xueqing; Eacker, Stephen M; Kam, Tae-In; Chen, Li; Yin, Xiling; Zhu, Juehua; Chi, Zhikai; Jiang, Haisong; Chen, Rong; Dawson, Ted M; Dawson, Valina L

    2016-04-06

    Translating neuroprotective treatments from discovery in cell and animal models to the clinic has proven challenging. To reduce the gap between basic studies of neurotoxicity and neuroprotection and clinically relevant therapies, we developed a human cortical neuron culture system from human embryonic stem cells or human inducible pluripotent stem cells that generated both excitatory and inhibitory neuronal networks resembling the composition of the human cortex. This methodology used timed administration of retinoic acid to FOXG1(+) neural precursor cells leading to differentiation of neuronal populations representative of the six cortical layers with both excitatory and inhibitory neuronal networks that were functional and homeostatically stable. In human cortical neuronal cultures, excitotoxicity or ischemia due to oxygen and glucose deprivation led to cell death that was dependent on N-methyl-D-aspartate (NMDA) receptors, nitric oxide (NO), and poly(ADP-ribose) polymerase (PARP) (a cell death pathway called parthanatos that is distinct from apoptosis, necroptosis, and other forms of cell death). Neuronal cell death was attenuated by PARP inhibitors that are currently in clinical trials for cancer treatment. This culture system provides a new platform for the study of human cortical neurotoxicity and suggests that PARP inhibitors may be useful for ameliorating excitotoxic and ischemic cell death in human neurons.

  4. Cell-surface expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) in heterogeneous cultures of marrow-derived mesenchymal stem cells.

    Science.gov (United States)

    Russell, Katie C; Tucker, H Alan; Bunnell, Bruce A; Andreeff, Michael; Schober, Wendy; Gaynor, Andrew S; Strickler, Karen L; Lin, Shuwen; Lacey, Michelle R; O'Connor, Kim C

    2013-10-01

    Cellular heterogeneity of mesenchymal stem cells (MSCs) impedes their use in regenerative medicine. The objective of this research is to identify potential biomarkers for the enrichment of progenitors from heterogeneous MSC cultures. To this end, the present study examines variation in expression of neuron-glial antigen 2 (NG2) and melanoma cell adhesion molecule (CD146) on the surface of MSCs derived from human bone marrow in response to culture conditions and among cell populations. Multipotent cells isolated from heterogeneous MSC cultures exhibit a greater than three-fold increase in surface expression for NG2 and greater than two-fold increase for CD146 as compared with parental and lineage-committed MSCs. For both antigens, surface expression is downregulated by greater than or equal to six-fold when MSCs become confluent. During serial passage, maximum surface expression of NG2 and CD146 is associated with minimum doubling time. Upregulation of NG2 and CD146 during loss of adipogenic potential at early passage suggests some limits to their utility as potency markers. A potential relationship between proliferation and antigen expression was explored by sorting heterogeneous MSCs into rapidly and slowly dividing groups. Fluorescence-activated cell sorting revealed that rapidly dividing MSCs display lower scatter and 50% higher NG2 surface expression than slowly dividing cells, but CD146 expression is comparable in both groups. Heterogeneous MSCs were sorted based on scatter properties and surface expression of NG2 and CD146 into high (HI) and low (LO) groups. Sc(LO)NG2(HI) and Sc(LO)NG2(HI)CD146(HI) MSCs have the highest proliferative potential of the sorted groups, with colony-forming efficiencies that are 1.5-2.2 times the value for the parental controls. The Sc(LO) gate enriches for rapidly dividing cells. Addition of the NG2(HI) gate increases cell survival to 1.5 times the parental control. Further addition of the CD146(HI) gate does not significantly

  5. Phagocytic microglia release cytokines and cytotoxins that regulate the survival of astrocytes and neurons in culture.

    Science.gov (United States)

    Giulian, D; Li, J; Leara, B; Keenen, C

    1994-09-01

    Numerous studies have now shown that microglia secrete factors which may influence the growth and survival of cells in the CNS. We employed glia-neuron co-cultures to investigate the net effect of soluble products from secretory microglia upon astroglia and neurons following microglial activation by a phagocytic signal. Stimulation of microglia produced soluble factors that both increase the number of astroglia and decrease the number of neurons. The astroglial proliferating activity was blocked when incubated with an interleukin-1 (IL-1) receptor antagonist while the neurotoxic effect was inhibited by a N-methyl-D-aspartate (NMDA) receptor antagonist. Recombinant IL-1 served as a potent mitogen for cultured astroglia and promoted neuron survival by indirect actions upon astrocytes. These observations suggest that reactive microglia mediate both astrogliosis and neuronal injury through the simultaneous release of cell growth factors and poisons. The net effect of secretion products from phagocytic microglia is to diminish neuronal survival.

  6. Barbed channels enhance unidirectional connectivity between neuronal networks cultured on multi electrode arrays.

    NARCIS (Netherlands)

    le Feber, Jakob; Postma, W.; de Weerd, Eemke Luurt; Weusthof, Marcel H.H.; Rutten, Wim

    2015-01-01

    Cultured neurons on multi electrode arrays (MEAs) have been widely used to study various as-pects of neuronal (network) functioning. A possible drawback of this approach is the lack of struc-ture in these networks. At the single cell level, several solutions have been proposed to enable di-rected co

  7. Cultured Vestibular Ganglion Neurons Demonstrate Latent HSV1 Reactivation

    Science.gov (United States)

    Roehm, Pamela C.; Camarena, Vladimir; Nayak, Shruti; Gardner, James B.; Wilson, Angus; Mohr, Ian; Chao, Moses V.

    2013-01-01

    Objectives/Hypothesis Vestibular neuritis is a common cause of both acute and chronic vestibular dysfunction. Multiple pathologies have been hypothesized to be the causative agent of vestibular neuritis; however, whether herpes simplex type I (HSV1) reactivation occurs within the vestibular ganglion has not been demonstrated previously by experimental evidence. We developed an in vitro system to study HSV1 infection of vestibular ganglion neurons (VGNs) using a cell culture model system. Study design basic science study. Results Lytic infection of cultured rat VGNs was observed following low viral multiplicity of infection (MOI). Inclusion of acyclovir suppressed lytic replication and allowed latency to be established. Upon removal of acyclovir, latent infection was confirmed with reverse-transcription polymerase chain reaction and by RNA fluorescent in situ hybridization for the latency-associated transcript (LAT). 29% cells in latently infected cultures were LAT positive. The lytic IPC27 transcript was not detected by reverse-transcription polymerase chain reaction (RT-PCR). Reactivation of HSV1 occurred at a high frequency in latently infected cultures following treatment with trichostatin A (TSA), a histone deactylase inhibitor. Conclusions VGNs can be both lytically and latently infected with HSV1. Furthermore, latently infected VGNs can be induced to reactivate using TSA. This demonstrates that reactivation of latent HSV1 infection in the vestibular ganglion can occur in a cell culture model, and suggests that reactivation of HSV1 infection a plausible etiologic mechanism of vestibular neuritis. PMID:21898423

  8. Isolated hippocampal neurons in cryopreserved long-term cultures: development of neuroarchitecture and sensitivity to NMDA.

    Science.gov (United States)

    Mattson, M P; Kater, S B

    1988-01-01

    Isolated neurons in long-term culture provide a unique opportunity to address important problems in neuronal development. In the present study we established conditions for cryopreservation and long-term primary culture of isolated embryonic hippocampal neurons. This culture system was then used for initial characterizations of the development of neuroarchitecture and neurotransmitter response systems. Cryoprotection with 8% dimethylsulfoxide, slow freezing, and rapid thawing provided high-yield cultures which appeared normal in terms of cell types, mitotic ability, axonal and dendritic outgrowth, and sensitivity to glutamate neurotoxicity. A reduced medium volume and moderate elevation in extracellular K+ to 20 mM promoted survival of isolated neurons through 3 weeks of culture. The outgrowth of axons and dendrites in pyramidal-like neurons was found to differ over a 3-week culture period such that axons continued to grow at a relatively constant rate while dendritic outgrowth slowed during the second week and ceased by the end of week 3. Developmental changes were also observed in the sensitivity of pyramidal neurons to glutamate neurotoxicity; functional kainate/quisqualate receptors were present during the first week of culture, while responses to N-methyl-D-aspartic acid (NMDA) did not appear until the second week. The technologies for cryopreservation and long-term culture of isolated hippocampal neurons reported here provide a useful system in which to address a variety of problems in development neuroscience.

  9. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    Science.gov (United States)

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  10. Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation.

    Science.gov (United States)

    Strasser, U; Fischer, G

    1995-04-01

    Organotypic hippocampal cultures were used to study cell degeneration during the recovery period after defined periods (30 and 60 min) of combined oxygen/glucose deprivation mimicking transient ischemic conditions. Staining with the fluorescent dye propidium iodide allowed detection of damaged cells. Fluorescence intensity was measured by an image analysis system and used to quantify cell damage at different time points during the recovery period (up to 22 h). At 30 min of oxygen/glucose deprivation cells in the CA1 area were relatively more sensitive compared to CA3 and dentate gyrus cells, with respect to the time course of degeneration and the percentage of affected cells. Expanding the oxygen/glucose deprivation period from 30 to 60 min drastically increased the percentage of cells dying in all hippocampal areas. Still, however, cells in CA1 degenerated faster compared to those in the CA3 area and dentate gyrus. A histological analysis of toluidine blue as well as MAP2-immunostained sections revealed that almost all neurons degenerated in all hippocampal areas following the 60-min deprivation period, whereas GFAP-stained astrocytes appeared to be unaffected. Therefore, neuronal degeneration could be quantified by taking the fluorescence intensity values 22 h after 60 min of oxygen/glucose deprivation as 100% neuronal damage. The possibility to quantify neuronal damage in organotypic cultures offers a useful tool for detailed studies on mechanisms of neuronal cell death in a cell culture system which is closer to in situ conditions than monolayer cell cultures.

  11. Effect of Methamidophos on cerebellar neuronal cells

    African Journals Online (AJOL)

    olayemitoyin

    TH-mediated cerebellar neuronal cell development and function, and consequently could interfere with TH-regulated neuronal ... 1972), decreased number of synapses between the. Purkinje .... 0.008%DNase and triturated in same solution to ...

  12. Protective effects of berberine against amyloid beta-induced toxicity in cultured rat cortical neurons

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Yanjun Zhang; Shuai Du; Mixia Zhang

    2011-01-01

    Berberine, a major constituent of Coptidis rhizoma, exhibits neural protective effects. The present study analyzed the potential protective effect of berberine against amyloid G-induced cytotoxicity in rat cerebral cortical neurons. Alzheimer's disease cell models were treated with 0.5 and 2 μmol/Lberberine for 36 hours to inhibit amyloid G-induced toxicity. Methyl thiazolyl tetrazolium assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining results showed that berberine significantly increased cell viability and reduced cell apoptosis in primary cultured rat cortical neurons. In addition, western blot analysis revealed a protective effect of berberine against amyloid β-induced toxicity in cultured cortical neurons, which coincided with significantly decreased abnormal up-regulation of activated caspase-3. These results showed that berberine exhibited a protective effect against amyloid 13-induced cytotoxicity in cultured rat cortical neurons.

  13. Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

    Science.gov (United States)

    Schwab, Andrew J; Ebert, Allison D

    2014-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

  14. Zinc Modulates Nanosilver-Induced Toxicity in Primary Neuronal Cultures.

    Science.gov (United States)

    Ziemińska, Elżbieta; Strużyńska, Lidia

    2016-02-01

    Silver nanoparticles (NAg) have recently become one of the most commonly used nanomaterials. Since the ability of nanosilver to enter the brain has been confirmed, there has been a need to investigate mechanisms of its neurotoxicity. We previously showed that primary neuronal cultures treated with nanosilver undergo destabilization of calcium homeostasis via a mechanism involving glutamatergic NMDA receptors. Considering the fact that zinc interacts with these receptors, the aim of the present study was to examine the role of zinc in mechanisms of neuronal cell death in primary cultures. In cells treated with nanosilver, we noted an imbalance between extracellular and intracellular zinc levels. Thus, the influence of zinc deficiency and supplementation on nanosilver-evoked cytotoxicity was investigated by treatment with TPEN (a chelator of zinc ions), or ZnCl(2), respectively. Elimination of zinc leads to complete death of nanosilver-treated CGCs. In contrast, supplementation with ZnCl(2) increases viability of CGCs in a dose-dependent manner. Addition of zinc provided protection against the extra/intracellular calcium imbalance in a manner similar to MK-801, an antagonist of NMDA receptors. Zinc chelation by TPEN decreases the mitochondrial potential and dramatically increases the rate of production of reactive oxygen species. Our results indicate that zinc supplementation positively influences nanosilver-evoked changes in CGCs. This is presumed to be due to an inhibitory effect on NMDA-sensitive calcium channels.

  15. Satellite glial cells can promote the extension of neuronal axons in vitro

    Institute of Scientific and Technical Information of China (English)

    Jiu-Hong Zhao; Yi-Di Huang; Xi-Nan Yi; Quan-Peng Zhang; Xian-Fang Zhang; Xu Dong; Gang Luo; Hai-Ying Zhang; Kun-Ju Wang; Mei-Li Lao

    2015-01-01

    Objective: To study the influence of satellite glial cells (SGCs) on the outgrowth of neuronal neurite and the role of Slit1 protein and the contact with neurons in this process, in vitro. Methods: Neurons culture and SGC-neuron co-culture were used as the cell models. The length of axons and dendrites were measured via immunofluorescence to observe the influence of SGCs on the outgrowth of neuronal neurite. The Slit1 protein was added into SGC-neuron co-culture model. The length of dendrites was measured via immunofluorescence at different point times. Result: The anatomical relationship between neurons and SGCs changed as culture period expand. At 12 h after culture, SGCs all surrounded neurons; by 72 h after culture, SGCs were all off neurons. SGCs can promote the growth of neuronal axos, but inhibit the growth of its dendrites; when SGCs closely contact with neurons, the effect of Slit1 on promoting the dendritic growth is not obvious, but when SGCs were off neurons, the effect of Slit1 on promoting the dendritic growth is significant. Conclusion: SGCs can promote the growth of neuronal axos, but inhibit the growth of its dendrites; Slit- Robo signaling pathways and contact with neurons play a role in this process.

  16. Chronaxie Measurements in Patterned Neuronal Cultures from Rat Hippocampus.

    Science.gov (United States)

    Stern, Shani; Agudelo-Toro, Andres; Rotem, Assaf; Moses, Elisha; Neef, Andreas

    2015-01-01

    Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD) curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields.

  17. Chronaxie Measurements in Patterned Neuronal Cultures from Rat Hippocampus.

    Directory of Open Access Journals (Sweden)

    Shani Stern

    Full Text Available Excitation of neurons by an externally induced electric field is a long standing question that has recently attracted attention due to its relevance in novel clinical intervention systems for the brain. Here we use patterned quasi one-dimensional neuronal cultures from rat hippocampus, exploiting the alignment of axons along the linear patterned culture to separate the contribution of dendrites to the excitation of the neuron from that of axons. Network disconnection by channel blockers, along with rotation of the electric field direction, allows the derivation of strength-duration (SD curves that characterize the statistical ensemble of a population of cells. SD curves with the electric field aligned either parallel or perpendicular to the axons yield the chronaxie and rheobase of axons and dendrites respectively, and these differ considerably. Dendritic chronaxie is measured to be about 1 ms, while that of axons is on the order of 0.1 ms. Axons are thus more excitable at short time scales, but at longer time scales dendrites are more easily excited. We complement these studies with experiments on fully connected cultures. An explanation for the chronaxie of dendrites is found in the numerical simulations of passive, realistically structured dendritic trees under external stimulation. The much shorter chronaxie of axons is not captured in the passive model and may be related to active processes. The lower rheobase of dendrites at longer durations can improve brain stimulation protocols, since in the brain dendrites are less specifically oriented than axonal bundles, and the requirement for precise directional stimulation may be circumvented by using longer duration fields.

  18. Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons

    Directory of Open Access Journals (Sweden)

    Fomina Alla F

    2008-09-01

    Full Text Available Abstract Background Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment. Results We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro. Conclusion This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.

  19. Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining.

    Science.gov (United States)

    Daniel, Bron; DeCoster, Mark A

    2004-08-01

    The terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) stain is in wide use for measuring apoptosis in neurons, as well as in other cell types. TUNEL may give false positive results due to variations in labeling technique as well as staining of cells that have undergone non-apoptotic DNA strand breaks. Therefore, in isolation, TUNEL is not a certain indicator of apoptosis. Recently, we have demonstrated the potent apoptotic effect of secreted phospholipase A2 from group III (sPLA2-III) on primary cortical neurons from rat. Here we describe a computer-assisted method for quantifying TUNEL-positive neurons after sPLA2-III induced apoptosis. Extent of TUNEL is normalized to total nuclear content using 4',6-diamidino-2-phenylindole (DAPI) staining. Furthermore, DAPI counterstaining allows for determination of a nuclear morphology indicator, based on nuclear size and roundness, which we call the nuclear area factor. We found that the nuclear area factor is an early indicator of cell death (significant after 4 h post treatment), while TUNEL staining is significant at later times (26 h). Thus, the independent staining techniques using TUNEL and DAPI complement each other, and with commercially available image analysis software, may be used to indicate early as well as delayed cell injury processes.

  20. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    de Turco, Elena B; Diemer, Nils Henrik; Bazan, Nicolas G

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H]A...

  1. Quantitation of peptide hormone in single cultured secretory neurons of the crab, Cardisoma carnifex.

    Science.gov (United States)

    Keller, R; Grau, S; Cooke, I M

    1995-09-01

    The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1-50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of approximately 150 peptidergic neurons that form the X-organ--sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having "high" (> 33 pg; mean 86 +/- 5, S.E., n = 47) or "low" (< or = 33 pg; mean 22 +/- 2.5; n = 17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross-reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2 +/- 2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing.

  2. Sugammadex, a neuromuscular blockade reversal agent, causes neuronal apoptosis in primary cultures.

    Science.gov (United States)

    Palanca, José M; Aguirre-Rueda, Diana; Granell, Manuel V; Aldasoro, Martin; Garcia, Alma; Iradi, Antonio; Obrador, Elena; Mauricio, Maria Dolores; Vila, Jose; Gil-Bisquert, Anna; Valles, Soraya L

    2013-01-01

    Sugammadex, a γ-cyclodextrin that encapsulates selectively steroidal neuromuscular blocking agents, such as rocuronium or vecuronium, has changed the face of clinical neuromuscular pharmacology. Sugammadex allows a rapid reversal of muscle paralysis. Sugammadex appears to be safe and well tolerated. Its blood-brain barrier penetration is poor (Sugammadex in neurons in primary culture. Here we show that clinically relevant sugammadex concentrations cause apoptotic/necrosis neuron death in primary cultures. Studies on the underlying mechanism revealed that sugammadex-induced activation of mitochondria-dependent apoptosis associates with depletion of neuronal cholesterol levels. Furthermore SUG increase CytC, AIF, Smac/Diablo and CASP-3 protein expression in cells in culture. Potential association of SUG-induced alteration in cholesterol homeostasis with oxidative stress and apoptosis activation occurs. Furthermore, resistance/sensitivity to oxidative stress differs between neuronal cell types.

  3. Rhynchophylline Protects Cultured Rat Neurons against Methamphetamine Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Dan Dan Xu

    2012-01-01

    Full Text Available Rhynchophylline (Rhy is an active component isolated from species of the genus Uncaria which has been used for the treatment of ailments to the central nervous system in traditional Chinese medicine. Besides acting as a calcium channel blocker, Rhy was also reported to be able to protect against glutamate-induced neuronal death. We thus hypothesize that Rhy may have neuroprotective activity against methamphetamine (MA. The primary neurons were cultured directly from the cerebral cortex of neonatal rats, acting as in vitro model in the present study. The neurotoxicity of MA and the protective effect of Rhy were evaluated by MTT assay. The effects of MA, Rhy or their combination on intracellular free calcium concentration ([Ca2+]i were determined in individual neocortical neurons by the Fluo-3/AM tracing method. The MTT assay demonstrated that MA has a dose-dependent neurotoxicity in neuronal cultures. The addition of Rhy prior to the exposure to MA prevented neuronal death. Time course studies with the Fluo-3/AM probe showed that Rhy significantly decreased neuronal [Ca2+]i which was elevated by the exposure to MA. Our results suggested that Rhy can protect the neuronal cultures against MA exposure and promptly attenuate intracellular calcium overload triggered by MA challenge. This is the first report demonstrating an inhibitory effect of Rhy against MA impairment in cultured neurons in vitro.

  4. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  5. Cell Culture Made Easy.

    Science.gov (United States)

    Dye, Frank J.

    1985-01-01

    Outlines steps to generate cell samples for observation and experimentation. The procedures (which use ordinary laboratory equipment) will establish a short-term primary culture of normal mammalian cells. Information on culture vessels and cell division and a list of questions to generate student interest and involvement in the topics are…

  6. Detoxification of ammonia in mouse cortical GABAergic cell cultures increases neuronal oxidative metabolism and reveals an emerging role for release of glucose-derived alanine.

    Science.gov (United States)

    Leke, Renata; Bak, Lasse K; Anker, Malene; Melø, Torun M; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Ott, Peter; Portela, Luis V; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2011-04-01

    Cerebral hyperammonemia is believed to play a pivotal role in the development of hepatic encephalopathy (HE), a debilitating condition arising due to acute or chronic liver disease. In the brain, ammonia is thought to be detoxified via the activity of glutamine synthetase, an astrocytic enzyme. Moreover, it has been suggested that cerebral tricarboxylic acid (TCA) cycle metabolism is inhibited and glycolysis enhanced during hyperammonemia. The aim of this study was to characterize the ammonia-detoxifying mechanisms as well as the effects of ammonia on energy-generating metabolic pathways in a mouse neuronal-astrocytic co-culture model of the GABAergic system. We found that 5 mM ammonium chloride affected energy metabolism by increasing the neuronal TCA cycle activity and switching the astrocytic TCA cycle toward synthesis of substrate for glutamine synthesis. Furthermore, ammonia exposure enhanced the synthesis and release of alanine. Collectively, our results demonstrate that (1) formation of glutamine is seminal for detoxification of ammonia; (2) neuronal oxidative metabolism is increased in the presence of ammonia; and (3) synthesis and release of alanine is likely to be important for ammonia detoxification as a supplement to formation of glutamine.

  7. A Neuronal Culture System to Detect Prion Synaptotoxicity.

    Directory of Open Access Journals (Sweden)

    Cheng Fang

    2016-05-01

    Full Text Available Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrPSc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrPC, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrPC molecule. Both protease-resistant and protease-sensitive forms of PrPSc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrPSc and testing potential therapeutic agents.

  8. Loss of glutathione homeostasis associated with neuronal senescence facilitates TRPM2 channel activation in cultured hippocampal pyramidal neurons

    Directory of Open Access Journals (Sweden)

    Belrose Jillian C

    2012-04-01

    Full Text Available Abstract Background Glutathione (GSH plays an important role in neuronal oxidant defence. Depletion of cellular GSH is observed in neurodegenerative diseases and thereby contributes to the associated oxidative stress and Ca2+ dysregulation. Whether depletion of cellular GSH, associated with neuronal senescence, directly influences Ca2+ permeation pathways is not known. Transient receptor potential melastatin type 2 (TRPM2 is a Ca2+ permeable non-selective cation channel expressed in several cell types including hippocampal pyramidal neurons. Moreover, activation of TRPM2 during oxidative stress has been linked to cell death. Importantly, GSH has been reported to inhibit TRPM2 channels, suggesting they may directly contribute to Ca2+ dysregulation associated with neuronal senescence. Herein, we explore the relation between cellular GSH and TRPM2 channel activity in long-term cultures of hippocampal neurons. Results In whole-cell voltage-clamp recordings, we observe that TRPM2 current density increases in cultured pyramidal neurons over time in vitro. The observed increase in current density was prevented by treatment with NAC, a precursor to GSH synthesis. Conversely, treatment of cultures maintained for 2 weeks in vitro with L-BSO, which depletes GSH by inhibiting its synthesis, augments TRPM2 currents. Additionally, we demonstrate that GSH inhibits TRPM2 currents through a thiol-independent mechanism, and produces a 3.5-fold shift in the dose-response curve generated by ADPR, the intracellular agonist for TRPM2. Conclusion These results indicate that GSH plays a physiologically relevant role in the regulation of TRPM2 currents in hippocampal pyramidal neurons. This interaction may play an important role in aging and neurological diseases associated with depletion of GSH.

  9. Ionic currents of morphologically distinct peptidergic neurons in defined culture.

    Science.gov (United States)

    Meyers, D E; Graf, R A; Cooke, I M

    1992-05-01

    1. The X-organ sinus gland is a major peptidergic neurosecretory system in Crustacea, analogous to the vertebrate hypothalamoneurohypophyseal system. Neuronal somata isolated from the crab (Cardisoma carnifex) X-organ and maintained in primary culture in unconditioned, fully defined medium show immediate regenerative outgrowth. Outgrowth occurring as broad lamellipodia ("veiled") distinguishes neurons consistently showing crustacean hyperglycemic hormone immunoreactivity. Neurons that are immunoreactive against molt-inhibiting hormone and red pigment concentrating hormone antisera give rise to branched neurites ("branched"). 2. The whole-cell variation of the patch-clamp technique was used to study the electrophysiology of these two cell types 24-48 h after plating. Under current clamp, only veiled neurons fired overshooting action potentials either spontaneously or in response to depolarization. 3. Under voltage clamp, net current was predominantly outward. When solutions that suppressed outward current were used, only veiled neurons showed significant inward current. These included a tetrodotoxin (TTX)-sensitive Na current and a slow (time to peak 6-10 ms at 0 mV) Cd-sensitive Ca current (ICa) that was activated at potentials less than -30 mV, was maximal at 0 to +20 mV, and did not reverse at potentials up to +60 mV. 4. In TTX, the form of the Ca current I(V) curve was unchanged by changes of holding potential between -40 and -80 mV, and 75-100% of ICa was available from -40 mV. 5. ICa inactivated slowly and incompletely. Analysis with two-pulse regimes suggested that both inactivation and facilitation mechanisms were present. 6. Outward current was examined in the presence and absence of 0.5 mM Cd2+ (1 microM TTX was always present in the external medium). Cd2+ ions slightly reduced the peak outward current, usually by less than 10% (Vc = -10 to +20 mV; Vh = -80 mV). All additional observations were in the presence of TTX and Cd2+. 7. Both cell types expressed

  10. Adipose tissue-derived stromal cells express neuronal phenotypes

    Institute of Scientific and Technical Information of China (English)

    杨立业; 刘相名; 孙兵; 惠国桢; 费俭; 郭礼和

    2004-01-01

    Background Adipose tissue-derived stromal cells (ADSCs) can be greatly expanded in vitro, and induced to differentiate into multiple mesenchymal cell types, including osteogenic, chondrogenic, myogenic, and adipogenic cells. This study was designed to investigate the possibility of ADSCs differentiating into neurons.Methods Adipose tissue from rats was digested with collagenase, and adherent stromal cells were cultured. A medium containing a low concentration of fetal bovine serum was adopted to induce the cells to differentiate. ADSCs were identified by immunocytochemistry, and semi-quantitative RT-PCR was applied to detect mRNA expression of neurofilament 1 (NF1), nestin, and neuron-specific enolase (NSE).Results Nestin-positive cells were found occasionally among ADSCs. ADSCs were found to express NSE mRNA and nestin mRNA, but not NF1 mRNA. ADSCs could differentiate into neuron-like cells in a medium composed of a low concentration of fetal bovine serum, and these differentiated cells displayed complicated neuron-like morphologies.Conclusions The data support the hypothesis that adipose tissue contains stem cells capable of differentiating into neurons. These stem cells can overcome their mesenchymal commitment, and may represent an alternative autologous stem cell source for CNS cell transplantation.

  11. Emergence of assortative mixing between clusters of cultured neurons.

    Directory of Open Access Journals (Sweden)

    Sara Teller

    2014-09-01

    Full Text Available The analysis of the activity of neuronal cultures is considered to be a good proxy of the functional connectivity of in vivo neuronal tissues. Thus, the functional complex network inferred from activity patterns is a promising way to unravel the interplay between structure and functionality of neuronal systems. Here, we monitor the spontaneous self-sustained dynamics in neuronal cultures formed by interconnected aggregates of neurons (clusters. Dynamics is characterized by the fast activation of groups of clusters in sequences termed bursts. The analysis of the time delays between clusters' activations within the bursts allows the reconstruction of the directed functional connectivity of the network. We propose a method to statistically infer this connectivity and analyze the resulting properties of the associated complex networks. Surprisingly enough, in contrast to what has been reported for many biological networks, the clustered neuronal cultures present assortative mixing connectivity values, meaning that there is a preference for clusters to link to other clusters that share similar functional connectivity, as well as a rich-club core, which shapes a 'connectivity backbone' in the network. These results point out that the grouping of neurons and the assortative connectivity between clusters are intrinsic survival mechanisms of the culture.

  12. Emergence of Assortative Mixing between Clusters of Cultured Neurons

    Science.gov (United States)

    Teller, Sara; Granell, Clara; De Domenico, Manlio; Soriano, Jordi; Gómez, Sergio; Arenas, Alex

    2014-01-01

    The analysis of the activity of neuronal cultures is considered to be a good proxy of the functional connectivity of in vivo neuronal tissues. Thus, the functional complex network inferred from activity patterns is a promising way to unravel the interplay between structure and functionality of neuronal systems. Here, we monitor the spontaneous self-sustained dynamics in neuronal cultures formed by interconnected aggregates of neurons (clusters). Dynamics is characterized by the fast activation of groups of clusters in sequences termed bursts. The analysis of the time delays between clusters' activations within the bursts allows the reconstruction of the directed functional connectivity of the network. We propose a method to statistically infer this connectivity and analyze the resulting properties of the associated complex networks. Surprisingly enough, in contrast to what has been reported for many biological networks, the clustered neuronal cultures present assortative mixing connectivity values, meaning that there is a preference for clusters to link to other clusters that share similar functional connectivity, as well as a rich-club core, which shapes a ‘connectivity backbone’ in the network. These results point out that the grouping of neurons and the assortative connectivity between clusters are intrinsic survival mechanisms of the culture. PMID:25188377

  13. Neuronize: a tool for building realistic neuronal cell morphologies

    Science.gov (United States)

    Brito, Juan P.; Mata, Susana; Bayona, Sofia; Pastor, Luis; DeFelipe, Javier; Benavides-Piccione, Ruth

    2013-01-01

    This study presents a tool, Neuronize, for building realistic three-dimensional models of neuronal cells from the morphological information extracted through computer-aided tracing applications. Neuronize consists of a set of methods designed to build 3D neural meshes that approximate the cell membrane at different resolution levels, allowing a balance to be reached between the complexity and the quality of the final model. The main contribution of the present study is the proposal of a novel approach to build a realistic and accurate 3D shape of the soma from the incomplete information stored in the digitally traced neuron, which usually consists of a 2D cell body contour. This technique is based on the deformation of an initial shape driven by the position and thickness of the first order dendrites. The addition of a set of spines along the dendrites completes the model, building a final 3D neuronal cell suitable for its visualization in a wide range of 3D environments. PMID:23761740

  14. Emergence of small-world anatomical networks in self-organizing clustered neuronal cultures

    CERN Document Server

    de Santos-Sierra, Daniel; Leyva, Inmaculada; Almendral, Juan A; Anava, Sarit; Ayali, Amir; Papo, David; Boccaletti, Stefano

    2014-01-01

    In vitro primary cultures of dissociated invertebrate neurons from locust ganglia are used to experimentally investigate the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. At all the different stages of the culture's development, identification of neurons' and neurites' location by means of a dedicated software allows to ultimately extract an adjacency matrix from each image of the culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main network's characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graph's micro- and meso-scale properties emerge. Finally, we identify the main physical processes ruling the culture's morphological transformati...

  15. Existence of glia mitigated ketamine-induced neurotoxicity in neuron-glia mixed cultures of neonatal rat cortex and the glia-mediated protective effect of 2-PMPA.

    Science.gov (United States)

    Zuo, Daiying; Wang, Chengna; Li, Zengqiang; Lin, Li; Duan, Zhenfang; Qi, Huan; Li, Lin; Sun, Feng; Wu, Yingliang

    2014-09-01

    The present study compared ketamine-induced neurotoxicity in the neuron-glia mixed cultures and neuronal cultures and further explored the neuroprotective effect of the NAAG peptidase inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA). Firstly, Rosenfeld's staining and immunofluorescence staining of microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) were used to address the difference of morphology in the mixed cultures and neuronal cultures. Our results showed that neurons and astrocytes grew in good conditions. The ratio of neurons and astrocytes in the mixed cultures was around 1:1, and the purity of neurons in the neuronal cultures is 91.3%. Furthermore, ketamine was used to test the hypothesis that the presence of a higher proportion of glia in the mixed cultures would be protective against ketamine-induced neurotoxicity in the mixed cultures compared with neuronal cultures. The results showed that ketamine-induced morphological changes, cell viability decrease and lactate dehydrogenase (LDH) levels increase were significantly mitigated in neuron-glia mixed cultures compared with neuronal cultures. Furthermore, 2-PMPA was included to further explore efficient protective drug for ketamine-induced neurotoxicity. Our results showed that 2-PMPA reduced ketamine-induced decrease of cell viability and increase of LDH levels in the mixed cultures but not in the neuronal cultures. Further morphological changes of neurons and astrocytes also indicated that 2-PMPA could improve ketamine damaged neurons in the mixed cultures instead of neuronal cultures. These results indicate that glia protect neurons from ketamine-induced neurotoxicity. These data further suggest that glia mediate the neuroprotective effect of 2-PMPA and 2-PMPA has the potential to treat ketamine-induced neurotoxicity in vivo. Delineating the mechanisms underlying the communication between neurons and glia and the neuroprotective effects of 2-PMPA in the mixed

  16. Chronic lithium treatment increased intracellular S100ß levels in rat primary neuronal culture.

    Directory of Open Access Journals (Sweden)

    Masoumeh Emamghoreishi

    2015-02-01

    Full Text Available S100ß a neurotrophic factor mainly released by astrocytes, has been implicated in the pathogenesis of bipolar disorder. Thus, lithium may exert its neuroprotective effects to some extent through S100ß. Furthermore, the possible effects of lithium on astrocytes as well as on interactions between neurons and astrocytes as a part of its mechanisms of actions are unknown. This study was undertaken to determine the effect of lithium on S100β in neurons, astrocytes and a mixture of neurons and astrocytes. Rat primary astrocyte, neuronal and mixed neuro-astroglia cultures were prepared from cortices of 18-day's embryos. Cell cultures were exposed to lithium (1mM or vehicle for 1day (acute or 7 days (chronic. RT-PCR and ELISA determined S100β mRNA and intra- and extracellular protein levels. Chronic lithium treatment significantly increased intracellular S100β in neuronal and neuro-astroglia cultures in comparison to control cultures (P<0.05. Acute and chronic lithium treatments exerted no significant effects on intracellular S100β protein levels in astrocytes, and extracellular S100β protein levels in three studied cultures as compared to control cultures. Acute and chronic lithium treatments did not significantly alter S100β mRNA levels in three studied cultures, compared to control cultures. Chronic lithium treatment increased intracellular S100ß protein levels in a cell-type specific manner which may favor its neuroprotective action. The findings of this study suggest that lithium may exert its neuroprotective action, at least partly, by increasing neuronal S100ß level, with no effect on astrocytes or interaction between neurons and astrocytes.

  17. Shenfu injection attenuates neurotoxicity of bupivacaine in cultured mouse spinal cord neurons

    Institute of Scientific and Technical Information of China (English)

    XIONG Li-ze; WANG Qiang; LIU Mu-yun; PENG Ye; LI Qing-bo; LU Zhi-hong; LEI Chong

    2007-01-01

    Background Our previous in vivo study in the rat demonstrates that Shenfu injection, a clinically used extract preparation from Chinese herbs, attenuates neural and cardiac toxicity induced by intravenous infusion of bupivacaine, a local anesthetic. This study was designed to investigate whether bupivacaine could induce a toxic effect in primary cultured mouse spinal cord neuron and if so, whether the Shenfu injection had a similar neuroprotective effect in the cell model. Methods The spinal cords from 11- to 14-day-old fetal mice were minced and incubated. Cytarabine was added into the medium to inhibit the proliferation of non-neuronal cells. The immunocytochemical staining of β-tubulin was used to determine the identity of cultured cells. The cultured neurons were randomly assigned into three sets treated with various doses of bupivacaine, Shenfu and bupivacaine+Shenfu, for 48 hours respectively. Cell viability in each group was analyzed by methyl thiazoleterazolium (MTT) assay. Results The viability of the cultured neurons treated with bupivacaine at concentrations of 0.01%, 0.02%, 0.04% and 0.08% was decreased in a dose-dependent manner. Although the Shenfu injection at concentrations ranging from 1/50 to 1/12.5 (V/V) had no significant influence on the viability of cultured neurons (P<0.05 vs control), the injection significantly increased the cellular viability of cultured neurons pretreated with 0.03% bupivacaine (P<0.05). Conclusion Although Shenfu injection itself has no effect on spinal neurons, it was able to reduce the bupivacaine induced neurotoxicity in vitro.

  18. Cultured neurons as model systems for biochemical and pharmacological studies on receptors for neurotransmitter amino acids

    DEFF Research Database (Denmark)

    Schousboe, A; Drejer, J; Hansen, Gert Helge

    1985-01-01

    By the use of primary cultures of neurons consisting of cerebral cortex interneurons or cerebellar granule cells it is possible to study biochemical and pharmacological aspects of receptors for GABA and glutamate. Cerebellar granule cells have been shown to express both high- and low-affinity GAB...

  19. NGF诱导PC12细胞分化的神经细胞行为观察%Observation on differentiated neuron behaviour induced by NGF in cultured PC12 cells

    Institute of Scientific and Technical Information of China (English)

    丁明杰; 史学义; 张娓; 丁一

    2001-01-01

    Aim:To study the cell division, neurite extension, migration of neurons differentiated by NGF in subcultured PC12 cells.Methods: The Giemsa staining, methyl green-pyronin staining and proliferating cell nuclear antigen immunohistochemical assay were used to study the subcultured PC12 cells.Results: The differentiated neuron appeared different patterns of amitotic figures such as transverse sever, longitudinal split, etc. The proliferating neurons divided in an asymmetric way morphologically and chemically. The differentiated neuron appeared an obvious movement of cell migration and neurite extension.Conclusion: The neurons differentiated by NGF in cultured PC12 cells can divide in amitotic way.The differentiated neuron can form junction by cell migration, neurite extension or cytoplasmic bridge.%目的:观察PC12细胞分化的神经细胞的分裂、突起延伸、迁徙和分化等细胞行为。方法:采用Giemsa、甲绿-派郎宁染色和增殖细胞核抗原(PCNA)免疫组织化学染色方法观察培养的PC12细胞。结果:培养分化的神经细胞呈现横裂、纵裂等不同方式的无丝分裂像;分裂中的神经细胞可呈现形态及化学不对称性;分化的神经细胞有明显的细胞迁徙运动和突起延伸。结论:NGF诱导PC12细胞分化的神经细胞能以无丝分裂方式增殖;分化神经元可以通过细胞迁徙、突起延伸及共同胞质桥等方式相连接。

  20. Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    Changqing Ye; Xiaodong Yuan; Hui Liu; Yanan Cai; Ya Ou

    2010-01-01

    β-mercaptoethanol induces in vitro adult adipose-derived stromal cells (ADSCs) to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptcethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker (nestin), a neuronal marker (neuron-specific enolase), and a glial marker (glial fibrillary acidic protein). In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

  1. High yield derivation of enriched glutamatergic neurons from suspension-cultured mouse ESCs for neurotoxicology research

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    Hubbard Kyle S

    2012-10-01

    Full Text Available Abstract Background Recently, there has been a strong emphasis on identifying an in vitro model for neurotoxicity research that combines the biological relevance of primary neurons with the scalability, reproducibility and genetic tractability of continuous cell lines. Derived neurons should be homotypic, exhibit neuron-specific gene expression and morphology, form functioning synapses and consistently respond to neurotoxins in a fashion indistinguishable from primary neurons. However, efficient methods to produce neuronal populations that are suitable alternatives to primary neurons have not been available. Methods With the objective of developing a more facile, robust and efficient method to generate enriched glutamatergic neuronal cultures, we evaluated the neurogenic capacity of three mouse embryonic stem cell (ESC lines (R1, C57BL/6 and D3 adapted to feeder-independent suspension culture. Neurogenesis and neuronal maturation were characterized as a function of time in culture using immunological, genomic, morphological and functional metrics. The functional responses of ESNs to neurotropic toxins with distinctly different targets and mechanisms of toxicity, such as glutamate, α-latrotoxin (LTX, and botulinum neurotoxin (BoNT, were also evaluated. Results Suspension-adapted ESCs expressed markers of pluripotency through at least 30 passages, and differentiation produced 97×106 neural progenitor cells (NPCs per 10-cm dish. Greater than 99% of embryonic stem cell-derived neurons (ESNs expressed neuron-specific markers by 96 h after plating and rapidly developed complex axodendritic arbors and appropriate compartmentalization of neurotypic proteins. Expression profiling demonstrated the presence of transcripts necessary for neuronal function and confirmed that ESN populations were predominantly glutamatergic. Furthermore, ESNs were functionally receptive to all toxins with sensitivities and responses consistent with primary neurons

  2. Leaders of neuronal cultures in a quorum percolation model

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    Jean-Pierre Eckmann

    2010-09-01

    Full Text Available We present a theoretical framework using quorum-percolation for describing the initiation of activity in a neural culture. The cultures are modeled as random graphs, whose nodes are neurons with $kin$ inputs and $kout$ outputs, and whose input degrees $kin=k$ obey given distribution functions $p_k$. We examine the firing activity of the population of neurons according to their input degree ($k$ classes and calculate for each class its firing probability $Phi_k(t$ as a function of $t$. The probability of a node to fire is found to be determined by its in-degree $k$, and the first-to-fire neurons are those that have a high $k$. A small minority of high-$k$ classes may be called ``Leaders,'' as they form an inter-connected subnetwork that consistently fires much before the rest of the culture. Once initiated, the activity spreads from the Leaders to the less connected majority of the culture. We then use the distribution of in-degree of the Leaders to study the growth rate of the number of neurons active in a burst, which was experimentally measured to be initially exponential. We find that this kind of growth rate is best described by a population that has an in-degree distribution that is a Gaussian centered around $k=75$ with width $sigma=31$ for the majority of the neurons, but also has a power law tail with exponent $-2$ for ten percent of the population. Neurons in the tail may have as many as $k=4,700$ inputs. We explore and discuss the correspondence between the degree distribution and a dynamic neuronal threshold, showing that from the functional point of view, structure and elementary dynamics are interchangeable. We discuss possible geometric origins of this distribution, and comment on the importance of size, or of having a large number of neurons, in the culture.

  3. Control of hair cell excitability by vestibular primary sensory neurons.

    OpenAIRE

    Brugeaud, Aurore; Travo, Cécile; Demêmes, Danielle; Lenoir, Marc; Llorens, Jordi; Puel, Jean-Luc; Chabbert, Christian

    2007-01-01

    International audience; In the rat utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat utricle in which the setting ...

  4. Progranulin is expressed within motor neurons and promotes neuronal cell survival

    Directory of Open Access Journals (Sweden)

    Kay Denis G

    2009-10-01

    Full Text Available Abstract Background Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival. Results In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months. This is mediated at least in part through

  5. Identification of neuronal network properties from the spectral analysis of calcium imaging signals in neuronal cultures.

    Science.gov (United States)

    Tibau, Elisenda; Valencia, Miguel; Soriano, Jordi

    2013-01-01

    Neuronal networks in vitro are prominent systems to study the development of connections in living neuronal networks and the interplay between connectivity, activity and function. These cultured networks show a rich spontaneous activity that evolves concurrently with the connectivity of the underlying network. In this work we monitor the development of neuronal cultures, and record their activity using calcium fluorescence imaging. We use spectral analysis to characterize global dynamical and structural traits of the neuronal cultures. We first observe that the power spectrum can be used as a signature of the state of the network, for instance when inhibition is active or silent, as well as a measure of the network's connectivity strength. Second, the power spectrum identifies prominent developmental changes in the network such as GABAA switch. And third, the analysis of the spatial distribution of the spectral density, in experiments with a controlled disintegration of the network through CNQX, an AMPA-glutamate receptor antagonist in excitatory neurons, reveals the existence of communities of strongly connected, highly active neurons that display synchronous oscillations. Our work illustrates the interest of spectral analysis for the study of in vitro networks, and its potential use as a network-state indicator, for instance to compare healthy and diseased neuronal networks.

  6. Identification of Neuronal Network Properties from the Spectral Analysis of Calcium Imaging Signals in Neuronal Cultures

    Directory of Open Access Journals (Sweden)

    Elisenda eTibau

    2013-12-01

    Full Text Available Neuronal networks in vitro are prominent systems to study the development of connections in living neuronal networks and the interplay between connectivity, activity and function. These cultured networks show a rich spontaneous activity that evolves concurrently with the connectivity of the underlying network. In this work we monitor the development of neuronal cultures, and record their activity using calcium fluorescence imaging. We use spectral analysis to characterize global dynamical and structural traits of the neuronal cultures. We first observe that the power spectrum can be used as a signature of the state of the network, for instance when inhibition is active or silent, as well as a measure of the network's connectivity strength. Second, the power spectrum identifies prominent developmental changes in the network such as GABAA switch. And third, the analysis of the spatial distribution of the spectral density, in experiments with a controlled disintegration of the network through CNQX, an AMPA-glutamate receptor antagonist in excitatory neurons, reveals the existence of communities of strongly connected, highly active neurons that display synchronous oscillations. Our work illustrates the interest of spectral analysis for the study of in vitro networks, and its potential use as a network-state indicator, for instance to compare healthy and diseased neuronal networks.

  7. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    Directory of Open Access Journals (Sweden)

    Costa Eunice M

    2009-09-01

    Full Text Available Abstract Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors.

  8. Inhibition of neuronal cell-cell adhesion measured by the microscopic aggregation assay and impedance sensing

    Science.gov (United States)

    Wiertz, R. W. F.; Marani, E.; Rutten, W. L. C.

    2010-10-01

    Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron-neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron-neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control of spatial arrangement of cells in culture. In the literature N-CAM, L1 and N-cadherin proteins are pointed out as main regulators of neuronal adhesion. In this study, these three main cell adhesion molecules were used to inhibit neuron-to-neuron adhesion and aggregation. Both soluble extracellular domains and antigen antibodies were added to these adhesion molecules. They were investigated for their blocking ability in neuronal cultures. First, in a 96 h aggregation assay on a low-adhesive substrate, the effect of inhibition of the three proteins on aggregation of cortical neurons was investigated optically. Both L1 antibody and L1 protein had no effect on the degree of aggregation. An N-cadherin antibody however was shown to be effective in aggregation inhibition at concentrations of 1 and 3 µg ml-1. Up to 96 h no aggregation occurred. A similar effect was achieved by the N-cadherin protein, although less distinct. N-CAM blocking revealed no inhibition of aggregation. Second, results from IS corresponded to those of the aggregation assays. In these experiments neuron-neuron adhesion was also inhibited by blocking N-CAM L1 and N-cadherin. Cortical neurons were cultured in small wells containing circular 100 µm diameter gold electrodes, so small changes in cell-cell interactions in monolayers of neurons could be monitored by IS. Impedances of neuron-covered electrodes were significantly lower in the presence of the N-cadherin antibody and protein at concentrations of 1, 3 and 10 µg ml-1, indicating a less profound binding between adjacent neurons. Results from the aggregation assays and impedance measurements demonstrate the applicability of blocking cell adhesion

  9. Optimizing NTS-polyplex as a tool for gene transfer to cultured dopamine neurons.

    Science.gov (United States)

    Hernandez-Baltazar, Daniel; Martinez-Fong, Daniel; Trudeau, Louis-Eric

    2012-01-01

    The study of signal transduction in dopamine (DA)-containing neurons as well as the development of new therapeutic approaches for Parkinson's disease requires the selective expression of transgenes in such neurons. Here we describe optimization of the use of the NTS-polyplex, a gene carrier system taking advantage of neurotensin receptor internalization, to transfect mouse DA neurons in primary culture. The plasmids DsRed2 (4.7 kbp) and VGLUT2-Venus (11 kbp) were used to compare the ability of this carrier system to transfect plasmids of different sizes. We examined the impact of age of the neurons (1, 3, 5 and 8 days after seeding), of culture media used during the transfection (Neurobasal with B27 vs. conditioned medium) and of three molar ratios of plasmid DNA to carrier. While the NTS-polyplex successfully transfected both plasmids in a control N1E-115 cell line, only the pDsRed2 plasmid could be transfected in primary cultured DA neurons. We achieved 20% transfection efficiency of pDsRed2 in DA neurons, with 80% cell viability. The transfection was demonstrated pharmacologically to be dependent on activation of neurotensin receptors and to be selective for DA neurons. The presence of conditioned medium for transfection was found to be required to insure cell viability. Highest transfection efficiency was achieved in the most mature neurons. In contrast, transfection with the VGLUT2-Venus plasmid produced cell damage, most likely due to the high molar ratios required, as evidenced by a 15% cell viability of DA neurons at the three molar ratios tested (1:36, 1:39 and 1:42). We conclude that, when used at molar ratios lower than 1:33, the NTS-polyplex can selectively transfect mature cultured DA neurons with only low levels of toxicity. Our results provide evidence that the NTS-polyplex has good potential for targeted gene delivery in cultured DA neurons, an in vitro system of great use for the screening of new therapeutic approaches for Parkinson's disease.

  10. Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor

    Directory of Open Access Journals (Sweden)

    Sherry L. Xu

    2012-01-01

    Full Text Available Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer’s disease and depression.

  11. Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor.

    Science.gov (United States)

    Xu, Sherry L; Choi, Roy C Y; Zhu, Kevin Y; Leung, Ka-Wing; Guo, Ava J Y; Bi, Dan; Xu, Hong; Lau, David T W; Dong, Tina T X; Tsim, Karl W K

    2012-01-01

    Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.

  12. Mammalian Cell Culture Simplified.

    Science.gov (United States)

    Moss, Robert; Solomon, Sondra

    1991-01-01

    A tissue culture experiment that does not require elaborate equipment and that can be used to teach sterile technique, the principles of animal cell line maintenance, and the concept of cell growth curves is described. The differences between cancerous and normal cells can be highlighted. The procedure is included. (KR)

  13. Reinnervation of hair cells by neural stem cell-derived neurons

    Institute of Scientific and Technical Information of China (English)

    Yuan Yasheng; Wang Yang; Chi Fanglu

    2014-01-01

    Background Replacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss.However,the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored.The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro.Methods NSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor.Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane.Results At Day 3,the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia.After 9-day co-culture,neurites of NSC-derived neurons predominantly elongated toward hair cells.Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells,indicating the formation of new synaptic connections.After 14-day culture,triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle),indicating the formation of new ribbon synapse.Conclusions NSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration.Whether the newly forming synapse is functional merits further electrophysiological study.

  14. Reinnervation of hair cells by neural stem cell-derived neurons.

    Science.gov (United States)

    Yuan, Yasheng; Wang, Yang; Chi, Fanglu

    2014-01-01

    Replacement of spiral ganglion neurons would be one prioritized step in an attempt to restore sensory neuronal hearing loss. However, the possibility that transplanted neurons could regenerate new synaptic connections to hair cells has not been explored. The objective of this study was to test whether neural stem cell (NSC)-derived neurons can form synaptic connections with hair cells in vitro. NSCs were mechanically separated from the hippocampus in SD rat embryos (E12-E14) and cultured in a serum-free medium containing basic fibroblast growth factor and epidermal growth factor. Rat NSCs were co-cultured with explants of cochlea sensory epithelia obtained from postnatal Day 3 rats under transway filter membrane. At Day 3, the NSCs began to show chemotactic differentiation and grew toward cochlea sensory epithelia. After 9-day co-culture, neurites of NSC-derived neurons predominantly elongated toward hair cells. Immunohistochemical analyses revealed the fibers overlapped with synapsin and hair cells, indicating the formation of new synaptic connections. After 14-day culture, triple staining revealed the fibers overlapped with PSD95 (postsynaptic density) which is juxtaposed with CtBP2 (presynaptic vesicle), indicating the formation of new ribbon synapse. NSC-derived neurons can make synaptic connections with hair cells and provide a model for studying synaptic plasticity and regeneration. Whether the newly forming synapse is functional merits further electrophysiological study.

  15. Fish stem cell cultures.

    Science.gov (United States)

    Hong, Ni; Li, Zhendong; Hong, Yunhan

    2011-04-13

    Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  16. Fish Stem Cell Cultures

    Directory of Open Access Journals (Sweden)

    Ni Hong, Zhendong Li, Yunhan Hong

    2011-01-01

    Full Text Available Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on “Fish Stem Cells and Nuclear Transfer”, we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.

  17. Astrocyte-neuron co-culture on microchips based on the model of SOD mutation to mimic ALS.

    Science.gov (United States)

    Kunze, Anja; Lengacher, Sylvain; Dirren, Elisabeth; Aebischer, Patrick; Magistretti, Pierre J; Renaud, Philippe

    2013-07-24

    Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.

  18. Efficient Differentiation of Embryonic Stem Cells into Neurons in Glial Cell-conditioned Medium under Attaching Conditions

    Institute of Scientific and Technical Information of China (English)

    Hai-Bin TIAN; Zeng-Liang BAI; Hong WANG; Jian-Quan CHEN; Guo-Xiang CHENG

    2005-01-01

    Embryonic stem (ES) cells can differentiate into neurons in vitro, which provides hope for the treatment of some neurodegenerative diseases through cell transplantation. However, it remains a challenge to efficiently induce ES cells to differentiate into neurons. Here, we show that murine ES cells can efficiently differentiate into neurons when cultured in glial cell- conditioned medium (GCM) under attaching conditions without the formation of embryoid bodies. In comparison with murine embryonic fibroblast-conditioned medium, we found that GCM has a positive effect on limiting the generation of non-neuronal cells, such as astrocytes. In addition, compared with suspension conditions, attaching conditions delay the differentiation process of ES cells.

  19. Time window characteristics of cultured rat hippocampal neurons subjected to ischemia and reperfusion

    Institute of Scientific and Technical Information of China (English)

    XU Zhong; XU Ru-xiang; LIU Bao-song; JIANG Xiao-dan; HUANG Tao; DING Lian-shu; YUAN Jun

    2005-01-01

    Objective: To explore cell death and apoptosis in rat hippocampal neurons at different time points after ischemia, hypoxia and reperfusion injury and to elucidate time window characteristics in ischemia neuronal injury.Methods: Hippocampal neurons were obtained from rat embryo and were cultured in vitro. The ischemia and reperfusion of cultured rat hippocampal neurons were simulated by oxygen-glucose deprivation (OGD) and recovery. OGD at different time points (0.25 h to 3.0 h) and then the same recovery (24 h) were prepared. Annexin V-PI staining and flow cytometry examined neuron death and apoptosis at different time after injury. Results: After OGD and recovery, both necrosis and apoptosis were observed. At different times after OGD, there were statistically significant differences in neuron necrosis rate (P0.05). At recovery, survival rate of hippocampal neurons further decreased while apoptosis rate increased. Furthermore, apoptosis rates of different time differed greatly (P<0.05). Apoptosis rate gradually increased with significant difference among those of different time points (P<0.05). However, 2 h after ischemia, apoptosis rate decreased markedly.Conclusions: Apoptosis is an important pathway of delayed neuron death. The therapeutic time window should be within 2 h after cerebral ischemia and hypoxia.

  20. Differential modulation of interleukin-6 expression by interleukin-1beta in neuronal and glial cultures.

    Science.gov (United States)

    Di Loreto, Silvia; Maccarone, Rita; Corvetti, Luigi; Sebastiani, Pierluigi; Piancatelli, Daniela; Adorno, Domenico

    2003-01-01

    We analysed the specific effects of IL-1beta immunoneutralization on the expression of IL-6 in different pure cultures of neurones and glia after both experimental subliminal hypoxia and recovery. Whereas the IL-1beta-deprivation signal induced a decrease in IL-6 expression and release of normoxic neurones, it provoked an increase in IL-6 protein in hypoxic neurones. Moreover, the direct correlation between IL-1beta and IL-6, observed in normal and recovering neuronal cultures, was reversed in hypoxic conditions. These reversals were not observed in glial cells, in which IL-1beta immunosuppression led to a decrease in IL-6 under all conditions considered. In conclusion, the IL-1beta modulates IL-6 in different ways according to the ambient physiological or pathological conditions, and also acts via different mechanisms, depending on the cellular phenotype.

  1. Maturation of spinal motor neurons derived from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tomonori Takazawa

    Full Text Available Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

  2. NRSF causes cAMP-sensitive suppression of sodium current in cultured hippocampal neurons

    Science.gov (United States)

    Nadeau, H.; Lester, H. A.

    2002-01-01

    The neuron restrictive silencer factor (NRSF/REST) has been shown to bind to the promoters of many neuron-specific genes and is able to suppress transcription of Na(+) channels in PC12 cells, although its functional effect in terminally differentiated neurons is unknown. We constructed lentiviral vectors to express NRSF as a bicistronic message with green fluorescent protein (GFP) and followed infected hippocampal neurons in culture over a period of 1-2 wk. NRSF-expressing neurons showed a time-dependent suppression of Na(+) channel function as measured by whole cell electrophysiology. Suppression was reversed or prevented by the addition of membrane-permeable cAMP analogues and enhanced by cAMP antagonists but not affected by increasing protein expression with a viral enhancer. Secondary effects, including altered sensitivity to glutamate and GABA and reduced outward K(+) currents, were duplicated by culturing GFP-infected control neurons in TTX. The striking similarity of the phenotypes makes NRSF potentially useful as a genetic "silencer" and also suggests avenues of further exploration that may elucidate the transcription factor's in vivo role in neuronal plasticity.

  3. Long-term culture of pluripotent stem-cell-derived human neurons on diamonds" A substrate for neurodegeneration research and therapy

    OpenAIRE

    Caldwell, Maeve

    2015-01-01

    PUBLISHED Brain Computer Interfaces (BCI) currently represent a field of intense research aimed both at understanding neural circuit physiology and at providing functional therapy for traumatic or degenerative neurological conditions. Due to its chemical inertness, biocompatibility and stability, diamond is currently being actively investigated as a potential substrate material for culturing cells and for use as the electrically active component of a neural sensor. Here we provide a protoc...

  4. Long-term culture of pluripotent stem-cell-derived human neurons on diamond - A substrate for neurodegeneration research and therapy

    OpenAIRE

    Nistor, Paul A.; May, Paul W.; Tamagnini, Francesco; Randall, Andrew D; Caldwell, Maeve A.

    2015-01-01

    Brain Computer Interfaces (BCI) currently represent a field of intense research aimed both at understanding neural circuit physiology and at providing functional therapy for traumatic or degenerative neurological conditions. Due to its chemical inertness, biocompatibility and stability, diamond is currently being actively investigated as a potential substrate material for culturing cells and for use as the electrically active component of a neural sensor. Here we provide a protocol for the di...

  5. Quantitative Analysis of Rat Dorsal Root Ganglion Neurons Cultured on Microelectrode Arrays Based on Fluorescence Microscopy Image Processing.

    Science.gov (United States)

    Mari, João Fernando; Saito, José Hiroki; Neves, Amanda Ferreira; Lotufo, Celina Monteiro da Cruz; Destro-Filho, João-Batista; Nicoletti, Maria do Carmo

    2015-12-01

    Microelectrode Arrays (MEA) are devices for long term electrophysiological recording of extracellular spontaneous or evocated activities on in vitro neuron culture. This work proposes and develops a framework for quantitative and morphological analysis of neuron cultures on MEAs, by processing their corresponding images, acquired by fluorescence microscopy. The neurons are segmented from the fluorescence channel images using a combination of segmentation by thresholding, watershed transform, and object classification. The positioning of microelectrodes is obtained from the transmitted light channel images using the circular Hough transform. The proposed method was applied to images of dissociated culture of rat dorsal root ganglion (DRG) neuronal cells. The morphological and topological quantitative analysis carried out produced information regarding the state of culture, such as population count, neuron-to-neuron and neuron-to-microelectrode distances, soma morphologies, neuron sizes, neuron and microelectrode spatial distributions. Most of the analysis of microscopy images taken from neuronal cultures on MEA only consider simple qualitative analysis. Also, the proposed framework aims to standardize the image processing and to compute quantitative useful measures for integrated image-signal studies and further computational simulations. As results show, the implemented microelectrode identification method is robust and so are the implemented neuron segmentation and classification one (with a correct segmentation rate up to 84%). The quantitative information retrieved by the method is highly relevant to assist the integrated signal-image study of recorded electrophysiological signals as well as the physical aspects of the neuron culture on MEA. Although the experiments deal with DRG cell images, cortical and hippocampal cell images could also be processed with small adjustments in the image processing parameter estimation.

  6. Statins induce differentiation and cell death in neurons and astroglia.

    Science.gov (United States)

    März, Pia; Otten, Uwe; Miserez, André R

    2007-01-01

    Statins are potent inhibitors of the hydroxy-methyl-glutaryl-coenzyme A reductase, the rate limiting enzyme for cholesterol biosynthesis. Experimental and clinical studies with statins suggest that they have beneficial effects on neurodegenerative disorders. Thus, it was of interest to characterize the direct effects of statins on CNS neurons and glial cells. We have treated defined cultures of neurons and astrocytes of newborn rats with two lipophilic statins, atorvastatin and simvastatin, and analyzed their effects on morphology and survival. Treatment of astrocytes with statins induced a time- and dose-dependent stellation, followed by apoptosis. Similarly, statins elicited programmed cell death of cerebellar granule neurons but with a higher sensitivity. Analysis of different signaling cascades revealed that statins fail to influence classical pathways such as Akt or MAP kinases, known to be activated in CNS cells. In addition, astrocyte stellation triggered by statins resembled dibutryl-cyclic AMP (db-cAMP) induced morphological differentiation. However, in contrast to db-cAMP, statins induced upregulation of low-density lipoprotein receptors, without affecting GFAP expression, indicating separate underlying mechanisms. Analysis of the cholesterol biosynthetic pathway revealed that lack of mevalonate and of its downstream metabolites, mainly geranylgeranyl-pyrophosphate (GGPP), is responsible for the statin-induced apoptosis of neurons and astrocytes. Moreover, astrocytic stellation triggered by statins was inhibited by mevalonate and GGPP. Interestingly, neuronal cell death was significantly reduced in astrocyte/neuron co-cultures treated with statins. We postulate that under these conditions signals provided by astrocytes, e.g., isoprenoids play a key role in neuronal survival.

  7. Controlled adhesion and growth of long term glial and neuronal cultures on Parylene-C.

    Directory of Open Access Journals (Sweden)

    Evangelos Delivopoulos

    Full Text Available This paper explores the long term development of networks of glia and neurons on patterns of Parylene-C on a SiO(2 substrate. We harvested glia and neurons from the Sprague-Dawley (P1-P7 rat hippocampus and utilized an established cell patterning technique in order to investigate cellular migration, over the course of 3 weeks. This work demonstrates that uncontrolled glial mitosis gradually disrupts cellular patterns that are established early during culture. This effect is not attributed to a loss of protein from the Parylene-C surface, as nitrogen levels on the substrate remain stable over 3 weeks. The inclusion of the anti-mitotic cytarabine (Ara-C in the culture medium moderates glial division and thus, adequately preserves initial glial and neuronal conformity to underlying patterns. Neuronal apoptosis, often associated with the use of Ara-C, is mitigated by the addition of brain derived neurotrophic factor (BDNF. We believe that with the right combination of glial inhibitors and neuronal promoters, the Parylene-C based cell patterning method can generate structured, active neural networks that can be sustained and investigated over extended periods of time. To our knowledge this is the first report on the concurrent application of Ara-C and BDNF on patterned cell cultures.

  8. New Insight in Neuron Regeneration: Induction of Glia Cell to Neuron Cell

    Directory of Open Access Journals (Sweden)

    Morteza Aliashrafi

    2016-04-01

    Full Text Available Induction neuron from a veriety of cell resource were remaining challeng in regenerative medicine, so finding the convenient method to reprogram different cells to neuron could be helpful. In this study, we analysis the transcriptome of glia and neuron cells to determine the gene expression in neuron that different when compare to glia cells. Then based on this transcriptom data seek the transcription factor and miRNA. Data extract from transcriptome database of mouse cells comprise cerebral cortex that generated by RNAseq technique. By comparison neuron against glia cells (astrocyte, oligodenderocyte and microglia determined different gene expression in neuron. By using genetrail2 database determined transcription factor and miRNA associated with neuron gene expression.Result determined the 500 genes with different expression in neuron in comparison with glia cells. 2 significant TF families, DLX and MSX, 3 TF, Sp1, Ctcf and Pax1, 85 miRNA release from analysis this 500 gene. Analysis the gene target of all identified miRNA represent the important biological process related to neurogenesis neurodevelopment, in addition to most important proteins like Dnm1, Gad1 and Grin1 were obtained by functional and structural of network analysis. Dnm1 and Grin1 regulated by Sp1. In sum up, since one of the methods to reprogramming resident glia cells to induce neuron is applying TF and miRNAs, TF like Sp1 lonely or in combination with other factors can be experimentally approved.

  9. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and ti...

  10. Sulfite triggers sustained calcium overload in cultured cortical neurons via a redox-dependent mechanism.

    Science.gov (United States)

    Wang, Xiao; Cao, Hui; Guan, Xin-Lei; Long, Li-Hong; Hu, Zhuang-Li; Ni, Lan; Wang, Fang; Chen, Jian-Guo; Wu, Peng-Fei

    2016-09-06

    Sulfite is a compound commonly used as preservative in foods and pharmaceuticals. Many studies have examined the neurotoxicity of sulfite, but its effect on neuronal calcium homeostasis has not yet been reported. Here, we observed the effect of sulfite on the cytosolic free calcium concentration ([Ca(2+)]i) in cultured cortical neurons using Fura-2/AM based calcium imaging technique. Sulfite (250-1000μM) caused a sustained increase in [Ca(2+)]i in the neurons via a dose-dependent manner. In Ca(2+)-free solution, sulfite failed to increase [Ca(2+)]i. After the depletion of the intracellular calcium store, the effect of sulfite on the [Ca(2+)]i was largely abolished. Pharmacological inhibition of phospholipase C (PLC)-inositol 1,4,5-triphosphate (IP3) signaling pathway blocked sulfite-induced increase of [Ca(2+)]i. Interestingly, antioxidants such as trolox and dithiothreitol, abolished the increase of [Ca(2+)]i induced by sulfite. Exposure to sulfite triggered generation of sulfur- and oxygen-centered free radicals in neurons and increased oxidative stress both in the cultured cortical neurons and the prefrontal cortex of rats. Furthemore, sulfite decreased cell viability in cultured cortical neurons via a calcium-dependent manner. Thus, our current study suggests that the redox-dependent calcium overload triggered by sulfite in cortical neuronsmay be involved in its neurotoxicity. Copyright © 2016. Published by Elsevier Ireland Ltd.

  11. Effects of inorganic lead on the differentiation and growth of cortical neurons in culture.

    Science.gov (United States)

    Kern, M; Audesirk, T; Audesirk, G

    1993-01-01

    Lead exposure has devastating effects on the developing nervous system, producing morphological, cognitive, and behavioral deficits. To elucidate some of the mechanisms of lead neurotoxicity, we have examined its effects on the differentiation of several types of cultured neurons. Previously, we reported the effects of inorganic lead on several parameters of growth and differentiation of E18 rat hippocampal neurons and two types of neuroblastoma cells cultured in medium with 2% fetal calf serum (FCS) (Audesirk et al., 1991). In the present study, we report the effects of concentrations of lead ranging from 1nM to 1 mM on the differentiation of hippocampal neurons cultured in medium containing 10% FCS. In addition, we investigated lead effects on neurons isolated from the motor cortex region of the E18 rat embryo. Cortical neurons were exposed to lead in concentrations ranging from 0.1 nM to 1 mM in medium with either 10% FCS or 2% FCS for 48 hr. The effects of lead tended to be multimodal. Neurite initiation, which is highly sensitive to neurotoxic compounds, was inhibited by lead at both high and low concentrations, with no effects at intermediate levels. Medium with 10% FCS enhanced certain growth parameters and tended to reduce the effects of lead. There was an overall consistency in the effects of lead on motor cortex and hippocampal neurons.

  12. CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors

    Directory of Open Access Journals (Sweden)

    Mihalescu-Maingot Maria

    2010-02-01

    Full Text Available Abstract Background Tumor initiating cells (TICs provide a new paradigm for developing original therapeutic strategies. Methods We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas. Results A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs. Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide. Conclusions Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

  13. Ontogeny of electrically excitable cells in cultured olfactory epithelium.

    OpenAIRE

    Schubert, D; Stallcup, W.; LaCorbiere, M; Kidokoro, Y; Orgel, L

    1985-01-01

    A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the di...

  14. Mild hypoxia affects synaptic connectivity in cultured neuronal networks.

    Science.gov (United States)

    Hofmeijer, Jeannette; Mulder, Alex T B; Farinha, Ana C; van Putten, Michel J A M; le Feber, Joost

    2014-04-01

    Eighty percent of patients with chronic mild cerebral ischemia/hypoxia resulting from chronic heart failure or pulmonary disease have cognitive impairment. Overt structural neuronal damage is lacking and the precise cause of neuronal damage is unclear. As almost half of the cerebral energy consumption is used for synaptic transmission, and synaptic failure is the first abrupt consequence of acute complete anoxia, synaptic dysfunction is a candidate mechanism for the cognitive deterioration in chronic mild ischemia/hypoxia. Because measurement of synaptic functioning in patients is problematic, we use cultured networks of cortical neurons from new born rats, grown over a multi-electrode array, as a model system. These were exposed to partial hypoxia (partial oxygen pressure of 150Torr lowered to 40-50Torr) during 3 (n=14) or 6 (n=8) hours. Synaptic functioning was assessed before, during, and after hypoxia by assessment of spontaneous network activity, functional connectivity, and synaptically driven network responses to electrical stimulation. Action potential heights and shapes and non-synaptic stimulus responses were used as measures of individual neuronal integrity. During hypoxia of 3 and 6h, there was a statistically significant decrease of spontaneous network activity, functional connectivity, and synaptically driven network responses, whereas direct responses and action potentials remained unchanged. These changes were largely reversible. Our results indicate that in cultured neuronal networks, partial hypoxia during 3 or 6h causes isolated disturbances of synaptic connectivity.

  15. Comparison of Ca2+ currents of peptidergic neurons developing differing morphology with time in culture.

    Science.gov (United States)

    Meyers, D E; Cooke, I M

    1997-02-01

    The whole-cell patch-clamp technique was used to examine Ca2+ currents (ICa) in mature neurons cultured in defined medium and derived from the principal neurosecretory system of decapod crustaceans, the X-organ-sinus gland. After 1 day in culture, X-organ neurons of the crab Cardisoma carnifex showed vigorous outgrowth characterized either by the production of broad lamellipodia (veils) or, from smaller somata, a branching morphology. The neurons developing veils (veilers) had a large ICa (approximately 650 pA) and ICa current density (approximately 5 microA cm-2) while other types of neuron had little or no ICa. This distinction between the two types was still present after 5-6 days in culture. However, morphologies observed after additional outgrowth, when correlated with the ICa responses, allowed four groups to be distinguished: (1) veilers and (2) branching veilers, which developed from veilers and had a similar ICa density (approximately 3 microA cm-2); and, developing from the 1 day branchers, (3) spiny branchers or (4) small cells (ICa density approximately 0.8 microA cm-2). Immunoreactivity indicative of the presence of crustacean hyperglycemic hormone was found in all veilers and branching veilers tested, while moltinhibiting hormone reactivity, when observed, was seen in cells having a robust ICa density (> or = 1.2 microA cm-2). Normalized average current-voltage curves for each morphological group were examined for changes with increasing time in culture. The curves were consistent with the ICa being produced by a population of high-voltage-activated Ca2+ channels whose properties are biophysically indistinguishable and unaffected by time in culture. The averaged peak current did not change, despite an increase in neuronal surface area as outgrowth proceeded, and this resulted in a reduction of ICa density. This indicated that net addition of Ca2+ channels did not match the addition of new membrane under our culturing conditions.

  16. Development of an artificial neuronal network with post-mitotic rat fetal hippocampal cells by polyethylenimine.

    Science.gov (United States)

    Liu, Bingfang; Ma, Jun; Gao, Erjing; He, Yu; Cui, Fuzhai; Xu, Qunyuan

    2008-03-14

    The selection of appropriate surface materials that promote cellular adhesion and growth is an important consideration when designing a simplified neuronal network in vitro. In the past, extracellular matrix proteins such as laminin (LN) or positively charged substances such as poly-l-lysine (PLL) have been used. In this study, we examined the ability of another positively charged polymer, polyethyleneimine (PEI), to promote neuronal adhesion, growth and the formation of a functional neuronal network in vitro. PEI, PLL and LN were used to produce grid-shape patterns on glass coverslips by micro-contact printing. Post-mitotic neurons from the rat fetal hippocampus were cultured on the different polymers and the viability and morphology of these neurons under serum-free culture conditions were observed using fluorescent microscopy and atomic force microscopy (AFM). We show that neurons cultured on the PEI- and PLL-coated surfaces adhered to and extended neurites along the grid-shape patterns, whereas neurons cultured on the LN-coated coverslips clustered into clumps of cells. In addition, we found that the neurons on the PEI and PLL-coated grids survived for more than 2 weeks in serum-free conditions, whereas most neurons cultured on the LN-coated grids died after 1 week. Using AFM, we observed some neurosynapse-like structures near the neuronal soma on PEI-coated coverslips. These findings indicate that PEI is a suitable surface for establishing a functional neuronal network in vitro.

  17. Novel cell separation method for molecular analysis of neuron-astrocyte cocultures

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    Andrea eGoudriaan

    2014-01-01

    Full Text Available Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that many astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-responsive astrocyte genes in vitro, we sought to establish an expedite technique for separation of neurons from co-cultured astrocytes. Our newly established method makes use of cold jet, which exploits different adhesion characteristics of subpopulations of cells (Jirsova et al., 1997, and is rapid, performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this purification method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis determined that many astrocytic mRNAs and biological processes are regulated by neuronal interaction. Our results validate the cold jet as an efficient method to separate astrocytes from neurons in co-culture, and reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

  18. Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures.

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    Dario Greco

    Full Text Available In the mammalian central nervous system (CNS an important contingent of dopaminergic neurons are localized in the substantia nigra and in the ventral tegmental area of the ventral midbrain. They constitute an anatomically and functionally heterogeneous group of cells involved in a variety of regulatory mechanisms, from locomotion to emotional/motivational behavior. Midbrain dopaminergic neuron (mDA primary cultures represent a useful tool to study molecular mechanisms involved in their development and maintenance. Considerable information has been gathered on the mDA neurons development and maturation in vivo, as well as on the molecular features of mDA primary cultures. Here we investigated in detail the gene expression differences between the tissue of origin and ventral midbrain primary cultures enriched in mDA neurons, using microarray technique. We integrated the results based on different re-annotations of the microarray probes. By using knowledge-based gene network techniques and promoter sequence analysis, we also uncovered mechanisms that might regulate the expression of CNS genes involved in the definition of the identity of specific cell types in the ventral midbrain. We integrate bioinformatics and functional genomics, together with developmental neurobiology. Moreover, we propose guidelines for the computational analysis of microarray gene expression data. Our findings help to clarify some molecular aspects of the development and differentiation of DA neurons within the midbrain.

  19. Culture of skin-derived precursors and their differentiation into neurons

    Institute of Scientific and Technical Information of China (English)

    杨立业; 郑佳坤; 刘相名; 惠国桢; 郭礼和

    2004-01-01

    Objective:To investigate the culture method of skin- derived precursors ( SKPs ) and to explore a new cell source for cell transplantation of central nervous system. Methods: Cells from skins of juvenile and adult mice were isolated and cultured in serum-free medium.A mechanical method was chosen to passage these cells and they were idemified by the immunocytochemistry assay. Results: SKPs could be isolated from adult and neonatal skins. They could be maintained in vitro for long periods with stable proliferation, and expanded as undifferentiated cells in culture for more than 12 passages. About 50% of SKPs expressed nestin and majority of these cells expressed fibronectin when they were plated on polyornithine and laminin coated plates. About 5% cells showed neuronal differentiation and expressed neurofilament-M (NF-M) and NSE when SKPs were plated in serun-containing medium, and these cells could also differentiate into adipocytes and fibroblast-like cells.Conclusions: The data support the hypothesis that adult skin contains stem cells capable of differentiating into neurons, adipocytes, and fibroblast-like cells. They may represent an alternative autologous stem cell source for CNS cell transplantation.

  20. Identification of a mouse synaptic glycoprotein gene in cultured neurons.

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    Yu, Albert Cheung-Hoi; Sun, Chun Xiao; Li, Qiang; Liu, Hua Dong; Wang, Chen Ran; Zhao, Guo Ping; Jin, Meilei; Lau, Lok Ting; Fung, Yin-Wan Wendy; Liu, Shuang

    2005-10-01

    Neuronal differentiation and aging are known to involve many genes, which may also be differentially expressed during these developmental processes. From primary cultured cerebral cortical neurons, we have previously identified various differentially expressed gene transcripts from cultured cortical neurons using the technique of arbitrarily primed PCR (RAP-PCR). Among these transcripts, clone 0-2 was found to have high homology to rat and human synaptic glycoprotein. By in silico analysis using an EST database and the FACTURA software, the full-length sequence of 0-2 was assembled and the clone was named as mouse synaptic glycoprotein homolog 2 (mSC2). DNA sequencing revealed transcript size of mSC2 being smaller than the human and rat homologs. RT-PCR indicated that mSC2 was expressed differentially at various culture days. The mSC2 gene was located in various tissues with higher expression in brain, lung, and liver. Functions of mSC2 in neurons and other tissues remain elusive and will require more investigation.

  1. Persistent dynamic attractors in activity patterns of cultured neuronal networks

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    Wagenaar, Daniel A.; Nadasdy, Zoltan; Potter, Steve M.

    2006-05-01

    Three remarkable features of the nervous system—complex spatiotemporal patterns, oscillations, and persistent activity—are fundamental to such diverse functions as stereotypical motor behavior, working memory, and awareness. Here we report that cultured cortical networks spontaneously generate a hierarchical structure of periodic activity with a strongly stereotyped population-wide spatiotemporal structure demonstrating all three fundamental properties in a recurring pattern. During these “superbursts,” the firing sequence of the culture periodically converges to a dynamic attractor orbit. Precursors of oscillations and persistent activity have previously been reported as intrinsic properties of the neurons. However, complex spatiotemporal patterns that are coordinated in a large population of neurons and persist over several hours—and thus are capable of representing and preserving information—cannot be explained by known oscillatory properties of isolated neurons. Instead, the complexity of the observed spatiotemporal patterns implies large-scale self-organization of neurons interacting in a precise temporal order even in vitro, in cultures usually considered to have random connectivity.

  2. Cell Culturing of Cytoskeleton

    Science.gov (United States)

    2004-01-01

    Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

  3. Functional P2X7 receptors at cultured hippocampal astrocytes but not neurons.

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    Rubini, Patrizia; Pagel, Gregor; Mehri, Soghra; Marquardt, Peter; Riedel, Thomas; Illes, Peter

    2014-10-01

    P2X7 receptors have been suggested to be located both on neurons and astrocytes of the central and peripheral nervous systems. In the present Ca(2+)-imaging and patch-clamp study, we reinvestigated these findings on mixed neuronal-astrocytic cell cultures prepared from embryonic or newborn rat hippocampi. We found in a Mg(2+)-free bath medium that the prototypic P2X7 receptor agonist dibenzoyl-adenosine triphosphate (Bz-ATP) increased the intracellular Ca(2+) concentration ([Ca(2+)]i) both in the neuronal cell bodies and in their axo-dendritic processes only to a very minor extent. However, Bz-ATP produced marked [Ca(2+)]i transients in the neuronal processes, when they grew above a glial carpet, which was uniformly sensitive to Bz-ATP. These glial signals might be misinterpreted as neuronal responses because of the poor focal discrimination by a fluorescent microscope. Most astrocytes had a polygonal shape without clearly circumscribable boundaries, but a subgroup of them had neuron-like appearance. The cellular processes of this astrocytic subgroup, just as their cell somata and their polygonal counterparts, appeared to possess a high density of functional P2X7 receptors. In contrast to astrocytes, in a low Ca(2+)/no Mg(2+)-containing bath medium, hippocampal neurons failed to respond to Bz-ATP with membrane currents. In addition, neither the amplitude nor the frequency of spontaneous excitatory postsynaptic currents, representing the quantal release of glutamate, was modified by Bz-ATP. We conclude that cultured hippocampal neurons, in contrast to astrocytes, possess P2X7 receptors, if at all, only at a low density.

  4. Stem cells decreased neuronal cell death after hypoxic stress in primary fetal rat neurons in vitro.

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    Sakai, Tetsuro; Xu, Yan

    2012-01-01

    To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h nad then were returned to a normoxic condition. The study group then received rat umbilical cord matrix-derived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rα (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK α1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

  5. Tff3 is Expressed in Neurons and Microglial Cells

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    Ting Fu

    2014-11-01

    Full Text Available Background/Aims: The trefoil factor family (TFF peptide TFF3 is typically secreted by mucous epithelia, but is also expressed in the immune system and the brain. It was the aim of this study to determine the cerebral cell types which express Tff3. Methods: Primary cultures from rat embryonic or neonatal cerebral cortex and hippocampus, respectively, were studied by means of RT-PCR and immunofluorescence. Moreover, Tff3 expression was localized by immunocytochemistry in sections of adult rat cerebellum. Results: Tff3 transcripts were detectable in neural cultures of both the cortex and the hippocampus as well as in glial cell-enriched cultures. Tff3 peptide co-localized with Map2 indicating an expression in neurons in vitro. The neuronal expression was confirmed by immunofluorescence studies of adult rat cerebellum. Furthermore, Tff3 peptide showed also a clear co-localization with Iba-1 in vitro typical of activated microglial cells. Conclusion: The neuronal expression of Tff3 is in line with a function of a typical neuropeptide influencing, e.g., fear, memory, depression and motoric skills. The expression in activated microglial cells, which is demonstrated here for the first time, points towards a possible function for Tff3 in immune reactions in the CNS. This opens a plethora of additional possible functions for Tff3 including synaptic plasticity and cognition as well as during neuroinflammatory diseases and psychiatric disorders.

  6. Oscillating Cell Culture Bioreactor

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    Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

    2010-01-01

    To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid

  7. Self-contained induction of neurons from human embryonic stem cells.

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    Tsuyoshi Okuno

    Full Text Available BACKGROUND: Neurons and glial cells can be efficiently induced from mouse embryonic stem (ES cells in a conditioned medium collected from rat primary-cultured astrocytes (P-ACM. However, the use of rodent primary cells for clinical applications may be hampered by limited supply and risk of contamination with xeno-proteins. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an alternative method for unimpeded production of human neurons under xeno-free conditions. Initially, neural stem cells in sphere-like clusters were induced from human ES (hES cells after being cultured in P-ACM under free-floating conditions. The resultant neural stem cells could circumferentially proliferate under subsequent adhesive culture, and selectively differentiate into neurons or astrocytes by changing the medium to P-ACM or G5, respectively. These hES cell-derived neurons and astrocytes could procure functions similar to those of primary cells. Interestingly, a conditioned medium obtained from the hES cell-derived astrocytes (ES-ACM could successfully be used to substitute P-ACM for induction of neurons. Neurons made by this method could survive in mice brain after xeno-transplantation. CONCLUSION/SIGNIFICANCE: By inducing astrocytes from hES cells in a chemically defined medium, we could produce human neurons without the use of P-ACM. This self-serving method provides an unlimited source of human neural cells and may facilitate clinical applications of hES cells for neurological diseases.

  8. Perampanel inhibition of AMPA receptor currents in cultured hippocampal neurons.

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    Chao-Yin Chen

    Full Text Available Perampanel is an aryl substituted 2-pyridone AMPA receptor antagonist that was recently approved as a treatment for epilepsy. The drug potently inhibits AMPA receptor responses but the mode of block has not been characterized. Here the action of perampanel on AMPA receptors was investigated by whole-cell voltage-clamp recording in cultured rat hippocampal neurons. Perampanel caused a slow (τ∼1 s at 3 µM, concentration-dependent inhibition of AMPA receptor currents evoked by AMPA and kainate. The rates of block and unblock of AMPA receptor currents were 1.5×105 M-1 s-1 and 0.58 s-1, respectively. Perampanel did not affect NMDA receptor currents. The extent of block of non-desensitizing kainate-evoked currents (IC50, 0.56 µM was similar at all kainate concentrations (3-100 µM, demonstrating a noncompetitive blocking action. Parampanel did not alter the trajectory of AMPA evoked currents indicating that it does not influence AMPA receptor desensitization. Perampanel is a selective negative allosteric AMPA receptor antagonist of high-affinity and slow blocking kinetics.

  9. Reduced synaptic activity in neuronal networks derived from embryonic stem cells of murine Rett syndrome model.

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    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E

    2014-01-01

    Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used in vitro cultures of neurons derived from murine embryonic stem cells missing the methyl-CpG-binding protein 2 (MECP2) gene (MeCP2-/y) and from wild type cells of the corresponding background. Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology.

  10. Genetic study of axon regeneration with cultured adult dorsal root ganglion neurons.

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    Saijilafu; Zhou, Feng-Quan

    2012-08-17

    It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS(1,2). In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries(3). Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved(4). Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth(5-7). Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection(6,8). By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred

  11. Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

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    Raymon, H K; Thode, S; Zhou, J; Friedman, G C; Pardinas, J R; Barrere, C; Johnson, R M; Sah, D W

    1999-07-01

    A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

  12. Addition of glutamate to serum free culture promotes recovery of electrical activity in adult hippocampal neurons in vitro

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    Edwards, Darin; Das, Mainak; Molnar, Peter; Hickman, James J.

    2010-01-01

    A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25 μM for 1 to 7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult derived neuronal systems, especially for traumatic brain injury (TBI). PMID:20452373

  13. Addition of glutamate to serum-free culture promotes recovery of electrical activity in adult hippocampal neurons in vitro.

    Science.gov (United States)

    Edwards, Darin; Das, Mainak; Molnar, Peter; Hickman, James J

    2010-07-15

    A long-term cell culture system utilizing normal adult hippocampal neurons would represent an important tool that could be useful in research on the mature brain, neurological disorders and age-related neurological diseases. Historically, in vitro neuronal systems are derived from embryonic rather than mature brain tissue, a practice predicated upon difficulties in supporting regeneration, functional recovery and long-term survival of adult neurons in vitro. A few studies have shown that neurons derived from the hippocampal tissue of adult rats can survive and regenerate in vitro under serum-free conditions. However, while the adult neurons regenerated morphologically under these conditions, both the electrical activity characteristic of in vivo neurons as well as long-term neuronal survival was not consistently recovered in vitro. In this study, we report on the development of a defined culture system with the ability to support functional recovery and long-term survival of adult rat hippocampal neurons. In this system, the cell-adhesive substrate, N-1 [3-(trimethoxysilyl) propyl]-diethylenetriamine, supported neuronal attachment, regeneration, and long-term survival of adult neurons for more than 80 days in vitro. Additionally, the excitatory neurotransmitter glutamate, applied at 25muM for 1-7 days after morphological neuronal regeneration in vitro, enabled full recovery of neuronal electrical activity. This low concentration of glutamate promoted the recovery of neuronal electrical activity but with minimal excitotoxicity. These improvements allowed electrically active adult neurons to survive in vitro for several months, providing a stable test-bed for the long-term study of regeneration in adult-derived neuronal systems, especially for traumatic brain injury (TBI). Copyright 2010 Elsevier B.V. All rights reserved.

  14. Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons.

    Science.gov (United States)

    Chandrasekaran, Vidya; Lea, Charlotte; Sosa, Jose Carlo; Higgins, Dennis; Lein, Pamela J

    2015-07-01

    Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons.

  15. Synaptic network activity induces neuronal differentiation of adult hippocampal precursor cells through BDNF signaling

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    Harish Babu

    2009-09-01

    Full Text Available Adult hippocampal neurogenesis is regulated by activity. But how do neural precursor cells in the hippocampus respond to surrounding network activity and translate increased neural activity into a developmental program? Here we show that long-term potential (LTP-like synaptic activity within a cellular network of mature hippocampal neurons promotes neuronal differentiation of newly generated cells. In co-cultures of precursor cells with primary hippocampal neurons, LTP-like synaptic plasticity induced by addition of glycine in Mg2+-free media for 5 min, produced synchronous network activity and subsequently increased synaptic strength between neurons. Furthermore, this synchronous network activity led to a significant increase in neuronal differentiation from the co-cultured neural precursor cells. When applied directly to precursor cells, glycine and Mg2+-free solution did not induce neuronal differentiation. Synaptic plasticity-induced neuronal differentiation of precursor cells was observed in the presence of GABAergic neurotransmission blockers but was dependent on NMDA-mediated Ca2+ influx. Most importantly, neuronal differentiation required the release of brain-derived neurotrophic factor (BDNF from the underlying substrate hippocampal neurons as well as TrkB receptor phosphorylation in precursor cells. This suggests that activity-dependent stem cell differentiation within the hippocampal network is mediated via synaptically evoked BDNF signaling.

  16. Transcriptional responses of cultured rat sympathetic neurons during BMP-7-induced dendritic growth.

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    Michelle M Garred

    Full Text Available BACKGROUND: Dendrites are the primary site of synapse formation in the vertebrate nervous system; however, relatively little is known about the molecular mechanisms that regulate the initial formation of primary dendrites. Embryonic rat sympathetic neurons cultured under defined conditions extend a single functional axon, but fail to form dendrites. Addition of bone morphogenetic proteins (BMPs triggers these neurons to extend multiple dendrites without altering axonal growth or cell survival. We used this culture system to examine differential gene expression patterns in naïve vs. BMP-treated sympathetic neurons in order to identify candidate genes involved in regulation of primary dendritogenesis. METHODOLOGY/PRINCIPAL FINDINGS: To determine the critical transcriptional window during BMP-induced dendritic growth, morphometric analysis of microtubule-associated protein (MAP-2-immunopositive processes was used to quantify dendritic growth in cultures exposed to the transcription inhibitor actinomycin-D added at varying times after addition of BMP-7. BMP-7-induced dendritic growth was blocked when transcription was inhibited within the first 24 hr after adding exogenous BMP-7. Thus, total RNA was isolated from sympathetic neurons exposed to three different experimental conditions: (1 no BMP-7 treatment; (2 treatment with BMP-7 for 6 hr; and (3 treatment with BMP-7 for 24 hr. Affymetrix oligonucleotide microarrays were used to identify differential gene expression under these three culture conditions. BMP-7 significantly regulated 56 unique genes at 6 hr and 185 unique genes at 24 hr. Bioinformatic analyses implicate both established and novel genes and signaling pathways in primary dendritogenesis. CONCLUSIONS/SIGNIFICANCE: This study provides a unique dataset that will be useful in generating testable hypotheses regarding transcriptional control of the initial stages of dendritic growth. Since BMPs selectively promote dendritic growth in

  17. Microglia activation and interaction with neuronal cells in a biochemical model of mevalonate kinase deficiency.

    Science.gov (United States)

    Tricarico, Paola Maura; Piscianz, Elisa; Monasta, Lorenzo; Kleiner, Giulio; Crovella, Sergio; Marcuzzi, Annalisa

    2015-08-01

    Mevalonate kinase deficiency is a rare disease whose worst manifestation, characterised by severe neurologic impairment, is called mevalonic aciduria. The progressive neuronal loss associated to cell death can be studied in vitro with a simplified model based on a biochemical block of the mevalonate pathway and a subsequent inflammatory trigger. The aim of this study was to evaluate the effect of the mevalonate blocking on glial cells (BV-2) and the following effects on neuronal cells (SH-SY5Y) when the two populations were cultured together. To better understand the cross-talk between glial and neuronal cells, as it happens in vivo, BV-2 and SH-SY5Y were co-cultured in different experimental settings (alone, transwell, direct contact); the effect of mevalonate pathway biochemical block by Lovastatin, followed by LPS inflammatory trigger, were evaluated by analysing programmed cell death and mitochondrial membrane potential, cytokines' release and cells' morphology modifications. In this experimental condition, glial cells underwent an evident activation, confirmed by elevated pro-inflammatory cytokines release, typical of these disorders, and a modification in morphology. Moreover, the activation induced an increase in apoptosis. When glial cells were co-cultured with neurons, their activation caused an increase of programmed cell death also in neuronal cells, but only if the two populations were cultured in direct contact. Our findings, being aware of the limitations related to the cell models used, represent a preliminary step towards understanding the pathological and neuroinflammatory mechanisms occurring in mevalonate kinase diseases. Contact co-culture between neuronal and microglial cells seems to be a good model to study mevalonic aciduria in vitro, and to contribute to the identification of potential drugs able to block microglial activation for this orphan disease. In fact, in such a pathological condition, we demonstrated that microglial cells are

  18. Identification of neurotoxic cytokines by profiling Alzheimer's disease tissues and neuron culture viability screening.

    Science.gov (United States)

    Wood, Levi B; Winslow, Ashley R; Proctor, Elizabeth A; McGuone, Declan; Mordes, Daniel A; Frosch, Matthew P; Hyman, Bradley T; Lauffenburger, Douglas A; Haigis, Kevin M

    2015-11-13

    Alzheimer's disease (AD) therapeutics based on the amyloid hypothesis have shown minimal efficacy in patients, suggesting that the activity of amyloid beta (Aβ) represents only one aspect of AD pathogenesis. Since neuroinflammation is thought to play an important role in AD, we hypothesized that cytokines may play a direct role in promoting neuronal death. Here, we profiled cytokine expression in a small cohort of human AD and control brain tissues. We identified AD-associated cytokines using partial least squares regression to correlate cytokine expression with quantified pathologic disease state and then used neuron cultures to test whether cytokines up-regulated in AD tissues could affect neuronal viability. This analysis identified cytokines that were associated with the pathological severity. Of the top correlates, only TNF-α reduced viability in neuron culture when applied alone. VEGF also reduced viability when applied together with Aβ, which was surprising because VEGF has been viewed as a neuro-protective protein. We found that this synthetic pro-death effect of VEGF in the context of Aβ was commensurate with VEGFR-dependent changes in multiple signaling pathways that govern cell fate. Our findings suggest that profiling of tissues combined with a culture-based screening approach can successfully identify new mechanisms driving neuronal death.

  19. Primary culture and characteristic morphologies of neurons from the cerebral ganglion of the mud crab, Scylla paramamosain.

    Science.gov (United States)

    Xu, Yan; Ye, Haihui; Ma, Jun; Huang, Huiyang; Wang, Guizhong

    2010-09-01

    Crustacean neurons, obtained from the cerebral ganglion of the mud crab Scylla paramamosain, were successfully cultured in vitro. They maintained typical morphological characteristics and showed better outgrowth in modified Medium 199 (M199) medium than that in Liebowitz's L-15 medium. Fetal bovine serum (FBS), muscle extracts, and hemolymph of the mud crab S. paramamosain were added as supplements. Only 20% FBS could promote neuron outgrowth, while muscle extracts and hemolymph of S. paramamosain did not improve neuron outgrowth. For cell dissociation, both collagenase type I and trypsin worked well as determined by initial cell viability and following cell outgrowth potential. More than six kinds of cells with different morphological characteristics were identified in the neuron outgrowth. They were "small cells", "veilers", "branchers", "multipolar cells", "super-large cell", and "bipolar cells". Among all of the cells, bipolar cells were identified for the first time in crustacean neurons culture and they could live longer than other cells. The neurons could grow for more than a week before retraction and eventual degradation.

  20. Enhancing neuronal growth from human endometrial stem cells derived neuron-like cells in three-dimensional fibrin gel for nerve tissue engineering.

    Science.gov (United States)

    Navaei-Nigjeh, Mona; Amoabedini, Ghasem; Noroozi, Abbas; Azami, Mahmoud; Asmani, Mohammad N; Ebrahimi-Barough, Somayeh; Saberi, Hooshang; Ai, Armin; Ai, Jafar

    2014-08-01

    Nerve tissue engineering (NTE) is one of the most promising methods to restore central nerve systems in human health care. Three-dimensional (3D) distribution and growth of cells within the porous scaffold composed of nanofibers are of clinical significance for NTE. In this study, an attempt was made to develop and characterize the use of fibrin gel and human endometrial stem cells (hEnSCs)-derived neuron-like cells simultaneously to support cell behavior especially neuron outgrowth. The structural and mechanical characteristics of fibrin gel scaffold were examined with SEM and rheometer. Also, hEnSCs-derived neuron-like cells were cultured in fibrin gel and were subsequently analyzed with immunofluorescent staining against neuronal markers. In parallel, the survival and growth rates of the cells were determined by MTT assay and neurite extension. At the end, cell-matrix interactions were investigated with SEM and TEM micrographs. Mechanical properties of fabricated scaffold were studied and results indicated appropriate choice of material, SEM and TEM showed excellent integration of cells with nanofibers regarding the relation between cells and fibrin gel. Immunofluorescent staining of fibrin gel after 6 days of cell seeding and culture demonstrated well expanded and incorporated network of neurons. In addition, viability, proliferation, and neuronal growth of seeded cells were analyzed at days 1, 3, and 6. Comparing those results with 2D culture of seeded cells showed positive effect of 3D culture. Taken together, the results suggest that fibrin can provide a suitable, three-dimensional scaffold for neuronal survival and outgrowth for regeneration of the central nervous system. © 2013 Wiley Periodicals, Inc.

  1. Therapeutic potential of mesenchymal stromal cells and MSC conditioned medium in Amyotrophic Lateral Sclerosis (ALS--in vitro evidence from primary motor neuron cultures, NSC-34 cells, astrocytes and microglia.

    Directory of Open Access Journals (Sweden)

    Hui Sun

    Full Text Available Administration of mesenchymal stromal cells (MSC improves functional outcome in the SOD1G93A mouse model of the degenerative motor neuron disorder amyotrophic lateral sclerosis (ALS as well as in models of other neurological disorders. We have now investigated the effect of the interaction between MSC and motor neurons (derived from both non-transgenic and mutant SOD1G93A transgenic mice, NSC-34 cells and glial cells (astrocytes, microglia (derived again from both non-transgenic and mutant SOD1G93A ALS transgenic mice in vitro. In primary motor neurons, NSC-34 cells and astrocytes, MSC conditioned medium (MSC CM attenuated staurosporine (STS - induced apoptosis in a concentration-dependent manner. Studying MSC CM-induced expression of neurotrophic factors in astrocytes and NSC-34 cells, we found that glial cell line-derived neurotrophic factor (GDNF and ciliary neurotrophic factor (CNTF gene expression in astrocytes were significantly enhanced by MSC CM, with differential responses of non-transgenic and mutant astrocytes. Expression of Vascular Endothelial Growth Factor (VEGF in NSC-34 cells was significantly upregulated upon MSC CM-treatment. MSC CM significantly reduced the expression of the cytokines TNFα and IL-6 and iNOS both in transgenic and non-transgenic astrocytes. Gene expression of the neuroprotective chemokine Fractalkine (CX3CL1 was also upregulated in mutant SOD1G93A transgenic astrocytes by MSC CM treatment. Correspondingly, MSC CM increased the respective receptor, CX3CR1, in mutant SOD1G93A transgenic microglia. Our data demonstrate that MSC modulate motor neuronal and glial response to apoptosis and inflammation. MSC therefore represent an interesting candidate for further preclinical and clinical evaluation in ALS.

  2. Functional crosstalk in culture between macrophages and trigeminal sensory neurons of a mouse genetic model of migraine

    Directory of Open Access Journals (Sweden)

    Franceschini Alessia

    2012-11-01

    Full Text Available Abstract Background Enhanced activity of trigeminal ganglion neurons is thought to underlie neuronal sensitization facilitating the onset of chronic pain attacks, including migraine. Recurrent headache attacks might establish a chronic neuroinflammatory ganglion profile contributing to the hypersensitive phenotype. Since it is difficult to study this process in vivo, we investigated functional crosstalk between macrophages and sensory neurons in primary cultures from trigeminal sensory ganglia of wild-type (WT or knock-in (KI mice expressing the Cacna1a gene mutation (R192Q found in familial hemiplegic migraine-type 1. After studying the number and morphology of resident macrophages in culture, the consequences of adding host macrophages on macrophage phagocytosis and membrane currents mediated by pain-transducing P2X3 receptors on sensory neurons were examined. Results KI ganglion cultures constitutively contained a larger number of active macrophages, although no difference in P2X3 receptor expression was found. Co-culturing WT or KI ganglia with host macrophages (active as much as resident cells strongly stimulated single cell phagocytosis. The same protocol had no effect on P2X3 receptor expression in WT or KI co-cultures, but it largely enhanced WT neuron currents that grew to the high amplitude constitutively seen for KI neurons. No further potentiation of KI neuronal currents was observed. Conclusions Trigeminal ganglion cultures from a genetic mouse model of migraine showed basal macrophage activation together with enhanced neuronal currents mediated by P2X3 receptors. This phenotype could be replicated in WT cultures by adding host macrophages, indicating an important functional crosstalk between macrophages and sensory neurons.

  3. Liposomes to target peripheral neurons and Schwann cells.

    Directory of Open Access Journals (Sweden)

    Sooyeon Lee

    Full Text Available While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral

  4. Achyranthes bidentata Blume extract promotes neuronal growth in cultured embryonic rat hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Xin Tang; Yiren Chen; Xiaosong Gu; Fei Ding

    2009-01-01

    We have prepared an aqueous extract of Achyranthes bidentata Blume,a commonly prescribed Chinese medicinal herb,and reported,in previous studies,that A.bidentata extract benefits nerve growth and prevents neuron apoptosis.In this study,we investigated the actions of ,4.bidentata extract on survival and growth of primarily cultured rat hippocampal neurons.The morphological observation revealed that neurite growth from hippocampal neurons was significantly enhanced by A.bidentata extract with similar effects to those induced by nerve growth factor (NGF),and the greatest neurite growth appeared on treatment with A.bidentata extract at 1 ttg/ml for 24 h.DNA microarray analysis indicated that there were 25 upregulated genes and 47 downregulated genes exhibiting significantly differential expression in hippocampal neurons treated with A.bidentata extract at 1 μg/ml for 6 h when compared to those in untreated hippocampal neurons.Real-time quantitative RT-PCR and Western blot analysis demonstrated that the expression of growth-associated protein-43 in hippocampal neurons was upregulated at both mRNA and protein levels after treatment with A.bidentata extract,and the optimal dosage of the extract was also 1 μg/ml.These data confirm that A.bidentata extract could promote in vitro hippocampal neuronal growth in a dose- and time-dependent manner.(C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences.Published by Elsevier Limited and Science in China Press.All rights reserved.

  5. Perfusion Based Cell Culture Chips

    Science.gov (United States)

    Heiskanen, A.; Emnéus, J.; Dufva, M.

    Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.

  6. Perfusion based cell culture chips

    DEFF Research Database (Denmark)

    Heiskanen, Arto; Emnéus, Jenny; Dufva, Martin

    2010-01-01

    and tissue like structuring of the cultures. However, as this chapter illustrates, many issues remain to be identified regarding perfusion cell culture such as design, material choice and how to use these systems before they will be widespread amongst biomedical researchers.......Performing cell culture in miniaturized perfusion chambers gives possibilities to experiment with cells under near in vivo like conditions. In contrast to traditional batch cultures, miniaturized perfusion systems provide precise control of medium composition, long term unattended cultures...

  7. Exposure to extremely low frequency electromagnetic fields alters the calcium dynamics of cultured entorhinal cortex neurons.

    Science.gov (United States)

    Luo, Fen-Lan; Yang, Nian; He, Chao; Li, Hong-Li; Li, Chao; Chen, Fang; Xiong, Jia-Xiang; Hu, Zhi-An; Zhang, Jun

    2014-11-01

    Previous studies have revealed that extremely low frequency electromagnetic field (ELF-EMF) exposure affects neuronal dendritic spine density and NMDAR and AMPAR subunit expressions in the entorhinal cortex (EC). Although calcium signaling has a critical role in control of EC neuronal functions, however, it is still unclear whether the ELF-EMF exposure affects the EC neuronal calcium homeostasis. In the present study, using whole-cell recording and calcium imaging, we record the whole-cell inward currents that contain the voltage-gated calcium currents and show that ELF-EMF (50Hz, 1mT or 3mT, lasting 24h) exposure does not influence these currents. Next, we specifically isolate the high-voltage activated (HVA) and low-voltage activated (LVA) calcium channels-induced currents. Similarly, the activation and inactivation characteristics of these membrane calcium channels are also not influenced by ELF-EMF. Importantly, ELF-EMF exposure reduces the maximum amplitude of the high-K(+)-evoked calcium elevation in EC neurons, which is abolished by thapsigargin, a Ca(2+) ATPase inhibitor, to empty the intracellular calcium stores of EC neurons. Together, these findings indicate that ELF-EMF exposure specifically influences the intracellular calcium dynamics of cultural EC neurons via a calcium channel-independent mechanism.

  8. Influence of Ginkgo Biloba extract on beta-secretase in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions

    Institute of Scientific and Technical Information of China (English)

    Xueneng Guan; Fuling Yan

    2008-01-01

    BACKGROUND: Preparation of Ginkgo leaf has been widely used to improve cognitive deficits and dementia, in particular in Alzheimer's disease patients. However, the precise mechanism of action of Ginkgo leaf remains unclear.OBJECTIVE: To explore the effect of Ginkgo Biloba extract (Egb761), Ginaton, on β-secretase expression in rat hippocampal neuronal cultures following chronic hypoxic and hypoglycemic conditions.DESIGN, TIME AND SETTNG: Completely by randomized, grouping study. The experiment was performed at the Laboratory of Molecular Imaging, Southeast University between August 2006 and August 2007.MATERIALS: A total of 128 Wistar rats aged 24 hours were selected, and hippocampal neurons were harvested for primary cultures.METHODS: On day 7, primary hippocampal neuronal cultures were treated with Egb761 (0, 25, 50, 100, 150, and 200 μ g/mL) under hypoxic/hypoglycemic or hypoglycemic culture conditions for 12, 24, and 36 hours, respectively. Hippocampal neurons cultured in primary culture medium served as control.MAIN OUTCOME MEASURES: Cell viability was assayed using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); fluorescence detection of β-secretase activity was performed; Western Blot was used to measure β -secretase expression.RESULTS: Cell viability under hypoxic/hypoglycemic or hypoglycemic culture conditions was significantly less than control cells (P 25 μ g/mL Egb761 induced greater cell viability (P 0.05). Α -secretase activity was increased after 12 hours in hypoxic/hypoglycemic culture (P 0.05). Β -secretase activity was greater after 12, 24, and 36 hours in hypoxic/hypoglycemic culture conditions, compared with control conditions (P < 0.05). Β-secretase activity was significantly decreased in neurons treated with Egb761 for 12, 24, or 36 hours, compared with the hypoxic/hypoglycemic group (P < 0.05).β-secretase protein expression was significantly up-regulated in neurons cultured in hypoxic/hypoglycemic conditions for

  9. The mast cell degranulator compound 48/80 directly activates neurons.

    Directory of Open Access Journals (Sweden)

    Michael Schemann

    Full Text Available BACKGROUND: Compound 48/80 is widely used in animal and tissue models as a "selective" mast cell activator. With this study we demonstrate that compound 48/80 also directly activates enteric neurons and visceral afferents. METHODOLOGY/PRINCIPAL FINDINGS: We used in vivo recordings from extrinsic intestinal afferents together with Ca(++ imaging from primary cultures of DRG and nodose neurons. Enteric neuronal activation was examined by Ca(++ and voltage sensitive dye imaging in isolated gut preparations and primary cultures of enteric neurons. Intraluminal application of compound 48/80 evoked marked afferent firing which desensitized on subsequent administration. In egg albumen-sensitized animals, intraluminal antigen evoked a similar pattern of afferent activation which also desensitized on subsequent exposure to antigen. In cross-desensitization experiments prior administration of compound 48/80 failed to influence the mast cell mediated response. Application of 1 and 10 µg/ml compound 48/80 evoked spike discharge and Ca(++ transients in enteric neurons. The same nerve activating effect was observed in primary cultures of DRG and nodose ganglion cells. Enteric neuron cultures were devoid of mast cells confirmed by negative staining for c-kit or toluidine blue. In addition, in cultured enteric neurons the excitatory action of compound 48/80 was preserved in the presence of histamine H(1 and H(2 antagonists. The mast cell stabilizer cromolyn attenuated compound 48/80 and nicotine evoked Ca(++ transients in mast cell-free enteric neuron cultures. CONCLUSIONS/SIGNIFICANCE: The results showed direct excitatory action of compound 48/80 on enteric neurons and visceral afferents. Therefore, functional changes measured in tissue or animal models may involve a mast cell independent effect of compound 48/80 and cromolyn.

  10. Phenotypic changes in satellite glial cells in cultured trigeminal ganglia.

    Science.gov (United States)

    Belzer, Vitali; Shraer, Nathanael; Hanani, Menachem

    2010-11-01

    Satellite glial cells (SGCs) are specialized cells that form a tight sheath around neurons in sensory ganglia. In recent years, there is increasing interest in SGCs and they have been studied in both intact ganglia and in tissue culture. Here we studied phenotypic changes in SGCs in cultured trigeminal ganglia from adult mice, containing both neurons and SGCs, using phase optics, immunohistochemistry and time-lapse photography. Cultures were followed for up to 14 days. After isolation virtually every sensory neuron is ensheathed by SGCs, as in the intact ganglia. After one day in culture, SGCs begin to migrate away from their parent neurons, but in most cases the neurons still retain an intact glial cover. At later times in culture, there is a massive migration of SGCs away from the neurons and they undergo clear morphological changes, and at 7 days they become spindle-shaped. At one day in culture SGCs express the glial marker glutamine synthetase, and also the purinergic receptor P2X7. From day 2 in culture the glutamine synthetase expression is greatly diminished, whereas that of P2X7 is largely unchanged. We conclude that SGCs retain most of their characteristics for about 24 h after culturing, but undergo major phenotypic changes at later times.

  11. EFFECT OF MELATONIN AGAINST GLUTAMATE-INDUCED EXCITOTOXICITY ON CULTURED CEREBRAL CORTICAL NEURONS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To research the effect of melatonin against glutamate excitotoxicity. Methods The model of glutamate-induced excitotoxic damage was built up in rat cerebral cortical cell culture. The effect of mela- tonin against excitotoxic injury was observed by determining the leakage rate of lactate dehydrogenase(LDH) from neurons. Results The leakage rate of LDH wasn't decreased markedly when cultures were exposed to melatonin be- fore, during or 6 h after glutamate treatment. The leakage rate of LDH was decreased significantly when melatonin was administered 0 h, 2 h or 4 h after the cultures were exposed to glutamate. The inhibitory function of melatonin on LDH leakage was most effective at 2 h and 4 h. Conclusion Melatonin has protective effects on neurons damaged by glutamate in a certain time limit.

  12. Microfluidic Cell Culture Device

    Science.gov (United States)

    Takayama, Shuichi (Inventor); Cabrera, Lourdes Marcella (Inventor); Heo, Yun Seok (Inventor); Smith, Gary Daniel (Inventor)

    2014-01-01

    Microfluidic devices for cell culturing and methods for using the same are disclosed. One device includes a substrate and membrane. The substrate includes a reservoir in fluid communication with a passage. A bio-compatible fluid may be added to the reservoir and passage. The reservoir is configured to receive and retain at least a portion of a cell mass. The membrane acts as a barrier to evaporation of the bio-compatible fluid from the passage. A cover fluid may be added to cover the bio-compatible fluid to prevent evaporation of the bio-compatible fluid.

  13. The neurotoxicity of hallucinogenic amphetamines in primary cultures of hippocampal neurons.

    Science.gov (United States)

    Capela, João Paulo; da Costa Araújo, Silvana; Costa, Vera Marisa; Ruscher, Karsten; Fernandes, Eduarda; Bastos, Maria de Lourdes; Dirnagl, Ulrich; Meisel, Andreas; Carvalho, Félix

    2013-01-01

    3,4-Methylenedioxymethamphetamine (MDMA or "Ecstasy") and 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) are hallucinogenic amphetamines with addictive properties. The hippocampus is involved in learning and memory and seems particularly vulnerable to amphetamine's neurotoxicity. We evaluated the neurotoxicity of DOI and MDMA in primary neuronal cultures of hippocampus obtained from Wistar rat embryos (E-17 to E-19). Mature neurons after 10 days in culture were exposed for 24 or 48 h either to MDMA (100-800 μM) or DOI (10-100 μM). Both the lactate dehydrogenase (LDH) release and the tetrazolium-based (MTT) assays revealed a concentration- and time-dependent neuronal death and mitochondrial dysfunction after exposure to both drugs. Both drugs promoted a significant increase in caspase-8 and caspase-3 activities. At concentrations that produced similar levels of neuronal death, DOI promoted a higher increase in the activity of both caspases than MDMA. In the mitochondrial fraction of neurons exposed 24h to DOI or MDMA, we found a significant increase in the 67 kDa band of apoptosis inducing factor (AIF) by Western blot. Moreover, 24h exposure to DOI promoted an increase in cytochrome c in the cytoplasmatic fraction of neurons. Pre-treatment with an antibody raised against the 5-HT(2A)-receptor (an irreversible antagonist) greatly attenuated neuronal death promoted by 48 h exposure to DOI or MDMA. In conclusion, hallucinogenic amphetamines promoted programmed neuronal death involving both the mitochondria machinery and the extrinsic cell death key regulators. Death was dependent, at least in part, on the stimulation of the 5-HT(2A)-receptors.

  14. Differentiation of Wharton’s jelly mesenchymal stem cells into neurons in alginate scaffold

    Institute of Scientific and Technical Information of China (English)

    Seyed Mojtaba Hosseini; Attiyeh Vasaghi; Newsha Nakhlparvar; Reza Roshanravan; Tahereh Talaei-khozani; Zahra Razi

    2015-01-01

    Alginate scaffold has been considered as an appropriate biomaterial for promoting the differ-entiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton’s jelly mesenchymal stem cells (WJMSCs) and can pro-mote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 mM CaCl2 in the presence of neurogenic medium containing 10 µM retinoic acid and 20 ng/mL basic ifbroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunolfuorescence staining was performed for detectingβ-tubulin (marker for WJMSCs-differentiated neuron) and CD271 (motor neuron marker).β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

  15. Phenotypic differentiation of neonatal rat cochlear spiral ganglion neurons following trypsin dissociation and culture

    Institute of Scientific and Technical Information of China (English)

    Dingjun Zha; Li Qiao; Lianjun Lu; Xue Gao; Tao Xue; Wenjuan Mi; Shunli Liu; Jianhua Qiu

    2008-01-01

    BACKGROUND: Under laboratory conditions, cochlear spiral ganglion neurons are commonly isolated and cultured by mechanical dissociation. However, these neurons are extremely fragile and survive for only a short time.OBJECTIVE: To establish a trypsin dissociation and culture method for studying neonatal rat cochlear spiral ganglion neurons. DESIGN: A single sample study. SETTING: Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA.MATERIALS: This study was performed at the central laboratory for Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA from February to May 2006. A total of 40 neonatal Sprague Dawley rats of either gender, aged 2-5 days, were provided by the Laboratory Animal Center of the Fourth Military Medical University of Chinese PLA. Trypsin and neuronal-specific nuclear protein (NeuN) monoclonal antibodies were purchased from Sigma Company, USA. Culture medium was synthesized using Dulbecco's modified Eagle's medium (DMEM)/F12 (Gibco Company, USA) supplemented with 10% fetal bovine serum (Sigma Company, USA), 100 000 U/L penicillin, and 1 mol/L NaOH. The following protocol was performed in accordance with ethical guidelines for the use and care of animals.METHODS: After anesthesia, rats were sacrificed by neck dislocation. A complete cochlear axis with spiral ganglion tissue was removed. The cochlear axis was rinsed three times in a culture dish with a diameter of 35 mm using Hank's balanced solution. After washings, the tissue was cut into pieces, digested with 0.25% trypsin for about 20 minutes, and incubated in a 37 ℃ water bath. The tissue was centrifuged, then mixed with serum-containing culture medium. Using a transfer pipette, the cell suspension was transferred to polylysine (0.1%)-treated culture dishes with a diameter of 35 mm. The culture dish was incubated at 37 ℃, with a 5% CO2-air environment. Once

  16. Brain-derived neurotrophic factor protects neurons from GdCl3-induced impairment in neuron-astrocyte co-cultures

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Gadolinium (Gd3+) complexes are important contrast agents in medical magnetic resonance imaging (MRI) and of great potential value in brain research. In order to better understand the mechanisms of the action of Gd3+ on neurons in the complex central nervous system (CNS), the neurotoxic actions of GdCl3 have been investigated in both neuron monoculture and astrocyte-neuron co-culture systems. Measurements of lactate dehydrogenase release showed that GdCl3 causes significant cell death of monocultured neurons as a result of reactive oxygen species (ROS) generation and down-regulation of brain-derived neurotrophic factor (BDNF). However, GdCl3 does not affect the viability and BDNF expression of astrocytes. Both co-culturing of neurons with astrocytes and addition of BDNF ameliorated GdCl3-induced neurotoxicity by decreasing ROS generation and facilitating recovery of BDNF levels. The results obtained suggest that astrocytes in the CNS may protect neurons from GdCl3-induced impairment through secreting BDNF and thus up-regulating BDNF expression and interfering with Gd3+-induced cell signaling in neurons. A possible molecular mechanism is suggested which should be helpful in understand- ing the neurotoxic actions of gadolinium probes .

  17. Neuroprotective effect of piperine on primarily cultured hippocampal neurons.

    Science.gov (United States)

    Fu, Min; Sun, Zhao-Hui; Zuo, Huan-Cong

    2010-01-01

    It was previously reported that piperine (PIP) significantly blocks convulsions induced by intracerebroventricular injection of threshold doses of kainate, but had no or only slight effects on convulsions induced by L-glutamate, N-methyl-D-aspartate and guanidinosuccinate. In traditional Chinese medicine, black pepper has been used for epileptic treatment; however, the exact mechanism is still unclear. We reported here in that appropriate concentration of PIP effectively inhibites the synchronized oscillation of intracellular calcium in rat hippocampal neuronal networks and represses spontaneous synaptic activities in terms of spontaneous synaptic currents (SSC) and spontaneous excitatory postsynaptic currents (sEPSC). Moreover, pretreatment with PIP expects protective effect on glutamate-induced decrease of cell viability and apoptosis of hippocampal neurons. These data suggest that the neuroprotective effects of PIP might be associated with suppression of synchronization of neuronal networks, presynaptic glutamic acid release, and Ca(2+) overloading.

  18. Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays.

    Science.gov (United States)

    Hahnewald, Stefan; Roccio, Marta; Tscherter, Anne; Streit, Jürg; Ambett, Ranjeeta; Senn, Pascal

    2016-10-19

    Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

  19. Chronic excitotoxin-induced axon degeneration in a compartmented neuronal culture model

    Directory of Open Access Journals (Sweden)

    Katherine A Hosie

    2012-02-01

    Full Text Available Glutamate excitotoxicity is a major pathogenic process implicated in many neurodegenerative conditions, including AD (Alzheimer's disease and following traumatic brain injury. Occurring predominantly from over-stimulation of ionotropic glutamate receptors located along dendrites, excitotoxic axonal degeneration may also occur in white matter tracts. Recent identification of axonal glutamate receptor subunits within axonal nanocomplexes raises the possibility of direct excitotoxic effects on axons. Individual neuronal responses to excitotoxicity are highly dependent on the complement of glutamate receptors expressed by the cell, and the localization of the functional receptors. To enable isolation of distal axons and targeted excitotoxicity, murine cortical neuron cultures were prepared in compartmented microfluidic devices, such that distal axons were isolated from neuronal cell bodies. Within the compartmented culture system, cortical neurons developed to relative maturity at 11 DIV (days in vitro as demonstrated by the formation of dendritic spines and clustering of the presynaptic protein synaptophysin. The isolated distal axons retained growth cone structures in the absence of synaptic targets, and expressed glutamate receptor subunits. Glutamate treatment (100 μM to the cell body chamber resulted in widespread degeneration within this chamber and degeneration of distal axons in the other chamber. Glutamate application to the distal axon chamber triggered a lesser degree of axonal degeneration without degenerative changes in the untreated somal chamber. These data indicate that in addition to current mechanisms of indirect axonal excitotoxicity, the distal axon may be a primary target for excitotoxicity in neurodegenerative conditions.

  20. Non-cell autonomous influence of the astrocyte system xc− on hypoglycaemic neuronal cell death

    Directory of Open Access Journals (Sweden)

    Sandra J Hewett

    2012-02-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc− – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−. Indeed, drugs known to inhibit system xc− ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc− (xCT. Finally, enhancement of astrocytic system xc− expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc− inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc−, have a direct, non-cell autonomous effect on cortical neuron survival.

  1. Non-cell autonomous influence of the astrocyte system xc- on hypoglycaemic neuronal cell death.

    Science.gov (United States)

    Jackman, Nicole A; Melchior, Shannon E; Hewett, James A; Hewett, Sandra J

    2012-02-08

    Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc---an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents--whereas addition of L-cystine restores--GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc-. Indeed, drugs known to inhibit system xc- ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc- (xCT). Finally, enhancement of astrocytic system xc- expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc- inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc-, have a direct, non-cell autonomous effect on cortical neuron survival.

  2. Non-Cell Autonomous Influence of the Astrocyte System xc − on Hypoglycaemic Neuronal Cell Death

    Directory of Open Access Journals (Sweden)

    Nicole A Jackman

    2012-01-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc −– – an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of L-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−–. Indeed, drugs known to inhibit system xc −– ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc −– (xCT. Finally, enhancement of astrocytic system xc −– expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of L-cystine and/or addition of system xc −– inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc −–, have a direct, non-cell autonomous effect on cortical neuron survival.

  3. Emergence of small-world anatomical networks in self-organizing clustered neuronal cultures.

    Directory of Open Access Journals (Sweden)

    Daniel de Santos-Sierra

    Full Text Available In vitro primary cultures of dissociated invertebrate neurons from locust ganglia are used to experimentally investigate the morphological evolution of assemblies of living neurons, as they self-organize from collections of separated cells into elaborated, clustered, networks. At all the different stages of the culture's development, identification of neurons' and neurites' location by means of a dedicated software allows to ultimately extract an adjacency matrix from each image of the culture. In turn, a systematic statistical analysis of a group of topological observables grants us the possibility of quantifying and tracking the progression of the main network's characteristics during the self-organization process of the culture. Our results point to the existence of a particular state corresponding to a small-world network configuration, in which several relevant graph's micro- and meso-scale properties emerge. Finally, we identify the main physical processes ruling the culture's morphological transformations, and embed them into a simplified growth model qualitatively reproducing the overall set of experimental observations.

  4. Low-cost, flexible polymer arrays for long-term neuronal culture.

    Science.gov (United States)

    Hogan, N Catherine; Talei-Franzesi, Giovanni; Abudayyeh, Omar; Taberner, Andrew; Hunter, Ian

    2012-01-01

    Conducting polymers are promising materials for fabrication of microelectrode arrays for both neural stimulation and recording. Our ability to engineer the morphology and composition of polypyrrole together with its suitability as an electrically addressable tissue/cell substrate have been used to develop an inexpensive, disposable three-dimensional polymeric array for use in neuronal culture and drug discovery. These arrays could be interfaced with a fixed, parallel stimulation and optical imaging system, amenable to automated handling and data analysis.

  5. Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening.

    Science.gov (United States)

    Gordon, Kent R; Wang, Yuli; Allbritton, Nancy L; Taylor, Anne Marion

    2015-10-01

    High-throughput screening (HTS) on neurons presents unique difficulties because they are postmitotic, limited in supply, and challenging to harvest from animals or generate from stem cells. These limitations have hindered neurological drug discovery, leaving an unmet need to develop cost-effective technology for HTS using neurons. Traditional screening methods use up to 20,000 neurons per well in 384-well plates. To increase throughput, we use "microraft" arrays, consisting of 1600 square, releasable, paramagnetic, polystyrene microelements (microrafts), each providing a culture surface for 500-700 neurons. These microrafts can be detached from the array and transferred to 384-well plates for HTS; however, they must be centered within wells for automated imaging. Here, we developed a magnet array plate, compatible with HTS fluid-handling systems, to center microrafts within wells. We used finite element analysis to select an effective size of the magnets and confirmed that adjacent magnetic fields do not interfere. We then experimentally tested the plate's centering ability and found a centering efficiency of 100%, compared with 4.35% using a flat magnet. We concluded that microrafts could be centered after settling randomly within the well, overcoming friction, and confirmed these results by centering microrafts containing hippocampal neurons cultured for 8 days.

  6. A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

    Science.gov (United States)

    Anderl, Janet L; Redpath, Stella; Ball, Andrew J

    2009-01-01

    High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with βIII-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture

  7. A neuronal and astrocyte co-culture assay for high content analysis of neurotoxicity.

    Science.gov (United States)

    Anderl, Janet L; Redpath, Stella; Ball, Andrew J

    2009-05-05

    High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with betaIII-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture

  8. Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

    Science.gov (United States)

    Christou, Y A; Moore, H D; Shaw, P J; Monk, P N

    2007-10-01

    Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease.

  9. Changing Numbers of Neuronal and Non-Neuronal Cells Underlie Postnatal Brain Growth in the Rat

    National Research Council Canada - National Science Library

    Fabiana Bandeira; Roberto Lent; Suzana Herculano-Houzel; Jon H. Kaas

    2009-01-01

    .... To test this hypothesis, here we investigate quantitatively the postnatal changes in the total number of neuronal and non-neuronal cells in the developing rat brain, and examine how these changes...

  10. Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non‐neuronal cells

    National Research Council Canada - National Science Library

    Grenier, Catherine; Bissonnette, Cyntia; Volkov, Leonid; Roucou, Xavier

    2006-01-01

    .... The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non‐neuronal cells...

  11. Differentiation of Mesenchymal Stem Cells Into Dopaminergic Neuron-like Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LI GUO; HONG-XUE FAN; FEI YIN; HONG-QI MENG; LING LING; TA-NA HU-HE; PENG LI; CHUN-XIA ZHANG; SHUN YU; DE-SHENG DUAN

    2005-01-01

    Objective To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. Methods MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days. Results MSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032±2.489%, 41.580±5.101% and 34.958±5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days. Conclusion MSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.

  12. Characterization of glutamate-induced formation of N- acylphosphatidylethanolamine and N-acylethanolamine in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Hansen, Harald S.; Lauritzen, L.; Strand, A.M.;

    1997-01-01

    Glutamate-induced formation of N-acylethanolamine (NAE) and N- acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [C]ethanolamine. The formation of these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate......-stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate-induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.......-quinoxaline-2,3-dione (CNQX). In 6-day-old cultures, exposure to NMDA (100 µM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40-50% neuronal death, as measured by a decrease in cellular formazan formation [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT...

  13. Mechanism of in Vitro Differentiation of Bone Marrow Stromal Cells into Neuron-like Cells

    Institute of Scientific and Technical Information of China (English)

    褚倩; 王亚平; 傅新巧; 张苏明

    2004-01-01

    Summary: In order to study whether marrow stromal cells (MSCs) can be induced into nerve-like cells in vitro, and the mechanism, the MSCs in Wistar rats were isolated and cultured, and then induced with DMSO and BHA in vitro. The expression of specific marking proteins in neurons, glia and neural stem cells were detected before preinduction, at 24 h of preinduction, at 6 h, 24 h, and 48 h of neuronal induction by using immunohistochemistry and Western blotting. The ultrastructural changes after the inducement were observed. The results showed that after the inducement, many MSCs turned into bipolar, multipolar and taper, and then intersected as network structure. At the same time, some MSCs had the typical neuron-like ultrastructure. Immunohistochemistry revealed that NeuN and Nestin expression was detectable after inducement, but there was no GFAP and CNP expression. Western blotting showed the expression of Nestin was strong at 6 h of neuronal induction, and decreased at 24 h, 48 h of the induction. NeuN was detectable at 6 h of neuronal induction, and increased at 24 h, 48 h of the induction. It was concluded MSCs were induced intc neural stem cells, and then differentiated into neuron-like cells in vitro.

  14. JNK抑制剂及NAC对氧糖剥离后复氧复糖所致脊髓神经元损伤的保护作用%The study of JNK inhibitor and NAC on oxygen-glucose deprivation/regain induced neuronal apoptosis in cultured spinal neuronal cells

    Institute of Scientific and Technical Information of China (English)

    华政哲; 张子韬; 张宁; 胡志毅; 蔡卫华; 殷国勇

    2011-01-01

    Objective:To study the protective effects of JNK (the c-Jun NH-tenninal kinase)inhibitor and NAC (N-acetylcysteine) on neuronal apoptosis induced by oxyen-glucose deprivation/regain in vitro. Methods:The oxygen-glucose deprivation/regain (OGD/ R) models were established in vitro. The neurons were exposed to OGD 4h before being placed into normal culture medium. Group A was taken as a blank control. Spinal cord neurons received the JNK inhibitor and NAC treatment before regaining the oxygen-glucose in group B and C, respectively. The neuronal apoptosis were detected by MTT assay in different time points (4 h, 12 h,24 h after regaining oxygen-glucose). Results:The model of OGD/R in spinal cord neurons was successfully established. Cell viability in MTT assay were higher in group B and group C than that in group A, and there was no significant difference between group B and group C. Conclusion:The JNK inhibitor and NAC can effectively protect neurons from apoptosis induced by OGD/R.%目的:初步探讨JNK(The c-Jun NH-terminal kinase)抑制剂及N-乙酰半胱氨酸(N-acetylcysteine,NAC)对脊髓神经细胞氧糖剥夺复糖复氧的保护作用。方法:离体脊髓神经元氧糖剥夺复糖复氧损伤(oxggen-glucose deprivation/regain,OGD/R)模型建立。随机分组:A组对照组(n=6);B组JNK抑制剂组(n=6);C组NAC处理组(n=6)。分别于脊髓神经元氧糖剥夺(oxygen-glucose deprivation,OGD)4 h后,再复氧复糖4、12、24 h,应用MTT法检测细胞凋亡。结果:成功建立OGD/R模型;脊髓神经元OGD/R后,B组和C组MTT各时间点吸光值均高于A组,但B组和C组间差异不显著。结论:JNK抑制剂及NAC能有效抑制体外培养的脊髓神经元氧糖剥夺复糖复氧所引起的凋亡。

  15. Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex.

    Science.gov (United States)

    Tang, Zheng-Quan; Lu, Yun-Gang; Chen, Lin

    2008-01-03

    Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.

  16. Cultured hypothalamic neurons are resistant to inflammation and insulin resistance induced by saturated fatty acids.

    Science.gov (United States)

    Choi, Sun Ju; Kim, Francis; Schwartz, Michael W; Wisse, Brent E

    2010-06-01

    Hypothalamic inflammation induced by high-fat feeding causes insulin and leptin resistance and contributes to the pathogenesis of obesity. Since in vitro exposure to saturated fatty acids causes inflammation and insulin resistance in many cultured cell types, we determined how cultured hypothalamic neurons respond to this stimulus. Two murine hypothalamic neuronal cell cultures, N43/5 and GT1-7, were exposed to escalating concentrations of saturated fatty acids for up to 24 h. Harvested cells were evaluated for activation of inflammation by gene expression and protein content. Insulin-treated cells were evaluated for induction of markers of insulin receptor signaling (p-IRS, p-Akt). In both hypothalamic cell lines, inflammation was induced by prototypical inflammatory mediators LPS and TNFalpha, as judged by induction of IkappaBalpha (3- to 5-fold) and IL-6 (3- to 7-fold) mRNA and p-IkappaBalpha protein, and TNFalpha pretreatment reduced insulin-mediated p-Akt activation by 30% (P saturated fatty acids on nonneuronal cells.

  17. Neurotoxin-induced DNA damage is persistentin SH-SY5Y cells and LC neurons

    Science.gov (United States)

    Wang, Yan; Musich, Phillip R.; Cui, Kui; Zou, Yue; Zhu, Meng-Yang

    2015-01-01

    Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathologic characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxincamptothecin (CPT)to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured LC and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficientin DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathologic characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insultsin vivo. PMID:25724887

  18. Primary neuron culture for nerve growth and axon guidance studies in zebrafish (Danio rerio.

    Directory of Open Access Journals (Sweden)

    Zheyan Chen

    Full Text Available Zebrafish (Danio rerio is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr(-1 s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day(-1 growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca(2+-imaging revealed local elevation of cytoplasmic Ca(2+ concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca(2+ signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development

  19. Runx1 promotes proliferation and neuronal differentiation in adult mouse neurosphere cultures

    Directory of Open Access Journals (Sweden)

    T.T. Logan

    2015-11-01

    Full Text Available Traumatic brain injury alters the signaling environment of the adult neurogenic niche and may activate unique proliferative cell populations that contribute to the post-injury neurogenic response. Runx1 is not normally expressed by adult neural stem or progenitor cells (NSPCs but is induced in a subpopulation of putative NSPCs after brain injury in adult mice. In order to investigate the role of Runx1 in NSPCs, we established neurosphere cultures of adult mouse subventricular zone NSPCs. We show that Runx1 is basally expressed in neurosphere culture. Removal of the mitogen bFGF or addition of 1% FBS decreased Runx1 expression. Inhibition of endogenous Runx1 activity with either Ro5-3335 or shRNA-mediated Runx1 knockdown inhibited NSPC proliferation without affecting differentiation. Lentiviral mediated over-expression of Runx1 in neurospheres caused a significant change in cell morphology without reducing proliferation. Runx1-overexpressing neurospheres changed from floating spheres to adherent colonies or individual unipolar or bipolar cells. Flow cytometry analysis indicated that Runx1 over-expression produced a significant increase in expression of the neuronal marker TuJ1 and a minor increase in the astrocytic marker S100β. Thus, Runx1 expression drove adult NSPC differentiation, predominantly toward a neuronal lineage. These data suggest that Runx1 could be manipulated after injury to promote neuronal differentiation to facilitate repair of the CNS.

  20. Tetramethylpyrazine suppresses transient oxygen-glucose deprivation-induced connexin32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathway in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Gu Gong

    Full Text Available Tetramethylpyrazine (TMP has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD. The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32 induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.

  1. Parallel expression of synaptophysin and evoked neurotransmitter release during development of cultured neurons

    DEFF Research Database (Denmark)

    Ehrhart-Bornstein, M; Treiman, M; Hansen, Gert Helge;

    1991-01-01

    and neurotransmitter release were measured in each of the culture types as a function of development for up to 8 days in vitro, using the same batch of cells for both sets of measurements to obtain optimal comparisons. The content and the distribution of synaptophysin in the developing cells were assessed...... by quantitative immunoblotting and light microscope immunocytochemistry, respectively. In both cell types, a close parallelism was found between the temporal pattern of development in synaptophysin expression and neurotransmitter release. This temporal pattern differed between the two types of neurons....... The cerebral cortex neurons showed a biphasic time course of increase in synaptophysin content, paralleled by a biphasic pattern of development in their ability to release [3H]GABA in response to depolarization by glutamate or elevated K+ concentrations. In contrast, a monophasic, approximately linear increase...

  2. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Giada Graziana Genchi

    2015-01-01

    Full Text Available Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g. Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  3. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds.

    Science.gov (United States)

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I

    2014-05-16

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

  4. Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds

    Science.gov (United States)

    Hsu, Tzu-Chia; Liu, Kuang-Kai; Chang, Huan-Cheng; Hwang, Eric; Chao, Jui-I.

    2014-05-01

    Nanodiamond is a promising carbon nanomaterial developed for biomedical applications. Here, we show fluorescent nanodiamond (FND) with the biocompatible properties that can be used for the labeling and tracking of neuronal differentiation and neuron cells derived from embryonal carcinoma stem (ECS) cells. The fluorescence intensities of FNDs were increased by treatment with FNDs in both the mouse P19 and human NT2/D1 ECS cells. FNDs were taken into ECS cells; however, FNDs did not alter the cellular morphology and growth ability. Moreover, FNDs did not change the protein expression of stem cell marker SSEA-1 of ECS cells. The neuronal differentiation of ECS cells could be induced by retinoic acid (RA). Interestingly, FNDs did not affect on the morphological alteration, cytotoxicity and apoptosis during the neuronal differentiation. Besides, FNDs did not alter the cell viability and the expression of neuron-specific marker β-III-tubulin in these differentiated neuron cells. The existence of FNDs in the neuron cells can be identified by confocal microscopy and flow cytometry. Together, FND is a biocompatible and readily detectable nanomaterial for the labeling and tracking of neuronal differentiation process and neuron cells from stem cells.

  5. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    Directory of Open Access Journals (Sweden)

    Junko Kimura-Kuroda

    2016-10-01

    Full Text Available Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of longterm (14 days and low dose (1 μM exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain.

  6. Roles and regulation of ketogenesis in cultured astroglia and neurons under hypoxia and hypoglycemia.

    Science.gov (United States)

    Takahashi, Shinichi; Iizumi, Takuya; Mashima, Kyoko; Abe, Takato; Suzuki, Norihiro

    2014-09-11

    Exogenous ketone bodies (KBs), acetoacetate (AA), and β-hydroxybutyrate (BHB) act as alternative energy substrates in neural cells under starvation. The present study examined the endogenous ketogenic capacity of astroglia under hypoxia with/without glucose and the possible roles of KBs in neuronal energy metabolism. Cultured neurons and astroglia were prepared from Sprague-Dawley rats. Palmitic acid (PAL) and l-carnitine (LC) were added to the assay medium. The 4- to 24-hr production of AA and BHB was measured using the cyclic thio-NADH method. (14)C-labeled acid-soluble products (KBs) and (14)CO2 produced from [1-(14)C]PAL were also measured. l-[U-(14)C]lactic acid ([(14)C]LAC), [1-(14)C]pyruvic acid ([(14)C]PYR), or β-[1-(14)C]hydroxybutyric acid ([(14)C]BHB) was used to compare the oxidative metabolism of the glycolysis end products with that of the KBs. Some cells were placed in a hypoxic chamber (1% O2). PAL and LC induced a higher production of KBs in astroglia than in neurons, while the CO2 production from PAL was less than 5% of the KB production in both astroglia and neurons. KB production in astroglia was augmented by the AMP-activated protein kinase activators, AICAR and metformin, as well as hypoxia with/without glucose. Neuronal KB production increased under hypoxia in the absence of PAL and LC. In neurons, [(14)C]LAC and [(14)C]PYR oxidation decreased after 24 hr of hypoxia, while [(14)C]BHB oxidation was preserved. Astroglia responds to ischemia in vitro by enhancing KB production, and astroglia-produced KBs derived from fatty acid might serve as a neuronal energy substrate for the tricarboxylic acid cycle instead of lactate, as pyruvate dehydrogenase is susceptible to ischemia.

  7. Roles and Regulation of Ketogenesis in Cultured Astroglia and Neurons Under Hypoxia and Hypoglycemia

    Directory of Open Access Journals (Sweden)

    Shinichi Takahashi

    2014-09-01

    Full Text Available Exogenous ketone bodies (KBs, acetoacetate (AA, and β-hydroxybutyrate (BHB act as alternative energy substrates in neural cells under starvation. The present study examined the endogenous ketogenic capacity of astroglia under hypoxia with/without glucose and the possible roles of KBs in neuronal energy metabolism. Cultured neurons and astroglia were prepared from Sprague-Dawley rats. Palmitic acid (PAL and l-carnitine (LC were added to the assay medium. The 4- to 24-hr production of AA and BHB was measured using the cyclic thio-NADH method. 14C-labeled acid-soluble products (KBs and 14CO2 produced from [1-14C]PAL were also measured. l-[U-14C]lactic acid ([14C]LAC, [1-14C]pyruvic acid ([14C]PYR, or β-[1-14C]hydroxybutyric acid ([14C]BHB was used to compare the oxidative metabolism of the glycolysis end products with that of the KBs. Some cells were placed in a hypoxic chamber (1% O2. PAL and LC induced a higher production of KBs in astroglia than in neurons, while the CO2 production from PAL was less than 5% of the KB production in both astroglia and neurons. KB production in astroglia was augmented by the AMP-activated protein kinase activators, AICAR and metformin, as well as hypoxia with/without glucose. Neuronal KB production increased under hypoxia in the absence of PAL and LC. In neurons, [14C]LAC and [14C]PYR oxidation decreased after 24 hr of hypoxia, while [14C]BHB oxidation was preserved. Astroglia responds to ischemia in vitro by enhancing KB production, and astroglia-produced KBs derived from fatty acid might serve as a neuronal energy substrate for the tricarboxylic acid cycle instead of lactate, as pyruvate dehydrogenase is susceptible to ischemia.

  8. Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system

    Directory of Open Access Journals (Sweden)

    Shu-Yong Wei

    2015-01-01

    Full Text Available The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer′s disease, Parkinson′s disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astrocytes in vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identified and co-cultured in a system based on Transwell membranes. The retention time and distribution of puerarin in each cell type was detected by fluorescence spectrophotometry and fluorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addition, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neurons than that observed in astrocytes.

  9. Cell culture's spider silk road.

    Science.gov (United States)

    Perkel, Jeffrey

    2014-06-01

    A number of synthetic and natural materials have been tried in cell culture and tissue engineering applications in recent years. Now Jeffrey Perkel takes a look at one new culture component that might surprise you-spider silk.

  10. Liraglutide is neurotrophic and neuroprotective in neuronal cultures and mitigates mild traumatic brain injury in mice.

    Science.gov (United States)

    Li, Yazhou; Bader, Miaad; Tamargo, Ian; Rubovitch, Vardit; Tweedie, David; Pick, Chaim G; Greig, Nigel H

    2015-12-01

    Traumatic brain injury (TBI), a brain dysfunction for which there is no present effective treatment, is often caused by a concussive impact to the head and affects an estimated 1.7 million Americans annually. Our laboratory previously demonstrated that exendin-4, a long-lasting glucagon-like peptide 1 receptor (GLP-1R) agonist, has neuroprotective effects in cellular and animal models of TBI. Here, we demonstrate neurotrophic and neuroprotective effects of a different GLP-1R agonist, liraglutide, in neuronal cultures and a mouse model of mild TBI (mTBI). Liraglutide promoted dose-dependent proliferation in SH-SY5Y cells and in a GLP-1R over-expressing cell line at reduced concentrations. Pre-treatment with liraglutide rescued neuronal cells from oxidative stress- and glutamate excitotoxicity-induced cell death. Liraglutide produced neurotrophic and neuroprotective effects similar to those of exendin-4 in vitro. The cAMP/PKA/pCREB pathway appears to play an important role in this neuroprotective activity of liraglutide. Furthermore, our findings in cell culture were well-translated in a weight drop mTBI mouse model. Post-treatment with a clinically relevant dose of liraglutide for 7 days in mice ameliorated memory impairments caused by mTBI when evaluated 7 and 30 days post trauma. These data cross-validate former studies of exendin-4 and suggest that liraglutide holds therapeutic potential for the treatment of mTBI. Exendin-4, a long-lasting glucagon-like peptide 1 receptor (GLP-1R) agonist, has neuroprotective effects in cellular and animal models of traumatic brain injury (TBI). Here, we demonstrate neurotrophic and neuroprotective effects of a different GLP-1R agonist, liraglutide, in neuronal cultures and a mouse model of mild TBI (mTBI). Liraglutide promoted dose-dependent proliferation in SH-SY5Y cells and in a GLP-1R over-expressing cell line at reduced concentrations. Pretreatment with liraglutide rescued neuronal cells from oxidative stress- and glutamate

  11. Liposome-siRNA-peptide complexes cross the blood-brain barrier and significantly decrease PrP on neuronal cells and PrP in infected cell cultures.

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    Bruce Pulford

    Full Text Available BACKGROUND: Recent advances toward an effective therapy for prion diseases employ RNA interference to suppress PrP(C expression and subsequent prion neuropathology, exploiting the phenomenon that disease severity and progression correlate with host PrP(C expression levels. However, delivery of lentivirus encoding PrP shRNA has demonstrated only modest efficacy in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a new siRNA delivery system incorporating a small peptide that binds siRNA and acetylcholine receptors (AchRs, acting as a molecular messenger for delivery to neurons, and cationic liposomes that protect siRNA-peptide complexes from serum degradation. CONCLUSIONS/SIGNIFICANCE: Liposome-siRNA-peptide complexes (LSPCs delivered PrP siRNA specifically to AchR-expressing cells, suppressed PrP(C expression and eliminated PrP(RES formation in vitro. LSPCs injected intravenously into mice resisted serum degradation and delivered PrP siRNA throughout the brain to AchR and PrP(C-expressing neurons. These data promote LSPCs as effective vehicles for delivery of PrP and other siRNAs specifically to neurons to treat prion and other neuropathological diseases.

  12. Label-free distinguishing between neurons and glial cells based on two-photon excited fluorescence signal of neuron perinuclear granules

    Science.gov (United States)

    Du, Huiping; Jiang, Liwei; Wang, Xingfu; Liu, Gaoqiang; Wang, Shu; Zheng, Liqin; Li, Lianhuang; Zhuo, Shuangmu; Zhu, Xiaoqin; Chen, Jianxin

    2016-08-01

    Neurons and glial cells are two critical cell types of brain tissue. Their accurate identification is important for the diagnosis of psychiatric disorders such as depression and schizophrenia. In this paper, distinguishing between neurons and glial cells by using the two-photon excited fluorescence (TPEF) signals of intracellular intrinsic sources was performed. TPEF microscopy combined with TUJ-1 and GFAP immunostaining and quantitative image analysis demonstrated that the perinuclear granules of neurons in the TPEF images of brain tissue and the primary cultured cortical cells were a unique characteristic of neurons compared to glial cells which can become a quantitative feature to distinguish neurons from glial cells. With the development of miniaturized TPEF microscope (‘two-photon fiberscopes’) imaging devices, TPEF microscopy can be developed into an effective diagnostic and monitoring tool for psychiatric disorders such as depression and schizophrenia.

  13. Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

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    Siebler Mario

    2009-08-01

    Full Text Available Abstract Background The present work was performed to investigate the ability of two different embryonic stem (ES cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs, progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. Results While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2, only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2–4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated

  14. Genetic strategies to investigate neuronal circuit properties using stem cell-derived neurons

    Directory of Open Access Journals (Sweden)

    Isabella eGarcia

    2012-12-01

    Full Text Available The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC and induced pluripotent stem cell (iPSC technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs towards investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches.

  15. Information in a Network of Neuronal Cells: Effect of Cell Density and Short-Term Depression

    KAUST Repository

    Onesto, Valentina

    2016-05-10

    Neurons are specialized, electrically excitable cells which use electrical to chemical signals to transmit and elaborate information. Understanding how the cooperation of a great many of neurons in a grid may modify and perhaps improve the information quality, in contrast to few neurons in isolation, is critical for the rational design of cell-materials interfaces for applications in regenerative medicine, tissue engineering, and personalized lab-on-a-chips. In the present paper, we couple an integrate-and-fire model with information theory variables to analyse the extent of information in a network of nerve cells. We provide an estimate of the information in the network in bits as a function of cell density and short-term depression time. In the model, neurons are connected through a Delaunay triangulation of not-intersecting edges; in doing so, the number of connecting synapses per neuron is approximately constant to reproduce the early time of network development in planar neural cell cultures. In simulations where the number of nodes is varied, we observe an optimal value of cell density for which information in the grid is maximized. In simulations in which the posttransmission latency time is varied, we observe that information increases as the latency time decreases and, for specific configurations of the grid, it is largely enhanced in a resonance effect.

  16. Differentiation of fetal pancreatic stem cells into neuron-like and islet-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Hua; Yanwei Wang; Peiwen Lian; Shouxin Zhang; Jianyuan Li; Haiyan Wang; Shulin Chen; Wei Gao

    2012-01-01

    Pancreatic stem cells were isolated and cultured from aborted human fetal pancreases of gestational age 14-20 weeks.They were seeded at a density of 1 × 104 in serum-free media for differentiation into neuron-like cells, expressing β-tubulin III and glial fibrillary acidic protein.These neuron-like cells displayed a synapse-like morphology and appeared to form a neuronal network.Pancreatic stem cells were also seeded at a density of 1 × 105 for differentiation into islet-like cells, expressing insulin and glucagon, with an islet-like morphology.These cells had glucose-stimulated secretion of human insulin and C-peptide.Results suggest that pancreatic stem cells can be differentiated into neuron-like and islet-like cells.

  17. Differential effects of synthetic progestagens on neuron survival and estrogen neuroprotection in cultured neurons.

    Science.gov (United States)

    Jayaraman, Anusha; Pike, Christian J

    2014-03-25

    Progesterone and other progestagens are used in combination with estrogens for clinical purposes, including contraception and postmenopausal hormone therapy. Progesterone and estrogens have interactive effects in brain, however interactions between synthetic progestagens and 17β-estradiol (E2) in neurons are not well understood. In this study, we investigated the effects of seven clinically relevant progestagens on estrogen receptor (ER) mRNA expression, E2-induced neuroprotection, and E2-induced BDNF mRNA expression. We found that medroxyprogesterone acetate decreased both ERα and ERβ expression and blocked E2-mediated neuroprotection and BDNF expression. Conversely, levonorgestrel and nesterone increased ERα and or ERβ expression, were neuroprotective, and failed to attenuate E2-mediated increases in neuron survival and BDNF expression. Other progestagens tested, including norethindrone, norethindrone acetate, norethynodrel, and norgestimate, had variable effects on the measured endpoints. Our results demonstrate a range of qualitatively different actions of progestagens in cultured neurons, suggesting significant variability in the neural effects of clinically utilized progestagens.

  18. Neuronal apoptosis: signal and cell diversity

    Directory of Open Access Journals (Sweden)

    Lina Vanessa Becerra

    2009-12-01

    Full Text Available Programmed cell death occurs as a physiological process during development. In the brain and spinal cord this event determines the number and location of the different cell types. In adulthood, programmed cell death or apoptosis is more restricted but it may play a major role in different acute and chronic pathological entities. However, in contrast to other tissues where apoptosis has been widely documented from a morphological point of view, in the central nervous system complete anatomical evidence of apoptosis is scanty. In spite of this there is consensus about the activation of different signal systems associated to programmed cell death. In the present article we attempt to summarize the main apoptotic pathways so far identified in nervous tissue. Considering that apoptotic pathways are multiple, the neuronal cell types are highly diverse and specialized and that neuronal response to injury and survival depends upon tissue context, (i.e., preservation of connectivity, glial integrity and cell matrix, blood supply and trophic factors availability what is relevant for the apoptotic process in a sector of the brain may not be important in another.

  19. Organic and inorganic lead inhibit neurite growth in vertebrate and invertebrate neurons in culture.

    Science.gov (United States)

    Audesirk, G; Shugarts, D; Nelson, G; Przekwas, J

    1989-12-01

    Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl2), or organic lead (triethyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 270 microM total lead, approximately 70 nM free Pb2+) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.24 microM) and the mean neurite length (extrapolated IC50 = 3.6 microM) but did not reduce the number of neurites per cell. In Lymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50 = 13 microM total lead; approximately 10 nM free Pb2+). Triethyl lead reduced the percentage of cells that grew neurites (IC50 = 0.4 microM) and exerted significant toxicity at 0.2 microM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechanisms of action are different.

  20. Transient maintenance in bioreactor improves health of neuronal cells.

    Science.gov (United States)

    Di Loreto, Silvia; Sebastiani, Pierluigi; Benedetti, Elisabetta; Zimmitti, Vincenzo; Caracciolo, Valentina; Amicarelli, Fernanda; Cimini, Annamaria; Adorno, Domenico

    2006-01-01

    To examine whether a neuronal cell suspension can be held in vitro for a relatively short period without compromising survival rates and functionality, we have set up an experimental protocol planning 24 h of suspension culture in a rotary wall vessel (RWV) bioreactor before plating in a conventional adherent system. Apoptosis measurement and activated caspase-8, -9, and -3 detection have demonstrated that survey of the cells was not affected. The activity of major antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was significantly decreased in RWV-maintained cells. A significant decrease of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) is coupled with a level of activated nuclear factor-kappaB (NF-kappaB) protein significantly lower in RVW cells than in the control. On the contrary, the level of IL-6 expression did not change between the test and the control. A significant up-regulation of growth-associated protein-43 (GAP-43), peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta), and acyl-CoA synthetase 2 (ACS2) in RWV cells has been detected. We provide the evidence that primary neuronal cells, at an early stage of development, can be maintained in a suspension condition before adherent plating. This experimental environment does not induce detrimental effects but may have an activator role, leading cells to development and maturation in a tridimensional state.

  1. SIRT1 Activating Compounds Reduce Oxidative Stress and Prevent Cell Death in Neuronal Cells

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    Reas S Khan

    2012-12-01

    Full Text Available Activation of SIRT1, an NAD+-dependent deacetylase, prevents retinal ganglion cell (RGC loss in optic neuritis, an inflammatory demyelinating optic nerve disease. While SIRT1 deacetylates numerous protein targets, downstream mechanisms of SIRT1 activation mediating this neuroprotective effect are unknown. SIRT1 increases mitochondrial function and reduces oxidative stress in muscle and other cells, and oxidative stress occurs in neuronal degeneration. We examined whether SIRT1 activators reduce oxidative stress and promote mitochondrial function in neuronal cells. Oxidative stress, marked by reactive oxygen species (ROS accumulation, was induced in RGC-5 cells by serum deprivation, or addition of doxorubicin or hydrogen peroxide, and resulted in significant cell loss. SIRT1 activators resveratrol and SRTAW04 reduced ROS levels, and promoted cell survival in RGC-5 cells as well as primary RGC cultures. Effects were blocked by SIRT1 siRNA. SIRT1 activators also increased expression of succinate dehydrogenase, a mitochondrial enzyme, and promoted deacetylation of PGC-1α, a co-enzyme involved in mitochondrial function. Results show SIRT1 activators prevent cell loss by reducing oxidative stress and promoting mitochondrial function in a neuronal cell line. Results suggest SIRT1 activators can mediate neuroprotective effects during optic neuritis by these mechanisms, and they have the potential to preserve neurons in other neurodegenerative diseases that involve oxidative stress.

  2. Brain-derived neurotrophic factor but not neurotrophin-3 enhances differentiation of somatostatin neurons in hypothalamic cultures.

    Science.gov (United States)

    Loudes, C; Petit, F; Kordon, C; Faivre-Bauman, A

    2000-09-01

    The present work investigated whether neurotrophins could differentially affect in vitro growth and maturation of two related subsets of hypothalamic neurons, hypophysiotropic somatostatin (SRIH) neurons projecting from the periventricular area and arcuate SRIH interneurons. For this purpose, the hypothalamus of 17-day-old rat fetuses was sampled and separated into a ventral and a dorsal fragment containing respectively periventricular and arcuate regions. Each fragment was dissociated and seeded separately in defined medium. Brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), two important members of the neurotrophin family involved in neuronal differentiation and plasticity, were added to the cultures at seeding time. After 6 or 11 days in vitro, neurons were labeled with an anti-SRIH antiserum and submitted to morphometric analysis. In parallel, SRIH mRNA was estimated by semiquantitative reverse-transcriptase-polymerase chain reaction, and neuronal SRIH content, basal and depolarisation-stimulated releases measured by radioimmunoassay. The response of control, non-labeled neurons was estimated by neuronal counts and by assaying glutamic acid decarboxylase, a marker of a large majority of hypothalamic neurons. BDNF markedly increased the size and the branching number of SRIH periventricular cell bodies. Expression of SRIH mRNA, as well as SRIH content and release into the culture medium, were also stimulated by the neurotrophin. Non-SRIH neurons were not affected by the treatment. Under the same conditions, arcuate neurons exhibited a weak, mostly transient response to BDNF. NT-3 was ineffective on either neuronal subset. Immunoneutralization of Trk receptors provided further evidence for BDNF effect specificity. The results indicate that BDNF is a selective activator of the differentiation of hypophysiotropic SRIH neurons in the periventricular area of the hypothalamus.

  3. Adult human nasal mesenchymal-like stem cells restore cochlear spiral ganglion neurons after experimental lesion.

    Science.gov (United States)

    Bas, Esperanza; Van De Water, Thomas R; Lumbreras, Vicente; Rajguru, Suhrud; Goss, Garrett; Hare, Joshua M; Goldstein, Bradley J

    2014-03-01

    A loss of sensory hair cells or spiral ganglion neurons from the inner ear causes deafness, affecting millions of people. Currently, there is no effective therapy to repair the inner ear sensory structures in humans. Cochlear implantation can restore input, but only if auditory neurons remain intact. Efforts to develop stem cell-based treatments for deafness have demonstrated progress, most notably utilizing embryonic-derived cells. In an effort to bypass limitations of embryonic or induced pluripotent stem cells that may impede the translation to clinical applications, we sought to utilize an alternative cell source. Here, we show that adult human mesenchymal-like stem cells (MSCs) obtained from nasal tissue can repair spiral ganglion loss in experimentally lesioned cochlear cultures from neonatal rats. Stem cells engraft into gentamicin-lesioned organotypic cultures and orchestrate the restoration of the spiral ganglion neuronal population, involving both direct neuronal differentiation and secondary effects on endogenous cells. As a physiologic assay, nasal MSC-derived cells engrafted into lesioned spiral ganglia demonstrate responses to infrared laser stimulus that are consistent with those typical of excitable cells. The addition of a pharmacologic activator of the canonical Wnt/β-catenin pathway concurrent with stem cell treatment promoted robust neuronal differentiation. The availability of an effective adult autologous cell source for inner ear tissue repair should contribute to efforts to translate cell-based strategies to the clinic.

  4. Fabrication of biocompatible free-standing nanopatterned films for primary neuronal cultures

    KAUST Repository

    Cesca, F.

    2014-09-10

    Devising and constructing biocompatible devices for nervous system regeneration is an extremely challenging task. Besides tackling the issue of biocompatibility, biomaterials for neuroscience applications should mimic the complex environment of the extracellular matrix, which in vivo provides neurons with a series of cues and signals to guide cells towards their appropriate targets. In this work, a novel nanopatterned biocompatible poly-ε-caprolactone (PCL) film is realized to assist the attachment and growth of primary hippocampal neurons. Costly and time-consuming processes can be avoided using plasma-surface nanotexturing obtained by a mixed gas SF6/Ar at −5 °C. The intrinsic composition and line topography of nanopatterned PCL ensure healthy development of the neuronal network, as shown by confocal microscopy, by analysing the expression of a range of neuronal markers typical of mature cultures, as well as by scanning electron microscopy. In addition, we show that surface nanopatterning improves differentiation of neurons compared to flat PCL films, while no neural growth was observed on either flat or nanopatterned substrates in the absence of a poly-D-lysine coating. Thus, we successfully optimized a nanofabrication protocol to obtain nanostructured PCL layers endowed with several mechanical and structural characteristics that make them a promising, versatile tool for future tissue engineering studies aimed at neural tissue regeneration.

  5. Recombinant AAV-mediated Expression of Human BDNF Protects Neurons against Cell Apoptosis in Aβ-induced Neuronal Damage Model

    Institute of Scientific and Technical Information of China (English)

    LIU Zhaohui; MA Dongliang; FENG Gaifeng; MA Yanbing; HU Haitao

    2007-01-01

    The human brain-derived neurotrophic factor (hBDNF) gene was cloned by polymerase chain reaction and the recombinant adeno-associated viral vector inserted with hBDNF gene (AAV-hBDNF) was constructed. Cultured rat hippocampal neurons were treated with Aβ25-35 and serued as the experimental Aβ-induced neuronal damage model (AD model), and the AD model was infected with AAV-hBDNF to explore neuroprotective effects of expression of BDNF. Cell viability was assayed by MTT. The expression of bcl-2 anti-apoptosis protein was detected by immunocytochemical staining. The change of intracellular free Ca ion ([Ca2+]i) was measured by laser scanning confocal microscopy. The results showed that BDNF had protective effects against Aβ-induced neuronal damage. The expression of the bcl-2 anti-apoptosis protein was raised significantly and the balance of [Ca2+]i was maintained in the AAV-hBDNF treatment group as compared with AD model group. These data suggested that recombinant AAV mediated a stable expression of hBDNF in cultured hippocampal neurons and resulted in significant neuron protective effects in AD model. The BDNF may reduce neuron apoptosis through increasing the expression of the bcl-2 anti-apoptosis protein and inhibiting intracellular calcium overload. The viral vector-mediated gene expression of BDNF may pave the way of a novel therapeutic strategy for the treatment of neurodegenerative diseases such as Alzheimer's disease.

  6. Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

    Science.gov (United States)

    Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E

    2001-01-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

  7. Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells

    DEFF Research Database (Denmark)

    Aldana, Blanca I; Zhang, Yu; Lihme, Maria Fog

    2017-01-01

    pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass...... acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U-(13)C]Glutamate and [U-(13)C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans...

  8. Differentiation of Human Cord Blood and Stromal Derived Stem Cells into Neuron Cells

    Directory of Open Access Journals (Sweden)

    Özlem Pamukçu Baran

    2007-01-01

    Full Text Available The most basic properties of stem cells are the capacities to self-renew indefinitely and to differentiate into multiple cell or tissue types. Umbilical cord blood has been utilized for human hematopoietic stem cell transplantation as an alternative source to bone marrow.The experiments show that Wharton’s jelly cells are easily attainable and can be expanded in vitro, maintained in culture, and induced to differentiate into neural cells. Almost recent studies it has been discovered that the cord blood-derived cells can differantiate not only to blood cells but also to various somatic cells like neuron or muscle cell with the signals taken from the envoirenment.Interestingly, neural cells obtained from umbilical cord blood show a relatively high spontaneous differentiation into oligodendrocytes, Embryonic stem cells proliferate indefinitely and can differentiate spontaneously into all tissue types.It has been shown that embryonic stem cells can be induced to differentiate into neurons and glia by treatment with retinoic acid or basic fibroblast growth factor. It has been studied that the diseases as Motor Neuron Disease, Parkinson, Alzheimer and degeneration of medulla spinalis and also paralysises could be treated with transplantation of cord blood-dericed stem cells.

  9. Effect of cholecystokinin-8 on in vitro cultured rat cortical neurons against apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ying Liu; Jiangbao Zhou

    2006-01-01

    BACKGROUND: Cholecystokinin (CCK-8) can regulate the synthesis of NO, release of amino acid substance and suppress Ca2+ inflow. It is unknown about neuroprotection of CCK-8 on neuronal apoptosis and its relationship with nerve growth factor (NGF).OBJECTryE: To investigate the protective effect of CCK-8 on in vitro cultured rat cortical neurons against apoptosis induced by glutamate, and explore its effect on expression of NGF in the neurons during apoptosis.DESIGN: Randomized controlled experiment on the basis of cells.SETTING: Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University.MATERIALS: Eighty SD rats of 1-day old; DMEM/F12 culture medium (Biochrom Company, Germany);Fetal bovine serum (TBD Company, Tianjin); CCK-8 (Sigma Company, USA). Glutamate (Bioengineering Company, Shanghai); TUNEL kit and NGF- in situ hybridization kit (Boster Bioengineering Company,Wuhan); anti-NGF polyclonal antibody (Santa-Cluz Company); NGF immunocytochemistry kit (Zhongshan Company, Beijing).METHODS: The experiments were carried out in Children's Research Institute Affiliated to Children Hospital of Chongqing Medical University from December 2004 to September 2005. Primary cultured cortical neurons from SD rats of 1-day oldwere incubated for 7 days. The cultured cells were divided randomly into 3 groups:experimental group, model group and control group. Neurons in experimental groups were added CCK-8 of 1 ×10-6, 1 ×10-7, 1 ×10-8 μ mol/L respectively, and then added 50 μmol/L glutamate solution a hour later. Neurons in model groups were treated with 50 μ mol/L glutamate solution. In the control group, cells were treated with normal medium. Apoptosis of cultured cortical neurons were observed by fluorescent microscope, the expression of NGF protein and mRNA were determined respectively by immunocytochemistry and in situ hybridization, and apoptosis of cortical neurons was detected with terminal deoxynucleotidyl transferase-mediated nick

  10. Nicotine inhibits potassium currents in Aplysia bag cell neurons.

    Science.gov (United States)

    White, Sean H; Sturgeon, Raymond M; Magoski, Neil S

    2016-06-01

    Acetylcholine and the archetypal cholinergic agonist, nicotine, are typically associated with the opening of ionotropic receptors. In the bag cell neurons, which govern the reproductive behavior of the marine snail, Aplysia californica, there are two cholinergic responses: a relatively large acetylcholine-induced current and a relatively small nicotine-induced current. Both currents are readily apparent at resting membrane potential and result from the opening of distinct ionotropic receptors. We now report a separate current response elicited by applying nicotine to cultured bag cell neurons under whole cell voltage-clamp. This current was ostensibly inward, best resolved at depolarized voltages, presented a noncooperative dose-response with a half-maximal concentration near 1.5 mM, and associated with a decrease in membrane conductance. The unique nicotine-evoked response was not altered by intracellular perfusion with the G protein blocker GDPβS or exposure to classical nicotinic antagonists but was occluded by replacing intracellular K(+) with Cs(+) Consistent with an underlying mechanism of direct inhibition of one or more K(+) channels, nicotine was found to rapidly reduce the fast-inactivating A-type K(+) current as well as both components of the delayed-rectifier K(+) current. Finally, nicotine increased bag cell neuron excitability, which manifested as reduction in spike threshold, greater action potential height and width, and markedly more spiking to continuous depolarizing current injection. In contrast to conventional transient activation of nicotinic ionotropic receptors, block of K(+) channels could represent a nonstandard means for nicotine to profoundly alter the electrical properties of neurons over prolonged periods of time.

  11. PCB 136 atropselectively alters morphometric and functional parameters of neuronal connectivity in cultured rat hippocampal neurons via ryanodine receptor-dependent mechanisms.

    Science.gov (United States)

    Yang, Dongren; Kania-Korwel, Izabela; Ghogha, Atefeh; Chen, Hao; Stamou, Marianna; Bose, Diptiman D; Pessah, Isaac N; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-04-01

    We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca²⁺-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (-)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (-)-PCB 136 is observed at concentrations ranging from 0.1 to 100 nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca²⁺-sensitive dye demonstrates that (-)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca²⁺ oscillations. Similarly, (-)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure.

  12. Reprogramming somatic cells to cells with neuronal characteristics by defined medium both in vitro and in vivo.

    Science.gov (United States)

    He, Songwei; Guo, Yiping; Zhang, Yixin; Li, Yuan; Feng, Chengqian; Li, Xiang; Lin, Lilong; Guo, Lin; Wang, Haitao; Liu, Chunhua; Zheng, Yi; Luo, Chuanming; Liu, Qiang; Wang, Fuhui; Sun, Hao; Liang, Lining; Li, Lingyu; Su, Huanxing; Chen, Jiekai; Pei, Duanqing; Zheng, Hui

    2015-01-01

    Currently, direct conversion from somatic cells to neurons requires virus-mediated delivery of at least one transcriptional factor or a combination of several small-molecule compounds. Delivery of transcriptional factors may affect genome stability, while small-molecule compounds may require more evaluations when applied in vivo. Thus, a defined medium with only conventional growth factors or additives for cell culture is desirable for inducing neuronal trans-differentiation. Here, we report that a defined medium (5C) consisting of basic fibroblast growth factor (bFGF), N2 supplement, leukemia inhibitory factor, vitamin C (Vc), and β-mercaptoethanol (βMe) induces the direct conversion of somatic cells to cells with neuronal characteristics. Application of 5C medium converted mouse embryonic fibroblasts (MEFs) into TuJ+ neuronal-like cells, which were capable of survival after being transplanted into the mouse brain. The same 5C medium could convert primary rat astrocytes into neuronal-like cells with mature electrophysiology characteristics in vitro and facilitated the recovery of brain injury, possibly by inducing similar conversions, when infused into the mouse brain in vivo. Crucially, 5C medium could also induce neuronal characteristics in several human cell types. In summary, this 5C medium not only provides a means to derive cells with neuronal characteristics without viral transfection in vitro but might also be useful to produce neurons in vivo for neurodegenerative disease treatment.

  13. Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, K.A.; Toledo, S.P. (Univ. of California-San Diego, La Jolla (USA))

    1989-09-01

    In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of (3H)leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of (3H)aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that (1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; (2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and (3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons.

  14. Neurotoxicity evaluation of three root canal sealers on cultured rat trigeminal ganglion neurons

    Science.gov (United States)

    Ayar, Ahmet; Kalkan, Omer-Faruk; Canpolat, Sinan; Tasdemir, Tamer; Ozan, Ulku

    2017-01-01

    Background The aim of this study was to investigate the possible neurotoxic effects of 3 root canal sealers (RCSs) (AH Plus, GuttaFlow, iRoot SP) on cultured rat trigeminal ganglion (TG) neurons. Material and Methods Primary cultures of TG neurons were obtained from 1 to 2-day old rats. Freshly mixed RCSs were incubated in sterile phosphate buffered saline and cells were incubated with supernatants of the RCSs for different time intervals (1-, 3-, 6- and 24-h; 1 or 1/10 diluted) and viability/cytotoxicity was tested by counting the number of live cells. Pair of dishes with cells from the same culture incubated with only culture medium was considered as negative controls. Cell images were captured and acquired at x200 magnification using a microscope equipped with a camera using special image program. The viable cells were manually counted assigned from the images for each dose and incubation duration. Data was analysed by using 1-way analysis of variance with Tukey post hoc tests. Results There was no significant change in cell viability after short duration of incubation (1- and 3-h) with the supernatant of any of RCSs, except for undiluted-AH Plus at 3-h. When AH Plus was compared with other RCSs, for diluted supernatants, there was only significant difference between iRoot SP and AH Plus at 24-h (P<0.05). Whereas undiluted-AH Plus was significantly more cytotoxic for 3-, 6- and 24-h periods as compared to respective incubation periods of undiluted other groups (P<0.05). GuttaFlow groups had similar neurotoxic effect on cells for all test periods. Conclusions All tested RCSs exhibited a variable degree of neurotoxicity on these primary sensory neurons of orofacial tissues, depending on their chemical compositions. GuttaFlow and iRoot SP evoked a less toxic response to TG cells than AH Plus. Key words:Neurotoxicity, trigeminal ganglia, cell culture, root canal sealer, AH Plus, GuttaFlow, iRoot SP. PMID:28149460

  15. Cell cycle markers have different expression and localization patterns in neuron-like PC12 cells and primary hippocampal neurons.

    Science.gov (United States)

    Negis, Yesim; Unal, Aysegul Yildiz; Korulu, Sirin; Karabay, Arzu

    2011-06-01

    Neuron-like PC12 cells are extensively used in place of neurons in published studies. Aim of this paper has been to compare mRNA and protein expressions of cell cycle markers; cyclinA, B, D, E; Cdk1, 2 and 4; and p27 in post-mitotic primary hippocampal neurons, mitotically active PC12 cells and NGF-differentiated post-mitotic PC12 cells. Contrary to PC12 cells, in neurons, the presence of all these markers was detected only at mRNA level; except for cyclinA, cyclinE and Cdk4, which were detectable also at protein levels. In both NGF-treated PC12 cells and neurons, cyclinE was localized only in the nucleus. In NGF-treated PC12 cells cyclinD and Cdk4 were localized in the nucleus while, in neurons cyclinD expression was not detectable; Cdk4 was localized in the cytoplasm. In neurons, cyclinA was nuclear, whereas in NGF-treated PC12 cells, it was localized in the cell body and along the processes. These results suggest that PC12 cells and primary neurons are different in terms of cell cycle protein expressions and localizations. Thus, it may not be very appropriate to use these cells as neuronal model system in order to understand neuronal physiological activities, upstream of where may lie cell cycle activation triggered events.

  16. A possible role of the non-GAT1 GABA transporters in transfer of GABA from GABAergic to glutamatergic neurons in mouse cerebellar neuronal cultures.

    Science.gov (United States)

    Suñol, C; Babot, Z; Cristòfol, R; Sonnewald, U; Waagepetersen, H S; Schousboe, A

    2010-09-01

    Cultures of dissociated cerebellum from 7-day-old mice were used to investigate the mechanism involved in synthesis and cellular redistribution of GABA in these cultures consisting primarily of glutamatergic granule neurons and a smaller population of GABAergic Golgi and stellate neurons. The distribution of GAD, GABA and the vesicular glutamate transporter VGlut-1 was assessed using specific antibodies combined with immunofluorescence microscopy. Additionally, tiagabine, SKF 89976-A, betaine, beta-alanine, nipecotic acid and guvacine were used to inhibit the GAT1, betaine/GABA (BGT1), GAT2 and GAT3 transporters. Only a small population of cells were immuno-stained for GAD while many cells exhibited VGlut-1 like immuno-reactivity which, however, never co-localized with GAD positive neurons. This likely reflects the small number of GABAergic neurons compared to the glutamatergic granule neurons constituting the majority of the cells. GABA uptake exhibited the kinetics of high affinity transport and could be partly (20%) inhibited by betaine (IC(50) 142 microM), beta-alanine (30%) and almost fully (90%) inhibited by SKF 89976-A (IC(50) 0.8 microM) or nipecotic acid and guvacine at 1 mM concentrations (95%). Essentially all neurons showed GABA like immunostaining albeit with differences in intensity. The results indicate that GABA which is synthesized in a small population of GAD-positive neurons is redistributed to essentially all neurons including the glutamatergic granule cells. GAT1 is not likely involved in this redistribution since addition of 15 microM tiagabine (GAT1 inhibitor) to the culture medium had no effect on the overall GABA content of the cells. Likewise the BGT1 transporter cannot alone account for the redistribution since inclusion of 3 mM betaine in the culture medium had no effect on the overall GABA content. The inhibitory action of beta-alanine and high concentrations of nipecotic acid and guvacine on GABA transport strongly suggests that also

  17. Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons.

    Science.gov (United States)

    Lööv, Camilla; Fernqvist, Maria; Walmsley, Adrian; Marklund, Niklas; Erlandsson, Anna

    2012-01-01

    Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

  18. Neutralization of LINGO-1 during in vitro differentiation of neural stem cells results in proliferation of immature neurons.

    Directory of Open Access Journals (Sweden)

    Camilla Lööv

    Full Text Available Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

  19. Effects of DDT and permethrin on neurite growth in cultured neurons of chick embryo brain and Lymnaea stagnalis.

    Science.gov (United States)

    Ferguson, C A; Audesirk, G

    1990-01-01

    The pesticides permethrin and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT), dissolved in either ethanol (EtOH) or dimethylsulphoxide (DMSO), were studied to determine their effect on neurite growth from cultured neurons of Lymnaea stagnalis and embryonic chicks. Both of these toxins decreased the percentage of neurons growing neurites, mean neurite length, and number of neurites/cell in a dose-dependent manner. DMSO increased the toxicity of permethrin and DDT in L. stagnalis neurons. EtOH was not used as a solvent with the embryonic chick cultures. Pre-existing neurites of L. stagnalis neurons exposed to permethrin regressed in a dose- and time-dependent manner. These two toxins may affect neurite outgrowth through interference with intracellular calcium regulation.

  20. Chronic homocysteine exposure causes changes in the intrinsic electrophysiological properties of cultured hippocampal neurons.

    Science.gov (United States)

    Schaub, Christina; Uebachs, Mischa; Beck, Heinz; Linnebank, Michael

    2013-04-01

    Homocystinuria is an inborn error of metabolism characterized by plasma homocysteine levels up to 500 μM, premature vascular events and mental retardation. Mild elevations of homocysteine plasma levels up to 25 μM, which are common in the general population, are associated with vascular disease, cognitive impairment and neurodegeneration. Several mechanisms of homocysteine neurotoxicity have been investigated. However, information on putative effects of hyperhomocysteinemia on the electrophysiology of neurons is limited. To screen for such effects, we examined primary cultures of mouse hippocampal neurons with the whole-cell patch-clamp technique. Homocysteine was applied intracellularly (100 μM), or cell cultures were incubated with 100 μM homocysteine for 24 h. Membrane voltage was measured in current-clamp mode, and action potential firing was induced with short and prolonged current injections. Single action potentials induced by short current injections (5 ms) were not altered by acute application or incubation of homocysteine. When we elicited trains of action potentials with prolonged current injections (200 ms), a broadening of action potentials during repetitive firing was observed in control neurons. This spike broadening was unaltered by acute application of homocysteine. However, it was significantly diminished when incubation with homocysteine was extended to 24 h prior to recording. Furthermore, the number of action potentials elicited by low current injections was reduced after long-term incubation with homocysteine, but not by the acute application. After 24 h of homocysteine incubation, the input resistance was reduced which might have contributed to the observed alterations in membrane excitability. We conclude that homocysteine exposure causes changes in the intrinsic electrophysiological properties of cultured hippocampal neurons as a mechanism of neurological symptoms of hyperhomocysteinemia.

  1. Distinct regulation of activity-dependent transcription of immediate early genes in cultured rat cortical neurons.

    Science.gov (United States)

    Fukuchi, Mamoru; Sanabe, Tomofumi; Watanabe, Toshifumi; Kubota, Takane; Tabuchi, Akiko; Tsuda, Masaaki

    2017-08-26

    The activity-regulated expression of immediate early genes (IEGs) contributes to long-lasting neuronal functions underlying long-term memory. However, their response properties following neuronal activity are unique and remain poorly understood. To address this knowledge gap, here we further investigated the response properties of two representative IEGs, c-fos and brain-derived neurotrophic factor (Bdnf). Treatment of cultured cortical cells with KCl produces a depolarization process that results in the increase of intracellular calcium concentration in a KCl concentration-dependent manner. Consistent with this increase, c-fos expression was induced in a KCl concentration-dependent manner. In contrast, however, Bdnf expression was optimally activated by both 25 and 50 mM concentration of KCl. Similar results were observed when the cells were treated with okadaic acid, which inhibits protein phosphatases and elicits the hyper-phosphorylation of signaling molecules. Thus, Bdnf expression is strictly regulated by a neuronal activity threshold in an all or nothing manner, whereas c-fos expression is activated in a neuronal activity-dependent manner. Our findings also suggest that these differential responses might be due to the presence or absence of a TATA box. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A fast flexible ink-jet printing method for patterning dissociated neurons in culture.

    Science.gov (United States)

    Sanjana, Neville E; Fuller, Sawyer B

    2004-07-30

    We present a new technique that uses a custom-built ink-jet printer to fabricate precise micropatterns of cell adhesion materials for neural cell culture. Other work in neural cell patterning has employed photolithography or "soft lithographic" techniques such as micro-stamping, but such approaches are limited by their use of an un-alterable master pattern such as a mask or stamp master and can be resource-intensive. In contrast, ink-jet printing, used in low-cost desktop printers, patterns material by depositing microscopic droplets under robotic control in a programmable and inexpensive manner. We report the use of ink-jet printing to fabricate neuron-adhesive patterns such as islands and other shapes using poly(ethylene) glycol as the cell-repulsive material and a collagen/poly-D-lysine (PDL) mixture as the cell-adhesive material. We show that dissociated rat hippocampal neurons and glia grown at low densities on such patterns retain strong pattern adherence for over 25 days. The patterned neurons are comparable to control, un-patterned cells in electrophysiological properties and in immunocytochemical measurements of synaptic density and inhibitory cell distributions. We suggest that an inexpensive desktop printer may be an accessible tool for making micro-island cultures and other basic patterns. We also suggest that ink-jet printing may be extended to a range of developmental neuroscience studies, given its ability to more easily layer materials, build substrate-bound gradients, construct out-of-plane structure, and deposit sources of diffusible factors.

  3. Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

    Directory of Open Access Journals (Sweden)

    Al-Zoubi A

    2016-06-01

    Full Text Available Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both. Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte

  4. Interpretation of cell culture phenomena.

    Science.gov (United States)

    Vierck, J L; Dodson, M V

    2000-03-01

    This paper discusses the dilemma of interpreting unusual or abnormal phenomena seen in cell cultures and is not intended to address the statistical design of experiments. Problems that can be encountered when growing cells in experimental situations include low or decreasing cell numbers, abnormal cell morphology, microbial contamination, and detachment of the cell monolayer. If any of these situations occur, it is not realistic to proceed with data analysis until the problem is corrected. The best policy is to attempt to standardize all types of cultures used for analysis and to avoid using any cultures that display atypical characteristics.

  5. Two cell circuits of oriented adult hippocampal neurons on self-assembled monolayers for use in the study of neuronal communication in a defined system.

    Science.gov (United States)

    Edwards, Darin; Stancescu, Maria; Molnar, Peter; Hickman, James J

    2013-08-21

    In this study, we demonstrate the directed formation of small circuits of electrically active, synaptically connected neurons derived from the hippocampus of adult rats through the use of engineered chemically modified culture surfaces that orient the polarity of the neuronal processes. Although synaptogenesis, synaptic communication, synaptic plasticity, and brain disease pathophysiology can be studied using brain slice or dissociated embryonic neuronal culture systems, the complex elements found in neuronal synapses makes specific studies difficult in these random cultures. The study of synaptic transmission in mature adult neurons and factors affecting synaptic transmission are generally studied in organotypic cultures, in brain slices, or in vivo. However, engineered neuronal networks would allow these studies to be performed instead on simple functional neuronal circuits derived from adult brain tissue. Photolithographic patterned self-assembled monolayers (SAMs) were used to create the two-cell "bidirectional polarity" circuit patterns. This pattern consisted of a cell permissive SAM, N-1[3-(trimethoxysilyl)propyl] diethylenetriamine (DETA), and was composed of two 25 μm somal adhesion sites connected with 5 μm lines acting as surface cues for guided axonal and dendritic regeneration. Surrounding the DETA pattern was a background of a non-cell-permissive poly(ethylene glycol) (PEG) SAM. Adult hippocampal neurons were first cultured on coverslips coated with DETA monolayers and were later passaged onto the PEG-DETA bidirectional polarity patterns in serum-free medium. These neurons followed surface cues, attaching and regenerating only along the DETA substrate to form small engineered neuronal circuits. These circuits were stable for more than 21 days in vitro (DIV), during which synaptic connectivity was evaluated using basic electrophysiological methods.

  6. Evaluation of PFOS-mediated neurotoxicity in rat primary neurons and astrocytes cultured separately or in co-culture.

    Science.gov (United States)

    Li, Zhenwei; Liu, Qi; Liu, Chang; Li, Chunna; Li, Yachen; Li, Shuangyue; Liu, Xiaohui; Shao, Jing

    2017-02-01

    Perfluorooctane sulfonate (PFOS) is a potential neurotoxicant reported by epidemiological investigations and experimental studies, while the underlying mechanisms are still unclear. Astrocytes not only support for the construction of neurons, but also conduct neuronal functions through glutamate-glutamine cycle in astrocyte-neuron crosstalk. In the present study, the effect of PFOS exposure on rat primary hippocampal neurons or cortex astrocytes was evaluated. Then the role of the astrocytes in PFOS-induced toxic effect on neurons was explored with astrocyte-neuron co-culture system. Exposure of rat primary hippocampal neurons to PFOS has led to oxidation-antioxidation imbalance, increased apoptosis and abnormal autophagy. The adverse effect of PFOS on rat primary cortex astrocytes manifested in the form of altered extracellular glutamate and glutamine concentrations, decreased glutamine synthase activity, as well as decreased gene expression of glutamine synthase, glutamate transporters and glutamine transporters in the glutamate-glutamine cycle. Especially, the alleviation of PFOS-inhibited neurite outgrowth in neurons could be observed in astrocyte-neuron co-culture system, though the ability of astrocytes in fostering neurite outgrowth was affected by PFOS. These results indicated that both astrocytes and neurons might be the targets of PFOS-induced neurotoxicity, and astrocytes could protect against PFOS-inhibited neurite outgrowth in primary cultured neurons. Our research might render some information in explaining the mechanisms of PFOS-induced neurotoxicity. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Molecular misreading in non-neuronal cells.

    Science.gov (United States)

    Van Leeuwen, F W; Hol, E M; Hermanussen, R W; Sonnemans, M A; Moraal, E; Fischer, D F; Evans, D A; Chooi, K F; Burbach, J P; Murphy, D

    2000-08-01

    +1 Frame-shifted proteins such as amyloid precursor protein(+1) and ubiquitin-B(+1) have been identified in the neuropathological hallmarks of Alzheimer's disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed 'molecular misreading'. A key question is whether this process is confined to neurons or whether it could also occur in non-neuronal cells. A transgenic mouse line (MV-B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV-LTR promotor was used to screen non-neuronal tissues for molecular misreading by means of detection of the rat vasopressin(+1) protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non-neural tissues too, could be affected by cellular derangements caused by molecular misreading.

  8. Beneficial effects of BV2 cell on proliferation and neuron-differentiating of mesenchymal stem cells in the circumstance of injured PC12 cell supernatant

    Institute of Scientific and Technical Information of China (English)

    Xiao-Guang LUO; Hong WANG; Jin ZHOU; Rong YAN; Zhe WU; Chao-Dong ZHANG; Qiu-Shuang WANG

    2006-01-01

    Objective The microglias is the representative of immune cells in the brain. It plays dual roles of both repairing and damaging in injured nervous system, and works as an inevitable component of the circumstance of injured neurons. This study was aiming at the effects of the microglias on the biological activities of mesenchymal stem cells (MSCs) inthe circumstance of injured neurons. Methods MSCs were obtained by primary culture. We adopted PC12 cells (PC12) and BV2 cells (BV2) to substitute for neurons and microglias, respectively. PC12 were injured by aged Aβ1-40 and the supernatant of the injured PC12 was used to set up the circumstance of injured neurons. Transwells were used for co-culture of BV2 and MSCs, which allowed the independent detection of cells after co-culture. Immunofluorescence was used to identify MSCs and neuron-differentiating cells with CD44 and neuron specific enolase (NSE) staining, respectively. MTT assay was adopted to measure the proliferation. Results In the circumstance of both BV2 presence and injured PC12 supernatant incubation, either the proliferation or the differentiation of MSCs reached the highest, which seemed to be contradictory, but we gave our explanations. With the BV2 co-culture, the proliferation of MSCs tend to be higher, but the neuron-differentiating MSCs were similar to those incubated without BV2 co-culture either in normal or injured in PC12 supernatant. With the incubation of injured PC12 supernatant, the neuron-differentiating cells were significantly higher than that of control (P < 0.05). Conclusion In the circumstance of injured neurons, microlgias tend to promote the MSCs proliferation. Although not helpful in neuron-differentiating, microglias did not exert any negative effect either.

  9. Phytic Acid Protects against 6-Hydroxydopamine-Induced Dopaminergic Neuron Apoptosis in Normal and Iron Excess Conditions in a Cell Culture Model.

    Science.gov (United States)

    Xu, Qi; Kanthasamy, Anumantha G; Reddy, Manju B

    2011-02-07

    Iron may play an important role in Parkinson's disease (PD) since it can induce oxidative stress-dependent neurodegeneration. The objective of this study was to determine whether the iron chelator, phytic acid (IP6) can protect against 6-hydroxydopamine- (6-OHDA-) induced apoptosis in immortalized rat mesencephalic dopaminergic cells under normal and iron-excess conditions. Caspase-3 activity was increased about 6-fold after 6-OHDA treatment (compared to control; P IP6 pretreatment decreased it by 38% (P IP6 pretreatment. Under iron-excess condition, a 6-fold increase in caspase-3 activity (P IP6. Together, our data suggest that IP6 protects against 6-OHDA-induced cell apoptosis in both normal and iron-excess conditions, and IP6 may offer neuroprotection in PD.

  10. Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology

    NARCIS (Netherlands)

    Frega, M.; Tedesco, M.; Massobrio, P.; Pesce, M.; Martinoia, S.

    2014-01-01

    Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These

  11. Neuroprotection and neuronal differentiation studies using substantia nigra dopaminergic cells derived from transgenic mouse embryos.

    Science.gov (United States)

    Son, J H; Chun, H S; Joh, T H; Cho, S; Conti, B; Lee, J W

    1999-01-01

    The major pathological lesion of Parkinson's disease (PD) is the selective cell death of dopaminergic (DA) neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to DA cell death underlying the PD process remain elusive, brain-derived neurotrophic factor (BDNF) is thought to exert neuroprotective as well as neurotrophic roles for the survival and differentiation of DA neurons in SN. Addressing molecular mechanisms of BDNF action in both primary embryonic mesencephalic cultures and in vivo animal models has been technically difficult because DA neurons in SN are relatively rare and present with many heterogeneous cell populations in midbrain. We have developed and characterized a DA neuronal cell line of embryonic SN origin that is more accessible to molecular analysis and can be used as an in vitro model system for studying SN DA neurons. A clonal SN DA neuronal progenitor cell line SN4741, arrested at an early DA developmental stage, was established from transgenic mouse embryos containing the targeted expression of the thermolabile SV40Tag in SN DA neurons. The phenotypic and morphological differentiation of the SN4741 cells could be manipulated by environmental cues in vitro. Exogenous BDNF treatment produced significant neuroprotection against 1-methyl-4-phenylpyridinium, glutamate, and nitric oxide-induced neurotoxicity in the SN4741 cells. Simultaneous phosphorylation of receptor tyrosine kinase B accompanied the neuroprotection. This SN DA neuronal cell line provides a unique model system to circumvent the limitations associated with primary mesencephalic cultures for the elucidation of molecular mechanisms of BDNF action on DA neurons of the SN.

  12. Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos, necroptosis, and apoptosis, but not autophagy.

    Science.gov (United States)

    Kong, Deyan; Zhu, Juehua; Liu, Qian; Jiang, Yongjun; Xu, Lily; Luo, Ning; Zhao, Zhenqiang; Zhai, Qijin; Zhang, Hao; Zhu, Mingyue; Liu, Xinfeng

    2017-03-01

    Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.

  13. Semiautomated analysis of dendrite morphology in cell culture.

    Science.gov (United States)

    Sweet, Eric S; Langhammer, Chris L; Kutzing, Melinda K; Firestein, Bonnie L

    2013-01-01

    Quantifying dendrite morphology is a method for determining the effect of biochemical pathways and extracellular agents on neuronal development and differentiation. Quantification can be performed using Sholl analysis, dendrite counting, and length quantification. These procedures can be performed on dendrite-forming cell lines or primary neurons grown in culture. In this protocol, we describe the use of a set of computer programs to assist in quantifying many aspects of dendrite morphology, including changes in total and localized arbor complexity.

  14. Long-term live imaging of neuronal circuits in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Gogolla, Nadine; Galimberti, Ivan; DePaola, Vincenzo; Caroni, Pico

    2006-01-01

    This protocol details a method for imaging organotypic slice cultures from the mouse hippocampus. The cultures are based on the interface method, which does not require special equipment, is easy to execute, and yields slice cultures that can be imaged repeatedly after they are isolated on postnatal day 6-9 and for up to 6 months in vitro. The preserved tissue architecture facilitates the analysis of defined hippocampal synapses, cells and entire projections. Time-lapse imaging is based on transgenes expressed in the mice, or on constructs introduced through transfection or viral vectors; it can reveal processes that develop over time periods ranging from seconds to months. Imaging can be repeated at least eight times without detectable morphological damage to neurons. Subsequent to imaging, the slices can be processed for immunocytochemistry or electron microscopy, to collect further information about the structures that have been imaged. This protocol can be completed in 35 min.

  15. Phytic Acid Protects against 6-Hydroxydopamine-Induced Dopaminergic Neuron Apoptosis in Normal and Iron Excess Conditions in a Cell Culture Model

    Directory of Open Access Journals (Sweden)

    Qi Xu

    2011-01-01

    Full Text Available Iron may play an important role in Parkinson's disease (PD since it can induce oxidative stress-dependent neurodegeneration. The objective of this study was to determine whether the iron chelator, phytic acid (IP6 can protect against 6-hydroxydopamine- (6-OHDA- induced apoptosis in immortalized rat mesencephalic dopaminergic cells under normal and iron-excess conditions. Caspase-3 activity was increased about 6-fold after 6-OHDA treatment (compared to control; <.001 and 30 μmol/L IP6 pretreatment decreased it by 38% (<.05. Similarly, a 63% protection (<.001 against 6-OHDA induced DNA fragmentation was observed with IP6 pretreatment. Under iron-excess condition, a 6-fold increase in caspase-3 activity (<.001 and a 42% increase in DNA fragmentation (<.05 with 6-OHDA treatment were decreased by 41% (<.01 and 27% (<.05, respectively, with 30 μmol/L IP6. Together, our data suggest that IP6 protects against 6-OHDA-induced cell apoptosis in both normal and iron-excess conditions, and IP6 may offer neuroprotection in PD.

  16. Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

    Science.gov (United States)

    Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

    2015-11-17

    Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

  17. Age-Dependent Glutamate Induction of Synaptic Plasticity in Cultured Hippocampal Neurons

    Science.gov (United States)

    Ivenshitz, Miriam; Segal, Menahem; Sapoznik, Stav

    2006-01-01

    A common denominator for the induction of morphological and functional plasticity in cultured hippocampal neurons involves the activation of excitatory synapses. We now demonstrate massive morphological plasticity in mature cultured hippocampal neurons caused by a brief exposure to glutamate. This plasticity involves a slow, 70%-80% increase in…

  18. Dissociated neurons and glial cells derived from rat inferior colliculi after digestion with papain.

    Science.gov (United States)

    Kaiser, Odett; Aliuos, Pooyan; Wissel, Kirsten; Lenarz, Thomas; Werner, Darja; Reuter, Günter; Kral, Andrej; Warnecke, Athanasia

    2013-01-01

    The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode-tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue-material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×10(4) cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.

  19. Multiple Modes of Communication between Neurons and Oligodendrocyte Precursor Cells

    NARCIS (Netherlands)

    Maldonado, Paloma P; Angulo, María Cecilia

    2015-01-01

    The surprising discovery of bona fide synapses between neurons and oligodendrocytes precursor cells (OPCs) 15 years ago placed these progenitors as real partners of neurons in the CNS. The role of these synapses has not been established yet, but a main hypothesis is that neuron-OPC synaptic activity

  20. Bone morphogenetic protein-5 (BMP-5 promotes dendritic growth in cultured sympathetic neurons

    Directory of Open Access Journals (Sweden)

    Higgins Dennis

    2001-09-01

    Full Text Available Abstract Background BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons. Results Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG during times of initial growth and rapid expansion of the dendritic arbor. Conclusions These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

  1. Prospects for replacement of auditory neurons by stem cells.

    Science.gov (United States)

    Shi, Fuxin; Edge, Albert S B

    2013-03-01

    Sensorineural hearing loss is caused by degeneration of hair cells or auditory neurons. Spiral ganglion cells, the primary afferent neurons of the auditory system, are patterned during development and send out projections to hair cells and to the brainstem under the control of largely unknown guidance molecules. The neurons do not regenerate after loss and even damage to their projections tends to be permanent. The genesis of spiral ganglion neurons and their synapses forms a basis for regenerative approaches. In this review we critically present the current experimental findings on auditory neuron replacement. We discuss the latest advances with a focus on (a) exogenous stem cell transplantation into the cochlea for neural replacement, (b) expression of local guidance signals in the cochlea after loss of auditory neurons, (c) the possibility of neural replacement from an endogenous cell source, and (d) functional changes from cell engraftment. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Alpha-synuclein cell-to-cell transfer and seeding in grafted dopaminergic neurons in vivo.

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    Elodie Angot

    Full Text Available Several people with Parkinson's disease have been treated with intrastriatal grafts of fetal dopaminergic neurons. Following autopsy, 10-22 years after surgery, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. Numerous studies have attempted to explain these findings in cell and animal models. In cell culture, α-synuclein has been found to transfer from one cell to another, via mechanisms that include exosomal transport and endocytosis, and in certain cases seed aggregation in the recipient cell. In animal models, transfer of α-synuclein from host brain cells to grafted neurons has been shown, but the reported frequency of the event has been relatively low and little is known about the underlying mechanisms as well as the fate of the transferred α-synuclein. We now demonstrate frequent transfer of α-synuclein from a rat brain engineered to overexpress human α-synuclein to grafted dopaminergic neurons. Further, we show that this model can be used to explore mechanisms underlying cell-to-cell transfer of α-synuclein. Thus, we present evidence both for the involvement of endocytosis in α-synuclein uptake in vivo, and for seeding of aggregation of endogenous α-synuclein in the recipient neuron by the transferred α-synuclein. Finally, we show that, at least in a subset of the studied cells, the transmitted α-synuclein is sensitive to proteinase K. Our new model system could be used to test compounds that inhibit cell-to-cell transfer of α-synuclein and therefore might retard progression of Parkinson neuropathology.

  3. Electrophysiology of embryonic, adult and aged rat hippocampal neurons in serum-free culture.

    Science.gov (United States)

    Evans, M S; Collings, M A; Brewer, G J

    1998-01-31

    Methods were recently developed for culturing neurons from adult rat hippocampus using the serum-free medium Neurobasal with B27 supplement. To determine whether adult cultured neurons have normal electrical properties, we studied cultures from rats of three age groups: (1) embryonic; (2) 10-11 months old and (3) 35-36 months old. Neurons had a polarized morphology with a large branching apical dendrite and small basal dendrites. Mean resting potentials were similar in the three age groups. All neurons had nonlinear current-voltage relationships, indicating the presence of voltage-sensitive ion channels. Most neurons had a voltage-sensitive inward current followed by a sustained voltage-sensitive outward current. Tetrodotoxin blocked the inward current, which is likely to be a sodium current. The sustained outward current, which is likely to be a potassium current, reversed at -71 mV. Most neurons exhibited anomalous rectification. Calcium currents were present in both embryonic and adult neurons. Embryonic neurons would sometimes fire multiple action potentials but adult neurons fired only single action potentials. Our results indicate that both embryonic and adult cultured neurons retain a clearly neuronal electrophysiological phenotype in Neurobasal/B27 serum-free medium.

  4. Isolation and culture of larval cells from C. elegans.

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    Sihui Zhang

    Full Text Available Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81% of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

  5. Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Hailer, Nils P; Vogt, Cornelia; Korf, Horst-Werner; Dehghani, Faramarz

    2005-05-01

    The effects of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N-methyl-D-aspartate (NMDA) and treated with IL-1beta (6 ng/mL) or IL-1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL-1beta did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA-lesioned OHSCs with IL-1beta exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA-lesioned cultures with IL-1ra significantly attenuated NMDA-induced neuronal damage and reduced the number of microglial cells, whereas application of IL-1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL-1beta directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL-1beta induces microglial activation but is not neurotoxic per se; (iii) IL-1beta enhances excitotoxic neuronal damage and microglial activation and (iv) IL-1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.

  6. S-Nitrosoglutathione and glutathione act as NMDA receptor agonists in cultured hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Ting-yu CHIN; Sheau-huei CHUEH; Pao-luh TAO

    2006-01-01

    Aim: To characterize the effect of combined pre- and postnatal morphine exposure on Af-methyl-D-aspartate receptor (NMDA) receptor signaling in hippocampal neurons of the offspring of morphine-addicted female rats. Methods: Cultured hippocampal neurons and synaptosomes were prepared from neonatal and 2-week-old offspring, respectively, of control or morphine-addicted female rats. The increase in the cytosolic Ca2+ concentration ([Ca2+]i) of cultured cells was measured using Fura-2, and glutamate release from synaptosomes was measured enzymatically. Results: Both glutamate and NMDA caused a dose-dependent increase in the [Ca2+]i. The nitric oxide (NO) donor, S-nitrosoglutathione (GSNO), but not 3-morpholinosydnonimine, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine, also induced a [Ca2+]i increase. GSNO and glutathione caused a dose-dependent increase in the [Ca2+]i with respective EC50 values of 56 and 414 μmol/L. Both effects were inhibited by Mg2+ or an NMDA receptor antagonist and were unaffected by the presence of a glutamate scavenger. The other glutathione derivatives, oxidized glutathione, S-methylglutathione, S-ethylglutathione, S-propylglutathione, and S-butylglutathione, the dipeptides, Glu-Cys and Cys-Gly, and the antioxidants, dithiothreitol and mercaptoethanol, failed to induce a [Ca2+]i increase. In addition, glutathione caused a dose-dependent increase in glutamate release from synaptosomes. The maximal responses and the EC50 values for the glutamate-, NMDA-, GSNO-, and glutathione-induced [Ca2+]i increases and the glutathione-induced glutamate release were indistinguishable in the neurons of the offspring from control and morphine-addicted female rats. Conclusion: GSNO and glutathione act as NMDA receptor agonists and, in contrast to hippocampal brain slice, combined pre- and postnatal morphine exposure does not modulate NMDA receptor signaling in the cultured hippocampal neurons.

  7. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons.

    Science.gov (United States)

    Wardyn, Joanna D; Sanderson, Chris; Swan, Laura E; Stagi, Massimiliano

    2015-08-15

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology.

  8. Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: effects of cytisine and its bromo derivatives.

    Science.gov (United States)

    Varas, Rodrigo; Valdés, Viviana; Iturriaga-Vásquez, Patricio; Cassels, Bruce K; Iturriaga, Rodrigo; Alcayaga, Julio

    2006-02-09

    Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although alpha4 and alpha7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5-10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine > acetylcholine approximately = cytisine > 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

  9. Susceptibility of primary culture neurons from rats of different ages to encephalomyocarditis (EMC) virus infection.

    Science.gov (United States)

    Su, Weiping; Ikegami, Hisashi; Nakayama, Yumi; Suzuki, Kazuhiko; Katayama, Kei-ichi; Nakayama, Hiroyuki; Doi, Kunio

    2003-10-01

    The changes in susceptibility of neurons to the D variant of EMC virus (EMC-D) (10(6) PFU/well) were investigated in developing hippocampal primary cultures from postnatal days of 1, 7, and 56 Fischer 344 rats (P1, P7, and P56) for up to 12 h after infection (12 HAI). The virus titer of primary culture neurons increased at 1 HAI, decreased at 2 HAI, increased at 3 HAI, peaked at 8 HAI, and decreased at 12 HAI in all age groups. The titers at 1 and 8 HAI were lowest in P56 cultures. The virus titer of neurons was always higher than that of culture media, especially at 1 HAI, in P1 cultures, whereas the former was lower than the latter from 2 to 3 HAI in P7 cultures and from 2 to 4 HAI in P56 cultures, respectively. Signals of viral RNA detected by in situ hybridization were first observed in the peripheral cytoplasm of neurons at 1 HAI in P1 and P7 cultures and at 4 HAI in P56 cultures, respectively. The signals spread to a large or whole area of cytoplasm and also to processes thereafter. The number of viral RNA-positive neurons and the amount of signals decreased with age. The present results indicated that the susceptibility of primary culture neurons to EMC-D decreased with age but viral replication still occurred in P56 cultures.

  10. Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Zhao HE; Jing-jing LI; Chang-hong ZHEN; Lin-ying FENG; Xiao-yan DING

    2006-01-01

    Aim: Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. Methods: In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB)was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-gem layers was compared. Results: Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. Conclusion:These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

  11. A transient, RCK4-like K+ current in cultured Xenopus olfactory bulb neurons.

    Science.gov (United States)

    Engel, J; Rabba, J; Schild, D

    1996-09-01

    A transient K+ current in cultured olfactory bulb neurons of Xenopus tadpoles was studied using the whole-cell patch-clamp technique. The current, which was resistant to 80 mM tetraethylammoniumchloride (TEA) and 10 nM charybdotoxin but blocked by 5 mM 4-aminopyridine (4-AP), activated between -60 and -40 mV and showed time- and voltage-dependent inactivation. Its peak amplitude was nearly independent of the extracellular K+ concentration ([K+]o) in the range of 0.05 to 10 mM, indicating that its conductance increased upon increasing [K+]o. The transient K+ current showed a slow recovery from inactivation with the time for half-maximum recovery from a conditioning pulse to 80 mV for 1 s varying from 100 ms to 500 ms. Complete recovery required as much as 5-10 s at -80 mV, but could be speeded up at hyperpolarized potentials. The current resembles the RCK4 (Kv1.4) current of rat neurons except that its recovery from inactivation was independent of [K+]o. High-frequency stimulation (20-67 Hz) of the neurons with short (5 ms) voltage pulses resulted in a frequency-dependent, progressive inactivation of the transient K+ current. This suggests that, during phasic responses of olfactory bulb neurons, inactivation of the transient K+ current occurs and may lead to lengthening of action potentials and facilitation of synaptic transmission.

  12. Comparison of the neurotoxicities between volatile organic compounds and fragrant organic compounds on human neuroblastoma SK-N-SH cells and primary cultured rat neurons

    Directory of Open Access Journals (Sweden)

    Yasue Yamada

    2015-01-01

    Full Text Available These are many volatile organic compounds (VOCs that are synthesized, produced from petroleum or derived from natural compounds, mostly plants. Fragrant and volatile organic compounds from plants have been used as food additives, medicines and aromatherapy. Several clinical and pathological studies have shown that chronic abuse of VOCs, mainly toluene, causes several neuropsychiatric disorders. Little is known about the mechanisms of neurotoxicity of the solvents. n-Octanal, nonanal, and 2-ethyl-1-hexanol, which are used catalyzers or intermediates of chemical reactions, are released into the environment. Essential oils have the functions of self-defense, sterilization, and antibiosis in plants. When volatile organic compounds enter the body, there is the possibility that they will pass through the blood–brain barrier (BBB and affect the central nervous system (CNS. However, the direct effects of volatile organic compounds on neural function and their toxicities are still unclear. We compared the toxicities of n-octanal, nonanal and 2-ethyl-1-hexanol with those of five naturally derived fragrant organic compounds (FOCs, linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and n-phenethyl alcohol. MTT assay of human neuroblastoma SK-N-SH cells showed that the IC50 values of linalool, cis-3-hexen-1-ol, isoamyl alcohol, n-propyl alcohol and phenethyl alcohol were 1.33, 2.3, >5, >5, and 2.39 mM, respectively, and the IC50 values of toluene, n-octanal, nonanal and 2-ethyl-1-hexanol were 850, 37.2, 8.31 and 15.1 μM, respectively. FOCs showed lower toxicities than those of VOCs. These results indicate that FOCs are safer than other compounds.

  13. The Ketone Body, β-Hydroxybutyrate Stimulates the Autophagic Flux and Prevents Neuronal Death Induced by Glucose Deprivation in Cortical Cultured Neurons.

    Science.gov (United States)

    Camberos-Luna, Lucy; Gerónimo-Olvera, Cristian; Montiel, Teresa; Rincon-Heredia, Ruth; Massieu, Lourdes

    2016-03-01

    Glucose is the major energy substrate in brain, however, during ketogenesis induced by starvation or prolonged hypoglycemia, the ketone bodies (KB), acetoacetate and β-hydroxybutyrate (BHB) can substitute for glucose. KB improve neuronal survival in diverse injury models, but the mechanisms by which KB prevent neuronal damage are still not well understood. In the present study we have investigated whether protection by the D isomer of BHB (D-BHB) against neuronal death induced by glucose deprivation (GD), is related to autophagy. Autophagy is a lysosomal-dependent degradation process activated during nutritional stress, which leads to the digestion of damaged proteins and organelles providing energy for cell survival. Results show that autophagy is activated in cortical cultured neurons during GD, as indicated by the increase in the levels of the lipidated form of the microtubule associated protein light chain 3 (LC3-II), and the number of autophagic vesicles. At early phases of glucose reintroduction (GR), the levels of p62 declined suggesting that the degradation of the autophagolysosomal content takes place at this time. In cultures exposed to GD and GR in the presence of D-BHB, the levels of LC3-II and p62 rapidly declined and remained low during GR, suggesting that the KB stimulates the autophagic flux preventing autophagosome accumulation and improving neuronal survival.

  14. Structured PDMS Chambers for Enhanced Human Neuronal Cell Activity on MEA Platforms

    Institute of Scientific and Technical Information of China (English)

    Joose Kreutzer; Laura Yl(a)-Outinen; Paula K(a)irn(a); Tiina Kaarela; Jarno Mikkonen; Heli Skottman; Susanna Narkilahti; Pasi Kallio

    2012-01-01

    Structured poly(dimethylsiloxane) (PDMS) chambers were designed and fabricated to enhance the signaling of human Embryonic Stem Cell (hESC) - derived neuronal networks on Microelectrode Array (MEA) platforms.The structured PDMS chambers enable cell seeding on restricted areas and thus,reduce the amount of needed coating materials and cells.In addition,the neuronal cells formed spontaneously active networks faster in the structured PDMS chambers than that in control chainbers.In the PDMS chambers,the neuronal networks were more active and able to develop their signaling into organized signal trains faster than control cultures.The PDMS chamber design enables much more repeatable analysis and rapid growth of functional neuronal network in vitro.Moreover,due to its easy and cheap fabrication process,new configurations can be easily fabricated based on investigator requirements.

  15. Nanoparticle Targeting to Neurons in a Rat Hippocampal Slice Culture Model

    Directory of Open Access Journals (Sweden)

    Ryan Walters

    2012-09-01

    Full Text Available We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots coated with PEG [poly(ethylene glycol]-appended DHLA (dihydrolipoic acid can bind AcWG(PalVKIKKP9GGH6 (Palm1 through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry, demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.

  16. Deoxyschisandrin modulates synchronized Ca2+ oscillations and spontaneous synaptic transmission of cultured hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Min FU; Zhao-hui SUN; Min ZONG; Xiang-ping HE; Huan-cong ZUO; Zuo-ping XIE

    2008-01-01

    Aim: Deoxyschisandrin is one of the most effective composites of Schisandra chinensis, a famous Chinese medicine widely used as an antistress, anti-aging, and neurological performance-improving herb. In this study, we examined its spe- cific mechanisms of action on cultured hippocampal neurons. Methods: Hippoc- ampal neurons, primarily cultured for 9-11 d in vitro, were used for this study. DS were dissolved in DMSO and applied to calcium imaging and whole-cell patch clamp. Results: The application of 3 mg/L DS decreased the frequency of sponta- neous and synchronous oscillations of intracellular Ca2+ to 72%±2% (mean±SEM), and the spontaneous inhibitory postsynaptic currents to 60%±3% (mean±SEM). The inhibitory concentraton 50% (IC50) for the effect of DS on calcium oscillations was 3.8 mg/L. DS also depressed the high voltage-gated Ca2+ channel and the voltage-gated Na+ channel currents at the same time point. It had no effect, however, on voltage-gated K+ and spontaneous excitatory postsynaptic currents. Conclusion: DS inhibited the spontaneous and synchronous oscillations of intra- cellular Ca2+ through the depression of influx of extracellular calcium and the initiation of action potential. By repressing the spontaneous neurotransmitter release, DS modulated the neuronal network activities.

  17. Nanoparticle targeting to neurons in a rat hippocampal slice culture model

    Directory of Open Access Journals (Sweden)

    Richard P Kraig

    2012-10-01

    Full Text Available We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots coated with PEG [poly(ethylene glycol]-appended DHLA (dihydrolipoic acid can bind AcWG(PalVKIKKP9GGH6 (Palm1 through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry, demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.

  18. Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle.

    Directory of Open Access Journals (Sweden)

    Takuya Isomura

    2015-12-01

    Full Text Available Blind source separation is the computation underlying the cocktail party effect--a partygoer can distinguish a particular talker's voice from the ambient noise. Early studies indicated that the brain might use blind source separation as a signal processing strategy for sensory perception and numerous mathematical models have been proposed; however, it remains unclear how the neural networks extract particular sources from a complex mixture of inputs. We discovered that neurons in cultures of dissociated rat cortical cells could learn to represent particular sources while filtering out other signals. Specifically, the distinct classes of neurons in the culture learned to respond to the distinct sources after repeating training stimulation. Moreover, the neural network structures changed to reduce free energy, as predicted by the free-energy principle, a candidate unified theory of learning and memory, and by Jaynes' principle of maximum entropy. This implicit learning can only be explained by some form of Hebbian plasticity. These results are the first in vitro (as opposed to in silico demonstration of neural networks performing blind source separation, and the first formal demonstration of neuronal self-organization under the free energy principle.

  19. Cell-surface marker signatures for the isolation of neural stem cells, glia and neurons derived from human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Shauna H Yuan

    Full Text Available BACKGROUND: Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC, glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS. METHODOLOGY/PRINCIPAL FINDINGS: We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+/CD271(-/CD44(-/CD24(+ from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC. Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-/CD44(-/CD15(LOW/CD24(+ and a population of glia that was CD184(+/CD44(+ were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural

  20. Preliminary Study on in Vitro Induced Differentiation of Embryonic Stem Cells into Neurons

    Institute of Scientific and Technical Information of China (English)

    JianGe; ShunongLi; 等

    2002-01-01

    Purpose;To study preliminarily in vitro induced differentiation of embryonic stem cells into neurons for further investigation of an alternative for the treatment of glaumatous neuropathy.Materials and methods:Supernatant of cultured Buffalo rat liver cells (buffalorat liver cell-conditioned medium,BRL-CM)was used for culturing embryonic stem cells(ES-D3 cell line)..Morphological features of undifferentiated ES cells were studied by HE staining and electron microscopy.Based on the methods used by Bain et al,we modified the methods and used retinoic,acid(RA) as an inducer to differentiate ES-D3 cells and cytosine arabinoside(Ara-C) as inhibitor of proliferative cells.The growth of the cells was observed under phase contrast microscope.Reuslts:ES-D3 cells cultured by BRL-CM grew in aggregates and remained undifferentiated.Electromicroscopy showed large nucleus and a large amount of mitochondria in undifferentiated ES cells and many processes on the surfaces.In the first day after the adding of retinoic acid,some neuron-like cells with one,two or more processes were present.In the second day after adding RA and the first day after the plus of 10μm Ara-C,a large amount of neuron-like cells appeared,with the formation of neuron-like networks.Conclusions:Combined use of RA and Ara-C can induce ES cells to different into neuron-like cells.Our present preliminary study might provide insights into an alternative for the treatment of glaucomatous neuropathy by the transplantation of embryonic stem cells.Eye Science 2000;16:1-6.

  1. L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture.

    Science.gov (United States)

    Wang, C; Sadovova, N; Ali, H K; Duhart, H M; Fu, X; Zou, X; Patterson, T A; Binienda, Z K; Virmani, A; Paule, M G; Slikker, W; Ali, S F

    2007-01-05

    1-Methyl-4-phenylpyridinium ion (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of L-carnitine against the toxicity of MPP+ in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 microM MPP+ alone or co-incubated with L-carnitine. MPP+ produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)-positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP+ with L-carnitine significantly reduced MPP+-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP+ decreased the ratio of BCL-X(L) to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. L-carnitine blocked these effects of MPP+ suggesting its potential therapeutic utility in degenerative disorders such as Parkinson's disease, Alzheimer's disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.

  2. Aged garlic extract and its components protect cultured rat hippocampal neurons from amyloid β—protein—in—duced neuronal death

    Institute of Scientific and Technical Information of China (English)

    ItoY; KosuY

    2002-01-01

    Aged garlic extract and its components such as S-allyl-L-cysteine (SAC) and sllixin have been shown to possess various biological effects including neurotrophic activity.We characterized the neuronal death induced by amyloid β-protein (Aβ),4-hydroxynoenal (HNE),tunicamycin(TM),and trophic factor-deprivation (TFD),and ivestigated whether these garlic compounds could prevent this in cultured PC12 cells and rat hippocampal neurons.Treatment with SAC protected these cells against Aβ- and TM-induced neuronal death.SAC also attenuated the processing of procaspase-12 induced by Aβ25-35 or TM.In contrast,allixin and its analogue,DHP,afforded no protection against Aβ-induced cell death.SAC afforded no protection against HNE- and TFD-induced cell death,which has been shown to be mediated by caspase-3 dependent pathway.These results suggest that SAC protect against the neuronal cell death that is triggered by ER dysfunction.

  3. Cortical cell and neuron density estimates in one chimpanzee hemisphere.

    Science.gov (United States)

    Collins, Christine E; Turner, Emily C; Sawyer, Eva Kille; Reed, Jamie L; Young, Nicole A; Flaherty, David K; Kaas, Jon H

    2016-01-19

    The density of cells and neurons in the neocortex of many mammals varies across cortical areas and regions. This variability is, perhaps, most pronounced in primates. Nonuniformity in the composition of cortex suggests regions of the cortex have different specializations. Specifically, regions with densely packed neurons contain smaller neurons that are activated by relatively few inputs, thereby preserving information, whereas regions that are less densely packed have larger neurons that have more integrative functions. Here we present the numbers of cells and neurons for 742 discrete locations across the neocortex in a chimpanzee. Using isotropic fractionation and flow fractionation methods for cell and neuron counts, we estimate that neocortex of one hemisphere contains 9.5 billion cells and 3.7 billion neurons. Primary visual cortex occupies 35 cm(2) of surface, 10% of the total, and contains 737 million densely packed neurons, 20% of the total neurons contained within the hemisphere. Other areas of high neuron packing include secondary visual areas, somatosensory cortex, and prefrontal granular cortex. Areas of low levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of cortex in humans and other primates.

  4. Secondary Ion Mass Spectrometry Imaging of Molecular Distributions in Cultured Neurons and Their Processes: Comparative Analysis of Sample Preparation

    Science.gov (United States)

    Tucker, Kevin R.; Li, Zhen; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2012-11-01

    Neurons often exhibit a complex chemical distribution and topography; therefore, sample preparation protocols that preserve structures ranging from relatively large cell somata to small neurites and growth cones are important factors in secondary ion mass spectrometry (SIMS) imaging studies. Here, SIMS was used to investigate the subcellular localization of lipids and lipophilic species in neurons from Aplysia californica. Using individual neurons cultured on silicon wafers, we compared and optimized several SIMS sampling approaches. After an initial step to remove the high salt culturing media, formaldehyde, paraformaldehyde, and glycerol, and various combinations thereof, were tested for their ability to achieve cell stabilization during and after the removal of extracellular media. These treatments improved the preservation of cellular morphology as visualized with SIMS imaging. For analytes >250 Da, coating the cell surface with a 3.2 nm-thick gold layer increased the ion intensity; multiple analytes previously not observed or observed at low abundance were detected, including intact cholesterol and vitamin E molecular ions. However, once a sample was coated, many of the lower molecular mass (cell stabilization with glycerol and 4 % paraformaldehyde. The sample preparation methods described here enhance SIMS imaging of processes of individual cultured neurons over a broad mass range with enhanced image contrast.

  5. Construction of a vector generating both siRNA and a fluorescent reporter: a siRNA study in cultured neurons.

    Science.gov (United States)

    Yoon, Seung Yong; Choi, Jung Eun; Hwang, Onyou; Hong, Hea Nam; Lee, Heuiran; Kim, Yoo Kyum; Cho, Sung-Woo; Kim, Hyun; Kim, DongHou

    2004-08-31

    RNA interference is an important tool for gene silencing. However, its application to primary cultured cells has been limited by low transfection efficiencies. In this work we developed a vector which encodes both siRNA and red fluorescent protein. Using this vector we could markedly suppress green fluorescent protein (GFP) and bim an endogenous gene. Primary cultured cortical neurons transfected with siRNA against doublecortin showed that doublecortin expression was significantly inhibited in nearly all the transfected neurons. This vector identifies the transfected cells and should be useful for loss-of-gene function studies in neurons.

  6. Unidirectional signal propagation in primary neurons micropatterned at a single-cell resolution

    Science.gov (United States)

    Yamamoto, H.; Matsumura, R.; Takaoki, H.; Katsurabayashi, S.; Hirano-Iwata, A.; Niwano, M.

    2016-07-01

    The structure and connectivity of cultured neuronal networks can be controlled by using micropatterned surfaces. Here, we demonstrate that the direction of signal propagation can be precisely controlled at a single-cell resolution by growing primary neurons on micropatterns. To achieve this, we first examined the process by which axons develop and how synapses form in micropatterned primary neurons using immunocytochemistry. By aligning asymmetric micropatterns with a marginal gap, it was possible to pattern primary neurons with a directed polarization axis at the single-cell level. We then examined how synapses develop on micropatterned hippocampal neurons. Three types of micropatterns with different numbers of short paths for dendrite growth were compared. A normal development in synapse density was observed when micropatterns with three or more short paths were used. Finally, we performed double patch clamp recordings on micropatterned neurons to confirm that these synapses are indeed functional, and that the neuronal signal is transmitted unidirectionally in the intended orientation. This work provides a practical guideline for patterning single neurons to design functional neuronal networks in vitro with the direction of signal propagation being controlled.

  7. Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

    Institute of Scientific and Technical Information of China (English)

    Robert H. LaMotte; Chao MA

    2008-01-01

    The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accom-panied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP- 1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.

  8. Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion.

    Science.gov (United States)

    LaMotte, Robert H; Ma, Chao

    2008-10-25

    The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accompanied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.

  9. Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Pei-xun Zhang; Xiao-rui Jiang; Lei Wang; Fang-min Chen; Lin Xu; Fei Huang

    2015-01-01

    Preliminary animal experiments have conifrmed that sensory nerve ifbers promote osteoblast differentiation, but motor nerve ifbers have no promotion effect. Whether sensory neurons pro-mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons (sensory neurons) from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green lfuorescent protein 3 weeks after osteo-genic differentiationin vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera-tion of bone marrow mesenchymal stem cell-derived osteoblasts at 3 and 5 days of co-culture, as observed by lfuorescence microscopy. The levels of mRNAs for osteogenic differentiation-re-lated factors (including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2) in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our ifndings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro-vides a theoretical basis forin vitro experiments aimed at constructing tissue-engineered bone.

  10. Encoding of High Frequencies Improves with Maturation of Action Potential Generation in Cultured Neocortical Neurons

    Science.gov (United States)

    Nikitin, Evgeny S.; Bal, Natalia V.; Malyshev, Aleksey; Ierusalimsky, Victor N.; Spivak, Yulia; Balaban, Pavel M.; Volgushev, Maxim

    2017-01-01

    The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials (APs). Experimental results support this idea, however mechanisms of fast onset of APs in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of AP generation and encoding. Here we characterize properties of AP encoding in neocortical neurons grown for 11–25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and AP generation. The onset of APs becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of APs as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of AP generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of AP generation and encoding, and their maturation. PMID

  11. Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

    Science.gov (United States)

    Yagami, Tatsurou; Ueda, Keiichi; Asakura, Kenji; Hata, Satoshi; Kuroda, Takayuki; Sakaeda, Toshiyuki; Takasu, Nobuo; Tanaka, Kazushige; Gemba, Takefumi; Hori, Yozo

    2002-01-01

    Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.

  12. Interaction of SH-SY5Y cells with nanogratings during neuronal differentiation: comparison with primary neurons.

    Science.gov (United States)

    Tonazzini, Ilaria; Cecchini, Alessandra; Elgersma, Ype; Cecchini, Marco

    2014-04-01

    Controlling neuronal cell adhesion, migration, and axonal outgrowth via contact interactions with biomaterials is a critical element for tissue engineering applications and for developing artificial neuronal interfaces. One promising approach relies on the exploitation of nanostructured surfaces. Here, the human neuroblastoma cell line SH-SY5Y is interfaced with plastic nanogratings (NGs; anisotropic topographies composed by alternating lines of grooves and ridges with sub-micrometer lateral dimension). The SH-SY5Y cells' (SHs) contact guidance is investigated under proliferating conditions and upon differentiation after treatment with retinoic acid (RA) and brain-derived neurotrophin factor (BDNF), and compared with mouse primary hippocampal neurons (HNs). Quantitative readouts are obtained by measuring changes in tubulin cytoskeleton organization and cell morphology induced by mechanotransduction. Results demonstrate that SHs effectively retrieve substrate topographical signals, in particular during differentiation. Remarkably, RA/BDNF improves SH responsiveness to NG directional cues, and significantly enhances the alignment to the NG lines. HNs behave similarly, showing a marked change in network organization if cultured on NGs. These results might help the rational engineering of neuro-regenerative scaffolds to improve peripheral nerve wound healing, as well as to investigate the basic mechanisms of neuronal wiring.

  13. Current status of neuronal cell xenotransplantation.

    Science.gov (United States)

    Vadori, Marta; Aron Badin, Romina; Hantraye, Philippe; Cozzi, Emanuele

    2015-11-01

    Neural cell transplantation has long been considered as an option for the treatment of neurodegenerative disorders. To date, several patients with Parkinson's and Huntington's diseases have been treated with human fetal-derived neurons with disparate results. However, the limited efficacy to date combined with the scarce availability of human fetal tissues and ethical concerns render this procedure inapplicable to a wide population scale. With a view to overcoming these shortcomings, transplantation of pig-derived cell precursors has been proposed and applied in preclinical and clinical trials. Recently long-term survival (more than 18 months) associated with clinical efficacy has been reported following transplantation of genetically engineered porcine neural precursors in fully immunosuppressed primate recipients. Despite the promising results obtained to date, several questions remain unanswered. In particular, the ideal xenogeneic cell-products to transplant, the extent of the immune response against the implanted xenograft and the most suitable therapeutic strategies to improve engraftment are all issues that still need to be thoroughly addressed. The present review describes the current knowledge in the pig-to-primate xenotransplantation field. In this context, recent data on human-to-nonhuman primate xenogeneic stem cell-based treatments for neurological disorders are discussed. Copyright © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

  14. Concentration-dependent effects of fullerenol on cultured hippocampal neuron viability

    Directory of Open Access Journals (Sweden)

    Zha YY

    2012-06-01

    Full Text Available Ying-ying Zha,1 Bo Yang,1 Ming-liang Tang,2 Qiu-chen Guo,1 Ju-tao Chen,1 Long-ping Wen,3 Ming Wang11CAS Key Laboratory of Brain Function and Disease, School of Life Sciences, University of Science and Technology of China, Hefei, 2Suzhou Institute of NanoTech and NanoBionics, Chinese Academy of Sciences, Suzhou, 3Laboratory of Nano-biology, School of Life Sciences, University of Science and Technology of China, Hefei, People's Republic of ChinaBackground: Recent studies have shown that the biological actions and toxicity of the water-soluble compound, polyhydroxyfullerene (fullerenol, are related to the concentrations present at a particular site of action. This study investigated the effects of different concentrations of fullerenol on cultured rat hippocampal neurons.Methods and results: Fullerenol at low concentrations significantly enhanced hippocampal neuron viability as tested by MTT assay and Hoechst 33342/propidium iodide double stain detection. At high concentrations, fullerenol induced apoptosis confirmed by Comet assay and assessment of caspase proteins.Conclusion: These findings suggest that fullerenol promotes cell death and protects against cell damage, depending on the concentration present. The concentration-dependent effects of fullerenol were mainly due to its influence on the reduction-oxidation pathway.Keywords: fullerenol, nanomaterial, neurotoxicity, neuroprotection, hippocampal neuron

  15. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

    Science.gov (United States)

    Machado, Carolina Barcellos; Kanning, Kevin C; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-02-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

  16. The presence of cortical neurons in striatal-cortical co-cultures alters the effects of dopamine and BDNF on Medium Spiny Neuron dendritic development

    Directory of Open Access Journals (Sweden)

    Rachel D Penrod

    2015-07-01

    Full Text Available Medium spiny neurons (MSNs are the major striatal neuron and receive synaptic input from both glutamatergic and dopaminergic afferents. These synapses are made on MSN dendritic spines, which undergo density and morphology changes in association with numerous disease and experience-dependent states. Despite wide interest in the structure and function of mature MSNs, relatively little is known about MSN development. Furthermore, most in vitro studies of MSN development have been done in simple striatal cultures that lack any type of non-autologous synaptic input, leaving open the question of how MSN development is affected by a complex environment that includes other types of neurons, glia, and accompanying secreted and cell-associated cues. Here we characterize the development of MSNs in striatal-cortical co-culture, including quantitative morphological analysis of dendritic arborization and spine development, describing progressive changes in density and morphology of developing spines. Overall, MSN growth is much more robust in the striatal-cortical co-culture compared to striatal mono-culture. Inclusion of dopamine in the co-culture further enhances MSN dendritic arborization and spine density, but the effects of dopamine on dendritic branching are only significant at later times in development. In contrast, exogenous Brain Derived Neurotrophic Factor (BDNF has only a minimal effect on MSN development in the co-culture, but significantly enhances MSN dendritic arborization in striatal mono-culture. Importantly, inhibition of NMDA receptors in the co-culture significantly enhances the effect of exogenous BDNF, suggesting that the efficacy of BDNF depends on the cellular environment. Combined, these studies identify specific periods of MSN development that may be particularly sensitive to perturbation by external factors and demonstrate the importance of studying MSN development in a complex signaling environment.

  17. Rhubarb extract has a protective role against radiation-induced brain injury and neuronal cell apoptosis.

    Science.gov (United States)

    Lu, Kui; Zhang, Cheng; Wu, Wenjun; Zhou, Min; Tang, Yamei; Peng, Ying

    2015-08-01

    Oxidative stress caused by ionizing radiation is involved in neuronal damage in a number of disorders, including trauma, stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Ionizing radiation can lead to the formation of free radicals, which cause neuronal apoptosis and have important roles in the development of some types of chronic brain disease. The present study evaluated the effects of varying concentrations (2, 5 and 10 µg/ml) of ethanolic rhubarb extract on the neuronal damage caused by irradiation in primary neuronal cultures obtained from the cortices of rat embryos aged 20 days. Brain damage was induced with a single dose of γ-irradiation that induced DNA fragmentation, increased lactate dehydrogenase release in neuronal cells and acted as a trigger for microglial cell proliferation. Treatment with rhubarb extract significantly decreased radiation-induced lactate dehydrogenase release and DNA fragmentation, which are important in the process of cell apoptosis. The rhubarb extract exhibited dose-dependent inhibition of lactate dehydrogenase release and neuronal cell apoptosis that were induced by the administration of ionizing radiation. The effect of a 10 µg/ml dose of rhubarb extract on the generation of reactive oxygen species (ROS) induced by radiation was also investigated. This dose led to significant inhibition of ROS generation. In conclusion, the present study showed a protective role of rhubarb extract against irradiation-induced apoptotic neuronal cell death and ROS generation.

  18. Phenolic Compounds Protect Cultured Hippocampal Neurons against Ethanol-Withdrawal Induced Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Marianna E. Jung

    2009-04-01

    Full Text Available Ethanol withdrawal is linked to elevated oxidative damage to neurons. Here we report our findings on the contribution of phenolic antioxidants (17β-estradiol, p-octyl-phenol and 2,6-di-tert-butyl-4-methylphenol to counterbalance sudden ethanol withdrawal-initiated oxidative events in hippocampus-derived cultured HT-22 cells. We showed that ethanol withdrawal for 4 h after 24-h ethanol treatment provoked greater levels of oxidative damage than the preceding ethanol exposure. Phenolic antioxidant treatment either during ethanol exposure or ethanol withdrawal only, however, dose-dependently reversed cellular oxidative damage, as demonstrated by the significantly enhanced cell viability, reduced malondialdehyde production and protein carbonylation, compared to untreated cells. Interestingly, the antioxidant treatment schedule had no significant impact on the observed neuroprotection. In addition, the efficacy of the three phenolic compounds was practically equipotent in protecting HT-22 cells in spite of predictions based on an in silico study and a cell free assay of lipid peroxidation. This finding implies that free-radical scavenging may not be the sole factor responsible for the observed neuroprotection and warrants further studies to establish, whether the HT-22 line is indeed a suitable model for in vitro screening of antioxidants against EW-related neuronal damage.

  19. Cell culture purity issues and DFAT cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Shengjuan [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States); Bergen, Werner G. [Program in Cellular and Molecular Biosciences/Department of Animal Sciences, Auburn University, Auburn, AL 36849 (United States); Hausman, Gary J. [Animal Science Department, University of Georgia, Athens, GA 30602-2771 (United States); Zan, Linsen, E-mail: zanls@yahoo.com.cn [College of Animal Science and Technology, Northwest A and F University, Yangling, Shaanxi Province 712100 (China); Dodson, Michael V., E-mail: dodson@wsu.edu [Department of Animal Sciences, Washington State University, Pullman, WA 99164 (United States)

    2013-04-12

    Highlights: •DFAT cells are progeny cells derived from dedifferentiated mature adipocytes. •Common problems in this research is potential cell contamination of initial cultures. •The initial cell culture purity is crucial in DFAT cell research field. -- Abstract: Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  20. Cooperative transcription activation by Nurr1 and Pitx3 induces embryonic stem cell maturation to the midbrain dopamine neuron phenotype

    DEFF Research Database (Denmark)

    Martinat, Cecile; Bacci, Jean-Jacques; Leete, Thomas

    2006-01-01

    Midbrain dopamine (DA) neurons play a central role in the regulation of voluntary movement, and their degeneration is associated with Parkinson's disease. Cell replacement therapies, and in particular embryonic stem (ES) cell-derived DA neurons, offer a potential therapeutic venue for Parkinson......'s disease. We sought to identify genes that can potentiate maturation of ES cell cultures to the midbrain DA neuron phenotype. A number of transcription factors have been implicated in the development of midbrain DA neurons by expression analyses and loss-of-function knockout mouse studies, including Nurr1......, Pitx3, Lmx1b, Engrailed-1, and Engrailed-2. However, none of these factors appear sufficient alone to induce the mature midbrain DA neuron phenotype in ES cell cultures in vitro, suggesting a more complex regulatory network. Here we show that Nurr1 and Pitx3 cooperatively promote terminal maturation...

  1. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    Science.gov (United States)

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  2. Arctigenin protects against neuronal hearing loss by promoting neural stem cell survival and differentiation.

    Science.gov (United States)

    Huang, Xinghua; Chen, Mo; Ding, Yan; Wang, Qin

    2016-12-27

    Neuronal hearing loss has become a prevalent health problem. This study focused on the function of arctigenin (ARC) in promoting survival and neuronal differentiation of mouse cochlear neural stem cells (NSCs), and its protection against gentamicin (GMC) induced neuronal hearing loss. Mouse cochlea was used to isolate NSCs, which were subsequently cultured in vitro. The effects of ARC on NSC survival, neurosphere formation, differentiation of NSCs, neurite outgrowth, and neural excitability in neuronal network in vitro were examined. Mechanotransduction ability demonstrated by intact cochlea, auditory brainstem response (ABR), and distortion product optoacoustic emissions (DPOAE) amplitude in mice were measured to evaluate effects of ARC on GMC-induced neuronal hearing loss. ARC increased survival, neurosphere formation, neuron differentiation of NSCs in mouse cochlear in vitro. ARC also promoted the outgrowth of neurites, as well as neural excitability of the NSC-differentiated neuron culture. Additionally, ARC rescued mechanotransduction capacity, restored the threshold shifts of ABR and DPOAE in our GMC ototoxicity murine model. This study supports the potential therapeutic role of ARC in promoting both NSCs proliferation and differentiation in vitro to functional neurons, thus supporting its protective function in the therapeutic treatment of neuropathic hearing loss in vivo.

  3. Histamine is required during neural stem cell proliferation to increase neuron differentiation.

    Science.gov (United States)

    Rodríguez-Martínez, G; Velasco, I; García-López, G; Solís, K H; Flores-Herrera, H; Díaz, N F; Molina-Hernández, A

    2012-08-02

    Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 μM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

  4. Creation of defined single cell resolution neuronal circuits on microelectrode arrays

    Science.gov (United States)

    Pirlo, Russell Kirk

    2009-12-01

    The way cell-cell organization of neuronal networks influences activity and facilitates function is not well understood. Microelectrode arrays (MEAs) and advancing cell patterning technologies have enabled access to and control of in vitro neuronal networks spawning much new research in neuroscience and neuroengineering. We propose that small, simple networks of neurons with defined circuitry may serve as valuable research models where every connection can be analyzed, controlled and manipulated. Towards the goal of creating such neuronal networks we have applied microfabricated elastomeric membranes, surface modification and our unique laser cell patterning system to create defined neuronal circuits with single-cell precision on MEAs. Definition of synaptic connectivity was imposed by the 3D physical constraints of polydimethylsiloxane elastomeric membranes. The membranes had 20mum clear-through holes and 2-3mum deep channels which when applied to the surface of the MEA formed microwells to confine neurons to electrodes connected via shallow tunnels to direct neurite outgrowth. Tapering and turning of channels was used to influence neurite polarity. Biocompatibility of the membranes was increased by vacuum baking, oligomer extraction, and autoclaving. Membranes were bound to the MEA by oxygen plasma treatment and heated pressure. The MEA/membrane surface was treated with oxygen plasma, poly-D-lysine and laminin to improve neuron attachment, survival and neurite outgrowth. Prior to cell patterning the outer edge of culture area was seeded with 5x10 5 cells per cm and incubated for 2 days. Single embryonic day 7 chick forebrain neurons were then patterned into the microwells and onto the electrodes using our laser cell patterning system. Patterned neurons successfully attached to and were confined to the electrodes. Neurites extended through the interconnecting channels and connected with adjacent neurons. These results demonstrate that neuronal circuits can be

  5. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons.

    Science.gov (United States)

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A

    2014-10-17

    During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia-NCAMs) modulate cell-cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia-NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb's to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell-cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders. Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.

  6. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture.

    Science.gov (United States)

    López, E; Arce, C; Oset-Gasque, M J; Cañadas, S; González, M P

    2006-03-15

    Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.

  7. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

    Science.gov (United States)

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

    2014-03-01

    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  8. Fibroblast growth factor-20 increases the yield of midbrain dopaminergic neurons derived from human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Ana Sofia Correia

    2007-12-01

    Full Text Available In the central nervous system, fibroblast growth factor (FGF-20 has been reported to act preferentially on midbrain dopaminergic neurons. It also promotes the dopaminergic differentiation of stem cells. We have analyzed the effects of FGF-20 on human embryonic stem cells (hESCs differentiation into dopaminergic neurons. We induced neuronal differentiation of hESCs by co-culturing those with PA6 mouse stromal cells for 3 weeks. When we supplemented the culture medium with FGF-20, the number of tyrosine hydroxylase (TH- expressing neurons increased fivefold, from 3% to 15% of the hESC-derived cells. The cultured cells also expressed other midbrain dopaminergic markers (PITX3, En1, Msx1, and Aldh1, suggesting that some had differentiated into midbrain dopaminergic neurons. We observed no effect of FGF-20 on the size of the soma area or neurite length of the TH-immunopositive neurons. Regardless of whether FGF-20 had been added or not, 17% of the hESC-derived cells expressed the pan-neuronal marker b-III-Tubulin. The proportion of proliferating cells positive for Ki-67 was also not affected by FGF-20 (7% of the hESC-derived cells. By contrast, after 3 weeks in culture FGF-20 significantly reduced the proportion of cells undergoing cell death, as revealed by immunoreactivity for cleaved caspase-8, Bcl-2 associated X protein (BAX and cleaved caspase-3 (2.5% to 1.2% of cleaved caspase-3-positive cells out of the hESC-derived cells. Taken together, our results indicate that FGF-20 specifically increases the yield of dopaminergic neurons from hESCs grown on PA6 feeder cells and at least part of this effect is due to a reduction in cell death.

  9. Acute and chronic effects of clozapine on cholinergic transmission in cultured mouse superior cervical ganglion neurons.

    Science.gov (United States)

    Saur, Taixiang; Cohen, Bruce M; Ma, Qi; Babb, Suzann M; Buttner, Edgar A; Yao, Wei-Dong

    Cholinergic dysfunction contributes to cognitive deficits in schizophrenia. The atypical antipsychotic clozapine improves cognition in patients with schizophrenia, possibly through modulation of the cholinergic system. However, little is known about specific underlying mechanisms. We investigated the acute and chronic effects of clozapine on cholinergic synaptic transmission in cultured superior cervical ganglion (SCG) neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) were detected and were reversibly inhibited by the nicotinic receptor antagonist d-tubocurarine, confirming that the synaptic responses were primarily mediated by nicotinic receptors. Bath application of clozapine at therapeutic concentrations rapidly and reversely inhibited both the amplitude and frequency of sEPSCs in a concentration-dependent manner, without changing either rise or decay time, suggesting that clozapine effects have both presynaptic and postsynaptic origins. The acute effects of clozapine on sEPSCs were recapitulated by chronic treatment of SCG cultures with similar concentrations of clozapine, as clozapine treatment for 4 d reduced the frequency and amplitude of sEPSCs without affecting their kinetics. Cell survival analysis indicated that SCG neuron cell counts after chronic clozapine treatment were comparable to the control group. These results demonstrate that therapeutic concentrations of clozapine suppress nicotinic synaptic transmission in SCG cholinergic synapses, a simple in vitro preparation of cholinergic transmission.

  10. Reduced Hyperpolarization-Activated Current Contributes to Enhanced Intrinsic Excitability in Cultured Hippocampal Neurons from PrP(-/-) Mice.

    Science.gov (United States)

    Fan, Jing; Stemkowski, Patrick L; Gandini, Maria A; Black, Stefanie A; Zhang, Zizhen; Souza, Ivana A; Chen, Lina; Zamponi, Gerald W

    2016-01-01

    Genetic ablation of cellular prion protein (PrP(C)) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrP(C) profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrP(C). The amplitude of voltage sag, a characteristic of activating HCN channel current (I h), was decreased in null mice. Moreover, I h peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrP(C). These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability.

  11. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    Energy Technology Data Exchange (ETDEWEB)

    Park, Kyoung Ho [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Yeo, Sang Won, E-mail: swyeo@catholic.ac.kr [Department of Otolaryngology Head and Neck Surgery, College of Medicine, Catholic University, Seoul (Korea, Republic of); Troy, Frederic A., E-mail: fatroy@ucdavis.edu [Department of Biochemistry and Molecular Medicine, University of California, School of Medicine, Davis, CA 95616 (United States); Xiamen University, School of Medicine, Xiamen City (China)

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  12. 5-HT7 Receptors Are Not Involved in Neuropeptide Release in Primary Cultured Rat Trigeminal Ganglion Neurons.

    Science.gov (United States)

    Wang, Xiaojuan; Hu, Rong; Liang, Jianbo; Li, Ze; Sun, Weiwen; Pan, Xiaoping

    2016-06-01

    Migraine is a common but complex neurological disorder. Its precise mechanisms are not fully understood. Increasing indirect evidence indicates that 5-HT7 receptors may be involved; however, their role remains unknown. Our previous in vivo study showed that selective blockade of 5-HT7 receptors caused decreased serum levels of calcitonin gene-related peptide (CGRP) in the external jugular vein following electrical stimulation of the trigeminal ganglion (TG) in an animal model of migraine. In the present study, we used an in vitro model of cultured TG cells to further investigate whether 5-HT7 receptors are directly responsible for the release of CGRP and substance P from TG neurons. We stimulated rat primary cultured TG neurons with capsaicin or potassium chloride (KCl) to mimic neurogenic inflammation, resulting in release of CGRP and substance P. 5-HT7 receptors were abundantly expressed in TG neurons. Greater than 93 % of 5-HT7 receptor-positive neurons co-expressed CGRP and 56 % co-expressed substance P. Both the capsaicin- and KCl-induced release of CGRP and substance P were unaffected by pretreatment of cultured TG cells with the selective 5-HT7 receptor agonist AS19 and antagonist SB269970. This study demonstrates for the first time that 5-HT7 receptors are abundantly co-expressed with CGRP and substance P in rat primary TG neurons and suggests that they are not responsible for the release of CGRP and substance P from cultured TG neurons evoked by capsaicin or KCl.

  13. Characterization of energy and neurotransmitter metabolism in cortical glutamatergic neurons derived from human induced pluripotent stem cells: A novel approach to study metabolism in human neurons.

    Science.gov (United States)

    Aldana, Blanca I; Zhang, Yu; Lihme, Maria Fog; Bak, Lasse K; Nielsen, Jørgen E; Holst, Bjørn; Hyttel, Poul; Freude, Kristine K; Waagepetersen, Helle S

    2017-02-24

    Alterations in the cellular metabolic machinery of the brain are associated with neurodegenerative disorders such as Alzheimer's disease. Novel human cellular disease models are essential in order to study underlying disease mechanisms. In the present study, we characterized major metabolic pathways in neurons derived from human induced pluripotent stem cells (hiPSC). With this aim, cultures of hiPSC-derived neurons were incubated with [U-(13)C]glucose, [U-(13)C]glutamate or [U-(13)C]glutamine. Isotopic labeling in metabolites was determined using gas chromatography coupled to mass spectrometry, and cellular amino acid content was quantified by high-performance liquid chromatography. Additionally, we evaluated mitochondrial function using real-time assessment of oxygen consumption via the Seahorse XF(e)96 Analyzer. Moreover, in order to validate the hiPSC-derived neurons as a model system, a metabolic profiling was performed in parallel in primary neuronal cultures of mouse cerebral cortex and cerebellum. These serve as well-established models of GABAergic and glutamatergic neurons, respectively. The hiPSC-derived neurons were previously characterized as being forebrain-specific cortical glutamatergic neurons. However, a comparable preparation of predominantly mouse cortical glutamatergic neurons is not available. We found a higher glycolytic capacity in hiPSC-derived neurons compared to mouse neurons and a substantial oxidative metabolism through the mitochondrial tricarboxylic acid (TCA) cycle. This finding is supported by the extracellular acidification and oxygen consumption rates measured in the cultured human neurons. [U-(13)C]Glutamate and [U-(13)C]glutamine were found to be efficient energy substrates for the neuronal cultures originating from both mice and humans. Interestingly, isotopic labeling in metabolites from [U-(13)C]glutamate was higher than that from [U-(13)C]glutamine. Although the metabolic profile of hiPSC-derived neurons in vitro was

  14. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  15. DIRECT VISUALIZATION OF THE DOPAMINE TRANSPORTER IN CULTURED NEWBORN RAT MIDBRAIN NEURONS USING THE FLUORESCENT COCAINE ANALOGUE JHC 1-64

    DEFF Research Database (Denmark)

    Rasmussen, Søren; Vægter, Christian Bjerggaard; Cha, J

    In this study we have established methods for visualization and tracking of the dopamine transporter (DAT) in cultured dopaminergic neurons in real time using a fluorescent cocaine analogue JHC 1-64 and confocal fluorescence microscopy. The initial binding experiments in HEK 293 cells stably......-DAT was internalized, corroborating the usefulness of this cocaine analogue as a tool for monitoring DAT trafficking. In the cultured neurons JHC 1-64 labeled the surface of almost the entire dopaminergic neurons including the cell body, although not as strongly as some of the neuronal extensions. This labeling by JHC...... 1-64 was prevented by excess concentrations of dopamine, cocaine, mazindol, or RTI-55, whereas the norepinephrine and/or serotonin transporter specific inhibitors desmethylimipramine and citalopram did not affect fluorescent labeling of the neurons. This strongly supports that JHC 1-64 specifically...

  16. Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

    OpenAIRE

    Saijilafu,; Zhou, Feng-Quan

    2012-01-01

    It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which ...

  17. Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems.

    Science.gov (United States)

    Woehrling, E K; Hill, E J; Coleman, M D

    2010-02-01

    Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

  18. Cell culture purity issues and DFAT cells.

    Science.gov (United States)

    Wei, Shengjuan; Bergen, Werner G; Hausman, Gary J; Zan, Linsen; Dodson, Michael V

    2013-04-12

    Dedifferentiation of mature adipocytes, in vitro, has been pursued/documented for over forty years. The subsequent progeny cells are named dedifferentiated adipocyte-derived progeny cells (DFAT cells). DFAT cells are proliferative and likely to possess mutilineage potential. As a consequence, DFAT cells and their progeny/daughter cells may be useful as a potential tool for various aspects of tissue engineering and as potential vectors for the alleviation of several disease states. Publications in this area have been increasing annually, but the purity of the initial culture of mature adipocytes has seldom been documented. Consequently, it is not always clear whether DFAT cells are derived from dedifferentiated mature (lipid filled) adipocytes or from contaminating cells that reside in an impure culture.

  19. Coculture of Primary Motor Neurons and Schwann Cells as a Model for In Vitro Myelination.

    Science.gov (United States)

    Hyung, Sujin; Yoon Lee, Bo; Park, Jong-Chul; Kim, Jinseok; Hur, Eun-Mi; Francis Suh, Jun-Kyo

    2015-10-12

    A culture system that can recapitulate myelination in vitro will not only help us better understand the mechanism of myelination and demyelination, but also find out possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC coculture system, MNs survived over 3 weeks and extended long axons. Both viability and axon growth of MNs in the coculture were markedly enhanced as compared to those of MN monoculture. Co-labeling of myelin basic proteins (MBPs) and neuronal microtubules revealed that SC formed myelin sheaths by wrapping around the axons of MNs. Furthermore, using the coculture system we found that treatment of an antioxidant substance coenzyme Q10 (Co-Q10) markedly facilitated myelination.

  20. Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Ward Manus W

    2007-02-01

    Full Text Available Abstract Background Bcl-2 homology domain (BH 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid, which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM. Results Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1–58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures. Conclusion Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

  1. Peptidergic neurons of the crab, Cardisoma carnifex, in defined culture maintain characteristic morphologies under a variety of conditions.

    Science.gov (United States)

    Grau, S M; Cooke, I M

    1992-11-01

    Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, "superlarge," found occasionally, has a soma diameter greater than 40 microns and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with L-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26 degrees C instead of 22 degrees C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K+]o medium ([K+]o = 15-110 mM; standard = 11 mM) has no long-term effect, but growth is arrested by [K+]o greater than 30 mM. Cultures were also grown in media in which [Ca2+]o ranged from 0.1 microM to 26 mM (standard = 13 mM). Outgrowth occurred from all neuronal types in all [Ca2+]o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.

  2. Lycopene Prevents Amyloid [Beta]-Induced Mitochondrial Oxidative Stress and Dysfunctions in Cultured Rat Cortical Neurons.

    Science.gov (United States)

    Qu, Mingyue; Jiang, Zheng; Liao, Yuanxiang; Song, Zhenyao; Nan, Xinzhong

    2016-06-01

    Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between β-amyloid (Aβ) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Aβ and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat cortical neurons exposed to Aβ. It was found that lycopene attenuated Aβ-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Aβ-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Aβ-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in mitochondria. Those results indicate that lycopene protects mitochondria against Aβ-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Aβ-induced neurotoxicity.

  3. Network dynamics of cultured hippocampal neurons in a multi-electrode array

    Science.gov (United States)

    Taguchi, Takahisa; Kudoh, Suguru N.

    2005-02-01

    The neurons in dissociation culture autonomously re-organized their functional neuronal networks, after the process for elongating neurites and establishing synaptic connections. The spatio-temporal patterns of activity in the networks might be a reflection of functional neuron assemblies. The functional connections were dynamically modified by synaptic potentiation and the process may be required for reorganization of the functional group of neurons. Such neuron assemblies are critical for information processing in brain. To visualize the functional connections between neurons, we have analyzed the autonomous activity of synaptically induced action potentials in the living neuronal networks on a multi-electrode array, using "connection map analysis" that we developed for this purpose. Moreover, we designed aan original wide area covering electrode array and succeeded in recording spontaneous action potentials from wider area than commercial multi electrode arrays.

  4. Simulation of Code Spectrum and Code Flow of Cultured Neuronal Networks.

    Science.gov (United States)

    Tamura, Shinichi; Nishitani, Yoshi; Hosokawa, Chie; Miyoshi, Tomomitsu; Sawai, Hajime

    2016-01-01

    It has been shown that, in cultured neuronal networks on a multielectrode, pseudorandom-like sequences (codes) are detected, and they flow with some spatial decay constant. Each cultured neuronal network is characterized by a specific spectrum curve. That is, we may consider the spectrum curve as a "signature" of its associated neuronal network that is dependent on the characteristics of neurons and network configuration, including the weight distribution. In the present study, we used an integrate-and-fire model of neurons with intrinsic and instantaneous fluctuations of characteristics for performing a simulation of a code spectrum from multielectrodes on a 2D mesh neural network. We showed that it is possible to estimate the characteristics of neurons such as the distribution of number of neurons around each electrode and their refractory periods. Although this process is a reverse problem and theoretically the solutions are not sufficiently guaranteed, the parameters seem to be consistent with those of neurons. That is, the proposed neural network model may adequately reflect the behavior of a cultured neuronal network. Furthermore, such prospect is discussed that code analysis will provide a base of communication within a neural network that will also create a base of natural intelligence.

  5. Human Neuron Cultures: Micropatterning Facilitates the Long-Term Growth and Analysis of iPSC-Derived Individual Human Neurons and Neuronal Networks (Adv. Healthcare Mater. 15/2016).

    Science.gov (United States)

    Burbulla, Lena F; Beaumont, Kristin G; Mrksich, Milan; Krainc, Dimitri

    2016-08-01

    Dimitri Krainc, Milan Mrksich, and co-workers demonstrate the utility of microcontact printing technology for culturing of human neurons in defined patterns over extended periods of time on page 1894. This approach facilitates studies of neuronal development, cellular trafficking, and related mechanisms that require assessment of individual neurons and neuronal networks.

  6. Hallmarks of Alzheimer's Disease in Stem-Cell-Derived Human Neurons Transplanted into Mouse Brain.

    Science.gov (United States)

    Espuny-Camacho, Ira; Arranz, Amaia M; Fiers, Mark; Snellinx, An; Ando, Kunie; Munck, Sebastian; Bonnefont, Jerome; Lambot, Laurie; Corthout, Nikky; Omodho, Lorna; Vanden Eynden, Elke; Radaelli, Enrico; Tesseur, Ina; Wray, Selina; Ebneth, Andreas; Hardy, John; Leroy, Karelle; Brion, Jean-Pierre; Vanderhaeghen, Pierre; De Strooper, Bart

    2017-03-08

    Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Transcriptional Elongation Regulator 1 Affects Transcription and Splicing of Genes Associated with Cellular Morphology and Cytoskeleton Dynamics and Is Required for Neurite Outgrowth in Neuroblastoma Cells and Primary Neuronal Cultures

    DEFF Research Database (Denmark)

    Muñoz-Cobo, Juan Pablo; Sánchez-Hernández, Noemí; Gutiérrez-Enríquez, Sara

    2017-01-01

    ) pathogenesis. At present, the molecular mechanism underlying TCERG1-mediated neuronal effects is unknown. Here, we show that TCERG1 depletion led to widespread alterations in mRNA processing that affected different types of alternative transcriptional or splicing events, indicating that TCERG1 plays a broad......TCERG1 is a highly conserved human protein implicated in interactions with the transcriptional and splicing machinery that is associated with neurodegenerative disorders. Biochemical, neuropathological, and genetic evidence suggests an important role for TCERG1 in Huntington’s disease (HD...... role in the regulation of alternative splicing. We observed considerable changes in the transcription and alternative splicing patterns of genes involved in cytoskeleton dynamics and neurite outgrowth. Accordingly, TCERG1 depletion in the neuroblastoma SH-SY5Y cell line and primary mouse neurons...

  8. Effects of combined BDNF and GDNF treatment on cultured dopaminergic midbrain neurons

    DEFF Research Database (Denmark)

    Sautter, J; Meyer, Morten; Spenger, C

    1998-01-01

    Neural transplantation is an experimental therapy for Parkinson's disease. Pretreatment of fetal donor tissue with neurotrophic factors may improve survival of grafted dopaminergic neurons. Free-floating roller tube cultures of fetal rat ventral mesencephalon were treated with brain...

  9. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    Science.gov (United States)

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  10. Gene expression profile of neuronal progenitor cells derived from hESCs: activation of chromosome 11p15.5 and comparison to human dopaminergic neurons.

    Directory of Open Access Journals (Sweden)

    William J Freed

    Full Text Available BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS. Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

  11. Caspase-Mediated Apoptosis in Sensory Neurons of Cultured Dorsal Root Ganglia in Adult Mouse

    Directory of Open Access Journals (Sweden)

    Hamid Reza Momeni

    2013-01-01

    Full Text Available Objective: Sensory neurons in dorsal root ganglia (DRG undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG.Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342 and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM and immunohistochemistry for activated caspase-3 were used.Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours.Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

  12. Huperzine A provides neuroprotection against several cell death inducers using in vitro model systems of motor neuron cell death.

    Science.gov (United States)

    Hemendinger, Richelle A; Armstrong, Edward J; Persinski, Rafal; Todd, Julianne; Mougeot, Jean-Luc; Volvovitz, Franklin; Rosenfeld, Jeffrey

    2008-01-01

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting from the progressive loss of motor neurons in the spinal cord and brain. To date, clinically effective neuroprotective agents have not been available. The current study demonstrates for the first time that huperzine A, a potential neuroprotective agent, has the ability to protect a motor neuron-like cell line and motor neurons in spinal cord organotypic cultures from toxin-induced cell death. The neuroblastoma-spinal motor neuron fusion cell line, NSC34 and rat spinal cord organotypic cultures (OTC) were exposed to cell death inducers for 24 h or 14 d, respectively, with and without pre-treatment with huperzine A. The inducers used here include: staurosporine, thapsigargin, hydrogen peroxide (H2O2), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and L-(-)-threo-3-hydroxyaspartic acid (THA). These agents were selected as they induce apoptosis/necrosis via mechanisms implicated in patients with generalized motor neuron disease. Cell death was determined in NSC34 cells by metabolic activity, caspase activity/expression and by nuclear morphology and in the OTCs, using immunohistochemistry and Western blot analysis. Nuclear staining of NSC34 cells revealed cell death induced by staurosporine, thapsigargin, H2O2 and CCCP. This induction was significantly reduced with 2 h pre-treatment with 10 microM huperzine A (maximum, 35% rescue; p 0.05) following exposure to staurosporine, thapsigargin and H2O2 but not with CCCP. These data were supported by the metabolic assays and caspase activity. In addition, pre-treatment with huperzine A dramatically improved motor neuron survival, based on choline acetyltransferase (ChAT) expression analysis in OTCs following exposure to THA, and compared to THA-treated control cultures. These studies are currently being extended to include other inducers and with additional compounds as potential drug therapies that could be used in combination for the treatment of

  13. miR-124 promotes the neuronal differentiation of mouse inner ear neural stem cells

    Science.gov (United States)

    Jiang, Di; Du, Jintao; Zhang, Xuemei; Zhou, Wei; Zong, Lin; Dong, Chang; Chen, Kaitian; Chen, Yu; Chen, Xihui; Jiang, Hongyan

    2016-01-01

    MicroRNAs (miRNAs or miRs) act as key regulators in neuronal development, synaptic morphogenesis and plasticity. However, their role in the neuronal differentiation of inner ear neural stem cells (NSCs) remains unclear. In this study, 6 miRNAs were selected and their expression patterns during the neuronal differentiation of inner ear NSCs were examined by RT-qPCR. We demonstrated that the culture of spiral ganglion stem cells present in the inner ears of newborn mice gave rise to neurons in vitro. The expression patterns of miR-124, miR-132, miR-134, miR-20a, miR-17-5p and miR-30a-5p were examined during a 14-day neuronal differentiation period. We found that miR-124 promoted the neuronal differentiation of and neurite outgrowth in mouse inner ear NSCs, and that the changes in the expression of tropomyosin receptor kinase B (TrkB) and cell division control protein 42 homolog (Cdc42) during inner ear NSC differentiation were associated with miR-124 expression. Our findings indicate that miR-124 plays a role in the neuronal differentiation of inner ear NSCs. This finding may lead to the development of novel strategies for restoring hearing in neurodegenerative diseases.

  14. Effects of cerebrolysin on motor-neuron-like NSC-34 cells

    Energy Technology Data Exchange (ETDEWEB)

    Keilhoff, Gerburg, E-mail: Gerburg.keilhoff@med.ovgu.de [Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg (Germany); Lucas, Benjamin; Pinkernelle, Josephine; Steiner, Michael [Institute of Biochemistry and Cell Biology, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg (Germany); Fansa, Hisham [Department of Plastic, Reconstructive and Aesthetic Surgery, Hand Surgery, Klinikum Bielefeld, Teutoburger Str. 50, D-33604 Bielefeld (Germany)

    2014-10-01

    Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL) – a proteolytic peptide fraction – were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries. - Highlights: • Cerebrolysin (CL) is anti-proliferative but initially neuroprotective in OGD-stressed NSC-34 cells. • CL amplified neurite reconstruction of NSC-34 cells. • CL affected calpain-1 expression and calpain-mediated spectrin cleavage as function of Src expression. • In organotypic spinal cord cultures, CL hampered motor neuron survival and

  15. Efficient differentiation of mouse embryonic stem cells into motor neurons.

    Science.gov (United States)

    Wu, C