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Sample records for cultured hypothalamic neurons

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

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    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 fatty acid (100, 250, or 500 microM for neurons, whereas they did in control muscle and endothelial cell lines. Despite the lack of evidence of inflammatory signaling, saturated fatty acid exposure in cultured hypothalamic neurons causes endoplasmic reticulum stress, induces mitogen-activated protein kinase, and causes apoptotic cell death with prolonged exposure. We conclude that saturated fatty acid exposure does not induce inflammatory signaling or insulin resistance in cultured hypothalamic neurons. Therefore, hypothalamic neuronal inflammation in the setting of DIO may involve an indirect mechanism mediated by saturated fatty acids on nonneuronal cells.

  2. Electrophysiology of Hypothalamic Magnocellular Neurons In vitro: A Rhythmic Drive in Organotypic Cultures and Acute Slices.

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    Israel, Jean-Marc; Oliet, Stéphane H; Ciofi, Philippe

    2016-01-01

    Hypothalamic neurohormones are released in a pulsatile manner. The mechanisms of this pulsatility remain poorly understood and several hypotheses are available, depending upon the neuroendocrine system considered. Among these systems, hypothalamo-neurohypophyseal magnocellular neurons have been early-considered models, as they typically display an electrical activity consisting of bursts of action potentials that is optimal for the release of boluses of the neurohormones oxytocin and vasopressin. The cellular mechanisms underlying this bursting behavior have been studied in vitro, using either acute slices of the adult hypothalamus, or organotypic cultures of neonatal hypothalamic tissue. We have recently proposed, from experiments in organotypic cultures, that specific central pattern generator networks, upstream of magnocellular neurons, determine their bursting activity. Here, we have tested whether a similar hypothesis can be derived from in vitro experiments in acute slices of the adult hypothalamus. To this aim we have screened our electrophysiological recordings of the magnocellular neurons, previously obtained from acute slices, with an analysis of autocorrelation of action potentials to detect a rhythmic drive as we recently did for organotypic cultures. This confirmed that the bursting behavior of magnocellular neurons is governed by central pattern generator networks whose rhythmic drive, and thus probably integrity, is however less satisfactorily preserved in the acute slices from adult brains.

  3. Electrophysiology of hypothalamic magnocellular neurons in vitro: a rhythmic drive in organotypic cultures and acute slices

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    Jean-Marc eIsrael

    2016-03-01

    Full Text Available Hypothalamic neurohormones are released in a pulsatile manner. The mechanisms of this pulsatility remain poorly understood and several hypotheses are available, depending upon the neuroendocrine system considered. Among these systems, hypothalamo-neurohypophyseal magnocellular neurons have been early-considered models, as they typically display an electrical activity consisting of bursts of action potentials that is optimal for the release of boluses of the neurohormones oxytocin and vasopressin. The cellular mechanisms underlying this bursting behavior have been studied in vitro, using either acute slices of the adult hypothalamus, or organotypic cultures of neonatal hypothalamic tissue. We have recently proposed, from experiments in organotypic cultures, that specific central pattern generator networks, upstream of magnocellular neurons, determine their bursting activity. Here, we have tested whether a similar hypothesis can be derived from in vitro experiments in acute slices of the adult hypothalamus. To this aim we have screened our electrophysiological recordings of the magnocellular neurons, previously obtained from acute slices, with an analysis of autocorrelation of action potentials to detect a rhythmic drive as we recently did for organotypic cultures. This confirmed that the bursting behavior of magnocellular neurons is governed by central pattern generator networks whose rhythmic drive, and thus probably integrity, is however less satisfactorily preserved in the acute slices from adult brains.

  4. Evolution of triiodothyronine nuclear binding sites in hypothalamic serum-free cultures: evidence for their presence in neurons and astrocytes

    International Nuclear Information System (INIS)

    Puymirat, J.; Faivre-Bauman, A.

    1986-01-01

    ( 125 I)Triiodothyronine (T 3 ) nuclear binding was studied in hypothalamic cultures from fetal mouse grown in serum-free medium. In enriched neuronal cultures, the apparent dissociation constant of the binding does not change with time in vitro (7 x 10 -11 M), but the maximum binding capacity (MBC) doubles between day 7 and day 14 in vitro. We show here for the first time that homologous astrocyte cell cultures, devoid of neurons as checked by tetanus toxin binding, also display T 3 nuclear binding, with the same affinity as neuronal cultures. However, their MBC is 3 times lower than that of neurons after a week in vitro, and increases more quickly thereafter (Author)

  5. Effect of acute ethanol on beta-endorphin secretion from rat fetal hypothalamic neurons in primary cultures

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    Sarkar, D.K.; Minami, S. (Washington State Univ., Pullman (USA))

    1990-01-01

    To characterize the effect of ethanol on the hypothalamic {beta}-endorphin-containing neurons, rat fetal hypothalamic neurons were maintained in primary culture, and the secretion of {beta}-endorphin ({beta}-EP) was determined after ethanol challenges. Constant exposure to ethanol at doses of 6-50 mM produced a dose-dependent increase in basal secretion of {beta}-EP from these cultured cells. These doses of ethanol did not produce any significant effect on cell viability, DNA or protein content. The stimulated secretion of {beta}-EP following constant ethanol exposure is short-lasting. However, intermittent ethanol exposures maintained the ethanol stimulatory action on {beta}-EP secretion for a longer time. The magnitude of the {beta}-EP response to 50 mM ethanol is similar to that of the {beta}-EP response to 56 mM of potassium. Ethanol-stimulated {beta}-EP secretion required extracellular calcium and was blocked by a calcium channel blocker; a sodium channel blocker did not affect ethanol-stimulated secretion. These results suggest that the neuron culture system is a useful model for studying the cellular mechanisms involved in the ethanol-regulated hypothalamic opioid secretion.

  6. Mechanosensing in hypothalamic osmosensory neurons.

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    Prager-Khoutorsky, Masha

    2017-11-01

    Osmosensory neurons are specialized cells activated by increases in blood osmolality to trigger thirst, secretion of the antidiuretic hormone vasopressin, and elevated sympathetic tone during dehydration. In addition to multiple extrinsic factors modulating their activity, osmosensory neurons are intrinsically osmosensitive, as they are activated by increased osmolality in the absence of neighboring cells or synaptic contacts. This intrinsic osmosensitivity is a mechanical process associated with osmolality-induced changes in cell volume. This review summarises recent findings revealing molecular mechanisms underlying the mechanical activation of osmosensory neurons and highlighting important roles of microtubules, actin, and mechanosensitive ion channels in this process. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Hypothalamic neurones governing glucose homeostasis.

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    Coppari, R

    2015-06-01

    The notion that the brain directly controls the level of glucose in the blood (glycaemia) independent of its known action on food intake and body weight has been known ever since 1849. That year, the French physiologist Dr Claude Bernard reported that physical puncture of the floor of the fourth cerebral ventricle rapidly leads to an increased level of sugar in the blood (and urine) in rabbits. Despite this important discovery, it took approximately 150 years before significant efforts aimed at understanding the underlying mechanism of brain-mediated control of glucose metabolism were made. Technological developments allowing for genetically-mediated manipulation of selected molecular pathways in a neurone-type-specific fashion unravelled the importance of specific molecules in specific neuronal populations. These neuronal pathways govern glucose metabolism in the presence and even in the absence of insulin. Also, a peculiarity of these pathways is that certain biochemically-defined neurones govern glucose metabolism in a tissue-specific fashion. © 2015 British Society for Neuroendocrinology.

  8. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells

    OpenAIRE

    Merkle, Florian T.; Maroof, Asif; Wataya, Takafumi; Sasai, Yoshiki; Studer, Lorenz; Eggan, Kevin; Schier, Alexander F.

    2015-01-01

    Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin...

  9. Rapid sensing of l-leucine by human and murine hypothalamic neurons: Neurochemical and mechanistic insights.

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    Heeley, Nicholas; Kirwan, Peter; Darwish, Tamana; Arnaud, Marion; Evans, Mark L; Merkle, Florian T; Reimann, Frank; Gribble, Fiona M; Blouet, Clemence

    2018-04-01

    Dietary proteins are sensed by hypothalamic neurons and strongly influence multiple aspects of metabolic health, including appetite, weight gain, and adiposity. However, little is known about the mechanisms by which hypothalamic neural circuits controlling behavior and metabolism sense protein availability. The aim of this study is to characterize how neurons from the mediobasal hypothalamus respond to a signal of protein availability: the amino acid l-leucine. We used primary cultures of post-weaning murine mediobasal hypothalamic neurons, hypothalamic neurons derived from human induced pluripotent stem cells, and calcium imaging to characterize rapid neuronal responses to physiological changes in extracellular l-Leucine concentration. A neurochemically diverse subset of both mouse and human hypothalamic neurons responded rapidly to l-leucine. Consistent with l-leucine's anorexigenic role, we found that 25% of mouse MBH POMC neurons were activated by l-leucine. 10% of MBH NPY neurons were inhibited by l-leucine, and leucine rapidly reduced AGRP secretion, providing a mechanism for the rapid leucine-induced inhibition of foraging behavior in rodents. Surprisingly, none of the candidate mechanisms previously implicated in hypothalamic leucine sensing (K ATP channels, mTORC1 signaling, amino-acid decarboxylation) were involved in the acute activity changes produced by l-leucine. Instead, our data indicate that leucine-induced neuronal activation involves a plasma membrane Ca 2+ channel, whereas leucine-induced neuronal inhibition is mediated by inhibition of a store-operated Ca 2+ current. A subset of neurons in the mediobasal hypothalamus rapidly respond to physiological changes in extracellular leucine concentration. Leucine can produce both increases and decreases in neuronal Ca 2+ concentrations in a neurochemically-diverse group of neurons, including some POMC and NPY/AGRP neurons. Our data reveal that leucine can signal through novel mechanisms to rapidly

  10. Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells.

    Science.gov (United States)

    Merkle, Florian T; Maroof, Asif; Wataya, Takafumi; Sasai, Yoshiki; Studer, Lorenz; Eggan, Kevin; Schier, Alexander F

    2015-02-15

    Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides, and are relevant to human diseases such as obesity, narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti-related peptide, hypocretin/orexin, melanin-concentrating hormone, oxytocin, arginine vasopressin, corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types, or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo, and are able to integrate into the mouse brain. These neurons could form the basis of cellular models, chemical screens or cellular therapies to study and treat common human diseases. © 2015. Published by The Company of Biologists Ltd.

  11. Differential sensitivity to nicotine among hypothalamic magnocellular neurons

    DEFF Research Database (Denmark)

    Mikkelsen, J D; Jacobsen, Julie; Kiss, Adrian Emil

    2012-01-01

    The magnocellular neurons in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON) either contain vasopressin or oxytocin. Even though both hormones are released after systemic administration of nicotine, the mechanism through which the two populations of neurons are activated...... is not known. This study was carried out in the rat to investigate the effect of increasing doses of nicotine on subsets of magnocellular neurons containing either oxytocin or vasopressin....

  12. Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neurons

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    Ammy M. Santiago

    2016-10-01

    Full Text Available Objective: Brain regulation of glucose homeostasis is sexually dimorphic; however, the impact sex hormones have on specific neuronal populations within the ventromedial hypothalamic nucleus (VMN, a metabolically sensitive brain region, has yet to be fully characterized. Glucose-excited (GE and -inhibited (GI neurons are located throughout the VMN and may play a critical role in glucose and energy homeostasis. Within the ventrolateral portion of the VMN (VL-VMN, glucose sensing neurons and estrogen receptor (ER distributions overlap. We therefore tested the hypothesis that VL-VMN glucose sensing neurons were sexually dimorphic and regulated by 17β-estradiol (17βE. Methods: Electrophysiological recordings of VL-VMN glucose sensing neurons in brain slices isolated from age- and weight-matched female and male mice were performed in the presence and absence of 17βE. Results: We found a new class of VL-VMN GI neurons whose response to low glucose was transient despite continued exposure to low glucose. Heretofore, we refer to these newly identified VL-VMN GI neurons as ‘adapting’ or AdGI neurons. We found a sexual dimorphic response to low glucose, with male nonadapting GI neurons, but not AdGI neurons, responding more robustly to low glucose than those from females. 17βE blunted the response of both nonadapting GI and AdGI neurons to low glucose in both males and females, which was mediated by activation of estrogen receptor β and inhibition of AMP-activated kinase. In contrast, 17βE had no impact on GE or non-glucose sensing neurons in either sex. Conclusion: These data suggest sex differences and estrogenic regulation of VMN hypothalamic glucose sensing may contribute to the sexual dimorphism in glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: 17β-estradiol, AMP-activated kinase, Glucose excited neurons, Glucose inhibited neurons, Ventromedial hypothalamic nucleus, Sexual dimorphism

  13. Brain pericyte-derived soluble factors enhance insulin sensitivity in GT1-7 hypothalamic neurons.

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    Takahashi, Hiroyuki; Takata, Fuyuko; Matsumoto, Junichi; Machida, Takashi; Yamauchi, Atsushi; Dohgu, Shinya; Kataoka, Yasufumi

    2015-02-20

    Insulin signaling in the hypothalamus plays an important role in food intake and glucose homeostasis. Hypothalamic neuronal functions are modulated by glial cells; these form an extensive network connecting the neurons and cerebral vasculature, known as the neurovascular unit (NVU). Brain pericytes are periendothelial accessory structures of the blood-brain barrier and integral members of the NVU. However, the interaction between pericytes and neurons is largely unexplored. Here, we investigate whether brain pericytes could affect hypothalamic neuronal insulin signaling. Our immunohistochemical observations demonstrated the existence of pericytes in the mouse hypothalamus, exhibiting immunoreactivity of platelet-derived growth factor receptor β (a pericyte marker), and laminin, a basal lamina marker. We then exposed a murine hypothalamic neuronal cell line, GT1-7, to conditioned medium obtained from primary cultures of rat brain pericytes. Pericyte-conditioned medium (PCM), but not astrocyte- or aortic smooth muscle cell-conditioned medium, increased the insulin-stimulated phosphorylation of Akt in GT1-7 cells in a concentration-dependent manner. PCM also enhanced insulin-stimulated tyrosine phosphorylation of insulin receptor β without changing its expression or localization in cytosolic or plasma membrane fractions. These results suggest that pericytes, rather than astrocytes, increase insulin sensitivity in hypothalamic neurons by releasing soluble factors under physiological conditions in the NVU. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Microglia Dictate the Impact of Saturated Fat Consumption on Hypothalamic Inflammation and Neuronal Function

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    Martin Valdearcos

    2014-12-01

    Full Text Available Diets rich in saturated fat produce inflammation, gliosis, and neuronal stress in the mediobasal hypothalamus (MBH. Here, we show that microglia mediate this process and its functional impact. Although microglia and astrocytes accumulate in the MBH of mice fed a diet rich in saturated fatty acids (SFAs, only the microglia undergo inflammatory activation, along with a buildup of hypothalamic SFAs. Enteric gavage specifically with SFAs reproduces microglial activation and neuronal stress in the MBH, and SFA treatment activates murine microglia, but not astrocytes, in culture. Moreover, depleting microglia abrogates SFA-induced inflammation in hypothalamic slices. Remarkably, depleting microglia from the MBH of mice abolishes inflammation and neuronal stress induced by excess SFA consumption, and in this context, microglial depletion enhances leptin signaling and reduces food intake. We thus show that microglia sense SFAs and orchestrate an inflammatory process in the MBH that alters neuronal function when SFA consumption is high.

  15. Glutamate and GABA as rapid effectors of hypothalamic peptidergic neurons

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    Cornelia eSchöne

    2012-11-01

    Full Text Available Vital hypothalamic neurons regulating hunger, wakefulness, reward-seeking, and body weight are often defined by unique expression of hypothalamus-specific neuropeptides. Gene-ablation studies show that some of these peptides, notably orexin/hypocretin (hcrt/orx, are themselves critical for stable states of consciousness and metabolic health. However, neuron-ablation studies often reveal more severe phenotypes, suggesting key roles for co-expressed transmitters. Indeed, most hypothalamic neurons, including hcrt/orx cells, contain fast transmitters glutamate and GABA, as well as several neuropeptides. What are the roles and relations between different transmitters expressed by the same neuron? Here, we consider signaling codes for releasing different transmitters in relation to transmitter and receptor diversity in behaviorally-defined, widely-projecting peptidergic neurons, such as hcrt/orx cells. We then discuss latest optogenetic studies of endogenous transmitter release from defined sets of axons in situ, which suggest that recently-characterized vital peptidergic neurons (e.g. hcrt/orx, proopiomelanocortin , and agouti-related peptide cells, as well as classical modulatory neurons (e.g. dopamine and acetylcholine cells, all use fast transmitters to control their postsynaptic targets. These optogenetic insights are complemented by recent observations of behavioral deficiencies caused by genetic ablation of fast transmission from specific neuropeptidergic and aminergic neurons. Powerful and fast (millisecond-scale GABAergic and glutamatergic signaling from neurons previously considered to be primarily modulatory raises new questions about the roles of slower co-transmitters they co-express.

  16. Neurogenin 3 Mediates Sex Chromosome Effects on the Generation of Sex Differences in Hypothalamic Neuronal Development

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    Maria Julia Scerbo

    2014-07-01

    Full Text Available The organizational action of testosterone during critical periods of development is the cause of numerous sex differences in the brain. However, sex differences in neuritogenesis have been detected in primary neuronal hypothalamic cultures prepared before the peak of testosterone production by fetal testis. In the present study we assessed the hypothesis of that cell-autonomous action of sex chromosomes can differentially regulate the expression of the neuritogenic gene neurogenin 3 (Ngn3 in male and female hypothalamic neurons, generating sex differences in neuronal development. Neuronal cultures were prepared from male and female E14 mouse hypothalami, before the fetal peak of testosterone. Female neurons showed enhanced neuritogenesis and higher expression of Ngn3 than male neurons. The silencing of Ngn3 abolished sex differences in neuritogenesis, decreasing the differentiation of female neurons. The sex difference in Ngn3 expression was determined by sex chromosomes, as demonstrated using the four core genotypes mouse model, in which a spontaneous deletion of the testis-determining gene Sry from the Y chromosome was combined with the insertion of the Sry gene onto an autosome. In addition, the expression of Ngn3, which is also known to mediate the neuritogenic actions of estradiol, was increased in the cultures treated with the hormone, but only in those from male embryos. Furthermore, the hormone reversed the sex differences in neuritogenesis promoting the differentiation of male neurons. These findings indicate that Ngn3 mediates both cell-autonomous actions of sex chromosomes and hormonal effects on neuritogenesis.

  17. Effects of endogenous pyrogen and prostaglandin E2 on hypothalamic neurons in rat brain slices.

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    Watanabe, T; Morimoto, A; Murakami, N

    1987-06-01

    We investigated the effects of endogenous pyrogen and prostaglandin E2 (PGE2) on the preoptic and anterior hypothalamic (POAH) neurons using brain slice preparations from the rat. Partially purified endogenous pyrogen did not change the activities of most of the neurons in the POAH region when applied locally through a micropipette attached to the recording electrode in proximity to the neurons. This indicates that partially purified endogenous pyrogen does not act directly on the neuronal activity in the POAH region. The partially purified endogenous pyrogen, applied into a culture chamber containing a brain slice, facilitated the activities in 24% of the total neurons tested, regardless of the thermal specificity of the neurons. Moreover, PGE2 added to the culture chamber facilitated 48% of the warm-responsive, 33% of the cold-responsive, and 29% of the thermally insensitive neurons. The direction of change in neuronal activity induced by partially purified endogenous pyrogen appears to be almost the same as that induced by PGE2 when these substances were applied by perfusion to the same neuron in the culture chamber. These results suggest that partially purified pyrogen applied to the perfusate of the culture chamber stimulates some constituents of brain tissue to synthesize and release prostaglandin, which in turn affects the neuronal activity of the POAH region.

  18. Characteristics and mechanisms of hypothalamic neuronal fatty acid sensing.

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    Le Foll, Christelle; Irani, Boman G; Magnan, Christophe; Dunn-Meynell, Ambrose A; Levin, Barry E

    2009-09-01

    We assessed the mechanisms by which specialized hypothalamic ventromedial nucleus (VMN) neurons utilize both glucose and long-chain fatty acids as signaling molecules to alter their activity as a potential means of regulating energy homeostasis. Fura-2 calcium (Ca(2+)) and membrane potential dye imaging, together with pharmacological agents, were used to assess the mechanisms by which oleic acid (OA) alters the activity of dissociated VMN neurons from 3- to 4-wk-old rats. OA excited up to 43% and inhibited up to 29% of all VMN neurons independently of glucose concentrations. In those neurons excited by both 2.5 mM glucose and OA, OA had a concentration-dependent effective excitatory concentration (EC(50)) of 13.1 nM. Neurons inhibited by both 2.5 mM glucose and OA had an effective inhibitory concentration (IC(50)) of 93 nM. At 0.5 mM glucose, OA had markedly different effects on these same neurons. Inhibition of carnitine palmitoyltransferase, reactive oxygen species formation, long-chain acetyl-CoA synthetase and ATP-sensitive K(+) channel activity or activation of uncoupling protein 2 (UCP2) accounted for only approximately 20% of OA's excitatory effects and approximately 40% of its inhibitory effects. Inhibition of CD36, a fatty acid transporter that can alter cell function independently of intracellular fatty acid metabolism, reduced the effects of OA by up to 45%. Thus OA affects VMN neuronal activity through multiple pathways. In glucosensing neurons, its effects are glucose dependent. This glucose-OA interaction provides a potential mechanism whereby such "metabolic sensing" neurons can respond to differences in the metabolic states associated with fasting and feeding.

  19. Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes*

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    Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

    2013-01-01

    Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance. PMID:24240094

  20. Glucose regulates hypothalamic long-chain fatty acid metabolism via AMP-activated kinase (AMPK) in neurons and astrocytes.

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    Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

    2013-12-27

    Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.

  1. Oral glucose intake inhibits hypothalamic neuronal activity more effectively than glucose infusion

    NARCIS (Netherlands)

    Smeets, P.A.M.; Vidarsdottir, S.; Graaf, de C.; Stafleu, A.; Osch, M.J.P.; Viergever, M.A.; Pijl, H.; Grond, van der J.

    2007-01-01

    Oral glucose intake inhibits hypothalamic neuronal activity more effectively than glucose infusion. Am J Physiol Endocrinol Metab 293: E754-E758, 2007. First published June 12, 2007; doi:10.1152/ajpendo.00231.2007. - We previously showed that hypothalamic neuronal activity, as measured by the blood

  2. Oral glucose intake inhibits hypothalamic neuronal activity more effectively than glucose infusion

    NARCIS (Netherlands)

    Smeets, P.A.M.; Vidarsdottir, S.; Graaf, C. de; Stafleu, A.; Osch, M.J.P. van; Viergever, M.A.; Pijl, H.; Grond, J. van der

    2007-01-01

    We previously showed that hypothalamic neuronal activity, as measured by the blood oxygen level-dependent (BOLD) functional MRI signal, declines in response to oral glucose intake. To further explore the mechanism driving changes in hypothalamic neuronal activity in response to an oral glucose load,

  3. Direct versus indirect actions of ghrelin on hypothalamic NPY neurons.

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    Hashiguchi, Hiroshi; Sheng, Zhenyu; Routh, Vanessa; Gerzanich, Volodymyr; Simard, J Marc; Bryan, Joseph

    2017-01-01

    Assess direct versus indirect action(s) of ghrelin on hypothalamic NPY neurons. Electrophysiology was used to measure ion channel activity in NPY-GFP neurons in slice preparations. Ca2+ imaging was used to monitor ghrelin activation of isolated NPY GFP-labeled neurons. Immunohistochemistry was used to localize Trpm4, SUR1 and Kir6.2 in the hypothalamus. Acylated ghrelin depolarized the membrane potential (MP) of NPY-GFP neurons in brain slices. Depolarization resulted from a decreased input resistance (IR) in ~70% of neurons (15/22) or an increased IR in the remainder (7/22), consistent with the opening or closing of ion channels, respectively. Although tetrodotoxin (TTX) blockade of presynaptic action potentials reduced ghrelin-induced changes in MP and IR, ghrelin still significantly depolarized the MP and decreased IR in TTX-treated neurons, suggesting that ghrelin directly opens cation channel(s) in NPY neurons. In isolated NPY-GFP neurons, ghrelin produced a sustained rise of [Ca2+]c, with an EC50 ~110 pM. Pharmacologic studies confirmed that the direct action of ghrelin was through occupation of the growth hormone secretagogue receptor, GHS-R, and demonstrated the importance of the adenylate cyclase/cAMP/protein kinase A (PKA) and phospholipase C/inositol triphosphate (PLC/IP3) pathways as activators of 5' AMP-activated protein kinase (AMPK). Activation of isolated neurons was not affected by CNQX or TTX, but reducing [Na+]o suppressed activation, suggesting a role for Na+-permeable cation channels. SUR1 and two channel partners, Kir6.2 and Trpm4, were identified immunologically in NPY-GFP neurons in situ. The actions of SUR1 and Trpm4 modulators were informative: like ghrelin, diazoxide, a SUR1 agonist, elevated [Ca2+]c and glibenclamide, a SUR1 antagonist, partially suppressed ghrelin action, while 9-phenanthrol and flufenamic acid, selective Trpm4 antagonists, blocked ghrelin actions on isolated neurons. Ghrelin activation was unaffected by nifedipine and

  4. Optogenetic identification of hypothalamic orexin neuron projections to paraventricular spinally projecting neurons.

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    Dergacheva, Olga; Yamanaka, Akihiro; Schwartz, Alan R; Polotsky, Vsevolod Y; Mendelowitz, David

    2017-04-01

    Orexin neurons, and activation of orexin receptors, are generally thought to be sympathoexcitatory; however, the functional connectivity between orexin neurons and a likely sympathetic target, the hypothalamic spinally projecting neurons (SPNs) in the paraventricular nucleus of the hypothalamus (PVN) has not been established. To test the hypothesis that orexin neurons project directly to SPNs in the PVN, channelrhodopsin-2 (ChR2) was selectively expressed in orexin neurons to enable photoactivation of ChR2-expressing fibers while examining evoked postsynaptic currents in SPNs in rat hypothalamic slices. Selective photoactivation of orexin fibers elicited short-latency postsynaptic currents in all SPNs tested ( n = 34). These light-triggered responses were heterogeneous, with a majority being excitatory glutamatergic responses (59%) and a minority of inhibitory GABAergic (35%) and mixed glutamatergic and GABAergic currents (6%). Both glutamatergic and GABAergic responses were present in the presence of tetrodotoxin and 4-aminopyridine, suggesting a monosynaptic connection between orexin neurons and SPNs. In addition to generating postsynaptic responses, photostimulation facilitated action potential firing in SPNs (current clamp configuration). Glutamatergic, but not GABAergic, postsynaptic currents were diminished by application of the orexin receptor antagonist almorexant, indicating orexin release facilitates glutamatergic neurotransmission in this pathway. This work identifies a neuronal circuit by which orexin neurons likely exert sympathoexcitatory control of cardiovascular function. NEW & NOTEWORTHY This is the first study to establish, using innovative optogenetic approaches in a transgenic rat model, that there are robust heterogeneous projections from orexin neurons to paraventricular spinally projecting neurons, including excitatory glutamatergic and inhibitory GABAergic neurotransmission. Endogenous orexin release modulates glutamatergic, but not

  5. Brain Innate Immunity Regulates Hypothalamic Arcuate Neuronal Activity and Feeding Behavior

    NARCIS (Netherlands)

    Reis, Wagner L.; Yi, Chun-Xia; Gao, Yuanqing; Tschöp, Mathias H.; Stern, Javier E.

    2015-01-01

    Hypothalamic inflammation, involving microglia activation in the arcuate nucleus (ARC), is proposed as a novel underlying mechanism in obesity, insulin and leptin resistance. However, whether activated microglia affects ARC neuronal activity, and consequently basal and hormonal-induced food intake,

  6. Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance

    OpenAIRE

    Kaushik, Susmita; Rodriguez-Navarro, Jose Antonio; Arias, Esperanza; Kiffin, Roberta; Sahu, Srabani; Schwartz, Gary J.; Cuervo, Ana Maria; Singh, Rajat

    2011-01-01

    Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here, we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the...

  7. Zebrafish adult-derived hypothalamic neurospheres generate gonadotropin-releasing hormone (GnRH neurons

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    Christian Cortés-Campos

    2015-09-01

    Full Text Available Gonadotropin-releasing hormone (GnRH is a hypothalamic decapeptide essential for fertility in vertebrates. Human male patients lacking GnRH and treated with hormone therapy can remain fertile after cessation of treatment suggesting that new GnRH neurons can be generated during adult life. We used zebrafish to investigate the neurogenic potential of the adult hypothalamus. Previously we have characterized the development of GnRH cells in the zebrafish linking genetic pathways to the differentiation of neuromodulatory and endocrine GnRH cells in specific regions of the brain. Here, we developed a new method to obtain neural progenitors from the adult hypothalamus in vitro. Using this system, we show that neurospheres derived from the adult hypothalamus can be maintained in culture and subsequently differentiate glia and neurons. Importantly, the adult derived progenitors differentiate into neurons containing GnRH and the number of cells is increased through exposure to either testosterone or GnRH, hormones used in therapeutic treatment in humans. Finally, we show in vivo that a neurogenic niche in the hypothalamus contains GnRH positive neurons. Thus, we demonstrated for the first time that neurospheres can be derived from the hypothalamus of the adult zebrafish and that these neural progenitors are capable of producing GnRH containing neurons.

  8. Proliferative hypothalamic neurospheres express NPY, AGRP, POMC, CART and Orexin-A and differentiate to functional neurons.

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    Lígia Sousa-Ferreira

    Full Text Available Some pathological conditions with feeding pattern alterations, including obesity and Huntington disease (HD are associated with hypothalamic dysfunction and neuronal cell death. Additionally, the hypothalamus is a neurogenic region with the constitutive capacity to generate new cells of neuronal lineage, in adult rodents. The aim of the present work was to evaluate the expression of feeding-related neuropeptides in hypothalamic progenitor cells and their capacity to differentiate to functional neurons which have been described to be affected by hypothalamic dysfunction. Our study shows that hypothalamic progenitor cells from rat embryos grow as floating neurospheres and express the feeding-related neuropeptides Neuropeptide Y (NPY, Agouti-related Protein (AGRP, Pro-OpioMelanocortin (POMC, Cocaine-and-Amphetamine Responsive Transcript (CART and Orexin-A/Hypocretin-1. Moreover the relative mRNA expression of NPY and POMC increases during the expansion of hypothalamic neurospheres in proliferative conditions.Mature neurons were obtained from the differentiation of hypothalamic progenitor cells including NPY, AGRP, POMC, CART and Orexin-A positive neurons. Furthermore the relative mRNA expression of NPY, CART and Orexin-A increases after the differentiation of hypothalamic neurospheres. Similarly to the adult hypothalamic neurons the neurospheres-derived neurons express the glutamate transporter EAAT3. The orexigenic and anorexigenic phenotype of these neurons was identified by functional response to ghrelin and leptin hormones, respectively. This work demonstrates the presence of appetite-related neuropeptides in hypothalamic progenitor cells and neurons obtained from the differentiation of hypothalamic neurospheres, including the neuronal phenotypes that have been described by others as being affected by hypothalamic neurodegeneration. These in vitro models can be used to study hypothalamic progenitor cells aiming a therapeutic intervention to

  9. Mechanisms underlying prorenin actions on hypothalamic neurons implicated in cardiometabolic control

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    Soledad Pitra

    2016-10-01

    Conclusions: We identified novel neuronal targets and cellular mechanisms underlying PR/PRR actions in critical hypothalamic neurons involved in cardiometabolic regulation. This fundamental mechanistic information regarding central PR/PRR actions is essential for the development of novel RAS-based therapeutic targets for the treatment of cardiometabolic disorders in obesity and hypertension.

  10. NEUROANATOMICAL ASSOCIATION OF HYPOTHALAMIC HSD2-CONTAINING NEURONS WITH ERα, CATECHOLAMINES, OR OXYTOCIN: IMPLICATIONS FOR FEEDING?

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    Maegan L. Askew

    2015-06-01

    Full Text Available This study used immunohistochemical methods to investigate the possibility that hypothalamic neurons that contain 11-β-hydroxysteroid dehydrogenase type 2 (HSD2 are involved in the control of feeding by rats via neuroanatomical associations with the α subtype of estrogen receptor (ERα, catecholamines, and/or oxytocin. An aggregate of HSD2-containing neurons is located laterally in the hypothalamus, and the numbers of these neurons were greatly increased by estradiol treatment in ovariectomized rats compared to numbers in male rats and in ovariectomized rats that were not given estradiol. However, HSD2-containing neurons were anatomically segregated from ERα-containing neurons in the Ventromedial Hypothalamus and the Arcuate Nucleus. There was an absence of oxytocin-immunolabeled fibers in the area of HSD2-labeled neurons. Taken together, these findings provide no support for direct associations between hypothalamic HSD2 and ERα or oxytocin neurons in the control of feeding. In contrast, there was catecholamine-fiber labeling in the area of HSD2-labeled neurons, and these fibers occasionally were in close apposition to HSD2-labeled neurons. Therefore, we cannot rule out interactions between HSD2 and catecholamines in the control of feeding; however, given the relative sparseness of the appositions, any such interaction would appear to be modest. Thus, these studies do not conclusively identify a neuroanatomical substrate by which HSD2-containing neurons in the hypothalamus may alter feeding, and leave the functional role of hypothalamic HSD2-containing neurons subject to further investigation.

  11. Anorexia and Impaired Glucose Metabolism in Mice With Hypothalamic Ablation of Glut4 Neurons

    OpenAIRE

    Ren, Hongxia; Lu, Taylor Y.; McGraw, Timothy E.; Accili, Domenico

    2014-01-01

    The central nervous system (CNS) uses glucose independent of insulin. Nonetheless, insulin receptors and insulin-responsive glucose transporters (Glut4) often colocalize in neurons (Glut4 neurons) in anatomically and functionally distinct areas of the CNS. The apparent heterogeneity of Glut4 neurons has thus far thwarted attempts to understand their function. To answer this question, we used Cre-dependent, diphtheria toxin?mediated cell ablation to selectively remove basal hypothalamic Glut4 ...

  12. Rapid shift in substrate utilization driven by hypothalamic Agrp neurons

    OpenAIRE

    Cavalcanti-De-Albuquerque, Joao; Dietrich, Marcelo; Bober, Jeremy; Zimmer, Marcelo

    2016-01-01

    Agrp neurons drive feeding. To what extend these neurons participate in the regulation of other homeostatic processes is not well understood. We investigated the role of Agrp neurons in substrate utilization in mice. Activation of Agrp neurons was sufficient to rapidly increase RER and carbohydrate utilization, while decreasing fat utilization. These metabolic changes were linearly correlated with carbohydrates ingested, but not protein or fat ingestion. However, even in the absence of ingest...

  13. Cellular activation of hypothalamic hypocretin/orexin neurons facilitates short-term spatial memory in mice.

    Science.gov (United States)

    Aitta-Aho, Teemu; Pappa, Elpiniki; Burdakov, Denis; Apergis-Schoute, John

    2016-12-01

    The hypothalamic hypocretin/orexin (HO) system holds a central role in the regulation of several physiological functions critical for food-seeking behavior including mnemonic processes for effective foraging behavior. It is unclear however whether physiological increases in HO neuronal activity can support such processes. Using a designer rM3Ds receptor activation approach increasing HO neuronal activity resulted in improved short-term memory for novel locations. When tested on a non-spatial novelty object recognition task no significant difference was detected between groups indicating that hypothalamic HO neuronal activation can selectively facilitate short-term spatial memory for potentially supporting memory for locations during active exploration. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Hypothalamic glucose-sensing: role of Glia-to-neuron signaling.

    Science.gov (United States)

    Tonon, M C; Lanfray, D; Castel, H; Vaudry, H; Morin, F

    2013-12-01

    The hypothalamus senses hormones and nutrients in order to regulate energy balance. In particular, detection of hypothalamic glucose levels has been shown to regulate both feeding behavior and peripheral glucose homeostasis, and impairment of this regulatory system is believed to be involved in the development of obesity and diabetes. Several data clearly demonstrate that glial cells are key elements in the perception of glucose, constituting with neurons a "glucose-sensing unit". Characterization of this interplay between glia and neurons represents an exciting challenge, and will undoubtedly contribute to identify new candidates for therapeutic intervention. The purpose of this review is to summarize the current data that stress the importance of glia in central glucose-sensing. The nature of the glia-to-neuron signaling is discussed, with a special focus on the endozepine ODN, a potent anorexigenic peptide that is highly expressed in hypothalamic glia. © Georg Thieme Verlag KG Stuttgart · New York.

  15. ERK1/2 mediates glucose-regulated POMC gene expression in hypothalamic neurons.

    Science.gov (United States)

    Zhang, Juan; Zhou, Yunting; Chen, Cheng; Yu, Feiyuan; Wang, Yun; Gu, Jiang; Ma, Lian; Ho, Guyu

    2015-04-01

    Hypothalamic glucose-sensing neurons regulate the expression of genes encoding feeding-related neuropetides POMC, AgRP, and NPY - the key components governing metabolic homeostasis. AMP-activated protein kinase (AMPK) is postulated to be the molecular mediator relaying glucose signals to regulate the expression of these neuropeptides. Whether other signaling mediator(s) plays a role is not clear. In this study, we investigated the role of ERK1/2 using primary hypothalamic neurons as the model system. The primary neurons were differentiated from hypothalamic progenitor cells. The differentiated neurons possessed the characteristic neuronal cell morphology and expressed neuronal post-mitotic markers as well as leptin-regulated orexigenic POMC and anorexigenic AgRP/NPY genes. Treatment of cells with glucose dose-dependently increased POMC and decreased AgRP/NPY expression with a concurrent suppression of AMPK phosphorylation. In addition, glucose treatment dose-dependently increased the ERK1/2 phosphorylation. Blockade of ERK1/2 activity with its specific inhibitor PD98059 partially (approximately 50%) abolished glucose-induced POMC expression, but had little effect on AgRP/NPY expression. Conversely, blockade of AMPK activity with its specific inhibitor produced a partial (approximately 50%) reversion of low-glucose-suppressed POMC expression, but almost completely blunted the low-glucose-induced AgRP/NPY expression. The results indicate that ERK1/2 mediated POMC but not AgRP/NPY expression. Confirming the in vitro findings, i.c.v. administration of PD98059 in rats similarly attenuated glucose-induced POMC expression in the hypothalamus, but again had little effect on AgRP/NPY expression. The results are indicative of a novel role of ERK1/2 in glucose-regulated POMC expression and offer new mechanistic insights into hypothalamic glucose sensing. © 2015 Society for Endocrinology.

  16. Glucose rapidly induces different forms of excitatory synaptic plasticity in hypothalamic POMC neurons.

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    Jun Hu

    Full Text Available Hypothalamic POMC neurons are required for glucose and energy homeostasis. POMC neurons have a wide synaptic connection with neurons both within and outside the hypothalamus, and their activity is controlled by a balance between excitatory and inhibitory synaptic inputs. Brain glucose-sensing plays an essential role in the maintenance of normal body weight and metabolism; however, the effect of glucose on synaptic transmission in POMC neurons is largely unknown. Here we identified three types of POMC neurons (EPSC(+, EPSC(-, and EPSC(+/- based on their glucose-regulated spontaneous excitatory postsynaptic currents (sEPSCs, using whole-cell patch-clamp recordings. Lowering extracellular glucose decreased the frequency of sEPSCs in EPSC(+ neurons, but increased it in EPSC(- neurons. Unlike EPSC(+ and EPSC(- neurons, EPSC(+/- neurons displayed a bi-phasic sEPSC response to glucoprivation. In the first phase of glucoprivation, both the frequency and the amplitude of sEPSCs decreased, whereas in the second phase, they increased progressively to the levels above the baseline values. Accordingly, lowering glucose exerted a bi-phasic effect on spontaneous action potentials in EPSC(+/- neurons. Glucoprivation decreased firing rates in the first phase, but increased them in the second phase. These data indicate that glucose induces distinct excitatory synaptic plasticity in different subpopulations of POMC neurons. This synaptic remodeling is likely to regulate the sensitivity of the melanocortin system to neuronal and hormonal signals.

  17. Glucose Rapidly Induces Different Forms of Excitatory Synaptic Plasticity in Hypothalamic POMC Neurons

    Science.gov (United States)

    Hu, Jun; Jiang, Lin; Low, Malcolm J.; Rui, Liangyou

    2014-01-01

    Hypothalamic POMC neurons are required for glucose and energy homeostasis. POMC neurons have a wide synaptic connection with neurons both within and outside the hypothalamus, and their activity is controlled by a balance between excitatory and inhibitory synaptic inputs. Brain glucose-sensing plays an essential role in the maintenance of normal body weight and metabolism; however, the effect of glucose on synaptic transmission in POMC neurons is largely unknown. Here we identified three types of POMC neurons (EPSC(+), EPSC(−), and EPSC(+/−)) based on their glucose-regulated spontaneous excitatory postsynaptic currents (sEPSCs), using whole-cell patch-clamp recordings. Lowering extracellular glucose decreased the frequency of sEPSCs in EPSC(+) neurons, but increased it in EPSC(−) neurons. Unlike EPSC(+) and EPSC(−) neurons, EPSC(+/−) neurons displayed a bi-phasic sEPSC response to glucoprivation. In the first phase of glucoprivation, both the frequency and the amplitude of sEPSCs decreased, whereas in the second phase, they increased progressively to the levels above the baseline values. Accordingly, lowering glucose exerted a bi-phasic effect on spontaneous action potentials in EPSC(+/−) neurons. Glucoprivation decreased firing rates in the first phase, but increased them in the second phase. These data indicate that glucose induces distinct excitatory synaptic plasticity in different subpopulations of POMC neurons. This synaptic remodeling is likely to regulate the sensitivity of the melanocortin system to neuronal and hormonal signals. PMID:25127258

  18. Glucose rapidly induces different forms of excitatory synaptic plasticity in hypothalamic POMC neurons.

    Science.gov (United States)

    Hu, Jun; Jiang, Lin; Low, Malcolm J; Rui, Liangyou

    2014-01-01

    Hypothalamic POMC neurons are required for glucose and energy homeostasis. POMC neurons have a wide synaptic connection with neurons both within and outside the hypothalamus, and their activity is controlled by a balance between excitatory and inhibitory synaptic inputs. Brain glucose-sensing plays an essential role in the maintenance of normal body weight and metabolism; however, the effect of glucose on synaptic transmission in POMC neurons is largely unknown. Here we identified three types of POMC neurons (EPSC(+), EPSC(-), and EPSC(+/-)) based on their glucose-regulated spontaneous excitatory postsynaptic currents (sEPSCs), using whole-cell patch-clamp recordings. Lowering extracellular glucose decreased the frequency of sEPSCs in EPSC(+) neurons, but increased it in EPSC(-) neurons. Unlike EPSC(+) and EPSC(-) neurons, EPSC(+/-) neurons displayed a bi-phasic sEPSC response to glucoprivation. In the first phase of glucoprivation, both the frequency and the amplitude of sEPSCs decreased, whereas in the second phase, they increased progressively to the levels above the baseline values. Accordingly, lowering glucose exerted a bi-phasic effect on spontaneous action potentials in EPSC(+/-) neurons. Glucoprivation decreased firing rates in the first phase, but increased them in the second phase. These data indicate that glucose induces distinct excitatory synaptic plasticity in different subpopulations of POMC neurons. This synaptic remodeling is likely to regulate the sensitivity of the melanocortin system to neuronal and hormonal signals.

  19. Understanding how discrete populations of hypothalamic neurons orchestrate complicated behavioral states

    Directory of Open Access Journals (Sweden)

    Allison eGraebner

    2015-08-01

    Full Text Available A major question in systems neuroscience is how a single population of neurons can interact with the rest of the brain to orchestrate complex behavioral states. The hypothalamus contains many such discrete neuronal populations that individually regulate arousal, feeding, and drinking. For example, hypothalamic neurons that express hypocretin (Hcrt neuropeptides can sense homeostatic and metabolic factors affecting wakefulness and orchestrate organismal arousal. Neurons that express agouti-related protein (AgRP can sense the metabolic needs of the body and orchestrate a state of hunger. The organum vasculosum of the lamina terminalis (OVLT can detect the hypertonicity of blood and orchestrate a state of thirst. Each hypothalamic population is sufficient to generate complicated behavioral states through the combined efforts of distinct efferent projections. The principal challenge to understanding these brain systems is therefore to determine the individual roles of each downstream projection for each behavioral state. In recent years, the development and application of temporally precise, genetically encoded tools have greatly improved our understanding of the structure and function of these neural systems. This review will survey recent advances in our understanding of how these individual hypothalamic populations can orchestrate complicated behavioral states due to the combined efforts of individual downstream projections.

  20. Anorexia and impaired glucose metabolism in mice with hypothalamic ablation of Glut4 neurons.

    Science.gov (United States)

    Ren, Hongxia; Lu, Taylor Y; McGraw, Timothy E; Accili, Domenico

    2015-02-01

    The central nervous system (CNS) uses glucose independent of insulin. Nonetheless, insulin receptors and insulin-responsive glucose transporters (Glut4) often colocalize in neurons (Glut4 neurons) in anatomically and functionally distinct areas of the CNS. The apparent heterogeneity of Glut4 neurons has thus far thwarted attempts to understand their function. To answer this question, we used Cre-dependent, diphtheria toxin-mediated cell ablation to selectively remove basal hypothalamic Glut4 neurons and investigate the resulting phenotypes. After Glut4 neuron ablation, mice demonstrate altered hormone and nutrient signaling in the CNS. Accordingly, they exhibit negative energy balance phenotype characterized by reduced food intake and increased energy expenditure, without locomotor deficits or gross neuronal abnormalities. Glut4 neuron ablation affects orexigenic melanin-concentrating hormone neurons but has limited effect on neuropeptide Y/agouti-related protein and proopiomelanocortin neurons. The food intake phenotype can be partially normalized by GABA administration, suggesting that it arises from defective GABAergic transmission. Glut4 neuron-ablated mice show peripheral metabolic defects, including fasting hyperglycemia and glucose intolerance, decreased insulin levels, and elevated hepatic gluconeogenic genes. We conclude that Glut4 neurons integrate hormonal and nutritional cues and mediate CNS actions of insulin on energy balance and peripheral metabolism. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  1. Dehydration-induced release of vasopressin involves activation of hypothalamic histaminergic neurons.

    Science.gov (United States)

    Kjaer, A; Knigge, U; Rouleau, A; Garbarg, M; Warberg, J

    1994-08-01

    The hypothalamic neurotransmitter histamine (HA) induces arginine vasopressin (AVP) release when administered centrally. We studied and characterized this effect of HA with respect to receptor involvement. In addition, we studied the possible role of hypothalamic histaminergic neurons in the mediation of a physiological stimulus (dehydration) for AVP secretion. Intracerebroventricular administration of HA, the H1-receptor agonists 2(3-bromophenyl)HA and 2-thiazolylethylamine, or the H2-receptor agonists amthamine or 4-methyl-HA stimulated AVP secretion. The stimulatory action of HA on AVP was inhibited by pretreatment with the H1-receptor antagonist mepyramine or the H2-receptor antagonist cimetidine. Twenty-four hours of dehydration elevated the plasma osmolality from 298 +/- 3 to 310 +/- 3 mmol/liter and increased the plasma AVP concentration 4-fold. The hypothalamic content of HA and its metabolite tele-methyl-HA was elevated in response to dehydration, indicating an increased synthesis and release of hypothalamic HA. Dehydration-induced AVP secretion was lowered when neuronal HA synthesis was inhibited by the administration of (S) alpha-fluoromethylhistidine or when the animals were pretreated with the H3-receptor agonist R(alpha)methylhistamine, which inhibits the release and synthesis of HA, the H1-receptor antagonists mepyramine and cetirizine, or the H2-receptor antagonists cimetidine and ranitidine. We conclude that HA, via activation of both H1- and H2-receptors, stimulates AVP release and that HA is a physiological regulator of AVP secretion.

  2. Bicarbonate Contributes to GABAA Receptor-Mediated Neuronal Excitation in Surgically-Resected Human Hypothalamic Hamartomas

    Science.gov (United States)

    Do-Young, Kim; Fenoglio, Kristina A.; Kerrigan, John F.; Rho, Jong M.

    2009-01-01

    SUMMARY The role of bicarbonate (HCO3-) in GABAA receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABAA receptors with muscimol (30 μM) provoked neuronal excitation in over 70% of large (18-22 μM) HH neurons in HCO3- buffer. Subsequent perfusion of HCO3--free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO3--free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO3- efflux through GABAA receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels. PMID:19022626

  3. On the acoustic wave sensor response to immortalized hypothalamic neurons at the device-liquid interface

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    Shilin Cheung

    2016-12-01

    Full Text Available The response of a thickness shear mode biosensor to immortalized murine hypothalamic neurons (mHypoE-38 and -46 cells under a variety of conditions and stimuli is discussed. Cellular studies which lead to the production of detectable neuronal responses include neuronal deposition, adhesion and proliferation, alteration in the extent of specific cell-surface interactions, actin filament and microtubule cytoskeletal disruptions, effects of cell depolarization, inhibition of the Na+-K+ pump via ouabain, effects of neuronal synchronization and the effects ligand-receptor interaction (glucagon. In the presence of cells, fs shifts are largely influenced by the damping of the TSM resonator. The formation of cell-surface interactions and hence the increase in coupling and acoustic energy dissipation can be modeled as an additional resistor in the BVD model. Further sensor and cellular changes can be obtained by negating the effects of damping from fs via the use of Rm and θmax. Keywords: Acoustic wave sensor, Hypothalamic neurons, Neuron cell-surface interaction

  4. An ATF4-ATG5 signaling in hypothalamic POMC neurons regulates obesity.

    Science.gov (United States)

    Xiao, Yuzhong; Deng, Yalan; Yuan, Feixiang; Xia, Tingting; Liu, Hao; Li, Zhigang; Chen, Shanghai; Liu, Zhixue; Ying, Hao; Liu, Yi; Zhai, Qiwei; Guo, Feifan

    2017-06-03

    ATF4 (activating transcription factor 4) is an important transcription factor that has many biological functions, while its role in hypothalamic POMC (pro-opiomelanocortin-α) neurons in the regulation of energy homeostasis has not been explored. We recently discovered that mice with an Atf4 deletion specific to POMC neurons (PAKO mice) are lean and have higher energy expenditure. Furthermore, these mice are resistant to high-fat diet (HFD)-induced obesity and obesity-related metabolic disorders. Mechanistically, we found the expression of ATG5 (autophagy-related 5) is upregulated in POMC neurons of PAKO mice, and ATF4 regulates ATG5 expression by binding directly to its promoter. Mice with Atf4 and Atg5 double knockout in POMC neurons have reduced energy expenditure and gain more fat mass compared with PAKO mice under a HFD. Finally, the effect of Atf4 knockout in POMC neurons is possibly mediated by enhanced ATG5-dependent macroautophagy/autophagy and α-melanocyte-stimulating hormone (α-MSH) production in the hypothalamus. Together, this work not only identifies a beneficial role for ATF4 in hypothalamic POMC neurons in the regulation of obesity, but also provides a new potential therapeutic target for obesity and obesity-related metabolic diseases.

  5. Metabolic regulation of lateral hypothalamic glucose-inhibited orexin neurons may influence midbrain reward neurocircuitry.

    Science.gov (United States)

    Sheng, Zhenyu; Santiago, Ammy M; Thomas, Mark P; Routh, Vanessa H

    2014-09-01

    Lateral hypothalamic area (LHA) orexin neurons modulate reward-based feeding by activating ventral tegmental area (VTA) dopamine (DA) neurons. We hypothesize that signals of peripheral energy status influence reward-based feeding by modulating the glucose sensitivity of LHA orexin glucose-inhibited (GI) neurons. This hypothesis was tested using electrophysiological recordings of LHA orexin-GI neurons in brain slices from 4 to 6week old male mice whose orexin neurons express green fluorescent protein (GFP) or putative VTA-DA neurons from C57Bl/6 mice. Low glucose directly activated ~60% of LHA orexin-GFP neurons in both whole cell and cell attached recordings. Leptin indirectly reduced and ghrelin directly enhanced the activation of LHA orexin-GI neurons by glucose decreases from 2.5 to 0.1mM by 53±12% (n=16, Pglucose sensitivity. Fasting increased activation of LHA orexin-GI neurons by decreased glucose, as would be predicted by these hormonal effects. We also evaluated putative VTA-DA neurons in a novel horizontal slice preparation containing the LHA and VTA. Decreased glucose increased the frequency of spontaneous excitatory post-synaptic currents (sEPSCs; 125 ± 40%, n=9, Pneurons. sEPSCs were completely blocked by AMPA and NMDA glutamate receptor antagonists (CNQX 20 μM, n=4; APV 20μM, n=4; respectively), demonstrating that these sEPSCs were mediated by glutamatergic transmission onto VTA DA neurons. Orexin-1 but not 2 receptor antagonism with SB334867 (10μM; n=9) and TCS-OX2-29 (2μM; n=5), respectively, blocks the effects of decreased glucose on VTA DA neurons. Thus, decreased glucose increases orexin-dependent excitatory glutamate neurotransmission onto VTA DA neurons. These data suggest that the glucose sensitivity of LHA orexin-GI neurons links metabolic state and reward-based feeding. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Brain innate immunity regulates hypothalamic arcuate neuronal activity and feeding behavior.

    Science.gov (United States)

    Reis, Wagner L; Yi, Chun-Xia; Gao, Yuanqing; Tschöp, Mathias H; Stern, Javier E

    2015-04-01

    Hypothalamic inflammation, involving microglia activation in the arcuate nucleus (ARC), is proposed as a novel underlying mechanism in obesity, insulin and leptin resistance. However, whether activated microglia affects ARC neuronal activity, and consequently basal and hormonal-induced food intake, is unknown. We show that lipopolysaccharide, an agonist of the toll-like receptor-4 (TLR4), which we found to be expressed in ARC microglia, inhibited the firing activity of the majority of orexigenic agouti gene-related protein/neuropeptide Y neurons, whereas it increased the activity of the majority of anorexigenic proopiomelanocortin neurons. Lipopolysaccharide effects in agouti gene-related protein/neuropeptide Y (but not in proopiomelanocortin) neurons were occluded by inhibiting microglia function or by blocking TLR4 receptors. Finally, we report that inhibition of hypothalamic microglia altered basal food intake, also preventing central orexigenic responses to ghrelin. Our studies support a major role for a TLR4-mediated microglia signaling pathway in the control of ARC neuronal activity and feeding behavior.

  7. Hypoglycemia: Role of Hypothalamic Glucose-Inhibited (GI) Neurons in Detection and Correction.

    Science.gov (United States)

    Zhou, Chunxue; Teegala, Suraj B; Khan, Bilal A; Gonzalez, Christina; Routh, Vanessa H

    2018-01-01

    Hypoglycemia is a profound threat to the brain since glucose is its primary fuel. As a result, glucose sensors are widely located in the central nervous system and periphery. In this perspective we will focus on the role of hypothalamic glucose-inhibited (GI) neurons in sensing and correcting hypoglycemia. In particular, we will discuss GI neurons in the ventromedial hypothalamus (VMH) which express neuronal nitric oxide synthase (nNOS) and in the perifornical hypothalamus (PFH) which express orexin. The ability of VMH nNOS-GI neurons to depolarize in low glucose closely parallels the hormonal response to hypoglycemia which stimulates gluconeogenesis. We have found that nitric oxide (NO) production in low glucose is dependent on oxidative status. In this perspective we will discuss the potential relevance of our work showing that enhancing the glutathione antioxidant system prevents hypoglycemia associated autonomic failure (HAAF) in non-diabetic rats whereas VMH overexpression of the thioredoxin antioxidant system restores hypoglycemia counterregulation in rats with type 1 diabetes.We will also address the potential role of the orexin-GI neurons in the arousal response needed for hypoglycemia awareness which leads to behavioral correction (e.g., food intake, glucose administration). The potential relationship between the hypothalamic sensors and the neurocircuitry in the hindbrain and portal mesenteric vein which is critical for hypoglycemia correction will then be discussed.

  8. Hypoglycemia: Role of Hypothalamic Glucose-Inhibited (GI Neurons in Detection and Correction

    Directory of Open Access Journals (Sweden)

    Chunxue Zhou

    2018-03-01

    Full Text Available Hypoglycemia is a profound threat to the brain since glucose is its primary fuel. As a result, glucose sensors are widely located in the central nervous system and periphery. In this perspective we will focus on the role of hypothalamic glucose-inhibited (GI neurons in sensing and correcting hypoglycemia. In particular, we will discuss GI neurons in the ventromedial hypothalamus (VMH which express neuronal nitric oxide synthase (nNOS and in the perifornical hypothalamus (PFH which express orexin. The ability of VMH nNOS-GI neurons to depolarize in low glucose closely parallels the hormonal response to hypoglycemia which stimulates gluconeogenesis. We have found that nitric oxide (NO production in low glucose is dependent on oxidative status. In this perspective we will discuss the potential relevance of our work showing that enhancing the glutathione antioxidant system prevents hypoglycemia associated autonomic failure (HAAF in non-diabetic rats whereas VMH overexpression of the thioredoxin antioxidant system restores hypoglycemia counterregulation in rats with type 1 diabetes.We will also address the potential role of the orexin-GI neurons in the arousal response needed for hypoglycemia awareness which leads to behavioral correction (e.g., food intake, glucose administration. The potential relationship between the hypothalamic sensors and the neurocircuitry in the hindbrain and portal mesenteric vein which is critical for hypoglycemia correction will then be discussed.

  9. Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

    Science.gov (United States)

    Chen, Yu; Zhou, Jun; Xie, Na; Huang, Chao; Zhang, Jun-qi; Hu, Zhuang-li; Ni, Lan; Jin, You; Wang, Fang; Chen, Jian-guo; Long, Li-hong

    2012-01-01

    Aim: To identify the mechanisms underlying the elevation of intracellular Ca2+ level ([Ca2+]i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons. Methods: Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca2+]i was measured using fura-2 AM. Ca2+ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay. Results: Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca2+]i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca2+]i in ARC neurons depended on extracellular Ca2+, and was blocked by P/Q-type Ca2+channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca2+ channel blocker nifedipine (10 μmol/L) or N-type Ca2+channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca2+ current in ARC neurons. The low-glucose induced elevation of [Ca2+]i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L). Conclusion: Lowering glucose level enhances the activity of P/Q type Ca2+channels and elevates [Ca2+]i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β. PMID:22504905

  10. Endothelial HIF-1α Enables Hypothalamic Glucose Uptake to Drive POMC Neurons.

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    Varela, Luis; Suyama, Shigetomo; Huang, Yan; Shanabrough, Marya; Tschöp, Matthias H; Gao, Xiao-Bing; Giordano, Frank J; Horvath, Tamas L

    2017-06-01

    Glucose is the primary driver of hypothalamic proopiomelanocortin (POMC) neurons. We show that endothelial hypoxia-inducible factor 1α (HIF-1α) controls glucose uptake in the hypothalamus and that it is upregulated in conditions of undernourishment, during which POMC neuronal activity is decreased. Endothelium-specific knockdown of HIF-1α impairs the ability of POMC neurons to adapt to the changing metabolic environment in vivo, resulting in overeating after food deprivation in mice. The impaired functioning of POMC neurons was reversed ex vivo or by parenchymal glucose administration. These observations indicate an active role for endothelial cells in the central control of metabolism and suggest that central vascular impairments may cause metabolic disorders. © 2017 by the American Diabetes Association.

  11. Hypothalamic Tuberomammillary Nucleus Neurons: Electrophysiological Diversity and Essential Role in Arousal Stability.

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    Fujita, Akie; Bonnavion, Patricia; Wilson, Miryam H; Mickelsen, Laura E; Bloit, Julien; de Lecea, Luis; Jackson, Alexander C

    2017-09-27

    Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons ( Hdc -Cre) followed by a systematic census of the membrane properties of both HA and non-HA neurons in the ventral TMN (TMNv) region. Through unsupervised hierarchical cluster analysis, we found electrophysiological diversity both between TMNv HA and non-HA neurons, and among HA neurons. To directly determine the impact of acute cessation of HA neuron activity on sleep-wake states in awake and behaving mice, we examined the effects of optogenetic silencing of TMNv HA neurons in vivo We found that acute silencing of HA neurons during wakefulness promotes slow-wave sleep, but not rapid eye movement sleep, during a period of low sleep pressure. Together, these data suggest that the tonic firing of HA neurons is necessary for the maintenance of wakefulness, and their silencing not only impairs arousal but is sufficient to rapidly and selectively induce slow-wave sleep. SIGNIFICANCE STATEMENT The function of monoaminergic systems and circuits that regulate sleep and wakefulness is often disrupted as part of the pathophysiology of many neuropsychiatric disorders. One such circuit is the posterior hypothalamic histamine (HA) system, implicated in supporting wakefulness and higher brain

  12. Systemic Glucoregulation by Glucose-Sensing Neurons in the Ventromedial Hypothalamic Nucleus (VMH).

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    Shimazu, Takashi; Minokoshi, Yasuhiko

    2017-05-01

    The ventromedial hypothalamic nucleus (VMH) regulates glucose production in the liver as well as glucose uptake and utilization in peripheral tissues, including skeletal muscle and brown adipose tissue, via efferent sympathetic innervation and neuroendocrine mechanisms. The action of leptin on VMH neurons also increases glucose uptake in specific peripheral tissues through the sympathetic nervous system, with improved insulin sensitivity. On the other hand, subsets of VMH neurons, such as those that express steroidogenic factor 1 (SF1), sense changes in the ambient glucose concentration and are characterized as glucose-excited (GE) and glucose-inhibited (GI) neurons whose action potential frequency increases and decreases, respectively, as glucose levels rise. However, how these glucose-sensing (GE and GI) neurons in the VMH contribute to systemic glucoregulation remains poorly understood. In this review, we provide historical background and discuss recent advances related to glucoregulation by VMH neurons. In particular, the article describes the role of GE neurons in the control of peripheral glucose utilization and insulin sensitivity, which depend on mitochondrial uncoupling protein 2 of the neurons, as well as that of GI neurons in the control of hepatic glucose production through hypoglycemia-induced counterregulatory mechanisms.

  13. Structure-activity studies with carboxy- and amino-terminal fragments of neurotensin on hypothalamic neurons in vitro.

    Science.gov (United States)

    Baldino, F; Davis, L G; Wolfson, B

    1985-09-09

    The purpose of this study was to determine the structural requirements for the activity of neurotensin (NT1-13) on preoptic/anterior hypothalamic (POAH) neurons in vitro. Standard explant culture electrophysiological techniques were employed. NT was administered to POAH cultures through the superfusion fluid, or, to the vicinity of individual neurons by pressure ejection (0.5-10 psi) from micropipettes. Computer-generated, peri-event histograms were used to quantitate neuronal responses. Pressure ejection of NT1-13 (50 pM to 1 microM) consistently produced an excitatory effect on 30 of 42 neurons. The remaining cells were either inhibited or unaffected. Application of the C-terminal hexapeptide, NT8-13, but not the N-terminal octapeptide, NT1-8 (less than or equal to 1 mM), produced an excitatory response in 21 of 30 neurons, but was less potent than NT1-13. Application of an N-acetylated NT8-13 fragment (NTAC8-13) produced a response that was similar to that produced by NT8-13. The excitatory effects of NT1-13 and NT8-13 were maintained in medium which effectively blocked synaptic transmission (0 mM Ca2+/12 mM Mg2+ 1 mM EGTA). These data indicate that the C-terminal hexapeptide, but not the N-terminal octapeptide, produces a dose-related, excitatory effect on single neurons in the POAH in vitro. The persistence of these effects in Ca2+-free medium supports a postsynaptic site of action for these peptides.

  14. Orexin A/Hypocretin Modulates Leptin Receptor-Mediated Signaling by Allosteric Modulations Mediated by the Ghrelin GHS-R1A Receptor in Hypothalamic Neurons.

    Science.gov (United States)

    Medrano, Mireia; Aguinaga, David; Reyes-Resina, Irene; Canela, Enric I; Mallol, Josefa; Navarro, Gemma; Franco, Rafael

    2018-06-01

    The hypothalamus is a key integrator of nutrient-seeking signals in the form of hormones and metabolites originated in both the central nervous system and the periphery. The main autocrine and paracrine target of orexinergic-related hormones such as leptin, orexin/hypocretin, and ghrelin are neuropeptide Y neurons located in the arcuate nucleus of the hypothalamus. The aim of this study was to investigate the expression and the molecular and functional relationships between leptin, orexin/hypocretin and ghrelin receptors. Biophysical studies in a heterologous system showed physical interactions between them, with potential formation of heterotrimeric complexes. Functional assays showed robust allosteric interactions particularly different when the three receptors are expressed together. Further biochemical and pharmacological assays provided evidence of heterotrimer functional expression in primary cultures of hypothalamic neurons. These findings constitute evidence of close relationships in the action of the three hormones already starting at the receptor level in hypothalamic cells.

  15. Phosphatidyl inositol 3-kinase signaling in hypothalamic proopiomelanocortin neurons contributes to the regulation of glucose homeostasis.

    Science.gov (United States)

    Hill, Jennifer W; Xu, Yong; Preitner, Frederic; Fukuda, Makota; Cho, You-Ree; Luo, Ji; Balthasar, Nina; Coppari, Roberto; Cantley, Lewis C; Kahn, Barbara B; Zhao, Jean J; Elmquist, Joel K

    2009-11-01

    Recent studies demonstrated a role for hypothalamic insulin and leptin action in the regulation of glucose homeostasis. This regulation involves proopiomelanocortin (POMC) neurons because suppression of phosphatidyl inositol 3-kinase (PI3K) signaling in these neurons blunts the acute effects of insulin and leptin on POMC neuronal activity. In the current study, we investigated whether disruption of PI3K signaling in POMC neurons alters normal glucose homeostasis using mouse models designed to both increase and decrease PI3K-mediated signaling in these neurons. We found that deleting p85alpha alone induced resistance to diet-induced obesity. In contrast, deletion of the p110alpha catalytic subunit of PI3K led to increased weight gain and adipose tissue along with reduced energy expenditure. Independent of these effects, increased PI3K activity in POMC neurons improved insulin sensitivity, whereas decreased PI3K signaling resulted in impaired glucose regulation. These studies show that activity of the PI3K pathway in POMC neurons is involved in not only normal energy regulation but also glucose homeostasis.

  16. Presynaptic inhibition of GABAergic synaptic transmission by adenosine in mouse hypothalamic hypocretin neurons.

    Science.gov (United States)

    Xia, J X; Xiong, J X; Wang, H K; Duan, S M; Ye, J N; Hu, Z A

    2012-01-10

    Hypocretin neurons in the lateral hypothalamus, a new wakefulness-promoting center, have been recently regarded as an important target involved in endogenous adenosine-regulating sleep homeostasis. The GABAergic synaptic transmissions are the main inhibitory afferents to hypocretin neurons, which play an important role in the regulation of excitability of these neurons. The inhibitory effect of adenosine, a homeostatic sleep-promoting factor, on the excitatory glutamatergic synaptic transmissions in hypocretin neurons has been well documented, whether adenosine also modulates these inhibitory GABAergic synaptic transmissions in these neurons has not been investigated. In this study, the effect of adenosine on inhibitory postsynaptic currents (IPSCs) in hypocretin neurons was examined by using perforated patch-clamp recordings in the acute hypothalamic slices. The findings demonstrated that adenosine suppressed the amplitude of evoked IPSCs in a dose-dependent manner, which was completely abolished by 8-cyclopentyltheophylline (CPT), a selective antagonist of adenosine A1 receptor but not adenosine A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl) xanthine. A presynaptic origin was suggested as following: adenosine increased paired-pulse ratio as well as reduced GABAergic miniature IPSC frequency without affecting the miniature IPSC amplitude. Further findings demonstrated that when the frequency of electrical stimulation was raised to 10 Hz, but not 1 Hz, a time-dependent depression of evoked IPSC amplitude was detected in hypocretin neurons, which could be partially blocked by CPT. However, under a higher frequency at 100 Hz stimulation, CPT had no action on the depressed GABAergic synaptic transmission induced by such tetanic stimulation in these hypocretin neurons. These results suggest that endogenous adenosine generated under certain stronger activities of synaptic transmissions exerts an inhibitory effect on GABAergic synaptic transmission in hypocretin

  17. Glucose and insulin induce Ca2+ signaling in nesfatin-1 neurons in the hypothalamic paraventricular nucleus.

    Science.gov (United States)

    Gantulga, Darambazar; Maejima, Yuko; Nakata, Masanori; Yada, Toshihiko

    2012-04-20

    Nucleobindin-2 derived nesfatin-1 in the hypothalamic paraventricular nucleus (PVN) plays a role in inhibition of feeding. The neural pathways downstream of PVN nesfatin-1 have been extensively investigated. However, regulation of the PVN nesfatin-1 neurons remains unclear. Since starvation decreases and refeeding stimulates nesfatin-1 expression specifically in the PVN, this study aimed to clarify direct effects of meal-evoked metabolic factors, glucose and insulin, on PVN nesfatin-1 neurons. High glucose (10mM) and insulin (10(-13)M) increased cytosolic calcium concentration ([Ca(2+)](i)) in 55 of 331 (16.6%) and 32 of 249 (12.9%) PVN neurons, respectively. Post [Ca(2+)](i) measurement immunocytochemistry identified that 58.2% of glucose-responsive and 62.5% of insulin-responsive neurons were immunoreactive to nesfatin-1. Furthermore, a fraction of the glucose-responsive nesfatin-1 neurons also responded to insulin, and vice versa. Some of the neurons that responded to neither glucose nor insulin were recruited to [Ca(2+)](i) increases by glucose and insulin in combination. Our data demonstrate that glucose and insulin directly interact with and increase [Ca(2+)](i) in nesfatin-1 neurons in the PVN, and that the nesfatin-1 neuron is the primary target for them in the PVN. The results suggest that high glucose- and insulin-induced activation of PVN nesfatin-1 neurons serves as a mechanism through which meal ingestion stimulates nesfatin-1 neurons in the PVN and thereby produces satiety. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. The Brain–to–Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions

    Science.gov (United States)

    Rosario, Wilfredo; Singh, Inderroop; Wautlet, Arnaud; Patterson, Christa; Flak, Jonathan; Becker, Thomas C.; Ali, Almas; Tamarina, Natalia; Philipson, Louis H.; Enquist, Lynn W.; Myers, Martin G.

    2016-01-01

    The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia. PMID:27207534

  19. The Brain-to-Pancreatic Islet Neuronal Map Reveals Differential Glucose Regulation From Distinct Hypothalamic Regions.

    Science.gov (United States)

    Rosario, Wilfredo; Singh, Inderroop; Wautlet, Arnaud; Patterson, Christa; Flak, Jonathan; Becker, Thomas C; Ali, Almas; Tamarina, Natalia; Philipson, Louis H; Enquist, Lynn W; Myers, Martin G; Rhodes, Christopher J

    2016-09-01

    The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia. © 2016 by the American Diabetes Association.

  20. Astrocytes Modulate a Postsynaptic NMDA–GABAA-Receptor Crosstalk in Hypothalamic Neurosecretory Neurons

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    Potapenko, Evgeniy S.; Biancardi, Vinicia C.; Zhou, Yiqiang

    2013-01-01

    A dynamic balance between the excitatory and inhibitory neurotransmitters glutamate and GABA is critical for maintaining proper neuronal activity in the brain. This balance is partly achieved via presynaptic interactions between glutamatergic and GABAAergic synapses converging into the same targets. Here, we show that in hypothalamic magnocellular neurosecretory neurons (MNCs), a direct crosstalk between postsynaptic NMDA receptors (NMDARs) and GABAA receptors (GABAARs) contributes to the excitatory/inhibitory balance in this system. We found that activation of NMDARs by endogenous glutamate levels controlled by astrocyte glutamate transporters, evokes a transient and reversible potentiation of postsynaptic GABAARs. This inter-receptor crosstalk is calcium-dependent and involves a kinase-dependent phosphorylation mechanism, but does not require nitric oxide as an intermediary signal. Finally, we found the NMDAR–GABAAR crosstalk to be blunted in rats with heart failure, a pathological condition in which the hypothalamic glutamate–GABA balance is tipped toward an excitatory predominance. Together, our findings support a novel form of glutamate–GABA interactions in MNCs, which involves crosstalk between NMDA and GABAA postsynaptic receptors, whose strength is controlled by the activity of local astrocytes. We propose this inter-receptor crosstalk to act as a compensatory, counterbalancing mechanism to dampen glutamate-mediated overexcitation. Finally, we propose that an uncoupling between NMDARs and GABAARs may contribute to exacerbated neuronal activity and, consequently, sympathohumoral activation in such disease conditions as heart failure. PMID:23303942

  1. Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit.

    Science.gov (United States)

    Larsen, P J; Seier, V; Fink-Jensen, A; Holst, J J; Warberg, J; Vrang, N

    2003-03-01

    Cocaine- and amphetamine-regulated transcript (CART) is present in a number of hypothalamic nuclei. Besides actions in circuits regulating feeding behaviour and stress responses, the hypothalamic functions of CART are largely unknown. We report that CART immunoreactivity is present in hypothalamic neuroendocrine neurones. Adult male rats received a systemic injection of the neuronal tracer Fluorogold (FG) 2 days before fixation, and subsequent double- and triple-labelling immunoflourescence analysis demonstrated that neuroendocrine CART-containing neurones were present in the anteroventral periventricular, supraoptic, paraventricular (PVN) and periventricular nuclei of the hypothalamus. In the PVN, CART-positive neuroendocrine neurones were found in all of cytoarchitectonically identified nuclei. In the periventricular nucleus, approximately one-third of somatostatin cells were also CART-immunoreactive. In the medial parvicellular subnucleus of the PVN, CART and FG coexisted with thyrotrophin-releasing hormone, whereas very few of the corticotrophin-releasing hormone containing cells were CART-immunoreactive. In the arcuate nucleus, CART was extensively colocalized with pro-opiomelanocortin in the ventrolateral part, but completely absent from neuroendocrine neurones of the dorsomedial part. To assess the possible role of CART as a hypothalamic-releasing factor, immunoreactive CART was measured in blood samples from the long portal vessels connecting the median eminence with the anterior pituitary gland. Adult male rats were anaesthetized and the infundibular stalk exposed via a transpharyngeal approach. The long portal vessels were transected and blood collected in 30-min periods (one prestimulatory and three poststimulatory periods). Compared to systemic venous plasma samples, baseline concentrations of immunoreactive CART were elevated in portal plasma. Exposure to sodium nitroprusside hypotension triggered a two-fold elevation of portal CART42

  2. Activation of hypothalamic RIP-Cre neurons promotes beiging of WAT via sympathetic nervous system.

    Science.gov (United States)

    Wang, Baile; Li, Ang; Li, Xiaomu; Ho, Philip Wl; Wu, Donghai; Wang, Xiaoqi; Liu, Zhuohao; Wu, Kelvin Kl; Yau, Sonata Sy; Xu, Aimin; Cheng, Kenneth Ky

    2018-04-01

    Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat-insulin-promoter-Cre (RIP-Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT Genetic ablation of APPL2 in RIP-Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP-Cre neurons, inactivation of VMH AMPK, or treatment with a β3-adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP-Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP-Cre neurons, in which the APPL2-AMPK signaling axis is crucial for this defending mechanism to cold and obesity. © 2018 The Authors.

  3. Neuropeptide co-expression in hypothalamic kisspeptin neurons of laboratory animals and the human

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    Katalin eSkrapits

    2015-02-01

    Full Text Available Hypothalamic peptidergic neurons using kisspeptin (KP and its co-transmitters for communication are critically involved in the regulation of mammalian reproduction and puberty. This article provides an overview of neuropeptides present in KP neurons, with a focus on the human species. Immunohistochemical studies reveal that large subsets of human KP neurons synthesize neurokinin B, as also shown in laboratory species. In contrast, dynorphin described in KP neurons of rodents and sheep is found rarely in KP cells of human males and postmenopausal females. Similarly, galanin is detectable in mouse, but not human, KP cells, whereas substance P, cocaine- and amphetamine-regulated transcript and proenkephalin-derived opioids are expressed in varying subsets of KP neurons in humans, but not reported in ARC of other species. Human KP neurons do not contain neurotensin, cholecystokinin, proopiomelanocortin-derivatives, agouti-related protein, neuropeptide Y, somatostatin or tyrosine hydroxylase (dopamine. These data identify the possible co-transmitters of human KP cells. Neurochemical properties distinct from those of laboratory species indicate that humans use considerably different neurotransmitter mechanisms to regulate fertility.

  4. To Ingest or Rest? Specialized Roles of Lateral Hypothalamic Area Neurons in Coordinating Energy Balance

    Directory of Open Access Journals (Sweden)

    Juliette A. Brown

    2015-02-01

    Full Text Available Survival depends on an organism’s ability to sense nutrient status and accordingly regulate intake and energy expenditure behaviors. Uncoupling of energy sensing and behavior, however, underlies energy balance disorders such as anorexia or obesity. The hypothalamus regulates energy balance, and in particular the lateral hypothalamic area (LHA is poised to coordinate peripheral cues of energy status and behaviors that impact weight, such as drinking, locomotor behavior, arousal/sleep and autonomic output. There are several populations of LHA neurons that are defined by their neuropeptide content and contribute to energy balance. LHA neurons that express the neuropeptides melanin-concentrating hormone (MCH or orexins/hypocretins (OX are best characterized and these neurons play important roles in regulating ingestion, arousal, locomotor behavior and autonomic function via distinct neuronal circuits. Recently, another population of LHA neurons containing the neuropeptide Neurotensin (Nts has been implicated in coordinating anorectic stimuli and behavior to regulate hydration and energy balance. Understanding the specific roles of MCH, OX and Nts neurons in harmonizing energy sensing and behavior thus has the potential to inform pharmacological strategies to modify behaviors and treat energy balance disorders.

  5. Leptin and insulin engage specific PI3K subunits in hypothalamic SF1 neurons

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    Jong-Woo Sohn

    2016-08-01

    Full Text Available Objective: The ventromedial hypothalamic nucleus (VMH regulates energy balance and glucose homeostasis. Leptin and insulin exert metabolic effects via their cognate receptors expressed by the steroidogenic factor 1 (SF1 neurons within the VMH. However, detailed cellular mechanisms involved in the regulation of these neurons by leptin and insulin remain to be identified. Methods: We utilized genetically-modified mouse models and performed patch-clamp electrophysiology experiments to resolve this issue. Results: We identified distinct populations of leptin-activated and leptin-inhibited SF1 neurons. In contrast, insulin uniformly inhibited SF1 neurons. Notably, we found that leptin-activated, leptin-inhibited, and insulin-inhibited SF1 neurons are distinct subpopulations within the VMH. Leptin depolarization of SF1 neuron also required the PI3K p110β catalytic subunit. This effect was mediated by the putative transient receptor potential C (TRPC channel. On the other hand, hyperpolarizing responses of SF1 neurons by leptin and insulin required either of the p110α or p110β catalytic subunits, and were mediated by the putative ATP-sensitive K+ (KATP channel. Conclusions: Our results demonstrate that specific PI3K catalytic subunits are responsible for the acute effects of leptin and insulin on VMH SF1 neurons, and provide insights into the cellular mechanisms of leptin and insulin action on VMH SF1 neurons that regulate energy balance and glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: Cellular mechanism, Conditional knockout mouse, Patch clamp technique, Functional heterogeneity, Homeostasis

  6. Developmental programming of hypothalamic neuronal circuits: impact on energy balance control

    Science.gov (United States)

    Gali Ramamoorthy, Thanuja; Begum, Ghazala; Harno, Erika; White, Anne

    2015-01-01

    The prevalence of obesity in adults and children has increased globally at an alarming rate. Mounting evidence from both epidemiological studies and animal models indicates that adult obesity and associated metabolic disorders can be programmed by intrauterine and early postnatal environment- a phenomenon known as “fetal programming of adult disease.” Data from nutritional intervention studies in animals including maternal under- and over-nutrition support the developmental origins of obesity and metabolic syndrome. The hypothalamic neuronal circuits located in the arcuate nucleus controlling appetite and energy expenditure are set early in life and are perturbed by maternal nutritional insults. In this review, we focus on the effects of maternal nutrition in programming permanent changes in these hypothalamic circuits, with experimental evidence from animal models of maternal under- and over-nutrition. We discuss the epigenetic modifications which regulate hypothalamic gene expression as potential molecular mechanisms linking maternal diet during pregnancy to the offspring's risk of obesity at a later age. Understanding these mechanisms in key metabolic genes may provide insights into the development of preventative intervention strategies. PMID:25954145

  7. Developmental programming of hypothalamic neuronal circuits: impact on energy balance control

    Directory of Open Access Journals (Sweden)

    Thanuja eGali Ramamoorthy

    2015-04-01

    Full Text Available The prevalence of obesity in adults and children has increased globally at an alarming rate. Mounting evidence from both epidemiological studies and animal models indicates that adult obesity and associated metabolic disorders can be programmed by intrauterine and early postnatal environment- a phenomenon known as fetal programming of adult disease. Data from nutritional intervention studies in animals including maternal under- and over-nutrition support the developmental origins of obesity and metabolic syndrome. The hypothalamic neuronal circuits located in the arcuate nucleus controlling appetite and energy expenditure are set early in life and are perturbed by maternal nutritional insults. In this review, we focus on the effects of maternal nutrition in programming permanent changes in these hypothalamic circuits, with experimental evidence from animal models of maternal under- and over-nutrition. We discuss the epigenetic modifications which regulate hypothalamic gene expression as potential molecular mechanisms linking maternal diet during pregnancy to the offspring’s risk of obesity at a later age. Understanding these mechanisms in key metabolic genes may provide insights into the development of preventative intervention strategies.

  8. Stress and Sucrose Intake Modulate Neuronal Activity in the Anterior Hypothalamic Area in Rats.

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    Mitra, Arojit; Guèvremont, Geneviève; Timofeeva, Elena

    2016-01-01

    The anterior hypothalamic area (AHA) is an important integrative relay structure for a variety of autonomic, endocrine, and behavioral responses including feeding behavior and response to stress. However, changes in the activity of the AHA neurons during stress and feeding in freely moving rats are not clear. The present study investigated the firing rate and burst activity of neurons in the central nucleus of the AHA (cAHA) during sucrose intake in non-stressful conditions and after acute stress in freely behaving rats. Rats were implanted with micro-electrodes into the cAHA, and extracellular multi-unit activity was recorded during 1-h access to 10% sucrose in non-stressful conditions or after acute foot shock stress. Acute stress significantly reduced sucrose intake, total sucrose lick number, and lick frequency in licking clusters, and increased inter-lick intervals. At the cluster start (CS) of sucrose licking, the cAHA neurons increased (CS-excited, 20% of the recorded neurons), decreased (CS-inhibited, 42% of the neurons) or did not change (CS-nonresponsive, 38% of the neurons) their firing rate. Stress resulted in a significant increase in the firing rate of the CS-inhibited neurons by decreasing inter-spike intervals within the burst firing of these neurons. This increase in the stress-induced firing rate of the CS-inhibited neurons was accompanied by a disruption of the correlation between the firing rate of CS-inhibited and CS-nonresponsive neurons that was observed in non-stressful conditions. Stress did not affect the firing rate of the CS-excited and CS-nonresponsive neurons. However, stress changed the pattern of burst firing of the CS-excited and CS-nonresponsive neurons by decreasing and increasing the burst number in the CS-excited and CS-nonresponsive neurons, respectively. These results suggest that the cAHA neurons integrate the signals related to stress and intake of palatable food and play a role in the stress- and eating-related circuitry.

  9. Activation of synaptic and extrasynaptic glycine receptors by taurine in preoptic hypothalamic neurons.

    Science.gov (United States)

    Bhattarai, Janardhan Prasad; Park, Soo Joung; Chun, Sang Woo; Cho, Dong Hyu; Han, Seong Kyu

    2015-11-03

    Taurine is an essential amino-sulfonic acid having a fundamental function in the brain, participating in both cell volume regulation and neurotransmission. Using a whole cell voltage patch clamp technique, the taurine-activated neurotransmitter receptors in the preoptic hypothalamic area (PHA) neurons were investigated. In the first set of experiments, different concentrations of taurine were applied on PHA neurons. Taurine-induced responses were concentration-dependent. Taurine-induced currents were action potential-independent and sensitive to strychnine, suggesting the involvement of glycine receptors. In addition, taurine activated not only α-homomeric, but also αβ-heteromeric glycine receptors in PHA neurons. Interestingly, a low concentration of taurine (0.5mM) activated glycine receptors, whereas a higher concentration (3mM) activated both glycine and gamma-aminobutyric acid A (GABAA) receptors in PHA neurons. These results suggest that PHA neurons are influenced by taurine and respond via glycine and GABAA receptors. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  10. Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning

    Science.gov (United States)

    Joly-Amado, Aurélie; Denis, Raphaël G P; Castel, Julien; Lacombe, Amélie; Cansell, Céline; Rouch, Claude; Kassis, Nadim; Dairou, Julien; Cani, Patrice D; Ventura-Clapier, Renée; Prola, Alexandre; Flamment, Melissa; Foufelle, Fabienne; Magnan, Christophe; Luquet, Serge

    2012-01-01

    Obesity-related diseases such as diabetes and dyslipidemia result from metabolic alterations including the defective conversion, storage and utilization of nutrients, but the central mechanisms that regulate this process of nutrient partitioning remain elusive. As positive regulators of feeding behaviour, agouti-related protein (AgRP) producing neurons are indispensible for the hypothalamic integration of energy balance. Here, we demonstrate a role for AgRP-neurons in the control of nutrient partitioning. We report that ablation of AgRP-neurons leads to a change in autonomic output onto liver, muscle and pancreas affecting the relative balance between lipids and carbohydrates metabolism. As a consequence, mice lacking AgRP-neurons become obese and hyperinsulinemic on regular chow but display reduced body weight gain and paradoxical improvement in glucose tolerance on high-fat diet. These results provide a direct demonstration of a role for AgRP-neurons in the coordination of efferent organ activity and nutrient partitioning, providing a mechanistic link between obesity and obesity-related disorders. PMID:22990237

  11. Nutrient sensing and insulin signaling in neuropeptide-expressing immortalized, hypothalamic neurons: A cellular model of insulin resistance.

    Science.gov (United States)

    Fick, Laura J; Belsham, Denise D

    2010-08-15

    Obesity and type 2 diabetes mellitus represent a significant global health crisis. These two interrelated diseases are typified by perturbed insulin signaling in the hypothalamus. Using novel hypothalamic cell lines, we have begun to elucidate the molecular and intracellular mechanisms involved in the hypothalamic control of energy homeostasis and insulin resistance. In this review, we present evidence of insulin and glucose signaling pathways that lead to changes in neuropeptide gene expression. We have identified some of the molecular mechanisms involved in the control of de novo hypothalamic insulin mRNA expression. And finally, we have defined key mechanisms involved in the etiology of cellular insulin resistance in hypothalamic neurons that may play a fundamental role in cases of high levels of insulin or saturated fatty acids, often linked to the exacerbation of obesity and diabetes.

  12. Direct projections from hypothalamic orexin neurons to brainstem cardiac vagal neurons.

    Science.gov (United States)

    Dergacheva, Olga; Yamanaka, Akihiro; Schwartz, Alan R; Polotsky, Vsevolod Y; Mendelowitz, David

    2016-12-17

    Orexin neurons are known to augment the sympathetic control of cardiovascular function, however the role of orexin neurons in parasympathetic cardiac regulation remains unclear. To test the hypothesis that orexin neurons contribute to parasympathetic control we selectively expressed channelrhodopsin-2 (ChR2) in orexin neurons in orexin-Cre transgenic rats and examined postsynaptic currents in cardiac vagal neurons (CVNs) in the dorsal motor nucleus of the vagus (DMV). Simultaneous photostimulation and recording in ChR2-expressing orexin neurons in the lateral hypothalamus resulted in reliable action potential firing as well as large whole-cell currents suggesting a strong expression of ChR2 and reliable optogenetic excitation. Photostimulation of ChR2-expressing fibers in the DMV elicited short-latency (ranging from 3.2ms to 8.5ms) postsynaptic currents in 16 out of 44 CVNs tested. These responses were heterogeneous and included excitatory glutamatergic (63%) and inhibitory GABAergic (37%) postsynaptic currents. The results from this study suggest different sub-population of orexin neurons may exert diverse influences on brainstem CVNs and therefore may play distinct functional roles in parasympathetic control of the heart. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Cocaine- and amphetamine-regulated transcript is present in hypothalamic neuroendocrine neurones and is released to the hypothalamic-pituitary portal circuit

    DEFF Research Database (Denmark)

    Larsen, P J; Seier, V; Fink-Jensen, A

    2003-01-01

    Cocaine- and amphetamine-regulated transcript (CART) is present in a number of hypothalamic nuclei. Besides actions in circuits regulating feeding behaviour and stress responses, the hypothalamic functions of CART are largely unknown. We report that CART immunoreactivity is present in hypothalami......, supraoptic, paraventricular (PVN) and periventricular nuclei of the hypothalamus. In the PVN, CART-positive neuroendocrine neurones were found in all of cytoarchitectonically identified nuclei. In the periventricular nucleus, approximately one-third of somatostatin cells were also CART......-immunoreactive. In the medial parvicellular subnucleus of the PVN, CART and FG coexisted with thyrotrophin-releasing hormone, whereas very few of the corticotrophin-releasing hormone containing cells were CART-immunoreactive. In the arcuate nucleus, CART was extensively colocalized with pro...

  14. Ventral medullary neurones excited from the hypothalamic and mid-brain defence areas.

    Science.gov (United States)

    Hilton, S M; Smith, P R

    1984-07-01

    In cats anaesthetised with chloralose, the ventral medulla was explored in and around the strip previously identified as the location of the efferent pathway from the hypothalamic and mid-brain defence areas to the spinal cord, in a search for neurones excited by electrical stimulation of the defence areas. Such units were found mostly in the caudal part of this strip, at a depth of not more than 500 microns from the surface. Nearly all were located in the ventral part of nucleus paragigantocellularis lateralis (PGL) at the level of the rostral pole of the inferior olive. There was evidence of temporal and spatial facilitation, indicating a convergent excitatory input from the defence areas onto neurones in PGL. This is consistent with earlier evidence of a synaptic relay in the efferent pathway at this site. When the pathway is blocked at this site, arterial blood pressure falls profoundly, so activity in these neurones may be essential for the normal level of sympathetic nerve activity.

  15. Hypothalamic vasopressinergic projections innervate central amygdala GABAergic neurons: implications for anxiety and stress coping

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    Vito Salvador Hernandez

    2016-11-01

    Full Text Available The arginine-vasopressin (AVP-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs are known for their role in hydro-electrolytic balance control via their projections to neurohypophysis. Recently, projections from these same neurons to hippocampus, habenula, and other brain regions, in which vasopressin infusion modulates contingent social and emotionally-affected behaviors, have been reported. Here, we present evidence that VPMNN collaterals also project to the amygdaloid complex, and establish synaptic connections with neurons in central amygdala (CeA. The density of AVP innervation in amygdala was substantially increased in adult rats that had experienced neonatal maternal separation (MS, consistent with our previous observations that MS enhances VPMNN number in the paraventricular (PVN and supraoptic (SON nuclei of the hypothalamus. In the CeA, V1a AVP receptor mRNA was only observed in GABAergic neurons, demonstrated by complete co-localization of V1a transcripts in neurons expressing Gad1 and Gad2 transcripts in CeA using the RNAscope method. V1b and V2 receptors mRNA were not detected, using the same method. Water-deprivation for 24 hrs, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze test, and this effect was mimicked by bilateral microinfusion of VP into the CeA. Anxious behavior induced by either water deprivation or VP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of central amygdala inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala.

  16. CART neurons in the arcuate nucleus and lateral hypothalamic area exert differential controls on energy homeostasis

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    Jackie Lau

    2018-01-01

    Full Text Available Objective: The cocaine- and amphetamine-regulated transcript (CART codes for a pivotal neuropeptide important in the control of appetite and energy homeostasis. However, limited understanding exists for the defined effector sites underlying CART function, as discrepant effects of central CART administration have been reported. Methods: By combining Cart-cre knock-in mice with a Cart adeno-associated viral vector designed using the flip-excision switch (AAV-FLEX technology, specific reintroduction or overexpression of CART selectively in CART neurons in the arcuate nucleus (Arc and lateral hypothalamic area (LHA, respectively, was achieved. The effects on energy homeostasis control were investigated. Results: Here we show that CART neuron-specific reintroduction of CART into the Arc and LHA leads to distinct effects on energy homeostasis control. Specifically, CART reintroduction into the Arc of otherwise CART-deficient Cartcre/cre mice markedly decreased fat mass and body weight, whereas CART reintroduction into the LHA caused significant fat mass gain and lean mass loss, but overall unaltered body weight. The reduced adiposity in ArcCART;Cartcre/cre mice was associated with an increase in both energy expenditure and physical activity, along with significantly decreased Npy mRNA levels in the Arc but with no change in food consumption. Distinctively, the elevated fat mass in LHACART;Cartcre/cre mice was accompanied by diminished insulin responsiveness and glucose tolerance, greater spontaneous food intake, and reduced energy expenditure, which is consistent with the observed decrease of brown adipose tissue temperature. This is also in line with significantly reduced tyrosine hydroxylase (Th and notably increased corticotropin-releasing hormone (Crh mRNA expressions in the paraventricular nucleus (PVN. Conclusions: Taken together, these results identify catabolic and anabolic effects of CART in the Arc and LHA, respectively, demonstrating for

  17. Effects of tamoxifen on neuronal morphology, connectivity and biochemistry of hypothalamic ventromedial neurons: Impact on the modulators of sexual behavior.

    Science.gov (United States)

    Sá, Susana I; Teixeira, Natércia; Fonseca, Bruno M

    2018-01-01

    Tamoxifen (TAM) is a selective estrogen receptor modulator, widely used in the treatment and prevention of estrogen-dependent breast cancer. Although with great clinical results, women on TAM therapy still report several side effects, such as sexual dysfunction, which impairs quality of life. The anatomo-functional substrates of the human sexual behavior are still unknown; however, these same substrates are very well characterized in the rodent female sexual behavior, which has advantage of being a very simple reflexive response, dependent on the activation of estrogen receptors (ERs) in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl). In fact, in the female rodent, the sexual behavior is triggered by increasing circulation levels of estradiol that changes the nucleus neurochemistry and modulates its intricate neuronal network. Therefore, we considered of notice the examination of the possible neurochemical alterations and the synaptic plasticity impairment in VMNvl neurons of estradiol-primed female rats treated with TAM that may be in the basis of this neurological disorder. Accordingly, we used stereological and biochemical methods to study the action of TAM in axospinous and axodendritic synaptic plasticity and on ER expression. The administration of TAM changed the VMNvl neurochemistry by reducing ERα mRNA and increasing ERβ mRNA expression. Furthermore, present results show that TAM induced neuronal atrophy and reduced synaptic connectivity, favoring electrical inactivity. These data suggest that these cellular and molecular changes may be a possible neuronal mechanism of TAM action in the disruption of the VMNvl network, leading to the development of behavioral disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. β-arrestin regulates estradiol membrane-initiated signaling in hypothalamic neurons.

    Directory of Open Access Journals (Sweden)

    Angela M Wong

    Full Text Available Estradiol (E2 action in the nervous system is the result of both direct nuclear and membrane-initiated signaling (EMS. E2 regulates membrane estrogen receptor-α (ERα levels through opposing mechanisms of EMS-mediated trafficking and internalization. While ß-arrestin-mediated mERα internalization has been described in the cortex, a role of ß-arrestin in EMS, which underlies multiple physiological processes, remains undefined. In the arcuate nucleus of the hypothalamus (ARH, membrane-initiated E2 signaling modulates lordosis behavior, a measure of female sexually receptivity. To better understand EMS and regulation of ERα membrane levels, we examined the role of ß-arrestin, a molecule associated with internalization following agonist stimulation. In the present study, we used an immortalized neuronal cell line derived from embryonic hypothalamic neurons, the N-38 line, to examine whether ß-arrestins mediate internalization of mERα. β-arrestin-1 (Arrb1 was found in the ARH and in N-38 neurons. In vitro, E2 increased trafficking and internalization of full-length ERα and ERαΔ4, an alternatively spliced isoform of ERα, which predominates in the membrane. Treatment with E2 also increased phosphorylation of extracellular-signal regulated kinases 1/2 (ERK1/2 in N-38 neurons. Arrb1 siRNA knockdown prevented E2-induced ERαΔ4 internalization and ERK1/2 phosphorylation. In vivo, microinfusions of Arrb1 antisense oligodeoxynucleotides (ODN into female rat ARH knocked down Arrb1 and prevented estradiol benzoate-induced lordosis behavior compared with nonsense scrambled ODN (lordosis quotient: 3 ± 2.1 vs. 85.0 ± 6.0; p < 0.0001. These results indicate a role for Arrb1 in both EMS and internalization of mERα, which are required for the E2-induction of female sexual receptivity.

  19. Oleate induces KATP channel-dependent hyperpolarization in mouse hypothalamic glucose-excited neurons without altering cellular energy charge.

    Science.gov (United States)

    Dadak, Selma; Beall, Craig; Vlachaki Walker, Julia M; Soutar, Marc P M; McCrimmon, Rory J; Ashford, Michael L J

    2017-03-27

    The unsaturated fatty acid, oleate exhibits anorexigenic properties reducing food intake and hepatic glucose output. However, its mechanism of action in the hypothalamus has not been fully determined. This study investigated the effects of oleate and glucose on GT1-7 mouse hypothalamic cells (a model of glucose-excited (GE) neurons) and mouse arcuate nucleus (ARC) neurons. Whole-cell and perforated patch-clamp recordings, immunoblotting and cell energy status measures were used to investigate oleate- and glucose-sensing properties of mouse hypothalamic neurons. Oleate or lowered glucose concentration caused hyperpolarization and inhibition of firing of GT1-7 cells by the activation of ATP-sensitive K + channels (K ATP ). This effect of oleate was not dependent on fatty acid oxidation or raised AMP-activated protein kinase activity or prevented by the presence of the UCP2 inhibitor genipin. Oleate did not alter intracellular calcium, indicating that CD36/fatty acid translocase may not play a role. However, oleate activation of K ATP may require ATP metabolism. The short-chain fatty acid octanoate was unable to replicate the actions of oleate on GT1-7 cells. Although oleate decreased GT1-7 cell mitochondrial membrane potential there was no change in total cellular ATP or ATP/ADP ratios. Perforated patch and whole-cell recordings from mouse hypothalamic slices demonstrated that oleate hyperpolarized a subpopulation of ARC GE neurons by K ATP activation. Additionally, in a separate small population of ARC neurons, oleate application or lowered glucose concentration caused membrane depolarization. In conclusion, oleate induces K ATP- dependent hyperpolarization and inhibition of firing of a subgroup of GE hypothalamic neurons without altering cellular energy charge. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Pressor response to L-cysteine injected into the cisterna magna of conscious rats involves recruitment of hypothalamic vasopressinergic neurons.

    Science.gov (United States)

    Takemoto, Yumi

    2013-03-01

    The sulfur-containing non-essential amino acid L-cysteine injected into the cisterna magna of adult conscious rats produces an increase in blood pressure. The present study examined if the pressor response to L-cysteine is stereospecific and involves recruitment of hypothalamic vasopressinergic neurons and medullary noradrenergic A1 neurons. Intracisternally injected D-cysteine produced no cardiovascular changes, while L-cysteine produced hypertension and tachycardia in freely moving rats, indicating the stereospecific hemodynamic actions of L-cysteine via the brain. The double labeling immunohistochemistry combined with c-Fos detection as a marker of neuronal activation revealed significantly higher numbers of c-Fos-positive vasopressinergic neurons both in the supraoptic and paraventricular nuclei and tyrosine hydroxylase containing medullary A1 neurons, of L-cysteine-injected rats than those injected with D-cysteine as iso-osmotic control. The results indicate that the cardiovascular responses to intracisternal injection of L-cysteine in the conscious rat are stereospecific and include recruitment of hypothalamic vasopressinergic neurons both in the supraoptic and paraventricular nuclei, as well as of medullary A1 neurons. The findings may suggest a potential function of L-cysteine as an extracellular signal such as neuromodulators in central regulation of blood pressure.

  1. Tuberal hypothalamic neurons secreting the satiety molecule Nesfatin-1 are critically involved in paradoxical (REM sleep homeostasis.

    Directory of Open Access Journals (Sweden)

    Sonia Jego

    Full Text Available The recently discovered Nesfatin-1 plays a role in appetite regulation as a satiety factor through hypothalamic leptin-independent mechanisms. Nesfatin-1 is co-expressed with Melanin-Concentrating Hormone (MCH in neurons from the tuberal hypothalamic area (THA which are recruited during sleep states, especially paradoxical sleep (PS. To help decipher the contribution of this contingent of THA neurons to sleep regulatory mechanisms, we thus investigated in rats whether the co-factor Nesfatin-1 is also endowed with sleep-modulating properties. Here, we found that the disruption of the brain Nesfatin-1 signaling achieved by icv administration of Nesfatin-1 antiserum or antisense against the nucleobindin2 (NUCB2 prohormone suppressed PS with little, if any alteration of slow wave sleep (SWS. Further, the infusion of Nesfatin-1 antiserum after a selective PS deprivation, designed for elevating PS needs, severely prevented the ensuing expected PS recovery. Strengthening these pharmacological data, we finally demonstrated by using c-Fos as an index of neuronal activation that the recruitment of Nesfatin-1-immunoreactive neurons within THA is positively correlated to PS but not to SWS amounts experienced by rats prior to sacrifice. In conclusion, this work supports a functional contribution of the Nesfatin-1 signaling, operated by THA neurons, to PS regulatory mechanisms. We propose that these neurons, likely releasing MCH as a synergistic factor, constitute an appropriate lever by which the hypothalamus may integrate endogenous signals to adapt the ultradian rhythm and maintenance of PS in a manner dictated by homeostatic needs. This could be done through the inhibition of downstream targets comprised primarily of the local hypothalamic wake-active orexin- and histamine-containing neurons.

  2. Transfection in Primary Cultured Neuronal Cells.

    Science.gov (United States)

    Marwick, Katie F M; Hardingham, Giles E

    2017-01-01

    Transfection allows the introduction of foreign nucleic acid into eukaryotic cells. It is an important tool in understanding the roles of NMDARs in neurons. Here, we describe using lipofection-mediated transfection to introduce cDNA encoding NMDAR subunits into postmitotic rodent primary cortical neurons maintained in culture.

  3. Sex difference in physical activity, energy expenditure and obesity driven by a subpopulation of hypothalamic POMC neurons

    Directory of Open Access Journals (Sweden)

    Luke K. Burke

    2016-03-01

    Full Text Available Objective: Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified. Methods: Given that a 5-hydroxytryptamine 2c receptor (5-HT2CR agonist is a current obesity medication and 5-HT2CR agonist's effects on appetite are primarily mediated via POMC neurons, we hypothesized that a critical source of POMC regulating food intake and body weight is specifically synthesized in cells containing 5-HT2CRs. To exclusively manipulate Pomc synthesis only within 5-HT2CR containing cells, we generated a novel 5-HT2CRCRE mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable PomcNEO mice to restrict Pomc synthesis to the subset of hypothalamic cells containing 5-HT2CRs. This provided a means to clarify the specific contribution of a defined subgroup of POMC peptides in energy balance and body weight. Results: Here we transform genetically programed obese and hyperinsulinemic male mice lacking hypothalamic Pomc with increased appetite, reduced physical activity and compromised brown adipose tissue (BAT into lean, healthy mice via targeted restoration of Pomc function only within 5-HT2CR expressing cells. Remarkably, the same metabolic transformation does not occur in females, who despite corrected feeding behavior and normalized insulin levels remain physically inactive, have lower energy expenditure, compromised BAT and develop obesity. Conclusions: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc

  4. Advances in 3D neuronal cell culture

    NARCIS (Netherlands)

    Frimat, Jean Philippe; Xie, Sijia; Bastiaens, Alex; Schurink, Bart; Wolbers, Floor; Den Toonder, Jaap; Luttge, Regina

    2015-01-01

    In this contribution, the authors present our advances in three-dimensional (3D) neuronal cell culture platform technology contributing to controlled environments for microtissue engineering and analysis of cellular physiological and pathological responses. First, a micromachined silicon sieving

  5. Postsynaptic Depolarization Enhances GABA Drive to Dorsomedial Hypothalamic Neurons through Somatodendritic Cholecystokinin Release.

    Science.gov (United States)

    Crosby, Karen M; Baimoukhametova, Dinara V; Bains, Jaideep S; Pittman, Quentin J

    2015-09-23

    Somatodendritically released peptides alter synaptic function through a variety of mechanisms, including autocrine actions that liberate retrograde transmitters. Cholecystokinin (CCK) is a neuropeptide expressed in neurons in the dorsomedial hypothalamic nucleus (DMH), a region implicated in satiety and stress. There are clear demonstrations that exogenous CCK modulates food intake and neuropeptide expression in the DMH, but there is no information on how endogenous CCK alters synaptic properties. Here, we provide the first report of somatodendritic release of CCK in the brain in male Sprague Dawley rats. CCK is released from DMH neurons in response to repeated postsynaptic depolarizations, and acts in an autocrine fashion on CCK2 receptors to enhance postsynaptic NMDA receptor function and liberate the retrograde transmitter, nitric oxide (NO). NO subsequently acts presynaptically to enhance GABA release through a soluble guanylate cyclase-mediated pathway. These data provide the first demonstration of synaptic actions of somatodendritically released CCK in the hypothalamus and reveal a new form of retrograde plasticity, depolarization-induced potentiation of inhibition. Significance statement: Somatodendritic signaling using endocannabinoids or nitric oxide to alter the efficacy of afferent transmission is well established. Despite early convincing evidence for somatodendritic release of neurohypophysial peptides in the hypothalamus, there is only limited evidence for this mode of release for other peptides. Here, we provide the first evidence for somatodendritic release of the satiety peptide cholecystokinin (CCK) in the brain. We also reveal a new form of synaptic plasticity in which postsynaptic depolarization results in enhancement of inhibition through the somatodendritic release of CCK. Copyright © 2015 the authors 0270-6474/15/3513160-11$15.00/0.

  6. Knockin of Cre Gene at Ins2 Locus Reveals No Cre Activity in Mouse Hypothalamic Neurons.

    Science.gov (United States)

    Li, Ling; Gao, Lin; Wang, Kejia; Ma, Xianhua; Chang, Xusheng; Shi, Jian-Hui; Zhang, Ye; Yin, Kai; Liu, Zhimin; Shi, Yuguang; Xie, Zhifang; Zhang, Weiping J

    2016-02-02

    The recombination efficiency and cell specificity of Cre driver lines are critical for exploring pancreatic β cell biology with the Cre/LoxP approach. Some commonly used Cre lines are based on the short Ins2 promoter fragment and show recombination activity in hypothalamic neurons; however, whether this stems from endogenous Ins2 promoter activity remains controversial. In this study, we generated Ins2-Cre knockin mice with a targeted insertion of IRES-Cre at the Ins2 locus and demonstrated with a cell lineage tracing study that the Ins2 gene is not transcriptionally active in the hypothalamus. The Ins2-Cre driver line displayed robust Cre expression and activity in pancreatic β cells without significant alterations in insulin expression. In the brain, Cre activity was mainly restricted to the choroid plexus, without significant recombination detected in the hippocampus or hypothalamus by the LacZ or fluorescent tdTomato reporters. Furthermore, Ins2-Cre mice exhibited normal glucose tolerance and insulin secretion upon glucose stimulation in vivo. In conclusion, this Ins2-Cre driver line allowed high-fidelity detection of endogenous Ins2 promoter activity in vivo, and the negative activity in the hypothalamus demonstrated that this system is a promising alternative tool for studying β cell biology.

  7. Protocol for culturing low density pure rat hippocampal neurons supported by mature mixed neuron cultures.

    Science.gov (United States)

    Yang, Qian; Ke, Yini; Luo, Jianhong; Tang, Yang

    2017-02-01

    primary hippocampal neuron cultures allow for subcellular morphological dissection, easy access to drug treatment and electrophysiology analysis of individual neurons, and is therefore an ideal model for the study of neuron physiology. While neuron and glia mixed cultures are relatively easy to prepare, pure neurons are particular hard to culture at low densities which are suitable for morphology studies. This may be due to a lack of neurotrophic factors such as brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and Glial cell line-derived neurotrophic factor (GDNF). In this study we used a two step protocol in which neuron-glia mixed cultures were initially prepared for maturation to support the growth of young neurons plated at very low densities. Our protocol showed that neurotrophic support resulted in physiologically functional hippocampal neurons with larger cell body, increased neurite length and decreased branching and complexity compared to cultures prepared using a conventional method. Our protocol provides a novel way to culture highly uniformed hippocampal neurons for acquiring high quality, neuron based data. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage.

    Science.gov (United States)

    Hsu, Chuan-Chih; Chen, Sheng-Hsien; Lin, Cheng-Hsien; Yung, Ming-Chi

    2014-10-01

    Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage

  9. A small population of hypothalamic neurons govern fertility: the critical role of VAX1 in GnRH neuron development and fertility maintenance.

    Science.gov (United States)

    Hoffmann, Hanne M; Mellon, Pamela L

    2016-01-01

    Fertility depends on the correct maturation and function of approximately 800 gonadotropin-releasing hormone (GnRH) neurons in the brain. GnRH neurons are at the apex of the hypothalamic-pituitary-gonadal axis that regulates fertility. In adulthood, GnRH neurons are scattered throughout the anterior hypothalamic area and project to the median eminence, where GnRH is released into the portal vasculature to stimulate release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary. LH and FSH then regulate gonadal steroidogenesis and gametogenesis. Absence of GnRH neurons or inappropriate GnRH release leads to infertility. Despite the critical role of GnRH neurons in fertility, we still have a limited understanding of the genes responsible for proper GnRH neuron development and function in adulthood. GnRH neurons originate in the olfactory placode then migrate into the brain. Homeodomain transcription factors expressed within GnRH neurons or along their migratory path are candidate genes for inherited infertility. Using a combined in vitro and in vivo approach, we have identified Ventral Anterior Homeobox 1 ( Vax1 ) as a novel homeodomain transcription factor responsible for GnRH neuron maturation and fertility. GnRH neuron counts in Vax1 knock-out embryos revealed Vax1 to be required for the presence of GnRH-expressing cells at embryonic day 17.5 (E17.5), but not at E13.5. To localize the effects of Vax1 on fertility, we generated Vax1 flox mice and crossed them with Gnrh cre mice to specifically delete Vax1 within GnRH neurons. GnRH staining in Vax1 flox/flox :GnRH cre mice show a total absence of GnRH expression in the adult. We performed lineage tracing in Vax1 flox/flox :GnRH cre :RosaLacZ mice which proved GnRH neurons to be alive, but incapable of expressing GnRH. The absence of GnRH leads to delayed puberty, hypogonadism and complete infertility in both sexes. Finally, using the immortalized model GnRH neuron cell lines, GN11 and

  10. Leptin activates oxytocin neurons of the hypothalamic paraventricular nucleus in both control and diet-induced obese rodents.

    Directory of Open Access Journals (Sweden)

    Mario Perello

    Full Text Available The adipocyte-derived hormone leptin acts in the brain to reduce body weight and fat mass. Recent studies suggest that parvocellular oxytocin (OXT neurons of the hypothalamic paraventricular nucleus (PVN can mediate body weight reduction through inhibition of food intake and increased energy expenditure. However, the role of OXT neurons of the PVN as a primary target of leptin has not been investigated. Here, we studied the potential role of OXT neurons of the PVN in leptin-mediated effects on body weight regulation in fasted rats. We demonstrated that intracerebroventricular (ICV leptin activates STAT3 phosphorylation in OXT neurons of the PVN, showed that this occurs in a subpopulation of OXT neurons that innervate the nucleus of the solitary tract (NTS, and provided further evidence suggesting a role of OXT to mediate leptin's actions on body weight. In addition, our results indicated that OXT neurons are responsive to ICV leptin and mediate leptin effects on body weight in diet induced obese (DIO rats, which are resistant to the anorectic effects of the hormone. Thus, we conclude that leptin targets a specific subpopulation of parvocellular OXT neurons of the PVN, and that this action may be important for leptin's ability to reduce body weight in both control and obese rats.

  11. CaMKII Regulates Synaptic NMDA Receptor Activity of Hypothalamic Presympathetic Neurons and Sympathetic Outflow in Hypertension.

    Science.gov (United States)

    Li, De-Pei; Zhou, Jing-Jing; Zhang, Jixiang; Pan, Hui-Lin

    2017-11-01

    NMDAR activity in the hypothalamic paraventricular nucleus (PVN) is increased and critically involved in heightened sympathetic vasomotor tone in hypertension. Calcium/calmodulin-dependent protein kinase II (CaMKII) binds to and modulates NMDAR activity. In this study, we determined the role of CaMKII in regulating NMDAR activity of PVN presympathetic neurons in male spontaneously hypertensive rats (SHRs). NMDAR-mediated EPSCs and puff NMDA-elicited currents were recorded in spinally projecting PVN neurons in SHRs and male Wistar-Kyoto (WKY) rats. The basal amplitude of evoked NMDAR-EPSCs and puff NMDA currents in retrogradely labeled PVN neurons were significantly higher in SHRs than in WKY rats. The CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP) normalized the increased amplitude of NMDAR-EPSCs and puff NMDA currents in labeled PVN neurons in SHRs but had no effect in WKY rats. Treatment with AIP also normalized the higher frequency of NMDAR-mediated miniature EPSCs of PVN neurons in SHRs. CaMKII-mediated phosphorylation level of GluN2B serine 1303 (S1303) in the PVN, but not in the hippocampus and frontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased level of phosphorylated GluN2B S1303 in the PVN. In addition, microinjection of AIP into the PVN significantly reduced arterial blood pressure and lumbar sympathetic nerve discharges in SHRs. Our findings suggest that CaMKII activity is increased in the PVN and contributes to potentiated presynaptic and postsynaptic NMDAR activity to elevate sympathetic vasomotor tone in hypertension. SIGNIFICANCE STATEMENT Heightened sympathetic vasomotor tone is a major contributor to the development of hypertension. Although glutamate NMDA receptor (NMDAR)-mediated excitatory drive in the hypothalamus plays a critical role in increased sympathetic output in hypertension, the molecular mechanism involved in

  12. Somato-dendritic localization and signaling by leptin receptors in hypothalamic POMC and AgRP neurons.

    Directory of Open Access Journals (Sweden)

    Sangdeuk Ha

    Full Text Available Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC and agouti-related peptide (AgRP/Neuropeptide Y (NPY/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb. Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM, confocal-laser scanning microscopy (CLSM, and electron microscopy (EM to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb (+/+ mice and in Leprb (db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin's central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.

  13. Hippocampal Ghrelin-positive neurons directly project to arcuate hypothalamic and medial amygdaloid nuclei. Could they modulate food-intake?

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    Russo, Cristina; Russo, Antonella; Pellitteri, Rosalia; Stanzani, Stefania

    2017-07-13

    Feeding is a process controlled by a complex of associations between external and internal stimuli. The processes that involve learning and memory seem to exert a strong control over appetite and food intake, which is modulated by a gastrointestinal hormone, Ghrelin (Ghre). Recent studies claim that Ghre is involved in cognitive and neurobiological mechanisms that underlie the conditioning of eating behaviors. The expression of Ghre increases in anticipation of food intake based on learned behaviors. The hippocampal Ghre-containing neurons neurologically influence the orexigenic hypothalamus and consequently the learned feeding behavior. The CA1 field of Ammon's horn of the hippocampus (H-CA1) constitutes the most important neural substrate to control both appetitive and ingestive behavior. It also innervates amygdala regions that in turn innervate the hypothalamus. A recent study also implies that Ghre effects on cue-potentiated feeding behavior occur, at the least, via indirect action on the amygdala. In the present study, we investigate the neural substrates through which endogenous Ghre communicates conditioned appetite and feeding behavior within the CNS. We show the existence of a neural Ghre dependent pathway whereby peripherally-derived Ghre activates H-CA1 neurons, which in turn activate Ghre-expressing hypothalamic and amygdaloid neurons to stimulate appetite and feeding behavior. To highlight this pathway, we use two fluorescent retrograde tracers (Fluoro Gold and Dil) and immunohistochemical detection of Ghre expression in the hippocampus. Triple fluorescent-labeling has determined the presence of H-CA1 Ghre-containing collateralized neurons that project to the hypothalamus and amygdala monosynaptically. We hypothesize that H-Ghre-containing neurons in H-CA1 modulate food-intake behavior through direct pathways to the arcuate hypothalamic nucleus and medial amygdaloid nucleus. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Chronic exposure to KATP channel openers results in attenuated glucose sensing in hypothalamic GT1-7 neurons.

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    Haythorne, Elizabeth; Hamilton, D Lee; Findlay, John A; Beall, Craig; McCrimmon, Rory J; Ashford, Michael L J

    2016-12-01

    Individuals with Type 1 diabetes (T1D) are often exposed to recurrent episodes of hypoglycaemia. This reduces hormonal and behavioural responses that normally counteract low glucose in order to maintain glucose homeostasis, with altered responsiveness of glucose sensing hypothalamic neurons implicated. Although the molecular mechanisms are unknown, pharmacological studies implicate hypothalamic ATP-sensitive potassium channel (K ATP ) activity, with K ATP openers (KCOs) amplifying, through cell hyperpolarization, the response to hypoglycaemia. Although initial findings, using acute hypothalamic KCO delivery, in rats were promising, chronic exposure to the KCO NN414 worsened the responses to subsequent hypoglycaemic challenge. To investigate this further we used GT1-7 cells to explore how NN414 affected glucose-sensing behaviour, the metabolic response of cells to hypoglycaemia and K ATP activity. GT1-7 cells exposed to 3 or 24 h NN414 exhibited an attenuated hyperpolarization to subsequent hypoglycaemic challenge or NN414, which correlated with diminished K ATP activity. The reduced sensitivity to hypoglycaemia was apparent 24 h after NN414 removal, even though intrinsic K ATP activity recovered. The NN414-modified glucose responsiveness was not associated with adaptations in glucose uptake, metabolism or oxidation. K ATP inactivation by NN414 was prevented by the concurrent presence of tolbutamide, which maintains K ATP closure. Single channel recordings indicate that NN414 alters K ATP intrinsic gating inducing a stable closed or inactivated state. These data indicate that exposure of hypothalamic glucose sensing cells to chronic NN414 drives a sustained conformational change to K ATP , probably by binding to SUR1, that results in loss of channel sensitivity to intrinsic metabolic factors such as MgADP and small molecule agonists. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Activation of the omega-3 fatty acid receptor GPR120 mediates anti-inflammatory actions in immortalized hypothalamic neurons.

    Science.gov (United States)

    Wellhauser, Leigh; Belsham, Denise D

    2014-03-27

    Overnutrition and the ensuing hypothalamic inflammation is a major perpetuating factor in the development of metabolic diseases, such as obesity and diabetes. Inflamed neurons of the CNS fail to properly regulate energy homeostasis leading to pathogenic changes in glucose handling, feeding, and body weight. Hypothalamic neurons are particularly sensitive to pro-inflammatory signals derived locally and peripherally, and it is these neurons that become inflamed first upon high fat feeding. Given the prevalence of metabolic disease, efforts are underway to identify therapeutic targets for this inflammatory state. At least in the periphery, omega-3 fatty acids and their receptor, G-protein coupled receptor 120 (GPR120), have emerged as putative targets. The role for GPR120 in the hypothalamus or CNS in general is poorly understood. Here we introduce a novel, immortalized cell model derived from the rat hypothalamus, rHypoE-7, to study GPR120 activation at the level of the individual neuron. Gene expression levels of pro-inflammatory cytokines were studied by quantitative reverse transcriptase-PCR (qRT-PCR) upon exposure to tumor necrosis factor α (TNFα) treatment in the presence or absence of the polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA). Signal transduction pathway involvement was also studied using phospho-specific antibodies to key proteins by western blot analysis. Importantly, rHypoE-7 cells exhibit a transcriptional and translational inflammatory response upon exposure to TNFα and express abundant levels of GPR120, which is functionally responsive to DHA. DHA pretreatment prevents the inflammatory state and this effect was inhibited by the reduction of endogenous GPR120 levels. GPR120 activates both AKT (protein kinase b) and ERK (extracellular signal-regulated kinase); however, the anti-inflammatory action of this omega-3 fatty acid (FA) receptor is AKT- and ERK-independent and likely involves the GPR120-transforming growth factor

  16. Chronic activation of hypothalamic oxytocin neurons improves cardiac function during left ventricular hypertrophy-induced heart failure.

    Science.gov (United States)

    Garrott, Kara; Dyavanapalli, Jhansi; Cauley, Edmund; Dwyer, Mary Kate; Kuzmiak-Glancy, Sarah; Wang, Xin; Mendelowitz, David; Kay, Matthew W

    2017-09-01

    A distinctive hallmark of heart failure (HF) is autonomic imbalance, consisting of increased sympathetic activity, and decreased parasympathetic tone. Recent work suggests that activation of hypothalamic oxytocin (OXT) neurons could improve autonomic balance during HF. We hypothesized that a novel method of chronic selective activation of hypothalamic OXT neurons will improve cardiac function and reduce inflammation and fibrosis in a rat model of HF. Two groups of male Sprague-Dawley rats underwent trans-ascending aortic constriction (TAC) to induce left ventricular (LV) hypertrophy that progresses to HF. In one TAC group, OXT neurons in the paraventricular nucleus of the hypothalamus were chronically activated by selective expression and activation of excitatory DREADDs receptors with daily injections of clozapine N-oxide (CNO) (TAC + OXT). Two additional age-matched groups received either saline injections (Control) or CNO injections for excitatory DREADDs activation (OXT NORM). Heart rate (HR), LV developed pressure (LVDP), and coronary flow rate were measured in isolated heart experiments. Isoproterenol (0.01 nM-1.0 µM) was administered to evaluate β-adrenergic sensitivity. We found that increases in cellular hypertrophy and myocardial collagen density in TAC were blunted in TAC + OXT animals. Inflammatory cytokine IL-1β expression was more than twice higher in TAC than all other hearts. LVDP, rate pressure product (RPP), contractility, and relaxation were depressed in TAC compared with all other groups. The response of TAC and TAC + OXT hearts to isoproterenol was blunted, with no significant increase in RPP, contractility, or relaxation. However, HR in TAC + OXT animals increased to match Control at higher doses of isoproterenol. Activation of hypothalamic OXT neurons to elevate parasympathetic tone reduced cellular hypertrophy, levels of IL-1β, and fibrosis during TAC-induced HF in rats. Cardiac contractility parameters were

  17. Graphene foam as a biocompatible scaffold for culturing human neurons

    Science.gov (United States)

    Mattei, Cristiana; Nasr, Babak; Hudson, Emma J.; Alshawaf, Abdullah J.; Chana, Gursharan; Everall, Ian P.; Dottori, Mirella; Skafidas, Efstratios

    2018-01-01

    In this study, we explore the use of electrically active graphene foam as a scaffold for the culture of human-derived neurons. Human embryonic stem cell (hESC)-derived cortical neurons fated as either glutamatergic or GABAergic neuronal phenotypes were cultured on graphene foam. We show that graphene foam is biocompatible for the culture of human neurons, capable of supporting cell viability and differentiation of hESC-derived cortical neurons. Based on the findings, we propose that graphene foam represents a suitable scaffold for engineering neuronal tissue and warrants further investigation as a model for understanding neuronal maturation, function and circuit formation. PMID:29657752

  18. Daidzein induces neuritogenesis in DRG neuronal cultures

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    Yang Shih-Hung

    2012-08-01

    Full Text Available Absract Background Daidzein, a phytoestrogen found in isoflavone, is known to exert neurotrophic and neuroprotective effects on the nervous system. Using primary rat dorsal root ganglion (DRG neuronal cultures, we have examined the potential neurite outgrowth effect of daidzein. Methods Dissociated dorsal root ganglia (DRG cultures were used to study the signaling mechanism of daidzein-induced neuritogenesis by immunocytochemistry and Western blotting. Results In response to daidzein treatment, DRG neurons showed a significant increase in total neurite length and in tip number per neuron. The neuritogenic effect of daidzein was significantly hampered by specific blockers for Src, protein kinase C delta (PKCδ and mitogen-activated protein kinase/extracellular signal-regulated kinase kinases (MEK/ERK, but not by those for estrogen receptor (ER. Moreover, daidzein induced phosphorylation of Src, PKCδ and ERK. The activation of PKCδ by daidzein was attenuated in the presence of a Src kinase inhibitor, and that of ERK by daidzein was diminished in the presence of either a Src or PKCδ inhibitor. Conclusion Daidzein may stimulate neurite outgrowth of DRG neurons depending on Src kinase, PKCδ and ERK signaling pathway.

  19. Enhanced astroglial GABA uptake attenuates tonic GABAA inhibition of the presympathetic hypothalamic paraventricular nucleus neurons in heart failure.

    Science.gov (United States)

    Pandit, Sudip; Jo, Ji Yoon; Lee, Sang Ung; Lee, Young Jae; Lee, So Yeong; Ryu, Pan Dong; Lee, Jung Un; Kim, Hyun-Woo; Jeon, Byeong Hwa; Park, Jin Bong

    2015-08-01

    γ-Aminobutyric acid (GABA) generates persistent tonic inhibitory currents (Itonic) and conventional inhibitory postsynaptic currents in the hypothalamic paraventricular nucleus (PVN) via activation of GABAA receptors (GABAARs). We investigated the pathophysiological significance of astroglial GABA uptake in the regulation of Itonic in the PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM). The Itonic of PVN-RVLM neurons were significantly reduced in heart failure (HF) compared with sham-operated (SHAM) rats. Reduced Itonic sensitivity to THIP argued for the decreased function of GABAAR δ subunits in HF, whereas similar Itonic sensitivity to benzodiazepines argued against the difference of γ2 subunit-containing GABAARs in SHAM and HF rats. HF Itonic attenuation was reversed by a nonselective GABA transporter (GAT) blocker (nipecotic acid, NPA) and a GAT-3 selective blocker, but not by a GAT-1 blocker, suggesting that astroglial GABA clearance increased in HF. Similar and minimal Itonic responses to bestrophin-1 blockade in SHAM and HF neurons further argued against a role for astroglial GABA release in HF Itonic attenuation. Finally, the NPA-induced inhibition of spontaneous firing was greater in HF than in SHAM PVN-RVLM neurons, whereas diazepam induced less inhibition of spontaneous firing in HF than in SHAM neurons. Overall, our results showed that combined with reduced GABAARs function, the enhanced astroglial GABA uptake-induced attenuation of Itonic in HF PVN-RVLM neurons explains the deficit in tonic GABAergic inhibition and increased sympathetic outflow from the PVN during heart failure. Copyright © 2015 the American Physiological Society.

  20. Brainstem projections of neurons located in various subdivisions of the dorsolateral hypothalamic area – an anterograde tract-tracing study

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    Rege Sugárka Papp

    2014-05-01

    Full Text Available The projections from the dorsolateral hypothalamic area (DLH to the lower brainstem have been investigated by using biotinylated dextran amine (BDA, an anterograde tracer in rats. The DLH can be divided into 3 areas (dorsomedial hypothalamus, perifornical area, lateral hypothalamic area, and further subdivided into 8 subdivisions. After unilateral stereotaxic injections of BDA into individual DLH subdivisions, the correct sites of injections were controlled histologically, and the distribution patterns of BDA-positive fibers were mapped on serial sections between the hypothalamus and spinal cord in 22 rats. BDA-labeled fibers were observable over 100 different brainstem areas, nuclei or subdivisions. Injections into the 8 DLH subdivisions established distinct topographical patterns. In general, the density of labeled fibers was low in the lower brainstem. High density of fibers was seen only 4 of the 116 areas: in the lateral and ventrolateral parts of the periaqueductal gray, the Barrington’s and the pedunculopontine tegmental nuclei. All of the biogenic amine cell groups in the lower brainstem (9 noradrenaline, 3 adrenaline and 9 serotonin cell groups received labeled fibers, some of them from all, or at least 7 DLH subdivisions, mainly from perifornical and ventral lateral hypothalamic neurons. Some of the tegmental nuclei and nuclei of the reticular formation were widely innervated, although the density of the BDA-labeled fibers was generally low. No definitive descending BDA-positive pathway, but long-run solitaire BDA-labeled fibers were seen in the lower brainstem. These descending fibers joined some of the large tracts or fasciculi in the brainstem. The distribution pattern of BDA-positive fibers of DLH origin throughout the lower brainstem was comparable to patterns of previously published orexin- or melanin-concentrating hormone-immunoreactive fibers with somewhat differences.

  1. An In Vitro System Comprising Immortalized Hypothalamic Neuronal Cells (GT1?7 Cells) for Evaluation of the Neuroendocrine Effects of Essential Oils

    OpenAIRE

    Mizuno, Dai; Konoha-Mizuno, Keiko; Mori, Miwako; Yamazaki, Kentaro; Haneda, Toshihiro; Koyama, Hironari; Kawahara, Masahiro

    2015-01-01

    Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for symptoms including anxiety. Furthermore, it was reportedly effective for the care of several diseases such as Alzheimer’s disease and depressive illness. To investigate the pharmacological effects of essential oils, we developed an in vitro assay system using immortalized hypothalamic neuronal cells (GT1–7 cells). In this study, we evaluated the effects of essential oils on neuronal deat...

  2. The alpha(2)-adrenoceptors do not modify the activity of tyrosine hydroxylase, corticoliberine, and neuropeptide Y producing hypothalamic magnocellular neurons ion the Long Evans and Brattleboro rats

    DEFF Research Database (Denmark)

    Bundzikova, J; Pirnik, Z; Zelena, D

    2010-01-01

    The hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei are activated by body salt-fluid variations. Stimulation of alpha(2)-adrenoceptors by an agonist-xylazine (XYL) activates oxytocinergic but not vasopressinergic magnocellular neurons. In this study, tyrosine hydroxylase (TH), cort...

  3. Essential Role for Hypothalamic Calcitonin Receptor‒Expressing Neurons in the Control of Food Intake by Leptin.

    Science.gov (United States)

    Pan, Warren; Adams, Jessica M; Allison, Margaret B; Patterson, Christa; Flak, Jonathan N; Jones, Justin; Strohbehn, Garth; Trevaskis, James; Rhodes, Christopher J; Olson, David P; Myers, Martin G

    2018-04-01

    The adipocyte-derived hormone leptin acts via its receptor (LepRb) on central nervous system neurons to communicate the repletion of long-term energy stores, to decrease food intake, and to promote energy expenditure. We generated mice that express Cre recombinase from the calcitonin receptor (Calcr) locus (Calcrcre mice) to study Calcr-expressing LepRb (LepRbCalcr) neurons, which reside predominantly in the arcuate nucleus (ARC). Calcrcre-mediated ablation of LepRb in LepRbCalcrknockout (KO) mice caused hyperphagic obesity. Because LepRb-mediated transcriptional control plays a crucial role in leptin action, we used translating ribosome affinity purification followed by RNA sequencing to define the transcriptome of hypothalamic Calcr neurons, along with its alteration in LepRbCalcrKO mice. We found that ARC LepRbCalcr cells include neuropeptide Y (NPY)/agouti-related peptide (AgRP)/γ-aminobutyric acid (GABA) ("NAG") cells as well as non-NAG cells that are distinct from pro-opiomelanocortin cells. Furthermore, although LepRbCalcrKO mice exhibited dysregulated expression of several genes involved in energy balance, neither the expression of Agrp and Npy nor the activity of NAG cells was altered in vivo. Thus, although direct leptin action via LepRbCalcr cells plays an important role in leptin action, our data also suggest that leptin indirectly, as well as directly, regulates these cells.

  4. Pubertally born neurons and glia are functionally integrated into limbic and hypothalamic circuits of the male Syrian hamster.

    Science.gov (United States)

    Mohr, Margaret A; Sisk, Cheryl L

    2013-03-19

    During puberty, the brain goes through extensive remodeling, involving the addition of new neurons and glia to brain regions beyond the canonical neurogenic regions (i.e., dentate gyrus and olfactory bulb), including limbic and hypothalamic cell groups associated with sex-typical behavior. Whether these pubertally born cells become functionally integrated into neural circuits remains unknown. To address this question, we gave male Syrian hamsters daily injections of the cell birthdate marker bromodeoxyuridine throughout puberty (postnatal day 28-49). Half of the animals were housed in enriched environments with access to a running wheel to determine whether enrichment increased the survival of pubertally born cells compared with the control environment. At 4 wk after the last BrdU injection, animals were allowed to interact with a receptive female and were then killed 1 h later. Triple-label immunofluorescence for BrdU, the mature neuron marker neuronal nuclear antigen, and the astrocytic marker glial fibrillary acidic protein revealed that a proportion of pubertally born cells in the medial preoptic area, arcuate nucleus, and medial amygdala differentiate into either mature neurons or astrocytes. Double-label immunofluorescence for BrdU and the protein Fos revealed that a subset of pubertally born cells in these regions is activated during sociosexual behavior, indicative of their functional incorporation into neural circuits. Enrichment affected the survival and activation of pubertally born cells in a brain region-specific manner. These results demonstrate that pubertally born cells located outside of the traditional neurogenic regions differentiate into neurons and glia and become functionally incorporated into neural circuits that subserve sex-typical behaviors.

  5. Hypothalamic neuronal toll-like receptor 2 protects against age-induced obesity

    OpenAIRE

    Shechter, Ravid; London, Anat; Kuperman, Yael; Ronen, Ayal; Rolls, Asya; Chen, Alon; Schwartz, Michal

    2013-01-01

    Toll-like receptors (TLRs) are traditionally associated with immune-mediated host defense. Here, we ascribe a novel extra-immune, hypothalamic-associated function to TLR2, a TLR-family member known to recognize lipid components, in the protection against obesity. We found that TLR2-deficient mice exhibited mature-onset obesity and susceptibility to high-fat diet (HFD)-induced weight gain, via modulation of food intake. Age-related obesity was still evident in chimeric mice, carrying comparabl...

  6. Non-Neuronal Cells in the Hypothalamic Adaptation to Metabolic Signals

    Science.gov (United States)

    Freire-Regatillo, Alejandra; Argente-Arizón, Pilar; Argente, Jesús; García-Segura, Luis Miguel; Chowen, Julie A.

    2017-01-01

    Although the brain is composed of numerous cell types, neurons have received the vast majority of attention in the attempt to understand how this organ functions. Neurons are indeed fundamental but, in order for them to function correctly, they rely on the surrounding “non-neuronal” cells. These different cell types, which include glia, epithelial cells, pericytes, and endothelia, supply essential substances to neurons, in addition to protecting them from dangerous substances and situations. Moreover, it is now clear that non-neuronal cells can also actively participate in determining neuronal signaling outcomes. Due to the increasing problem of obesity in industrialized countries, investigation of the central control of energy balance has greatly increased in attempts to identify new therapeutic targets. This has led to interesting advances in our understanding of how appetite and systemic metabolism are modulated by non-neuronal cells. For example, not only are nutrients and hormones transported into the brain by non-neuronal cells, but these cells can also metabolize these metabolic factors, thus modifying the signals reaching the neurons. The hypothalamus is the main integrating center of incoming metabolic and hormonal signals and interprets this information in order to control appetite and systemic metabolism. Hence, the factors transported and released from surrounding non-neuronal cells will undoubtedly influence metabolic homeostasis. This review focuses on what is known to date regarding the involvement of different cell types in the transport and metabolism of nutrients and hormones in the hypothalamus. The possible involvement of non-neuronal cells, in particular glial cells, in physiopathological outcomes of poor dietary habits and excess weight gain are also discussed. PMID:28377744

  7. PROTECTIVE EFFECTS OF HYPOTHALAMIC BETA-ENDORPHIN NEURONS AGAINST ALCOHOL-INDUCED LIVER INJURIES AND LIVER CANCERS IN RAT ANIMAL MODELS

    Science.gov (United States)

    Murugan, Sengottuvelan; Boyadjieva, Nadka; Sarkar, Dipak K.

    2014-01-01

    Background Recently, retrograde tracing has provided evidence for an influence of hypothalamic β-endorphin (BEP) neurons on the liver, but functions of these neurons are not known. We evaluated the effect of BEP neuronal activation on alcohol-induced liver injury and hepatocellular cancer. Methods Male rats received either BEP neuron transplants or control transplants in the hypothalamus and randomly assigned to feeding alcohol-containing liquid diet or control liquid diet for 8 weeks or to treatment of a carcinogen diethylnitrosamine (DEN). Liver tissues of these animals were analyzed histochemically and biochemically for tissue injuries or cancer. Results Alcohol-feeding increased liver weight and induced several histopathological changes such as prominent microvesicular steatosis and hepatic fibrosis. Alcohol feeding also increased protein levels of triglyceride, hepatic stellate cell activation factors and catecholamines in the liver and endotoxin levels in the plasma. However, these effects of alcohol on the liver were reduced in animals with BEP neuron transplants. BEP neuron transplants also suppressed carcinogen-induced liver histopathologies such as extensive fibrosis, large focus of inflammatory infiltration, hepatocelluar carcinoma, collagen deposition, numbers of preneoplastic foci, levels of hepatic stellate cell activation factors and catecholamines, as well as inflammatory milieu and the levels of NK cell cytotoxic factors in the liver. Conclusion These findings are the first evidence for a role of hypothalamic BEP neurons in influencing liver functions. Additionally, the data identify that BEP neuron transplantation prevents hepatocellular injury and hepatocellular carcinoma formation possibly via influencing the immune function. PMID:25581653

  8. Calcitonin gene-related peptide alters the firing rates of hypothalamic temperature sensitive and insensitive neurons

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    Grimm Eleanor R

    2008-07-01

    Full Text Available Abstract Background Transient hyperthermic shifts in body temperature have been linked to the endogenous hormone calcitonin gene-related peptide (CGRP, which can increase sympathetic activation and metabolic heat production. Recent studies have demonstrated that these centrally mediated responses may result from CGRP dependent changes in the activity of thermoregulatory neurons in the preoptic and anterior regions of the hypothalamus (POAH. Results Using a tissue slice preparation, we recorded the single-unit activity of POAH neurons from the adult male rat, in response to temperature and CGRP (10 μM. Based on the slope of firing rate as a function of temperature, neurons were classified as either warm sensitive or temperature insensitive. All warm sensitive neurons responded to CGRP with a significant decrease in firing rate. While CGRP did not alter the firing rates of some temperature insensitive neurons, responsive neurons showed an increase in firing rate. Conclusion With respect to current models of thermoregulatory control, these CGRP dependent changes in firing rate would result in hyperthermia. This suggests that both warm sensitive and temperature insensitive neurons in the POAH may play a role in producing this hyperthermic shift in temperature.

  9. 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.

  10. Spike Code Flow in Cultured Neuronal Networks

    Directory of Open Access Journals (Sweden)

    Shinichi Tamura

    2016-01-01

    Full Text Available 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.

  11. Isolation and culture of adult mouse vestibular nucleus neurons

    Science.gov (United States)

    Him, Aydın; Altuntaş, Serap; Öztürk, Gürkan; Erdoğan, Ender; Cengiz, Nureddin

    2017-12-19

    Background/aim: Isolated cell cultures are widely used to study neuronal properties due to their advantages. Although embryonic animals are preferred for culturing, their morphological or electrophysiological properties may not reflect adult neurons, which may be important in neurodegenerative diseases. This paper aims to develop a method for preparing isolated cell cultures of medial vestibular nucleus (MVN) from adult mice and describe its morphological and electrophysiological properties.Materials and methods: Vestibular nucleus neurons were mechanically and enzymatically isolated and cultured using a defined medium with known growth factors. Cell survival was measured with propidium iodide, and electrophysiological properties were investigated with current-clamp recording.Results: Vestibular neurons grew neurites in cultures, gaining adult-like morphological properties, and stayed viable for 3 days in culture. Adding bovine calf serum, nerve growth factor, or insulin-like growth factor into the culture medium enhanced neuronal viability. Current-clamp recording of the cultured neurons revealed tonic and phasic-type neurons with similar input resistance, resting membrane potential, action potential amplitude, and duration. Conclusion: Vestibular neurons from adult mice can be cultured, and regenerate axons in a medium containing appropriate growth factors. Culturing adult vestibular neurons provides a new method to study age-related pathologies of the vestibular system.

  12. Lipopolysaccharide (LPS) stimulates adipokine and socs3 gene expression in mouse brain and pituitary gland in vivo, and in N-1 hypothalamic neurons in vitro.

    Science.gov (United States)

    Brown, Russell; Imran, Syed A; Wilkinson, Michael

    2009-04-30

    Adipokines that modulate metabolic and inflammatory responses, such as resistin (rstn) and fasting-induced adipose factor (fiaf), are also expressed in mouse brain and pituitary gland. Since lipopolysaccharide (LPS)-induced endotoxinemia provokes an anorectic response via a hypothalamic-dependent mechanism we hypothesized that LPS would also modify hypothalamic adipokine expression. Challenging male CD-1 mice with LPS (5 mg/kg; s.c.) significantly reduced bodyweight (24 h) and realtime RT-PCR revealed time- and tissue-dependent increases in rstn, fiaf and suppressor of cytokine signaling-3 (socs-3) mRNA in hypothalamic, pituitary, cortical and adipose tissues. Gene expression was rapidly increased (3-6 h) in the hypothalamus and pituitary, but returned to normal within 24 h. In contrast, with the exception of rstn in fat, the expression of target genes remained elevated in cortex and visceral fat at 24 h post-injection. In order to more specifically examine the hypothalamic response to LPS we investigated its effects directly on N-1 hypothalamic neurons in vitro. LPS (25 microg/mL; 3 h) had no effect on rstn mRNA, but significantly stimulated fiaf and socs-3 expression. Although various toll-like receptor 4 (TLR4) antagonists (parthenolide, PD098059, and SB202190) did not prevent the LPS-induced increases in fiaf and socs-3, they did partially attenuate its stimulatory effects. We conclude that LPS treatment increases the expression of central, and possibly neuronal, adipokine genes which may influence local tissue repair and function, but could also have downstream consequences on the hypothalamic control of appetite and energy metabolism following an inflammatory insult.

  13. Neural Damage in Experimental Trypanosoma brucei gambiense Infection: Hypothalamic Peptidergic Sleep and Wake-Regulatory Neurons

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    Claudia Laperchia

    2018-02-01

    Full Text Available Neuron populations of the lateral hypothalamus which synthesize the orexin (OX/hypocretin or melanin-concentrating hormone (MCH peptides play crucial, reciprocal roles in regulating wake stability and sleep. The disease human African trypanosomiasis (HAT, also called sleeping sickness, caused by extracellular Trypanosoma brucei (T. b. parasites, leads to characteristic sleep-wake cycle disruption and narcoleptic-like alterations of the sleep structure. Previous studies have revealed damage of OX and MCH neurons during systemic infection of laboratory rodents with the non-human pathogenic T. b. brucei subspecies. No information is available, however, on these peptidergic neurons after systemic infection with T. b. gambiense, the etiological agent of 97% of HAT cases. The present study was aimed at the investigation of immunohistochemically characterized OX and MCH neurons after T. b. gambiense or T. b. brucei infection of a susceptible rodent, the multimammate mouse, Mastomysnatalensis. Cell counts and evaluation of OX fiber density were performed at 4 and 8 weeks post-infection, when parasites had entered the brain parenchyma from the periphery. A significant decrease of OX neurons (about 44% reduction and MCH neurons (about 54% reduction was found in the lateral hypothalamus and perifornical area at 8 weeks in T. b. gambiense-infected M. natalensis. A moderate decrease (21% and 24% reduction, respectively, which did not reach statistical significance, was found after T. b. brucei infection. In two key targets of diencephalic orexinergic innervation, the peri-suprachiasmatic nucleus (SCN region and the thalamic paraventricular nucleus (PVT, densitometric analyses showed a significant progressive decrease in the density of orexinergic fibers in both infection paradigms, and especially during T. b. gambiense infection. Altogether the findings provide novel information showing that OX and MCH neurons are highly vulnerable to chronic

  14. Lateral hypothalamic thyrotropin-releasing hormone neurons: distribution and relationship to histochemically defined cell populations in the rat.

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    Horjales-Araujo, E; Hellysaz, A; Broberger, C

    2014-09-26

    The lateral hypothalamic area (LHA) constitutes a large component of the hypothalamus, and has been implicated in several aspects of motivated behavior. The LHA is of particular relevance to behavioral state control and the maintenance of arousal. Due to the cellular heterogeneity of this region, however, only some subpopulations of LHA cells have been properly anatomically characterized. Here, we have focused on cells expressing thyrotropin-releasing hormone (TRH), a peptide found in the LHA that has been implicated as a promoter of arousal. Immunofluorescence and in situ hybridization were used to map the LHA TRH population in the rat, and cells were observed to form a large ventral cluster that extended throughout almost the entire rostro-caudal axis of the hypothalamus. Almost no examples of coexistence were seen when sections were double-stained for TRH and markers of other LHA populations, including the peptides hypocretin/orexin, melanin-concentrating hormone and neurotensin. In the juxtaparaventricular area, however, a discrete group of TRH-immunoreactive cells were also stained with antisera against enkephalin and urocortin 3. Innervation from the metabolically sensitive hypothalamic arcuate nucleus was investigated by double-staining for peptide markers of the two centrally projecting groups of arcuate neurons, agouti gene-related peptide and α-melanocyte-stimulating hormone, respectively; both populations of terminals were observed forming close appositions on TRH cells in the LHA. The present study indicates that TRH-expressing cells form a unique population in the LHA that may serve as a link between metabolic signals and the generation of arousal. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

    Touzeau, G.; Kato, A.C.

    1983-01-01

    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 3 H-acetylcholine using 3 H-choline as precursor. ALS sera cause a small decrease in these three parameters, but this difference is not significant

  16. Changes in responsiveness to serotonin on rat ventromedial hypothalamic neurons after food deprivation.

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    Nishimura, F; Nishihara, M; Torii, K; Takahashi, M

    1996-07-01

    The effects of food deprivation on responsiveness of neurons in the ventromedial nucleus of the hypothalamus (VMH) to serotonin (5-HT), norepinephrine (NE), gamma-aminobutyric acid (GABA), and neuropeptide Y (NPY) were investigated using brain slices in vitro along with behavioral changes in vivo during fasting. Adult male rats were fasted for 48 h starting at the beginning of the dark phase (lights on: 0700-1900 h). The animals showed a significant loss of body weight on the second day of fasting and an increase in food consumption on the first day of refeeding. During fasting, voluntary locomotor activity was significantly increased in the light phase but not during the dark phase. Plasma catecholamine levels were not affected by fasting. In vitro electrophysiological study showed that, in normally fed rats, 5-HT and NE induced both excitatory and inhibitory responses, while GABA and NPY intensively suppressed unit activity in the VMH. Food deprivation for 48 h significantly changed the responsiveness of VMH neurons to 5-HT, for instance, the ratio of neurons whose activity was facilitated by 5-HT was significantly decreased. The responsiveness of VMH neurons to NE, GABA, and NPY was not affected by food deprivation. These results suggest that food deprivation decreases the facilitatory response of VMH neurons to 5-HT, and that this change in responsiveness to 5-HT is at least partially involved in the increase in food intake motivation and locomotor activity during fasting.

  17. Male mice ultrasonic vocalizations enhance female sexual approach and hypothalamic kisspeptin neuron activity.

    Science.gov (United States)

    Asaba, Akari; Osakada, Takuya; Touhara, Kazushige; Kato, Masahiro; Mogi, Kazutaka; Kikusui, Takefumi

    2017-08-01

    Vocal communication in animals is important for ensuring reproductive success. Male mice emit song-like "ultrasonic vocalizations (USVs)" when they encounter female mice, and females show approach to the USVs. However, it is unclear whether USVs of male mice trigger female behavioral and endocrine responses in reproduction. In this study, we first investigated the relationship between the number of deliveries in breeding pairs for 4months and USVs syllables emitted from those paired males during 3min of sexual encounter with unfamiliar female mice. There was a positive correlation between these two indices, which suggests that breeding pairs in which males could emit USVs more frequently had more offspring. Further, we examined the effect of USVs of male mice on female sexual behavior. Female mice showed more approach behavior towards vocalizing males than devocalized males. Finally, to determine whether USVs of male mice could activate the neural system governing reproductive function in female mice, the activation of kisspeptin neurons, key neurons to drive gonadotropin-releasing hormone neurons in the hypothalamus, was examined using dual-label immunocytochemistry with cAMP response element-binding protein phosphorylation (pCREB). In the arcuate nucleus (Arc), the number of kisspeptin neurons expressing pCREB significantly increased after exposure to USVs of male as compared with noise exposure group. In conclusion, our results suggest that USVs of male mice promote fertility in female mice by activating both their approaching behavior and central kisspeptin neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Histaminergic responses by hypothalamic neurons that regulate lordosis and their modulation by estradiol.

    Science.gov (United States)

    Dupré, Christophe; Lovett-Barron, Matthew; Pfaff, Donald W; Kow, Lee-Ming

    2010-07-06

    How do fluctuations in the level of generalized arousal of the brain affect the performance of specific motivated behaviors, such as sexual behaviors that depend on sexual arousal? A great deal of previous work has provided us with two important starting points in answering this question: (i) that histamine (HA) serves generalized CNS arousal and (ii) that heightened electrical activity of neurons in the ventromedial nucleus of the hypothalamus (VMN) is necessary and sufficient for facilitating the primary female sex behavior in laboratory animals, lordosis behavior. Here we used patch clamp recording technology to analyze HA effects on VMN neuronal activity. The results show that HA acting through H1 receptors (H1R) depolarizes these neurons. Further, acute administration of estradiol, an estrogen necessary for lordosis behavior to occur, heightens this effect. Hyperpolarization, which tends to decrease excitability and enhance inhibition, was not affected by acute estradiol or mediated by H1R but was mediated by other HA receptor subtypes, H2 and H3. Sampling of mRNA from individual VMN neurons showed colocalization of expression of H1 receptor mRNA with estrogen receptor (ER)-alpha mRNA but also revealed ER colocalization with the other HA receptor subtypes and colocalization of different subtypes with each other. The latter finding provides the molecular basis for complex "push-pull" regulation of VMN neuronal excitability by HA. Thus, in the simplest causal route, HA, acting on VMN neurons through H1R provides a mechanism by which elevated states of generalized CNS arousal can foster a specific estrogen-dependent, aroused behavior, sexual behavior.

  19. Hyperosmotic stimulus induces reversible angiogenesis within the hypothalamic magnocellular nuclei of the adult rat: a potential role for neuronal vascular endothelial growth factor

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    Vincent Anne

    2005-03-01

    Full Text Available Abstract Background In mammals, the CNS vasculature is established during the postnatal period via active angiogenesis, providing different brain regions with capillary networks of various densities that locally supply adapted metabolic support to neurons. Thereafter this vasculature remains essentially quiescent excepted for specific pathologies. In the adult rat hypothalamus, a particularly dense network of capillary vessels is associated with the supraoptic (SON and paraventricular (PVN nuclei containing the magnocellular neurons secreting vasopressin and oxytocin, two neurohormones involved in the control of the body fluid homoeostasis. In the seventies, it was reported that proliferation of astrocytes and endothelial cells occurs within these hypothalamic nuclei when strong metabolic activation of the vasopressinergic and oxytocinergic neurons was induced by prolonged hyperosmotic stimulation. The aim of the present study was to determine whether such proliferative response to osmotic stimulus is related to local angiogenesis and to elucidate the cellular and molecular mechanisms involved. Results Our results provide evidence that cell proliferation occurring within the SON of osmotically stimulated adult rats corresponds to local angiogenesis. We show that 1 a large majority of the SON proliferative cells is associated with capillary vessels, 2 this proliferative response correlates with a progressive increase in density of the capillary network within the nucleus, and 3 SON capillary vessels exhibit an increased expression of nestin and vimentin, two markers of newly formed vessels. Contrasting with most adult CNS neurons, hypothalamic magnocellular neurons were found to express vascular endothelial growth factor (VEGF, a potent angiogenic factor whose production was increased by osmotic stimulus. When VEGF was inhibited by dexamethasone treatment or by the local application of a blocking antibody, the angiogenic response was strongly

  20. Inhibitory neurons modulate spontaneous signaling in cultured cortical neurons: density-dependent regulation of excitatory neuronal signaling

    International Nuclear Information System (INIS)

    Serra, Michael; Guaraldi, Mary; Shea, Thomas B

    2010-01-01

    Cortical neuronal activity depends on a balance between excitatory and inhibitory influences. Culturing of neurons on multi-electrode arrays (MEAs) has provided insight into the development and maintenance of neuronal networks. Herein, we seeded MEAs with murine embryonic cortical/hippocampal neurons at different densities ( 1000 cells mm −2 ) and monitored resultant spontaneous signaling. Sparsely seeded cultures displayed a large number of bipolar, rapid, high-amplitude individual signals with no apparent temporal regularity. By contrast, densely seeded cultures instead displayed clusters of signals at regular intervals. These patterns were observed even within thinner and thicker areas of the same culture. GABAergic neurons (25% of total neurons in our cultures) mediated the differential signal patterns observed above, since addition of the inhibitory antagonist bicuculline to dense cultures and hippocampal slice cultures induced the signal pattern characteristic of sparse cultures. Sparsely seeded cultures likely lacked sufficient inhibitory neurons to modulate excitatory activity. Differential seeding of MEAs can provide a unique model for analyses of pertubation in the interaction between excitatory and inhibitory function during aging and neuropathological conditions where dysregulation of GABAergic neurons is a significant component

  1. NMDA receptors induce somatodendritic secretion in hypothalamic neurones of lactating female rats

    NARCIS (Netherlands)

    de Kock, C.P.J.; Burnashev, N.; Lodder, J.C.; Mansvelder, H.D.; Brussaard, A.B.

    2004-01-01

    Many neurones in the mammalian brain are known to release the content of their vesicles from somatodendritic locations. These vesicles usually contain retrograde messengers that modulate network properties. The back-propagating action potential is thought to be the principal physiological stimulus

  2. Recent Advances in the Cellular and Molecular Mechanisms of Hypothalamic Neuronal Glucose Detection.

    Science.gov (United States)

    Fioramonti, Xavier; Chrétien, Chloé; Leloup, Corinne; Pénicaud, Luc

    2017-01-01

    The hypothalamus have been recognized for decades as one of the major brain centers for the control of energy homeostasis. This area contains specialized neurons able to detect changes in nutrients level. Among them, glucose-sensing neurons use glucose as a signaling molecule in addition to its fueling role. In this review we will describe the different sub-populations of glucose-sensing neurons present in the hypothalamus and highlight their nature in terms of neurotransmitter/neuropeptide expression. This review will particularly discuss whether pro-opiomelanocortin (POMC) neurons from the arcuate nucleus are directly glucose-sensing. In addition, recent observations in glucose-sensing suggest a subtle system with different mechanisms involved in the detection of changes in glucose level and their involvement in specific physiological functions. Several data point out the critical role of reactive oxygen species (ROS) and mitochondria dynamics in the detection of increased glucose. This review will also highlight that ATP-dependent potassium (K ATP ) channels are not the only channels mediating glucose-sensing and discuss the new role of transient receptor potential canonical channels (TRPC). We will discuss the recent advances in the determination of glucose-sensing machinery and propose potential line of research needed to further understand the regulation of brain glucose detection.

  3. Recent Advances in the Cellular and Molecular Mechanisms of Hypothalamic Neuronal Glucose Detection

    Directory of Open Access Journals (Sweden)

    Xavier Fioramonti

    2017-11-01

    Full Text Available The hypothalamus have been recognized for decades as one of the major brain centers for the control of energy homeostasis. This area contains specialized neurons able to detect changes in nutrients level. Among them, glucose-sensing neurons use glucose as a signaling molecule in addition to its fueling role. In this review we will describe the different sub-populations of glucose-sensing neurons present in the hypothalamus and highlight their nature in terms of neurotransmitter/neuropeptide expression. This review will particularly discuss whether pro-opiomelanocortin (POMC neurons from the arcuate nucleus are directly glucose-sensing. In addition, recent observations in glucose-sensing suggest a subtle system with different mechanisms involved in the detection of changes in glucose level and their involvement in specific physiological functions. Several data point out the critical role of reactive oxygen species (ROS and mitochondria dynamics in the detection of increased glucose. This review will also highlight that ATP-dependent potassium (KATP channels are not the only channels mediating glucose-sensing and discuss the new role of transient receptor potential canonical channels (TRPC. We will discuss the recent advances in the determination of glucose-sensing machinery and propose potential line of research needed to further understand the regulation of brain glucose detection.

  4. The number of hypothalamic hypocretin (orexin) neurons is not affected in Prader-Willi syndrome.

    NARCIS (Netherlands)

    Fronczek, R.; Lammers, G.J.; Balesar, R.; Unm, U.A.hopa; Swaab, D.F.

    2005-01-01

    CONTEXT: Narcoleptic patients with cataplexy have a general loss of hypocretin (orexin) in the lateral hypothalamus, possibly due to an autoimmune-mediated degeneration of the hypocretin neurons. In addition to excessive daytime sleepiness, Prader-Willi syndrome (PWS) patients may show

  5. The number of hypothalamic hypocretin (orexin) neurons is not affected in Prader-Willi syndrome

    NARCIS (Netherlands)

    Fronczek, Rolf; Lammers, Gert Jan; Balesar, Rawien; Unmehopa, Unga A.; Swaab, Dick F.

    2005-01-01

    Narcoleptic patients with cataplexy have a general loss of hypocretin (orexin) in the lateral hypothalamus, possibly due to an autoimmune-mediated degeneration of the hypocretin neurons. In addition to excessive daytime sleepiness, Prader-Willi syndrome (PWS) patients may show narcolepsy-like

  6. Visualization of oxytocin release that mediates paired pulse facilitation in hypothalamic pathways to brainstem autonomic neurons.

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    Ramón A Piñol

    Full Text Available Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2 expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV. We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs, neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection.

  7. Visualization of Oxytocin Release that Mediates Paired Pulse Facilitation in Hypothalamic Pathways to Brainstem Autonomic Neurons

    Science.gov (United States)

    Piñol, Ramón A.; Jameson, Heather; Popratiloff, Anastas; Lee, Norman H.; Mendelowitz, David

    2014-01-01

    Recent work has shown that oxytocin is involved in more than lactation and uterine contraction. The paraventricular nucleus of the hypothalamus (PVN) contains neuroendocrine neurons that control the release of hormones, including vasopressin and oxytocin. Other populations of PVN neurons do not release hormones, but rather project to and release neurotransmitters onto other neurons in the CNS involved in fluid retention, thermoregulation, sexual behavior and responses to stress. Activation of oxytocin receptors can be cardioprotective and reduces the adverse cardiovascular consequences of anxiety and stress, yet how oxytocin can affect heart rate and cardiac function is unknown. While anatomical work has shown the presence of peptides, including oxytocin, in the projections from the PVN to parasympathetic nuclei, electrophysiological studies to date have only demonstrated release of glutamate and activation of fast ligand gated receptors in these pathways. In this study, using rats, we directly show, using sniffer CHO cells that express oxytocin receptors and the Ca2+ indicator R-GECO, that optogenetic activation of channelrhodopsin-2 (ChR2) expressing PVN fibers in the brainstem activates oxytocin receptors in the dorsomotor nucleus of the vagus (DMNV). We also demonstrate that while a single photoactivation of PVN terminals only activates glutamatergic receptors in brainstem cardiac vagal neurons (CVNs), neurons that dominate the neural control of heart rate, both the paired pulse facilitation, and sustained enhancement of glutamate release in this pathway is mediated by activation of oxytocin receptors. Our results provide direct evidence that a pathway from the PVN likely releases oxytocin and enhances short-term plasticity of this critical autonomic connection. PMID:25379676

  8. Regulation of hypothalamic neuronal sensing and food intake by ketone bodies and fatty acids.

    Science.gov (United States)

    Le Foll, Christelle; Dunn-Meynell, Ambrose A; Miziorko, Henri M; Levin, Barry E

    2014-04-01

    Metabolic sensing neurons in the ventromedial hypothalamus (VMH) alter their activity when ambient levels of metabolic substrates, such as glucose and fatty acids (FA), change. To assess the relationship between a high-fat diet (HFD; 60%) intake on feeding and serum and VMH FA levels, rats were trained to eat a low-fat diet (LFD; 13.5%) or an HFD in 3 h/day and were monitored with VMH FA microdialysis. Despite having higher serum levels, HFD rats had lower VMH FA levels but ate less from 3 to 6 h of refeeding than did LFD rats. However, VMH β-hydroxybutyrate (β-OHB) and VMH-to-serum β-OHB ratio levels were higher in HFD rats during the first 1 h of refeeding, suggesting that VMH astrocyte ketone production mediated their reduced intake. In fact, using calcium imaging in dissociated VMH neurons showed that ketone bodies overrode normal FA sensing, primarily by exciting neurons that were activated or inhibited by oleic acid. Importantly, bilateral inhibition of VMH ketone production with a 3-hydroxy-3-methylglutaryl-CoA synthase inhibitor reversed the 3- to 6-h HFD-induced inhibition of intake but had no effect in LFD-fed rats. These data suggest that a restricted HFD intake regimen inhibits caloric intake as a consequence of FA-induced VMH ketone body production by astrocytes.

  9. Norepinephrine metabolism in neuronal cultures is increased by angiotensin II

    International Nuclear Information System (INIS)

    Sumners, C.; Shalit, S.L.; Kalberg, C.J.; Raizada, M.K.

    1987-01-01

    In this study the authors have examined the actions of angiotensin II (ANG II) on catecholamine metabolism in neuronal brain cell cultures prepared from the hypothalamus and brain stem. Neuronal cultures prepared from the brains of 1-day-old Sprague-Dawley rats exhibit specific neuronal uptake mechanisms for both norepinephrine (NE) and dopamine (DA), and also monoamine oxidase (MAO) and catechol O-methyltransferase (COMT) activity. Separate neuronal uptake sites for NE and DA were identified by using specific neuronal uptake inhibitors for each amine. In previous studies, they determined that ANG II (10 nM-1 μM) stimulates increased neuronal [ 3 H]NE uptake by acting as specific receptors. They have confirmed these results here and in addition have shown that ANG II has not significant effects on neuronal [ 3 H]DA uptake. These results suggest that the actions of ANG II are restricted to the NE transporter in neuronal cultures. It is possible that ANG II stimulates the intraneuronal metabolism of at least part of the NE that is taken up, because the peptide stimulates MAO activity, an effect mediated by specific ANG II receptors. ANG II had no effect on COMT activity in neuronal cultures. Therefore, the use of neuronal cultures of hypothalamus and brain stem they have determined that ANG II can specifically alter NE metabolism in these areas, while apparently not altering DA metabolism

  10. Altered astrocyte glutamate transporter regulation of hypothalamic neurosecretory neurons in heart failure rats.

    Science.gov (United States)

    Potapenko, Evgeniy S; Biancardi, Vinicia C; Zhou, Yiqiang; Stern, Javier E

    2012-08-01

    Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-D-aspartate receptor (NMDAR)-mediated inward current (tonic I(NMDA)) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic I(NMDA) activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.

  11. Development of posterior hypothalamic neurons enlightens a switch in the prosencephalic basic plan.

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    Sophie Croizier

    Full Text Available In rats and mice, ascending and descending axons from neurons producing melanin-concentrating hormone (MCH reach the cerebral cortex and spinal cord. However, these ascending and descending projections originate from distinct sub-populations expressing or not "Cocaine-and-Amphetamine-Regulated-Transcript" (CART peptide. Using a BrdU approach, MCH cell bodies are among the very first generated in the hypothalamus, within a longitudinal cell cord made of earliest delaminating neuroblasts in the diencephalon and extending from the chiasmatic region to the ventral midbrain. This region also specifically expresses the regulatory genes Sonic hedgehog (Shh and Nkx2.2. First MCH axons run through the tractus postopticus (tpoc which gathers pioneer axons from the cell cord and courses parallel to the Shh/Nkx2.2 expression domain. Subsequently generated MCH neurons and ascending MCH axons differentiate while neurogenesis and mantle layer differentiation are generalized in the prosencephalon, including telencephalon. Ascending MCH axons follow dopaminergic axons of the mesotelencephalic tract, both being an initial component of the medial forebrain bundle (mfb. Netrin1 and Slit2 proteins that are involved in the establishment of the tpoc and mfb, respectively attract or repulse MCH axons.We conclude that first generated MCH neurons develop in a diencephalic segment of a longitudinal Shh/Nkx2.2 domain. This region can be seen as a prosencephalic segment of a medial neurogenic column extending from the chiasmatic region through the ventral neural tube. However, as the telencephalon expends, it exerts a trophic action and the mfb expands, inducing a switch in the longitudinal axial organization of the prosencephalon.

  12. Sweet taste receptor serves to activate glucose- and leptin-responsive neurons in the hypothalamic arcuate nucleus and participates in glucose responsiveness.

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    Daisuke Kohno

    2016-11-01

    Full Text Available The hypothalamic feeding center plays an important role in energy homeostasis. In the feeding center, whole-body energy signals including hormones and nutrients are sensed, processed, and integrated. As a result, food intake and energy expenditure are regulated. Two types of glucose-sensing neurons exist in the hypothalamic arcuate nucleus (ARC: glucose-excited neurons and glucose-inhibited neurons. While some molecules are known to be related to glucose sensing in the hypothalamus, the mechanism underlying glucose sensing in the hypothalamus are not fully understood. The sweet taste receptor is a heterodimer of taste type 1 receptor 2 (T1R2 and taste type 1 receptor 3 (T1R3 and senses sweet tastes. T1R2 and T1R3 receptors are distributed in multiple organs including the tongue, pancreas, adipose tissue, and hypothalamus. However, the role of sweet taste receptors in the ARC remains to be clarified. To examine the role of sweet taste receptors in the ARC, cytosolic Ca2+ concentration ([Ca2+]i in isolated single ARC neurons were measured using Fura-2 fluorescent imaging. An artificial sweetener, sucralose at 10-5 M-10-2 M dose dependently increased [Ca2+]i in 12-16% of ARC neurons. The sucralose-induced [Ca2+]i increase was suppressed by a sweet taste receptor inhibitor, gurmarin. The sucralose-induced [Ca2+]i increase was inhibited under an extracellular Ca2+-free condition and in the presence of an L-type Ca2+ channel blocker, nitrendipine. Sucralose-responding neurons were activated by high-concentration of glucose. This response to glucose was markedly suppressed by gurmarin. More than half of sucralose-responding neurons were activated by leptin but not ghrelin. Percentage of proopiomelanocortin (POMC neurons among sucralose-responding neurons and sweet taste receptor expressing neurons were low, suggesting that majority of sucralose-responding neurons are non-POMC neurons. These data suggest that sweet taste receptor-mediated cellular

  13. Sweet Taste Receptor Serves to Activate Glucose- and Leptin-Responsive Neurons in the Hypothalamic Arcuate Nucleus and Participates in Glucose Responsiveness.

    Science.gov (United States)

    Kohno, Daisuke; Koike, Miho; Ninomiya, Yuzo; Kojima, Itaru; Kitamura, Tadahiro; Yada, Toshihiko

    2016-01-01

    The hypothalamic feeding center plays an important role in energy homeostasis. In the feeding center, whole-body energy signals including hormones and nutrients are sensed, processed, and integrated. As a result, food intake and energy expenditure are regulated. Two types of glucose-sensing neurons exist in the hypothalamic arcuate nucleus (ARC): glucose-excited neurons and glucose-inhibited neurons. While some molecules are known to be related to glucose sensing in the hypothalamus, the mechanisms underlying glucose sensing in the hypothalamus are not fully understood. The sweet taste receptor is a heterodimer of taste type 1 receptor 2 (T1R2) and taste type 1 receptor 3 (T1R3) and senses sweet tastes. T1R2 and T1R3 are distributed in multiple organs including the tongue, pancreas, adipose tissue, and hypothalamus. However, the role of sweet taste receptors in the ARC remains to be clarified. To examine the role of sweet taste receptors in the ARC, cytosolic Ca 2+ concentration ([Ca 2+ ] i ) in isolated single ARC neurons were measured using Fura-2 fluorescent imaging. An artificial sweetener, sucralose at 10 -5 -10 -2 M dose dependently increased [Ca 2+ ] i in 12-16% of ARC neurons. The sucralose-induced [Ca 2+ ] i increase was suppressed by a sweet taste receptor inhibitor, gurmarin. The sucralose-induced [Ca 2+ ] i increase was inhibited under an extracellular Ca 2+ -free condition and in the presence of an L-type Ca 2+ channel blocker, nitrendipine. Sucralose-responding neurons were activated by high-concentration of glucose. This response to glucose was markedly suppressed by gurmarin. More than half of sucralose-responding neurons were activated by leptin but not ghrelin. Percentages of proopiomelanocortin (POMC) neurons among sucralose-responding neurons and sweet taste receptor expressing neurons were low, suggesting that majority of sucralose-responding neurons are non-POMC neurons. These data suggest that sweet taste receptor-mediated cellular activation

  14. Colocalization of neurotensin receptors and of the neurotensin-degrading enzyme endopeptidase 24-16 in primary cultures of neurons

    International Nuclear Information System (INIS)

    Chabry, J.; Checler, F.; Vincent, J.P.; Mazella, J.

    1990-01-01

    This paper compares the localization of neurotensin receptors and of endopeptidase 24-16, a peptidase likely involved in the inactivation of neurotensin in primary cultures of neurons. Neurotensin binding sites were radiolabeled with 125 I-Tyr3-neurotensin, whereas endopeptidase 24-16 was stained by immunohistochemical techniques using a monospecific polyclonal antibody. Endopeptidase 24-16 is present in 80-85% of the nondifferentiated neurons. The proportion of immunoreactive neurons decreased during maturation to reach 35-40% after 4-8 d of culture. By contrast, neurotensin receptors were not detectable in nondifferentiated cells and appear during maturation. Specific 125 I-Tyr3-neurotensin labeling is maximal after 4 d of culture and is located on about 10% of differentiated neurons. Double-labeling experiments show that about 90% of cortical, hypothalamic, and mesencephalic neurons bearing the neurotensin receptor also contained endopeptidase 24-16, supporting the hypothesis that one of the functions of endopeptidase 24-16 is the physiological inactivation of neurotensin. However, the presence of endopeptidase 24-16 on numerous neurons that do not contain neurotensin receptors also suggests that the enzyme could be involved in the degradation and/or maturation of other neuropeptides

  15. Short-term enrichment makes male rats more attractive, more defensive and alters hypothalamic neurons.

    Directory of Open Access Journals (Sweden)

    Rupshi Mitra

    Full Text Available Innate behaviors are shaped by contingencies built during evolutionary history. On the other hand, environmental stimuli play a significant role in shaping behavior. In particular, a short period of environmental enrichment can enhance cognitive behavior, modify effects of stress on learned behaviors and induce brain plasticity. It is unclear if modulation by environment can extend to innate behaviors which are preserved by intense selection pressure. In the present report we investigate this issue by studying effects of relatively short (14-days environmental enrichment on two prominent innate behaviors in rats, avoidance of predator odors and ability of males to attract mates. We show that enrichment has strong effects on both the innate behaviors: a enriched males were more avoidant of a predator odor than non-enriched controls, and had a greater rise in corticosterone levels in response to the odor; and b had higher testosterone levels and were more attractive to females. Additionally, we demonstrate decrease in dendritic length of neurons of ventrolateral nucleus of hypothalamus, important for reproductive mate-choice and increase in the same in dorsomedial nucleus, important for defensive behavior. Thus, behavioral and hormonal observations provide evidence that a short period of environmental manipulation can alter innate behaviors, providing a good example of gene-environment interaction.

  16. 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.

  17. Hypothalamic growth hormone receptor (GHR) controls hepatic glucose production in nutrient-sensing leptin receptor (LepRb) expressing neurons.

    Science.gov (United States)

    Cady, Gillian; Landeryou, Taylor; Garratt, Michael; Kopchick, John J; Qi, Nathan; Garcia-Galiano, David; Elias, Carol F; Myers, Martin G; Miller, Richard A; Sandoval, Darleen A; Sadagurski, Marianna

    2017-05-01

    The GH/IGF-1 axis has important roles in growth and metabolism. GH and GH receptor (GHR) are active in the central nervous system (CNS) and are crucial in regulating several aspects of metabolism. In the hypothalamus, there is a high abundance of GH-responsive cells, but the role of GH signaling in hypothalamic neurons is unknown. Previous work has demonstrated that the Ghr gene is highly expressed in LepRb neurons. Given that leptin is a key regulator of energy balance by acting on leptin receptor (LepRb)-expressing neurons, we tested the hypothesis that LepRb neurons represent an important site for GHR signaling to control body homeostasis. To determine the importance of GHR signaling in LepRb neurons, we utilized Cre/loxP technology to ablate GHR expression in LepRb neurons (Lepr EYFPΔGHR ). The mice were generated by crossing the Lepr cre on the cre-inducible ROSA26-EYFP mice to GHR L/L mice. Parameters of body composition and glucose homeostasis were evaluated. Our results demonstrate that the sites with GHR and LepRb co-expression include ARH, DMH, and LHA neurons. Leptin action was not altered in Lepr EYFPΔGHR mice; however, GH-induced pStat5-IR in LepRb neurons was significantly reduced in these mice. Serum IGF-1 and GH levels were unaltered, and we found no evidence that GHR signaling regulates food intake and body weight in LepRb neurons. In contrast, diminished GHR signaling in LepRb neurons impaired hepatic insulin sensitivity and peripheral lipid metabolism. This was paralleled with a failure to suppress expression of the gluconeogenic genes and impaired hepatic insulin signaling in Lepr EYFPΔGHR mice. These findings suggest the existence of GHR-leptin neurocircuitry that plays an important role in the GHR-mediated regulation of glucose metabolism irrespective of feeding.

  18. Estimation of in vitro activity of tuberoinfundibular dopaminergic neurons by measurement of DOPA synthesis in the median eminence of hypothalamic slices.

    Science.gov (United States)

    Arita, J; Kimura, F

    1984-12-01

    A new method for estimation of in vitro neurosecretory activity of tuberoinfundibular dopaminergic (TIDA) neurons was developed by measuring the rate of synthesis of dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic slices. Sagittal hypothalamic slices of ovariectomized rats were incubated in a medium containing 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of DOPA decarboxylase. DOPA accumulated in the median eminence following incubation with NSD 1015 was determined by high-performance liquid chromatography with electro-chemical detection. The amount of DOPA accumulated in vitro in the median eminence was maximal in a medium containing 10 mM NSD 1015 and linear up to 120 min at 37 degrees C. Increasing the concentration of tyrosine in medium stimulated the synthesis of DOPA in the median eminence. The synthesis of DOPA was blocked by 1 mM alpha-methyltyrosine, an inhibitor of tyrosine hydroxylase. The rate of in vitro synthesis of DOPA in the median eminence was 33% of that of in vivo synthesis. Incubation in a medium containing 50 mM K+ to depolarize neurons caused a 2.4-fold increase in DOPA synthesis in the median eminence. The high K+-induced increase in DOPA synthesis was blocked by omission of Ca2+ and addition of 1 mM EGTA into the medium, suggesting Ca2+ dependency of depolarization-activated DOPA synthesis. These results indicate that this in vitro assay is a useful means to study the regulatory mechanisms of TIDA neurons.

  19. Brainstem projections of neurons located in various subdivisions of the dorsolateral hypothalamic area – an anterograde tract-tracing study

    OpenAIRE

    Rege Sugárka Papp; Rege Sugárka Papp; Miklos ePalkovits; Miklos ePalkovits

    2014-01-01

    The projections from the dorsolateral hypothalamic area (DLH) to the lower brainstem have been investigated by using biotinylated dextran amine (BDA), an anterograde tracer in rats. The DLH can be divided into 3 areas (dorsomedial hypothalamus, perifornical area, lateral hypothalamic area), and further subdivided into 8 subdivisions. After unilateral stereotaxic injections of BDA into individual DLH subdivisions, the correct sites of injections were controlled histologically, and the distribu...

  20. Brainstem projections of neurons located in various subdivisions of the dorsolateral hypothalamic area—an anterograde tract-tracing study

    OpenAIRE

    Papp, Rege S.; Palkovits, Miklós

    2014-01-01

    The projections from the dorsolateral hypothalamic area (DLH) to the lower brainstem have been investigated by using biotinylated dextran amine (BDA), an anterograde tracer in rats. The DLH can be divided into 3 areas (dorsomedial hypothalamus, perifornical area, lateral hypothalamic area), and further subdivided into 8 subdivisions. After unilateral stereotaxic injections of BDA into individual DLH subdivisions, the correct sites of injections were controlled histologically, and the distribu...

  1. Estradiol target neurons in the hypothalamic arcuate nucleus and lateral ventromedial nucleus of young adult, reproductively senescent, and monosodium glutamate-lesioned female golden hamsters

    International Nuclear Information System (INIS)

    Blaha, G.C.; Lamperti, A.A.

    1983-01-01

    Histoautoradiographic methods were used to assess estrogen target neurons in the hypothalamic arcuate nucleus (ARC) and ventromedial nucleus, lateral portion (LVM), comparing young adult and aged female golden hamsters. A subgroup of young adult females had ARC lesions induced by monosodium glutamate at neonatal day 8. All were ovariectomized to remove endogenous estrogens. Controls were given nonradioactive estradiol. After 3 H-estradiol ( 3 H-E2) was injected intravenously, hypothalami were removed, frozen, and processed for histoautoradiography. In the ARC and LVM the ratio of 3 H-E2 labelled neurons to total neurons counted was significantly lower in the older animals. Young females with ARC lesions had very few 3 H-E2 labelled neurons remaining in the ARC but had a normal complement in the LVM. Although 3 H-E2 labelled ARC neurons were notably decreased in old females, those ARC neurons that were labelled in the old had virtually the same frequency distribution of the labelling index as in the young, suggesting no change in the average estrogen uptake per target cell

  2. Increasing fatty acid oxidation remodels the hypothalamic neurometabolome to mitigate stress and inflammation.

    Directory of Open Access Journals (Sweden)

    Joseph W McFadden

    Full Text Available Modification of hypothalamic fatty acid (FA metabolism can improve energy homeostasis and prevent hyperphagia and excessive weight gain in diet-induced obesity (DIO from a diet high in saturated fatty acids. We have shown previously that C75, a stimulator of carnitine palmitoyl transferase-1 (CPT-1 and fatty acid oxidation (FAOx, exerts at least some of its hypophagic effects via neuronal mechanisms in the hypothalamus. In the present work, we characterized the effects of C75 and another anorexigenic compound, the glycerol-3-phosphate acyltransferase (GPAT inhibitor FSG67, on FA metabolism, metabolomics profiles, and metabolic stress responses in cultured hypothalamic neurons and hypothalamic neuronal cell lines during lipid excess with palmitate. Both compounds enhanced palmitate oxidation, increased ATP, and inactivated AMP-activated protein kinase (AMPK in hypothalamic neurons in vitro. Lipidomics and untargeted metabolomics revealed that enhanced catabolism of FA decreased palmitate availability and prevented the production of fatty acylglycerols, ceramides, and cholesterol esters, lipids that are associated with lipotoxicity-provoked metabolic stress. This improved metabolic signature was accompanied by increased levels of reactive oxygen species (ROS, and yet favorable changes in oxidative stress, overt ER stress, and inflammation. We propose that enhancing FAOx in hypothalamic neurons exposed to excess lipids promotes metabolic remodeling that reduces local inflammatory and cell stress responses. This shift would restore mitochondrial function such that increased FAOx can produce hypothalamic neuronal ATP and lead to decreased food intake and body weight to improve systemic metabolism.

  3. Ankyrin repeat and SOCS box containing protein 4 (Asb-4 colocalizes with insulin receptor substrate 4 (IRS4 in the hypothalamic neurons and mediates IRS4 degradation

    Directory of Open Access Journals (Sweden)

    Xia Zefeng

    2011-09-01

    Full Text Available Abstract Background The arcuate nucleus of the hypothalamus regulates food intake. Ankyrin repeat and SOCS box containing protein 4 (Asb-4 is expressed in neuropeptide Y and proopiomelanocortin (POMC neurons in the arcuate nucleus, target neurons in the regulation of food intake and metabolism by insulin and leptin. However, the target protein(s of Asb-4 in these neurons remains unknown. Insulin receptor substrate 4 (IRS4 is an adaptor molecule involved in the signal transduction by both insulin and leptin. In the present study we examined the colocalization and interaction of Asb-4 with IRS4 and the involvement of Asb-4 in insulin signaling. Results In situ hybridization showed that the expression pattern of Asb-4 was consistent with that of IRS4 in the rat brain. Double in situ hybridization showed that IRS4 colocalized with Asb-4, and both Asb-4 and IRS4 mRNA were expressed in proopiomelanocortin (POMC and neuropeptide Y (NPY neurons within the arcuate nucleus of the hypothalamus. In HEK293 cells co-transfected with Myc-tagged Asb-4 and Flag-tagged IRS4, Asb-4 co-immunoprecipitated with IRS4; In these cells endogenous IRS4 also co-immunoprecipitated with transfected Myc-Asb-4; Furthermore, Asb-4 co-immunoprecipitated with IRS4 in rat hypothalamic extracts. In HEK293 cells over expression of Asb-4 decreased IRS4 protein levels and deletion of the SOCS box abolished this effect. Asb-4 increased the ubiquitination of IRS4; Deletion of SOCS box abolished this effect. Expression of Asb-4 decreased both basal and insulin-stimulated phosphorylation of AKT at Thr308. Conclusions These data demonstrated that Asb-4 co-localizes and interacts with IRS4 in hypothalamic neurons. The interaction of Asb-4 with IRS4 in cell lines mediates the degradation of IRS4 and decreases insulin signaling.

  4. NeuronMetrics: software for semi-automated processing of cultured neuron images.

    Science.gov (United States)

    Narro, Martha L; Yang, Fan; Kraft, Robert; Wenk, Carola; Efrat, Alon; Restifo, Linda L

    2007-03-23

    Using primary cell culture to screen for changes in neuronal morphology requires specialized analysis software. We developed NeuronMetrics for semi-automated, quantitative analysis of two-dimensional (2D) images of fluorescently labeled cultured neurons. It skeletonizes the neuron image using two complementary image-processing techniques, capturing fine terminal neurites with high fidelity. An algorithm was devised to span wide gaps in the skeleton. NeuronMetrics uses a novel strategy based on geometric features called faces to extract a branch number estimate from complex arbors with numerous neurite-to-neurite contacts, without creating a precise, contact-free representation of the neurite arbor. It estimates total neurite length, branch number, primary neurite number, territory (the area of the convex polygon bounding the skeleton and cell body), and Polarity Index (a measure of neuronal polarity). These parameters provide fundamental information about the size and shape of neurite arbors, which are critical factors for neuronal function. NeuronMetrics streamlines optional manual tasks such as removing noise, isolating the largest primary neurite, and correcting length for self-fasciculating neurites. Numeric data are output in a single text file, readily imported into other applications for further analysis. Written as modules for ImageJ, NeuronMetrics provides practical analysis tools that are easy to use and support batch processing. Depending on the need for manual intervention, processing time for a batch of approximately 60 2D images is 1.0-2.5 h, from a folder of images to a table of numeric data. NeuronMetrics' output accelerates the quantitative detection of mutations and chemical compounds that alter neurite morphology in vitro, and will contribute to the use of cultured neurons for drug discovery.

  5. Leaders of neuronal cultures in a quorum percolation model

    Directory of Open Access Journals (Sweden)

    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.

  6. Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure.

    Science.gov (United States)

    Ciriello, J; Caverson, M M; McMurray, J C; Bruckschwaiger, E B

    2013-10-10

    Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which L-glutamate (0.25 M; 10 nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207 ± 78), 32.3 ± 2.3% co-expressed L-Enk immunoreactivity and of these, 2.6 ± 1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo

  7. 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.

  8. Reduced Synaptic Vesicle Recycling during Hypoxia in Cultured Cortical Neurons

    OpenAIRE

    Fedorovich, Sergei; Hofmeijer, Jeannette; van Putten, Michel Johannes Antonius Maria; le Feber, Jakob

    2017-01-01

    Improvement of neuronal recovery in the ischemic penumbra, an area around the core of a brain infarct with some remaining perfusion, has a large potential for the development of therapy against acute ischemic stroke. However, mechanisms that lead to either recovery or secondary damage in the penumbra largely remain unclear. Recent studies in cultured networks of cortical neurons showed that failure of synaptic transmission (referred to as synaptic failure) is a critical factor in the penumbra...

  9. Metabolism of Mannose in Cultured Primary Rat Neurons.

    Science.gov (United States)

    Rastedt, Wiebke; Blumrich, Eva-Maria; Dringen, Ralf

    2017-08-01

    Glucose is the main peripheral substrate for energy production in the brain. However, as other hexoses are present in blood and cerebrospinal fluid, we have investigated whether neurons have the potential to metabolize, in addition to glucose, also the hexoses mannose, fructose or galactose. Incubation of primary cerebellar granule neurons in the absence of glucose caused severe cell toxicity within 24 h, which could not be prevented by application of galactose or fructose, while the cells remained viable during incubation in the presence of either mannose or glucose. In addition, cultured neurons produced substantial and almost identical amounts of lactate after exposure to either glucose or mannose, while lactate production was low in the presence of fructose and hardly detectable during incubations without hexoses or with galactose as carbon source. Determination of the K M values of hexokinase in lysates of cultured neurons for the hexoses revealed values in the micromolar range for mannose (32 ± 2 µM) and glucose (59 ± 10 µM) and in the millimolar range for fructose (4.4 ± 2.3 mM), demonstrating that mannose is efficiently phosphorylated by neuronal hexokinase. Finally, cultured neurons contained reasonable specific activity of the enzyme phosphomannose isomerase, which is required for isomerization of the hexokinase product mannose-6-phosphate into the glycolysis intermediate fructose-6-phosphate. These data demonstrate that cultured cerebellar granule neurons have the potential and express the required enzymes to efficiently metabolize mannose, while galactose and fructose serve at best poorly as extracellular carbon sources for neurons.

  10. Hidden neuronal correlations in cultured networks

    International Nuclear Information System (INIS)

    Segev, Ronen; Baruchi, Itay; Hulata, Eyal; Ben-Jacob, Eshel

    2004-01-01

    Utilization of a clustering algorithm on neuronal spatiotemporal correlation matrices recorded during a spontaneous activity of in vitro networks revealed the existence of hidden correlations: the sequence of synchronized bursting events (SBEs) is composed of statistically distinguishable subgroups each with its own distinct pattern of interneuron spatiotemporal correlations. These findings hint that each of the SBE subgroups can serve as a template for coding, storage, and retrieval of a specific information

  11. Hypothalamic growth hormone receptor (GHR controls hepatic glucose production in nutrient-sensing leptin receptor (LepRb expressing neurons

    Directory of Open Access Journals (Sweden)

    Gillian Cady

    2017-05-01

    Full Text Available Objective: The GH/IGF-1 axis has important roles in growth and metabolism. GH and GH receptor (GHR are active in the central nervous system (CNS and are crucial in regulating several aspects of metabolism. In the hypothalamus, there is a high abundance of GH-responsive cells, but the role of GH signaling in hypothalamic neurons is unknown. Previous work has demonstrated that the Ghr gene is highly expressed in LepRb neurons. Given that leptin is a key regulator of energy balance by acting on leptin receptor (LepRb-expressing neurons, we tested the hypothesis that LepRb neurons represent an important site for GHR signaling to control body homeostasis. Methods: To determine the importance of GHR signaling in LepRb neurons, we utilized Cre/loxP technology to ablate GHR expression in LepRb neurons (LeprEYFPΔGHR. The mice were generated by crossing the Leprcre on the cre-inducible ROSA26-EYFP mice to GHRL/L mice. Parameters of body composition and glucose homeostasis were evaluated. Results: Our results demonstrate that the sites with GHR and LepRb co-expression include ARH, DMH, and LHA neurons. Leptin action was not altered in LeprEYFPΔGHR mice; however, GH-induced pStat5-IR in LepRb neurons was significantly reduced in these mice. Serum IGF-1 and GH levels were unaltered, and we found no evidence that GHR signaling regulates food intake and body weight in LepRb neurons. In contrast, diminished GHR signaling in LepRb neurons impaired hepatic insulin sensitivity and peripheral lipid metabolism. This was paralleled with a failure to suppress expression of the gluconeogenic genes and impaired hepatic insulin signaling in LeprEYFPΔGHR mice. Conclusion: These findings suggest the existence of GHR-leptin neurocircuitry that plays an important role in the GHR-mediated regulation of glucose metabolism irrespective of feeding. Keywords: Growth hormone receptor, Hypothalamus, Leptin receptor, Glucose production, Liver

  12. 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...... 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....

  13. Arcuate hypothalamic AgRP and putative POMC neurons show opposite changes in spiking across multiple timescales

    Science.gov (United States)

    Mandelblat-Cerf, Yael; Ramesh, Rohan N; Burgess, Christian R; Patella, Paola; Yang, Zongfang; Lowell, Bradford B; Andermann, Mark L

    2015-01-01

    Agouti-related-peptide (AgRP) neurons—interoceptive neurons in the arcuate nucleus of the hypothalamus (ARC)—are both necessary and sufficient for driving feeding behavior. To better understand the functional roles of AgRP neurons, we performed optetrode electrophysiological recordings from AgRP neurons in awake, behaving AgRP-IRES-Cre mice. In free-feeding mice, we observed a fivefold increase in AgRP neuron firing with mounting caloric deficit in afternoon vs morning recordings. In food-restricted mice, as food became available, AgRP neuron firing dropped, yet remained elevated as compared to firing in sated mice. The rapid drop in spiking activity of AgRP neurons at meal onset may reflect a termination of the drive to find food, while residual, persistent spiking may reflect a sustained drive to consume food. Moreover, nearby neurons inhibited by AgRP neuron photostimulation, likely including satiety-promoting pro-opiomelanocortin (POMC) neurons, demonstrated opposite changes in spiking. Finally, firing of ARC neurons was also rapidly modulated within seconds of individual licks for liquid food. These findings suggest novel roles for antagonistic AgRP and POMC neurons in the regulation of feeding behaviors across multiple timescales. DOI: http://dx.doi.org/10.7554/eLife.07122.001 PMID:26159614

  14. 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

    neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system...

  15. 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

  16. Deletion of Suppressor of Cytokine Signaling 3 from Forebrain Neurons Delays Infertility and Onset of Hypothalamic Leptin Resistance in Response to a High Caloric Diet.

    Science.gov (United States)

    McEwen, Hayden J L; Inglis, Megan A; Quennell, Janette H; Grattan, David R; Anderson, Greg M

    2016-07-06

    The cellular processes that cause high caloric diet (HCD)-induced infertility are poorly understood but may involve upregulation of suppressor of cytokine signaling (SOCS-3) proteins that are associated with hypothalamic leptin resistance. Deletion of SOCS-3 from brain cells is known to protect mice from diet-induced obesity, but the effects on HCD-induced infertility are unknown. We used neuron-specific SOCS3 knock-out mice to elucidate this and the effects on regional hypothalamic leptin resistance. As expected, male and female neuron-specific SOCS3 knock-out mice were protected from HCD-induced obesity. While female wild-type mice became infertile after 4 months of HCD feeding, infertility onset in knock-out females was delayed by 4 weeks. Similarly, knock-out mice had delayed leptin resistance development in the medial preoptic area and anteroventral periventricular nucleus, regions important for generation of the surge of GnRH and LH that induces ovulation. We therefore tested whether the suppressive effects of HCD on the estradiol-induced GnRH/LH surge were overcome by neuron-specific SOCS3 knock-out. Although only 20% of control HCD-mice experienced a preovulatory-like LH surge, LH surges could be induced in almost all neuron-specific SOCS3 knock-out mice on this diet. In contrast to females, HCD-fed male mice did not exhibit any fertility decline compared with low caloric diet-fed males despite their resistance to the satiety effects of leptin. These data show that deletion of SOCS3 delays the onset of leptin resistance and infertility in HCD-fed female mice, but given continued HCD feeding this state does eventually occur, presumably in response to other mechanisms inhibiting leptin signal transduction. Obesity is commonly associated with infertility in humans and other animals. Treatments for human infertility show a decreased success rate with increasing body mass index. A hallmark of obesity is an increase in circulating leptin levels; despite this, the

  17. An In Vitro System Comprising Immortalized Hypothalamic Neuronal Cells (GT1-7 Cells) for Evaluation of the Neuroendocrine Effects of Essential Oils.

    Science.gov (United States)

    Mizuno, Dai; Konoha-Mizuno, Keiko; Mori, Miwako; Yamazaki, Kentaro; Haneda, Toshihiro; Koyama, Hironari; Kawahara, Masahiro

    2015-01-01

    Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for symptoms including anxiety. Furthermore, it was reportedly effective for the care of several diseases such as Alzheimer's disease and depressive illness. To investigate the pharmacological effects of essential oils, we developed an in vitro assay system using immortalized hypothalamic neuronal cells (GT1-7 cells). In this study, we evaluated the effects of essential oils on neuronal death induced by hydrogen peroxide (H2O2), aluminum, zinc, or the antagonist of estrogen receptor (tamoxifen). Among tests of various essential oils, we found that H2O2-induced neuronal death was attenuated by the essential oils of damask rose, eucalyptus, fennel, geranium, ginger, kabosu, mandarin, myrrh, and neroli. Damask rose oil had protective effects against aluminum-induced neurotoxicity, while geranium and rosemary oil showed protective activity against zinc-induced neurotoxicity. In contrast, geranium oil and ginger oil enhanced the neurotoxicity of tamoxifen. Our in vitro assay system could be useful for the neuropharmacological and endocrine pharmacological studies of essential oils.

  18. An In Vitro System Comprising Immortalized Hypothalamic Neuronal Cells (GT1–7 Cells for Evaluation of the Neuroendocrine Effects of Essential Oils

    Directory of Open Access Journals (Sweden)

    Dai Mizuno

    2015-01-01

    Full Text Available Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for symptoms including anxiety. Furthermore, it was reportedly effective for the care of several diseases such as Alzheimer’s disease and depressive illness. To investigate the pharmacological effects of essential oils, we developed an in vitro assay system using immortalized hypothalamic neuronal cells (GT1–7 cells. In this study, we evaluated the effects of essential oils on neuronal death induced by hydrogen peroxide (H2O2, aluminum, zinc, or the antagonist of estrogen receptor (tamoxifen. Among tests of various essential oils, we found that H2O2-induced neuronal death was attenuated by the essential oils of damask rose, eucalyptus, fennel, geranium, ginger, kabosu, mandarin, myrrh, and neroli. Damask rose oil had protective effects against aluminum-induced neurotoxicity, while geranium and rosemary oil showed protective activity against zinc-induced neurotoxicity. In contrast, geranium oil and ginger oil enhanced the neurotoxicity of tamoxifen. Our in vitro assay system could be useful for the neuropharmacological and endocrine pharmacological studies of essential oils.

  19. Dietary sugars, not lipids, drive hypothalamic inflammation

    Directory of Open Access Journals (Sweden)

    Yuanqing Gao

    2017-08-01

    Conclusions: Combined overconsumption of fat and sugar, but not the overconsumption of fat per se, leads to excessive CML production in hypothalamic neurons, which, in turn, stimulates hypothalamic inflammatory responses such as microgliosis and eventually leads to neuronal dysfunction in the control of energy metabolism.

  20. 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...

  1. Signal transfer within a cultured asymmetric cortical neuron circuit.

    Science.gov (United States)

    Isomura, Takuya; Shimba, Kenta; Takayama, Yuzo; Takeuchi, Akimasa; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-12-01

    Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate. The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction. When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity. These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

  2. Signal transfer within a cultured asymmetric cortical neuron circuit

    Science.gov (United States)

    Isomura, Takuya; Shimba, Kenta; Takayama, Yuzo; Takeuchi, Akimasa; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-12-01

    Objective. Simplified neuronal circuits are required for investigating information representation in nervous systems and for validating theoretical neural network models. Here, we developed patterned neuronal circuits using micro fabricated devices, comprising a micro-well array bonded to a microelectrode-array substrate. Approach. The micro-well array consisted of micrometre-scale wells connected by tunnels, all contained within a silicone slab called a micro-chamber. The design of the micro-chamber confined somata to the wells and allowed axons to grow through the tunnels bidirectionally but with a designed, unidirectional bias. We guided axons into the point of the arrow structure where one of the two tunnel entrances is located, making that the preferred direction. Main results. When rat cortical neurons were cultured in the wells, their axons grew through the tunnels and connected to neurons in adjoining wells. Unidirectional burst transfers and other asymmetric signal-propagation phenomena were observed via the substrate-embedded electrodes. Seventy-nine percent of burst transfers were in the forward direction. We also observed rapid propagation of activity from sites of local electrical stimulation, and significant effects of inhibitory synapse blockade on bursting activity. Significance. These results suggest that this simple, substrate-controlled neuronal circuit can be applied to develop in vitro models of the function of cortical microcircuits or deep neural networks, better to elucidate the laws governing the dynamics of neuronal networks.

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

    Science.gov (United States)

    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.

  4. A low-density culture method of cerebellar granule neurons with paracrine support applicable for the study of neuronal morphogenesis.

    Science.gov (United States)

    Kubota, Kenta; Seno, Takeshi; Konishi, Yoshiyuki

    2013-11-20

    Cerebellar granule neuronal cultures have been used to study the molecular mechanisms underlying neuronal functions, including neuronal morphogenesis. However, a limitation of this system is the difficulty to analyze isolated neurons because these are required to be maintained at a high density. Therefore, in the present study, we aimed to develop a simple and cost-effective method for culturing low-density cerebellar granule neurons. Cerebellar granule cells at two different densities (low- and high-density) were co-cultivated in order for the low-density culture to be supported by the paracrine signals from the high-density culture. This method enabled morphology analysis of isolated cerebellar granule neurons without astrocytic feeder cultures or supplements such as B27. Using this method, we investigated the function of a polarity factor. Studies using hippocampal neurons suggested that glycogen synthase kinase-3 (GSK-3) is an essential regulator of neuronal polarity, and inhibition of GSK-3 results in the formation of multiple axons. Pharmacological inhibitors for GSK-3 (6-bromoindirubin-3'-oxime and lithium chloride) did not cause the formation of multiple axons of cerebellar granule neurons but significantly reduced their length. Consistent results were obtained by introducing kinase-dead form of GSK-3 beta (K85A). These results indicated that GSK-3 is not directly involved in the control of neuronal polarity in cerebellar granule neurons. Overall, this study provides a simple method for culturing low-density cerebellar granule neurons and insights in to the neuronal-type dependent function of GSK-3 in neuronal morphogenesis. © 2013 Elsevier B.V. All rights reserved.

  5. Desipramine inhibits histamine H1 receptor-induced Ca2+ signaling in rat hypothalamic cells.

    Directory of Open Access Journals (Sweden)

    Ji-Ah Kang

    Full Text Available The hypothalamus in the brain is the main center for appetite control and integrates signals from adipose tissue and the gastrointestinal tract. Antidepressants are known to modulate the activities of hypothalamic neurons and affect food intake, but the cellular and molecular mechanisms by which antidepressants modulate hypothalamic function remain unclear. Here we have investigated how hypothalamic neurons respond to treatment with antidepressants, including desipramine and sibutramine. In primary cultured rat hypothalamic cells, desipramine markedly suppressed the elevation of intracellular Ca(2+ evoked by histamine H1 receptor activation. Desipramine also inhibited the histamine-induced Ca(2+ increase and the expression of corticotrophin-releasing hormone in hypothalamic GT1-1 cells. The effect of desipramine was not affected by pretreatment with prazosin or propranolol, excluding catecholamine reuptake activity of desipramine as an underlying mechanism. Sibutramine which is also an antidepressant but decreases food intake, had little effect on the histamine-induced Ca(2+ increase or AMP-activated protein kinase activity. Our results reveal that desipramine and sibutramine have different effects on histamine H1 receptor signaling in hypothalamic cells and suggest that distinct regulation of hypothalamic histamine signaling might underlie the differential regulation of food intake between antidepressants.

  6. Alcohol alters hypothalamic glial-neuronal communications involved in the neuroendocrine control of puberty: In vivo and in vitro assessments.

    Science.gov (United States)

    Dees, W L; Hiney, J K; Srivastava, V K

    2015-11-01

    The onset of puberty is the result of the increased secretion of hypothalamic luteinizing hormone-releasing hormone (LHRH). The pubertal process can be altered by substances that can affect the prepubertal secretion of this peptide. Alcohol is one such substance known to diminish LHRH secretion and delay the initiation of puberty. The increased secretion of LHRH that normally occurs at the time of puberty is due to a decrease of inhibitory tone that prevails prior to the onset of puberty, as well as an enhanced development of excitatory inputs to the LHRH secretory system. Additionally, it has become increasingly clear that glial-neuronal communications are important for pubertal development because they play an integral role in facilitating the pubertal rise in LHRH secretion. Thus, in recent years attempts have been made to identify specific glial-derived components that contribute to the development of coordinated communication networks between glia and LHRH cell bodies, as well as their nerve terminals. Transforming growth factor-α and transforming growth factor-β1 are two such glial substances that have received attention in this regard. This review summarizes the use of multiple neuroendocrine research techniques employed to assess these glial-neuronal communication pathways involved in regulating prepubertal LHRH secretion and the effects that alcohol can have on their respective functions. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Neurons of the A5 region are required for the tachycardia evoked by electrical stimulation of the hypothalamic defence area in anaesthetized rats.

    Science.gov (United States)

    López-González, M V; Díaz-Casares, A; Peinado-Aragonés, C A; Lara, J P; Barbancho, M A; Dawid-Milner, M S

    2013-08-01

    In order to assess the possible interactions between the pontine A5 region and the hypothalamic defence area (HDA), we have examined the pattern of double staining for c-Fos protein immunoreactivity (c-Fos-ir) and tyrosine hydroxylase, throughout the rostrocaudal extent of the A5 region in spontaneously breathing anaesthetized male Sprague-Dawley rats during electrical stimulation of the HDA. Activation of the HDA elicited a selective increase in c-Fos-ir with an ipsilateral predominance in catecholaminergic and non-catecholaminergic A5 somata (P HDA. Cardiorespiratory changes were analysed in response to electrical stimulation of the HDA before and after ipsilateral microinjection of muscimol within the A5 region. Stimulation of the HDA evoked an inspiratory facilitatory response, consisting of an increase in respiratory rate (P HDA stimulation were reduced (P HDA and the A5 region, extracellular recordings of putative A5 neurones were obtained during HDA stimulation. Seventy-five A5 cells were recorded, 35 of which were affected by the HDA (47%). These results indicate that neurones of the A5 region participate in the cardiovascular response evoked from the HDA. The possible mechanisms involved in these interactions are discussed.

  8. Efferent connections from the lateral hypothalamic region and the lateral preoptic area to the hypothalamic paraventricular nucleus of the rat

    DEFF Research Database (Denmark)

    Larsen, P J; Hay-Schmidt, Anders; Mikkelsen, J D

    1994-01-01

    , iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin were delivered into distinct areas of the lateral hypothalamic region. Neurons of the intermediate hypothalamic area projected mainly to the PVN subnuclei, which contained parvicellular neuroendocrine cells. In contrast...

  9. Fetal alcohol exposure disrupts metabolic signaling in hypothalamic proopiomelanocortin neurons via a circadian mechanism in male mice.

    Science.gov (United States)

    Agapito, Maria A; Zhang, Changqing; Murugan, Sengottuvelan; Sarkar, Dipak K

    2014-07-01

    Early-life ethanol feeding (ELAF) alters the metabolic function of proopiomelanocortin (POMC)-producing neurons and the circadian expression of clock regulatory genes in the hypothalamus. We investigated whether the circadian mechanisms control the action of ELAF on metabolic signaling genes in POMC neurons. Gene expression measurements of Pomc and a selected group of metabolic signaling genes, Stat3, Sirt1, Pgc1-α, and Asb4 in laser-captured microdissected POMC neurons in the hypothalamus of POMC-enhanced green fluorescent protein mice showed circadian oscillations under light/dark and constant darkness conditions. Ethanol programmed these neurons such that the adult expression of Pomc, Stat3, Sirt, and Asb4 gene transcripts became arrhythmic. In addition, ELAF dampened the circadian peak of gene expression of Bmal1, Per1, and Per2 in POMC neurons. We crossed Per2 mutant mice with transgenic POMC-enhanced green fluorescent protein mice to determine the role of circadian mechanism in ELAF-altered metabolic signaling in POMC neurons. We found that ELAF failed to alter arrhythmic expression of most circadian genes, with the exception of the Bmal1 gene and metabolic signaling regulating genes in Per2 mutant mice. Comparison of the ELAF effects on the circadian blood glucose in wild-type and Per2 mutant mice revealed that ELAF dampened the circadian peak of glucose, whereas the Per2 mutation shifted the circadian cycle and prevented the ELAF dampening of the glucose peak. These data suggest the possibility that the Per2 gene mutation may regulate the ethanol actions on Pomc and the metabolic signaling genes in POMC neurons in the hypothalamus by blocking circadian mechanisms.

  10. Water deprivation increases Fos expression in hypothalamic corticotropin-releasing factor neurons induced by right atrial distension in awake rats.

    Science.gov (United States)

    Benedetti, Mauricio; Rorato, Rodrigo; Castro, Margaret; Machado, Benedito H; Antunes-Rodrigues, Jose; Elias, Lucila L K

    2008-11-01

    Atrial mechanoreceptors, sensitive to stretch, contribute in regulating heart rate and intravascular volume. The information from those receptors reaches the nucleus tractus solitarius and then the paraventricular nucleus (PVN), known to have a crucial role in the regulation of cardiovascular function. Neurons in the PVN synthesize CRF, AVP, and oxytocin (OT). Stimulation of atrial mechanoreceptors was performed in awake rats implanted with a balloon at the junction of the superior vena cava and right atrium. Plasma ACTH, AVP, and OT concentrations and Fos, CRF, AVP, and OT immunolabeling in the PVN were determined after balloon inflation in hydrated and water-deprived rats. The distension of the balloon increased the plasma ACTH concentrations, which were higher in water-deprived than in hydrated rats (P neurons in the parvocellular PVN, which was higher in the water-deprived than in the hydrated group (P neurons after distension in hydrated and water-deprived groups, compared with respective controls. In conclusion, parvocellular CRF neurons showed an increase of Fos expression induced by stimulation of right atrial mechanoreceptors, suggesting that CRF participates in the cardiovascular reflex adjustments elicited by volume loading. Activation of CRF neurons in the PVN by cardiovascular reflex is affected by osmotic stimulation.

  11. Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

    Directory of Open Access Journals (Sweden)

    Martin Aldasoro

    Full Text Available Ranolazine (Rn is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M. Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-β and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 β and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.

  12. Hypothalamic lipophagy and energetic balance

    OpenAIRE

    Singh, Rajat

    2011-01-01

    Autophagy is a conserved cellular turnover process that degrades unwanted cytoplasmic material within lysosomes. Through ?in bulk? degradation of cytoplasmic proteins and organelles, including lipid droplets, autophagy helps provide an alternative fuel source, in particular, when nutrients are scarce. Recent work demonstrates a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in regulation of food intake and energy balance. The induction of autophagy in hypothalamic ne...

  13. Neural input is critical for arcuate hypothalamic neurons to mount intracellular signaling responses to systemic insulin and deoxyglucose challenges in male rats: implications for communication within feeding and metabolic control networks.

    Science.gov (United States)

    Khan, Arshad M; Walker, Ellen M; Dominguez, Nicole; Watts, Alan G

    2014-02-01

    The hypothalamic arcuate nucleus (ARH) controls rat feeding behavior in part through peptidergic neurons projecting to the hypothalamic paraventricular nucleus (PVH). Hindbrain catecholaminergic (CA) neurons innervate both the PVH and ARH, and ablation of CA afferents to PVH neuroendocrine neurons prevents them from mounting cellular responses to systemic metabolic challenges such as insulin or 2-deoxy-d-glucose (2-DG). Here, we asked whether ablating CA afferents also limits their ARH responses to the same challenges or alters ARH connectivity with the PVH. We examined ARH neurons for three features: (1) CA afferents, visualized by dopamine-β-hydroxylase (DBH)- immunoreactivity; (2) activation by systemic metabolic challenge, as measured by increased numbers of neurons immunoreactive (ir) for phosphorylated ERK1/2 (pERK1/2); and (3) density of PVH-targeted axons immunoreactive for the feeding control peptides Agouti-related peptide and α-melanocyte-stimulating hormone (αMSH). Loss of PVH DBH immunoreactivity resulted in concomitant ARH reductions of DBH-ir and pERK1/2-ir neurons in the medial ARH, where AgRP neurons are enriched. In contrast, pERK1/2 immunoreactivity after systemic metabolic challenge was absent in αMSH-ir ARH neurons. Yet surprisingly, axonal αMSH immunoreactivity in the PVH was markedly increased in CA-ablated animals. These results indicate that (1) intrinsic ARH activity is insufficient to recruit pERK1/2-ir ARH neurons during systemic metabolic challenges (rather, hindbrain-originating CA neurons are required); and (2) rats may compensate for a loss of CA innervation to the ARH and PVH by increased expression of αMSH. These findings highlight the existence of a hierarchical dependence for ARH responses to neural and humoral signals that influence feeding behavior and metabolism.

  14. Effects of chronic restraint stress on social behaviors and the number of hypothalamic oxytocin neurons in male rats.

    Science.gov (United States)

    Li, Jin; Li, Han-Xia; Shou, Xiao-Jing; Xu, Xin-Jie; Song, Tian-Jia; Han, Song-Ping; Zhang, Rong; Han, Ji-Sheng

    2016-12-01

    Oxytocin (OXT) and vasopressin (AVP) are considered to be related to mammalian social behavior and the regulation of stress responses. The present study investigated the effects of chronic homotypic restraint stress (CHRS) on social behaviors and anxiety, as well as its repercussions on OXT- and AVP-positive neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) nuclei in rat. Male Sprague-Dawley rats receiving CHRS were exposed to repeated restraint stress of 30min per day for 10days. Changes in social approach behaviors were evaluated with the three-chambered social approach task. Changes in anxiety-like behaviors were evaluated in the light-dark box test. The number of neurons expressing oxytocin and/or vasopressin in PVN and SON were examined by immunohistochemistry techniques. The results demonstrated that social approach was increased and anxiety was decreased following 10-day exposure to CHRS. Furthermore, the number of OXT-immunoreactive cells in PVN was increased significantly, whereas no change in SON was seen. The number of AVP immunoreactive cells either in PVN or SON was unaffected. The results of this study suggest that certain types of stress could be effective in the treatment of social dysfunction in persons with mental disorders such as autism, social anxiety disorder. The therapeutic effects may be mediated by changes in the function of OXT neurons in PVN. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Control of energy balance by hypothalamic gene circuitry involving two nuclear receptors, neuron-derived orphan receptor 1 and glucocorticoid receptor.

    Science.gov (United States)

    Kim, Sun-Gyun; Lee, Bora; Kim, Dae-Hwan; Kim, Juhee; Lee, Seunghee; Lee, Soo-Kyung; Lee, Jae W

    2013-10-01

    Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

  16. Impact of Triclosan on Female Reproduction through Reducing Thyroid Hormones to Suppress Hypothalamic Kisspeptin Neurons in Mice

    Directory of Open Access Journals (Sweden)

    Xin-Yuan Cao

    2018-01-01

    Full Text Available Triclosan (TCS, a broad-spectrum antimicrobial agent, is widely used in clinical settings and various personal care products. The aim of this study was to evaluate the influence of TCS on reproductive endocrine and function. Here, we show that the exposure of adult female mice to 10 or 100 mg/kg/day TCS caused prolongation of diestrus, and decreases in antral follicles and corpora lutea within 2 weeks. TCS mice showed decreases in the levels of serum luteinizing hormone (LH, follicle-stimulating hormone (FSH and progesterone, and gonadotrophin-releasing hormone (GnRH mRNA with the lack of LH surge and elevation of prolactin (PRL. TCS mice had lower kisspeptin immunoreactivity and kiss1 mRNA in anteroventral periventricular nucleus (AVPV and arcuate nucleus (ARC. Moreover, the estrogen (E2-enhanced AVPV-kisspeptin expression was reduced in TCS mice. In addition, the serum thyroid hormones (triiodothyronine (T3 and thyroxine (T4 in TCS mice were reduced with increases in levels of thyroid stimulating hormone (TSH and thyroid releasing hormone (TRH. In TCS mice, the treatment with Levothyroxine (L-T4 corrected the increases in PRL, TSH and TRH; the administration of L-T4 or type-2 dopamine receptors agonist quinpirole inhibiting PRL release could rescue the decline of kisspeptin expression in AVPV and ARC; the treatment with L-T4, quinpirole or the GPR45 agonist kisspeptin-10 recovered the levels of serum LH and FSH and progesterone, and GnRH mRNA. Furthermore, TCS mice treated with L-T4 or quinpirole resumed regular estrous cycling, follicular development and ovulation. Together, these results indicate that exposing adult female mice to TCS (≥10 mg/kg reduces thyroid hormones causing hyperprolactinemia that then suppresses hypothalamic kisspeptin expression, leading to deficits in reproductive endocrine and function.

  17. Recruitment of hypothalamic orexin neurons after formalin injections in adult male rats exposed to a neonatal immune challenge

    Directory of Open Access Journals (Sweden)

    Erin Jane Campbell

    2015-03-01

    Full Text Available Exposure to early life physiological stressors, such as infection, is thought to contribute to the onset of psychopathology in adulthood. In animal models, injections of the bacterial immune challenge, lipopolysaccharide (LPS, during the neonatal period has been shown to alter both neuroendocrine function and behavioural pain responses in adulthood. Interestingly, recent evidence suggests a role for the lateral hypothalamic peptide orexin in stress and nociceptive processing. However, whether neonatal LPS exposure affects the reactivity of the orexin system to formalin-induced inflammatory pain in later life remains to be determined. Male Wistar rats (n=13 were exposed to either LPS or saline (0.05mg/kg, i.p on postnatal days (PND 3 and 5. On PND 80-97, all rats were exposed to a subcutaneous hindpaw injection of 2.25% formalin. Following behavioural testing, animals were perfused and brains processed for Fos-protein and orexin immunohistochemistry. Rats treated with LPS during the neonatal period exhibited decreased licking behaviours during the interphase of the formalin test, the period typically associated with the active inhibition of pain, and increased grooming responses to formalin in adulthood. Interestingly, these behavioural changes were accompanied by an increase in the percentage of Fos-positive orexin cells in the dorsomedial and perifornical hypothalamus in LPS-exposed animals. Similar increases in Fos-protein were also observed in stress and pain sensitive brain regions that receive orexinergic inputs. These findings highlight a potential role for orexin in the behavioural responses to pain and provide further evidence that early life stress can prime the circuitry responsible for these responses in adulthood.

  18. Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.

    OpenAIRE

    Flavell, D M; Wells, T; Wells, S E; Carmignac, D F; Thomas, G B; Robinson, I C

    1996-01-01

    Expression of human growth hormone (hGH) was targeted to growth hormone-releasing (GRF) neurons in the hypothalamus of transgenic rats. This induced dominant dwarfism by local feedback inhibition of GRF. One line, bearing a single copy of a GRF-hGH transgene, has been characterized in detail, and has been termed Tgr (for Transgenic growth-retarded). hGH was detected by immunocytochemistry in the brain, restricted to the median eminence of the hypothalamus. Low levels were also detected in the...

  19. Topography of subnuclei of the hypothalamic paraventricular nucleus in rats and sensitivity of their neurons to insulin defficiency

    International Nuclear Information System (INIS)

    Goufman, E.I.

    1985-01-01

    This investigation was undertaken to study the reaction of paraventricular nuclei (PVN) subnuclei to insulin deficiency and to elevation of the blood glucose level under conditions of experimental alloxan diabetes. Experiments were carried out on 15 control and 15 experimental mature male Wistar rats. The state of the carbohydrate metabolism of the diabetic and control animals was judged by the blood glucose and radioimmune insulin levels. The results of these investigations show that both magnocellular and parvocellular neurons of PVN react to alloxan diabetes, which supports the hypothesis that PVN of the hypothalamus participates in the control of carbohydrate metabolism

  20. 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. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. 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.

  2. Influence of serial electrical stimulations of perifornical and posterior hypothalamic orexin-containing neurons on regulation of sleep homeostasis and sleep-wakefulness cycle recovery from experimental comatose state and anesthesia-induced deep sleep.

    Science.gov (United States)

    Chijavadze, E; Chkhartishvili, E; Babilodze, M; Maglakelidze, N; Nachkebia, N

    2013-11-01

    The work was aimed for the ascertainment of following question - whether Orexin-containing neurons of dorsal and lateral hypothalamic, and brain Orexinergic system in general, are those cellular targets which can speed up recovery of disturbed sleep homeostasis and accelerate restoration of sleep-wakefulness cycle phases during some pathological conditions - experimental comatose state and/or deep anesthesia-induced sleep. Study was carried out on white rats. Modeling of experimental comatose state was made by midbrain cytotoxic lesions at intra-collicular level.Animals were under artificial respiration and special care. Different doses of Sodium Ethaminal were used for deep anesthesia. 30 min after comatose state and/or deep anesthesia induced sleep serial electrical stimulations of posterior and/or perifornical hypothalamus were started. Stimulation period lasted for 1 hour with the 5 min intervals between subsequent stimulations applied by turn to the left and right side hypothalamic parts.EEG registration of cortical and hippocampal electrical activity was started immediately after experimental comatose state and deep anesthesia induced sleep and continued continuously during 72 hour. According to obtained new evidences, serial electrical stimulations of posterior and perifornical hypothalamic Orexin-containing neurons significantly accelerate recovery of sleep homeostasis, disturbed because of comatose state and/or deep anesthesia induced sleep. Speed up recovery of sleep homeostasis was manifested in acceleration of coming out from comatose state and deep anesthesia induced sleep and significant early restoration of sleep-wakefulness cycle behavioral states.

  3. Caudal fourth ventricular administration of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside regulates glucose and counterregulatory hormone profiles, dorsal vagal complex metabolosensory neuron function, and hypothalamic Fos expression.

    Science.gov (United States)

    Ibrahim, Baher A; Tamrakar, Pratistha; Gujar, Amit D; Cherian, Ajeesh Koshy; Briski, Karen P

    2013-09-01

    This study investigated the hypothesis that estrogen controls hindbrain AMP-activated protein kinase (AMPK) activity and regulation of blood glucose, counterregulatory hormone secretion, and hypothalamic nerve cell transcriptional status. Dorsal vagal complex A2 noradrenergic neurons were laser microdissected from estradiol benzoate (E)- or oil (O)-implanted ovariectomized female rats after caudal fourth ventricular (CV4) delivery of the AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR), for Western blot analysis. E advanced AICAR-induced increases in A2 phospho-AMPK (pAMPK) expression and in blood glucose levels and was required for augmentation of Fos, estrogen receptor-α (ERα), monocarboxylate transporter-2, and glucose transporter-3 protein in A2 neurons and enhancement of corticosterone secretion by this treatment paradigm. CV4 AICAR also resulted in site-specific modifications in Fos immunolabeling of hypothalamic metabolic structures, including the paraventricular, ventromedial, and arcuate nuclei. The current studies demonstrate that estrogen regulates AMPK activation in caudal hindbrain A2 noradrenergic neurons during pharmacological replication of energy shortage in this area of the brain, and that this sensor is involved in neural regulation of glucostasis, in part, through control of corticosterone secretion. The data provide unique evidence that A2 neurons express both ERα and -β proteins and that AMPK upregulates cellular sensitivity to ERα-mediated signaling during simulated energy insufficiency. The results also imply that estrogen promotes glucose and lactate uptake by these cells under those conditions. Evidence for correlation between hindbrain AMPK and hypothalamic nerve cell genomic activation provides novel proof for functional connectivity between this hindbrain sensor and higher order metabolic brain loci while demonstrating a modulatory role for estrogen in this interaction. Copyright © 2013 Wiley Periodicals, Inc.

  4. A combined Bodian-Nissl stain for improved network analysis in neuronal cell culture.

    Science.gov (United States)

    Hightower, M; Gross, G W

    1985-11-01

    Bodian and Nissl procedures were combined to stain dissociated mouse spinal cord cells cultured on coverslips. The Bodian technique stains fine neuronal processes in great detail as well as an intracellular fibrillar network concentrated around the nucleus and in proximal neurites. The Nissl stain clearly delimits neuronal cytoplasm in somata and in large dendrites. A combination of these techniques allows the simultaneous depiction of neuronal perikarya and all afferent and efferent processes. Costaining with little background staining by either procedure suggests high specificity for neurons. This procedure could be exploited for routine network analysis of cultured neurons.

  5. [Establishment of oxygen and glucose deprive model of in vitro cultured hippocampal neuron and effect of ligustrazine on intracellular Ca+ level in model neurons].

    Science.gov (United States)

    Wan, Hai-tong; Wang, Yu; Yang, Jie-hong

    2007-03-01

    To establish the oxygen and glucose deprive (OGD) model in cultured hippocampal neuron and study the effect of ligustrazine on intracellular Ca2+ level in the model neurons. The OGD model was established in cultured hippocampal neuron, and the intracellular Ca2+ level in it was detected by laser scanning confocal microscope (LSCM). The OGD model was successfully established in cultured hippocampal neurons; the intracellular Ca2+ level in the OGD model group was significantly higher than that in the blank control group (P neuron, which could be antagonized by ligustrazine, indicating that ligustrazine has a protective effect on hippocampal neuron from hypoxic-ischemic injury.

  6. Immunocytochemical indications for neuronal co-localization of GABA and aspartate in cultured neocortex explants

    NARCIS (Netherlands)

    de Jong, B. M.; Ruijter, J. M.; Buijs, R. M.

    1989-01-01

    The application of postembedding immunocytochemistry on serial semithin plastic sections, revealed the presence of gamma-aminobutyric acid (GABA)-positive and aspartate-positive neurons in cultured neocortex explants. GABA-positive neurons were found in all layers of the cultured cortex, whereas

  7. Single-cell axotomy of cultured hippocampal neurons integrated in neuronal circuits.

    Science.gov (United States)

    Gomis-Rüth, Susana; Stiess, Michael; Wierenga, Corette J; Meyn, Liane; Bradke, Frank

    2014-05-01

    An understanding of the molecular mechanisms of axon regeneration after injury is key for the development of potential therapies. Single-cell axotomy of dissociated neurons enables the study of the intrinsic regenerative capacities of injured axons. This protocol describes how to perform single-cell axotomy on dissociated hippocampal neurons containing synapses. Furthermore, to axotomize hippocampal neurons integrated in neuronal circuits, we describe how to set up coculture with a few fluorescently labeled neurons. This approach allows axotomy of single cells in a complex neuronal network and the observation of morphological and molecular changes during axon regeneration. Thus, single-cell axotomy of mature neurons is a valuable tool for gaining insights into cell intrinsic axon regeneration and the plasticity of neuronal polarity of mature neurons. Dissociation of the hippocampus and plating of hippocampal neurons takes ∼2 h. Neurons are then left to grow for 2 weeks, during which time they integrate into neuronal circuits. Subsequent axotomy takes 10 min per neuron and further imaging takes 10 min per neuron.

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

    Science.gov (United States)

    Hui, Chin Wai; Zhang, Yang; Herrup, Karl

    2016-01-01

    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.

  9. Miniature excitatory synaptic currents in cultured hippocampal neurons.

    Science.gov (United States)

    Finch, D M; Fisher, R S; Jackson, M B

    1990-06-04

    We performed patch clamp recordings in the whole cell mode from cultured embryonic mouse hippocampal neurons. In bathing solutions containing tetrodotoxin (TTX), the cells showed spontaneous inward currents (SICs) ranging in size from 1 to 100 pA. Several observations indicated that the SICs were miniature excitatory synaptic currents mediated primarily by non-NMDA (N-methyl-D-aspartate) excitatory amino acid receptors: the rising phase of SICs was fast (1 ms to half amplitude at room temperature) and smooth, suggesting unitary events. The SICs were blocked by the broad-spectrum glutamate receptor antagonist gamma-D-glutamylglycine (DGG), but not by the selective NMDA-receptor antagonist D-2-amino-5-phosphonovaleric acid (5-APV). SICs were also blocked by desensitizing concentrations of quisqualate. Incubating cells in tetanus toxin, which blocks exocytotic transmitter release, eliminated SICs. The presence of SICs was consistent with the morphological arrangement of glutamatergic innervation in the cell cultures demonstrated immunohistochemically. Spontaneous outward currents (SOCs) were blocked by bicuculline and presumed to be mediated by GABAA receptors. This is consistent with immunohistochemical demonstration of GABAergic synapses. SIC frequency was increased in a calcium dependent manner by bathing the cells in a solution high in K+, and application of the dihydropyridine L-type calcium channel agonist BAY K 8644 increased the frequency of SICs. Increases in SIC frequency produced by high K+ solutions were reversed by Cd2+ and omega-conotoxin GVIA, but not by the selective L-type channel antagonist nimodipine. This suggested that presynaptic L-type channels were in a gating mode that was not blocked by nimodipine, and/or that another class of calcium channel makes a dominant contribution to excitatory transmitter release.

  10. Neuronal Culture and labelling of receptors of rat brain by a radioactive molecule labelled with technetium

    International Nuclear Information System (INIS)

    Barhoumi, C; Mejri, N.; Saidi, M.; Coulais, Y.; Dunia, D.; Masmoudi, O.; Amri, M.

    2009-01-01

    Alzheimer's disease is a neurodegenerative disease of the brain which causes progressive and irreversible loss of mental function. It is characterized by a decrease of serotoninergic neurons that carry the 5HT1A receptors. In our study, we performed cultures of hippocampal and cortical neurons from brains of young rats. After the differentiation of these neurons, some wells of cell culture were incubated with 8 OH DPAT, a 5HT1A agonist of serotonin, which are located on the surface of neurons.The neurons were then incubated with a molecule labelled with technetium 99m Tc. These neurons are lysed and the radioactivity is read. The results show that for the culture of neurons in the hippocampus, we have levels of radioactivity of cells treated with agonist, below the level of radioactivity of cells treated with the radioactive molecule. Cortical neurons show the same level of radioactivity of cells treated with agonist and for cells treated only with the labelled molecule. Our results show a decrease in the fixation of the labelled molecule on serotoninergic neurons in the hippocampus compared to neurons in the cortex. This work will be continued in humans in order to achieve early diagnosis of Alzheimer's disease

  11. Over a century of neuron culture: from the hanging drop to microfluidic devices.

    Science.gov (United States)

    Millet, Larry J; Gillette, Martha U

    2012-12-01

    The brain is the most intricate, energetically active, and plastic organ in the body. These features extend to its cellular elements, the neurons and glia. Understanding neurons, or nerve cells, at the cellular and molecular levels is the cornerstone of modern neuroscience. The complexities of neuron structure and function require unusual methods of culture to determine how aberrations in or between cells give rise to brain dysfunction and disease. Here we review the methods that have emerged over the past century for culturing neurons in vitro, from the landmark finding by Harrison (1910) - that neurons can be cultured outside the body - to studies utilizing culture vessels, micro-islands, Campenot and brain slice chambers, and microfluidic technologies. We conclude with future prospects for neuronal culture and considerations for advancement. We anticipate that continued innovation in culture methods will enhance design capabilities for temporal control of media and reagents (chemotemporal control) within sub-cellular environments of three-dimensional fluidic spaces (microfluidic devices) and materials (e.g., hydrogels). They will enable new insights into the complexities of neuronal development and pathology.

  12. [3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes

    International Nuclear Information System (INIS)

    Gray, D.B.; Tuttle, J.B.

    1987-01-01

    Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation

  13. Deficiency of leptin receptor in myeloid cells disrupts hypothalamic metabolic circuits and causes body weight increase

    Directory of Open Access Journals (Sweden)

    Yuanqing Gao

    2018-01-01

    Conclusions: Myeloid cell leptin receptor deficient mice partially replicate the db/db phenotype. Leptin signaling in hypothalamic microglia is important for microglial function and a correct formation of the hypothalamic neuronal circuit regulating metabolism.

  14. Effect of correlating adjacent neurons for identifying communications: Feasibility experiment in a cultured neuronal network

    OpenAIRE

    Yoshi Nishitani; Chie Hosokawa; Yuko Mizuno-Matsumoto; Tomomitsu Miyoshi; Shinichi Tamura

    2017-01-01

    Neuronal networks have fluctuating characteristics, unlike the stable characteristics seen in computers. The underlying mechanisms that drive reliable communication among neuronal networks and their ability to perform intelligible tasks remain unknown. Recently, in an attempt to resolve this issue, we showed that stimulated neurons communicate via spikes that propagate temporally, in the form of spike trains. We named this phenomenon “spike wave propagation”. In these previous studies, using ...

  15. Pyrethroid insecticide accumulation in primary cultures of cortical neurons in vitro

    Science.gov (United States)

    Primary cultures of neurons have been widely utilized to study the actions of pyrethroids and other neurotoxicants, with the presumption that the media concentration accurately reflects the dose received by the cells. However, recent studies have demonstrated that lipophilic comp...

  16. Induction of neuronal axon outgrowth by Shati/Nat8l by energy metabolism in mice cultured neurons.

    Science.gov (United States)

    Sumi, Kazuyuki; Uno, Kyosuke; Matsumura, Shohei; Miyamoto, Yoshiaki; Furukawa-Hibi, Yoko; Muramatsu, Shin-Ichi; Nabeshima, Toshitaka; Nitta, Atsumi

    2015-09-09

    A novel N-acetyltransferase, Shati/Nat8l, was identified in the nucleus accumbens of mice repeatedly treated with methamphetamine (METH). Shati/Nat8l has been reported to inhibit the pharmacological action induced by METH. Shati/Nat8l produces N-acetylaspartate from aspartate and acetyl-CoA. Previously, we reported that overexpression of Shati/Nat8l in nucleus accumbens attenuates the response to METH by N-acetylaspartylglutamate (which is derived from N-acetylaspartate)-mGluR3 signaling in the mice brain. In the present study, to clarify the type of cells that produce Shati/Nat8l, we carried out in-situ hybridization for the detection of Shati/Nat8l mRNA along with immunohistochemical studies using serial sections of mice brain. Shati/Nat8l mRNA was detected in neuronal cells, but not in astrocytes or microglia cells. Next, we investigated the function of Shati/Nat8l in the neuronal cells in mice brain; then, we used an adeno-associated virus vector containing Shati/Nat8l for transfection and overexpression of Shati/Nat8l protein into the primary cultured neurons to investigate the contribution toward the neuronal activity of Shati/Nat8l. Overexpression of Shati/Nat8l in the mice primary cultured neurons induced axonal growth, but not dendrite elongation at day 1.5 (DIV). This finding indicated that Shati/Nat8l contributes toward neuronal development. LY341495, a selective group II mGluRs antagonist, did not abolish this axonal growth, and N-acetylaspartylglutamate itself did not abolish axon outgrowth in the same cultured system. The cultured neurons overexpressing Shati/Nat8l contained high ATP, suggesting that axon outgrowth is dependent on energy metabolism. This study shows that Shati/Nat8l in the neuron may induce axon outgrowth by ATP synthesis and not through mGluR3 signaling.

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

    International Nuclear Information System (INIS)

    Witte, H; Warnke, C; Krost, A; Voigt, T; De Lima, A; Ivanov, I; Vidakovic-Koch, T R; Sundmacher, K

    2011-01-01

    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 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.

  18. 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.

  19. Optogenetic control of human neurons in organotypic brain cultures

    DEFF Research Database (Denmark)

    Andersson, My; Avaliani, Natalia; Svensson, Andreas

    2016-01-01

    Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof......-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies....

  20. Rapid metabolism of exogenous angiotensin II by catecholaminergic neuronal cells in culture media.

    Science.gov (United States)

    Basu, Urmi; Seravalli, Javier; Madayiputhiya, Nandakumar; Adamec, Jiri; Case, Adam J; Zimmerman, Matthew C

    2015-02-01

    Angiotensin II (AngII) acts on central neurons to increase neuronal firing and induce sympathoexcitation, which contribute to the pathogenesis of cardiovascular diseases including hypertension and heart failure. Numerous studies have examined the precise AngII-induced intraneuronal signaling mechanism in an attempt to identify new therapeutic targets for these diseases. Considering the technical challenges in studying specific intraneuronal signaling pathways in vivo, especially in the cardiovascular control brain regions, most studies have relied on neuronal cell culture models. However, there are numerous limitations in using cell culture models to study AngII intraneuronal signaling, including the lack of evidence indicating the stability of AngII in culture media. Herein, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Using liquid chromatography-tandem mass spectrometry, we measured levels of AngII and its metabolites, Ang III, Ang IV, and Ang-1-7, in neuronal cell culture media after administration of exogenous AngII (100 nmol/L) to a neuronal cell culture model (CATH.a neurons). AngII levels rapidly declined in the media, returning to near baseline levels within 3 h of administration. Additionally, levels of Ang III and Ang-1-7 acutely increased, while levels of Ang IV remained unchanged. Replenishing the media with exogenous AngII every 3 h for 24 h resulted in a consistent and significant increase in AngII levels for the duration of the treatment period. These data indicate that AngII is rapidly metabolized in neuronal cell culture media, and replenishing the media at least every 3 h is needed to sustain chronically elevated levels. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  1. Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons.

    Science.gov (United States)

    Halim, Nader D; Mcfate, Thomas; Mohyeldin, Ahmed; Okagaki, Peter; Korotchkina, Lioubov G; Patel, Mulchand S; Jeoung, Nam Ho; Harris, Robert A; Schell, Michael J; Verma, Ajay

    2010-08-01

    Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed. (c) 2010 Wiley-Liss, Inc.

  2. 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.

  3. Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

    Science.gov (United States)

    Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

    2015-01-01

    Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

  4. Primary Neuron/Astrocyte Co-Culture on Polyelectrolyte Multilayer Films: A Template for Studying Astrocyte-Mediated Oxidative Stress in Neurons**

    OpenAIRE

    Kidambi, Srivatsan; Lee, Ilsoon; Chan, Christina

    2008-01-01

    We engineered patterned co-cultures of primary neurons and astrocytes on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the oxidative stress mediated by astrocytes on neuronal cells. A number of studies have explored engineering co-culture of neurons and astrocytes predominantly using cell lines rather than primary cells owing to the difficulties involved in attaching primary cells onto synthetic surfaces. To our knowledge this is the first demons...

  5. PYRETHROID MODULATION OF SPONTANEOUS NEURONAL EXCITABILITY AND NEUROTRANSMISSION IN HIPPOCAMPAL NEURONS IN CULTURE

    Science.gov (United States)

    Pyrethroid insecticides have potent actions on voltage-gated sodium channels, inhibiting inactivation and increasing channel open times. These are thought to underlie, at least in part, the clinical symptoms of pyrethroid intoxication. However, disruption of neuronal activity at ...

  6. Additivity of Pyrethroid Actions on Sodium Influx in Cortical Neurons in Cerebrocortical Neurons in Primary Culture

    Science.gov (United States)

    BACKGROUND: Pyrethroid insecticides bind to voltage-gated sodium channels and modify their gating kinetics, thereby disrupting neuronal function. Although previous work has tested the additivity of pyrethroids in vivo, this has not been assessed directly at the primary molecular ...

  7. Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons

    DEFF Research Database (Denmark)

    Jensen, Pia; Ducray, A D; Widmer, H R

    2015-01-01

    shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10days......, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells....... to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which...

  8. 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. PMID:23983586

  9. 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.

  10. Resveratrol Produces Neurotrophic Effects on Cultured Dopaminergic Neurons through Prompting Astroglial BDNF and GDNF Release

    Directory of Open Access Journals (Sweden)

    Feng Zhang

    2012-01-01

    Full Text Available Increasing evidence indicated astroglia-derived neurotrophic factors generation might hold a promising therapy for Parkinson’s disease (PD. Resveratrol, naturally present in red wine and grapes with potential benefit for health, is well known to possess a number of pharmacological activities. Besides the antineuroinflammatory properties, we hypothesized the neuroprotective potency of resveratrol is partially due to its additional neurotrophic effects. Here, primary rat midbrain neuron-glia cultures were applied to investigate the neurotrophic effects mediated by resveratrol on dopamine (DA neurons and further explore the role of neurotrophic factors in its actions. Results showed resveratrol produced neurotrophic effects on cultured DA neurons. Additionally, astroglia-derived neurotrophic factors release was responsible for resveratrol-mediated neurotrophic properties as evidenced by the following observations: (1 resveratrol failed to exert neurotrophic effects on DA neurons in the cultures without astroglia; (2 the astroglia-conditioned medium prepared from astroglia-enriched cultures treated with resveratrol produced neurotrophic effects in neuron-enriched cultures; (3 resveratrol increased neurotrophic factors release in the concentration- and time-dependent manners; (4 resveratrol-mediated neurotrophic effects were suppressed by blocking the action of the neurotrophic factors. Together, resveratrol could produce neurotrophic effects on DA neurons through prompting neurotrophic factors release, and these effects might open new alternative avenues for neurotrophic factor-based therapy targeting PD.

  11. Palmitoylethanolamide Blunts Amyloid-β42-Induced Astrocyte Activation and Improves Neuronal Survival in Primary Mouse Cortical Astrocyte-Neuron Co-Cultures.

    Science.gov (United States)

    Beggiato, Sarah; Borelli, Andrea Celeste; Ferraro, Luca; Tanganelli, Sergio; Antonelli, Tiziana; Tomasini, Maria Cristina

    2018-01-01

    Based on the pivotal role of astrocytes in brain homeostasis and the strong metabolic cooperation existing between neurons and astrocytes, it has been suggested that astrocytic dysfunctions might cause and/or contribute to neuroinflammation and neurodegenerative processes. Therapeutic approaches aimed at both neuroprotection and neuroinflammation reduction may prove particularly effective in slowing the progression of these diseases. The endogenous lipid mediator palmitoylethanolamide (PEA) displayed neuroprotective and anti(neuro)inflammatory properties, and demonstrated interesting potential as a novel treatment for Alzheimer's disease. We firstly evaluated whether astrocytes could participate in regulating the Aβ42-induced neuronal damage, by using primary mouse astrocytes cell cultures and mixed astrocytes-neurons cultures. Furthermore, the possible protective effects of PEA against Aβ42-induced neuronal toxicity have also been investigated by evaluating neuronal viability, apoptosis, and morphometric parameters. The presence of astrocytes pre-exposed to Aβ42 (0.5μM; 24 h) induced a reduction of neuronal viability in primary mouse astrocytes-neurons co-cultures. Furthermore, under these experimental conditions, an increase in the number of neuronal apoptotic nuclei and a decrease in the number of MAP-2 positive neurons were observed. Finally, astrocytic Aβ42 pre-exposure induced an increase in the number of neurite aggregations/100μm as compared to control (i.e., untreated) astrocytes-neurons co-cultures. These effects were not observed in neurons cultured in the presence of astrocytes pre-exposed to PEA (0.1μM), applied 1 h before and maintained during Aβ42 treatment. Astrocytes contribute to Aβ42-induced neurotoxicity and PEA, by blunting Aβ42-induced astrocyte activation, improved neuronal survival in mouse astrocyte-neuron co-cultures.

  12. Establishment of mouse neuron and microglial cell co-cultured models and its action mechanism.

    Science.gov (United States)

    Zhang, Bo; Yang, Yunfeng; Tang, Jun; Tao, Yihao; Jiang, Bing; Chen, Zhi; Feng, Hua; Yang, Liming; Zhu, Gang

    2017-06-27

    The objective of this study is to establish a co-culture model of mouse neurons and microglial cells, and to analyze the mechanism of action of oxygen glucose deprivation (OGD) and transient oxygen glucose deprivation (tOGD) preconditioning cell models. Mouse primary neurons and BV2 microglial cells were successfully cultured, and the OGD and tOGD models were also established. In the co-culture of mouse primary neurons and microglial cells, the cell number of tOGD mouse neurons and microglial cells was larger than the OGD cell number, observed by a microscope. CCK-8 assay result showed that at 1h after treatment, the OD value in the control group is lower compared to all the other three groups (P control group compared to other three groups (P neurons cells were cultured. In the meantime mouse BV2 microglia cells were cultured. Two types of cells were co-cultured, and OGD and tOGD cell models were established. There were four groups in the experiment: control group (OGD), treatment group (tOGD+OGD), placebo group (tOGD+OGD+saline) and minocycline intervention group (tOGD+OGD+minocycline). CCK-8 kit was used to detect cell viability and flow cytometry was used to detect apoptosis. In this study, mouse primary neurons and microglial cells were co-cultured. The OGD and tOGD models were established successfully. tOGD was able to effectively protect neurons and microglial cells from damage, and inhibit the apoptosis caused by oxygen glucose deprivation.

  13. Differential radiosensitivity of mouse embryonic neurons and glia in cell culture

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1977-01-01

    The responses of neurons and glial cells to ultraviolet and γ-radiation were studied in cell cultures of embryonic mouse brains. A decrease in the ratio of glia to neurons occurred after both forms of irradiation. [ 3 H]thymidine labelling followed by autoradiography revealed that all glia were capable of replication, whereas 70 percent of neurons were non-replicating under the conditions of the study. Ultraviolet radiation caused a decrease in the proportion of replicating neurons but did not affect the proportion of replicating glia, whereas γ-radiation caused a decrease in DNA replication in both cell types. Levels of ultraviolet radiation-induced unscheduled DNA synthesis were lower in neurons than in glia. It is concluded that sensitivity to both ionizing and ultraviolet radiation of neurons and glial cells in embryonic brain cultures is determined primarily by the capacity for and state of DNA replication. Neurons which have already reached the stage of terminal differentiation are more resistant than replicating neurons of glial cells

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

    DEFF Research Database (Denmark)

    Suñol, C; Babot, Z; Cristòfol, R

    2010-01-01

    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......3 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......M 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...

  15. 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.

  16. Comparative Analysis of Human and Rodent Brain Primary Neuronal Culture Spontaneous Activity Using Micro-Electrode Array Technology.

    Science.gov (United States)

    Napoli, Alessandro; Obeid, Iyad

    2016-03-01

    Electrical activity in embryonic brain tissue has typically been studied using Micro Electrode Array (MEA) technology to make dozens of simultaneous recordings from dissociated neuronal cultures, brain stem cell progenitors, or brain slices from fetal rodents. Although these rodent neuronal primary culture electrical properties are mostly investigated, it has not been yet established to what extent the electrical characteristics of rodent brain neuronal cultures can be generalized to those of humans. A direct comparison of spontaneous spiking activity between rodent and human primary neurons grown under the same in vitro conditions using MEA technology has never been carried out before and will be described in the present study. Human and rodent dissociated fetal brain neuronal cultures were established in-vitro by culturing on a glass grid of 60 planar microelectrodes neurons under identical conditions. Three different cultures of human neurons were produced from tissue sourced from a single aborted fetus (at 16-18 gestational weeks) and these were compared with seven different cultures of embryonic rat neurons (at 18 gestational days) originally isolated from a single rat. The results show that the human and rodent cultures behaved significantly differently. Whereas the rodent cultures demonstrated robust spontaneous activation and network activity after only 10 days, the human cultures required nearly 40 days to achieve a substantially weaker level of electrical function. These results suggest that rat neuron preparations may yield inferences that do not necessarily transfer to humans. © 2015 Wiley Periodicals, Inc.

  17. Hypothalamic pathogenesis of type 2 diabetes.

    Science.gov (United States)

    Koshiyama, Hiroyuki; Hamamoto, Yoshiyuki; Honjo, Sachiko; Wada, Yoshiharu; Lkeda, Hiroki

    2006-01-01

    There have recently been increasing experimental and clinical evidences suggesting that hypothalamic dysregulation may be one of the underlying mechanisms of abnormal glucose metabolism. First, increased hypothalamic-pituitary-adrenal axis activity induced by uncontrollable excess stress may cause diabetes mellitus as well as dyslipidemia, visceral obesity, and osteoporosis with some resemblance to Cushing's disease. Second, several molecules are known to be expressed both in pancreas and hypothalamus; adenosine triphosphate-sensitive potassium channels, malonyl-CoA, glucokinase, and AMP-activated protein kinase. Those molecules appear to form an integrated hypothalamic system, which may sense hypothalamic fuel status, especially glucose level, and inhibit action of insulin on hepatic gluconeogenesis, thereby forming a brain-liver circuit. Third, hypothalamic resistance to insulin as an adiposity signal may be involved in pathogenesis of peripheral insulin resistance. The results with mice with a neuron-specific disruption of the insulin receptor gene or those lacking insulin receptor substrate 2 in hypothalamus supported this possibility. Finally, it has very recently been suggested that dysregulation of clock genes in hypothalamus may cause abnormal glucose metabolism. Taken together, it is plausible that some hypothalamic abnormality may underlie at least some portion of type 2 diabetes or insulin resistance in humans, and this viewpoint of hypothalamic pathogenesis of type 2 diabetes may lead to the development of new drugs for type 2 diabetes.

  18. ELECTROPHYSIOLOGICAL CHARACTERIZATION OF DOPAMINERGIC AND NONDOPAMINERGIC NEURONS IN ORGANOTYPIC SLICE CULTURES OF THE RAT VENTRAL MESENCEPHALON

    DEFF Research Database (Denmark)

    STEENSEN, BH; NEDERGAARD, S; OSTERGAARD, K

    1995-01-01

    -old organotypic slice cultures of the ventral mesencephalon prepared from newborn rats. Dopaminergic neurones were distinguished from non-dopaminergic neurones by staining with the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and briefly viewing the preparation with short exposures to ultraviolet...... 81 M Omega), were silent or fired spontaneously at a low frequency (0-9 Hz), and no spontaneous GABA(A)-ergic inhibitory postsynaptic potentials or inward rectification were present. In contrast, non-dopaminergic neurones had fast action potentials (0.6-3.2 ms), low input resistance (mean 32 M Omega...

  19. Effects of Electrical Stimulation in Sympathetic Neuron-Cardiomyocyte Co-cultures

    Science.gov (United States)

    Takeuchi, Akimasa; Tani, Hiromasa; Mori, Masahide; Moriguchi, Hiroyuki; Kotani, Kiyoshi; Lee, Jong-Kook; Noshiro, Makoto; Jimbo, Yasuhiko

    The sympathetic nervous system is one of the principal sources for regulating cardiovascular functions. Little is known, however, about the network-level interactions between sympathetic neurons and cardiomyocytes. In this study, a semi-separated co-culture system of superior cervical ganglion (SCG) neurons and ventricular myocytes (VMs) was developed by using a polydimethylsyloxane (PDMS) chamber placed on a microelectrode-array (MEA) substrate. Neurites of SCG neurons passed through a conduit of the chamber and reached VMs. Evoked activities of SCG neurons were observed from several electrodes immediately after applying constant-voltage stimulation (1 V, 1 ms, biphasic square pulses) to SCG neurons by using 32 electrodes. Furthermore, this stimulation was applied to SCG neurons at the frequency of 1, 5 and 10 Hz. After applying these three kinds of stimulations, mean minute contraction rate of VMs increased with an increase in the frequency of stimulation. These results suggest that changes in contraction rate of VMs after applying electrical stimulations to SCG neurons depend on frequencies of these stimulations and that the heart-regulating mechanisms as well as that in the body were formed in this co-culture system.

  20. Primary cell culture of LHRH neurones from embryonic olfactory placode in the sheep (Ovis aries).

    Science.gov (United States)

    Duittoz, A H; Batailler, M; Caldani, M

    1997-09-01

    The aim of this study was to establish an in vitro model of ovine luteinizing hormone-releasing hormone (LHRH) neurones. Olfactory placodes from 26 day-old sheep embryos (E26) were used for explant culture. Cultures were maintained successfully up to 35 days, but were usually used at 17 days for immunocytochemistry. LHRH and neuronal markers such as neurofilament (NF) were detected by immunocytochemistry within and/or outside the explant. Three main types of LHRH positive cells are described: (1) neuroblastic LHRH and NF immunoreactive cells with round cell body and very short neurites found mainly within the explant, (2) migrating LHRH bipolar neurones with an fusiform cell body, found outside the explant, (3) network LHRH neuron, bipolar or multipolar with long neurites connecting other LHRH neurons. Cell morphology was very similar to that which has been described in the adult sheep brain. These results strongly suggest that LHRH neurones in the sheep originate from the olfactory placode. This mode may represent a useful tool to study LHRH neurones directly in the sheep.

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

    Directory of Open Access Journals (Sweden)

    Daniel Hernandez-Baltazar

    Full Text Available 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

  2. Different Influences of Lipofection and Electrotransfection on In Vitro Gene Delivery to Primary Cultured Cortex Neurons.

    Science.gov (United States)

    Zhang, Xui-Si; Huang, Jing; Zhan, Cong-Qing; Chen, Jing; Li, Tao; Kaye, Alan D; Wu, Sheng-Xi; Xiao, Lan

    2016-03-01

    Many pain states are linked to central nervous system (CNS) diseases involving the dysfunction of dendritic arborization, making restoration a promising therapeutic strategy. Transfection of primary cortex neurons offers the possibility to study mechanisms which are important for the restoration of proper arborization. Its progress is, however, limited at present due to the lack of suitable gene transfer techniques. To obtain better insight into the transfection potential of currently used techniques, 2 non-viral transfection methods, lipofection and gene electrotransfer (GET), were compared. This is a comparison study performed on cultured cells. The transfection efficiency and neuronal viability, as well as the neuronal dendritic arborization after lipofection or GET, were compared. Primary cultured cortex neurons were transfected with the pEGFP-N1 plasmid, either using Lipofectamine 2000 (2, 3, or 4µL) or with electroporation, with our previously optimized protocol (200V/25 ms). Transfection efficiency and cell viability were inversely proportional for lipofection. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection. Although GET offered higher transfection efficiency, it could not induce complex dendritic arborization, which made it unsuitable for in vitro gene transfer into cortex neurons. Limitations include species variability and translational applicability for CNS diseases and pain states related to potential toxicity. Based on these findings, lipofection might be advantageous for in vitro application to primary cultured cortex neurons. Pain states, stress mediated pathogenesis, and certain CNS diseases might potentially utilize this important technique in the future as a therapeutic modality.

  3. Glia co-culture with neurons in microfluidic platforms promotes the formation and stabilization of synaptic contacts.

    Science.gov (United States)

    Shi, Mingjian; Majumdar, Devi; Gao, Yandong; Brewer, Bryson M; Goodwin, Cody R; McLean, John A; Li, Deyu; Webb, Donna J

    2013-08-07

    Two novel microfluidic cell culture schemes, a vertically-layered set-up and a four chamber set-up, were developed for co-culturing central nervous system (CNS) neurons and glia. The cell chambers in these devices were separated by pressure-enabled valve barriers, which permitted us to control communication between the two cell types. The unique design of these devices facilitated the co-culture of glia with neurons in close proximity (∼50-100 μm), differential transfection of neuronal populations, and dynamic visualization of neuronal interactions, such as the development of synapses. With these co-culture devices, initial synaptic contact between neurons transfected with different fluorescent markers, such as green fluorescent protein (GFP) and mCherry-synaptophysin, was imaged using high-resolution fluorescence microscopy. The presence of glial cells had a profound influence on synapses by increasing the number and stability of synaptic contacts. Interestingly, as determined by liquid chromatography-ion mobility-mass spectrometry, neuron-glia co-cultures produced elevated levels of soluble factors compared to that secreted by individual neuron or glia cultures, suggesting a potential mechanism by which neuron-glia interactions could modulate synaptic function. Collectively, these results show that communication between neurons and glia is critical for the formation and stability of synapses and point to the importance of developing neuron-glia co-culture systems such as the microfluidic platforms described in this study.

  4. Hypothalamic demyelination causing panhypopituitarism.

    Science.gov (United States)

    Dixon-Douglas, Julia; Burgess, John; Dreyer, Michael

    2018-05-01

    Hypothalamic involvement in multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) is rare and endocrinopathies involving the hypothalamic-pituitary axis in patients with demyelinating conditions have rarely been reported. We present two cases of MS/NMOSD with associated hypothalamic-pituitary involvement and subsequent hypopituitarism, including the first report of a patient with hypothalamic demyelination causing panhypopituitarism. Differential diagnoses, including alemtuzumab-related and primary pituitary pathology are discussed. © 2018 Royal Australasian College of Physicians.

  5. Voltage-sensitive dye recording from networks of cultured neurons

    Science.gov (United States)

    Chien, Chi-Bin

    This thesis describes the development and testing of a sensitive apparatus for recording electrical activity from microcultures of rat superior cervical ganglion (SCG) neurons by using voltage-sensitive fluorescent dyes.The apparatus comprises a feedback-regulated mercury arc light source, an inverted epifluorescence microscope, a novel fiber-optic camera with discrete photodiode detectors, and low-noise preamplifiers. Using an NA 0.75 objective and illuminating at 10 W/cm2 with the 546 nm mercury line, a typical SCG neuron stained with the styryl dye RH423 gives a detected photocurrent of 1 nA; the light source and optical detectors are quiet enough that the shot noise in this photocurrent--about.03% rms--dominates. The design, theory, and performance of this dye-recording apparatus are discussed in detail.Styryl dyes such as RH423 typically give signals of 1%/100 mV on these cells; the signals are linear in membrane potential, but do not appear to arise from a purely electrochromic mechanism. Given this voltage sensitivity and the noise level of the apparatus, it should be possible to detect both action potentials and subthreshold synaptic potentials from SCG cell bodies. In practice, dye recording can easily detect action potentials from every neuron in an SCG microculture, but small synaptic potentials are obscured by dye signals from the dense network of axons.In another microculture system that does not have such long and complex axons, this dye-recording apparatus should be able to detect synaptic potentials, making it possible to noninvasively map the synaptic connections in a microculture, and thus to study long-term synaptic plasticity.

  6. Neuroprotective effects of phytochemicals on dopaminergic neuron cultures.

    Science.gov (United States)

    Sandoval-Avila, S; Diaz, N F; Gómez-Pinedo, U; Canales-Aguirre, A A; Gutiérrez-Mercado, Y K; Padilla-Camberos, E; Marquez-Aguirre, A L; Díaz-Martínez, N E

    2016-06-21

    Parkinson's disease is a progressive neurodegenerative disorder characterised by a loss of dopaminergic neurons in the substantia nigra pars compacta, which results in a significant decrease in dopamine levels and consequent functional motor impairment. Although its aetiology is not fully understood, several pathogenic mechanisms, including oxidative stress, have been proposed. Current therapeutic approaches are based on dopamine replacement drugs; these agents, however, are not able to stop or even slow disease progression. Novel therapeutic approaches aimed at acting on the pathways leading to neuronal dysfunction and death are under investigation. In recent years, such natural molecules as polyphenols, alkaloids, and saponins have been shown to have a neuroprotective effect due to their antioxidant and anti-inflammatory properties. The aim of our review is to analyse the most relevant studies worldwide addressing the benefits of some phytochemicals used in in vitro models of Parkinson's disease. Copyright © 2016 Sociedad Española de Neurología. Published by Elsevier España, S.L.U. All rights reserved.

  7. Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium.

    Science.gov (United States)

    O'Mullane, Lauren M; Keast, Janet R; Osborne, Peregrine B

    2013-08-01

    Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells. Sensory neurons and urothelial cells obtained from dorsal root ganglia and bladders dissected from adult female Sprague-Dawley® rats were isolated by enzyme treatment and mechanical dissociation. They were plated together or separately on collagen coated substrate and cultured in keratinocyte medium for 48 to 72 hours. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to that in neuronal monocultures. The growth promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor K252a but upstream inhibition of nerve growth factor signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to adenosine triphosphate (100 μM) and activation of TRPV4 (4α-PDD, 10 μM) but not TRPV1 (capsaicin, 1 μM), TRPV3 (farnesyl pyrophosphate, 1 μM) or TRPA1 (mustard oil, 100 μM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Results suggest that neuronal properties are maintained in the presence of urothelium and neurite growth is potentiated by a nerve growth factor independent mechanism. Copyright © 2013 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  8. Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

    Science.gov (United States)

    Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

    2018-01-01

    It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

  9. Sexual behavior reduces hypothalamic androgen receptor immunoreactivity

    NARCIS (Netherlands)

    Fernandez-Guasti, Alonso; Swaab, Dick; Rodríguez-Manzo, Gabriela

    2003-01-01

    Male sexual behavior is regulated by limbic areas like the medial preoptic nucleus (MPN), the bed nucleus of the stria terminalis (BST), the nucleus accumbens (nAcc) and the ventromedial hypothalamic nucleus (VMN). Neurons in these brain areas are rich in androgen receptors (AR) and express

  10. Graph-based unsupervised segmentation algorithm for cultured neuronal networks' structure characterization and modeling.

    Science.gov (United States)

    de Santos-Sierra, Daniel; Sendiña-Nadal, Irene; Leyva, Inmaculada; Almendral, Juan A; Ayali, Amir; Anava, Sarit; Sánchez-Ávila, Carmen; Boccaletti, Stefano

    2015-06-01

    Large scale phase-contrast images taken at high resolution through the life of a cultured neuronal network are analyzed by a graph-based unsupervised segmentation algorithm with a very low computational cost, scaling linearly with the image size. The processing automatically retrieves the whole network structure, an object whose mathematical representation is a matrix in which nodes are identified neurons or neurons' clusters, and links are the reconstructed connections between them. The algorithm is also able to extract any other relevant morphological information characterizing neurons and neurites. More importantly, and at variance with other segmentation methods that require fluorescence imaging from immunocytochemistry techniques, our non invasive measures entitle us to perform a longitudinal analysis during the maturation of a single culture. Such an analysis furnishes the way of individuating the main physical processes underlying the self-organization of the neurons' ensemble into a complex network, and drives the formulation of a phenomenological model yet able to describe qualitatively the overall scenario observed during the culture growth. © 2014 International Society for Advancement of Cytometry.

  11. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. The BDNF val-66-met Polymorphism Affects Neuronal Morphology and Synaptic Transmission in Cultured Hippocampal Neurons from Rett Syndrome Mice

    Directory of Open Access Journals (Sweden)

    Xin Xu

    2017-07-01

    Full Text Available Brain-derived neurotrophic factor (Bdnf has been implicated in several neurological disorders including Rett syndrome (RTT, an X-linked neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional modulator methyl-CpG-binding protein 2 (MECP2. The human BDNF gene has a single nucleotide polymorphism (SNP—a methionine (met substitution for valine (val at codon 66—that affects BDNF’s trafficking and activity-dependent release and results in cognitive dysfunction. Humans that are carriers of the met-BDNF allele have subclinical memory deficits and reduced hippocampal volume and activation. It is still unclear whether this BDNF SNP affects the clinical outcome of RTT individuals. To evaluate whether this BDNF SNP contributes to RTT pathophysiology, we examined the consequences of expression of either val-BDNF or met-BDNF on dendrite and dendritic spine morphology, and synaptic function in cultured hippocampal neurons from wildtype (WT and Mecp2 knockout (KO mice. Our findings revealed that met-BDNF does not increase dendritic growth and branching, dendritic spine density and individual spine volume, and the number of excitatory synapses in WT neurons, as val-BDNF does. Furthermore, met-BDNF reduces dendritic complexity, dendritic spine volume and quantal excitatory synaptic transmission in Mecp2 KO neurons. These results suggest that the val-BDNF variant contributes to RTT pathophysiology, and that BDNF-based therapies should take into consideration the BDNF genotype of the RTT individuals.

  14. Metabolic Benefit of Chronic Caloric Restriction and Activation of Hypothalamic AGRP/NPY Neurons in Male Mice Is Independent of Ghrelin

    Science.gov (United States)

    Rogers, Nicole H.; Walsh, Heidi; Alvarez-Garcia, Oscar; Park, Seongjoon; Gaylinn, Bruce; Thorner, Michael O.

    2016-01-01

    Aging is associated with attenuated ghrelin signaling. During aging, chronic caloric restriction (CR) produces health benefits accompanied by enhanced ghrelin production. Ghrelin receptor (GH secretagogue receptor 1a) agonists administered to aging rodents and humans restore the young adult phenotype; therefore, we tested the hypothesis that the metabolic benefits of CR are mediated by endogenous ghrelin. Three month-old male mice lacking ghrelin (Ghrelin−/−) or ghrelin receptor (Ghsr−/−), and their wild-type (WT) littermates were randomly assigned to 2 groups: ad libitum (AL) fed and CR, where 40% food restriction was introduced gradually to allow Ghrelin−/− and Ghsr−/− mice to metabolically adapt and avoid severe hypoglycemia. Twelve months later, plasma ghrelin, metabolic parameters, ambulatory activity, hypothalamic and liver gene expression, as well as body composition were measured. CR increased plasma ghrelin and des-acyl ghrelin concentrations in WT and Ghsr−/− mice. CR of WT, Ghsr−/−, and Ghrelin−/− mice markedly improved metabolic flexibility, enhanced ambulatory activity, and reduced adiposity. Inactivation of Ghrelin or Ghsr had no effect on AL food intake or food anticipatory behavior. In contrast to the widely held belief that endogenous ghrelin regulates food intake, CR increased expression of hypothalamic Agrp and Npy, with reduced expression of Pomc across genotypes. In the AL context, ablation of ghrelin signaling markedly inhibited liver steatosis, which correlated with reduced Pparγ expression and enhanced Irs2 expression. Although CR and administration of GH secretagogue receptor 1a agonists both benefit the aging phenotype, we conclude the benefits of chronic CR are a consequence of enhanced metabolic flexibility independent of endogenous ghrelin or des-acyl ghrelin signaling. PMID:26812158

  15. Ketamine-induced apoptosis in cultured rat cortical neurons

    International Nuclear Information System (INIS)

    Takadera, Tsuneo; Ishida, Akira; Ohyashiki, Takao

    2006-01-01

    Recent data suggest that anesthetic drugs cause neurodegeneration during development. Ketamine is frequently used in infants and toddlers for elective surgeries. The purpose of this study is to determine whether glycogen synthase kinase-3 (GSK-3) is involved in ketamine-induced apoptosis. Ketamine increased apoptotic cell death with morphological changes which were characterized by cell shrinkage, nuclear condensation or fragmentation. In addition, insulin growth factor-1 completely blocked the ketamine-induced apoptotic cell death. Ketamine decreased Akt phosphorylation. GSK-3 is known as a downstream target of Akt. The selective inhibitors of GSK-3 prevented the ketamine-induced apoptosis. Moreover, caspase-3 activation was accompanied by the ketamine-induced cell death and inhibited by the GSK-3 inhibitors. These results suggest that activation of GSK-3 is involved in ketamine-induced apoptosis in rat cortical neurons

  16. Noise focusing and the emergence of coherent activity in neuronal cultures

    Science.gov (United States)

    Orlandi, Javier G.; Soriano, Jordi; Alvarez-Lacalle, Enrique; Teller, Sara; Casademunt, Jaume

    2013-09-01

    At early stages of development, neuronal cultures in vitro spontaneously reach a coherent state of collective firing in a pattern of nearly periodic global bursts. Although understanding the spontaneous activity of neuronal networks is of chief importance in neuroscience, the origin and nature of that pulsation has remained elusive. By combining high-resolution calcium imaging with modelling in silico, we show that this behaviour is controlled by the propagation of waves that nucleate randomly in a set of points that is specific to each culture and is selected by a non-trivial interplay between dynamics and topology. The phenomenon is explained by the noise focusing effect--a strong spatio-temporal localization of the noise dynamics that originates in the complex structure of avalanches of spontaneous activity. Results are relevant to neuronal tissues and to complex networks with integrate-and-fire dynamics and metric correlations, for instance, in rumour spreading on social networks.

  17. 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...

  18. Network bursts in cortical neuronal cultures: 'noise - versus pacemaker'- driven neural network simulations

    NARCIS (Netherlands)

    Gritsun, T.; Stegenga, J.; le Feber, Jakob; Rutten, Wim

    2009-01-01

    In this paper we address the issue of spontaneous bursting activity in cortical neuronal cultures and explain what might cause this collective behavior using computer simulations of two different neural network models. While the common approach to acivate a passive network is done by introducing

  19. 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

  20. Neuroprotective effects of ginsenoside Rg1 against oxygen–glucose deprivation in cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Qing He

    2014-03-01

    Conclusion: Ginsenoside Rg1 has neuroprotective effect on ischemia–reperfusion injury in cultured hippocampal cells mediated by blocking calcium over-influx into neuronal cells and decreasing the nNOS activity after OGD exposure. We infer that ginsenoside Rg1 may serve as a potential therapeutic agent for cerebral ischemia injury.

  1. Altered Loyalties of Neuronal Markers in Cultured Slices of Resected Human Brain Tissue

    NARCIS (Netherlands)

    Verwer, Ronald W. H.; Sluiter, Arja A.; Balesar, Rawien A.; Baayen, Johannes C.; Speijer, Dave; Idema, Sander; Swaab, Dick F.

    2016-01-01

    Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic

  2. Repeated Stimulation of Cultured Networks of Rat Cortical Neurons Induces Parallel Memory Traces

    Science.gov (United States)

    le Feber, Joost; Witteveen, Tim; van Veenendaal, Tamar M.; Dijkstra, Jelle

    2015-01-01

    During systems consolidation, memories are spontaneously replayed favoring information transfer from hippocampus to neocortex. However, at present no empirically supported mechanism to accomplish a transfer of memory from hippocampal to extra-hippocampal sites has been offered. We used cultured neuronal networks on multielectrode arrays and…

  3. Agmatine protects against cell damage induced by NMDA and glutamate in cultured hippocampal neurons

    Science.gov (United States)

    Wang, Wei-Ping; Iyo, Abiye H.; Miguel-Hidalgo, Javier; Regunathan, Soundar; Zhu, Meng-Yang

    2010-01-01

    Agmatine is a polyamine and has been considered as a novel neurotransmitter or neuromodulator in the central nervous system. In the present study, the neuroprotective effect of agmatine against cell damage caused by N-methyl-d-aspartate (NMDA) and glutamate was investigated in cultured rat hippocampal neurons. Lactate dehydrogenase (LDH) activity assay, β-tubulin III immunocytochemical staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) assay were conducted to detect cell damage. Exposure of 12-day neuronal cultures of rat hippocampus to NMDA or glutamate for 1 h caused a concentration-dependent neurotoxicity, as indicated by the significant increase in released LDH activities. Addition of 100 µM agmatine into media ablated the neurotoxicity induced by NMDA or glutamate, an effect also produced by the specific NMDA receptor antagonist dizocilpine hydrogen maleate (MK801). Arcaine, an analog of agmatine with similar structure as agmatine, fully prevented the NMDA- or glutamate-induced neuronal damage. Spermine and putrescine, the endogenous polyamine and metabolic products of agmatine without the guanidine moiety of agmatine, failed to show this effect, indicating a structural relevance for this neuroprotection. Immunocytochemical staining and TUNEL assay confirmed the findings in the LDH measurement. That is, agmatine and MK801 markedly attenuated NMDA-induced neuronal death and significantly reduced TUNEL-positive cell numbers induced by exposure of cultured hippocampal neurons to NMDA. Taken together, these results demonstrate that agmatine can protect cultured hippocampal neurons from NMDA- or glutamate-induced excitotoxicity, through a possible blockade of the NMDA receptor channels or a potential anti-apoptotic property. PMID:16546145

  4. 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-derived neuro......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......-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), or a combination of both. Dopamine content of the culture medium, the number of tyrosine hydroxylase-immunoreactive neurons, and culture volumes were moderately increased in the BDNF- and GDNF-treated cultures but significantly...... increased by 6.8-, 3.2- and 2.4-fold, respectively after treatment with the combination of both factors. We conclude that pretreatment of dopaminergic tissue in culture with a combination of BDNF and GDNF may be an effective means to improve the quality of tissue prior to grafting....

  5. Purinergic modulation of adult guinea pig cardiomyocytes in long term cultures and co-cultures with extracardiac or intrinsic cardiac neurones.

    Science.gov (United States)

    Horackova, M; Huang, M H; Armour, J A

    1994-05-01

    To determine the capacity of ATP to modify cardiomyocytes directly or indirectly via peripheral autonomic neurones, the effects of various purinergic agents were studied on long term cultures of adult guinea pig ventricular myocytes and their co-cultures with extracardiac (stellate ganglion) or intrinsic cardiac neurones. Ventricular myocytes and cardiac neurones were enzymatically dissociated and plated together or alone (myocytes only). Myocyte cultures were used for experiments after three to six weeks. The electrical and contractile properties of cultured myocytes and myocyte-neuronal networks were investigated. The spontaneous beating frequency of ventricular myocytes co-cultured with stellate ganglion neurones increased by approximately 140% (p under control conditions, but when beta adrenergic receptors of tetrodotoxin sensitive neural responses were blocked, ATP induced greater augmentation (> 100%). In contrast, ATP induced much smaller effects in non-innervated myocyte cultures (approximately 26%, p UTP > MSATP > beta gamma ATP > alpha beta ATP. Adenosine (10(-4) M) attenuated the beating frequency of myocytes in both types of co-culture, while not significantly affecting non-innervated myocyte cultures. The experimental model used in this study showed that extrinsic and intrinsic cardiac neurones which possess P2 receptors can greatly enhance cardiac myocyte contractile rate when activated by ATP. Since adenosine reduced contractile rate in both types of co-cultures while not affecting non-innervated myocytes, it is concluded that some of these neurones possess P1 receptors.

  6. 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.

  7. Direct Conversion of Equine Adipose-Derived Stem Cells into Induced Neuronal Cells Is Enhanced in Three-Dimensional Culture.

    Science.gov (United States)

    Petersen, Gayle F; Hilbert, Bryan J; Trope, Gareth D; Kalle, Wouter H J; Strappe, Padraig M

    2015-12-01

    The ability to culture neurons from horses may allow further investigation into equine neurological disorders. In this study, we demonstrate the generation of induced neuronal cells from equine adipose-derived stem cells (EADSCs) using a combination of lentiviral vector expression of the neuronal transcription factors Brn2, Ascl1, Myt1l (BAM) and NeuroD1 and a defined chemical induction medium, with βIII-tubulin-positive induced neuronal cells displaying a distinct neuronal morphology of rounded and compact cell bodies, extensive neurite outgrowth, and branching of processes. Furthermore, we investigated the effects of dimensionality on neuronal transdifferentiation, comparing conventional two-dimensional (2D) monolayer culture against three-dimensional (3D) culture on a porous polystyrene scaffold. Neuronal transdifferentiation was enhanced in 3D culture, with evenly distributed cells located on the surface and throughout the scaffold. Transdifferentiation efficiency was increased in 3D culture, with an increase in mean percent conversion of more than 100% compared to 2D culture. Additionally, induced neuronal cells were shown to transit through a Nestin-positive precursor state, with MAP2 and Synapsin 2 expression significantly increased in 3D culture. These findings will help to increase our understanding of equine neuropathogenesis, with prospective roles in disease modeling, drug screening, and cellular replacement for treatment of equine neurological disorders.

  8. Oscillating from Neurosecretion to Multitasking Dopamine Neurons

    Directory of Open Access Journals (Sweden)

    David R. Grattan

    2016-04-01

    Full Text Available In this issue of Cell Reports, Stagkourakis et al. (2016 report that oscillating hypothalamic TIDA neurons, previously thought to be simple neurosecretory neurons controlling pituitary prolactin secretion, control dopamine output via autoregulatory mechanisms and thus could potentially regulate other physiologically important hypothalamic neuronal circuits.

  9. Ghrelin stimulates growth hormone release from the pituitary via hypothalamic growth hormone-releasing hormone neurons in the cichlid, Oreochromis niloticus

    Science.gov (United States)

    Ghrelin, a gastric peptide, is implicated in a multiplicity of biological functions, including energy homeostasis and reproduction. Neuronal systems that are involved in energy homeostasis as well as reproduction traverse the hypothalamus, however, the mechanism by which they control energy homeosta...

  10. Metabolic changes of cultured DRG neurons induced by adenosine using confocal microscopy imaging

    Science.gov (United States)

    Zheng, Liqin; Huang, Yimei; Chen, Jiangxu; Wang, Yuhua; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2012-12-01

    Adenosine exerts multiple effects on pain transmission in the peripheral nervous system. This study was performed to use confocal microscopy to evaluate whether adenosine could affect dorsal root ganglia (DRG) neurons in vitro and test which adenosine receptor mediates the effect of adenosine on DRG neurons. After adding adenosine with different concentration, we compared the metabolic changes by the real time imaging of calcium and mitochondria membrane potential using confocal microscopy. The results showed that the effect of 500 μM adenosine on the metabolic changes of DRG neurons was more significant than others. Furthermore, four different adenosine receptor antagonists were used to study which receptor mediated the influences of adenosine on the cultured DRG neurons. All adenosine receptor antagonists especially A1 receptor antagonist (DPCPX) had effect on the Ca2+ and mitochondria membrane potential dynamics of DRG neurons. The above studies demonstrated that the effect of adenosine which may be involved in the signal transmission on the sensory neurons was dose-dependent, and all the four adenosine receptors especially the A1R may mediate the transmission.

  11. 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.

  12. Effect of chondroitin sulfate proteoglycans on neuronal cell adhesion, spreading and neurite growth in culture

    Directory of Open Access Journals (Sweden)

    Jingyu Jin

    2018-01-01

    Full Text Available As one major component of extracellular matrix (ECM in the central nervous system, chondroitin sulfate proteoglycans (CSPGs have long been known as inhibitors enriched in the glial scar that prevent axon regeneration after injury. Although many studies have shown that CSPGs inhibited neurite outgrowth in vitro using different types of neurons, the mechanism by which CSPGs inhibit axonal growth remains poorly understood. Using cerebellar granule neuron (CGN culture, in this study, we evaluated the effects of different concentrations of both immobilized and soluble CSPGs on neuronal growth, including cell adhesion, spreading and neurite growth. Neurite length decreased while CSPGs concentration arised, meanwhile, a decrease in cell density accompanied by an increase in cell aggregates formation was observed. Soluble CSPGs also showed an inhibition on neurite outgrowth, but it required a higher concentration to induce cell aggregates formation than coated CSPGs. We also found that growth cone size was significantly reduced on CSPGs and neuronal cell spreading was restrained by CSPGs, attributing to an inhibition on lamellipodial extension. The effect of CSPGs on neuron adhesion was further evidenced by interference reflection microscopy (IRM which directly demonstrated that both CGNs and cerebral cortical neurons were more loosely adherent to a CSPG substrate. These data demonstrate that CSPGs have an effect on cell adhesion and spreading in addition to neurite outgrowth.

  13. Enhancement of synaptic transmission induced by BDNF in cultured cortical neurons

    Science.gov (United States)

    He, Jun; Gong, Hui; Zeng, Shaoqun; Li, Yanling; Luo, Qingming

    2005-03-01

    Brain-derived neurotrophic factor (BDNF), like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, BDNF has been reported to exert an acute potentiation of synaptic activity and are critically involved in long-term potentiation (LTP). We found that BDNF rapidly induced potentiation of synaptic activity and an increase in the intracellular Ca2+ concentration in cultured cortical neurons. Within minutes of BDNF application to cultured cortical neurons, spontaneous firing rate was dramatically increased as were the frequency and amplitude of excitatory spontaneous postsynaptic currents (EPSCs). Fura-2 recordings showed that BDNF acutely elicited an increase in intracellular calcium concentration ([Ca2+]c). This effect was partially dependent on [Ca2+]o; The BDNF-induced increase in [Ca2+]c can not be completely blocked by Ca2+-free solution. It was completely blocked by K252a and partially blocked by Cd2+ and TTX. The results demonstrate that BDNF can enhances synaptic transmission and that this effect is accompanied by a rise in [Ca2+]c that requires two route: the release of Ca2+ from intracellular calcium stores and influx of extracellular Ca2+ through voltage-dependent Ca2+ channels in cultured cortical neurons.

  14. Role of developmental factors in hypothalamic function

    Directory of Open Access Journals (Sweden)

    Jakob eBiran

    2015-04-01

    Full Text Available The hypothalamus is a brain region which regulates homeostasis by mediating endocrine, autonomic and behavioral functions. It is comprised of several nuclei containing distinct neuronal populations producing neuropeptides and neurotransmitters that regulate fundamental body functions including temperature and metabolic rate, thirst and hunger, sexual behavior and reproduction, circadian rhythm, and emotional responses. The identity, number and connectivity of these neuronal populations are established during the organism’s development and are of crucial importance for normal hypothalamic function. Studies have suggested that developmental abnormalities in specific hypothalamic circuits can lead to obesity, sleep disorders, anxiety, depression and autism. At the molecular level, the development of the hypothalamus is regulated by transcription factors, secreted growth factors, neuropeptides and their receptors. Recent studies in zebrafish and mouse have demonstrated that some of these molecules maintain their expression in the adult brain and subsequently play a role in the physiological functions that are regulated by hypothalamic neurons. Here, we summarize the involvement of some of the key developmental factors in hypothalamic development and function by focusing on the mouse and zebrafish genetic model organisms.

  15. 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.

  16. Establishment of a long-term spiral ganglion neuron culture with reduced glial cell number: Effects of AraC on cell composition and neurons.

    Science.gov (United States)

    Schwieger, Jana; Esser, Karl-Heinz; Lenarz, Thomas; Scheper, Verena

    2016-08-01

    Sensorineural deafness is mainly caused by damage to hair cells and degeneration of the spiral ganglion neurons (SGN). Cochlear implants can functionally replace lost hair cells and stimulate the SGN electrically. The benefit from cochlear implantation depends on the number and excitability of these neurons. To identify potential therapies for SGN protection, in vitro tests are carried out on spiral ganglion cells (SGC). A glial cell-reduced and neuron-enhanced culture of neonatal rat SGC under mitotic inhibition (cytarabine (AraC)) for up to seven days is presented. Serum containing and neurotrophin-enriched cultures with and without AraC-addition were analyzed after 4 and 7 days. The total number of cells was significantly reduced, while the proportion of neurons was greatly increased by AraC-treatment. Cell type-specific labeling demonstrated that nearly all fibroblasts and most of the glial cells were removed. Neither the neuronal survival, nor the neurite outgrowth or soma diameter were negatively affected. Additionally neurites remain partly free of surrounding non-neuronal cells. Recent culture conditions allow only for short-term cultivation of neonatal SGC and lack information on the influence of non-neuronal cells on SGN and of direct contact of neurites with test-materials. AraC-addition reduces the number of non-neuronal cells and increases the ratio of SGN in culture, without negative impact on neuronal viability. This treatment allows longer-term cultivation of SGC and provides deeper insight into SGN-glial cell interaction and the attachment of neurites on test-material surfaces. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia.

    Science.gov (United States)

    Tsunekawa, Taku; Banno, Ryoichi; Mizoguchi, Akira; Sugiyama, Mariko; Tominaga, Takashi; Onoue, Takeshi; Hagiwara, Daisuke; Ito, Yoshihiro; Iwama, Shintaro; Goto, Motomitsu; Suga, Hidetaka; Sugimura, Yoshihisa; Arima, Hiroshi

    2017-02-01

    Protein tyrosine phosphatase 1B (PTP1B) regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD), remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3) was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO) on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT). In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Deficiency of PTP1B Attenuates Hypothalamic Inflammation via Activation of the JAK2-STAT3 Pathway in Microglia

    Directory of Open Access Journals (Sweden)

    Taku Tsunekawa

    2017-02-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B regulates leptin signaling in hypothalamic neurons via the JAK2-STAT3 pathway. PTP1B has also been implicated in the regulation of inflammation in the periphery. However, the role of PTP1B in hypothalamic inflammation, which is induced by a high-fat diet (HFD, remains to be elucidated. Here, we showed that STAT3 phosphorylation (p-STAT3 was increased in microglia in the hypothalamic arcuate nucleus of PTP1B knock-out mice (KO on a HFD, accompanied by decreased Tnf and increased Il10 mRNA expression in the hypothalamus compared to wild-type mice (WT. In hypothalamic organotypic cultures, incubation with TNFα led to increased p-STAT3, accompanied by decreased Tnf and increased Il10 mRNA expression, in KO compared to WT. Incubation with p-STAT3 inhibitors or microglial depletion eliminated the differences in inflammation between genotypes. These data indicate an important role of JAK2-STAT3 signaling negatively regulated by PTP1B in microglia, which attenuates hypothalamic inflammation under HFD conditions.

  19. Study of the protective effects of nootropic agents against neuronal damage induced by amyloid-beta (fragment 25-35) in cultured hippocampal neurons.

    Science.gov (United States)

    Sendrowski, Krzysztof; Sobaniec, Wojciech; Stasiak-Barmuta, Anna; Sobaniec, Piotr; Popko, Janusz

    2015-04-01

    Alzheimer's disease (AD) is a common neurodegenerative disorder, in which progressive neuron loss, mainly in the hippocampus, is observed. The critical events in the pathogenesis of AD are associated with accumulation of β-amyloid (Aβ) peptides in the brain. Deposits of Aβ initiate a neurotoxic "cascade" leading to apoptotic death of neurons. Aim of this study was to assess a putative neuroprotective effects of two nootropic drugs: piracetam (PIR) and levetiracetam (LEV) on Aβ-injured hippocampal neurons in culture. Primary cultures of rat's hippocampal neurons at 7 day in vitro were exposed to Aβ(25-35) in the presence or absence of nootropics in varied concentrations. Flow cytometry with Annexin V/PI staining was used for counting and establishing neurons as viable, necrotic or apoptotic. Additionally, release of lactate dehydrogenase (LDH) to the culture medium, as a marker of cell death, was evaluated. Aβ(25-35) caused concentration-dependent death of about one third number of hippocampal neurons, mainly through an apoptotic pathway. In drugs-containing cultures, number of neurons injured with 20 μM Aβ(25-35) was about one-third lesser for PIR and almost two-fold lesser for LEV. When 40 μM Aβ(25-35) was used, only LEV exerted beneficial neuroprotective action, while PIR was ineffective. Our results suggest the protective potential of both studied nootropics against Aβ-induced death of cultured hippocampal neurons with more powerful neuroprotective effects of LEV. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  20. A Relationship between Reduced Nucleus Accumbens Shell and Enhanced Lateral Hypothalamic Orexin Neuronal Activation in Long-Term Fructose Bingeing Behavior

    Science.gov (United States)

    Rorabaugh, Jacki M.; Stratford, Jennifer M.; Zahniser, Nancy R.

    2014-01-01

    Fructose accounts for 10% of daily calories in the American diet. Fructose, but not glucose, given intracerebroventricularly stimulates homeostatic feeding mechanisms within the hypothalamus; however, little is known about how fructose affects hedonic feeding centers. Repeated ingestion of sucrose, a disaccharide of fructose and glucose, increases neuronal activity in hedonic centers, the nucleus accumbens (NAc) shell and core, but not the hypothalamus. Rats given glucose in the intermittent access model (IAM) display signatures of hedonic feeding including bingeing and altered DA receptor (R) numbers within the NAc. Here we examined whether substituting fructose for glucose in this IAM produces bingeing behavior, alters DA Rs and activates hedonic and homeostatic feeding centers. Following long-term (21-day) exposure to the IAM, rats given 8–12% fructose solutions displayed fructose bingeing but unaltered DA D1R or D2R number. Fructose bingeing rats, as compared to chow bingeing controls, exhibited reduced NAc shell neuron activation, as determined by c-Fos-immunoreactivity (Fos-IR). This activation was negatively correlated with orexin (Orx) neuron activation in the lateral hypothalamus/perifornical area (LH/PeF), a brain region linking homeostatic to hedonic feeding centers. Following short-term (2-day) access to the IAM, rats exhibited bingeing but unchanged Fos-IR, suggesting only long-term fructose bingeing increases Orx release. In long-term fructose bingeing rats, pretreatment with the Ox1R antagonist SB-334867 (30 mg/kg; i.p.) equally reduced fructose bingeing and chow intake, resulting in a 50% reduction in calories. Similarly, in control rats, SB-334867 reduced chow/caloric intake by 60%. Thus, in the IAM, Ox1Rs appear to regulate feeding based on caloric content rather than palatability. Overall, our results, in combination with the literature, suggest individual monosaccharides activate distinct neuronal circuits to promote feeding behavior

  1. A relationship between reduced nucleus accumbens shell and enhanced lateral hypothalamic orexin neuronal activation in long-term fructose bingeing behavior.

    Directory of Open Access Journals (Sweden)

    Jacki M Rorabaugh

    Full Text Available Fructose accounts for 10% of daily calories in the American diet. Fructose, but not glucose, given intracerebroventricularly stimulates homeostatic feeding mechanisms within the hypothalamus; however, little is known about how fructose affects hedonic feeding centers. Repeated ingestion of sucrose, a disaccharide of fructose and glucose, increases neuronal activity in hedonic centers, the nucleus accumbens (NAc shell and core, but not the hypothalamus. Rats given glucose in the intermittent access model (IAM display signatures of hedonic feeding including bingeing and altered DA receptor (R numbers within the NAc. Here we examined whether substituting fructose for glucose in this IAM produces bingeing behavior, alters DA Rs and activates hedonic and homeostatic feeding centers. Following long-term (21-day exposure to the IAM, rats given 8-12% fructose solutions displayed fructose bingeing but unaltered DA D1R or D2R number. Fructose bingeing rats, as compared to chow bingeing controls, exhibited reduced NAc shell neuron activation, as determined by c-Fos-immunoreactivity (Fos-IR. This activation was negatively correlated with orexin (Orx neuron activation in the lateral hypothalamus/perifornical area (LH/PeF, a brain region linking homeostatic to hedonic feeding centers. Following short-term (2-day access to the IAM, rats exhibited bingeing but unchanged Fos-IR, suggesting only long-term fructose bingeing increases Orx release. In long-term fructose bingeing rats, pretreatment with the Ox1R antagonist SB-334867 (30 mg/kg; i.p. equally reduced fructose bingeing and chow intake, resulting in a 50% reduction in calories. Similarly, in control rats, SB-334867 reduced chow/caloric intake by 60%. Thus, in the IAM, Ox1Rs appear to regulate feeding based on caloric content rather than palatability. Overall, our results, in combination with the literature, suggest individual monosaccharides activate distinct neuronal circuits to promote feeding behavior

  2. Comprehensive Method for Culturing Embryonic Dorsal Root Ganglion Neurons for Seahorse Extracellular Flux XF24 Analysis.

    Science.gov (United States)

    Lange, Miranda; Zeng, Yan; Knight, Andrew; Windebank, Anthony; Trushina, Eugenia

    2012-01-01

    Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG) neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15) rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

  3. Comprehensive method for culturing embryonic dorsal root ganglion neurons for Seahorse Extracellular Flux XF24 Analysis

    Directory of Open Access Journals (Sweden)

    Miranda L. Lange

    2012-12-01

    Full Text Available Changes in mitochondrial dynamics and function contribute to progression of multiple neurodegenerative diseases including peripheral neuropathies. The Seahorse Extracellular Flux XF24 analyzer provides a comprehensive assessment of the relative state of glycolytic and aerobic metabolism in live cells making this method instrumental in assessing mitochondrial function. One of the most important steps in the analysis of mitochondrial respiration using the Seahorse XF24 analyzer is plating a uniform monolayer of firmly attached cells. However, culturing of primary dorsal root ganglion (DRG neurons is associated with multiple challenges, including their propensity to form clumps and detach from the culture plate. This could significantly interfere with proper analysis and interpretation of data. We have tested multiple cell culture parameters including coating substrates, culture medium, XF24 microplate plastics, and plating techniques in order to optimize plating conditions. Here we describe a highly reproducible method to obtain neuron-enriched monolayers of securely attached dissociated primary embryonic (E15 rat DRG neurons suitable for analysis with the Seahorse XF24 platform.

  4. 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

    Primary cultures of GABAergic cerebral cortex neurons and glutamatergic cerebellar granule cells were used to study the expression of synaptophysin, a synaptic vesicle marker protein, along with the ability of each cell type to release neurotransmitter upon stimulation. The synaptophysin expression...... 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...

  5. Glucose Alters Per2 Rhythmicity Independent of AMPK, Whereas AMPK Inhibitor Compound C Causes Profound Repression of Clock Genes and AgRP in mHypoE-37 Hypothalamic Neurons.

    Directory of Open Access Journals (Sweden)

    Johanneke E Oosterman

    Full Text Available Specific neurons in the hypothalamus are regulated by peripheral hormones and nutrients to maintain proper metabolic control. It is unclear if nutrients can directly control clock gene expression. We have therefore utilized the immortalized, hypothalamic cell line mHypoE-37, which exhibits robust circadian rhythms of core clock genes. mHypoE-37 neurons were exposed to 0.5 or 5.5 mM glucose, comparable to physiological levels in the brain. Per2 and Bmal1 mRNAs were assessed every 3 hours over 36 hours. Incubation with 5.5 mM glucose significantly shortened the period and delayed the phase of Per2 mRNA levels, but had no effect on Bmal1. Glucose had no significant effect on phospho-GSK3β, whereas AMPK phosphorylation was altered. Thus, the AMPK inhibitor Compound C was utilized, and mRNA levels of Per2, Bmal1, Cryptochrome1 (Cry1, agouti-related peptide (AgRP, carnitine palmitoyltransferase 1C (Cpt1c, and O-linked N-acetylglucosamine transferase (Ogt were measured. Remarkably, Compound C dramatically reduced transcript levels of Per2, Bmal1, Cry1, and AgRP, but not Cpt1c or Ogt. Because AMPK was not inhibited at the same time or concentrations as the clock genes, we suggest that the effect of Compound C on gene expression occurs through an AMPK-independent mechanism. The consequences of inhibition of the rhythmic expression of clock genes, and in turn downstream metabolic mediators, such as AgRP, could have detrimental effects on overall metabolic processes. Importantly, the effects of the most commonly used AMPK inhibitor Compound C should be interpreted with caution, considering its role in AMPK-independent repression of specific genes, and especially clock gene rhythm dysregulation.

  6. Glucose Alters Per2 Rhythmicity Independent of AMPK, Whereas AMPK Inhibitor Compound C Causes Profound Repression of Clock Genes and AgRP in mHypoE-37 Hypothalamic Neurons.

    Science.gov (United States)

    Oosterman, Johanneke E; Belsham, Denise D

    2016-01-01

    Specific neurons in the hypothalamus are regulated by peripheral hormones and nutrients to maintain proper metabolic control. It is unclear if nutrients can directly control clock gene expression. We have therefore utilized the immortalized, hypothalamic cell line mHypoE-37, which exhibits robust circadian rhythms of core clock genes. mHypoE-37 neurons were exposed to 0.5 or 5.5 mM glucose, comparable to physiological levels in the brain. Per2 and Bmal1 mRNAs were assessed every 3 hours over 36 hours. Incubation with 5.5 mM glucose significantly shortened the period and delayed the phase of Per2 mRNA levels, but had no effect on Bmal1. Glucose had no significant effect on phospho-GSK3β, whereas AMPK phosphorylation was altered. Thus, the AMPK inhibitor Compound C was utilized, and mRNA levels of Per2, Bmal1, Cryptochrome1 (Cry1), agouti-related peptide (AgRP), carnitine palmitoyltransferase 1C (Cpt1c), and O-linked N-acetylglucosamine transferase (Ogt) were measured. Remarkably, Compound C dramatically reduced transcript levels of Per2, Bmal1, Cry1, and AgRP, but not Cpt1c or Ogt. Because AMPK was not inhibited at the same time or concentrations as the clock genes, we suggest that the effect of Compound C on gene expression occurs through an AMPK-independent mechanism. The consequences of inhibition of the rhythmic expression of clock genes, and in turn downstream metabolic mediators, such as AgRP, could have detrimental effects on overall metabolic processes. Importantly, the effects of the most commonly used AMPK inhibitor Compound C should be interpreted with caution, considering its role in AMPK-independent repression of specific genes, and especially clock gene rhythm dysregulation.

  7. Temporally coordinated spiking activity of human induced pluripotent stem cell-derived neurons co-cultured with astrocytes.

    Science.gov (United States)

    Kayama, Tasuku; Suzuki, Ikuro; Odawara, Aoi; Sasaki, Takuya; Ikegaya, Yuji

    2018-01-01

    In culture conditions, human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks, which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons, the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study, we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Response of Cultured Neuronal Network Activity After High-Intensity Power Frequency Magnetic Field Exposure

    Directory of Open Access Journals (Sweden)

    Atsushi Saito

    2018-03-01

    Full Text Available High-intensity and low frequency (1–100 kHz time-varying electromagnetic fields stimulate the human body through excitation of the nervous system. In power frequency range (50/60 Hz, a frequency-dependent threshold of the external electric field-induced neuronal modulation in cultured neuronal networks was used as one of the biological indicator in international guidelines; however, the threshold of the magnetic field-induced neuronal modulation has not been elucidated. In this study, we exposed rat brain-derived neuronal networks to a high-intensity power frequency magnetic field (hPF-MF, and evaluated the modulation of synchronized bursting activity using a multi-electrode array (MEA-based extracellular recording technique. As a result of short-term hPF-MF exposure (50–400 mT root-mean-square (rms, 50 Hz, sinusoidal wave, 6 s, the synchronized bursting activity was increased in the 400 mT-exposed group. On the other hand, no change was observed in the 50–200 mT-exposed groups. In order to clarify the mechanisms of the 400 mT hPF-MF exposure-induced neuronal response, we evaluated it after blocking inhibitory synapses using bicuculline methiodide (BMI; subsequently, increase in bursting activity was observed with BMI application, and the response of 400 mT hPF-MF exposure disappeared. Therefore, it was suggested that the response of hPF-MF exposure was involved in the inhibitory input. Next, we screened the inhibitory pacemaker-like neuronal activity which showed autonomous 4–10 Hz firing with CNQX and D-AP5 application, and it was confirmed that the activity was reduced after 400 mT hPF-MF exposure. Comparison of these experimental results with estimated values of the induced electric field (E-field in the culture medium revealed that the change in synchronized bursting activity occurred over 0.3 V/m, which was equivalent to the findings of a previous study that used the external electric fields. In addition, the results suggested that

  9. Amniotic fluid promotes the appearance of neural retinal progenitors and neurons in human RPE cell cultures.

    Science.gov (United States)

    Davari, Maliheh; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Sanie-Jahromi, Fateme; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Akrami, Hassan; Haghighi, Massoud; Javidi-Azad, Fahimeh

    2013-01-01

    Retinal pigment epithelial (RPE) cells are capable of differentiating into retinal neurons when induced by the appropriate growth factors. Amniotic fluid contains a variety of growth factors that are crucial for the development of a fetus. In this study, the effects of human amniotic fluid (HAF) on primary RPE cell cultures were evaluated. RPE cells were isolated from the globes of postnatal human cadavers. The isolated cells were plated and grown in DMEM/F12 with 10% fetal bovine serum. To confirm the RPE identity of the cultured cells, they were immunocytochemically examined for the presence of the RPE cell-specific marker RPE65. RPE cultures obtained from passages 2-7 were treated with HAF and examined morphologically for 1 month. To determine whether retinal neurons or progenitors developed in the treated cultures, specific markers for bipolar (protein kinase C isomer α, PKCα), amacrine (cellular retinoic acid-binding protein I, CRABPI), and neural progenitor (NESTIN) cells were sought, and the amount of mRNA was quantified using real-time PCR. Treating RPE cells with HAF led to a significant decrease in the number of RPE65-positive cells, while PKCα- and CRABPI-positive cells were detected in the cultures. Compared with the fetal bovine serum-treated cultures, the levels of mRNAs quantitatively increased by 2-, 20- and 22-fold for NESTIN, PKCα, and CRABPI, respectively. The RPE cultures treated with HAF established spheres containing both pigmented and nonpigmented cells, which expressed neural progenitor markers such as NESTIN. This study showed that HAF can induce RPE cells to transdifferentiate into retinal neurons and progenitor cells, and that it provides a potential source for cell-based therapies to treat retinal diseases.

  10. Prolonged Minocycline Treatment Impairs Motor Neuronal Survival and Glial Function in Organotypic Rat Spinal Cord Cultures

    Science.gov (United States)

    Pinkernelle, Josephine; Fansa, Hisham; Ebmeyer, Uwe; Keilhoff, Gerburg

    2013-01-01

    Background Minocycline, a second-generation tetracycline antibiotic, exhibits anti-inflammatory and neuroprotective effects in various experimental models of neurological diseases, such as stroke, Alzheimer’s disease, amyotrophic lateral sclerosis and spinal cord injury. However, conflicting results have prompted a debate regarding the beneficial effects of minocycline. Methods In this study, we analyzed minocycline treatment in organotypic spinal cord cultures of neonatal rats as a model of motor neuron survival and regeneration after injury. Minocycline was administered in 2 different concentrations (10 and 100 µM) at various time points in culture and fixed after 1 week. Results Prolonged minocycline administration decreased the survival of motor neurons in the organotypic cultures. This effect was strongly enhanced with higher concentrations of minocycline. High concentrations of minocycline reduced the number of DAPI-positive cell nuclei in organotypic cultures and simultaneously inhibited microglial activation. Astrocytes, which covered the surface of the control organotypic cultures, revealed a peripheral distribution after early minocycline treatment. Thus, we further analyzed the effects of 100 µM minocycline on the viability and migration ability of dispersed primary glial cell cultures. We found that minocycline reduced cell viability, delayed wound closure in a scratch migration assay and increased connexin 43 protein levels in these cultures. Conclusions The administration of high doses of minocycline was deleterious for motor neuron survival. In addition, it inhibited microglial activation and impaired glial viability and migration. These data suggest that especially high doses of minocycline might have undesired affects in treatment of spinal cord injury. Further experiments are required to determine the conditions for the safe clinical administration of minocycline in spinal cord injured patients. PMID:23967343

  11. Neuroprotective effects of ginsenoside Rg1 against oxygen-glucose deprivation in cultured hippocampal neurons.

    Science.gov (United States)

    He, Qing; Sun, Jianguo; Wang, Qin; Wang, Wei; He, Bin

    2014-03-01

    Ginsenoside Rg1 (Rg1) is believed to be one of the main active principles in ginseng, a traditional Chinese medicine extensively used to enhance stamina and deal with fatigue as well as physical stress. It has been reported that Rg1 performs multiple biological activities, including neuroprotective activity. In this study, we investigated the efficacy of ginsenoside Rg1 on ischemia-reperfusion injury in cultured hippocampal cells and also probed its possible mechanisms. To establish a model of oxygen-glucose deprivation (OGD) and reperfusion, cultured hippocampal neurons were exposed to OGD for 2.5 hours, followed by a 24-hour reoxygenation. Cultured hippocampal neurons were randomly divided into control group, model group (vehicle), and ginsenoside Rg1 treatment groups (5μM, 20μM, 60μM). At 24 hours post-OGD, the intracellular free calcium concentration was detected using Furo-3/AM-loaded hippocampal neurons deprived of oxygen and glucose. Neuronal nitric oxide synthase (nNOS) activity was measured by chemical colorimetry. Cell apoptosis was evaluated by Hoechst staining, and the neuron viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Excitotoxic neuronal injury of OGD was demonstrated by the increase of intracellular free calcium concentrations and elevated nNOS activity in the model group compared with the control group. The intracellular free calcium concentrations and the nNOS activity in the groups receiving intermediate and high dose of ginsenoside Rg1 were significantly lower than those of the control group (p cell viability loss (p cell apoptosis induced by OGD. Ginsenoside Rg1 has neuroprotective effect on ischemia-reperfusion injury in cultured hippocampal cells mediated by blocking calcium over-influx into neuronal cells and decreasing the nNOS activity after OGD exposure. We infer that ginsenoside Rg1 may serve as a potential therapeutic agent for cerebral ischemia injury. Copyright © 2014

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

    International Nuclear Information System (INIS)

    Heidenreich, K.A.; Toledo, S.P.

    1989-01-01

    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

  13. Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture.

    Science.gov (United States)

    Sabado, Virginie; Vienne, Ludovic; Nagoshi, Emi

    2017-01-01

    Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN) generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Drosophila circadian clock by monitoring transcriptional and post-transcriptional rhythms of individual clock neurons in dispersed culture with time-lapse microscopy. Expression patterns of the transcriptional reporter show that CLOCK/CYCLE (CLK/CYC)-mediated transcription is constantly active in dissociated clock neurons. In contrast, the expression profile of the post-transcriptional reporter indicates that PERIOD (PER) protein levels fluctuate and ~10% of cells display rhythms in PER levels with periods in the circadian range. Nevertheless, PER and TIM are enriched in the cytoplasm and no periodic PER nuclear accumulation was observed. These results suggest that repression of CLK/CYC-mediated transcription by nuclear PER is impaired, and thus the negative feedback loop of the molecular clock is incomplete in isolated clock neurons. We further demonstrate that, by pharmacological assays using the non-amidated form of neuropeptide pigment-dispersing factor (PDF), which could be specifically secreted from larval LNvs and adult s-LNvs, downstream events of the PDF signaling are partly impaired in dissociated larval clock neurons. Although non-amidated PDF is likely to be less active than the amidated one, these results point out the possibility that alteration in PDF downstream signaling may play a role in dampening of molecular rhythms in isolated clock neurons. Taken together, our results suggest that Drosophila clocks are weak oscillators that need to be in the intact circadian

  14. Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture

    Directory of Open Access Journals (Sweden)

    Virginie Sabado

    2017-10-01

    Full Text Available Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Drosophila circadian clock by monitoring transcriptional and post-transcriptional rhythms of individual clock neurons in dispersed culture with time-lapse microscopy. Expression patterns of the transcriptional reporter show that CLOCK/CYCLE (CLK/CYC-mediated transcription is constantly active in dissociated clock neurons. In contrast, the expression profile of the post-transcriptional reporter indicates that PERIOD (PER protein levels fluctuate and ~10% of cells display rhythms in PER levels with periods in the circadian range. Nevertheless, PER and TIM are enriched in the cytoplasm and no periodic PER nuclear accumulation was observed. These results suggest that repression of CLK/CYC-mediated transcription by nuclear PER is impaired, and thus the negative feedback loop of the molecular clock is incomplete in isolated clock neurons. We further demonstrate that, by pharmacological assays using the non-amidated form of neuropeptide pigment-dispersing factor (PDF, which could be specifically secreted from larval LNvs and adult s-LNvs, downstream events of the PDF signaling are partly impaired in dissociated larval clock neurons. Although non-amidated PDF is likely to be less active than the amidated one, these results point out the possibility that alteration in PDF downstream signaling may play a role in dampening of molecular rhythms in isolated clock neurons. Taken together, our results suggest that Drosophila clocks are weak oscillators that need to be in the

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

    KAUST Repository

    Cesca, F.; Limongi, T.; Accardo, A.; Rocchi, A.; Orlando, M.; Shalabaeva, V.; Di Fabrizio, Enzo M.; Benfenati, F.

    2014-01-01

    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.

  16. 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.

  17. 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......, in addition to isolating the neural tissue from nutrient delivery and to creating unwanted gradients (2). This means that typical techniques used to investigate neurodegenerative diseases cannot mimic in vivo conditions, as closely as desired. We have developed a novel microfluidic system for culturing PC12...... cells, neuronal cells, astrocytes cultures and brain slices. The microfluidic system provides efficient nutrient delivery, waste removal, access to oxygen, fine control over the neurochemical environment and access to modern microscopy. Additionally, the setup consists of an in vitro culturing...

  18. Effect of acute stretch injury on action potential and network activity of rat neocortical neurons in culture.

    Science.gov (United States)

    Magou, George C; Pfister, Bryan J; Berlin, Joshua R

    2015-10-22

    The basis for acute seizures following traumatic brain injury (TBI) remains unclear. Animal models of TBI have revealed acute hyperexcitablility in cortical neurons that could underlie seizure activity, but studying initiating events causing hyperexcitability is difficult in these models. In vitro models of stretch injury with cultured cortical neurons, a surrogate for TBI, allow facile investigation of cellular changes after injury but they have only demonstrated post-injury hypoexcitability. The goal of this study was to determine if neuronal hyperexcitability could be triggered by in vitro stretch injury. Controlled uniaxial stretch injury was delivered to a spatially delimited region of a spontaneously active network of cultured rat cortical neurons, yielding a region of stretch-injured neurons and adjacent regions of non-stretched neurons that did not directly experience stretch injury. Spontaneous electrical activity was measured in non-stretched and stretch-injured neurons, and in control neuronal networks not subjected to stretch injury. Non-stretched neurons in stretch-injured cultures displayed a three-fold increase in action potential firing rate and bursting activity 30-60 min post-injury. Stretch-injured neurons, however, displayed dramatically lower rates of action potential firing and bursting. These results demonstrate that acute hyperexcitability can be observed in non-stretched neurons located in regions adjacent to the site of stretch injury, consistent with reports that seizure activity can arise from regions surrounding the site of localized brain injury. Thus, this in vitro procedure for localized neuronal stretch injury may provide a model to study the earliest cellular changes in neuronal function associated with acute post-traumatic seizures. Copyright © 2015. Published by Elsevier B.V.

  19. The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes.

    Science.gov (United States)

    Johansen, Maja L; Bak, Lasse K; Schousboe, Arne; Iversen, Peter; Sørensen, Michael; Keiding, Susanne; Vilstrup, Hendrik; Gjedde, Albert; Ott, Peter; Waagepetersen, Helle S

    2007-06-01

    Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and alpha-ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the alpha-ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the alpha-ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-13C]glucose (2.5 mM) and isoleucine (1 mM) or [U-13C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of 13C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-13C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-13C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-13C]glucose. Labeling in glutamate and aspartate from [U-13C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially

  20. Functional Maturation of Human Stem Cell-Derived Neurons in Long-Term Cultures.

    Directory of Open Access Journals (Sweden)

    Rebecca S Lam

    Full Text Available Differentiated neurons can be rapidly acquired, within days, by inducing stem cells to express neurogenic transcription factors. We developed a protocol to maintain long-term cultures of human neurons, called iNGNs, which are obtained by inducing Neurogenin-1 and Neurogenin-2 expression in induced pluripotent stem cells. We followed the functional development of iNGNs over months and they showed many hallmark properties for neuronal maturation, including robust electrical and synaptic activity. Using iNGNs expressing a variant of channelrhodopsin-2, called CatCh, we could control iNGN activity with blue light stimulation. In combination with optogenetic tools, iNGNs offer opportunities for studies that require precise spatial and temporal resolution. iNGNs developed spontaneous network activity, and these networks had excitatory glutamatergic synapses, which we characterized with single-cell synaptic recordings. AMPA glutamatergic receptor activity was especially dominant in postsynaptic recordings, whereas NMDA glutamatergic receptor activity was absent from postsynaptic recordings but present in extrasynaptic recordings. Our results on long-term cultures of iNGNs could help in future studies elucidating mechanisms of human synaptogenesis and neurotransmission, along with the ability to scale-up the size of the cultures.

  1. 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.

  2. Neuron-specific enolase is a useful maker of neuroendocrine origin in pheochromocytoma cell culture

    International Nuclear Information System (INIS)

    Abelin, N.; Dahia, P.L.M.; Martin, R.; Kato, S.; Toledo, S.P.A.

    1994-01-01

    Neuron-specific enolase (NSE) has been used as a marker for neuroendocrine tumors either in immunocytochemical studies or in serum measurements. In this paper NSE levels were determined in cultured pheochromocytoma cells to test whether it is also a useful marker in cell culture of tumors derived from neuroendocrine system. Cultured pheochromocytoma cells came from a primary explant and were grown in RPMI supplemented with 20% fetal calf serum, 100 μg/mL ampicillin and 100 μ/mL streptomycin. NSE was measured in culture medium and cell homogenates. Samples from different pheochromocytoma cultures were analyzed and compared to normal cultured fibroblast cells derived from human skin. NSE was measured by a commercially available radioimmunoassay kit. NSE levels were higher in cell homogenates as compared to those in culture medium, reaching levels as high as 6-fold in the former in TE cell line (26.46 ng/mL and 4.39 ng/mL, respectively). Serial measurements in culture medium from TE cell line evidenced decreasing values in subsequential subcultures (from 9.24 ng/mL during primary explant to 1.7 ng/mL in the tenth subculture). In cultured normal fibroblasts, NSE levels in cultured media were definitely lower than those obtained from pheochromocytoma cultures. These preliminary data suggest that NSE may be a useful marker of neuroendocrine derived tumors, such as pheochromocytoma, in culture. Thus, the simplicity and availability of NSE radioimmunoassay provides an alternative to catecholamine measurement to better characterize pheochromocytoma cell lines in culture, with the advantage of faster result at lower costs. (author). 18 refs, 2 tabs

  3. GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

    DEFF Research Database (Denmark)

    Ducray, Angélique; Krebs, Sandra H:; Schaller, Benoft

    2006-01-01

    Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about...... factors for VM GABAergic and serotonergic neurons, demonstrating characteristic individual action profiles emphasizing their important and distinct roles during brain development....

  4. 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.

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

    Science.gov (United States)

    Franceschini, Alessia; Nair, Asha; Bele, Tanja; van den Maagdenberg, Arn Mjm; Nistri, Andrea; Fabbretti, Elsa

    2012-11-21

    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. 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. 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.

  6. Delineating the regulation of energy homeostasis using hypothalamic cell models.

    Science.gov (United States)

    Wellhauser, Leigh; Gojska, Nicole M; Belsham, Denise D

    2015-01-01

    Attesting to its intimate peripheral connections, hypothalamic neurons integrate nutritional and hormonal cues to effectively manage energy homeostasis according to the overall status of the system. Extensive progress in the identification of essential transcriptional and post-translational mechanisms regulating the controlled expression and actions of hypothalamic neuropeptides has been identified through the use of animal and cell models. This review will introduce the basic techniques of hypothalamic investigation both in vivo and in vitro and will briefly highlight the key advantages and challenges of their use. Further emphasis will be place on the use of immortalized models of hypothalamic neurons for in vitro study of feeding regulation, with a particular focus on cell lines proving themselves most fruitful in deciphering fundamental basics of NPY/AgRP, Proglucagon, and POMC neuropeptide function. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Proteomic Analysis of the Neurotrophic Effect of Gelidium amansii in Primary Cultured Neurons.

    Science.gov (United States)

    Hannan, Md Abdul; Mohibbullah, Md; Hong, Yong-Ki; Moon, Il Soo

    2017-03-01

    Gelidium amansii is an edible and economically important red alga consumed in South Eastern Asia. In previous studies, we reported that the ethanol extracts of G. amansii (GAE) has promising modulatory activity with respect to the morphological and functional maturation of hippocampal neurons in culture. In this study, we show that the chloroform (CHCl 3 ) subfraction of GAE and the ethyl acetate (EtOAc) fraction dose-dependently promoted neurite outgrowth, and their effects were comparable with that of GAE. We further assessed in cultured cortical neurons, proteins differentially expressed in the presence/absence of the GAE, CHCl 3 subfraction, and the EtOAc fraction by 2D-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proteomic data revealed that a number of proteins responsible for multiple cellular and biochemical functions vital for neuronal development and maturation were significantly upregulated in neurons treated with the GAE, CHCl 3 subfraction, and the EtOAc fraction. Of the identified proteins, profilin 2a, septin 7, cdc42, protein phosphatase 2A, DA11, eukaryotic translation initiation factor 5A-1, and γ-enolase are known to play important roles in neuritogenesis and dendritic arborization. Immunofluorescence data demonstrate that GAE-treated hippocampal neurons showed greater intensity ratios in the expressions of the septin 7 and cdc42 compared to vehicle control, validating their proteomic profiles. Together these results suggest that the GAE/CHCl 3 subfraction and EtOAc fraction promote neurite development by up or downregulating several key proteins.

  8. Microglia in Glia-Neuron Co-cultures Exhibit Robust Phagocytic Activity Without Concomitant Inflammation or Cytotoxicity.

    Science.gov (United States)

    Adams, Alexandra C; Kyle, Michele; Beaman-Hall, Carol M; Monaco, Edward A; Cullen, Matthew; Vallano, Mary Lou

    2015-10-01

    A simple method to co-culture granule neurons and glia from a single brain region is described, and microglia activation profiles are assessed in response to naturally occurring neuronal apoptosis, excitotoxin-induced neuronal death, and lipopolysaccharide (LPS) addition. Using neonatal rat cerebellar cortex as a tissue source, glial proliferation is regulated by omission or addition of the mitotic inhibitor cytosine arabinoside (AraC). After 7-8 days in vitro, microglia in AraC(-) cultures are abundant and activated based on their amoeboid morphology, expressions of ED1 and Iba1, and ability to phagocytose polystyrene beads and the majority of neurons undergoing spontaneous apoptosis. Microglia and phagocytic activities are sparse in AraC(+) cultures. Following exposure to excitotoxic kainate concentrations, microglia in AraC(-) cultures phagocytose most dead neurons within 24 h without exacerbating neuronal loss or mounting a strong or sustained inflammatory response. LPS addition induces a robust inflammatory response, based on microglial expressions of TNF-α, COX-2 and iNOS proteins, and mRNAs, whereas these markers are essentially undetectable in control cultures. Thus, the functional effector state of microglia is primed for phagocytosis but not inflammation or cytotoxicity even after kainate exposure that triggers death in the majority of neurons. This model should prove useful in studying the progressive activation states of microglia and factors that promote their conversion to inflammatory and cytotoxic phenotypes.

  9. Insulin stimulates choline acetyltransferase activity in cultured embryonic chicken retina neurons

    International Nuclear Information System (INIS)

    Kyriakis, J.M.; Hausman, R.E.; Peterson, S.W.

    1987-01-01

    The effect of insulin on the appearance of the enzyme choline acetyltransferase in embryonic chicken retina neurons cultured in defined medium was studied. In the presence of a minimal level of insulin (1 ng/ml), ChoAcT activity increased with time in culture. A correspondence between the insulin concentration in the defined medium (1-100 ng/ml) and both the rate of increase and maximum attained level of ChoAcT activity was observed. Maximal ChoAcT activity was 2- to 3-fold greater in cells cultured in the presence of 100 ng of insulin per ml than in cells cultured in the presence of 1 ng of insulin per ml. To elicit maximum ChoAcT activity, insulin at 100 ng/ml was required in the medium for only the first 4 days of the culture period, at which time insulin could be reduced to maintenance levels (10 ng/ml) without affecting ChoAcT activity. Insulin binding assays performed during a 7-day culture period revealed that irrespective of the 125 I-insulin concentration in the medium during culture, cell-surface insulin receptors decreased by ≅ 90% between 4 and 7 days in culture. This decrease in insulin binding corresponded to the observed decrease in the sensitivity of ChoAcT activity to insulin. The findings suggest that insulin plays a role in mediating cholinergic differentiation in the embryonic chicken retina

  10. Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

    Science.gov (United States)

    Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

    2014-06-04

    Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

  11. Increased hypothalamic serotonin turnover in inflammation-induced anorexia.

    Science.gov (United States)

    Dwarkasing, J T; Witkamp, R F; Boekschoten, M V; Ter Laak, M C; Heins, M S; van Norren, K

    2016-05-20

    Anorexia can occur as a serious complication of disease. Increasing evidence suggests that inflammation plays a major role, along with a hypothalamic dysregulation characterized by locally elevated serotonin levels. The present study was undertaken to further explore the connections between peripheral inflammation, anorexia and hypothalamic serotonin metabolism and signaling pathways. First, we investigated the response of two hypothalamic neuronal cell lines to TNFα, IL-6 and LPS. Next, we studied transcriptomic changes and serotonergic activity in the hypothalamus of mice after intraperitoneal injection with TNFα, IL-6 or a combination of TNFα and IL-6. In vitro, we showed that hypothalamic neurons responded to inflammatory mediators by releasing cytokines. This inflammatory response was associated with an increased serotonin release. Mice injected with TNFα and IL-6 showed decreased food intake, associated with altered expression of inflammation-related genes in the hypothalamus. In addition, hypothalamic serotonin turnover showed to be elevated in treated mice. Overall, our results underline that peripheral inflammation reaches the hypothalamus where it affects hypothalamic serotoninergic metabolism. These hypothalamic changes in serotonin pathways are associated with decreased food intake, providing evidence for a role of serotonin in inflammation-induced anorexia.

  12. Hypothalamic inflammation: a double-edged sword to nutritional diseases

    Science.gov (United States)

    Cai, Dongsheng; Liu, Tiewen

    2015-01-01

    The hypothalamus is one of the master regulators of various physiological processes, including energy balance and nutrient metabolism. These regulatory functions are mediated by discrete hypothalamic regions that integrate metabolic sensing with neuroendocrine and neural controls of systemic physiology. Neurons and non-neuronal cells in these hypothalamic regions act supportively to execute metabolic regulations. Under conditions of brain and hypothalamic inflammation, which may result from overnutrition-induced intracellular stresses or disease-associated systemic inflammatory factors, extracellular and intracellular environments of hypothalamic cells are disrupted, leading to central metabolic dysregulations and various diseases. Recent research has begun to elucidate the effects of hypothalamic inflammation in causing diverse components of metabolic syndrome leading to diabetes and cardiovascular disease. These new understandings have provocatively expanded previous knowledge on the cachectic roles of brain inflammatory response in diseases, such as infections and cancers. This review describes the molecular and cellular characteristics of hypothalamic inflammation in metabolic syndrome and related diseases as opposed to cachectic diseases, and also discusses concepts and potential applications of inhibiting central/hypothalamic inflammation to treat nutritional diseases. PMID:22417140

  13. Brain-derived neurotrophic factor/neurotrophin 3 regulate axon initial segment location and affect neuronal excitability in cultured hippocampal neurons.

    Science.gov (United States)

    Guo, Yu; Su, Zi-Jun; Chen, Yi-Kun; Chai, Zhen

    2017-07-01

    Plasticity of the axon initial segment (AIS) has aroused great interest in recent years because it regulates action potential initiation and neuronal excitability. AIS plasticity manifests as modulation of ion channels or variation in AIS structure. However, the mechanisms underlying structural plasticity of the AIS are not well understood. Here, we combined immunofluorescence, patch-clamp recordings, and pharmacological methods in cultured hippocampal neurons to investigate the factors participating in AIS structural plasticity during development. With lowered neuronal density, the distance between the AIS and the soma increased, while neuronal excitability decreased, as shown by the increased action potential threshold and current threshold for firing an action potential. This variation in the location of the AIS was associated with cellular secretory substances, including brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3). Indeed, blocking BDNF and NT3 with TrkB-Fc eliminated the effect of conditioned medium collected from high-density cultures on AIS relocation. Elevating the extracellular concentration of BDNF or NT3 promoted movement of the AIS proximally to the soma and increased neuronal excitability. Furthermore, knockdown of neurotrophin receptors TrkB and TrkC caused distal movement of the AIS. Our results demonstrate that BDNF and NT3 regulate AIS location and neuronal excitability. These regulatory functions of neurotrophic factors provide insight into the molecular mechanisms underlying AIS biology. © 2017 International Society for Neurochemistry.

  14. Morphine Preconditioning Downregulates MicroRNA-134 Expression Against Oxygen-Glucose Deprivation Injuries in Cultured Neurons of Mice.

    Science.gov (United States)

    Meng, Fanjun; Li, Yan; Chi, Wenying; Li, Junfa

    2016-07-01

    Brain protection by narcotics such as morphine is clinically relevant due to the extensive use of narcotics in the perioperative period. Morphine preconditioning induces neuroprotection in neurons, but it remains uncertain whether microRNA-134 (miR-134) is involved in morphine preconditioning against oxygen-glucose deprivation-induced injuries in primary cortical neurons of mice. The present study examined this issue. After cortical neurons of mice were cultured in vitro for 6 days, the neurons were transfected by respective virus vector, such as lentiviral vector (LV)-miR-control-GFP, LV-pre-miR-134-GFP, LV-pre-miR-134-inhibitor-GFP for 24 hours; after being normally cultured for 3 days again, morphine preconditioning was performed by incubating the transfected primary neurons with morphine (3 μM) for 1 hour, and then neuronal cells were exposed to oxygen-glucose deprivation (OGD) for 1 hour and oxygen-glucose recovery for 12 hours. The neuronal cells survival rate and the amount of apoptotic neurons were determined by MTT assay or TUNEL staining at designated time; and the expression levels of miR-134 were detected using real-time reverse transcription polymerase chain reaction at the same time. The neuronal cell survival rate was significantly higher, and the amount of apoptotic neurons was significantly decreased in neurons preconditioned with morphine before OGD than that of OGD alone. The neuroprotection induced by morphine preconditioning was partially blocked by upregulating miR-134 expression, and was enhanced by downregulating miR-134 expression. The expression of miR-134 was significantly decreased in morphine-preconditioned neurons alone without transfection. By downregulating miR-134 expression, morphine preconditioning protects primary cortical neurons of mice against injuries induced by OGD.

  15. Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons

    DEFF Research Database (Denmark)

    Bak, Lasse K; Schousboe, Arne; Sonnewald, Ursula

    2006-01-01

    Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was inves......Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis...... was investigated. Cultured cerebellar (primarily glutamatergic) neurons were superfused in medium containing [U-13C]glucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or glucose (2.5 mmol/L) and [U-13C]lactate (1 mmol/L), and exposed to pulses of N-methyl-D-aspartate (300 micromol/L), leading to synaptic activity...... significantly during induced depolarization. In contrast, at both concentrations of extracellular lactate, the metabolism of [U-13C]glucose was increased during neuronal depolarization. The role of glucose and lactate as energy substrates during vesicular release as well as transporter-mediated influx...

  16. Effects of exposure to high glucose on primary cultured hippocampal neurons: involvement of intracellular ROS accumulation.

    Science.gov (United States)

    Liu, Di; Zhang, Hong; Gu, Wenjuan; Zhang, Mengren

    2014-06-01

    Recent studies showed that hyperglycemia is the main trigger of diabetic cognitive impairment and can cause hippocampus abnormalities. The goal of this study is to explore the effects of different concentrations of high glucose for different exposure time on cell viability as well as intracellular reactive oxygen species (ROS) generation of primary cultured hippocampal neurons. Hippocampal neurons were exposed to different concentrations of high glucose (50, 75, 100, 125, and 150 mM) for 24, 48, 72 and 96 h. Cell viability and nuclear morphology were evaluated by MTT and Hoechst assays, respectively. Intracellular ROS were monitored using the fluorescent probe DCFH-DA. The results showed that, compared with control group, the cell viability of all high glucose-treated groups decreased significantly after 72 h and there also was a significant increase of apoptotic nuclei in high glucose-treated groups from 72 to 96 h. Furthermore, 50 mM glucose induced a peak rise in ROS generation at 24 h and the intracellular ROS levels of 50 mM glucose group were significantly higher than the corresponding control group from 6 to 72 h. These results suggest that hippocampal neurons could be injured by high glucose exposure and the neuronal injury induced by high glucose is potentially mediated through intracellular ROS accumulation.

  17. Cultured Cortical Neurons Can Perform Blind Source Separation According to the Free-Energy Principle

    Science.gov (United States)

    Isomura, Takuya; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-01-01

    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. PMID:26690814

  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. GDNF family ligands display distinct action profiles on cultured GABAergic and serotonergic neurons of rat ventral mesencephalon

    DEFF Research Database (Denmark)

    Ducray, Angélique; Krebs, Sandra H:; Schaller, Benoft

    2006-01-01

    the effects of GFLs on other neuronal populations in the VM is essential for their potential application as therapeutic molecules for Parkinson's disease. Hence, in a comparative study, we investigated the effects of GFLs on cell densities and morphological differentiation of gamma-aminobutyric acid......Glial-cell-line-derived neurotrophic factor (GDNF), neurturin (NRTN), artemin (ARTN) and persephin (PSPN), known as the GDNF family ligands (GFLs), influence the development, survival and differentiation of cultured dopaminergic neurons from ventral mesencephalon (VM). Detailed knowledge about......-immunoreactive (GABA-ir) and serotonin-ir (5-HT-ir) neurons in primary cultures of E14 rat VM. We observed that all GFLs [10 ng/ml] significantly increased GABA-ir cell densities (1.6-fold) as well as neurite length/neuron. However, only GDNF significantly increased the number of primary neurites/neuron, and none...

  20. Monocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures.

    Science.gov (United States)

    Gao, Chen; Zhou, Liya; Zhu, Wenxia; Wang, Hongyun; Wang, Ruijuan; He, Yunfei; Li, Zhiyun

    2015-05-06

    Hypoxic and low-glucose stressors contribute to neuronal death in many brain diseases. Astrocytes are anatomically well-positioned to shield neurons from hypoxic injury. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy. This process is mediated by monocarboxylate transporters (MCTs) in the central nervous system. In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary cultures of neurons, astrocytes, and astrocytes-neurons derived from rat hippocampus were subjected to OGD, MCT inhibition with small interfering (si)RNA. Cell survival and expression of MCT4, MCT2, glial fibrillary acidic protein, and neuronal nuclear antigen were evaluated. OGD significantly increased cell death in neuronal cultures and up-regulated MCT4 expression in astrocyte cultures, but no increased cell death was observed in neuron-astrocyte co-cultures or astrocyte cultures. However, neuronal cell death in co-cultures was increased by exposure to MCT4- or MCT2-specific siRNA, and this effect was attenuated by the addition of lactate into the extracellular medium of neuronal cultures prior to OGD. These findings demonstrate that resistance to OGD injury in astrocyte-neuron co-cultures occurs via an MCT-dependent mechanism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. High Content Analysis of Hippocampal Neuron-Astrocyte Co-cultures Shows a Positive Effect of Fortasyn Connect on Neuronal Survival and Postsynaptic Maturation.

    Science.gov (United States)

    van Deijk, Anne-Lieke F; Broersen, Laus M; Verkuyl, J Martin; Smit, August B; Verheijen, Mark H G

    2017-01-01

    Neuronal and synaptic membranes are composed of a phospholipid bilayer. Supplementation with dietary precursors for phospholipid synthesis -docosahexaenoic acid (DHA), uridine and choline- has been shown to increase neurite outgrowth and synaptogenesis both in vivo and in vitro . A role for multi-nutrient intervention with specific precursors and cofactors has recently emerged in early Alzheimer's disease, which is characterized by decreased synapse numbers in the hippocampus. Moreover, the medical food Souvenaid, containing the specific nutrient combination Fortasyn Connect (FC), improves memory performance in early Alzheimer's disease patients, possibly via maintaining brain connectivity. This suggests an effect of FC on synapses, but the underlying cellular mechanism is not fully understood. Therefore, we investigated the effect of FC (consisting of DHA, eicosapentaenoic acid (EPA), uridine, choline, phospholipids, folic acid, vitamins B12, B6, C and E, and selenium), on synaptogenesis by supplementing it to primary neuron-astrocyte co-cultures, a cellular model that mimics metabolic dependencies in the brain. We measured neuronal developmental processes using high content screening in an automated manner, including neuronal survival, neurite morphology, as well as the formation and maturation of synapses. Here, we show that FC supplementation resulted in increased numbers of neurons without affecting astrocyte number. Furthermore, FC increased postsynaptic PSD95 levels in both immature and mature synapses. These findings suggest that supplementation with FC to neuron-astrocyte co-cultures increased both neuronal survival and the maturation of postsynaptic terminals, which might aid the functional interpretation of FC-based intervention strategies in neurological diseases characterized by neuronal loss and impaired synaptic functioning.

  2. High Content Analysis of Hippocampal Neuron-Astrocyte Co-cultures Shows a Positive Effect of Fortasyn Connect on Neuronal Survival and Postsynaptic Maturation

    Directory of Open Access Journals (Sweden)

    Anne-Lieke F. van Deijk

    2017-08-01

    Full Text Available Neuronal and synaptic membranes are composed of a phospholipid bilayer. Supplementation with dietary precursors for phospholipid synthesis –docosahexaenoic acid (DHA, uridine and choline– has been shown to increase neurite outgrowth and synaptogenesis both in vivo and in vitro. A role for multi-nutrient intervention with specific precursors and cofactors has recently emerged in early Alzheimer's disease, which is characterized by decreased synapse numbers in the hippocampus. Moreover, the medical food Souvenaid, containing the specific nutrient combination Fortasyn Connect (FC, improves memory performance in early Alzheimer's disease patients, possibly via maintaining brain connectivity. This suggests an effect of FC on synapses, but the underlying cellular mechanism is not fully understood. Therefore, we investigated the effect of FC (consisting of DHA, eicosapentaenoic acid (EPA, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C and E, and selenium, on synaptogenesis by supplementing it to primary neuron-astrocyte co-cultures, a cellular model that mimics metabolic dependencies in the brain. We measured neuronal developmental processes using high content screening in an automated manner, including neuronal survival, neurite morphology, as well as the formation and maturation of synapses. Here, we show that FC supplementation resulted in increased numbers of neurons without affecting astrocyte number. Furthermore, FC increased postsynaptic PSD95 levels in both immature and mature synapses. These findings suggest that supplementation with FC to neuron-astrocyte co-cultures increased both neuronal survival and the maturation of postsynaptic terminals, which might aid the functional interpretation of FC-based intervention strategies in neurological diseases characterized by neuronal loss and impaired synaptic functioning.

  3. Localization and pharmacological characterization of voltage dependent calcium channels in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Timmermann, D B; Lund, Trine Meldgaard; Belhage, B

    2001-01-01

    The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured...... channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively...... most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise...

  4. Cell culture chamber with gas supply for prolonged recording of human neuronal cells on microelectrode array.

    Science.gov (United States)

    Kreutzer, Joose; Ylä-Outinen, Laura; Mäki, Antti-Juhana; Ristola, Mervi; Narkilahti, Susanna; Kallio, Pasi

    2017-03-15

    Typically, live cell analyses are performed outside an incubator in an ambient air, where the lack of sufficient CO 2 supply results in a fast change of pH and the high evaporation causes concentration drifts in the culture medium. That limits the experiment time for tens of minutes. In many applications, e.g. in neurotoxicity studies, a prolonged measurement of extracellular activity is, however, essential. We demonstrate a simple cell culture chamber that enables stable culture conditions during prolonged extracellular recordings on a microelectrode array (MEA) outside an incubator. The proposed chamber consists of a gas permeable silicone structure that enables gas transfer into the chamber. We show that the culture chamber supports the growth of the human embryonic stem cell (hESC)-derived neurons both inside and outside an incubator. The structure provides very low evaporation, stable pH and osmolarity, and maintains strong signaling of hESC-derived neuronal networks over three-day MEA experiments. Existing systems are typically complex including continuous perfusion of medium or relatively large amount of gas to supply. The proposed chamber requires only a supply of very low flow rate (1.5ml/min) of non-humidified 5% CO 2 gas. Utilizing dry gas supply makes the proposed chamber simple to use. Using the proposed culture structure on top of MEA, we can maintain hESC-derived neural networks over three days outside an incubator. Technically, the structure requires very low flow rate of dry gas supporting, however, low evaporation and maintaining the pH of the culture. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Oligomeric forms of the metastasis-related Mts1 (S100A4) protein stimulate neuronal differentiation in cultures of rat hippocampal neurons

    DEFF Research Database (Denmark)

    Novitskaya, V; Grigorian, M; Kriajevska, M

    2000-01-01

    protein family. The oligomeric but not the dimeric form of Mts1 strongly induces differentiation of cultured hippocampal neurons. A mutant with a single Y75F amino acid substitution, which stabilizes the dimeric form of Mts1, is unable to promote neurite extension. Disulfide bonds do not play an essential...

  6. 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.

    2014-01-01

    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 100nM 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. PMID:24385416

  7. 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.

  8. 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. © The Author(s) 2014 Reprints and permissions: sagepub.com/journalsPermissions.nav.

  9. Localized Induced Current Stimulation to Neuronal Culture Using Soft Magnetic Material

    Science.gov (United States)

    Saito, Atsushi; Saito, Aki; Moriguchi, Hiroyuki; Kotani, Kiyoshi; Jimbo, Yasuhiko

    To establish precisely focused magnetic stimulation, we developed a Mu-meal based low-frequency localized induced current (LIC) stimulation system with micro-fabricated dual cell-culture chamber. The dual cell-culture chamber was arranged in a concentric circle manner. Between the inner and outer chambers, 4 or 8 connecting micro-channels were fabricated using polydimethylsiloxane (PDMS). Rat cortical neurons were separately cultured in outer and inner chambers. Through the micro-channels, functional synaptic connections were formed. Mu-metal that has very high magnetic permeability was aligned along the outer circle, which allowed us of LIC stimulation to the cells in the outer chamber. Applying low-frequency magnetic fields to the Mu-metal, induced currents were generated and the electrical activity of the cells in the outer chamber was modified depending on the stimulation intensity. Following the modified activity in the outer circles, the cells in the inner chamber also showed slightly depressed activity patterns. These results suggested that our system would be promising for localized stimulation of neuronal networks and highly regulation of network activities.

  10. Culturing of primary rat neurons and glia on ultra-thin parylene-C

    International Nuclear Information System (INIS)

    Unsworth, C.P.; Delivopoulos, E.; Murray, A.F.

    2010-01-01

    Full text: In this article, we will describe how we have successfully cultured dissociated embryonic cortical neurons and glia from the postnatal rat hippocampus on extremely thin layers (up to 10 nm) of Parylene-C on a silicon dioxide substrate. Silicon wafers were oxidised, deposited with the biomaterial, Parylene-C, photo-lithographically patterned and plasma etched to produce chips that consisted of lines of Paryl ene-C with varying widths, thickness and lengths. The chips produced were then immersed in Horse Serum and plated with the cells. Ratios of Neurons; Glia; Cell Body were measured on, adjacent to and away from the Parylene-C. Our initial results show how these ratios remained roughly constant for ultra-thin Parylene-C thicknesses of 10 nm as compared to a benchmark thickness of 100 nm (where such cells are known to grow well). Thus, our findings demonstrate that it is possible to culture primary rat neurons and glia to practically cell membrane thicknesses of Parylene-C. Being able to culture cells on such ultra thin levels of Parylene-C will open up the possibility to develop Multi-Electrode Arrays (MEA) that can capacitively couple embedded electrodes through the parylene to the cells on its surface. Thus, providing a neat, insulated passive electrode. Only the ultra-thin thicknesses of Parylene demonstrated here would allow for the rea isation of such a technology. Hence, the outcome of this work, will be of great interest to the Neuroengineering and the Multi-Electrode Array (MEA) community, as an alternative material for the fabric tion of passive electrodes, used in capacitive coupling mode.

  11. 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.

  12. 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.

  13. Characterization of cortical neuronal and glial alterations during culture of organotypic whole brain slices from neonatal and mature mice.

    Science.gov (United States)

    Staal, Jerome A; Alexander, Samuel R; Liu, Yao; Dickson, Tracey D; Vickers, James C

    2011-01-01

    Organotypic brain slice culturing techniques are extensively used in a wide range of experimental procedures and are particularly useful in providing mechanistic insights into neurological disorders or injury. The cellular and morphological alterations associated with hippocampal brain slice cultures has been well established, however, the neuronal response of mouse cortical neurons to culture is not well documented. In the current study, we compared the cell viability, as well as phenotypic and protein expression changes in cortical neurons, in whole brain slice cultures from mouse neonates (P4-6), adolescent animals (P25-28) and mature adults (P50+). Cultures were prepared using the membrane interface method. Propidium iodide labeling of nuclei (due to compromised cell membrane) and AlamarBlue™ (cell respiration) analysis demonstrated that neonatal tissue was significantly less vulnerable to long-term culture in comparison to the more mature brain tissues. Cultures from P6 animals showed a significant increase in the expression of synaptic markers and a decrease in growth-associated proteins over the entire culture period. However, morphological analysis of organotypic brain slices cultured from neonatal tissue demonstrated that there were substantial changes to neuronal and glial organization within the neocortex, with a distinct loss of cytoarchitectural stratification and increased GFAP expression (pglial limitans and, after 14 DIV, displayed substantial cellular protrusions from slice edges, including cells that expressed both glial and neuronal markers. In summary, we present a substantial evaluation of the viability and morphological changes that occur in the neocortex of whole brain tissue cultures, from different ages, over an extended period of culture.

  14. Cultured subventricular zone progenitor cells transduced with neurogenin-2 become mature glutamatergic neurons and integrate into the dentate gyrus.

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    Xia Chen

    Full Text Available We have previously shown that transplantation of immature DCX+/NeuN+/Prox1+ neurons (found in the neonatal DG, but not undifferentiated neuronal progenitor cells (NPCs from ventral subventricular zone (SVZ, results in neuronal maturation in vivo within the dentate niche. Here we investigated whether we could enhance the integration of SVZ NPCs by forced expression of the proneural gene Neurogenin 2 (NEUROG2. NPCs cultured from neonatal GFP-transgenic rat SVZ for 7 days in a non-differentiating medium were transduced with a retrovirus encoding NEUROG2 and DsRed or the DsRed reporter gene alone (control. By 3 days post-transduction, the NEUROG2-transduced cells maintained in culture contained mostly immature neurons (91% DCX+; 76% NeuN+, whereas the control virus-transduced cells remained largely undifferentiated (30% DCX+; <1% NeuN+. At 6 weeks following transplantation into the DG of adult male rats, there were no neurons among the transplanted cells treated with the control virus but the majority of the NEUROG2-transduced DsRed+ SVZ cells became mature neurons (92% NeuN+; DCX-negative. Although the NEUROG2-transduced SVZ cells did not express the dentate granule neuron marker Prox1, most of the NEUROG2-transduced SVZ cells (78% expressed the glutamatergic marker Tbr1, suggesting the acquisition of a glutamatergic phenotype. Moreover, some neurons extended dendrites into the molecular layer, grew axons containing Ankyrin G+ axonal initial segments, and projected into the CA3 region, thus resembling mature DG granule neurons. A proportion of NEUROG2 transduced cells also expressed c-Fos and P-CREB, two markers of neuronal activation. We conclude that NEUROG2-transduction is sufficient to promote neuronal maturation and integration of transplanted NPCs from SVZ into the DG.

  15. 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.

  16. Low concentrations of ketamine initiate dendritic atrophy of differentiated GABAergic neurons in culture

    International Nuclear Information System (INIS)

    Vutskits, Laszlo; Gascon, Eduardo; Potter, Gael; Tassonyi, Edomer; Kiss, Jozsef Z.

    2007-01-01

    Administration of subanesthetic concentrations of ketamine, a noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) type of glutamate receptors, is a widely accepted therapeutic modality in perioperative and chronic pain management. Although extensive clinical use has demonstrated its safety, recent human histopathological observations as well as laboratory data suggest that ketamine can exert adverse effects on central nervous system neurons. To further investigate this issue, the present study was designed to evaluate the effects of ketamine on the survival and dendritic arbor architecture of differentiated γ-aminobutyric acidergic (GABAergic) interneurons in vitro. We show that short-term exposure of cultures to ketamine at concentrations of ≥20 μg/ml leads to a significant cell loss of differentiated cells and that non-cell death-inducing concentrations of ketamine (10 μg/ml) can still initiate long-term alterations of dendritic arbor in differentiated neurons, including dendritic retraction and branching point elimination. Most importantly, we also demonstrate that chronic (>24 h) administration of ketamine at concentrations as low as 0.01 μg/ml can interfere with the maintenance of dendritic arbor architecture. These results raise the possibility that chronic exposure to low, subanesthetic concentrations of ketamine, while not affecting cell survival, could still impair neuronal morphology and thus might lead to dysfunctions of neural networks

  17. Ethanol enhances GABA-induced 36Cl-influx in primary spinal cord cultured neurons

    International Nuclear Information System (INIS)

    Ticku, M.K.; Lowrimore, P.; Lehoullier, P.

    1986-01-01

    Ethanol has a pharmacological profile similar to other centrally acting drugs, which facilitate GABAergic transmission. GABA is known to produce its effects by increasing the conductance to Cl- ions. In this study, we have examined the effect of ethanol on GABA-induced 36Cl-influx in primary spinal cord cultured neurons. GABA produces a concentration-dependent, and saturable effect on 36Cl-influx in these neurons. Ethanol potentiates the effect of GABA on 36Cl-influx in these neurons. GABA (20 microM) increased the 36Cl-influx by 75% over the basal value, and in the presence of 50 mM ethanol, the observed increase was 142%. Eadie-Hoffstee analysis of the saturation curves indicated that ethanol decreases the Km value of GABA (10.6 microM to 4.2 microM), and also increases the Vmax. Besides potentiating the effect of GABA, ethanol also appears to have a direct effect in the absence of added GABA. These results suggest that ethanol enhances GABA-induced 36Cl-influx and indicate a role of GABAergic system in the actions of ethanol. These results also support the behavioral and electrophysiological studies, which have implicated GABA systems in the actions of ethanol. The potential mechanism(s) and the role of direct effect of ethanol is not clear at this time, but is currently being investigated

  18. Availability of neurotransmitter glutamate is diminished when beta-hydroxybutyrate replaces glucose in cultured neurons.

    Science.gov (United States)

    Lund, Trine M; Risa, Oystein; Sonnewald, Ursula; Schousboe, Arne; Waagepetersen, Helle S

    2009-07-01

    Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-(13)C]beta-hydroxybutyrate to that of [1,6-(13)C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate-glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of (13)C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-(13)C]beta-hydroxybutyrate as opposed to [1,6-(13)C]glucose. Our results suggest that the change in aspartate-glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate-aspartate shuttle activity in neurons using beta-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only beta-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing beta-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate-aspartate shuttle.

  19. 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

  20. 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.

  1. Hypothalamic glucose sensing: making ends meet

    Directory of Open Access Journals (Sweden)

    Vanessa eRouth

    2014-12-01

    Full Text Available The neuroendocrine system governs essential survival and homeostatic functions. For example, growth is needed for development. Thermoregulation maintains optimal core temperature in a changing environment. Reproduction ensures species survival. Stress and immune responses enable an organism to overcome external and internal threats. The circadian system regulates arousal and sleep such that vegetative and active functions do not overlap. All of these functions require a significant portion of the body’s energy. As the integrator of the neuroendocrine system, the hypothalamus carefully assesses the energy status of the body in order to appropriately partition resources to provide for each system without compromising the others. While doing so the hypothalamus must ensure that adequate glucose levels are preserved for brain function since glucose is the primary fuel of the brain. To this end, the hypothalamus contains specialized glucose sensing neurons which are scattered throughout the nuclei controlling distinct neuroendocrine functions. We hypothesize that these neurons play a key role in enabling the hypothalamus to partition energy to meet these peripheral survival needs without endangering the brain’s glucose supply. The goal of this review is to describe the varied mechanisms underlying glucose sensing in neurons within discrete hypothalamic nuclei. We will then evaluate the way in which peripheral energy status regulates glucose sensitivity. For example, during energy deficit such as fasting specific hypothalamic glucose sensing neurons become sensitized to decreased glucose. This increases the gain of the information relay when glucose availability is a greater concern for the brain. Finally, changes in glucose sensitivity under pathological conditions (e.g., recurrent insulin-hypoglycemia, diabetes will be addressed. The overall goal of this review is to place glucose sensing neurons within the context of hypothalamic control of

  2. BDNF regulates the expression and distribution of vesicular glutamate transporters in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Carlos V Melo

    Full Text Available BDNF is a pro-survival protein involved in neuronal development and synaptic plasticity. BDNF strengthens excitatory synapses and contributes to LTP, presynaptically, through enhancement of glutamate release, and postsynaptically, via phosphorylation of neurotransmitter receptors, modulation of receptor traffic and activation of the translation machinery. We examined whether BDNF upregulated vesicular glutamate receptor (VGLUT 1 and 2 expression, which would partly account for the increased glutamate release in LTP. Cultured rat hippocampal neurons were incubated with 100 ng/ml BDNF, for different periods of time, and VGLUT gene and protein expression were assessed by real-time PCR and immunoblotting, respectively. At DIV7, exogenous application of BDNF rapidly increased VGLUT2 mRNA and protein levels, in a dose-dependent manner. VGLUT1 expression also increased but only transiently. However, at DIV14, BDNF stably increased VGLUT1 expression, whilst VGLUT2 levels remained low. Transcription inhibition with actinomycin-D or α-amanitine, and translation inhibition with emetine or anisomycin, fully blocked BDNF-induced VGLUT upregulation. Fluorescence microscopy imaging showed that BDNF stimulation upregulates the number, integrated density and intensity of VGLUT1 and VGLUT2 puncta in neurites of cultured hippocampal neurons (DIV7, indicating that the neurotrophin also affects the subcellular distribution of the transporter in developing neurons. Increased VGLUT1 somatic signals were also found 3 h after stimulation with BDNF, further suggesting an increased de novo transcription and translation. BDNF regulation of VGLUT expression was specifically mediated by BDNF, as no effect was found upon application of IGF-1 or bFGF, which activate other receptor tyrosine kinases. Moreover, inhibition of TrkB receptors with K252a and PLCγ signaling with U-73122 precluded BDNF-induced VGLUT upregulation. Hippocampal neurons express both isoforms during

  3. Glutamate decarboxylase immunoreactivity and gamma-[3H] aminobutyric acid accumulation within the same neurons in dissociated cell cultures of cerebral cortex

    International Nuclear Information System (INIS)

    Neale, E.A.; Oertel, W.H.; Bowers, L.M.; Weise, V.K.

    1983-01-01

    In order to evaluate the reliability of high affinity [ 3 H]GABA accumulation as a marker for GABAergic neurons, murine cerebral cortical neurons were studied in dissociated cell culture. Cultures which had been incubated in [ 3 H]GABA were stained immunohistochemically for the GABA-synthesizing enzyme, glutamate decarboxylase, fixed with paraformaldehyde, and subsequently processed for radioautography. In mature cultures, there was an 84 to 94% correlation between the presence of the enzyme and [ 3 H]GABA uptake within the same cortical neurons. These data provide direct evidence that those neurons which synthesize GABA are the same neurons which are labeled by high affinity [ 3 H]GABA uptake

  4. Effect of 670-nm Light-Emitting Diode Light On Neuronal Cultures

    Science.gov (United States)

    Wong-Riley, Margaret T. T.; Whelan, Harry T.

    2002-01-01

    Light close to and within the near infrared range has documented benefits for promoting wound healing in human and animal studies. Our preliminary results using light-emitting diodes (LEDs) in this range have also demonstrated two-to five-fold increases in growth-phase-specific DNA synthesis in normal fibroblasts, muscle cells, osteoblasts, and mucosal epithelial cells in tissue cultures. However, the mechanisms of action of such light on cells are poorly understood. We hypothesized that the therapeutic effects of such light result from the stimulation of cellular events associated with increases in cytochrome oxidase activity. As a first step in testing our hypothesis, we subjected primary neuronal cultures to impulse blockade by tetrodotoxin (TTX), a voltage-dependent sodium channel blocker, and applied LED light at 670 nm to determine if it could partially or fully reverse the reduction of cytochrome oxidase activity by TTX. The wavelength and parameters were previously tested to be beneficial for wound healing.

  5. E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

    Science.gov (United States)

    Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

    2017-10-02

    Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

  6. Chlorpyrifos exerts opposing effects on axonal and dendritic growth in primary neuronal cultures

    International Nuclear Information System (INIS)

    Howard, Angela S.; Bucelli, Robert; Jett, David A.; Bruun, Donald; Yang, Dongren; Lein, Pamela J.

    2005-01-01

    Evidence that children are widely exposed to organophosphorus pesticides (OPs) and that OPs cause developmental neurotoxicity in animal models raises significant concerns about the risks these compounds pose to the developing human nervous system. Critical to assessing this risk is identifying specific neurodevelopmental events targeted by OPs. Observations that OPs alter brain morphometry in developing rodents and inhibit neurite outgrowth in neural cell lines suggest that OPs perturb neuronal morphogenesis. However, an important question yet to be answered is whether the dysmorphogenic effect of OPs reflects perturbation of axonal or dendritic growth. We addressed this question by quantifying axonal and dendritic growth in primary cultures of embryonic rat sympathetic neurons derived from superior cervical ganglia (SCG) following in vitro exposure to chlorpyrifos (CPF) or its metabolites CPF-oxon (CPFO) and trichloropyridinol (TCP). Axon outgrowth was significantly inhibited by CPF or CPFO, but not TCP, at concentrations ≥0.001 μM or 0.001 nM, respectively. In contrast, all three compounds enhanced BMP-induced dendritic growth. Acetylcholinesterase was inhibited only by the highest concentrations of CPF (≥1 μM) and CPFO (≥1 nM); TCP had no effect on this parameter. In summary, these compounds perturb neuronal morphogenesis via opposing effects on axonal and dendritic growth, and both effects are independent of acetylcholinesterase inhibition. These findings have important implications for current risk assessment practices of using acetylcholinesterase inhibition as a biomarker of OP neurotoxicity and suggest that OPs may disrupt normal patterns of neuronal connectivity in the developing nervous system

  7. Live imaging of dense-core vesicles in primary cultured hippocampal neurons.

    Science.gov (United States)

    Kwinter, David M; Silverman, Michael A; Kwinter, David; Michael, Silverman

    2009-05-29

    Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is

  8. Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons.

    Science.gov (United States)

    Bak, Lasse K; Schousboe, Arne; Sonnewald, Ursula; Waagepetersen, Helle S

    2006-10-01

    Glucose is the primary energy substrate for the adult mammalian brain. However, lactate produced within the brain might be able to serve this purpose in neurons. In the present study, the relative significance of glucose and lactate as substrates to maintain neurotransmitter homeostasis was investigated. Cultured cerebellar (primarily glutamatergic) neurons were superfused in medium containing [U-13C]glucose (2.5 mmol/L) and lactate (1 or 5 mmol/L) or glucose (2.5 mmol/L) and [U-13C]lactate (1 mmol/L), and exposed to pulses of N-methyl-D-aspartate (300 micromol/L), leading to synaptic activity including vesicular release. The incorporation of 13C label into intracellular lactate, alanine, succinate, glutamate, and aspartate was determined by mass spectrometry. The metabolism of [U-13C]lactate under non-depolarizing conditions was high compared with that of [U-13C]glucose; however, it decreased significantly during induced depolarization. In contrast, at both concentrations of extracellular lactate, the metabolism of [U-13C]glucose was increased during neuronal depolarization. The role of glucose and lactate as energy substrates during vesicular release as well as transporter-mediated influx and efflux of glutamate was examined using preloaded D-[3H]aspartate as a glutamate tracer and DL-threo-beta-benzyloxyaspartate to inhibit glutamate transporters. The results suggest that glucose is essential to prevent depolarization-induced reversal of the transporter (efflux), whereas vesicular release was unaffected by the choice of substrate. In conclusion, the present study shows that glucose is a necessary substrate to maintain neurotransmitter homeostasis during synaptic activity and that synaptic activity does not induce an upregulation of lactate metabolism in glutamatergic neurons.

  9. Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

    Science.gov (United States)

    Yamamoto, H; Karasawa, J; Sagi, N; Takahashi, S; Horikomi, K; Okuyama, S; Nukada, T; Sora, I; Yamamoto, T

    2001-08-03

    Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In

  10. Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

    International Nuclear Information System (INIS)

    Zeng, Xiang Jun; Yu, Shan Ping; Zhang, Like; Wei, Ling

    2010-01-01

    The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca 2+ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.

  11. Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

    Science.gov (United States)

    Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

    2017-08-01

    CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

  12. Transcriptomic Analysis of Ciguatoxin-Induced Changes in Gene Expression in Primary Cultures of Mice Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Juan Andrés Rubiolo

    2018-05-01

    Full Text Available Ciguatoxins are polyether marine toxins that act as sodium channel activators. These toxins cause ciguatera, one of the most widespread nonbacterial forms of food poisoning, which presents several symptoms in humans including long-term neurological alterations. Earlier work has shown that both acute and chronic exposure of primary cortical neurons to synthetic ciguatoxin CTX3C have profound impacts on neuronal function. Thus, the present work aimed to identify relevant neuronal genes and metabolic pathways that could be altered by ciguatoxin exposure. To study the effect of ciguatoxins in primary neurons in culture, we performed a transcriptomic analysis using whole mouse genome microarrays, for primary cortical neurons exposed during 6, 24, or 72 h in culture to CTX3C. Here, we have shown that the effects of the toxin on gene expression differ with the exposure time. The results presented here have identified several relevant genes and pathways related to the effect of ciguatoxins on neurons and may assist in future research or even treatment of ciguatera. Moreover, we demonstrated that the effects of the toxin on gene expression were exclusively consequential of its action as a voltage-gated sodium channel activator, since all the effects of CTX3C were avoided by preincubation of the neurons with the sodium channel blocker tetrodotoxin.

  13. Indirect evidence for decreased hypothalamic somatostatinergic tone in anorexia nervosa

    DEFF Research Database (Denmark)

    Støving, R K; Andersen, M; Flyvbjerg, A

    2002-01-01

    in the central feeding mechanism in anorexia nervosa (AN). Peripheral administration of pyridostigmine (PD) minimizes the release of hypothalamic SRIH. DESIGN: To study the influence of hypothalamic somatostatinergic inhibition on the exaggerated somatotroph responsiveness to GHRH in patients with severe AN, two...... indirectly to greater SRIH withdrawal and greater GHRH release in anorexia nervosa. Moreover, hypothalamic SRIH activity seems to be inversely related to cortisol levels, indirectly supporting the hypothesis that SRIH and CRH neuronal activity are inversely related in anorexia nervosa. Leptin, which...... is believed to act on hypothalamic feeding mechanisms, seems to be positively related to SRIH activity. Finally, the present data demonstrate that the potentiating effect of pyridostigmine in anorexia nervosa is related to body mass index and increases upon weight gain, suggesting that the low...

  14. Slice cultures of the imprinting-relevant forebrain area MNH of the domestic chick: quantitative characterization of neuronal morphology.

    Science.gov (United States)

    Hofmann, H; Braun, K

    1995-05-26

    The persistence of morphological features of neurons in slice cultures of the imprinting-relevant forebrain area MNH (mediorostral neostriatum and hyperstriatum ventrale) of the domestic chick was analysed at 7, 14, 21 and 28 days in vitro. After having been explanted and kept in culture the neurons in vitro have larger soma areas, longer and more extensively branched dendritic trees and lower spine frequencies compared to the neurons in vivo. During the analyzed culturing period, the parameters soma area, total and mean dendritic length, number of dendrites, number of dendritic nodes per dendrite and per neuron as well as the spine densities in different dendritic segments showed no significant differences between early and late periods. Highly correlated in every age group were the total dendritic length and the number of dendritic nodes per neuron, indicating regular ramification during dendritic growth. Since these morphological parameters remain stable during the first 4 weeks in vitro, this culture system may provide a suitable model to investigate experimentally induced morphological changes.

  15. ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

    Science.gov (United States)

    Gomes, P; Chevalier, J; Boesmans, W; Roosen, L; van den Abbeel, V; Neunlist, M; Tack, J; Vanden Berghe, P

    2009-08-01

    The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

  16. Neuroglobin overexpression inhibits oxygen-glucose deprivation-induced mitochondrial permeability transition pore opening in primary cultured mouse cortical neurons.

    Science.gov (United States)

    Yu, Zhanyang; Liu, Ning; Li, Yadan; Xu, Jianfeng; Wang, Xiaoying

    2013-08-01

    Neuroglobin (Ngb) is an endogenous neuroprotective molecule against hypoxic/ischemic brain injury, but the underlying mechanisms remain largely undefined. Our recent study revealed that Ngb can bind to voltage-dependent anion channel (VDAC), a regulator of mitochondria permeability transition (MPT). In this study we examined the role of Ngb in MPT pore (mPTP) opening following oxygen-glucose deprivation (OGD) in primary cultured mouse cortical neurons. Co-immunoprecipitation (Co-IP) and immunocytochemistry showed that the binding between Ngb and VDAC was increased after OGD compared to normoxia, indicating the OGD-enhanced Ngb-VDAC interaction. Ngb overexpression protected primary mouse cortical neurons from OGD-induced neuronal death, to an extent comparable to mPTP opening inhibitor, cyclosporine A (CsA) pretreatment. We further measured the role of Ngb in OGD-induced mPTP opening using Ngb overexpression and knockdown approaches in primary cultured neurons, and recombinant Ngb exposure to isolated mitochondria. Same as CsA pretreatment, Ngb overexpression significantly reduced OGD-induced mPTP opening markers including mitochondria swelling, mitochondrial NAD(+) release, and cytochrome c (Cyt c) release in primary cultured neurons. Recombinant Ngb incubation significantly reduced OGD-induced NAD(+) release and Cyt c release from isolated mitochondria. In contrast, Ngb knockdown significantly increased OGD-induced neuron death, and increased OGD-induced mitochondrial NAD(+) release and Cyt c release as well, and these outcomes could be rescued by CsA pretreatment. In summary, our results demonstrated that Ngb overexpression can inhibit OGD-induced mPTP opening in primary cultured mouse cortical neurons, which may be one of the molecular mechanisms of Ngb's neuroprotection. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Resveratrol protects primary cortical neuron cultures from transient oxygen-glucose deprivation by inhibiting MMP-9.

    Science.gov (United States)

    Gao, Dakuan; Huang, Tao; Jiang, Xiaofan; Hu, Shijie; Zhang, Lei; Fei, Zhou

    2014-06-01

    It was recently shown that resveratrol exerts neuroprotective effects against cerebral ischemia in mice. The aim of the present study was to further confirm these effects in in vitro primary cortical neuron cultures with transient oxygen-glucose deprivation (OGD), and to investigate whether these effects are due to the inhibition of matrix metalloproteinase-9 (MMP-9) and of cell apoptosis. Neuronal primary cultures of cerebral cortex were prepared from BALB/c mice embryos (13-15 days). Cells from 14- to 16-day cultures were subjected to OGD for 3 h, followed by 21 h of reoxygenation to simulate transient ischemia. Different doses of resveratrol were added into the culture medium during the simulation of transient ischemia. The effect of the extracellular signal-regulated kinase (ERK) inhibitor U0126 was studied by adding U0126 (5 µg/µl, 4 µl) into the culture medium during transient ischemia; as a control, we used treatment of cells with 50 µM of resveratrol. Cell viability was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Cell apoptosis was assessed by flow cytometry. The effects of resveratrol on the expression of MMP-9 were analyzed by western blotting and reverse transcription-polymerase chain reaction (RT-PCR), while the levels of ERK, phosphorylated (p)-ERK, cleaved caspase-3, Bax and Bcl-2 were measured by western blotting. The results of the MTT assay showed that cell viability is significantly reduced by transient OGD. OGD induced cell apoptosis, the expression of Bax and the activation of caspase-3 and ERK, inhibited the expression of Bcl-2 and increased the expression of MMP-9, while these effects were reversed by treatment with resveratrol. The therapeutic efficacy of resveratrol was shown to be dose-dependent, with the most suitable dose range determined at 50-100 µM. Treatment with U0126 inhibited MMP-9 and Bax expression and caspase-3 activation, while it further promoted the

  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. 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.

  20. 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.

  1. Glutamate stimulates the formation of N-acylphosphatidylethanolamine in cortical neurons in culture

    DEFF Research Database (Denmark)

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

    1995-01-01

    The formation of anandamide (N-arachidonoylethanolamine), N-acylethanolamine, and N-acylphosphatidylethanolamine was studied in primary cultures of rat cortical neurons. The cells were incubated for 22 h with [C]ethanolamine, [U-C]arachidonic acid, [H]arachidonic acid, [P]phosphate, [C]stearic acid......-acylethanolamine. Compound I could be labelled with [C]stearic acid and [H]myristic acid, but not with [H]- or [C]arachidonic acid. Exogenous [H]anandamide was metabolised with a t( 1/2 ) of 2.6 h. The labelling of the two compounds identified as N-acylethanolamine and N-acylphosphatidylethanolamine were more pronounced......, or [H]myristic acid. The lipids from the cells and media were separated by thin layer chromatography. [C]Ethanolamine labelling revealed two compounds (I and II), which on different thin layer chromatography systems migrated as N-acylethanolamine (0.06-0.55% of total radioactivity) and N...

  2. Functional MRI of human hypothalamic responses following glucose ingestion

    NARCIS (Netherlands)

    Smeets, P.A.M.; Graaf, C. de; Stafleu, A.; Osch, M.J.P. van; Grond, J. van der

    2005-01-01

    The hypothalamus is intimately involved in the regulation of food intake, integrating multiple neural and hormonal signals. Several hypothalamic nuclei contain glucose-sensitive neurons, which play a crucial role in energy homeostasis. Although a few functional magnetic resonance imaging (fMRI)

  3. Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures

    OpenAIRE

    Goudriaan, Andrea; Camargo, Nutabi; Carney, Karen E.; Oliet, Stéphane H. R.; Smit, August B.; Verheijen, Mark H. G.

    2014-01-01

    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 expedited technique for separation of neuro...

  4. Growth dynamics explain the development of spatiotemporal burst activity of young cultured neuronal networks in detail.

    Directory of Open Access Journals (Sweden)

    Taras A Gritsun

    Full Text Available A typical property of isolated cultured neuronal networks of dissociated rat cortical cells is synchronized spiking, called bursting, starting about one week after plating, when the dissociated cells have sufficiently sent out their neurites and formed enough synaptic connections. This paper is the third in a series of three on simulation models of cultured networks. Our two previous studies [26], [27] have shown that random recurrent network activity models generate intra- and inter-bursting patterns similar to experimental data. The networks were noise or pacemaker-driven and had Izhikevich-neuronal elements with only short-term plastic (STP synapses (so, no long-term potentiation, LTP, or depression, LTD, was included. However, elevated pre-phases (burst leaders and after-phases of burst main shapes, that usually arise during the development of the network, were not yet simulated in sufficient detail. This lack of detail may be due to the fact that the random models completely missed network topology .and a growth model. Therefore, the present paper adds, for the first time, a growth model to the activity model, to give the network a time dependent topology and to explain burst shapes in more detail. Again, without LTP or LTD mechanisms. The integrated growth-activity model yielded realistic bursting patterns. The automatic adjustment of various mutually interdependent network parameters is one of the major advantages of our current approach. Spatio-temporal bursting activity was validated against experiment. Depending on network size, wave reverberation mechanisms were seen along the network boundaries, which may explain the generation of phases of elevated firing before and after the main phase of the burst shape.In summary, the results show that adding topology and growth explain burst shapes in great detail and suggest that young networks still lack/do not need LTP or LTD mechanisms.

  5. Mesenchymal stem cells support neuronal fiber growth in an organotypic brain slice co-culture model.

    Science.gov (United States)

    Sygnecka, Katja; Heider, Andreas; Scherf, Nico; Alt, Rüdiger; Franke, Heike; Heine, Claudia

    2015-04-01

    Mesenchymal stem cells (MSCs) have been identified as promising candidates for neuroregenerative cell therapies. However, the impact of different isolation procedures on the functional and regenerative characteristics of MSC populations has not been studied thoroughly. To quantify these differences, we directly compared classically isolated bulk bone marrow-derived MSCs (bulk BM-MSCs) to the subpopulation Sca-1(+)Lin(-)CD45(-)-derived MSCs(-) (SL45-MSCs), isolated by fluorescence-activated cell sorting from bulk BM-cell suspensions. Both populations were analyzed with respect to functional readouts, that are, frequency of fibroblast colony forming units (CFU-f), general morphology, and expression of stem cell markers. The SL45-MSC population is characterized by greater morphological homogeneity, higher CFU-f frequency, and significantly increased nestin expression compared with bulk BM-MSCs. We further quantified the potential of both cell populations to enhance neuronal fiber growth, using an ex vivo model of organotypic brain slice co-cultures of the mesocortical dopaminergic projection system. The MSC populations were cultivated underneath the slice co-cultures without direct contact using a transwell system. After cultivation, the fiber density in the border region between the two brain slices was quantified. While both populations significantly enhanced fiber outgrowth as compared with controls, purified SL45-MSCs stimulated fiber growth to a larger degree. Subsequently, we analyzed the expression of different growth factors in both cell populations. The results show a significantly higher expression of brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor in the SL45-MSCs population. Altogether, we conclude that MSC preparations enriched for primary MSCs promote neuronal regeneration and axonal regrowth, more effectively than bulk BM-MSCs, an effect that may be mediated by a higher BDNF secretion.

  6. Hypothalamic glioma masquerading as craniopharyngioma

    Directory of Open Access Journals (Sweden)

    Sameer Vyas

    2013-01-01

    Full Text Available Hypothalamic glioma account for 10-15% of supratentorial tumors in children. They usually present earlier (first 5 years of age than craniopharyngioma. Hypothalamic glioma poses a diagnostic dilemma with craniopharyngioma and other hypothalamic region tumors, when they present with atypical clinical or imaging patterns. Neuroimaging modalities especially MRI plays a very important role in scrutinizing the lesions in the hypothalamic region. We report a case of a hypothalamic glioma masquerading as a craniopharyngioma on imaging along with brief review of both the tumors.

  7. DIDS prevents ischemic membrane degradation in cultured hippocampal neurons by inhibiting matrix metalloproteinase release.

    Directory of Open Access Journals (Sweden)

    Matthew E Pamenter

    Full Text Available During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra. Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs, which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed. DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA. Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS

  8. A Non-Destructive Culturing and Cell Sorting Method for Cardiomyocytes and Neurons Using a Double Alginate Layer

    Science.gov (United States)

    Terazono, Hideyuki; Kim, Hyonchol; Hayashi, Masahito; Hattori, Akihiro; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

    2012-01-01

    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. PMID:22870332

  9. 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.

  10. Generation of Otic Sensory Neurons from Mouse Embryonic Stem Cells in 3D Culture

    Directory of Open Access Journals (Sweden)

    Michael Perny

    2017-12-01

    Full Text Available The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs. The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs toward otic sensory neurons (OSNs. The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP signaling and concomitant inhibition of transforming growth factor beta (TGFβ signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2. Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF and neurotrophin-3 (NT-3 was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression

  11. Lipopolysaccharide (LPS Accumulates in Neocortical Neurons of Alzheimer’s Disease (AD Brain and Impairs Transcription in Human Neuronal-Glial Primary Co-cultures

    Directory of Open Access Journals (Sweden)

    Yuhai Zhao

    2017-12-01

    Full Text Available Several independent laboratories have recently reported the detection of bacterial nucleic acid sequences or bacterial-derived neurotoxins, such as highly inflammatory lipopolysaccharide (LPS, within Alzheimer’s disease (AD affected brain tissues. Whether these bacterial neurotoxins originate from the gastrointestinal (GI tract microbiome, a possible brain microbiome or some dormant pathological microbiome is currently not well understood. Previous studies indicate that the co-localization of pro-inflammatory LPS with AD-affected brain cell nuclei suggests that there may be a contribution of this neurotoxin to genotoxic events that support inflammatory neurodegeneration and failure in homeostatic gene expression. In this report we provide evidence that in sporadic AD, LPS progressively accumulates in neuronal parenchyma and appears to preferentially associate with the periphery of neuronal nuclei. Run-on transcription studies utilizing [α-32P]-uridine triphosphate incorporation into newly synthesized total RNA further indicates that human neuronal-glial (HNG cells in primary co-culture incubated with LPS exhibit significantly reduced output of DNA transcription products. These studies suggest that in AD LPS may impair the efficient readout of neuronal genetic information normally required for the homeostatic operation of brain cell function and may contribute to a progressive disruption in the read-out of genetic information.

  12. Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

    Science.gov (United States)

    Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

    Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Zolpidem, a selective GABA(A) receptor alpha1 subunit agonist, induces comparable Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat

    DEFF Research Database (Denmark)

    Kiss, Alexander; Søderman, Andreas; Bundzikova, Jana

    2006-01-01

    Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immun...

  14. 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...

  15. Role of GluR2 expression in AMPA-induced toxicity in cultured murine cerebral cortical neurons

    DEFF Research Database (Denmark)

    Jensen, Jette Bisgaard; Lund, Trine Meldgaard; Timmermann, Daniel B.

    2001-01-01

    of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during...

  16. /sup 125/I-labelled tetanus toxin as a neuronal marker in tissue cultures derived from embryonic CNS

    Energy Technology Data Exchange (ETDEWEB)

    Dimpfel, W; Neale, J H; Habermann, E [Giessen Univ. (F.R. Germany). Pharmakologisches Inst.; National Inst. of Child Health and Human Development, Bethesda, Md. (USA). Behavioural Biology Branch)

    1975-01-01

    Primary cultures derived from embryonic mouse brain and spinal cord were exposed to /sup 125/I-labelled tetanus toxin and subjected to autoradioraphy. Cells with neuronal, but not glial, morphology selectively accumulated the toxin. The distribution of the grains over these cells and their processes was not uniform, discrete processes showing heavier labelling.

  17. Inhibition of calcineurin inhibits the desensitization of capsaicin evoked currents in cultured dorsal root ganglion neurones from adult rats

    NARCIS (Netherlands)

    Docherty, RJ; Yeats, JC; Bevan, S; Boddeke, HWGM

    Capsaicin activates a non-specific cation conductance in mammalian sensory neurones. If capsaicin is applied continuously or repeatedly then there is a progressive decline in responsiveness. We have studied the mechanism of this desensitization using electrophysiological methods in cultured dorsal

  18. Neuroprotective effects of oxysophocarpine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion.

    Science.gov (United States)

    Zhu, Qing-Luan; Li, Yu-Xiang; Zhou, Ru; Ma, Ning-Tian; Chang, Ren-Yuan; Wang, Teng-Fei; Zhang, Yi; Chen, Xiao-Ping; Hao, Yin-Ju; Jin, Shao-Ju; Ma, Lin; Du, Juan; Sun, Tao; Yu, Jian-Qiang

    2014-08-01

    Oxysophocarpine (OSC), a quinolizidine alkaloid extracted from leguminous plants of the genus Robinia, is traditionally used for various diseases including neuronal disorders. This study investigated the protective effects of OSC on neonatal rat primary-cultured hippocampal neurons were injured by oxygen-glucose deprivation and reperfusion (OGD/RP). Cultured hippocampal neurons were exposed to OGD for 2 h followed by a 24 h RP. OSC (1, 2, and 5 μmol/L) and nimodipine (Nim) (12 μmol/L) were added to the culture after OGD but before RP. The cultures of the control group were not exposed to OGD/RP. MTT and LDH assay were used to evaluate the protective effects of OSC. The concentration of intracellular-free calcium [Ca(2+)]i and mitochondrial membrane potential (MMP) were determined to evaluate the degree of neuronal damage. Morphologic changes of neurons following OGD/RP were observed with a microscope. The expression of caspase-3 and caspase-12 mRNA was examined by real-time quantitative PCR. The IC50 of OSC was found to be 100 μmol/L. Treatment with OSC (1, 2, and 5 μmol/L) attenuated neuronal damage (p < 0.001), with evidence of increased cell viability (p < 0.001) and decreased cell morphologic impairment. Furthermore, OSC increased MMP (p < 0.001), but it inhibited [Ca(2+)]i (p < 0.001) elevation in a dose-dependent manner at OGD/RP. OSC (5 μmol/L) also decreased the expression of caspase-3 (p < 0.05) and caspase-12 (p < 0.05). The results suggested that OSC has significant neuroprotective effects that can be attributed to inhibiting endoplasmic reticulum (ER) stress-induced apoptosis.

  19. EFFECTS OF THALLIUM SALTS ON NEURONAL MITOCHONDRIA IN ORGANOTYPIC CORD-GANGLIA-MUSCLE COMBINATION CULTURES

    Science.gov (United States)

    Spencer, Peter S.; Peterson, Edith R.; Madrid A., Ricardo; Raine, Cedric S.

    1973-01-01

    A functionally coupled organotypic complex of cultured dorsal root ganglia, spinal cord peripheral nerve, and muscle has been employed in an experimental approach to the investigation of the neurotoxic effects of thallium. Selected cultures, grown for up to 12 wk in vitro, were exposed to thallous salts for periods ranging up to 4 days. Cytopathic effects were first detected after 2 h of exposure with the appearance of considerably enlarged mitochondria in axons of peripheral nerve fibers. With time, the matrix space of these mitochondria became progressively swollen, transforming the organelle into an axonal vacuole bounded by the original outer mitochondrial membrane. Coalescence of adjacent axonal vacuoles produced massive internal axon compartments, the membranes of which were shown by electron microprobe mass spectrometry to have an affinity for thallium. Other axoplasmic components were displaced within a distended but intact axolemma. The resultant fiber swelling caused myelin retraction from nodes of Ranvier but no degeneration. Impulses could still propagate along the nerve fibers throughout the time course of the experiment. Comparable, but less severe changes were seen in dorsal root ganglion neurons and in central nerve fibers. Other cell types showed no mitochondrial change. It is uncertain how these findings relate to the neurotoxic effects of thallium in vivo, but a sensitivity of the nerve cell and especially its axon to thallous salts is indicated. PMID:4125375

  20. Intracellular recording, sensory field mapping, and culturing identified neurons in the leech, Hirudo medicinalis.

    Science.gov (United States)

    Titlow, Josh; Majeed, Zana R; Nicholls, John G; Cooper, Robin L

    2013-11-04

    The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

  1. Hypothalamic control of energy and glucose metabolism.

    Science.gov (United States)

    Sisley, Stephanie; Sandoval, Darleen

    2011-09-01

    The central nervous system (CNS), generally accepted to regulate energy homeostasis, has been implicated in the metabolic perturbations that either cause or are associated with obesity. Normally, the CNS receives hormonal, metabolic, and neuronal input to assure adequate energy levels and maintain stable energy homeostasis. Recent evidence also supports that the CNS uses these same inputs to regulate glucose homeostasis and this aspect of CNS regulation also becomes impaired in the face of dietary-induced obesity. This review focuses on the literature surrounding hypothalamic regulation of energy and glucose homeostasis and discusses how dysregulation of this system may contribute to obesity and T2DM.

  2. Glutamate reduces glucose utilization while concomitantly enhancing AQP9 and MCT2 expression in cultured rat hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Fabio eTescarollo

    2014-08-01

    Full Text Available The excitatory neurotransmitter glutamate has been reported to have a major impact on brain energy metabolism. Using primary cultures of rat hippocampal neurons, we observed that glutamate reduces glucose utilization in this cell type, suggesting alteration in mitochondrial oxidative metabolism. The aquaglyceroporin AQP9 and the monocarboxylate transporter MCT2, two transporters for oxidative energy substrates, appear to be present in mitochondria of these neurons. Moreover, they not only co-localize but they interact with each other as they were found to co-immunoprecipitate from hippocampal neuron homogenates. Exposure of cultured hippocampal neurons to glutamate 100 µM for 1 hour led to enhanced expression of both AQP9 and MCT2 at the protein level without any significant change at the mRNA level. In parallel, a similar increase in the protein expression of LDHA was evidenced without an effect on the mRNA level. These data suggest that glutamate exerts an influence on neuronal energy metabolism likely through a regulation of the expression of some key mitochondrial proteins.

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

    Science.gov (United States)

    le Feber, Joost; Postma, Wybren; de Weerd, Eddy; Weusthof, Marcel; Rutten, Wim L. C.

    2015-01-01

    Cultured neurons on multi electrode arrays (MEAs) have been widely used to study various aspects of neuronal (network) functioning. A possible drawback of this approach is the lack of structure in these networks. At the single cell level, several solutions have been proposed to enable directed connectivity, and promising results were obtained. At the level of connected sub-populations, a few attempts have been made with promising results. First assessment of the designs' functionality, however, suggested room for further improvement. We designed a two chamber MEA aiming to create a unidirectional connection between the networks in both chambers (“emitting” and “receiving”). To achieve this unidirectionality, all interconnecting channels contained barbs that hindered axon growth in the opposite direction (from receiving to emitting chamber). Visual inspection showed that axons predominantly grew through the channels in the promoted direction. This observation was confirmed by spontaneous activity recordings. Cross-correlation between the signals from two electrodes inside the channels suggested signal propagation at ≈2 m/s from emitting to receiving chamber. Cross-correlation between the firing patterns in both chambers indicated that most correlated activity was initiated in the emitting chamber, which was also reflected by a significantly lower fraction of partial bursts (i.e., a one-chamber-only burst) in the emitting chamber. Finally, electrical stimulation in the emitting chamber induced a fast response in that chamber, and a slower response in the receiving chamber. Stimulation in the receiving chamber evoked a fast response in that chamber, but no response in the emitting chamber. These results confirm the predominantly unidirectional nature of the connecting channels from emitting to receiving chamber. PMID:26578869

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

    Directory of Open Access Journals (Sweden)

    Joost eLe Feber

    2015-11-01

    Full Text Available 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 structure in these networks. At the single cell level, several solutions have been proposed to ena-ble directed connectivity, and promising results were obtained. At the level of connected sub-populations, a few attempts have been made with promising results. First assessment of the de-signs’ functionality, however, suggested room for further improvement.We designed a two chamber MEA aiming to create a unidirectional connection between the net-works in both chambers (‘emitting’ and ‘receiving’. To achieve this unidirectionality, all inter-connecting channels contained barbs that hindered axon growth in the opposite direction (from receiving to emitting chamber. Visual inspection showed that axons predominantly grew through the channels in the promoted direction . This observation was confirmed by spontaneous activity recordings. Cross-correlation between the signals from two electrodes inside the channels suggested signal propagation at ≈2 m/s from emitting to receiving chamber. Cross-correlation between the firing patterns in both chambers indicated that most correlated activity was initiated in the emitting chamber, which was also reflected by a significantly lower fraction of partial bursts (e. a one-chamber-only burst in the emitting chamber. Finally, electrical stimulation in the emitting chamber induced a fast response in that chamber, and a slower response in the receiving chamber. Stimulation in the receiving chamber evoked a fast response in that chamber, but no response in the emitting chamber. These results confirm the predominantly unidirectional nature of the connecting channels from emitting to receiving chamber.

  5. Phase transition approach to bursting in neuronal cultures: quorum percolation models

    Science.gov (United States)

    Monceau, P.; Renault, R.; Métens, S.; Bottani, S.; Fardet, T.

    2017-10-01

    The Quorum Percolation model has been designed in the context of neurobiology to describe bursts of activity occurring in neuronal cultures from the point of view of statistical physics rather than from a dynamical synchronization approach. It is based upon information propagation on a directed graph with a threshold activation rule; this leads to a phase diagram which exhibits a giant percolation cluster below some critical value mC of the excitability. We describe the main characteristics of the original model and derive extensions according to additional relevant biological features. Firstly, we investigate the effects of an excitability variability on the phase diagram and show that the percolation transition can be destroyed by a sufficient amount of such a disorder; we stress the weakly averaging character of the order parameter and show that connectivity and excitability can be seen as two overlapping aspects of the same reality. Secondly, we elaborate a discrete time stochastic model taking into account the decay originating from ionic leakage through the membrane of neurons and synaptic depression; we give evidence that the decay softens and shifts the transition, and conjecture than decay destroys the transition in the thermodynamical limit. We were able to develop mean-field theories associated with each of the two effects; we discuss the framework of their agreement with Monte Carlo simulations. It turns out that the the critical point mC from which information on the connectivity of the network can be inferred is affected by each of these additional effects. Lastly, we show how dynamical simulations of bursts with an adaptive exponential integrateand- fire model can be interpreted in terms of Quorum Percolation. Moreover, the usefulness of the percolation model including the set of sophistication we investigated can be extended to many scientific fields involving information propagation, such as the spread of rumors in sociology, ethology, ecology.

  6. A spatially resolved network spike in model neuronal cultures reveals nucleation centers, circular traveling waves and drifting spiral waves.

    Science.gov (United States)

    Paraskevov, A V; Zendrikov, D K

    2017-03-23

    We show that in model neuronal cultures, where the probability of interneuronal connection formation decreases exponentially with increasing distance between the neurons, there exists a small number of spatial nucleation centers of a network spike, from where the synchronous spiking activity starts propagating in the network typically in the form of circular traveling waves. The number of nucleation centers and their spatial locations are unique and unchanged for a given realization of neuronal network but are different for different networks. In contrast, if the probability of interneuronal connection formation is independent of the distance between neurons, then the nucleation centers do not arise and the synchronization of spiking activity during a network spike occurs spatially uniform throughout the network. Therefore one can conclude that spatial proximity of connections between neurons is important for the formation of nucleation centers. It is also shown that fluctuations of the spatial density of neurons at their random homogeneous distribution typical for the experiments in vitro do not determine the locations of the nucleation centers. The simulation results are qualitatively consistent with the experimental observations.

  7. Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures

    NARCIS (Netherlands)

    Goudriaan, A.; Camargo, N.K.; Carney, K.E.; Oliet, S.H.R.; Smit, A.B.; Verheijen, M.H.G.

    2014-01-01

    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

  8. Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants.

    Directory of Open Access Journals (Sweden)

    Timo Schomann

    Full Text Available Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs are promising candidates for this type of therapy, because they (1 have migratory properties, enabling migration after transplantation, (2 can differentiate into sensory neurons and glial cells, and (3 can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP. Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 μm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair.

  9. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    International Nuclear Information System (INIS)

    Kim, Hyun-Suk; Guo, Chunlu; Thompson, Eric L.; Jiang, Yanlin; Kelley, Mark R.; Vasko, Michael R.; Lee, Suk-Hee

    2015-01-01

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons

  10. APE1, the DNA base excision repair protein, regulates the removal of platinum adducts in sensory neuronal cultures by NER

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyun-Suk [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Guo, Chunlu; Thompson, Eric L. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Jiang, Yanlin [Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Kelley, Mark R. [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States); Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Department of Pediatrics and Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Vasko, Michael R. [Department of Pharmacology and Toxicology, Indianapolis, IN 46202 (United States); Lee, Suk-Hee, E-mail: slee@iu.edu [Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202 (United States)

    2015-09-15

    Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24 h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.

  11. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    Science.gov (United States)

    Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

    2012-01-01

    Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

  12. Computed tomography in hypothalamic hamartoma

    International Nuclear Information System (INIS)

    Mori, Koreaki; Takeuchi, Juji; Hanakita, Junya; Handa, Hajime; Nakano, Yoshihisa.

    1981-01-01

    Two cases of hypothalamic hamartoma were reported. Hypothalamic hamartoma is a rate tumor. The onset of symptoms is in infancy and early childhood. Clinical symptoms are composed of convulsive seizures, laughing spells and precocious puberty. CT finding of hypothalamic hamartoma is a mass in the suprasellar and interpeduncular cisterns which has the same density as the surrounding normal brain. The mass is not enhanced by injection of the contrast material and is easily differentiated from other masses in the suprasellar region. (author)

  13. MR appearance of hypothalamic hamartoma

    Energy Technology Data Exchange (ETDEWEB)

    Hahn, F.J.; Leibrock, L.G.; Huseman, C.A.; Makos, M.M.

    1988-02-01

    Hypothalamic hamartoma is the most common detectable cerebral lesion causing precocious puberty. Two histologically confirmed cases were studied by computerized tomography (CT) and magnetic resonance (MR) imaging. T2 weighted, sagittal MR images were superior to CT in delineating the tumor from surrounding grey matter. The lesion was isointense to grey matter on T1 weighted images allowing exclusion of other hypothalamic tumors. MR will undoubtedly become the imaging modality of choice in the detection of hypothalamic hamartoma.

  14. 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.

  15. Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons.

    Science.gov (United States)

    Bak, Lasse K; Obel, Linea F; Walls, Anne B; Schousboe, Arne; Faek, Sevan A A; Jajo, Farah S; Waagepetersen, Helle S

    2012-04-05

    We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca2+ during activation cause influx of Ca2+ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate-aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca2+ (i.e. independent of synaptic activity) on neuronal energy metabolism employing 13C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca2+ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca2+ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca2+-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca2+ signalling.

  16. Live-Cell, Label-Free Identification of GABAergic and Non-GABAergic Neurons in Primary Cortical Cultures Using Micropatterned Surface

    Science.gov (United States)

    Kono, Sho; Kushida, Takatoshi; Hirano-Iwata, Ayumi; Niwano, Michio; Tanii, Takashi

    2016-01-01

    Excitatory and inhibitory neurons have distinct roles in cortical dynamics. Here we present a novel method for identifying inhibitory GABAergic neurons from non-GABAergic neurons, which are mostly excitatory glutamatergic neurons, in primary cortical cultures. This was achieved using an asymmetrically designed micropattern that directs an axonal process to the longest pathway. In the current work, we first modified the micropattern geometry to improve cell viability and then studied the axon length from 2 to 7 days in vitro (DIV). The cell types of neurons were evaluated retrospectively based on immunoreactivity against GAD67, a marker for inhibitory GABAergic neurons. We found that axons of non-GABAergic neurons grow significantly longer than those of GABAergic neurons in the early stages of development. The optimal threshold for identifying GABAergic and non-GABAergic neurons was evaluated to be 110 μm at 6 DIV. The method does not require any fluorescence labelling and can be carried out on live cells. The accuracy of identification was 98.2%. We confirmed that the high accuracy was due to the use of a micropattern, which standardized the development of cultured neurons. The method promises to be beneficial both for engineering neuronal networks in vitro and for basic cellular neuroscience research. PMID:27513933

  17. Mangiferin Upregulates Glyoxalase 1 Through Activation of Nrf2/ARE Signaling in Central Neurons Cultured with High Glucose.

    Science.gov (United States)

    Liu, Yao-Wu; Cheng, Ya-Qin; Liu, Xiao-Li; Hao, Yun-Chao; Li, Yu; Zhu, Xia; Zhang, Fan; Yin, Xiao-Xing

    2017-08-01

    Mangiferin, a natural C-glucoside xanthone, has anti-inflammatory, anti-oxidative, neuroprotective actions. Our previous study showed that mangiferin could attenuate diabetes-associated cognitive impairment of rats by enhancing the function of glyoxalase 1 (Glo-1) in brain. The aim of this study was to investigate whether Glo-1 upregulation by mangiferin in central neurons exposed to chronic high glucose may be related to activation of Nrf2/ARE pathway. Compared with normal glucose (25 mmol/L) culture, Glo-1 protein, mRNA, and activity levels were markedly decreased in primary hippocampal and cerebral cortical neurons cultured with high glucose (50 mmol/L) for 72 h, accompanied by the declined Nrf2 nuclear translocation and protein expression of Nrf2 in cell nucleus, as well as protein expression and mRNA level of γ-glutamylcysteine synthetase (γ-GCS) and superoxide dismutase activity, target genes of Nrf2/ARE signaling. Nonetheless, high glucose cotreating with mangiferin or sulforaphane, a typical inducer of Nrf2 activation, attenuated the above changes in both central neurons. In addition, mangiferin and sulforaphane significantly prevented the formation of advanced glycation end-products (AGEs) reflecting Glo-1 activity, while elevated the level of glutathione, a cofactor of Glo-1 activity and production of γ-GCS, in high glucose cultured central neurons. These findings demonstrated that Glo-1 was greatly downregulated in central neurons exposed to chronic high glucose, which is expected to lead the formation of AGEs and oxidative stress damages. We also proved that mangiferin enhanced the function of Glo-1 under high glucose condition by inducing activation of Nrf2/ARE signaling pathway.

  18. IL-10 Promotes Neurite Outgrowth and Synapse Formation in Cultured Cortical Neurons after the Oxygen-Glucose Deprivation via JAK1/STAT3 Pathway.

    Science.gov (United States)

    Chen, Hongbin; Lin, Wei; Zhang, Yixian; Lin, Longzai; Chen, Jianhao; Zeng, Yongping; Zheng, Mouwei; Zhuang, Zezhong; Du, Houwei; Chen, Ronghua; Liu, Nan

    2016-07-26

    As a classic immunoregulatory and anti-inflammatory cytokine, interleukin-10 (IL-10) provides neuroprotection in cerebral ischemia in vivo or oxygen-glucose deprivation (OGD)-induced injury in vitro. However, it remains blurred whether IL-10 promotes neurite outgrowth and synapse formation in cultured primary cortical neurons after OGD injury. In order to evaluate its effect on neuronal apoptosis, neurite outgrowth and synapse formation, we administered IL-10 or IL-10 neutralizing antibody (IL-10NA) to cultured rat primary cortical neurons after OGD injury. We found that IL-10 treatment activated the Janus kinase 1 (JAK1)/signal transducers and activators of transcription 3 (STAT3) signaling pathway. Moreover, IL-10 attenuated OGD-induced neuronal apoptosis by down-regulating the Bax expression and up-regulating the Bcl-2 expression, facilitated neurite outgrowth by increasing the expression of Netrin-1, and promoted synapse formation in cultured primary cortical neurons after OGD injury. These effects were partly abolished by JAK1 inhibitor GLPG0634. Contrarily, IL-10NA produced opposite effects on the cultured cortical neurons after OGD injury. Taken together, our findings suggest that IL-10 not only attenuates neuronal apoptosis, but also promotes neurite outgrowth and synapse formation via the JAK1/STAT3 signaling pathway in cultured primary cortical neurons after OGD injury.

  19. [The hypothalamic-pituitary-adrenal axis and depressive disorder: recent progress].

    Science.gov (United States)

    Kunugi, Hiroshi; Hori, Hiroaki; Numakawa, Tadahiro; Ota, Miho

    2012-08-01

    Depression is a stress-induced disorder and there is compelling evidence for the involvement of hypothalamic-pituitary-adrenal (HPA) axis abnormalities in the disease. Chronic hyperactivity of the HPA axis and resultant excessive glucocorticoid (hypercortisolism) may be causal to depression. We demonstrated that the dexamethasone (DEX)/CRH test is a sensitive state-dependent marker to monitor HPA axis abnormalities. Restoration from HPA axis abnormalities occurs with clinical responses to treatment. Brain-derived neurotrophic factor (BDNF) has also been implicated in depression. We found that glucocorticoid (DEX) suppresses BDNF-induced dendrite outgrowth and synaptic formation via blocking the MAPK pathway in early-developing cultured hippocampal neurons. Furthermore, we demonstrated that glucocorticoid receptor (GR) and TrkB (a specific receptor of BDNF) interact and that DEX acutely suppresses BDNF-induced glutamate release by affecting the PLC-gamma pathway in cultured cortical neurons, indicating a mechanism underlying the effect of excessive glucocorticoid on BDNF function and resultant damage in cortical neurons. In a macroscopic view using magnetic resonance imaging (MRI), we found that individuals with hypercortisolism detected by the DEX/CRH test demonstrated volume loss in gray matter and reduced neural network assessed with diffusion tensor imaging in several brain regions. Finally, we observed that individuals with hypocortisolism detected by the DEX/CRH test tend to present more distress symptoms, maladaptive coping styles, and schizotypal personality traits than their counterparts, which points to the important role of hypocortisolism as well as hypercortisolism in depression spectrum disorders.

  20. Role of laser fluence in protein synthesis of cultured DRG neurons following low-level laser irradiation

    Science.gov (United States)

    Zheng, Liqin; Qiu, Caimin; Wang, Yuhua; Zeng, Yixiu; Yang, Hongqin; Zhang, Yanding; Xie, Shusen

    2014-11-01

    Low-level lasers have been used to relieve pain in clinical for many years. But the mechanism is not fully clear. In animal models, nitric oxide (NO) has been reported involving in the transmission and modulation of nociceptive signals. So the objective of this study was to establish whether low-level laser with different fluence could stimulate the production of nitric oxide synthese (NOS), which produces NO in cultured primary dorsal root ganglion neurons (DRG neurons). The primary DRG neurons were isolated from healthy Sprague Dawley rats (8-12 weeks of age) and spread on 35 mm culture dishes specially used for confocal microscopy. 24 hours after spreading, cells were irradiated with 658 nm laser for two consecutive days at the energy density of 20, 40, 60 and 80 mJ·cm-2 respectively. Control groups were not exposed to the laser, but were kept under the same conditions as the irradiated ones. The synthesis of NOS after laser irradiation was detected by immunofluorescence assay, and the changes of NOS were evaluated using confocal microscopy and Image J software. The results showed that all the laser fluence could promote the production of NOS in DRG neurons, especially the 60 mJ·cm-2 . These results demonstrated that low-level laser irradiation could modify protein synthesis in a dose- or fluence- dependent manner, and indicated that low-level laser irradiation might achieve the analgesic effect through modulation of NO production.

  1. Caloric restriction mimetic 2-deoxyglucose maintains cytoarchitecture and reduces tau phosphorylation in primary culture of mouse hippocampal pyramidal neurons.

    Science.gov (United States)

    Bele, M S; Gajare, K A; Deshmukh, A A

    2015-06-01

    Typical form of neurons is crucially important for their functions. This is maintained by microtubules and associated proteins like tau. Hyperphosphorylation of tau is a major concern in neurodegenerative diseases. Glycogen synthase kinase3β (GSK3β) and cyclin-dependent protein kinase 5 (Cdk5) are the enzymes that govern tau phosphorylation. Currently, efforts are being made to target GSK3β and Cdk5 as possible therapeutic avenues to control tau phosphorylation and treat neurodegenerative diseases related to taupathies. In a number of studies, caloric restriction mimetic 2-deoxyglucose (C6H12O5) was found to be beneficial in improving the brain functions. However, no reports are available on the effect of 2-deoxyglucose 2-DG on tau phosphorylation. In the present study, hippocampal pyramidal neurons from E17 mouse embryos were isolated and cultured on poly-L-lysine-coated coverslips. Neurons from the experimental group were treated with 10 mM 2-deoxyglucose. The treatment of 2-DG resulted in healthier neuronal morphology in terms of significantly lower number of cytoplasmic vacuoles, little or no membrane blebbings, maintained axon hillock and intact neurites. There were decreased immunofluorescence signals for GSK3β, pTau at Ser262, Cdk5 and pTau at Ser235 suggesting decreased tau phosphorylation, which was further confirmed by Western blotting. The results indicate the beneficial effects of 2-DG in controlling the tau phosphorylation and maintaining the healthy neuronal cytoarchitecture.

  2. Hypothalamic mTOR signaling regulates food intake.

    Science.gov (United States)

    Cota, Daniela; Proulx, Karine; Smith, Kathi A Blake; Kozma, Sara C; Thomas, George; Woods, Stephen C; Seeley, Randy J

    2006-05-12

    The mammalian Target of Rapamycin (mTOR) protein is a serine-threonine kinase that regulates cell-cycle progression and growth by sensing changes in energy status. We demonstrated that mTOR signaling plays a role in the brain mechanisms that respond to nutrient availability, regulating energy balance. In the rat, mTOR signaling is controlled by energy status in specific regions of the hypothalamus and colocalizes with neuropeptide Y and proopiomelanocortin neurons in the arcuate nucleus. Central administration of leucine increases hypothalamic mTOR signaling and decreases food intake and body weight. The hormone leptin increases hypothalamic mTOR activity, and the inhibition of mTOR signaling blunts leptin's anorectic effect. Thus, mTOR is a cellular fuel sensor whose hypothalamic activity is directly tied to the regulation of energy intake.

  3. Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons

    DEFF Research Database (Denmark)

    Bak, Lasse Kristoffer; Johansen, Maja L.; Schousboe, Arne

    2012-01-01

    Synthesis of neuronal glutamate from a-ketoglutarate for neurotransmission necessitates an amino group nitrogen donor; however, it is not clear which amino acid(s) serves this role. Thus, the ability of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, to act as amino...... group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 µ......]valine was able to maintain the amount of vesicular glutamate during synaptic activity. This indicates that, among the BCAAs, only valine supports the increased need for synthesis of vesicular glutamate. © 2012 Wiley Periodicals, Inc....

  4. Neuronal differentiation and long-term culture of the human neuroblastoma line SH-SY5Y.

    Science.gov (United States)

    Constantinescu, R; Constantinescu, A T; Reichmann, H; Janetzky, B

    2007-01-01

    Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder in industrialized countries. Present cell culture models for PD rely on either primary cells or immortal cell lines, neither of which allow for long-term experiments on a constant population, a crucial requisite for a realistic model of slowly progressing neurodegenerative diseases. We differentiated SH-SY5Y human dopaminergic neuroblastoma cells to a neuronal-like state in a perfusion culture system using a combination of retinoic acid and mitotic inhibitors. The cells could be cultivated for two months without the need for passage. We show, by various means, that the differentiated cells exhibit, at the molecular level, many neuronal properties not characteristic to the starting line. This approach opens the possibility to develop chronic models, in which the effect of perturbations and putative counteracting strategies can be monitored over long periods of time in a quasi-stable cell population.

  5. High level over-expression of different NCX isoforms in HEK293 cell lines and primary neuronal cultures is protective following oxygen glucose deprivation.

    Science.gov (United States)

    Cross, Jane L; Boulos, Sherif; Shepherd, Kate L; Craig, Amanda J; Lee, Sharon; Bakker, Anthony J; Knuckey, Neville W; Meloni, Bruno P

    2012-07-01

    In this study we have assessed sodium-calcium exchanger (NCX) protein over-expression on cell viability in primary rat cortical neuronal and HEK293 cell cultures when subjected to oxygen-glucose deprivation (OGD). In cortical neuronal cultures, NCX2 and NCX3 over-expression was achieved using adenoviral vectors, and following OGD increased neuronal survival from ≈20% for control vector treated cultures to ≈80% for both NCX isoforms. In addition, we demonstrated that NCX2 and NCX3 over-expression in cortical neuronal cultures enables neurons to maintain intracellular calcium at significantly lower levels than control vector treated cultures when exposed to high (9mM) extracellular calcium challenge. Further assessment of NCX activity during OGD was performed using HEK293 cell lines generated to over-express NCX1, NCX2 or NCX3 isoforms. While it was shown that NCX isoform expression differed considerably in the different HEK293 cell lines, high levels of NCX over-expression was associated with increased resistance to OGD. Taken together, our findings show that high levels of NCX over-expression increases neuronal and HEK293 cell survival following OGD, improves calcium management in neuronal cultures and provides additional support for NCX as a therapeutic target to reduce ischemic brain injury. Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  6. 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

  7. Hypocretin/orexin loss changes the hypothalamic immune response.

    Science.gov (United States)

    Tanaka, Susumu; Takizawa, Nae; Honda, Yoshiko; Koike, Taro; Oe, Souichi; Toyoda, Hiromi; Kodama, Tohru; Yamada, Hisao

    2016-10-01

    Hypocretin, also known as orexin, maintains the vigilance state and regulates various physiological processes, such as arousal, sleep, food intake, energy expenditure, and reward. Previously, we found that when wild-type mice and hypocretin/ataxin-3 littermates (which are depleted of hypothalamic hypocretin-expressing neurons postnatally) were administered lipopolysaccharide (LPS), the two genotypes exhibited significant differences in their sleep/wake cycle, including differences in the degree of increase in sleep periods and in recovery from sickness behaviour. In the present study, we examined changes in the hypothalamic vigilance system and in the hypothalamic expression of inflammatory factors in response to LPS in hypocretin/ataxin-3 mice. Peripheral immune challenge with LPS affected the hypothalamic immune response and vigilance states. This response was altered by the loss of hypocretin. Hypocretin expression was inhibited after LPS injection in both hypocretin/ataxin-3 mice and their wild-type littermates, but expression was completely abolished only in hypocretin/ataxin-3 mice. Increases in the number of histidine decarboxylase (HDC)-positive cells and in Hdc mRNA expression were found in hypocretin/ataxin-3 mice, and this increase was suppressed by LPS. Hypocretin loss did not impact the change in expression of hypothalamic inflammatory factors in response to LPS, except for interferon gamma and colony stimulating factor 3. The number of c-Fos-positive/HDC-positive cells in hypocretin/ataxin-3 mice administered LPS injections was elevated, even during the rest period, in all areas, suggesting that there is an increase in the activity of histaminergic neurons in hypocretin/ataxin-3 mice following LPS injection. Taken together, our results suggest a novel role for hypocretin in the hypothalamic response to peripheral immune challenge. Our findings contribute to the understanding of the pathophysiology of narcolepsy. Copyright © 2016 Elsevier Inc. All

  8. MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction.

    Directory of Open Access Journals (Sweden)

    Christian Cortés-Campos

    Full Text Available Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes.

  9. Investigation of the quenched surfaces of visibly luminescent macro/nanoporous silicon under the exposure of typical neuron culture media

    International Nuclear Information System (INIS)

    Unal, B.

    2015-01-01

    In this research paper, the quenching effects of visible photoluminescence of porous silicon relevant to doping types under an exposure of culture media such as Dulbecco's Modified Eagle's Medium and Phosphate-Buffered Saline have been studied extensively in order to realize the application of a cell culture growth technique for porous silicon, in which biocompatibility is directly based on its size-dependent structures and morphologies. This could restrain the combination of either macro or micro-/nano-dimensional silicon morphologies by stain-etching single crystalline Si surfaces. The dopant-related quenching effect of well-known neuron culture media over visible photoluminescent porous silicon surface is found to be quite obvious for the two culture media mentioned above. Scanning electron microscope images of the cultured neuron cells over porous Si show how they have been linked to, and communicated with, each other, and directed along porous channels, fabricated by a photo lithographic technique. (authors)

  10. Target recognition and synapse formation by ciliary-ganglion neurons in tissue culture

    NARCIS (Netherlands)

    Stevens, W.F.; Slaaf, D.W.; Hooisma, J.; Magchielse, T.; Meeter, E.

    1978-01-01

    A less complicated source of neurons suitable for this type of studies is the parasympathetic ciliary ganglion. In the pigeon and in the chick this ganglion is known to contain only two classes of neurons, both of which are cholinoceptive and cholinergic and that innervate the muscle fibres of the

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

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    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...... mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living...

  12. The Relevance of AgRP Neuron-Derived GABA Inputs to POMC Neurons Differs for Spontaneous and Evoked Release

    OpenAIRE

    Rau, Andrew R.; Hentges, Shane T.

    2017-01-01

    Hypothalamic agouti-related peptide (AgRP) neurons potently stimulate food intake, whereas proopiomelanocortin (POMC) neurons inhibit feeding. Whether AgRP neurons exert their orexigenic actions, at least in part, by inhibiting anorexigenic POMC neurons remains unclear. Here, the connectivity between GABA-releasing AgRP neurons and POMC neurons was examined in brain slices from male and female mice. GABA-mediated spontaneous IPSCs (sIPSCs) in POMC neurons were unaffected by disturbing GABA re...

  13. Toxicity evaluation of new agricultural fungicides in primary cultured cortical neurons.

    Science.gov (United States)

    Regueiro, Jorge; Olguín, Nair; Simal-Gándara, Jesús; Suñol, Cristina

    2015-07-01

    Fungicides are crucial for food protection as well as for the production of crops of suitable quality and quantity to provide a viable economic return. Like other pesticides, fungicides are widely sprayed on agricultural land, especially in wine-growing areas, from where they can move-off after application. Furthermore, residues of these agrochemicals can remain on crops after harvest and even after some food processing operations, being a major exposure pathway. Although a relatively low toxicity has been claimed for this kind of compounds, information about their neurotoxicity is still scarce. In the present study, nine fungicides recently approved for agricultural uses in the EU - ametoctradin, boscalid, cyazofamid, dimethomorph, fenhexamid, kresoxim-methyl, mepanipyrim, metrafenone and pyraclostrobin - have been evaluated for their toxicity in primary cultured mouse cortical neurons. Exposure to 0.1-100µM for 7 days in vitro resulted in a dose-dependent toxicity in the MTT cell viability assay. Strobilurin fungicides kresoxim-methyl (KR) and pyraclostrobin (PY) were the most neurotoxic compounds (lethal concentration 50 were in the low micromolar and nanomolar levels, respectively) causing a rapid raise in intracellular calcium [Ca(2+)]i and strong depolarization of mitochondrial membrane potential. KR- and PY-induced cell death was reversed by the calcium channels blockers MK-801 and verapamil, suggesting that calcium entry through NMDA receptors and voltage-operated calcium channels are involved in KR- and PY-induced neurotoxicity. These results highlight the need for further evaluation of their neurotoxic effects in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Hypothalamic Circuits for Predation and Evasion.

    Science.gov (United States)

    Li, Yi; Zeng, Jiawei; Zhang, Juen; Yue, Chenyu; Zhong, Weixin; Liu, Zhixiang; Feng, Qiru; Luo, Minmin

    2018-02-21

    The interactions between predator and prey represent some of the most dramatic events in nature and constitute a matter of life and death for both sides. The hypothalamus has been implicated in driving predation and evasion; however, the exact hypothalamic neural circuits underlying these behaviors remain poorly defined. Here, we demonstrate that inhibitory and excitatory projections from the mouse lateral hypothalamus (LH) to the periaqueductal gray (PAG) in the midbrain drive, respectively, predation and evasion. LH GABA neurons were activated during predation. Optogenetically stimulating PAG-projecting LH GABA neurons drove strong predatory attack, and inhibiting these cells reversibly blocked predation. In contrast, LH glutamate neurons were activated during evasion. Stimulating PAG-projecting LH glutamate neurons drove evasion and inhibiting them impeded predictive evasion. Therefore, the seemingly opposite behaviors of predation and evasion are tightly regulated by two dissociable modular command systems within a single neural projection from the LH to the PAG. VIDEO ABSTRACT. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. 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.

  16. Bilateral descending hypothalamic projections to the spinal trigeminal nucleus caudalis in rats.

    Directory of Open Access Journals (Sweden)

    Khaled Abdallah

    Full Text Available Several lines of evidence suggest that the hypothalamus is involved in trigeminal pain processing. However, the organization of descending hypothalamic projections to the spinal trigeminal nucleus caudalis (Sp5C remains poorly understood. Microinjections of the retrograde tracer, fluorogold (FG, into the Sp5C, in rats, reveal that five hypothalamic nuclei project to the Sp5C: the paraventricular nucleus, the lateral hypothalamic area, the perifornical hypothalamic area, the A11 nucleus and the retrochiasmatic area. Descending hypothalamic projections to the Sp5C are bilateral, except those from the paraventricular nucleus which exhibit a clear ipsilateral predominance. Moreover, the density of retrogradely FG-labeled neurons in the hypothalamus varies according to the dorso-ventral localization of the Sp5C injection site. There are much more labeled neurons after injections into the ventrolateral part of the Sp5C (where ophthalmic afferents project than after injections into its dorsomedial or intermediate parts (where mandibular and maxillary afferents, respectively, project. These results demonstrate that the organization of descending hypothalamic projections to the spinal dorsal horn and Sp5C are different. Whereas the former are ipsilateral, the latter are bilateral. Moreover, hypothalamic projections to the Sp5C display somatotopy, suggesting that these projections are preferentially involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in rats. Therefore, our results suggest that the control of trigeminal and spinal dorsal horn processing of nociceptive information by hypothalamic neurons is different and raise the question of the role of bilateral, rather than unilateral, hypothalamic control.

  17. Hypothalamic BOLD response to glucose intake and hypothalamic volume are similar in anorexia nervosa and healthy control subjects

    Directory of Open Access Journals (Sweden)

    Anna M Van Opstal

    2015-05-01

    Full Text Available Background. Inconsistent findings about the neurobiology of Anorexia Nervosa (AN hinder the development of effective treatments for this severe mental disorder. Therefore the need arises for elucidation of neurobiological factors involved in the pathophysiology of AN. The hypothalamus plays a key role in the neurobiological processes that govern food intake and energy homeostasis, processes that are disturbed in anorexia nervosa (AN. The present study will assess the hypothalamic response to energy intake and the hypothalamic structure in patients with AN and healthy controls. Methods. 10 women aged 18-30 years diagnosed with AN and 11 healthy, lean (BMI <23 kg/m2 women in the same age range were recruited. We used functional magnetic resonance imaging (MRI to determine function of the hypothalamus in response to glucose. Structural MRI was used to determine differences in hypothalamic volume and local grey volume using manual segmentation and voxel-based morphometry.Results. No differences were found in hypothalamic volume and neuronal activity in response to a glucose load between the patients and controls. Whole brain structural analysis showed a significant decrease in grey matter volume in the cingulate cortex in the AN patients, bilaterally.Conclusions. We argue that in spite of various known changes in the hypothalamus the direct hypothalamic response to glucose intake is similar in AN patients and healthy controls.

  18. Differential induction of heme oxygenase and other stress proteins in cultured hippocampal astrocytes and neurons by inorganic lead

    International Nuclear Information System (INIS)

    Cabell, Leigh; Ferguson, Charles; Luginbill, Deana; Kern, Marcey; Weingart, Adam; Audesirk, Gerald

    2004-01-01

    We examined the effects of exposure to inorganic lead (Pb 2+ ) on the induction of stress proteins in cultured hippocampal neurons and astrocytes, with particular emphasis on the induction of heme oxygenase-1 (HO-1). In radiolabeled neuronal cultures, Pb 2+ exposure had no significant effect on the synthesis of any protein at any concentration (up to 250 μM) or duration of exposure (up to 4 days). In radiolabeled astrocyte cultures, however, Pb 2+ exposure (100 nM to 100 μM; 1-4 days) increased synthesis of proteins with approximate molecular weights of 23, 32, 45, 57, 72, and 90 kDa. Immunoblot experiments showed that Pb 2+ exposure (100 nM to 10 μM, 1-14 days) induces HO-1 synthesis in astrocytes, but not in neurons; this is probably the 32-kDa protein. The other heme oxygenase isoform, HO-2, is present in both neurons and astrocytes, but is not inducible by Pb 2+ at concentrations up to 100 μM. HO-1 can be induced by a variety of stimuli. We found that HO-1 induction in astrocytes is increased by combined exposure to Pb 2+ and many other stresses, including heat, nitric oxide, H 2 O 2 , and superoxide. One of the stimuli that may induce HO-1 is oxidative stress. Lead exposure causes oxidative stress in many cell types, including astrocytes. Induction of HO-1 by Pb 2+ is reduced by the hydroxyl radical scavengers dimethylthiourea (DMTU) and mannitol, but not by inhibitors of calmodulin, calmodulin-dependent protein kinases, protein kinase C, or extracellular signal-regulated kinases (ERK). Therefore, we conclude that oxidative stress is an important mechanism by which Pb 2+ induces HO-1 synthesis in astrocytes

  19. Dopamine elevates intracellular zinc concentration in cultured rat embryonic cortical neurons through the cAMP-nitric oxide signaling cascade.

    Science.gov (United States)

    Hung, Hui-Hsing; Kao, Lung-Sen; Liu, Pei-Shan; Huang, Chien-Chang; Yang, De-Ming; Pan, Chien-Yuan

    2017-07-01

    Zinc ion (Zn 2+ ), the second most abundant transition metal after iron in the body, is essential for neuronal activity and also induces toxicity if the concentration is abnormally high. Our previous results show that exposure of cultured cortical neurons to dopamine elevates intracellular Zn 2+ concentrations ([Zn 2+ ] i ) and induces autophagosome formation but the mechanism is not clear. In this study, we characterized the signaling pathway responsible for the dopamine-induced elevation of [Zn 2+ ] i and the effect of [Zn 2+ ] i in modulating the autophagy in cultured rat embryonic cortical neurons. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a membrane-permeable Zn 2+ chelator, could rescue the cell death and suppress the autophagosome puncta number induced by dopamine. Dopamine treatment increased the lipidation level of the endogenous microtubule-associated protein 1A/1B-light chain 3 (LC3 II), an autophagosome marker. TPEN added 1h before, but not after, dopamine treatment suppressed the dopamine-induced elevation of LC3 II level. Inhibitors of the dopamine D1-like receptor, protein kinase A (PKA), and NOS suppressed the dopamine-induced elevation of [Zn 2+ ] i . PKA activators and NO generators directly increased [Zn 2+ ] i in cultured neurons. Through cell fractionation, proteins with m.w. values between 5 and 10kD were found to release Zn 2+ following NO stimulation. In addition, TPEN pretreatment and an inhibitor against PKA could suppress the LC3 II level increased by NO and dopamine, respectively. Therefore, our results demonstrate that dopamine-induced elevation of [Zn 2+ ] i is mediated by the D1-like receptor-PKA-NO pathway and is important in modulating the cell death and autophagy. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Demonstration of extensive GABA synthesis in the small population of GAD positive neurons in cerebellar cultures by the use of pharmacological tools

    DEFF Research Database (Denmark)

    Sonnewald, Ursula; Kortner, Trond M; Qu, Hong

    2006-01-01

    by labeling from [U-(13)C]glutamine added on day 7. Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 microM AOAA, GABA synthesis from [U-(13)C]glutamine was not affected, indicating that transaminases are not involved in GABA...... that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period....

  1. The Effect of a Short-term Glucose Deprivation on Neuron Net Functioning of Hippocampus Primary Culture on a Multi-electrode Matrix

    OpenAIRE

    Vedunova M.V.; Korotchenko S.A.; Balashova A.N.; Isakova A.O.; Khaspekov L.G.; Kazantsev V.B.; Mukhina I.V.

    2011-01-01

    There has been studied the effect of a short-term glucose deprivation on neuron net functioning of hippocampus primary culture developing within 32 days on a multi-electrode matrix MED64 (Alpha MED Sciences Company, Japan) in an early and remote periods after deprivation. A short-term glucose deprivation (20 min) has been shown to result in the increase of electrobiological activity of neuron net of hippocampus primary culture, with the cascade of metabolic reactions being activated leading t...

  2. Effects of continuous hypoxia on energy metabolism in cultured cerebro-cortical neurons.

    Science.gov (United States)

    Malthankar-Phatak, Gauri H; Patel, Anant B; Xia, Ying; Hong, Soonsun; Chowdhury, Golam M I; Behar, Kevin L; Orina, Isaac A; Lai, James C K

    2008-09-10

    Mechanisms underlying hypoxia-induced neuronal adaptation have not been fully elucidated. In the present study we investigated glucose metabolism and the activities of glycolytic and TCA cycle enzymes in cerebro-cortical neurons exposed to hypoxia (3 days in 1% of O2) or normoxia (room air). Hypoxia led to increased activities of LDH (194%), PK (90%), and HK (24%) and decreased activities of CS (15%) and GDH (34%). Neurons were incubated with [1-(13)C]glucose for 45 and 120 min under normoxic or hypoxic (120 min only) conditions and 13C enrichment determined in the medium and cell extract using 1H-{13C}-NMR. In hypoxia-treated neurons [3-(13)C]lactate release into the medium was 428% greater than in normoxia-treated controls (45-min normoxic incubation) and total flux through lactate was increased by 425%. In contrast glucose oxidation was reduced significantly in hypoxia-treated neurons, even when expressed relative to total cellular protein, which correlated with the reduced activities of the measured mitochondrial enzymes. The results suggest that surviving neurons adapt to prolonged hypoxia by up-regulation of glycolysis and down-regulation of oxidative energy metabolism, similar to certain other cell types. The factors leading to adaptation and survival for some neurons but not others remain to be determined.

  3. Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

    Science.gov (United States)

    Nordström, Viola; Willershäuser, Monja; Herzer, Silke; Rozman, Jan; von Bohlen Und Halbach, Oliver; Meldner, Sascha; Rothermel, Ulrike; Kaden, Sylvia; Roth, Fabian C; Waldeck, Clemens; Gretz, Norbert; de Angelis, Martin Hrabě; Draguhn, Andreas; Klingenspor, Martin; Gröne, Hermann-Josef; Jennemann, Richard

    2013-01-01

    Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.

  4. Green Tea Polyphenols Attenuated Glutamate Excitotoxicity via Antioxidative and Antiapoptotic Pathway in the Primary Cultured Cortical Neurons

    Directory of Open Access Journals (Sweden)

    Lin Cong

    2016-01-01

    Full Text Available Green tea polyphenols are a natural product which has antioxidative and antiapoptotic effects. It has been shown that glutamate excitotoxicity induced oxidative stress is linked to neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. In this study we explored the neuroprotective effect of green teen polyphenols against glutamate excitotoxicity in the primary cultured cortical neurons. We found that green tea polyphenols protected against glutamate induced neurotoxicity in the cortical neurons as measured by MTT and TUNEL assays. Green tea polyphenols were then showed to inhibit the glutamate induced ROS release and SOD activity reduction in the neurons. Furthermore, our results demonstrated that green tea polyphenols restored the dysfunction of mitochondrial pro- or antiapoptotic proteins Bax, Bcl-2, and caspase-3 caused by glutamate. Interestingly, the neuroprotective effect of green tea polyphenols was abrogated when the neurons were incubated with siBcl-2. Taken together, these results demonstrated that green tea polyphenols protected against glutamate excitotoxicity through antioxidative and antiapoptotic pathways.

  5. 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.

  6. Nano-Ampere Stimulation Window for Cultured Neurons on Micro-Electrode Arrays

    National Research Council Canada - National Science Library

    Buitenweg, J

    2001-01-01

    From experiments, it appears to be possible to stimulate a neuron by depolarization of the lower membrane patch, the sealing part of the membrane, using a nano-ampere current through the extracellular...

  7. Hypothalamic FTO is associated with the regulation of energy intake not feeding reward

    Directory of Open Access Journals (Sweden)

    Radomska Katarzyna J

    2009-10-01

    Full Text Available Abstract Background Polymorphism in the FTO gene is strongly associated with obesity, but little is known about the molecular bases of this relationship. We investigated whether hypothalamic FTO is involved in energy-dependent overconsumption of food. We determined FTO mRNA levels in rodent models of short- and long-term intake of palatable fat or sugar, deprivation, diet-induced increase in body weight, baseline preference for fat versus sugar as well as in same-weight animals differing in the inherent propensity to eat calories especially upon availability of diverse diets, using quantitative PCR. FTO gene expression was also studied in organotypic hypothalamic cultures treated with anorexigenic amino acid, leucine. In situ hybridization (ISH was utilized to study FTO signal in reward- and hunger-related sites, colocalization with anorexigenic oxytocin, and c-Fos immunoreactivity in FTO cells at initiation and termination of a meal. Results Deprivation upregulated FTO mRNA, while leucine downregulated it. Consumption of palatable diets or macronutrient preference did not affect FTO expression. However, the propensity to ingest more energy without an effect on body weight was associated with lower FTO mRNA levels. We found that 4-fold higher number of FTO cells displayed c-Fos at meal termination as compared to initiation in the paraventricular and arcuate nuclei of re-fed mice. Moreover, ISH showed that FTO is present mainly in hunger-related sites and it shows a high degree of colocalization with anorexigenic oxytocin. Conclusion We conclude that FTO mRNA is present mainly in sites related to hunger/satiation control; changes in hypothalamic FTO expression are associated with cues related to energy intake rather than feeding reward. In line with that, neurons involved in feeding termination express FTO. Interestingly, baseline FTO expression appears linked not only with energy intake but also energy metabolism.

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

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

    The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system for cultur...... with mass production. The last part of this thesis also includes perspectives on how to expand the latest designed device to facilitate culturing of tissue and co-culturing of cells....

  9. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

    Directory of Open Access Journals (Sweden)

    Hai Zhang

    2011-07-01

    Full Text Available Hypoxia-inducible factor (HIF is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  10. Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J.; Park, Sung-min; Cai, Dongsheng

    2011-01-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance. PMID:21814490

  11. Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture

    OpenAIRE

    Sabado, Virginie; Vienne, Ludovic; Nagoshi, Emi

    2017-01-01

    Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN) generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Droso...

  12. Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid β toxicity

    Directory of Open Access Journals (Sweden)

    Rodriguez-Tebar Alfredo

    2011-02-01

    Full Text Available Abstract Background Amyloid beta (Aβ is the main agent responsible for the advent and progression of Alzheimer's disease. This peptide can at least partially antagonize nerve growth factor (NGF signalling in neurons, which may be responsible for some of the effects produced by Aβ. Accordingly, better understanding the NGF signalling pathway may provide clues as to how to protect neurons from the toxic effects of Aβ. Results We show here that Aβ activates the RhoA GTPase by binding to p75NTR, thereby preventing the NGF-induced activation of protein tyrosine phosphatase 1B (PTP1B that is required for neuron survival. We also show that the inactivation of RhoA GTPase and the activation of PTP1B protect cultured hippocampal neurons against the noxious effects of Aβ. Indeed, either pharmacological inhibition of RhoA with C3 ADP ribosyl transferase or the transfection of cultured neurons with a dominant negative form of RhoA protects cultured hippocampal neurons from the effects of Aβ. In addition, over-expression of PTP1B also prevents the deleterious effects of Aβ on cultured hippocampal neurons. Conclusion Our findings indicate that potentiating the activity of NGF at the level of RhoA inactivation and PTP1B activation may represent a new means to combat the noxious effects of Aβ in Alzheimer's disease.

  13. Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures

    DEFF Research Database (Denmark)

    Bak, Lasse K; Sickmann, Helle M; Schousboe, Arne

    2004-01-01

    The glutamate-glutamine cycle describes the neuronal release of glutamate into the synaptic cleft, astrocytic uptake, and conversion into glutamine, followed by release for use as a neuronal glutamate precursor. This only explains the fate of the carbon atoms, however, and not that of the ammonia....... Recently, a role for alanine has been proposed in transfer of ammonia between glutamatergic neurons and astrocytes, denoted the lactate-alanine shuttle (Waagepetersen et al. [ 2000] J. Neurochem. 75:471-479). The role of alanine in this context has been studied further using cerebellar neuronal cultures...... and corresponding neuronal-astrocytic cocultures. A superfusion paradigm was used to induce repetitively vesicular glutamate release by N-methyl-D-aspartate (NMDA) in the neurons, allowing the relative activity dependency of the lactate-alanine shuttle to be assessed. [(15)N]Alanine (0.2 mM), [2-(15)N]/[5-(15)N...

  14. Folate and S-adenosylmethionine modulate synaptic activity in cultured cortical neurons: acute differential impact on normal and apolipoprotein-deficient mice

    International Nuclear Information System (INIS)

    Serra, Michael; Chan, Amy; Dubey, Maya; Shea, Thomas B; Gilman, Vladimir

    2008-01-01

    Folate deficiency is accompanied by a decline in the cognitive neurotransmitter acetylcholine and a decline in cognitive performance in mice lacking apolipoprotein E (ApoE−/− mice), a low-density lipoprotein that regulates aspects of lipid metabolism. One direct consequence of folate deficiency is a decline in S-adenosylmethionine (SAM). Since dietary SAM supplementation maintains acetylcholine levels and cognitive performance in the absence of folate, we examined herein the impact of folate and SAM on neuronal synaptic activity. Embryonic cortical neurons from mice expressing or lacking ApoE (ApoE+/+ or −/−, respectively) were cultured for 1 month on multi-electrode arrays, and signaling was recorded. ApoE+/+ cultures displayed significantly more frequent spontaneous signals than ApoE−/− cultures. Supplementation with 166 µm SAM (not normally present in culture medium) increased signal frequency and decreased signal amplitude in ApoE+/+ cultures. SAM also increased the frequency of tightly clustered signal bursts. Folate deprivation reversibly reduced signal frequency in ApoE+/+ cultures; SAM supplementation maintained signal frequency despite folate deprivation. These findings support the importance of dietary supplementation with folate and SAM on neuronal health. Supplementation with 166 µm SAM did not alter signaling in ApoE−/− cultures, which may be a reflection of the reduced SAM levels in ApoE−/− mice. The differential impact of SAM on ApoE+/+ and −/− neurons underscores the combined impact of nutritional and genetic deficiencies on neuronal homeostasis. (communication)

  15. Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

    Science.gov (United States)

    Kaneko, Ai; Sankai, Yoshiyuki

    2014-01-01

    The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4) cells/mL (8.9×10(3) cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

  16. Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.

    Directory of Open Access Journals (Sweden)

    Ai Kaneko

    Full Text Available The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×10(4 cells/mL (8.9×10(3 cells/cm2 without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm, greater cell viability (≥30% for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks.

  17. Medical therapy of hypothalamic diseases

    International Nuclear Information System (INIS)

    Werder, K. von; Mueller, O.A.

    1985-01-01

    Hormonal disturbances caused by hypothalamic pathology can be treated effectively by target hormone replacement in the case of failure of glandotropic hormone secretion. Hyposomatotropism in children has to be substituted by parenteral administration of growth hormone. In addition gonadotropins respectively gonadotropin releasing factor have to be given in order to restore fertility in hypothalamic hypogonadism. Posterior pituitary failure can be adequately replaced by administration of analogues of antidiuretic hormone. Hypothalamic pathology causing hypersecretion of anterior pituitary hormones may also be accessable to medical treatment. This pertains particularly to hyperprolactinemia and precocious puberty. However, there is no medical therapy so far for hypothalamic disturbances leading to veterative dysfunction like disturbances of temperature regulation and control of thirst and polyphagia. In this situation symptomatic correction of the abnormality represents the only possibility to keep these patients alive. (Author)

  18. Exogenous α-synuclein hinders synaptic communication in cultured cortical primary rat neurons.

    Science.gov (United States)

    Hassink, G C; Raiss, C C; Segers-Nolten, I M J; van Wezel, R J A; Subramaniam, V; le Feber, J; Claessens, M M A E

    2018-01-01

    Amyloid aggregates of the protein α-synuclein (αS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up αS. Here we aimed to get more insight into the toxicity mechanism associated with high extracellular αS concentrations (50-100 μM). High extracellular αS concentrations resulted in a reduction of the firing rate of the neuronal network by disrupting synaptic transmission, while the neuronal ability to fire action potentials was still intact. Furthermore, many cells developed αS deposits larger than 500 nm within five days, but otherwise appeared healthy. Synaptic dysfunction clearly occurred before the establishment of large intracellular deposits and neuronal death, suggesting that an excessive extracellular αS concentration caused synaptic failure and which later possibly contributed to neuronal death.

  19. Pulsed infrared radiation excites cultured neonatal spiral and vestibular ganglion neurons by modulating mitochondrial calcium cycling.

    Science.gov (United States)

    Lumbreras, Vicente; Bas, Esperanza; Gupta, Chhavi; Rajguru, Suhrud M

    2014-09-15

    Cochlear implants are currently the most effective solution for profound sensorineural hearing loss, and vestibular prostheses are under development to treat bilateral vestibulopathies. Electrical current spread in these neuroprostheses limits channel independence and, in some cases, may impair their performance. In comparison, optical stimuli that are spatially confined may result in a significant functional improvement. Pulsed infrared radiation (IR) has previously been shown to elicit responses in neurons. This study analyzes the response of neonatal rat spiral and vestibular ganglion neurons in vitro to IR (wavelength = 1,863 nm) using Ca(2+) imaging. Both types of neurons responded consistently with robust intracellular Ca(2+) ([Ca(2+)]i) transients that matched the low-frequency IR pulses applied (4 ms, 0.25-1 pps). Radiant exposures of ∼637 mJ/cm(2) resulted in continual neuronal activation. Temperature or [Ca(2+)] variations in the media did not alter the IR-evoked transients, ruling out extracellular Ca(2+) involvement or primary mediation by thermal effects on the plasma membrane. While blockage of Na(+), K(+), and Ca(2+) plasma membrane channels did not alter the IR-evoked response, blocking of mitochondrial Ca(2+) cycling with CGP-37157 or ruthenium red reversibly inhibited the IR-evoked [Ca(2+)]i transients. Additionally, the magnitude of the IR-evoked transients was dependent on ryanodine and cyclopiazonic acid-dependent Ca(2+) release. These results suggest that IR modulation of intracellular calcium cycling contributes to stimulation of spiral and vestibular ganglion neurons. As a whole, the results suggest selective excitation of neurons in the IR beam path and the potential of IR stimulation in future auditory and vestibular prostheses. Copyright © 2014 the American Physiological Society.

  20. Neurochemical and toxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridine to rat serotonin neurons in dissociated cell cultures

    International Nuclear Information System (INIS)

    Friedman, L.K.; Mytilineou, C.

    1990-01-01

    Dissociated cell cultures from the pontine area of embryonic rat brain were used to study the sensitivity of serotonin (5-hydroxy-tryptamine (5-HT)) neurons to the neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine (MPP+). Treatment with MPTP (up to 100 microM) for 7 days did not cause degeneration of 5-HT neurons. A 50% inhibition of [3H]5-HT uptake caused by 100 microM MPTP was a direct effect on the 5-HT uptake carrier, reversed by washing for 7 days. Incubation of cultures with MPTP increased the intraneuronal levels of 5-HT and reduced the levels of 5-hydroxyindoleacetic acid, suggesting a reduction in 5-HT metabolism. MPTP reduced monoamine oxidase activity in the cultures, which probably led to the reduction in 5-HT metabolism. Exposure to MPP+ (0.5-10 microM) for 4 to 7 days decreased [3H]5-HT uptake and induced loss of neurons stained with antibodies against 5-HT. Comparison between 5-HT and dopamine (DA) neurons indicated a differential sensitivity to MPP+ toxicity with DA neurons being more susceptible. Analysis of the competition of MPP+ with the natural substrates for uptake sites of 5-HT and DA neurons demonstrated higher affinity of MPP+ for DA compared to 5-HT neurons. The lower affinity of MPP+ for 5-HT neurons could be responsible for the accumulation of lower MPP+ levels observed in pontine cultures and explain the resistance of 5-HT neurons to this toxin

  1. [Protective effect of pretreatment of Salvia miltiorrhiza Bunge. f. alba plasma against oxygen-glucose deprivation-induced injury of cultured rat hippocampal neurons by inhibiting apoptosis].

    Science.gov (United States)

    Li, Mei-Yi; Zhang, Yan-Bo; Zuo, Huan; Liu, Li-Li; Niu, Jing-Zhong

    2012-02-25

    The present study was to investigate the effect of Salvia miltiorrhiza Bunge. f. alba (SMA) pharmacological pretreatment on apoptosis of cultured hippocampal neurons from neonate rats under oxygen-glucose deprivation (OGD). Cultured hippocampal neurons were randomly divided into five groups (n = 6): normal plasma group, low dose SMA plasma (2.5%) group, middle dose SMA plasma (5%) group, high dose SMA plasma (10%) group and control group. The hippocampal neurons were cultured and treated with plasma from adult Wistar rats intragastrically administered with saline or aqueous extract of SMA. The apoptosis of neurons was induced by glucose-free Earle's solution containing 1 mmol/L Na2S2O4 and labeled by MTT and Annexin V/PI double staining. Moreover, protein expressions of Bcl-2 and Bax were detected by immunofluorescence. The results showed that few apoptotic cells were observed in control group, whereas the number of apoptotic cells was greatly increased in normal plasma group and low dose SMA plasma group. Both middle and high dose SMA plasma could protect cultured hippocampal neurons from apoptosis induced by OGD (P control, normal plasma and low dose SMA plasma groups, middle and high dose SMA plasma groups both showed significantly higher levels of Bcl-2 (P neurons by up-regulating the expression of Bcl-2 and down-regulating the expression of Bax.

  2. [Functional hypothalamic amenorrhea].

    Science.gov (United States)

    Stárka, Luboslav; Dušková, Michaela

    2015-10-01

    Functional hypothalamic amenorrhea (FHA) besides pregnancy and syndrome of polycystic ovary is one of the most common causes of secondary amenorrhea. FHA results from the aberrations in pulsatile gonadotropin-releasing hormone (GnRH) secretion, which in turn causes impairment of the gonadotropins (follicle-stimulating hormone and luteinizing hormone). FHA is a form of the defence of organism in situations where life functions are more important than reproductive function. FHA is reversible; it can be normalized after ceasing the stress situation. There are three types of FHA: weight loss related, stress-related, and exercise-related amenorrhea. The final consequences are complex hormonal changes manifested by profound hypoestrogenism. Additionally, these patients present mild hypercortisolemia, low serum insulin levels, low insulin-like growth factor 1 (IGF-1) and low total triiodothyronine. Women health in this disorder is disturbed in several aspects including the skeletal system, cardiovascular system, and mental problems. Patients manifest a decrease in bone mass density, which is related to an increase in fracture risk. Therefore, osteopenia and osteoporosis are the main long-term complications of FHA. Cardiovascular complications include endothelial dysfunction and abnormal changes in the lipid profile. FHA patients present significantly higher depression and anxiety and also sexual problems compared to healthy subjects.

  3. Accumulation of pyrethroid compounds in primary cultures of rat cortical neurons

    Science.gov (United States)

    Recent studies have demonstrated that lipophilic compounds (e.g. methylmercury, polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs)) rapidly accumulate in cells in culture to concentrations much higher than in the surrounding media. Primary cultures of neur...

  4. Chronic zinc exposure decreases the surface expression of NR2A-containing NMDA receptors in cultured hippocampal neurons.

    Directory of Open Access Journals (Sweden)

    Jia Zhu

    Full Text Available Zinc distributes widely in the central nervous system, especially in the hippocampus, amygdala and cortex. The dynamic balance of zinc is critical for neuronal functions. Zinc modulates the activity of N-methyl-D-aspartate receptors (NMDARs through the direct inhibition and various intracellular signaling pathways. Abnormal NMDAR activities have been implicated in the aetiology of many brain diseases. Sustained zinc accumulation in the extracellular fluid is known to link to pathological conditions. However, the mechanism linking this chronic zinc exposure and NMDAR dysfunction is poorly understood.We reported that chronic zinc exposure reduced the numbers of NR1 and NR2A clusters in cultured hippocampal pyramidal neurons. Whole-cell and synaptic NR2A-mediated currents also decreased. By contrast, zinc did not affect NR2B, suggesting that chronic zinc exposure specifically influences NR2A-containg NMDARs. Surface biotinylation indicated that zinc exposure attenuated the membrane expression of NR1 and NR2A, which might arise from to the dissociation of the NR2A-PSD-95-Src complex.Chronic zinc exposure perturbs the interaction of NR2A to PSD-95 and causes the disorder of NMDARs in hippocampal neurons, suggesting a novel action of zinc distinct from its acute effects on NMDAR activity.

  5. Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing.

    Science.gov (United States)

    Pistollato, Francesca; Canovas-Jorda, David; Zagoura, Dimitra; Price, Anna

    2017-06-09

    Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

  6. Conditional intrinsic voltage oscillations in mature vertebrate neurons undergo specific changes in culture

    DEFF Research Database (Denmark)

    Guertin, Pierre A; Hounsgaard, Jørn

    2006-01-01

    cord of adult turtles maintainable for several weeks in culture conditions. N-methyl-D-aspartate (NMDA)-induced-tetrodotoxin (TTX)-resistant voltage oscillations in motoneurons were approximately 10 times faster in culture than in acute preparations. Oscillations in culture were abolished by NMDA...

  7. Exogenous α-synuclein hinders synaptic communication in cultured cortical primary rat neurons

    NARCIS (Netherlands)

    Hassink, G. C.; Raiss, C. C.; Segers-Nolten, I. M.J.; Van Wezel, R. J.A.; Subramaniam, V.; Le Feber, J.; Claessens, M. M.A.E.

    2018-01-01

    Amyloid aggregates of the protein a-synuclein (aS) called Lewy Bodies (LB) and Lewy Neurites (LN) are the pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. We have previously shown that high extracellular αS concentrations can be toxic to cells and that neurons take up

  8. Comparison of P2X and TRPV1 receptors in ganglia or primary culture of trigeminal neurons and their modulation by NGF or serotonin

    Directory of Open Access Journals (Sweden)

    Giniatullin Rashid

    2006-03-01

    Full Text Available Abstract Background Cultured sensory neurons are a common experimental model to elucidate the molecular mechanisms of pain transduction typically involving activation of ATP-sensitive P2X or capsaicin-sensitive TRPV1 receptors. This applies also to trigeminal ganglion neurons that convey pain inputs from head tissues. Little is, however, known about the plasticity of these receptors on trigeminal neurons in culture, grown without adding the neurotrophin NGF which per se is a powerful algogen. The characteristics of such receptors after short-term culture were compared with those of ganglia. Furthermore, their modulation by chronically-applied serotonin or NGF was investigated. Results Rat or mouse neurons in culture mainly belonged to small and medium diameter neurons as observed in sections of trigeminal ganglia. Real time RT-PCR, Western blot analysis and immunocytochemistry showed upregulation of P2X3 and TRPV1 receptors after 1–4 days in culture (together with their more frequent co-localization, while P2X2 ones were unchanged. TRPV1 immunoreactivity was, however, lower in mouse ganglia and cultures. Intracellular Ca2+ imaging and whole-cell patch clamping showed functional P2X and TRPV1 receptors. Neurons exhibited a range of responses to the P2X agonist α, β-methylene-adenosine-5'-triphosphate indicating the presence of homomeric P2X3 receptors (selectively antagonized by A-317491 and heteromeric P2X2/3 receptors. The latter were observed in 16 % mouse neurons only. Despite upregulation of receptors in culture, neurons retained the potential for further enhancement of P2X3 receptors by 24 h NGF treatment. At this time point TRPV1 receptors had lost the facilitation observed after acute NGF application. Conversely, chronically-applied serotonin selectively upregulated TRPV1 receptors rather than P2X3 receptors. Conclusion Comparing ganglia and cultures offered the advantage of understanding early adaptive changes of nociception

  9. Neuron cell positioning on polystyrene in culture by silver-negative ion implantation and region control of neural outgrowth

    International Nuclear Information System (INIS)

    Tsuji, Hiroshi; Sato, Hiroko; Baba, Takahiro; Ikemura, Shin'ichi; Gotoh, Yasuhito; Ishikawa, Junzo

    2000-01-01

    A new method to control the position of neuron cell attachment and extension region of neural outgrowth has been developed by using a pattering ion implantation with silver-negative ions into polystyrene dishes. This technique offers a promising method to form an artificially designed neural network in cell culture in vitro. Silver-negative ions were implanted into non-treated polystyrene dishes (NTPS) at conditions of 20 keV and 3x10 15 ions/cm 2 through a pattering mask, which had as many as 67 slits of 60 μm in width and 4 mm in length with a spacing of 60 μm. For cell culture in vitro, nerve cells of PC-12h (rat adrenal phechromocytoma) were used because they respond to a nerve growth factor (NGF). In the first 2 days in culture without NGF, we observed a selective cell attachment only to the ion-implanted region in patterning Ag - implanted polystyrene sample (p-Ag/NTPS). In another 2 days in culture with NGF, the nerve cells expanded neurites only over the ion-implanted region. For collagen-coated p-Ag/NTPS sample of which collagen was coated after the ion implantation (Collagen/p-Ag/NTPS), most nerve cells were also attached on the ion-implanted region. However, neurites expanded in both ion-implanted and unimplanted regions. The contact angle of NTPS decreased after the ion implantation from 86 deg. to 74 deg. . The region selectivity of neuron attachment and neurite extension is considered to be due to contact angle lowering by the ion implantation as radiation effect on the surface

  10. Role of IFN-gamma and LPS on Neuron/Glial Co-Cultures Infected by Neospora caninum

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    Erica Etelvina Viana De Jesus

    2014-10-01

    Full Text Available Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 μg/mL of LPS on infected rat neuron/glial co-cultures. After 72 hours of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not, meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001, this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%. The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in CNS

  11. Dependence of synchronized bursting activity on medium stirring and the perfusion rate in a cultured network of neurons

    Science.gov (United States)

    Heo, Ryoun; Kim, Hyun; Lee, Kyoung J.

    2016-05-01

    A cultured network of neurons coupled with a multi-electrode-array (MEA) recording system has been a useful platform for investigating various issues in neuroscience and engineering. The neural activity supported by the system can be sensitive to environmental fluctuations, for example, in the medium's nutrient composition, ph, and temperature, and to mechanical disturbances, yet this issue has not been the subject. Especially, a normal practice in maintaining neuronal cell cultures involves an intermittent sequence of medium exchanges, typically at a time interval of a few days, and one such sudden medium exchange is unavoidably accompanied by many unintended disturbances. Here, based on a quantitative time-series analysis of synchronized bursting events, we explicitly demonstrate that such a medium exchange can, indeed, bring a huge change in the existing neural activity. Subsequently, we develop a medium perfusion-stirring system and an ideal protocol that can be used in conjunction with a MEA recording system, providing long-term stability. Specifically, we systematically evaluate the effects of medium stirring and perfusion rates. Unexpectedly, even some vigorous mechanical agitations do not have any impacts on neural activity. On the other hand, too much replenishment ( e.g., 1.8 ml/day for a 1.8-ml dish) of neurobasal medium results in an excitotoxicity.

  12. Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+-induced neuronal death in cultured hippocampal neurons

    International Nuclear Information System (INIS)

    Li Chenchen; Xing Tairan; Tang Mingliang; Yong Wu; Yan Dan; Deng Hongmin; Wang Huili; Wang Ming; Chen Jutao; Ruan Diyun

    2008-01-01

    Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb 2+ causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb 2+ . Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb 2+ -induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb 2+ treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 μM) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb 2+ . And that Pb 2+ -elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb 2+ and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death

  13. Determination of the phospholipid precursor of anandamide and other N- acylethanolamine phospholipids before and after sodium azide-induced toxicity in cultured neocortical neurons

    DEFF Research Database (Denmark)

    Hansen, H.H.; Schousboe, A.; Hansen, Harald S.

    2000-01-01

    Phospholipase D-mediated hydrolysis of N-acylethanolamine phospholipids (NAPEs) releases anandamide and other N-acylethanolamines, resulting in different actions at cellular targets in the CNS. Recently, we have demonstrated that these N-acyl lipids accumulate in cultured neocortical neurons subj...... method, neuronal NAPE species can be identified and quantified with respect to N-acyl composition, including a trans-isomer of the anandamide precursor. The anandamide precursor is up-regulated to the same extent as other NAPEs upon neuronal injury....

  14. Crambescidin 816 induces calcium influx though glutamate receptors in primary cultures of cortical neurons

    Directory of Open Access Journals (Sweden)

    Víctor Martín Vázquez

    2014-06-01

    In summary, our data suggest that the cytotoxic effect of 10 μM Cramb816 in cortical neurons may be related to an increase in the cytosolic calcium concentration elicited by the toxin, which is shown to be mediated by glutamate receptor activation. Further studies analyzing the effect of glutamate receptor blockers on the cytotoxic effect of Cramb816 are needed to confirm this hypothesis.

  15. Cellular changes in motor neuron cell culture produced by cytotoxic cerebrospinal fluid from patients with amyotrophic lateral sclerosis.

    Science.gov (United States)

    Gomez-Pinedo, U; Yáñez, M; Matías-Guiu, J; Galán, L; Guerrero-Sola, A; Benito-Martin, M S; Vela, A; Arranz-Tagarro, J A; García, A G

    2014-01-01

    The neurotoxic effects of cerebrospinal fluid (CSF) from patients with amyotrophic lateral sclerosis (ALS) have been reported by various authors who have attributed this neurotoxicity to the glutamate in CSF-ALS. Cultures of rat embryonic cortical neurons were exposed to CSF from ALS patients during an incubation period of 24 hours. Optical microscopy was used to compare cellular changes to those elicited by exposure to 100μm glutamate, and confocal microscopy was used to evaluate immunohistochemistry for caspase-3, TNFα, and peripherin. In the culture exposed to CSF-ALS, we observed cells with nuclear fragmentation and scarce or null structural modifications to the cytoplasmic organelles or to plasma membrane maintenance. This did not occur in the culture exposed to glutamate. The culture exposed to CSF-ALS also demonstrated increases in caspase-3, TNFα, and in peripherin co-locating with caspase-3, but not with TNFα, suggesting that TNFα may play an early role in the process of apoptosis. CFS-ALS cytotoxicity is not related to glutamate. It initially affects the nucleus without altering the cytoplasmic membrane. It causes cytoplasmic apoptosis that involves an increase in caspase-3 co-located with peripherin, which is also overexpressed. Copyright © 2013 Sociedad Española de Neurología. Published by Elsevier Espana. All rights reserved.

  16. Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

    Science.gov (United States)

    Heap, Lucy A.; Vanwalleghem, Gilles C.; Thompson, Andrew W.; Favre-Bulle, Itia; Rubinsztein-Dunlop, Halina; Scott, Ethan K.

    2018-01-01

    The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil. PMID:29403362

  17. Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

    Directory of Open Access Journals (Sweden)

    Lucy A. Heap

    2018-01-01

    Full Text Available The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC/stratum griseum periventriculare (SPV, and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil.

  18. Reduced α-MSH Underlies Hypothalamic ER-Stress-Induced Hepatic Gluconeogenesis.

    Science.gov (United States)

    Schneeberger, Marc; Gómez-Valadés, Alicia G; Altirriba, Jordi; Sebastián, David; Ramírez, Sara; Garcia, Ainhoa; Esteban, Yaiza; Drougard, Anne; Ferrés-Coy, Albert; Bortolozzi, Analía; Garcia-Roves, Pablo M; Jones, John G; Manadas, Bruno; Zorzano, Antonio; Gomis, Ramon; Claret, Marc

    2015-07-21

    Alterations in ER homeostasis have been implicated in the pathophysiology of obesity and type-2 diabetes (T2D). Acute ER stress induction in the hypothalamus produces glucose metabolism perturbations. However, the neurobiological basis linking hypothalamic ER stress with abnormal glucose metabolism remains unknown. Here, we report that genetic and induced models of hypothalamic ER stress are associated with alterations in systemic glucose homeostasis due to increased gluconeogenesis (GNG) independent of body weight changes. Defective alpha melanocyte-stimulating hormone (α-MSH) production underlies this metabolic phenotype, as pharmacological strategies aimed at rescuing hypothalamic α-MSH content reversed this phenotype at metabolic and molecular level. Collectively, our results posit defective α-MSH processing as a fundamental mediator of enhanced GNG in the context of hypothalamic ER stress and establish α-MSH deficiency in proopiomelanocortin (POMC) neurons as a potential contributor to the pathophysiology of T2D. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  19. A treasure trove of hypothalamic neurocircuitries governing body weight homeostasis.

    Science.gov (United States)

    Vianna, Claudia R; Coppari, Roberto

    2011-01-01

    Changes in physical activities and feeding habits have transformed the historically rare disease of obesity into a modern metabolic pandemic. Obesity occurs when energy intake exceeds energy expenditure over time. This energy imbalance significantly increases the risk for cardiovascular disease and type 2 diabetes mellitus and as such represents an enormous socioeconomic burden and health threat. To combat obesity, a better understanding of the molecular mechanisms and neurocircuitries underlying normal body weight homeostasis is required. In the 1940s, pioneering lesion experiments unveiled the importance of medial and lateral hypothalamic structures. In the 1980s and 1990s, several neuropeptides and peripheral hormones critical for appropriate feeding behavior, energy expenditure, and hence body weight homeostasis were identified. In the 2000s, results from metabolic analyses of genetically engineered mice bearing mutations only in selected neuronal groups greatly advanced our knowledge of the peripheral/brain feedback-loop modalities by which central neurons control energy balance. In this review, we will summarize these recent progresses with particular emphasis on the biochemical identities of hypothalamic neurons and molecular components underlying normal appetite, energy expenditure, and body weight homeostasis. We will also parse which of those neurons and molecules are critical components of homeostatic adaptive pathways against obesity induced by hypercaloric feeding.

  20. Effects of acetylpuerarin on hippocampal neurons and intracellular free calcium subjected to oxygen-glucose deprivation/reperfusion in primary culture.

    Science.gov (United States)

    Liu, Rui; Wei, Xin-bing; Zhang, Xiu-Mei

    2007-05-25

    This study was undertaken to find out the effects of acetylpuerarin on hippocampal neurons and intracellular free calcium in primary culture subjected to oxygen-glucose deprivation/reperfusion. According to different reperfusion time (1 h, 6 h, 12 h, 24 h), three concentrations (1.6 micromol l(-1), 0.4 micromol l(-1), 0.1 micromol l(-1)) of acetylpuerarin, and MK-801 (10 micromol l(-1)), a positive control drug, neurons were randomly divided into 21 groups. Each group was observed by inverted phase contrast microscope; neuron viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); intracellular Ca(2+) was observed by Fura-2/AM ester through fluorospectrophotometer. The injured neurons were protected and degeneration and necrosis were alleviated in treatment groups of acetylpuerarin and MK-801. Acetylpuerarin increased the neuron viability at high, middle and low concentrations. Fluorescence detection results showed that the calcium concentration in the group treated with acetylpuerarin and MK-801 was lowered in each reperfusion time. Our results demonstrated that acetylpuerarin could protect the hippocampal neurons from ischemia-reperfusion injury in rats by alleviating the morphological damage, increasing neuron viability and decreasing calcium concentration in neuron.

  1. Hypoxia preferentially destroys GABAergic neurons in developing rat neocortex explants in culture

    NARCIS (Netherlands)

    Romijn, H. J.; Ruijter, J. M.; Wolters, P. S.

    1988-01-01

    The hypothesis that hypoxic ischemia before or during the human birth process preferentially destroys GABAergic nerve cells, particularly in the neocortex, was tested in a tissue culture model system. To that end, rat neocortex explants dissected from 6-day-old rat pups and cultured to a

  2. Implications of mitochondrial dynamics on neurodegeneration and on hypothalamic dysfunction

    Directory of Open Access Journals (Sweden)

    Antonio eZorzano

    2015-06-01

    Full Text Available Mitochondrial dynamics is a term that encompasses the movement of mitochondria along the cytoskeleton, regulation of their architecture, and connectivity mediated by tethering and fusion/fission. The importance of these events in cell physiology and pathology has been partially unraveled with the identification of the genes responsible for the catalysis of mitochondrial fusion and fission. Mutations in two mitochondrial fusion genes (MFN2 and OPA1 cause neurodegenerative diseases, namely Charcot-Marie Tooth type 2A and autosomal dominant optic atrophy. Alterations in mitochondrial dynamics may be involved in the pathophysiology of prevalent neurodegenerative conditions. Moreover, impairment of the activity of mitochondrial fusion proteins dysregulates the function of hypothalamic neurons, leading to alterations in food intake and in energy homeostasis. Here we review selected findings in the field of mitochondrial dynamics and their relevance for neurodegeneration and hypothalamic dysfunction.

  3. Dissecting the hypothalamic pathways that underlie innate behaviors.

    Science.gov (United States)

    Zha, Xi; Xu, Xiaohong

    2015-12-01

    Many complex behaviors that do not require learning are displayed and are termed innate. Although traditionally the subject matter of ethology, innate behaviors offer a unique entry point for neuroscientists to dissect the physiological mechanisms governing complex behaviors. Since the last century, converging evidence has implicated the hypothalamus as the central brain area that controls innate behaviors. Recent studies using cutting-edge tools have revealed that genetically-defined populations of neurons residing in distinct hypothalamic nuclei and their associated neural pathways regulate the initiation and maintenance of diverse behaviors including feeding, sleep, aggression, and parental care. Here, we review the newly-defined hypothalamic pathways that regulate each innate behavior. In addition, emerging general principles of the neural control of complex behaviors are discussed.

  4. Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

    DEFF Research Database (Denmark)

    Dadsetan, Sherry; Bak, Lasse Kristoffer; Sørensen, Michael

    2011-01-01

    study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5h with [U-(13)C]glucose to monitor de novo......It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present...

  5. Recording Spikes Activity in Cultured Hippocampal Neurons Using Flexible or Transparent Graphene Transistors

    Directory of Open Access Journals (Sweden)

    Farida Veliev

    2017-08-01

    Full Text Available The emergence of nanoelectronics applied to neural interfaces has started few decades ago, and aims to provide new tools for replacing or restoring disabled functions of the nervous systems as well as further understanding the evolution of such complex organization. As the same time, graphene and other 2D materials have offered new possibilities for integrating micro and nano-devices on flexible, transparent, and biocompatible substrates, promising for bio and neuro-electronics. In addition to many bio-suitable features of graphene interface, such as, chemical inertness and anti-corrosive properties, its optical transparency enables multimodal approach of neuronal based systems, the electrical layer being compatible with additional microfluidics and optical manipulation ports. The convergence of these fields will provide a next generation of neural interfaces for the reliable detection of single spike and record with high fidelity activity patterns of neural networks. Here, we report on the fabrication of graphene field effect transistors (G-FETs on various substrates (silicon, sapphire, glass coverslips, and polyimide deposited onto Si/SiO2 substrates, exhibiting high sensitivity (4 mS/V, close to the Dirac point at VLG < VD and low noise level (10−22 A2/Hz, at VLG = 0 V. We demonstrate the in vitro detection of the spontaneous activity of hippocampal neurons in-situ-grown on top of the graphene sensors during several weeks in a millimeter size PDMS fluidics chamber (8 mm wide. These results provide an advance toward the realization of biocompatible devices for reliable and high spatio-temporal sensing of neuronal activity for both in vitro and in vivo applications.