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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 saturated fatty acids on nonneuronal cells.

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

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

    2000-09-01

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

  3. Application of the Co-culture Membrane System Pointed to a Protective Role of Catestatin on Hippocampal Plus Hypothalamic Neurons Exposed to Oxygen and Glucose Deprivation.

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    Mele, Maria; Morelli, Sabrina; Fazzari, Gilda; Avolio, Ennio; Alò, Raffaella; Piscioneri, Antonella; De Bartolo, Loredana; Facciolo, Rosa Maria; Canonaco, Marcello

    2016-11-05

    Depletion of oxygen and glucose even for brief periods is sufficient to cause cerebral ischemia, which is a predominant worldwide cause of motor deficits with the reduction of life quality and subsequently death. Hence, more insights regarding protective measures against ischemic events are becoming a major research goal. Among the many neuronal factors, N-methyl-D-aspartate receptors (NMDAR), orexinergic neuroreceptors (ORXR), and sympatho-inhibitory neuropeptide catestatin (CST) are widely involved with ischemic episodes. In this study, it was possible to induce in vitro ischemic conditions of the hamster (Mesocricetus auratus) hippocampal and hypothalamic neuronal cultures, grown on a newly compartmentalized membrane system, via oxygen and glucose deprivation (OGD). These cultures displayed notably differentiated NMDARergic and ORXergic receptor expression activities along with evident brain-derived neurotrophic factor (BDNF) plus orexin A (ORX-A) secretion, especially under co-cultured conditions. Interestingly, addition of CST in OGD-insulted hippocampal cells accounted for upregulated GluN1 and ORX1R transcripts that in the case of the latter neuroreceptor was very strongly (p BDNF and ORX-A secretion in the presence of hippocampal cells. Overall, the preferential CST effects on BDNF plus ORX-A production together with altered NMDAR and ORXR levels, especially in co-cultured hypothalamic cells pointed to ORX-containing neurons as major protective constituents against ischemic damages thus opening new scenarios on the cross-talking roles of CST during ischemic disorders.

  4. Leptin signalling pathways in hypothalamic neurons.

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    Kwon, Obin; Kim, Ki Woo; Kim, Min-Seon

    2016-04-01

    Leptin is the most critical hormone in the homeostatic regulation of energy balance among those so far discovered. Leptin primarily acts on the neurons of the mediobasal part of hypothalamus to regulate food intake, thermogenesis, and the blood glucose level. In the hypothalamic neurons, leptin binding to the long form leptin receptors on the plasma membrane initiates multiple signaling cascades. The signaling pathways known to mediate the actions of leptin include JAK-STAT signaling, PI3K-Akt-FoxO1 signaling, SHP2-ERK signaling, AMPK signaling, and mTOR-S6K signaling. Recent evidence suggests that leptin signaling in hypothalamic neurons is also linked to primary cilia function. On the other hand, signaling molecules/pathways mitigating leptin actions in hypothalamic neurons have been extensively investigated in an effort to treat leptin resistance observed in obesity. These include SOCS3, tyrosine phosphatase PTP1B, and inflammatory signaling pathways such as IKK-NFκB and JNK signaling, and ER stress-mitochondrial signaling. In this review, we discuss leptin signaling pathways in the hypothalamus, with a particular focus on the most recently discovered pathways.

  5. Hypothalamic leptin-neurotensin-hypocretin neuronal networks in zebrafish.

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    Levitas-Djerbi, Talia; Yelin-Bekerman, Laura; Lerer-Goldshtein, Tali; Appelbaum, Lior

    2015-04-01

    Neurotensin (NTS) is a 13 amino acid neuropeptide that is expressed in the hypothalamus. In mammals, NTS-producing neurons that express leptin receptor (LepRb) regulate the function of hypocretin/orexin (HCRT) and dopamine neurons. Thus, the hypothalamic leptin-NTS-HCRT neuronal network orchestrates key homeostatic output, including sleep, feeding, and reward. However, the intricate mechanisms of the circuitry and the unique role of NTS-expressing neurons remain unclear. We studied the NTS neuronal networks in zebrafish and cloned the genes encoding the NTS neuropeptide and receptor (NTSR). Similar to mammals, the ligand is expressed primarily in the hypothalamus, while the receptor is expressed widely throughout the brain in zebrafish. A portion of hypothalamic nts-expressing neurons are inhibitory and some coexpress leptin receptor (lepR1). As in mammals, NTS and HCRT neurons are localized adjacently in the hypothalamus. To track the development and axonal projection of NTS neurons, the NTS promoter was isolated. Transgenesis and double labeling of NTS and HCRT neurons showed that NTS axons project toward HCRT neurons, some of which express ntsr. Moreover, another target of NTS neurons is ntsr-expressing dopaminergeric neurons. These findings suggest structural circuitry between leptin, NTS, and hypocretinergic or dopaminergic neurons and establish the zebrafish as a model to study the role of these neuronal circuits in the regulation of feeding, sleep, and reward.

  6. Differential sensitivity to nicotine among hypothalamic magnocellular neurons

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    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....

  7. 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....

  8. 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.

  9. 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.

  10. Direct Cellular Peptidomics of Hypothalamic Neurons

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    Mitchell, Jennifer W.; Atkins, Norman; Sweedler, Jonathan V.; Gillette, Martha U.

    2011-01-01

    The chemical complexity of cell-to-cell communication has emerged as a fundamental challenge to understanding brain systems. This is certainly true for the hypothalamus, where neuropeptide signals are heterogeneous, localized and dynamic. Thus far, most hypothalamic peptidomic studies have centered on the entire structure; however, recent advances in collection strategies and analytical technologies have enabled direct, high-resolution peptidomic profiles focused on two regions of interest, the suprachiasmatic and supraoptic nuclei, including their subregions and individual cells. Suites of peptides now can be identified and probed for function. High spatial and analytical sensitivities reveal that discrete hypothalamic nuclei have distinct peptidomic signatures. Peptidomic discovery not only reveals unanticipated complexity, but also peptides previously unknown that act as key circuit components. Analysis of tissue releasates identifies peptides secreted into the extracellular environment and available for transmitting intercellular signals. Direct sampling techniques define peptide-releasate profiles in spatial, temporal and event-dependent patterns. These approaches are providing remarkable new insights into the complexity of neuropeptidergic cell-to-cell signaling central to neuroendocrine physiology. PMID:21334363

  11. [Conditioned trace reactions of hypothalamic neurons following exposure to vibration].

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    Mednikova, Iu S

    1977-01-01

    Trace reactions were studied in 112 hypothalamic units in rabbits during motor defensive conditioning to time after vibration action of two intensities. A later formation of conditioned trace reactions was observed as compared to the control. Vibration of lesser intensity improved the course of trace processes and slightly increased the number of neurones responding to the conditioned stimulus (45% as against 43% in the control); after a stronger vibration action, conditioned reactions to time were recorded only in 29% of the hypothalamic cells eith a simultaneous diminution of the trace response. It has been assumed that the observed transformations result from changes of the significance of the reinforcing factor in the formation of a motor defensive conditioned reflex after the action of vibration.

  12. 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,

  13. Tibolone Rapidly Attenuates the GABAB Response in Hypothalamic Neurones

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    Qiu, Jian; Bosch, Martha A.; Rønnekleiv, Oline K.; Kloosterboer, Helenius J.; Kelly, Martin J.

    2008-01-01

    Tibolone is primarily used for the treatment of climacteric symptoms. Tibolone is rapidly converted into three major metabolites: 3α- and 3β-hydroxy-tibolone (3α- and 3βOH-tibolone), which have oestrogenic effects, and the Δ4-isomer (Δ4-tibolone), which has progestogenic and androgenic effects. Since tibolone is effective in treating climacteric symptoms, the effects on the brain may be explained by the oestrogenic activity of tibolone. Previously using whole-cell patch clamp recording, we found that 17β-oestradiol (E2) rapidly altered GABA neurotransmission in hypothalamic neurones through a membrane oestrogen receptor (mER). E2 reduced the potency of the GABAB receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K+ channels in hypothalamic neurones. Therefore, we hypothesized that tibolone may have some rapid effects through the mER and sought to elucidate the signalling pathway of tibolone’s action using selective inhibitors and whole cell recording in ovariectomized female guinea pigs and mice. A sub-population of neurones was identified post hoc as proopiomelanocortin (POMC) neurones by immunocytochemical staining. Similar to E2, we have found that tibolone and its active metabolite 3βOH-tibolone rapidly reduced the potency of the GABAB receptor agonist baclofen to activate GIRK channels in POMC neurones. The effects were blocked by the ER antagonist ICI 182,780. Other metabolites of tibolone (3αOH-tibolone and Δ4-tibolone) had no effect. Furthermore, tibolone (and 3βOH-tibolone) was fully efficacious in ERαKO and ERβKO mice to attenuate GABAB responses. The effects of tibolone were blocked by phospholipase C inhibitor U73122. However, in contrast to E2, the effects of tibolone were not blocked by protein kinase C inhibitors or protein kinase A inhibitors. It appears that tibolone (and 3βOH-tibolone) activates phospholipase C leading to PIP2 metabolism and direct alteration of GIRK channel function. Therefore, tibolone

  14. Synaptic contact between median preoptic neurons and subfornical organ neurons projecting to the paraventricular hypothalamic nucleus.

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    Kawano, Hitoshi

    2017-04-01

    It is known that the median preoptic nucleus (POMe) sends dense projections to the subfornical organ (SFO). However, the functional significance of these projections have not been well discussed. In this electron microscopic study, we investigated the presence of synapses between POMe-derived axon terminals and SFO neurons that project to the paraventricular hypothalamic nucleus (PVN). After injection of a retrograde tracer, wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex, into the PVN, many labeled neurons were found in the SFO. In contrast, after injection of an anterograde tracer, biotinylated dextran amine, in the POMe, abundant labeled axon varicosities were observed in the SFO. Using electron microscopy, synapses were identified between retrogradely labeled dendrites and cell bodies, and anterogradely labeled axon terminals, indicating that POMe neurons innervate SFO neurons projecting to the PVN. The possibility that POMe neurons play multiple roles in the neuronal circuit responsible for vasopressin release and/or cardiovascular regulation is also discussed.

  15. Membrane-initiated Estrogen Signaling in Hypothalamic Neurons

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    Kelly, Martin J.; Rønnekleiv, Oline K.

    2008-01-01

    Summary It is well known that many of the actions of 17β-estradiol (E2) in the central nervous system are mediated via intracellular receptor/transcription factors that interact with steroid response elements on target genes. However, there is compelling evidence for membrane steroid receptors for estrogen in hypothalamic and other brain neurons. But it is not well understood how estrogen signals via membrane receptors, and how these signals impact not only membrane excitability but also gene transcription in neurons. Indeed, it has been known for sometime that E2 can rapidly alter neuronal activity within seconds, indicating that some cellular effects can occur via membrane delimited events. In addition, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell signaling. Therefore, this review will consider our current knowledge of rapid membrane-initiated and intracellular signaling by E2 in the hypothalamus, the nature of receptors involved and how they contribute to homeostatic functions. PMID:18538919

  16. Culturing rat hippocampal neurons.

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    Audesirk, G; Audesirk, T; Ferguson, C

    2001-01-01

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

  17. Lipoprotein Lipase is an Important Modulator of Lipid Uptake and Storage in Hypothalamic Neurons

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    Libby, Andrew E.; Wang, Hong; Mittal, Richa; Sungelo, Mitchell; Potma, Eric; Eckel, Robert H.

    2015-01-01

    LPL is the rate-limiting enzyme for uptake of TG-derived FFA in peripheral tissues, and the enzyme is expressed in the brain and CNS. We previously created a mouse which lacks neuronal LPL. This animal becomes obese on a standard chow, and we observed reduced lipid uptake in the hypothalamus at 3 months preceding obesity. In our present study, we replicated the animal phenotype in an immortalized mouse hypothalamic cell line (N41) to examine how LPL affects expression of AgRP as well as entry and storage of lipids into neurons. We show that LPL is able to modulate levels of the orexigenic peptide AgRP. LPL also exerts effects on lipid uptake into culture neurons, and that uptake of neutral lipid can be enhanced even by mutant LPL lacking catalytic activity. N41 cells also accumulate neutral lipid in droplets, and this is at least in part regulated by LPL. These data in addition to those published in mice with neuron-specific deletion of LPL suggest that neuronal LPL is an important regulator of lipid homeostasis in neurons and that alterations in LPL levels may have important effects on systemic metabolism and neuronal lipid biology. PMID:26265042

  18. 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 GABAergic

  19. Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions.

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    Mittag, Jens; Lyons, David J; Sällström, Johan; Vujovic, Milica; Dudazy-Gralla, Susi; Warner, Amy; Wallis, Karin; Alkemade, Anneke; Nordström, Kristina; Monyer, Hannah; Broberger, Christian; Arner, Anders; Vennström, Björn

    2013-01-01

    Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone's central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

  20. Activation of Strychnine-Sensitive Glycine Receptors by Shilajit on Preoptic Hypothalamic Neurons of Juvenile Mice.

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    Bhattarai, Janardhan Prasad; Cho, Dong Hyu; Han, Seong Kyu

    2016-02-29

    Shilajit, a mineral pitch, has been used in Ayurveda and Siddha system of medicine to treat many human ailments, and is reported to contain at least 85 minerals in ionic form. This study examined the possible mechanism of Shilajit action on preoptic hypothalamic neurons using juvenile mice. The hypothalamic neurons are the key regulator of many hormonal systems. In voltage clamp mode at a holding potential of -60 mV, and under a high chloride pipette solution, Shilajit induced dose-dependent inward current. Shilajit-induced inward currents were reproducible and persisted in the presence of 0.5 μM tetrodotoxin (TTX) suggesting a postsynaptic action of Shilajit on hypothalamic neurons. The currents induced by Shilajit were almost completely blocked by 2 μM strychnine (Stry), a glycine receptor antagonist. In addition, Shilajit-induced inward currents were partially blocked by bicuculline. Under a gramicidin-perforated patch clamp mode, Shilajit induced membrane depolarization on juvenile neurons. These results show that Shilajit affects hypothalamic neuronal activities by activating the Stry-sensitive glycine receptor with α₂/α₂β subunit. Taken together, these results suggest that Shilajit contains some ingredients with possible glycine mimetic activities and might influence hypothalamic neurophysiology through activation of Stry-sensitive glycine receptor-mediated responses on hypothalamic neurons postsynaptically.

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

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    Cortés-Campos, Christian; Letelier, Joaquín; Ceriani, Ricardo; Whitlock, Kathleen E.

    2015-01-01

    ABSTRACT 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. PMID:26209533

  2. 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.

  3. 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

  4. Crucial role of zebrafish prox1 in hypothalamic catecholaminergic neurons development

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    Del Giacco Luca

    2008-03-01

    Full Text Available Abstract Background Prox1, the vertebrate homolog of prospero in Drosophila melanogaster, is a divergent homeogene that regulates cell proliferation, fate determination and differentiation during vertebrate embryonic development. Results Here we report that, in zebrafish, prox1 is widely expressed in several districts of the Central Nervous System (CNS. Specifically, we evidenced prox1 expression in a group of neurons, already positive for otp1, located in the hypothalamus at the level of the posterior tuberculum (PT. Prox1 knock-down determines the severe loss of hypothalamic catecholaminergic (CA neurons, identified by tyrosine hydroxylase (TH expression, and the synergistic prox1/otp1 overexpression induces the appearance of hypothalamic supernumerary TH-positive neurons and ectopic TH-positive cells on the yolk epitelium. Conclusion Our findings indicate that prox1 activity is crucial for the proper development of the otp1-positive hypothalamic neuronal precursors to their terminal CA phenotype.

  5. 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.

  6. InsR/FoxO1 signaling curtails hypothalamic POMC neuron number.

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    Leona Plum

    Full Text Available Insulin receptor (InsR signaling through transcription factor FoxO1 is important in the development of hypothalamic neuron feeding circuits, but knowledge about underlying mechanisms is limited. To investigate the role of InsR/FoxO1 signaling in the development and maintenance of these circuits, we surveyed the pool of hypothalamic neurons expressing Pomc mRNA in different mouse models of impaired hypothalamic InsR signaling. InsR ablation in the entire hypothalamus did not affect Pomc-neuron number at birth, but resulted in a 25% increase, most notably in the middle arcuate nucleus region, in young adults. Selective restoration of InsR expression in POMC neurons in these mice partly reversed the abnormality, resulting in a 10% decrease compared to age-matched controls. To establish whether FoxO1 signaling plays a role in this process, we examined POMC neuron number in mice with POMC-specific deletion of FoxO1, and detected a 23% decrease in age-matched animals, consistent with a cell-autonomous role of InsR/FoxO1 signaling in regulating POMC neuron number, distinct from its established role to activate Pomc transcription. These changes in Pomc cells occurred in the absence of marked changes in humoral factors or hypothalamic NPY neurons.

  7. Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons.

    Science.gov (United States)

    Palomba, Letizia; Silvestri, Cristoforo; Imperatore, Roberta; Morello, Giovanna; Piscitelli, Fabiana; Martella, Andrea; Cristino, Luigia; Di Marzo, Vincenzo

    2015-05-29

    The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL(-/-) mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.

  8. [Effect of the intermittent hypoxic training on the functioning of peptidergic neurons of the paraventricular hypothalamic nucleus and brain stem neurons in rats].

    Science.gov (United States)

    Abramov, A V

    1998-03-01

    Internittent hypoxic training (IHT) increased the quantity and secretory activity of peptidergic neurons of the paraventricular hypothalamic nucleus (PHN) and activated neurons of the dorsal motor nucleus of n.vagus. These structures seem to take part in realisation of the IHT activating effect on condition of the pancreatic delta-cells. The effect involves insulin-stimulating and insuloprotective effects realised via hypothalamic and neuro-conducting ways of regulation of the endocrine pancreas with a direct participation of hypothalamic neuropeptides.

  9. Leptin Acts via Lateral Hypothalamic Area Neurotensin Neurons to Inhibit Orexin Neurons by Multiple GABA-Independent Mechanisms

    Science.gov (United States)

    Goforth, Paulette B.; Leinninger, Gina M.; Patterson, Christa M.

    2014-01-01

    The adipocyte-derived hormone leptin modulates neural systems appropriately for the status of body energy stores. Leptin inhibits lateral hypothalamic area (LHA) orexin (OX; also known as hypocretin)-producing neurons, which control feeding, activity, and energy expenditure, among other parameters. Our previous results suggest that GABAergic LHA leptin receptor (LepRb)-containing and neurotensin (Nts)-containing (LepRbNts) neurons lie in close apposition with OX neurons and control Ox mRNA expression. Here, we show that, similar to leptin, activation of LHA Nts neurons by the excitatory hM3Dq DREADD (designer receptor exclusively activated by designer drugs) hyperpolarizes membrane potential and suppresses action potential firing in OX neurons in mouse hypothalamic slices. Furthermore, ablation of LepRb from Nts neurons abrogated the leptin-mediated inhibition, demonstrating that LepRbNts neurons mediate the inhibition of OX neurons by leptin. Leptin did not significantly enhance GABAA-mediated inhibitory synaptic transmission, and GABA receptor antagonists did not block leptin-mediated inhibition of OX neuron activity. Rather, leptin diminished the frequency of spontaneous EPSCs onto OX neurons. Furthermore, leptin indirectly activated an ATP-sensitive potassium (KATP) channel in OX neurons, which was required for the hyperpolarization of OX neurons by leptin. Although Nts did not alter OX activity, galanin, which is coexpressed in LepRbNts neurons, inhibited OX neurons, whereas the galanin receptor antagonist M40 (galanin-(1–12)-Pro3-(Ala-Leu)2-Ala amide) prevented the leptin-induced hyperpolarization of OX cells. These findings demonstrate that leptin indirectly inhibits OX neurons by acting on LHA LepRbNts neurons to mediate two distinct GABA-independent mechanisms of inhibition: the presynaptic inhibition of excitatory neurotransmission and the opening of KATP channels. PMID:25143620

  10. Effect of copper on extracellular levels of key pro-inflammatory molecules in hypothalamic GN11 and primary neurons.

    Science.gov (United States)

    Spisni, Enzo; Valerii, Maria Chiara; Manerba, Marcella; Strillacci, Antonio; Polazzi, Elisabetta; Mattia, Toni; Griffoni, Cristiana; Tomasi, Vittorio

    2009-07-01

    Copper dyshomeostasis is responsible for the neurological symptoms observed in the genetically inherited copper-dependent disorders (e.g., Menkes' and Wilson's diseases), but it has been also shown to have an important role in neurodegenerative diseases such as Alzheimer disease, prion diseases, Parkinson's disease and amyotrophic lateral sclerosis. It is widely accepted that increased extracellular copper levels contribute to neuronal pathogenic process by increasing the production of dangerous radical oxygen species, but the existence of other molecular mechanisms explaining copper neurotoxicity has not been investigated yet. By using a cellular model based on hypothalamic GN11 cultured neurons exposed to copper supplementation and by analysing the cell conditioned media, we try here to identify new molecular events explaining the association between extracellular copper accumulation and neuronal damages. We show here that increased extracellular copper levels produce a wide complex of alterations in the neuronal extracellular environment. In particular, copper affects the secretion of molecules involved in the protection of neurons against oxidative stress, such as cyclophilin A (CypA), or of molecules capable of shifting neuronal cells towards a pro-inflammatory state, such as IL-1alpha, IL-12, Rantes, neutrophil gelatinase-associated lipocalin (NGAL) and secreted protein acidic and rich in cysteine (SPARC). Copper pro-inflammatory properties have been confirmed by using primary neurons.

  11. Differential effects of histamine on the activity of hypothalamic dopaminergic neurons in the rat.

    Science.gov (United States)

    Fleckenstein, A E; Lookingland, K J; Moore, K E

    1994-01-01

    The effect of intracerebroventricular administration of histamine on hypothalamic dopaminergic neuronal activity was estimated in male rats by measuring concentrations of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in brain regions containing terminals or perikarya of these neurons. Three distinct, regionally specific neurochemical responses were apparent. In the median eminence and intermediate lobe of the pituitary, histamine affected neither DOPAC nor dopamine concentrations, suggesting no effect on tuberoinfundibular or periventricular-hypophysial dopaminergic neuronal activity. In the medial zona incerta and in the dorsomedial, rostral periventricular and medial preoptic hypothalamic nuclei, histamine effected a dose- and time-related increase in both DOPAC and dopamine concentrations; these effects were blocked by destruction of noradrenergic neurons projecting to these regions, suggesting that these changes are attributable to noradrenergic neuronal activation, and that histamine does not affect the activity of incertohypothalamic or periventricular-preoptic dopaminergic neurons located in these brain regions. In the suprachiasmatic, caudal periventricular and paraventricular hypothalamic nuclei, histamine effected a dose- and time-related increase in DOPAC, but not dopamine, concentrations; these effects were blocked by the H1 antagonist mepyramine, but not the H2 antagonist zolantidine. Destruction of noradrenergic neurons projecting to these regions did not prevent the histamine-induced increases in DOPAC concentrations. These data indicate that histamine increases the activity of dopaminergic neurons projecting to the suprachiasmatic, caudal periventricular and paraventricular nuclei via an action at H1 receptors. Overall, these results reveal that i.c.v. administration of histamine differentially affects the activity of the various dopaminergic neuronal systems of the rat hypothalamus.

  12. Glut4 expression defines an insulin-sensitive hypothalamic neuronal population.

    Science.gov (United States)

    Ren, Hongxia; Yan, Shijun; Zhang, Baifang; Lu, Taylor Y; Arancio, Ottavio; Accili, Domenico

    2014-07-01

    Insulin signaling in the CNS modulates satiety and glucose metabolism, but insulin target neurons are poorly defined. We have previously shown that ablation of insulin receptors (InsR) in Glut4-expressing tissues results in systemic abnormalities of insulin action. We propose that Glut4 neurons constitute an insulin-sensitive neuronal subset. We determined their gene expression profiles using flow-sorted hypothalamic Glut4 neurons. Gene ontology analyses demonstrated that Glut4 neurons are enriched in olfacto-sensory receptors, M2 acetylcholine receptors, and pathways required for the acquisition of insulin sensitivity. Following genetic ablation of InsR, transcriptome profiling of Glut4 neurons demonstrated impairment of the insulin, peptide hormone, and cAMP signaling pathways, with a striking upregulation of anion homeostasis pathway. Accordingly, hypothalamic InsR-deficient Glut4 neurons showed reduced firing activity. The molecular signature of Glut4 neurons is consistent with a role for this neural population in the integration of olfacto-sensory cues with hormone signaling to regulate peripheral metabolism.

  13. Identification of hypothalamic neuron-derived neurotrophic factor as a novel factor modulating appetite.

    Science.gov (United States)

    Byerly, Mardi S; Swanson, Roy D; Semsarzadeh, Nina N; McCulloh, Patrick S; Kwon, Kiwook; Aja, Susan; Moran, Timothy H; Wong, G William; Blackshaw, Seth

    2013-06-15

    Disruption of finely coordinated neuropeptide signals in the hypothalamus can result in altered food intake and body weight. We identified neuron-derived neurotrophic factor (NENF) as a novel secreted protein through a large-scale screen aimed at identifying novel secreted hypothalamic proteins that regulate food intake. We observed robust Nenf expression in hypothalamic nuclei known to regulate food intake, and its expression was altered under the diet-induced obese (DIO) condition relative to the fed state. Hypothalamic Nenf mRNA was regulated by brain-derived neurotrophic factor (BDNF) signaling, itself an important regulator of appetite. Delivery of purified recombinant BDNF into the lateral cerebral ventricle decreased hypothalamic Nenf expression, while pharmacological inhibition of trkB signaling increased Nenf mRNA expression. Furthermore, recombinant NENF administered via an intracerebroventricular cannula decreased food intake and body weight and increased hypothalamic Pomc and Mc4r mRNA expression. Importantly, the appetite-suppressing effect of NENF was abrogated in obese mice fed a high-fat diet, demonstrating a diet-dependent modulation of NENF function. We propose the existence of a regulatory circuit involving BDNF, NENF, and melanocortin signaling. Our study validates the power of using an integrated experimental and bioinformatic approach to identify novel CNS-derived proteins with appetite-modulating function and reveals NENF as an important central modulator of food intake.

  14. 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.

  15. Hypothalamic neuron projection to autonomic preganglionic levels related with glucose metabolism: a fluorescent labelling study in the rat.

    Science.gov (United States)

    Portillo, F; Carrasco, M; Vallo, J J

    1996-06-01

    The location of hypothalamic paraventricular neurons projecting to sympathetic preganglionic levels and related to the autonomic regulation of various organs involved in glucose metabolism (OGM) was determined by ipsilateral injections of two fluorescent tracers, Diamidino Yellow into the left dorsal motor nucleus of the vagus and Fast Blue into the left intermediolateral cell column of the T8-T9 spinal cord. Hypothalamospinal neurons were mainly located in the dorsal part of the paraventricular hypothalamic nucleus (PVH) and the hypothalamobulbar neurons were most abundant in the ventral, medial and extreme lateral parts of the PVH. No double-labelled neurons were found in the hypothalamus. These results can help the knowledge of the neural hypothalamic network related with the autonomic hypothalamic control.

  16. Neuronal glucoprivation enhances hypothalamic histamine turnover in rats.

    Science.gov (United States)

    Oohara, A; Yoshimatsu, H; Kurokawa, M; Oishi, R; Saeki, K; Sakata, T

    1994-08-01

    Histamine (HA) turnover in the rat hypothalamus following insufficient energy supply due to glucoprivation was examined after administration of insulin or 2-deoxy-D-glucose (2-DG). HA turnover was assessed by accumulation of tele-methylhistamine (t-MH), a major metabolite of brain HA, following administration of pargyline. Intraperitoneal injection of 1, 2, and 4 U/kg of insulin, which had no influence on steady-state levels of HA and t-MH, increased pargyline-induced accumulation of t-MH. Accumulation of t-MH due to pargyline was inversely related to the concomitant plasma glucose concentration after different doses of insulin. The level of t-MH accumulated by pargyline did not change compared with that of controls, when a euglycemic condition was maintained or insulin at a dose of 6 mU per rat was infused into the third cerebroventricle. Intracerebroventricular infusion of 24 mumol per rat of 2-DG, which had no influence on steady-state levels of HA and t-MH, increased the level of t-MH enhanced by pargyline. The results indicate that an increase in hypothalamic HA turnover in response to glucoprivation may be involved in homeostatic regulation of energy metabolism in the brain.

  17. Depolarizing actions of hydrogen sulfide on hypothalamic paraventricular nucleus neurons.

    Directory of Open Access Journals (Sweden)

    C Sahara Khademullah

    Full Text Available Hydrogen sulfide (H2S is a novel neurotransmitter that has been shown to influence cardiovascular functions as well and corticotrophin hormone (CRH secretion. Since the paraventricular nucleus of the hypothalamus (PVN is a central relay center for autonomic and endocrine functions, we sought to investigate the effects of H2S on the neuronal population of the PVN. Whole cell current clamp recordings were acquired from the PVN neurons and sodium hydrosulfide hydrate (NaHS was bath applied at various concentrations (0.1, 1, 10, and 50 mM. NaHS (1, 10, and 50 mM elicited a concentration-response relationship from the majority of recorded neurons, with almost exclusively depolarizing effects following administration. Cells responded and recovered from NaHS administration quickly and the effects were repeatable. Input differences from baseline and during the NaHS-induced depolarization uncovered a biphasic response, implicating both a potassium and non-selective cation conductance. The results from the neuronal population of the PVN shed light on the possible physiological role that H2S has in autonomic and endocrine function.

  18. 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.

  19. Monosodium glutamate-sensitive hypothalamic neurons contribute to the control of bone mass

    Science.gov (United States)

    Elefteriou, Florent; Takeda, Shu; Liu, Xiuyun; Armstrong, Dawna; Karsenty, Gerard

    2003-01-01

    Using chemical lesioning we previously identified hypothalamic neurons that are required for leptin antiosteogenic function. In the course of these studies we observed that destruction of neurons sensitive to monosodium glutamate (MSG) in arcuate nuclei did not affect bone mass. However MSG treatment leads to hypogonadism, a condition inducing bone loss. Therefore the normal bone mass of MSG-treated mice suggested that MSG-sensitive neurons may be implicated in the control of bone mass. To test this hypothesis we assessed bone resorption and bone formation parameters in MSG-treated mice. We show here that MSG-treated mice display the expected increase in bone resorption and that their normal bone mass is due to a concomitant increase in bone formation. Correction of MSG-induced hypogonadism by physiological doses of estradiol corrected the abnormal bone resorptive activity in MSG-treated mice and uncovered their high bone mass phenotype. Because neuropeptide Y (NPY) is highly expressed in MSG-sensitive neurons we tested whether NPY regulates bone formation. Surprisingly, NPY-deficient mice had a normal bone mass. This study reveals that distinct populations of hypothalamic neurons are involved in the control of bone mass and demonstrates that MSG-sensitive neurons control bone formation in a leptin-independent manner. It also indicates that NPY deficiency does not affect bone mass.

  20. A Thalamo-Hypothalamic Pathway That Activates Oxytocin Neurons in Social Contexts in Female Rats.

    Science.gov (United States)

    Cservenák, Melinda; Keller, Dávid; Kis, Viktor; Fazekas, Emese A; Öllös, Hanna; Lékó, András H; Szabó, Éva R; Renner, Éva; Usdin, Ted B; Palkovits, Miklós; Dobolyi, Árpád

    2017-02-01

    Oxytocin is released from neurons in the paraventricular hypothalamic nucleus (PVN) in mothers upon suckling and during adult social interactions. However, neuronal pathways that activate oxytocin neurons in social contexts are not yet established. Neurons in the posterior intralaminar complex of the thalamus (PIL), which contain tuberoinfundibular peptide 39 (TIP39) and are activated by pup exposure in lactating mothers, provide a candidate projection. Innervation of oxytocin neurons by TIP39 neurons was examined by double labeling in combination with electron microscopy and retrograde tract-tracing. Potential classic neurotransmitters in TIP39 neurons were investigated by in situ hybridization histochemistry. Neurons activated after encounter with a familiar conspecific female in a familiar environment were mapped with the c-Fos technique. PVN and the supraoptic nucleus oxytocin neurons were closely apposed by an average of 2.0 and 0.4 TIP39 terminals, respectively. Asymmetric (presumed excitatory) synapses were found between TIP39 terminals and cell bodies of oxytocin neurons. In lactating rats, PIL TIP39 neurons were retrogradely labeled from the PVN. TIP39 neurons expressed vesicular glutamate transporter 2 but not glutamic acid decarboxylase 67. PIL contained a markedly increased number of c-Fos-positive neurons in response to social encounter with a familiar conspecific female. Furthermore, the PIL received ascending input from the spinal cord and the inferior colliculus. Thus, TIP39 neurons in the PIL may receive sensory input in response to social interactions and project to the PVN to innervate and excite oxytocin neurons, suggesting that the PIL-PVN projection contributes to the activation of oxytocin neurons in social contexts. Copyright © 2017 by the Endocrine Society.

  1. 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.

    2015-01-01

    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 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. PMID:25187366

  2. Relative number and distribution of murine hypothalamic proopiomelanocortin neurons innervating distinct target sites.

    Directory of Open Access Journals (Sweden)

    Connie M King

    Full Text Available Proopiomelanocortin (POMC neurons send projections widely throughout the brain consistent with their role in regulating numerous homeostatic processes and mediating analgesia and reward. Recent data suggest that POMC neurons located in the rostral and caudal extents of the arcuate nucleus of the hypothalamus may mediate selective actions, however it is not clear if POMC neurons in these regions of the arcuate nucleus innervate specific target sites. In the present study, fluorescent microspheres and cholera toxin B were used to retrogradely label POMC neurons in POMC-DsRed transgenic mice. The number and location of POMC cells projecting to the supraoptic nucleus, periaqueductal gray, ventral tegmental area, paraventricular nucleus, lateral hypothalamic nucleus, amygdala and the dosal vagal complex was determined. Tracer injected unilaterally labeled POMC neurons in both sides of the arcuate nucleus. While the total number of retrogradely labeled cells in the arcuate nucleus varied by injection site, less than 10% of POMC neurons were labeled with tracer injected into any target area. Limited target sites appear to be preferentially innervated by POMC neurons that reside in the rostral or caudal extremes of the arcuate nucleus, whereas the majority of target sites are innervated by diffusely distributed POMC neurons. The modest number of cells projecting to each target site indicates that relatively few POMC neurons may mediate potent and specific physiologic responses and therefore disturbed signaling in a very few POMC neurons may have significant consequences.

  3. Relative number and distribution of murine hypothalamic proopiomelanocortin neurons innervating distinct target sites.

    Science.gov (United States)

    King, Connie M; Hentges, Shane T

    2011-01-01

    Proopiomelanocortin (POMC) neurons send projections widely throughout the brain consistent with their role in regulating numerous homeostatic processes and mediating analgesia and reward. Recent data suggest that POMC neurons located in the rostral and caudal extents of the arcuate nucleus of the hypothalamus may mediate selective actions, however it is not clear if POMC neurons in these regions of the arcuate nucleus innervate specific target sites. In the present study, fluorescent microspheres and cholera toxin B were used to retrogradely label POMC neurons in POMC-DsRed transgenic mice. The number and location of POMC cells projecting to the supraoptic nucleus, periaqueductal gray, ventral tegmental area, paraventricular nucleus, lateral hypothalamic nucleus, amygdala and the dosal vagal complex was determined. Tracer injected unilaterally labeled POMC neurons in both sides of the arcuate nucleus. While the total number of retrogradely labeled cells in the arcuate nucleus varied by injection site, less than 10% of POMC neurons were labeled with tracer injected into any target area. Limited target sites appear to be preferentially innervated by POMC neurons that reside in the rostral or caudal extremes of the arcuate nucleus, whereas the majority of target sites are innervated by diffusely distributed POMC neurons. The modest number of cells projecting to each target site indicates that relatively few POMC neurons may mediate potent and specific physiologic responses and therefore disturbed signaling in a very few POMC neurons may have significant consequences.

  4. Hypothalamic Non-AgRP, Non-POMC GABAergic Neurons Are Required for Postweaning Feeding and NPY Hyperphagia.

    Science.gov (United States)

    Kim, Eun Ran; Wu, Zhaofei; Sun, Hao; Xu, Yuanzhong; Mangieri, Leandra R; Xu, Yong; Tong, Qingchun

    2015-07-22

    The hypothalamus is critical for feeding and body weight regulation. Prevailing studies focus on hypothalamic neurons that are defined by selectively expressing transcription factors or neuropeptides including those expressing proopiomelanocortin (POMC) and agouti-related peptides (AgRP). The Cre expression driven by the pancreas-duodenum homeobox 1 promoter is abundant in several hypothalamic nuclei but not in AgRP or POMC neurons. Using this line, we generated mice with disruption of GABA release from a major subset of non-POMC, non-AgRP GABAergic neurons in the hypothalamus. These mice exhibited a reduction in postweaning feeding and growth, and disrupted hyperphagic responses to NPY. Disruption of GABA release severely diminished GABAergic input to the paraventricular hypothalamic nucleus (PVH). Furthermore, disruption of GABA-A receptor function in the PVH also reduced postweaning feeding and blunted NPY-induced hyperphagia. Given the limited knowledge on postweaning feeding, our results are significant in identifying GABA release from a major subset of less appreciated hypothalamic neurons as a key mediator for postweaning feeding and NPY hyperphagia, and the PVH as one major downstream site that contributes significantly to the GABA action. Significance statement: Prevalent studies on feeding in the hypothalamus focus on well characterized, selective groups neurons [e.g., proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons], and as a result, the role of the majority of other hypothalamic neurons is largely neglected. Here, we demonstrated an important role for GABAergic projections from non-POMC non-AgRP neurons to the paraventricular hypothalamic nucleus in promoting postweaning (mainly nocturnal) feeding and mediating NPY-induced hyperphagia. Thus, these results signify an importance to study those yet to be defined hypothalamic neurons in the regulation of energy balance and reveal a neural basis for postweaning (nocturnal) feeding and

  5. Near-Perfect Synaptic Integration by Nav1.7 in Hypothalamic Neurons Regulates Body Weight.

    Science.gov (United States)

    Branco, Tiago; Tozer, Adam; Magnus, Christopher J; Sugino, Ken; Tanaka, Shinsuke; Lee, Albert K; Wood, John N; Sternson, Scott M

    2016-06-16

    Neurons are well suited for computations on millisecond timescales, but some neuronal circuits set behavioral states over long time periods, such as those involved in energy homeostasis. We found that multiple types of hypothalamic neurons, including those that oppositely regulate body weight, are specialized as near-perfect synaptic integrators that summate inputs over extended timescales. Excitatory postsynaptic potentials (EPSPs) are greatly prolonged, outlasting the neuronal membrane time-constant up to 10-fold. This is due to the voltage-gated sodium channel Nav1.7 (Scn9a), previously associated with pain-sensation but not synaptic integration. Scn9a deletion in AGRP, POMC, or paraventricular hypothalamic neurons reduced EPSP duration, synaptic integration, and altered body weight in mice. In vivo whole-cell recordings in the hypothalamus confirmed near-perfect synaptic integration. These experiments show that integration of synaptic inputs over time by Nav1.7 is critical for body weight regulation and reveal a mechanism for synaptic control of circuits regulating long term homeostatic functions.

  6. Firing behavior and network activity of single neurons in human epileptic hypothalamic hamartoma

    Directory of Open Access Journals (Sweden)

    Peter N. Steinmetz

    2013-12-01

    Full Text Available Objective: Human hypothalamic hamartomas (HH are intrinsically epileptogenic and are associated with treatment-resistant gelastic seizures. The basic cellular mechanisms responsible for seizure onset within HH are unknown. We used intra-operative microwire recordings of single neuron activity to measure the spontaneous firing rate of neurons and the degree of functional connection between neurons within the tumor.Technique: Fourteen patients underwent transventricular endoscopic resection of HH for treatment-resistant epilepsy. Prior to surgical resection, single neuron recordings from bundled microwires (total of 9 contacts were obtained from HH tissue. Spontaneous activity was recorded for two or three 5-minute epochs under steady-state general anesthesia. Off-line analysis included cluster analysis of single unit activity and probability analysis of firing relationships between pairs of neurons.Results: Altogether, 222 neurons were identified (mean 6 neurons per recording epoch. Cluster analysis of single neuron firing utilizing a mixture of Gaussians model identified two distinct populations on the basis of firing rate (median firing frequency 0.6 versus 15.0 spikes per second; p<10-5. Cluster analysis identified three populations determined by levels of burst-firing (median burst indices of 0.015, 0.18, and 0.39; p<10-15. Unbiased analysis of spontaneous single unit behavior showed that 51% of all possible neuron pairs within each recording epoch had a significant level of firing synchrony (p<10-15. The subgroup of neurons with higher median firing frequencies was more likely to demonstrate synchronous firing (p<10-7. Conclusions: HH tissue in-vivo contains neurons which fire spontaneously. The activity of single neurons is diverse but distributes into at least two electrophysiological phenoytpes. Functional linkage between single neurons suggests that HH neurons exist within local networks that may contribute to ictogenesis.

  7. Exercise training normalizes an increased neuronal excitability of NTS-projecting neurons of the hypothalamic paraventricular nucleus in hypertensive rats.

    Science.gov (United States)

    Stern, Javier E; Sonner, Patrick M; Son, Sook Jin; Silva, Fabiana C P; Jackson, Keshia; Michelini, Lisete C

    2012-05-01

    Elevated sympathetic outflow and altered autonomic reflexes, including impaired baroreflex function, are common findings observed in hypertensive disorders. Although a growing body of evidence supports a contribution of preautonomic neurons in the hypothalamic paraventricular nucleus (PVN) to altered autonomic control during hypertension, the precise underlying mechanisms remain unknown. Here, we aimed to determine whether the intrinsic excitability and repetitive firing properties of preautonomic PVN neurons that innervate the nucleus tractus solitarii (PVN-NTS neurons) were altered in spontaneously hypertensive rats (SHR). Moreover, given that exercise training is known to improve and/or correct autonomic deficits in hypertensive conditions, we evaluated whether exercise is an efficient behavioral approach to correct altered neuronal excitability in hypertensive rats. Patch-clamp recordings were obtained from retrogradely labeled PVN-NTS neurons in hypothalamic slices obtained from sedentary (S) and trained (T) Wistar-Kyoto (WKY) and SHR rats. Our results indicate an increased excitability of PVN-NTS neurons in SHR-S rats, reflected by an enhanced input-output function in response to depolarizing stimuli, a hyperpolarizing shift in Na(+) spike threshold, and smaller hyperpolarizing afterpotentials. Importantly, we found exercise training in SHR rats to restore all these parameters back to those levels observed in WKY-S rats. In several cases, exercise evoked opposing effects in WKY-S rats compared with SHR-S rats, suggesting that exercise effects on PVN-NTS neurons are state dependent. Taken together, our results suggest that elevated preautonomic PVN-NTS neuronal excitability may contribute to altered autonomic control in SHR rats and that exercise training efficiently corrects these abnormalities.

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

    Science.gov (United States)

    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.

  9. Hypothalamic neuronal histamine modulates febrile response but not anorexia induced by lipopolysaccharide.

    Science.gov (United States)

    Chiba, Seiichi; Itateyama, Emi; Oka, Kyoko; Masaki, Takayuki; Sakata, Toshiie; Yoshimatsu, Hironobu

    2005-05-01

    This study examined the contribution of hypothalamic neuronal histamine (HA) to the anorectic and febrile responses induced by lipopolysaccharide (LPS), an exogenous pyrogen, and the endogenous pyrogens interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Intraperitoneal (ip) injection of LPS, IL-1beta, or TNF-alpha suppressed 24-hr cumulative food intake and increased rectal temperature in rats. To analyze the histaminergic contribution, rats were pretreated with intracerebroventricular (icv) injection of 2.44 mmol/kg or ip injection of 244 mmol/kg of alpha-fluoromethylhistidine (FMH), a suicide inhibitor of histidine decarboxylase (HDC), to deplete neural HA. The depletion of neural HA augmented the febrile response to ip injection of LPS and IL-1beta and alleviated the anorectic response to ip injection of IL-1beta. However, the depletion of neural HA did not modify the LPS-induced anorectic response or TNF-alpha-induced febrile and anorectic responses. Consistent with these results, the rate of hypothalamic HA turnover, assessed by the accumulation of tele-methylhistamine (t-MH), was elevated with ip injections of LPS and IL-1beta, but unaffected by TNF-alpha at equivalent doses. This suggests that (i) LPS and IL-1beta activate hypothalamic neural HA turnover; (ii) hypothalamic neural HA suppresses the LPS- and IL-1beta-induced febrile responses and accelerates the IL-1beta-induced anorectic response; and (iii) TNF-alpha modulates the febrile and anorectic responses via a neural HA-independent pathway. Therefore, hypothalamic neural HA is involved in the IL-1beta-dominant pathway, rather than the TNF-alpha-dominant pathway, preceding the systemic inflammatory response induced by exogenous pyrogens, such as LPS. Further research on this is needed.

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

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    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.

  11. 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

  12. 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.

  13. 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.

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

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    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.

  15. 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, PFasting 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, P<0.05) and action potentials (n=9; P<0.05) in 45% (9/20) of VTA DA neurons. 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.

  16. Serotonin 5-HT2C receptor-mediated inhibition of the M-current in hypothalamic POMC neurons

    OpenAIRE

    Roepke, T. A.; Smith, A W; Rønnekleiv, O. K.; Kelly, M. J.

    2012-01-01

    Hypothalamic proopiomelanocortin (POMC) neurons are controlled by many central signals, including serotonin. Serotonin increases POMC activity and reduces feeding behavior via serotonion [5-hydroxytryptamine (5-HT)] receptors by modulating K+ currents. A potential K+ current is the M-current, a noninactivating, subthreshold outward K+ current. Previously, we found that M-current activity was highly reduced in fasted vs. fed states in neuropeptide Y neurons. Because POMC neurons also respond t...

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

    Science.gov (United States)

    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

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

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    Luke K. Burke

    2016-03-01

    Conclusions: These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient to regulate energy intake and insulin sensitivity in male and female mice. However, an unexpected sex difference in the function of this subset of POMC neurons was identified with regard to energy expenditure. We reveal that a large sex difference in physical activity, energy expenditure and the development of obesity is driven by this subpopulation, which constitutes approximately 40% of all POMC neurons in the hypothalamic arcuate nucleus. This may have broad implications for strategies utilized to combat obesity, which at present largely ignore the sex of the obese individual.

  19. Evidence suggesting phosphodiesterase-3B regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.

    Science.gov (United States)

    Anamthathmakula, Prashanth; Sahu, Maitrayee; Sahu, Abhiram

    2015-09-14

    Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (∼60-70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.

  20. Short-term caloric restriction normalizes hypothalamic neuronal responsiveness to glucose ingestion in patients with type 2 diabetes

    NARCIS (Netherlands)

    Teeuwisse, W.M.; Widya, R.L.; Paulides, M.; Lamb, H.J.; Smit, J.W.A.; Roos, A. de; Buchem, M.A. van; Pijl, H.; Grond, J. van der

    2012-01-01

    The hypothalamus is critically involved in the regulation of feeding. Previous studies have shown that glucose ingestion inhibits hypothalamic neuronal activity. However, this was not observed in patients with type 2 diabetes. Restoring energy balance by reducing caloric intake and losing weight are

  1. Intracisternally Injected L-Proline Activates Hypothalamic Supraoptic, but Not Paraventricular, Vasopressin-Expressing Neurons in Conscious Rats

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    Yumi Takemoto

    2011-01-01

    Full Text Available When injected into specific rat brain regions, the neurotransmitter candidate L-proline produces various cardiovascular changes through ionotropic excitatory amino acid receptors. The present study used an immunohistochemical double-labeling approach to determine whether intracisternally injected L-proline in freely moving rats, which increases blood pressure, activates hypothalamic vasopressin-expressing neurons and ventral medullary tyrosine-hydroxylase- (TH- containing neurons. Following injection of L-proline, the number of activated hypothalamic neurons that coexpressed vasopressin and c-Fos was much greater in the supraoptic nucleus (SON than in the paraventricular nucleus (PVN of rats with increased blood pressure. The number of activated TH-containing neurons was significantly greater following L-proline treatment than following control injections of artificial cerebrospinal fluid (ACSF. These results clearly demonstrate that intracisternally injected L-proline activates hypothalamic supraoptic, but not paraventricular, vasopressin-expressing neurons and medullary TH-containing (A1/C1 neurons in freely moving rats.

  2. Hypothalamic Vasopressinergic Projections Innervate Central Amygdala GABAergic Neurons: Implications for Anxiety and Stress Coping

    Science.gov (United States)

    Hernández, Vito S.; Hernández, Oscar R.; Perez de la Mora, Miguel; Gómora, María J.; Fuxe, Kjell; Eiden, Lee E.; Zhang, Limei

    2016-01-01

    The arginine-vasopressin (AVP)-containing hypothalamic magnocellular neurosecretory neurons (VPMNNs) are known for their role in hydro-electrolytic balance control via their projections to the 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 receptor mRNAs were not detected, using the same method. Water-deprivation (WD) for 24 h, which increased the metabolic activity of VPMNNs, also increased anxiety-like behavior measured using the elevated plus maze (EPM) test, and this effect was mimicked by bilateral microinfusion of AVP into the CeA. Anxious behavior induced by either WD or AVP infusion was reversed by CeA infusion of V1a antagonist. VPMNNs are thus a newly discovered source of CeA inhibitory circuit modulation, through which both early-life and adult stress coping signals are conveyed from the hypothalamus to the amygdala. PMID:27932956

  3. 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.

  4. A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons.

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    Imre Kalló

    Full Text Available Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.

  5. Magel2 is required for leptin-mediated depolarization of POMC neurons in the hypothalamic arcuate nucleus in mice.

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    Rebecca E Mercer

    Full Text Available Prader-Willi Syndrome is the most common syndromic form of human obesity and is caused by the loss of function of several genes, including MAGEL2. Mice lacking Magel2 display increased weight gain with excess adiposity and other defects suggestive of hypothalamic deficiency. We demonstrate Magel2-null mice are insensitive to the anorexic effect of peripherally administered leptin. Although their excessive adiposity and hyperleptinemia likely contribute to this physiological leptin resistance, we hypothesized that Magel2 may also have an essential role in intracellular leptin responses in hypothalamic neurons. We therefore measured neuronal activation by immunohistochemistry on brain sections from leptin-injected mice and found a reduced number of arcuate nucleus neurons activated after leptin injection in the Magel2-null animals, suggesting that most but not all leptin receptor-expressing neurons retain leptin sensitivity despite hyperleptinemia. Electrophysiological measurements of arcuate nucleus neurons expressing the leptin receptor demonstrated that although neurons exhibiting hyperpolarizing responses to leptin are present in normal numbers, there were no neurons exhibiting depolarizing responses to leptin in the mutant mice. Additional studies demonstrate that arcuate nucleus pro-opiomelanocortin (POMC expressing neurons are unresponsive to leptin. Interestingly, Magel2-null mice are hypersensitive to the anorexigenic effects of the melanocortin receptor agonist MT-II. In Prader-Willi Syndrome, loss of MAGEL2 may likewise abolish leptin responses in POMC hypothalamic neurons. This neural defect, together with increased fat mass, blunted circadian rhythm, and growth hormone response pathway defects that are also linked to loss of MAGEL2, could contribute to the hyperphagia and obesity that are hallmarks of this disorder.

  6. 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.

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

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    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.

  8. Stiff substrates enhance cultured neuronal network activity.

    Science.gov (United States)

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

    2014-08-28

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

  9. Influence of leptin and GABAB-receptor agonist and antagonist on neurons of the hypothalamic infundibular nucleus in the chicken.

    Science.gov (United States)

    Bogatyrev, S; Yakimova, K S; Tzschentke, B

    2017-04-01

    In birds and mammals, the neuroendocrine regulation of energy balance is conserved in many aspects. Despite significant similarities between the two groups, differences in the regulatory mechanisms were detected. The present study was performed to carry out investigations of the influence of human leptin and GABAB-receptor agonist and antagonist on the firing rate of neurons of the Nucleus infundibuli hypothalami in brain slices from juvenile chickens. For the first time, we demonstrated a clear, dose-related change in the firing rate of hypothalamic neurons in juvenile chickens after the acute application of recombinant human leptin (1, 10, and 100 nM). All investigated neurons increased their subsequent firing rate. Application of GABAB-receptor agonist baclofen (1 µM) blocked, while antagonist CGP 35348 (10 µM) increased the spontaneous neuronal activity. Simultaneous application of baclofen and leptin reduced the effect observed from single leptin application. This was not found after simultaneously application of leptin and CGP. Altogether, our results indicate that in bird brain slices, and exemplarily in those of the chicken, hypothalamic neurons show mammalian-like responsiveness after acute leptin and GABA application. GABAB-mechanisms involved in GABA release play a likely important role in the leptin-mediated effects on NI neurons via functional leptin receptors.

  10. Culturing conditions determine neuronal and glial excitability.

    Science.gov (United States)

    Stoppelkamp, Sandra; Riedel, Gernot; Platt, Bettina

    2010-12-15

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

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

    Science.gov (United States)

    Burke, Luke K; Doslikova, Barbora; D'Agostino, Giuseppe; Greenwald-Yarnell, Megan; Georgescu, Teodora; Chianese, Raffaella; Martinez de Morentin, Pablo B; Ogunnowo-Bada, Emmanuel; Cansell, Celine; Valencia-Torres, Lourdes; Garfield, Alastair S; Apergis-Schoute, John; Lam, Daniel D; Speakman, John R; Rubinstein, Marcelo; Low, Malcolm J; Rochford, Justin J; Myers, Martin G; Evans, Mark L; Heisler, Lora K

    2016-03-01

    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. 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-HT 2C R (CRE) mouse line and intercrossed it with Cre recombinase-dependent and hypothalamic specific reactivatable Pomc (NEO) 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. 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. These data provide support for the functional heterogeneity of hypothalamic POMC neurons, revealing that Pomc expression within 5-HT2CR expressing neurons is sufficient

  12. 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

    -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 CART42-89 immunoreactivity throughout the 90-min stimulation period. In contrast, the concentration of portal plasma CART immunoreactivity dropped in the vehicle infused rats...

  13. The nutritional induction of COUP-TFII gene expression in ventromedial hypothalamic neurons is mediated by the melanocortin pathway.

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    Lina Sabra-Makke

    Full Text Available BACKGROUND: The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. METHODOLOGY/PRINCIPAL FINDINGS: Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. CONCLUSIONS/SIGNIFICANCE: These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.

  14. Differential role of hypothalamic orexin/hypocretin neurons in reward seeking motivated by cocaine versus palatable food.

    Science.gov (United States)

    Martin-Fardon, Rémi; Cauvi, Gabrielle; Kerr, Tony M; Weiss, Friedbert

    2016-08-24

    Hypothalamic orexin/hypocretin (Orx/Hcrt) neurons are thought to mediate both food-reinforced behaviors and behavior motivated by drugs of abuse. However, the relative role of the Orx/Hcrt system in behavior motivated by food versus drugs of abuse remains unclear. This investigation addressed this question by contrasting hypothalamic Orx/Hcrt neuronal activation associated with reinstatement of reward seeking induced by stimuli conditioned to cocaine (COC) versus highly palatable food reward, sweetened condensed milk (SCM). Orx/Hcrt neuronal activation in the lateral hypothalamus, dorsomedial hypothalamus and perifornical area, determined by dual c-fos/orx immunocytochemistry, was quantified in rat brains, following reinstatement of reward seeking induced by a discriminative stimulus (S(+) ) conditioned to COC or SCM. The COC S(+) and SCM S(+) initially produced the same magnitude of reward seeking. However, over four subsequent tests, behavior induced by the SCM S(+) decreased to extinction levels, whereas reinstatement induced by the COC S(+) perseverated at undiminished levels. Following both the first and fourth tests, the percentage of Orx/Hcrt cells expressing Fos was significantly increased in all hypothalamic subregions in rats tested with the COC S(+) but not rats tested with the SCM S(+) . These findings point toward a role for the Orx/Hcrt system in perseverating, compulsive-like COC seeking but not behavior motivated by palatable food. Moreover, analysis of the Orx/Hcrt recruitment patterns suggests that failure of Orx/Hcrt neurons in the lateral hypothalamus to respond to inhibitory inputs from Orx/Hcrt neurons in the dorsomedial hypothalamus/perifornical area may contribute to the perseverating nature of COC seeking.

  15. Female Mice Lacking Estrogen Receptor-α in Hypothalamic Proopiomelanocortin (POMC) Neurons Display Enhanced Estrogenic Response on Cortical Bone Mass.

    Science.gov (United States)

    Farman, H H; Windahl, S H; Westberg, L; Isaksson, H; Egecioglu, E; Schele, E; Ryberg, H; Jansson, J O; Tuukkanen, J; Koskela, A; Xie, S K; Hahner, L; Zehr, J; Clegg, D J; Lagerquist, M K; Ohlsson, C

    2016-08-01

    Estrogens are important regulators of bone mass and their effects are mainly mediated via estrogen receptor (ER)α. Central ERα exerts an inhibitory role on bone mass. ERα is highly expressed in the arcuate (ARC) and the ventromedial (VMN) nuclei in the hypothalamus. To test whether ERα in proopiomelanocortin (POMC) neurons, located in ARC, is involved in the regulation of bone mass, we used mice lacking ERα expression specifically in POMC neurons (POMC-ERα(-/-)). Female POMC-ERα(-/-) and control mice were ovariectomized (OVX) and treated with vehicle or estradiol (0.5 μg/d) for 6 weeks. As expected, estradiol treatment increased the cortical bone thickness in femur, the cortical bone mechanical strength in tibia and the trabecular bone volume fraction in both femur and vertebrae in OVX control mice. Importantly, the estrogenic responses were substantially increased in OVX POMC-ERα(-/-) mice compared with the estrogenic responses in OVX control mice for cortical bone thickness (+126 ± 34%, P mass, ERα was silenced using an adeno-associated viral vector. Silencing of ERα in hypothalamic VMN resulted in unchanged bone mass. In conclusion, mice lacking ERα in POMC neurons display enhanced estrogenic response on cortical bone mass and mechanical strength. We propose that the balance between inhibitory effects of central ERα activity in hypothalamic POMC neurons in ARC and stimulatory peripheral ERα-mediated effects in bone determines cortical bone mass in female mice.

  16. Dehydration-induced drinking decreases Fos expression in hypothalamic paraventricular neurons expressing vasopressin but not corticotropin-releasing hormone.

    Science.gov (United States)

    Wotus, Cheryl; Arnhold, Michelle M; Engeland, William C

    2007-03-01

    Water-restricted (WR) rats exhibit a rapid suppression of plasma corticosterone following drinking. The present study monitored Fos-like immunoreactivity (Fos) to assess the effect of WR-induced drinking on the activity of vasopressin (VP)-positive magnocellular and parvocellular neurons and corticotropin-releasing hormone (CRH)-positive parvocellular neurons in the paraventricular nucleus of the hypothalamus. Adult male rats received water for 30 min (WR) in the post meridiem (PM) each day for 6 days and were killed without receiving water or at 1 h after receiving water for 15 min. In WR rats, Fos increased in VP magnocellular and parvocellular neurons but not CRH neurons. After drinking, Fos was reduced in VP magnocellular and parvocellular neurons but did not change in CRH neurons. To assess the severity of osmotic stress, rats were sampled throughout the final day of WR. Plasma osmolality, hematocrit and plasma VP were increased throughout the day before PM rehydration, and plasma ACTH and corticosterone were elevated at 1230 and 1430, respectively, showing that WR activates hypothalamic-pituitary-adrenal activity during the early PM before the time of rehydration. To determine the effects of WR-induced drinking on CRH neurons activated by acute stress, WR rats underwent restraint. Restraint increased plasma ACTH and corticosterone and Fos in CRH neurons; although rehydration reduced plasma ACTH and Fos expression in VP neurons, Fos in CRH neurons was not affected. These results suggest that inhibition of VP magnocellular and parvocellular neurons, but not CRH parvocellular neurons, contributes to the suppression of corticosterone after WR-induced drinking.

  17. 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

  18. 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.

  19. Short photoperiod-induced decrease of histamine H3 receptors facilitates activation of hypothalamic neurons in the Siberian hamster.

    Science.gov (United States)

    Barrett, P; van den Top, M; Wilson, D; Mercer, J G; Song, C K; Bartness, T J; Morgan, P J; Spanswick, D

    2009-08-01

    Nonhibernating seasonal mammals have adapted to temporal changes in food availability through behavioral and physiological mechanisms to store food and energy during times of predictable plenty and conserve energy during predicted shortage. Little is known, however, of the hypothalamic neuronal events that lead to a change in behavior or physiology. Here we show for the first time that a shift from long summer-like to short winter-like photoperiod, which induces physiological adaptation to winter in the Siberian hamster, including a body weight decrease of up to 30%, increases neuronal activity in the dorsomedial region of the arcuate nucleus (dmpARC) assessed by electrophysiological patch-clamping recording. Increased neuronal activity in short days is dependent on a photoperiod-driven down-regulation of H3 receptor expression and can be mimicked in long-day dmpARC neurons by the application of the H3 receptor antagonist, clobenproprit. Short-day activation of dmpARC neurons results in increased c-Fos expression. Tract tracing with the trans-synaptic retrograde tracer, pseudorabies virus, delivered into adipose tissue reveals a multisynaptic neuronal sympathetic outflow from dmpARC to white adipose tissue. These data strongly suggest that increased activity of dmpARC neurons, as a consequence of down-regulation of the histamine H3 receptor, contributes to the physiological adaptation of body weight regulation in seasonal photoperiod.

  20. Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat.

    Science.gov (United States)

    Wang, Q; Bing, C; Al-Barazanji, K; Mossakowaska, D E; Wang, X M; McBay, D L; Neville, W A; Taddayon, M; Pickavance, L; Dryden, S; Thomas, M E; McHale, M T; Gloyer, I S; Wilson, S; Buckingham, R; Arch, J R; Trayhurn, P; Williams, G

    1997-03-01

    Leptin acts on the brain to inhibit feeding, increase thermogenesis, and decrease body weight. Neuropeptide Y (NPY)-ergic neurons of the hypothalamic arcuate nucleus (ARC) that project to the paraventricular nuclei (PVN) and dorsomedial nuclei (DMH) are postulated to control energy balance by stimulating feeding and inhibiting thermogenesis, especially under conditions of energy deficit. We investigated whether leptin's short-term effects on energy balance are mediated by inhibition of the NPY neurons. Recombinant murine leptin (11 microg) injected into the lateral ventricle of fasted adult Wistar rats inhibited food intake by 20-25% between 2 and 6 h after administration, compared with saline-treated controls (P ARC, PVN, and DMH and significantly decreased hypothalamic NPY mRNA levels (0.61 +/- 0.02 vs. 0.78 +/- 0.03 arbitrary units; P 0.1), but plasma leptin levels were significantly higher (4.88 +/- 0.66 vs. 2.85 +/- 0.20 ng/ml; P ARC-PVN projection; reduced NPY release in the PVN may mediate leptin's hypophagic and thermogenic actions. Conversely, NPY-induced obesity results in raised circulating leptin concentrations. Leptin and the NPY-ergic ARC-PVN neurons may interact in a homeostatic loop to regulate body fat mass and energy balance.

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

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    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.

  2. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K(+) channels.

    Science.gov (United States)

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-10-15

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K(+) (K(ATP)) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP](c)) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of K(ATP) channels in GR neurons, glucose (3 or 15 mM) caused no detectable increase in [ATP](c), monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25-luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mM) with a significant increase in [ATP](c). Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mM glucose with increases in [ATP](c). Further implicating an ATP-independent mechanism of K(ATP) channel closure in hypothalamic neurons, removal of extracellular glucose (10 mM) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mM) of intracellular ATP. Neurons from both brain regions responded to 5 mM lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP](c). High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed K(ATP) channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells.

  3. Dynamic imaging of free cytosolic ATP concentration during fuel sensing by rat hypothalamic neurones: evidence for ATP-independent control of ATP-sensitive K+ channels

    Science.gov (United States)

    Ainscow, Edward K; Mirshamsi, Shirin; Tang, Teresa; Ashford, Michael L J; Rutter, Guy A

    2002-01-01

    Glucose-responsive (GR) neurons from hypothalamic nuclei are implicated in the regulation of feeding and satiety. To determine the role of intracellular ATP in the closure of ATP-sensitive K+ (KATP) channels in these cells and associated glia, the cytosolic ATP concentration ([ATP]c) was monitored in vivo using adenoviral-driven expression of recombinant targeted luciferases and bioluminescence imaging. Arguing against a role for ATP in the closure of KATP channels in GR neurons, glucose (3 or 15 mm) caused no detectable increase in [ATP]c, monitored with cytosolic luciferase, and only a small decrease in the concentration of ATP immediately beneath the plasma membrane, monitored with a SNAP25–luciferase fusion protein. In contrast to hypothalamic neurons, hypothalamic glia responded to glucose (3 and 15 mm) with a significant increase in [ATP]c. Both neurons and glia from the cerebellum, a glucose-unresponsive region of the brain, responded robustly to 3 or 15 mm glucose with increases in [ATP]c. Further implicating an ATP-independent mechanism of KATP channel closure in hypothalamic neurons, removal of extracellular glucose (10 mm) suppressed the electrical activity of GR neurons in the presence of a fixed, high concentration (3 mm) of intracellular ATP. Neurons from both brain regions responded to 5 mm lactate (but not pyruvate) with an oligomycin-sensitive increase in [ATP]c. High levels of the plasma membrane lactate-monocarboxylate transporter, MCT1, were found in both cell types, and exogenous lactate efficiently closed KATP channels in GR neurons. These data suggest that (1) ATP-independent intracellular signalling mechanisms lead to the stimulation of hypothalamic neurons by glucose, and (2) these effects may be potentiated in vivo by the release of lactate from neighbouring glial cells. PMID:12381816

  4. Interactions between hormonal and environmental signals on hypothalamic neurons molecular mechanisms signaling environmental events.

    Science.gov (United States)

    Zhu, Y S; Dellovade, T L; Pfaff, D W

    1997-04-01

    It is axiomatic that the central nervous system must manage the integration of several environmental factors with steroid hormonal influences for the biologically adaptive performance of reproductive behavior. Launching from established behavioral investigations and from hormonal influences on gene function in the brain, we review here studies on how synaptic inputs and sex hormone influences codetermine hypothalamic gene expression. A particularly exciting implication of results on the ability of thyroid hormone receptors to interfere with estrogen receptor-dependent neuroendocrine function is that environmentally stimulated changes in thyroid hormone levels could influence hypothalamic transcriptional mechanisms important for behavior. If so, this would unite naturalistic environmental thinking with molecular neurobiological thinking important for the hypothalamic control of reproduction. (Trends Endocrinol Metab 1997;8:111-115). (c) 1997, Elsevier Science Inc.

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

    Science.gov (United States)

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

    2014-01-01

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

  6. Reciprocal connections between CART-immunoreactive, hypothalamic paraventricular neurons and serotonergic dorsal raphe cells in the rat: Light microscopic study.

    Science.gov (United States)

    Lee, Ji S; Lee, Hyun S

    2014-04-29

    Based on the overlapping physiological roles of cocaine- and amphetamine-regulated transcript (CART) peptides and serotonin, the present study examined the anatomical connection between the hypothalamic paraventricular nucleus (PVN) and the dorsal raphe (DR). The first series of experiments were performed to investigate descending projections from the CART-immunoreactive (CART-ir) PVN to serotonergic DR cells. CART-ir varicosities made contact with serotonergic DR neurons. An anterograde tracing study revealed that varicosities originating from the PVN formed close appositions to serotonergic neuronal profiles along the entire rostro-caudal extent of the DR. A retrograde study demonstrated that CART neurons projecting to the DR were mainly localized in the caudal parvicellular PVN, comprising approximately 3.0%±0.4% (n=8) of total CART cells. A second series of experiments was performed to investigate ascending projections from the DR to CART-ir PVN cells. Serotonin transporter-ir boutons made contact with CART-ir PVN neurons. Anterograde tracing revealed that varicosities originating from the DR formed close appositions to CART-ir PVN cells. Retrograde examination demonstrated that serotonergic neurons projecting to the parvicellular PVN were located along the entire rostro-caudal extent of the DR. The present observation provided an anatomical basis for accumulating evidence in the literature that suggests a functional interaction between the CART and serotonin systems during the regulation of energy balance, emotional behavior, and arousal.

  7. Neuronal androgen receptor regulates insulin sensitivity via suppression of hypothalamic NF-κB-mediated PTP1B expression.

    Science.gov (United States)

    Yu, I-Chen; Lin, Hung-Yun; Liu, Ning-Chun; Sparks, Janet D; Yeh, Shuyuan; Fang, Lei-Ya; Chen, Lumin; Chang, Chawnshang

    2013-02-01

    Clinical investigations highlight the increased incidence of metabolic syndrome in prostate cancer (PCa) patients receiving androgen deprivation therapy (ADT). Studies using global androgen receptor (AR) knockout mice demonstrate that AR deficiency results in the development of insulin resistance in males. However, mechanisms by which AR in individual organs coordinately regulates insulin sensitivity remain unexplored. Here we tested the hypothesis that functional AR in the brain contributes to whole-body insulin sensitivity regulation and to the metabolic abnormalities developed in AR-deficient male mice. The mouse model selectively lacking AR in the central nervous system and AR-expressing GT1-7 neuronal cells were established and used to delineate molecular mechanisms in insulin signaling modulated by AR. Neuronal AR deficiency leads to reduced insulin sensitivity in middle-aged mice. Neuronal AR regulates hypothalamic insulin signaling by repressing nuclear factor-κB (NF-κB)-mediated induction of protein-tyrosine phosphatase 1B (PTP1B). Hypothalamic insulin resistance leads to hepatic insulin resistance, lipid accumulation, and visceral obesity. The functional deficiency of AR in the hypothalamus leads to male mice being more susceptible to the effects of high-fat diet consumption on PTP1B expression and NF-κB activation. These findings suggest that in men with PCa undergoing ADT, reduction of AR function in the brain may contribute to insulin resistance and visceral obesity. Pharmacotherapies targeting neuronal AR and NF-κB may be developed to combat the metabolic syndrome in men receiving ADT and in elderly men with age-associated hypogonadism.

  8. General overview of neuronal cell culture.

    Science.gov (United States)

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

    2013-01-01

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

  9. Regulation of Energy Balance via BDNF Expressed in Nonparaventricular Hypothalamic Neurons.

    Science.gov (United States)

    Yang, Haili; An, Juan Ji; Sun, Chao; Xu, Baoji

    2016-05-01

    Brain-derived neurotrophic factor (BDNF) expressed in the paraventricular hypothalamus (PVH) has been shown to play a key role in regulating energy intake and energy expenditure. BDNF is also expressed in other hypothalamic nuclei; however, the role in the control of energy balance for BDNF produced in these structures remains largely unknown. We found that deleting the Bdnf gene in the ventromedial hypothalamus (VMH) during embryogenesis using the Sf1-Cre transgene had no effect on body weight in mice. In contrast, deleting the Bdnf gene in the adult VMH using Cre-expressing virus led to significant hyperphagia and obesity. These observations indicate that the lack of a hyperphagia phenotype in the Sf1-Cre/Bdnf mutant mice is likely due to developmental compensation. To investigate the role of BDNF expressed in other hypothalamic areas, we employed the hypothalamus-specific Nkx2.1-Cre transgene to delete the Bdnf gene. We found that the Nkx2.1-Cre transgene could abolish BDNF expression in many hypothalamic nuclei, but not in the PVH, and that the resulting mutant mice developed modest obesity due to reduced energy expenditure. Thus, BDNF produced in the VMH plays a role in regulating energy intake. Furthermore, BDNF expressed in hypothalamic areas other than PVH and VMH is also involved in the control of energy expenditure.

  10. The cytokine ciliary neurotrophic factor (CNTF activates hypothalamic urocortin-expressing neurons both in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Matthew J Purser

    Full Text Available Ciliary neurotrophic factor (CNTF induces neurogenesis, reduces feeding, and induces weight loss. However, the central mechanisms by which CNTF acts are vague. We employed the mHypoE-20/2 line that endogenously expresses the CNTF receptor to examine the direct effects of CNTF on mRNA levels of urocortin-1, urocortin-2, agouti-related peptide, brain-derived neurotrophic factor, and neurotensin. We found that treatment of 10 ng/ml CNTF significantly increased only urocortin-1 mRNA by 1.84-fold at 48 h. We then performed intracerebroventricular injections of 0.5 mg/mL CNTF into mice, and examined its effects on urocortin-1 neurons post-exposure. Through double-label immunohistochemistry using specific antibodies against c-Fos and urocortin-1, we showed that central CNTF administration significantly activated urocortin-1 neurons in specific areas of the hypothalamus. Taken together, our studies point to a potential role for CNTF in regulating hypothalamic urocortin-1-expressing neurons to mediate its recognized effects on energy homeostasis, neuronal proliferaton/survival, and/or neurogenesis.

  11. The effect of moxonidine on feeding and body fat in obese Zucker rats: role of hypothalamic NPY neurones

    Science.gov (United States)

    Bing, Chen; King, Peter; Pickavance, Lucy; Brown, Michael; Ziegler, Dieter; Kaan, Elbert; Williams, Gareth

    1999-01-01

    The antihypertensive agent moxonidine, an imidazoline Ii-receptor agonist, also induces hypophagia and lowers body weight in the obese spontaneously hypertensive rat, but the central mediation of this action and the neuronal pathways that moxonidine may interact with are not known. We studied whether moxonidine has anti-obesity effects in the genetically-obese and insulin-resistant fa/fa Zucker rat, and whether these are mediated through inhibition of the hypothalamic neuropeptide Y (NPY) neurones.Lean and obese Zucker rats were given moxonidine (3 mg kg−1 day−1) or saline by gavage for 21 days.Moxonidine decreased food intake throughout by 20% in obese rats (P<0.001) and by 8% in lean rats (P<0.001), and reduced weight gain that final body weight was 15% lower in obese (P<0.001) and 7% lower in lean (P<0.01) rats than their untreated controls. Plasma insulin and leptin levels were decreased in moxonidine-treated obese rats (P<0.01 and P<0.05), but unchanged in treated lean rats. Uncoupling protein-1 gene expression in brown adipose tissue was stimulated by 40–50% (P⩽0.05) in both obese and lean animals given moxonidine. Obese animals given moxonidine showed a 37% reduction in hypothalamic NPY mRNA levels (P=0.01), together with significantly increased NPY concentrations in the paraventricular nucleus (P<0.05), but no changes in the arcuate nucleus or other nuclei; this is consistent with reduced NPY synthesis in the arcuate nucleus and blocked release of NPY in the paraventricular nucleus. In lean animals, moxonidine did not affect NPY levels or NPY mRNA.The hypophagic, thermogenic and anti-obesity effects of moxonidine in obese Zucker rats may be partly due to inhibition of the NPY neurones, whose inappropriate overactivity may underlie obesity in this model. PMID:10369453

  12. [Impregnoarchitectonic study of neurons in the caudal hypothalamic area in sheep].

    Science.gov (United States)

    Rajtová, V

    1985-06-01

    The impregnoarchitectonics of neurons in the regio hypothalamica caudalis was studied by the methods after Ramón-Moliner and Golgi-Cox. Neuron types, morphology of synaptic conjugations and orientation of dendrites and axons are indicated for each nucleus. Small spindle-shaped neurons are the most frequent neuron forms occurring in the regio hypothalamica caudalis. It is only in the nucl. hypothalamicus perifornicalis that neuron processes and the long cell axes are oriented around the columna fornicis. One especially large neuron "occupies" the dorsal part of this nucleus.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers.

    Science.gov (United States)

    Hrabovszky, Erik; Wittmann, Gábor; Kalló, Imre; Füzesi, Tamás; Fekete, Csaba; Liposits, Zsolt

    2012-04-01

    Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.

  15. 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...

  16. Gender difference in age-related number of corticotropin-releasing hormone-expressing neurons in the human hypothalamic paraventricular nucleus and the role of sex hormones

    NARCIS (Netherlands)

    Bao, A.-M.; Swaab, D.F.

    2007-01-01

    Previous studies have shown that the total number of corticotropin-releasing hormone (CRH)-stained neurons in the human hypothalamic paraventricular nucleus (PVN) increases with age. To determine whether this age-related change depends on gender and whether circulating sex hormones play a role, we

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

    Science.gov (United States)

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

    2016-04-01

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

  18. Projection patterns of lateral hypothalamic, cocaine- and amphetamine-regulated transcript (CART) neurons to the dorsal raphe and/or the locus coeruleus in the rat.

    Science.gov (United States)

    Yoon, Ye S; Lee, Hyun S

    2013-02-04

    The present study was designed to reveal the projection patterns of lateral hypothalamic (LH), cocaine- and amphetamine-regulated transcript (CART) neurons to the dorsal raphe (DR) and/or the locus coeruleus (LC) in the rat. After the injection of Red or Green Retrobeads into the DR or LC, LH sections were immunostained for CART and/or melanin-concentrating hormone (MCH). First, CART-immunoreactive axon terminals formed close appositions to the DR (or LC) neuronal profiles. Second, a subpopulation of CART neurons containing MCH projected to the monoaminergic nuclei; the majority of labeled neurons were observed in the dorsal hypothalamic area, the dorsal part of the posterior hypothalamic area, and the zona incerta. Cells were also observed in the perifornical part of the LH, the dorsomedial hypothalamic nucleus, the peduncular and the magnocellular parts of the LH. Of the total population of DR (or LC)-projecting cells, CART/MCH co-containing neurons were 9.5% ± 1.6% (or 10.8% ± 1.3% for LC). Finally, a subset of CART (or MCH) neurons provided divergent axon collaterals to the DR and the LC. Of the entire CART (or MCH) cell population, 3.9% ± 0.8% (or 5.6% ± 1.0% for MCH) sent axon collaterals to the DR/LC. CART/MCH co-containing neurons projecting to the DR or LC might be involved in the feeding-related regulation of arousal, stress-related responses, and emotional behaviors. Thus, CART (or MCH) cells that send divergent axon collaterals to the DR/LC might have a simultaneous (and possibly more efficient) way to exert their specific influences on the aminergic nuclei.

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

    Science.gov (United States)

    Nakagawa, A; Iwama, A

    1995-12-01

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

  20. 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

  1. Reduced fasting-induced activation of hypothalamic arcuate neurons is associated with hyperleptinemia and increased leptin sensitivity in obese mice.

    Science.gov (United States)

    Becskei, Csilla; Lutz, Thomas A; Riediger, Thomas

    2010-08-01

    Fasting increases c-Fos expression in neuropeptide Y (NPY) neurons of the hypothalamic arcuate nucleus (ARC) in lean, but not in hyperleptinemic mice with late-onset obesity (LOO). Although obesity is associated with leptin resistance, we hypothesized that under fasting conditions, leptin sensitivity might be restored and that hyperleptinemia may counteract the neuronal response to fasting. We investigated whether the reduced fasting response of ARC neurons in LOO is paralleled by an increase in leptin sensitivity, as measured by leptin-induced STAT-3 phosphorylation. To assess leptin's role in the modulation of the fasting-induced ARC activation, we investigated c-Fos responses and hormone and metabolite levels in hyperleptinemic diet-induced obese (DIO) and in leptin-deficient ob/ob mice. Leptin induced a stronger STAT-3 phosphorylation in fasted LOO and lean mice than in ad libitum-fed animals. Similar to LOO, hyperleptinemic DIO mice showed no c-Fos response after fasting, while ob/ob mice showed a stronger response than lean control mice. Mimicking hyperleptinemia by repeated leptin injections in lean mice during fasting attenuated the fasting-induced c-Fos expression. Our findings indicate that high leptin levels prevent the fasting-induced activation of ARC neurons in mice. Moreover, leptin sensitivity is dynamic in obese subjects and depends on the feeding status. During short-term increases in leptin sensitivity, e.g., during fasting, leptin signaling appears to be effective, even in hyperleptinemic obesity. As reflected by the blockade of the fasting-induced ARC activation, fasting seems to interfere with the responsiveness of the ARC to signals related to the status of energy intake.

  2. Lateral Hypothalamic Stimulation Reduces Hyperalgesia Through Spinally Descending Orexin-A Neurons in Neuropathic Pain.

    Science.gov (United States)

    Wardach, Jacob; Wagner, Monica; Jeong, Younhee; Holden, Janean E

    2016-03-01

    No evidence to date shows that lateral hypothalamic (LH) stimulation produces orexin-A-mediated antinociception in the spinal cord dorsal horn (SCDH) in a model of neuropathic pain. We conducted experiments to examine the effect of orexin-A-mediated LH stimulation in female rats with chronic constriction injury (CCI) on thermal hyperalgesia. Rats receiving carbachol into the LH demonstrated antinociception on both the left CCI and right nonligated paws (p orexin-1 (OX1) receptor antagonist SB-334867, which blocked LH-induced antinociception compared with control groups (p orexin-A connection between the LH and the SCDH. Identification of this pathway may lead to studies using orexins to manage clinical pain.

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

    Institute of Scientific and Technical Information of China (English)

    LIUYin; CHENJing-Hong; GONGZe-Hui

    2004-01-01

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

  4. Cholesterol secoaldehyde, an ozonation product of cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons.

    Science.gov (United States)

    Sathishkumar, K; Xi, Xiaochun; Martin, Roy; Uppu, Rao M

    2007-06-01

    Aldehydic products from ozonation of cholesterol and peroxidation of phospholipids have been shown to accelerate aggregation of amyloid-beta (Abeta) in vitro. Here, we show that 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (ChSeco), an ozonation product of cholesterol, induces Abeta aggregation, generation of reactive oxygen species (ROS), and cytotoxicity in murine GT1-7 hypothalamic neurons. The formation of Abeta aggregates in situ was dose-dependent at ChSeco concentrations ranging from 1 to 20 microM. The increase in insoluble Abeta aggregates at increasing concentrations of ChSeco was accompanied by a decrease in soluble Abeta as evidenced by Western blot analysis. The formation of ROS in neuronal cells was found to be dose- and time-dependent with the magnitude being higher at 20 microM compared to 10 microM ChSeco or untreated controls. The increase in ROS was associated with depletion of GSH. The cytotoxicity induced by ChSeco involved changes in phosphatidylserine translocation, DNA fragmentation, and caspase 3/7 activity that are characteristic of apoptosis. Pretreatment of neuronal cells with Trolox, a water-soluble analog of alpha-tocopherol offered partial, but significant protection against ChSeco-induced cell death, whereas, N-acetyl-L-cysteine (NAC) completely prevented the cytotoxic effects of ChSeco. NAC and Trolox were without any effects on ChSeco-induced Abeta aggregation. Fibrillogenesis inhibitors, which inhibited Abeta aggregation, did not inhibit cell death induced by ChSeco, implying that ROS generation, and not Abeta aggregation, plays a major role in the observed cytotoxicity. However, since Alzheimer's and other neurodegenerative diseases are slow and progressive, the formation of Abeta aggregates in vivo by ChSeco may have long-term pathological consequences.

  5. Neuropeptide FF, but not prolactin-releasing peptide, mRNA is differentially regulated in the hypothalamic and medullary neurons after salt loading.

    Science.gov (United States)

    Kalliomäki, M-L; Panula, P

    2004-01-01

    Hypothalamic paraventricular and supraoptic nuclei are involved in the body fluid homeostasis. Especially vasopressin peptide and mRNA levels are regulated by hypo- and hyperosmolar stimuli. Other neuropeptides such as dynorphin, galanin and neuropeptide FF are coregulated with vasopressin. In this study neuropeptide FF and another RF-amide peptide, the prolactin-releasing peptide mRNA levels were studied by quantitative in situ hybridization after chronic salt loading, a laboratory model of chronic dehydration. The neuropeptide FF mRNA expressing cells virtually disappeared from the hypothalamic supraoptic and paraventricular nuclei after salt loading, suggesting that hyperosmolar stress downregulated the NPFF gene transcription. The neuropeptide FF mRNA signal levels were returned to control levels after the rehydration period of 7 days. No changes were observed in those medullary nuclei that express neuropeptide FF mRNA. No significant changes were observed in the hypothalamic or medullary prolactin-releasing peptide mRNA levels. Neuropeptide FF mRNA is drastically downregulated in the hypothalamic magnocellular neurons after salt loading. Other neuropeptides studied in this model are concomitantly coregulated with vasopressin: i.e. their peptide levels are downregulated and mRNA levels are upregulated which is in contrast to neuropeptide FF regulation. It can thus be concluded that neuropeptide FF is not regulated through the vasopressin regulatory system but via an independent pathway. The detailed mechanisms underlying the downregulation of neuropeptide FF mRNA in neurons remain to be clarified.

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

    Directory of Open Access Journals (Sweden)

    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

  7. Hypothalamic glial-to-neuronal signaling during puberty: influence of alcohol.

    Science.gov (United States)

    Srivastava, Vinod K; Hiney, Jill K; Dees, W Les

    2011-07-01

    Mammalian puberty requires complex interactions between glial and neuronal regulatory systems within the hypothalamus that results in the timely increase in the secretion of luteinizing hormone releasing hormone (LHRH). Assessing the molecules required for the development of coordinated communication networks between glia and LHRH neuron terminals in the basal hypothalamus, as well as identifying substances capable of affecting cell-cell communication are important. One such pathway involves growth factors of the epidermal growth factor (EGF) family that bind to specific erbB receptors. Activation of this receptor results in the release of prostaglandin-E(2) (PGE(2)) from adjacent glial cells, which then acts on the nearby LHRH nerve terminals to elicit release of the peptide. Another pathway involves novel genes which synthesize adhesion/signaling proteins responsible for the structural integrity of bi-directional glial-neuronal communication. In this review, we will discuss the influence of these glial-neuronal communication pathways on the prepubertal LHRH secretory system, and furthermore, discuss the actions and interactions of alcohol on these two signaling processes.

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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

  10. Suprachiasmatic nuclei and Circadian rhythms. The role of suprachiasmatic nuclei on rhythmic activity of neurons in the lateral hypothalamic area, ventromedian nuclei and pineal gland

    Science.gov (United States)

    Nishino, H.

    1977-01-01

    Unit activity of lateral hypothalamic area (LHA) and Ventromedian nuclei (VMN) was recorded in urethane anesthetized male rats. A 5 to 10 sec. a 3-5 min and a circadian rhythmicity were observed. In about 15% of all neurons, spontaneous activity of LHA and VMN showed reciprocal relationships. Subthreshold stimuli applied at a slow rate in the septum and the suprachiasmatic nuclei (SCN) suppressed the rhythms without changing firing rates. On the other hand, stimulation of the optic nerve at a rate of 5 to 10/sec increased firing rates in 1/3 of neurons of SCN. Iontophoretically applied acetylcholine increased 80% of tested neurons of SCN, whereas norepinephrine, dopamine and 5 HT inhibited 64, 60 and 75% of SCN neurons respectively. These inhibitions were much stronger in neurons, the activity of which was increased by optic nerve stimulation. Stimulation of the SCN inhibited the tonic activity in cervical sympathetic nerves.

  11. 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.

  12. Production of compartmented cultures of rat sympathetic neurons.

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Touzeau, G.; Kato, A.C.

    1983-03-01

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

  15. 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.

  16. Interactions of histaminergic and serotonergic neurons in the hypothalamic regulation of prolactin and ACTH secretion.

    Science.gov (United States)

    Jørgensen, H; Knigge, U; Kjaer, A; Warberg, J

    1996-11-01

    Serotonergic and histaminergic neuronal systems are both involved in mediation of the stress-induced release of the pituitary hormones prolactin (PRL) and ACTH. We investigated the possibility of an interaction between serotonin (5-HT) and histamine (HA) in regulation of PRL and ACTH secretion in conscious male rats. Animals were pretreated systemically with antagonists to 5-HT1, 5-HT2 or 5-HT3 receptors prior to intracerebroventricular (icv) administration of HA. The 5-HT1 + 2 receptor antagonist methysergide prevented and the 5-HT2 receptor antagonist LY 53857 attenuated the HA-induced PRL release while the 5-HT3 receptor antagonist ondansetron had no effect on this response. None of the three 5-HT receptor antagonists affected the ACTH response to HA. Specific blockade of HA synthesis by alpha-fluoromethylhistidine or blockade of postsynaptic HA receptors by icv infusion of the H1 receptor antagonist mepyramine or the H2 receptor antagonist cimetidine inhibited the PRL response to 5-HT or to the 5-HT precursor 5-hydroxytryptophan (5- HTP) given in combination with the 5-HT reuptake inhibitor fluoxetine (Flx). Blockade of the histaminergic system had no effect on the ACTH response to serotonergic stimulation. The H3 receptors are inhibitory HA receptors. Systemic pretreatment with the H3 receptor agonist R(alpha)methylhistamine, or the H3 receptor antagonist thioperamide had no effect on the hormone response to activation of the serotonergic system by 5-HTP plus Flx. We conclude that the serotonergic and histaminergic neuronal systems interact in their stimulation of PRL secretion, but not in their stimulation of ACTH secretion. This interaction involves serotonergic 5-HT1 and 5-HT2 receptors and histaminergic H1 and H2 receptors. Furthermore, the previously observed inhibitory effect of the H3 receptor agonist R(alpha)methylhistamine on stress-induced PRL and ACTH release seems not to be exerted by activation of presynaptic H3 receptors located on serotonergic

  17. Properties of native P2X receptors in large multipolar neurons dissociated from rat hypothalamic arcuate nucleus.

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    Wakamori, Minoru; Sorimachi, Masaru

    2004-04-16

    ATP, the ligand of P2X receptors, is a candidate of neurotransmitter or co-transmitter in the peripheral and the central nervous systems. Anatomical studies have revealed the wide distribution of P2X receptors in the brain. So far, P2X-mediated small synaptic responses have been recorded in some brain regions. To determine the physiological significance of postsynaptic ATP receptors in the brain, we have investigated the P2X responses in rat dissociated hypothalamic arcuate neurons by using the patch-clamp technique. ATP evoked inward currents in a concentration-dependent manner (EC(50)=42 microM) at a holding potential of -70 mV. The current-voltage relationship showed a marked inward rectification starting around -10 mV. Although neither 300 microM alphabeta-methylene-ATP nor 300 microM betagamma-methylene-ATP induced any currents, 100 microM ATPgammaS and 100 microM 2-methylthio-ATP evoked inward currents of which amplitude was about 60% of the control currents evoked by 100 microM ATP. PPADS, one of P2 receptor antagonists, inhibited the ATP-evoked currents in a time- and a concentration-dependent manners (IC(50)=19 microM at 2 min). Permeant Ca(2+) inhibited the ATP-evoked currents in the range of millimolars (IC(50)=7 mM); however, Cd(2+) (1-300 microM), a broad cation channel blocker, facilitated the currents with slow off-response. Zn(2+) in the range of 1-100 microM facilitated the currents whereas Zn(2+) at the concentrations over 100 microM inhibited the currents. These observations suggest that functional P2X receptors are expressed in the hypothalamic arcuate nucleus. The most likely subunit combinations of the P2X receptors are P2X(2)-homomultimer and P2X(2)/P2X(6)-heteromultimer.

  18. Fasted/fed states regulate postsynaptic hub protein DYNLL2 and glutamatergic transmission in oxytocin neurons in the hypothalamic paraventricular nucleus.

    Science.gov (United States)

    Suyama, Shigetomo; Kodaira-Hirano, Misato; Otgon-Uul, Zesemdorj; Ueta, Yoichi; Nakata, Masanori; Yada, Toshihiko

    2016-04-01

    The neurons in the hypothalamus regulate food intake and energy metabolism on reception of systemic energy states. Accumulating evidences have indicated that synaptic transmission on the hypothalamic neurons is modulated by the metabolic condition related to fasted/fed states, and that this modulation of synaptic plasticity plays a role in regulation of feeding. It has been shown that oxytocin (Oxt) neurons in the paraventricular nucleus (PVN) of the hypothalamus sense and integrate various peripheral and central signals and thereby induce satiety. However, whether metabolic conditions regulate the synaptic transmission on Oxt neurons in PVN remains unclear. The present study examined whether the fasted/fed states regulate synaptic transmission on Oxt neurons in PVN. The miniature excitatory postsynaptic currents (mEPSCs) onto Oxt neurons in PVN were increased under ad lib fed condition compared to 24h fasted condition. Furthermore, the NMDA receptor-mediated EPSC on Oxt neurons was increased under fed, compared to fasted, condition. In Oxt neurons, dynein light chain 2 (DYNLL2), a protein suggested to be implicated in the NMDA receptor trafficking to the postsynaptic site, was increased under fed, compared to fasted, condition. The present results suggest that feeding increases excitatory synaptic input on PVN Oxt neurons via mechanisms involving DYNLL2 upregulation and NMDA receptor-mediated synaptic reorganization.

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

    Institute of Scientific and Technical Information of China (English)

    ZHANGYu-Yang; YUQing-Hai; YOUSong; SHENGLi

    2004-01-01

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

  20. 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 Ca2+ concentration ([Ca2+]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 [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. 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 mainly

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

    Science.gov (United States)

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

    2006-07-15

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

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

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    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.

  3. Activation of hypothalamic neuronal nitric oxide synthase in lithium-induced diabetes insipidus rats.

    Science.gov (United States)

    Anai, H; Ueta, Y; Serino, R; Nomura, M; Nakashima, Y; Yamashita, H

    2001-02-01

    The expression of the neuronal nitric oxide synthase (nNOS) gene in the paraventricular (PVN) and supraoptic nuclei (SON) in rats with lithium (Li)-induced polyuria was examined by using in situ hybridization histochemistry. The state of the thyroid axis in these rats was also examined by in situ hybridization histochemistry for thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH) mRNAs and radioimmunoassay for circulating thyroid hormones. Adult male Wistar rats consuming a diet that contained LiCl (60 mmol/kg) for 4 weeks developed remarkable polyuria. The urine in the Li-treated rats was hypotonic and had a large volume and low ionic concentration. The nNOS mRNA in the PVN and SON was significantly increased in the Li-treated rats in comparison with that in control. The increased levels of the nNOS mRNA in the PVN and SON were confirmed by NADPH-diaphorase histochemical staining. There were no differences of TRH mRNA in the PVN, TSH mRNA in the anterior pituitary and plasma concentrations of free T3 and free T4 between Li-treated rats and control rats. These results suggest that Li-induced diabetes insipidus may activate nNOS in the PVN and SON without change of the thyroid axis.

  4. Central action of FGF19 reduces hypothalamic AGRP/NPY neuron activity and improves glucose metabolism.

    Science.gov (United States)

    Marcelin, Geneviève; Jo, Young-Hwan; Li, Xiaosong; Schwartz, Gary J; Zhang, Ying; Dun, Nae J; Lyu, Rong-Ming; Blouet, Clémence; Chang, Jaw K; Chua, Streamson

    2014-02-01

    Tight control of glucose excursions has been a long-standing goal of treatment for patients with type 2 diabetes mellitus in order to ameliorate the morbidity and mortality associated with hyperglycemia. Fibroblast growth factor (FGF) 19 is a hormone-like enterokine released postprandially that emerged as a potential therapeutic agent for metabolic disorders, including diabetes and obesity. Remarkably, FGF19 treatment has hypoglycemic actions that remain potent in models of genetic and acquired insulin resistance. Here, we provided evidence that the central nervous system responds to FGF19 administered in the periphery. Then, in two mouse models of insulin resistance, leptin-deficiency and high-fat diet feeding, third intra-cerebro-ventricular infusions of FGF19 improved glycemic status, reduced insulin resistance and potentiated insulin signaling in the periphery. In addition, our study highlights a new mechanism of central FGF19 action, involving the suppression of AGRP/NPY neuronal activity. Overall, our work unveils novel regulatory pathways induced by FGF19 that will be useful in the design of novel strategies to control diabetes in obesity.

  5. Refeeding-activated glutamatergic neurons in the hypothalamic paraventricular nucleus (PVN) mediate effects of melanocortin signaling in the nucleus tractus solitarius (NTS).

    Science.gov (United States)

    Singru, Praful S; Wittmann, Gábor; Farkas, Erzsébet; Zséli, Györgyi; Fekete, Csaba; Lechan, Ronald M

    2012-08-01

    We previously demonstrated that refeeding after a prolonged fast activates a subset of neurons in the ventral parvocellular subdivision of the paraventricular nucleus (PVNv) as a result of increased melanocortin signaling. To determine whether these neurons contribute to satiety by projecting to the nucleus tractus solitarius (NTS), the retrogradely transported marker substance, cholera toxin-β (CTB), was injected into the dorsal vagal complex of rats that were subsequently fasted and refed for 2 h. By double-labeling immunohistochemistry, CTB accumulation was found in the cytoplasm of the majority of refeeding-activated c-Fos neurons in the ventral parvocellular subdivision of the hypothalamic paraventricular nucleus (PVNv). In addition, a large number of refeeding-activated c-Fos-expressing neurons were observed in the lateral parvocellular subdivision (PVNl) that also contained CTB and were innervated by axon terminals of proopiomelanocortin neurons. To visualize the location of neuronal activation within the NTS by melanocortin-activated PVN neurons, α-MSH was focally injected into the PVN, resulting in an increased number of c-Fos-containing neurons in the PVN and in the NTS, primarily in the medial and commissural parts. All refeeding-activated neurons in the PVNv and PVNl expressed the mRNA of the glutamatergic marker, type 2 vesicular glutamate transporter (VGLUT2), indicating their glutamatergic phenotype, but only rare neurons contained oxytocin. These data suggest that melanocortin-activated neurons in the PVNv and PVNl may contribute to refeeding-induced satiety through effects on the NTS and may alter the sensitivity of NTS neurons to vagal satiety inputs via glutamate excitation.

  6. Hypothalamic neuronal targets activated by neuropeptide S%神经肽S激活下丘脑靶神经元的分布

    Institute of Scientific and Technical Information of China (English)

    闫琼; 邵玉峰; 赵鹏; 孔祥攀; 姜信诚; 侯一平

    2013-01-01

    Objective To identify the distributions of neuropeptide S (NPS) induced neuronal activation in hypothalamus in rats because NPS,a newly identified neuropeptide,was presumed to activate the hypothalamic neurons through its receptors to participate hypothalamic physiological regulation.Methods Fos immunohistochemistry was employed to label the activated neurons following intracerebroventricular (i.c.v.) injection of NPS (1 nmol,n=6) and saline (n=6).The distribution and number of fos immunoreactive (-IR) neurons in the hypothalamus were observed,counted and statistically analyzed.Results The number of fos-IR neurons induced by NPS in the suprachiasmatic nucleus,paraventricular nucleus,dorsomedial hypothalamic nucleus,ventromedial hypothalamic nucleus,arcuate nucleus,perifornical nucleus,and the ventral and dorsal tuberomammillary nuclei and lateral hypothalamic area were respectively increased by 322%,108%,274%,126%,267%,520%,641%,586% and 378% compared with saline (P <0.0001).Conclusion NPS-induced a large number of active neurons in the hypothalamus suggest that NPS is involved in hypothalamic physiological regulation including sleep-wake cycle,emotion,feeding,circadian rhythm,temperature and neuroendocrine.%目的 推测新鉴定的神经肽S (NPS)经其受体激活下丘脑神经元参与下丘脑生理调节,运用神经功能活动形态定位法确定NPS在下丘脑靶神经元的分布.方法 注射NPS(1 nmol,n=6)和生理盐水(n=6),c-fos免疫组化学分别标记大鼠中枢fos免疫反应神经元在下丘脑的分布,并统计分析fos免疫反应神经元数量在各核的变化.结果 与生理盐水比较,NPS增加fos免疫反应神经元,数量分别为下丘脑视交叉上核322%,室旁核108%,背内侧核274%,腹内侧核126%,弓状核267%,穹窿周核520%,结节乳头体腹侧核641%,背侧核586%,外侧区378% (P <0.0001).结论 NPS激活下丘脑上述靶神经元,可能与下丘脑睡眠觉醒周期、

  7. Insulin directly regulates NPY and AgRP gene expression via the MAPK MEK/ERK signal transduction pathway in mHypoE-46 hypothalamic neurons.

    Science.gov (United States)

    Mayer, Christopher M; Belsham, Denise D

    2009-08-13

    Insulin plays a key role in the maintenance of nutrient homeostasis through central regulation of neuropeptides. Neuropeptide Y (NPY) and agouti-related peptide (AgRP) are vital orexigenic peptides that are regulated by insulin, although the processes utilized are unknown. Using a hypothalamic, clonal cell line, mHypoE-46, which endogenously expresses NPY, AgRP and the insulin receptor, we studied the mechanisms involved in the regulation of the NPY/AgRP neuron by insulin. We determined that insulin has direct actions on the neurons and acts to repress NPY/AgRP gene expression through a MAPK MEK/ERK-dependent pathway. Transient transfection analysis determined that human NPY and AgRP 5' flanking gene regions were not regulated by insulin in the mouse cell line, while sequence comparison analysis indicated only a 50% sequence similarity between human and mouse NPY and AgRP 5' flanking regions. These experiments indicate that insulin acts directly on specific hypothalamic neurons to regulate neuropeptide transcription.

  8. 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.

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

    Science.gov (United States)

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

    2008-01-01

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

  10. 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.

  11. Rhynchophylline Protects Cultured Rat Neurons against Methamphetamine Cytotoxicity

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    Dan Dan Xu

    2012-01-01

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

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

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    Sara Teller

    2014-09-01

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

  13. Emergence of Assortative Mixing between Clusters of Cultured Neurons

    Science.gov (United States)

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

    2014-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2007-01-01

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

  15. Hypothalamic dysfunction

    Science.gov (United States)

    ... common causes of hypothalamic dysfunction are surgery, traumatic brain injury, tumors, and radiation. Other causes include: Anorexia nervosa or bulimia Bleeding Genetic disorders that cause iron ...

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

    Science.gov (United States)

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

    2004-01-01

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

  17. 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.

  18. Novel culturing platform for brain slices and neuronal cells

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  19. Separate populations of neurons within the paraventricular hypothalamic nucleus of the rat project to vagal and thoracic autonomic preganglionic levels and express c-Fos protein induced by lithium chloride.

    Science.gov (United States)

    Portillo, F; Carrasco, M; Vallo, J J

    1998-03-01

    The role of different hypothalamic nuclei, particularly the paraventricular nucleus (PVN), in the control of food intake and feeding behaviour is well known. It is also well established that lithium chloride (LiCl) causes various disorders in feeding behaviour. In this study, we analyzed the precise distribution of hypothalamic neurons activated by i.p. LiCl administration (LCA neurons) and compared it to that of hypothalamic neurons which project to autonomic preganglionic levels (HAP neurons). We also analysed the possibility that some neurons belong to both populations of nerve cells. To this end, a multiple-labelling technique, using two retrograde fluorescent tracers together with c-Fos-like immunohistochemistry, was performed. Fast Blue was injected in the dorsal motor nucleus of the vagus and Fluorogold (FG) in the thoracic intermedial-lateral cell column, to trace parasympathetic and sympathetic pathways, respectively. LiCl was used as stimulus for c-Fos-like immunohistochemistry. HAP neurons were located mainly in the dorsal, ventral and lateral regions of the parvocellular PVN, while LCA neurons were observed predominantly in the magnocellular region of the PVN rostrally to HAP neurons. A significant number of FG/Fos double-labelled neurons were located in the dorsal parvocellular subnucleus of the PVN (dp) in the LiCl-stimulated rats. We concluded that there is a clear segregation of LCA neurons from HAP neurons within the PVN. The presence of FG/Fos double-labelled neurons in the dp suggests that this nucleus could mediate a sympathetic response after LiCl administration.

  20. Retrograde study of CART- or NPY-neuronal projection from the hypothalamic arcuate nucleus to the dorsal raphe and/or the locus coeruleus in the rat.

    Science.gov (United States)

    Yoon, Ye S; Lee, Ji S; Lee, Hyun S

    2013-06-26

    The present study was designed to reveal cocaine- and amphetamine-regulated transcript (CART)- or neuropeptide Y (NPY)-immunoreactive neuronal projections from the hypothalamic arcuate nucleus (Arc) to the dorsal raphe (DR) and/or the locus coeruleus (LC) in the rat. Our results demonstrated that CART or NPY axon terminals formed close appositions to the neuronal profiles in the DR and the LC. Thus, arcuate sections were immunostained for the CART or NPY after the injections of green RetroBeads(™) into the DR and red tracer into the LC (or vice versa). First, retrogradely-labeled CART cells were mainly observed in the lateral Arc without colchicine. Of the total population of arcuate CART neurons, DR- and LC-projecting cells were 5.7% ± 0.9% and 6.6% ± 0.7%, respectively. In addition, a subset (3.3% ± 0.7%) of CART neurons provided divergent axon collaterals to the DR and the LC. Second, retrogradely-labeled NPY cells were observed in lateral or ventral borders of the medial Arc only after colchicine injection. Of the entire NPY cell population, DR- and LC-projecting neurons were 1.5% ± 0.3% and 1.3% ± 0.3%, respectively. Only a scanty proportion (0.1% ± 0.0%) sent axon collaterals to the DR and the LC. These observations suggested that arcuate CART or NPY system might have a potential influence on the brainstem monoaminergic nuclei, modulating their roles in feeding, nociception, emotional behaviors, arousal, and stress responses. Furthermore, a portion of arcuate CART neurons (along with only a few NPY cells) sending divergent axon collaterals to the DR/LC might have a simultaneous (and possibly more efficient) way to exert their specific influences on the monoaminergic nuclei.

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

    Directory of Open Access Journals (Sweden)

    Stephane ePages

    2011-10-01

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

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    Elisenda eTibau

    2013-12-01

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

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

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  5. 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

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

    Directory of Open Access Journals (Sweden)

    Sui-Yi Xu

    2012-01-01

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

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

    Science.gov (United States)

    McCarthy, K D; Partlow, L M

    1976-09-24

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

  8. 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.

  9. Prenatal exposure to ethanol stimulates hypothalamic CCR2 chemokine receptor system: Possible relation to increased density of orexigenic peptide neurons and ethanol drinking in adolescent offspring.

    Science.gov (United States)

    Chang, G-Q; Karatayev, O; Leibowitz, S F

    2015-12-01

    Clinical and animal studies indicate that maternal consumption of ethanol during pregnancy increases alcohol drinking in the offspring. Possible underlying mechanisms may involve orexigenic peptides, which are stimulated by prenatal ethanol exposure and themselves promote drinking. Building on evidence that ethanol stimulates neuroimmune factors such as the chemokine CCL2 that in adult rats is shown to colocalize with the orexigenic peptide, melanin-concentrating hormone (MCH) in the lateral hypothalamus (LH), the present study sought to investigate the possibility that CCL2 or its receptor CCR2 in LH is stimulated by prenatal ethanol exposure, perhaps specifically within MCH neurons. Our paradigm of intraoral administration of ethanol to pregnant rats, at low-to-moderate doses (1 or 3g/kg/day) during peak hypothalamic neurogenesis, caused in adolescent male offspring twofold increase in drinking of and preference for ethanol and reinstatement of ethanol drinking in a two-bottle choice paradigm under an intermittent access schedule. This effect of prenatal ethanol exposure was associated with an increased expression of MCH and density of MCH(+) neurons in LH of preadolescent offspring. Whereas CCL2(+) cells at this age were low in density and unaffected by ethanol, CCR2(+) cells were dense in LH and increased by prenatal ethanol, with a large percentage (83-87%) identified as neurons and found to colocalize MCH. Prenatal ethanol also stimulated the genesis of CCR2(+) and MCH(+) neurons in the embryo, which co-labeled the proliferation marker, BrdU. Ethanol also increased the genesis and density of neurons that co-expressed CCR2 and MCH in LH, with triple-labeled CCR2(+)/MCH(+)/BrdU(+) neurons that were absent in control rats accounting for 35% of newly generated neurons in ethanol-exposed rats. With both the chemokine and MCH systems believed to promote ethanol consumption, this greater density of CCR2(+)/MCH(+) neurons in the LH of preadolescent rats suggests that

  10. 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.

  11. Dual projections of single orexin- or CART-immunoreactive, lateral hypothalamic neurons to the paraventricular thalamic nucleus and nucleus accumbens shell in the rat: Light microscopic study.

    Science.gov (United States)

    Lee, Eun Y; Lee, Hyun S

    2016-03-01

    The paraventricular thalamic nucleus (PVT) is a major relay station to the limbic forebrain areas such as the nucleus accumbens shell (AcbSh). Both PVT and AcbSh are known to receive feeding/arousal-related peptidergic fibers including orexin (ORX) and cocaine- and amphetamine-regulated transcript (CART) peptide. In the first series of experiments, we examined the peptidergic fiber distribution in the AcbSh; the density of ORX (or CART) fibers in the AcbSh was substantially lower than that in the PVT. At the light microscopic level, ORX (or CART) terminals formed close appositions to choline acetyltransferase (ChAT)-, glutamate decarboxylase (GAD)-, or enkephalin (Enk)-immunoreactive neuronal elements in the AcbSh. In the second series of experiments, we addressed the question of whether single ORX (or CART) cells in the hypothalamus provided divergent axon collaterals to the PVT and AcbSh. ORX neurons with dual projections were found in the medial, central, and lateral subdivisions of the lateral hypothalamus (LH), which amounted to an average of 1.6% of total ORX cells. CART neurons with divergent axon collaterals were observed in the LH, zona incerta, dorsal hypothalamic area, and retrochiasmatic nucleus, which represented a mean of 2.5% of total CART cells. None of arcuate CART cells sent dual projections. These data suggested that a portion of ORX (or CART) neurons in the hypothalamus, via divergent axon collaterals, might concurrently modulate the activity of PVT and AcbSh cells to affect feeding and drug-seeking behaviors.

  12. Lateral hypothalamic area orexin-A influence the firing activity of gastric distension-sensitive neurons and gastric motility in rats.

    Science.gov (United States)

    Hao, Heling; Luan, Xiao; Guo, Feifei; Sun, Xiangrong; Gong, Yanling; Xu, Luo

    2016-06-01

    The orexins system consists of two G-protein coupled receptors (the orexin-1 and the orexin-2 receptor) and two neuropeptides, orexin-A and orexin-B. Orexin-A is an excitatory neuropeptide that regulates arousal, wakefulness and appetite. Recent studies have shown that orexin-A may promote gastric motility. We aim to explore the effects of orexin-A on the gastric -distension (GD) sensitive neurons and gastric motility in the lateral hypothalamic area (LHA), and the possible regulation by the paraventricular nucleus (PVN). Extracellular single unit discharges were recorded and the gastric motility was monitored by administration of orexin-A into the LHA and electrical stimulation of the PVN. There were GD neurons in the LHA, and administration of orexin-A to the LHA could increase the firing rate of both GD-excitatory (GD-E) and GD-inhibited (GD-I) neurons. The gastric motility was significantly enhanced by injection of orexin-A into the LHA with a dose dependent manner, which could be completely abolished by pre-treatment with orexin-A receptor antagonist SB334867. Electrical stimulation of the PVN could significantly increase the firing rate of GD neurons responsive to orexin-A in the LHA as well as promote gastric motility of rats. However, those effects could be partly blocked by pre-treatment with SB334867 in the LHA. It is suggested that orexin-A plays an important role in promoting gastric motility via LHA. The PVN may be involved in regulation of LHA on gastric motility.

  13. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-02-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  16. Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure.

    Science.gov (United States)

    Hussy, N; Deleuze, C; Desarménien, M G; Moos, F C

    2000-10-01

    Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.

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

    DEFF Research Database (Denmark)

    Bakmand, Tanya; Waagepetersen, Helle S.

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

  18. 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...

  19. 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.

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

    Science.gov (United States)

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

    2006-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Sun Hee Kim

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

  2. Parvalbumin-Neurons of the Ventrolateral Hypothalamic Parvafox Nucleus Receive a Glycinergic Input: A Gene-Microarray Study

    Science.gov (United States)

    Szabolcsi, Viktoria; Albisetti, Gioele W.; Celio, Marco R.

    2017-01-01

    The ventrolateral hypothalamic parvafox (formerly called PV1-Foxb1) nucleus is an anatomical entity of recent discovery and unknown function. With a view to gaining an insight into its putative functional role(s), we conducted a gene-microarray analysis and, armed with the forthcoming data, controlled the results with the Allen databases and the murine BrainStars (B*) database. The parvafox nucleus was specifically sampled by laser-capture microdissection and the transcriptome was subjected to a microarray analysis on Affymetrix chips. Eighty-two relevant genes were found to be potentially more expressed in this brain region than in either the cerebral cortex or the hippocampus. When the expression patterns of these genes were counterchecked in the Allen-Database of in-situ hybridizations and in the B*-microarray database, their localization in the parvafox region was confirmed for thirteen. For nine novel genes, which are particularly interesting because of their possible involvement in neuromodulation, the expression was verified by quantitative real time-PCR. Of particular functional importance may be the occurrence of glycine receptors, the presence of which indicates that the activity of the parvafox nucleus is under ascending inhibitory control. PMID:28167900

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

    Institute of Scientific and Technical Information of China (English)

    WANG Jian; LU Chuanzhen; JIANG Yuping

    2000-01-01

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

  4. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Xin Tang; Yiren Chen; Xiaosong Gu; Fei Ding

    2009-01-01

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

  6. 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.

  7. Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

    Science.gov (United States)

    Fick, Laura J; Fick, Gordon H; Belsham, Denise D

    2011-09-30

    The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

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

    Science.gov (United States)

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

    2009-04-01

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

  9. MCT2 expression and lactate influx in anorexigenic and orexigenic neurons of the arcuate nucleus.

    Directory of Open Access Journals (Sweden)

    Christian Cortes-Campos

    Full Text Available Hypothalamic neurons of the arcuate nucleus control food intake, releasing orexigenic and anorexigenic neuropeptides in response to changes in glucose concentration. Several studies have suggested that the glucosensing mechanism is governed by a metabolic interaction between neurons and glial cells via lactate flux through monocarboxylate transporters (MCTs. Hypothalamic glial cells (tanycytes release lactate through MCT1 and MCT4; however, similar analyses in neuroendocrine neurons have yet to be undertaken. Using primary rat hypothalamic cell cultures and fluorimetric assays, lactate incorporation was detected. Furthermore, the expression and function of MCT2 was demonstrated in the hypothalamic neuronal cell line, GT1-7, using kinetic and inhibition assays. Moreover, MCT2 expression and localization in the Sprague Dawley rat hypothalamus was analyzed using RT-PCR, in situ hybridization and Western blot analyses. Confocal immunohistochemistry analyses revealed MCT2 localization in neuronal but not glial cells. Moreover, MCT2 was localized to ∼90% of orexigenic and ~60% of anorexigenic neurons as determined by immunolocalization analysis of AgRP and POMC with MCT2-positives neurons. Thus, MCT2 distribution coupled with lactate uptake by hypothalamic neurons suggests that hypothalamic neurons control food intake using lactate to reflect changes in glucose levels.

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

    National Research Council Canada - National Science Library

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

    2013-01-01

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

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

    Science.gov (United States)

    Shimizu, Saori; Abt, Anna; Meucci, Olimpia

    2011-11-12

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Shani Stern

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

  14. 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.

  15. Cultured Vestibular Ganglion Neurons Demonstrate Latent HSV1 Reactivation

    Science.gov (United States)

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

    2013-01-01

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

  16. 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.

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

    Science.gov (United States)

    Ziemińska, Elżbieta; Strużyńska, Lidia

    2016-02-01

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

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

    OpenAIRE

    Cai, Dongsheng; Liu, Tiewen

    2011-01-01

    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 o...

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

    Science.gov (United States)

    Turrigiano, G; LeMasson, G; Marder, E

    1995-05-01

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

  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.

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

    Science.gov (United States)

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

    2010-11-24

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

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

    Science.gov (United States)

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

    1992-05-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

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

    Science.gov (United States)

    Shi, Ying; Zhou, Mi; Jiang, Min

    2013-02-25

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

  5. Tyrosine hydroxylase is short-term regulated by the ubiquitin-proteasome system in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats: possible implications in hypertension.

    Directory of Open Access Journals (Sweden)

    Nadia A Congo Carbajosa

    Full Text Available Aberrations in the ubiquitin-proteasome system (UPS are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.

  6. Age-Dependent Glutamate Induction of Synaptic Plasticity in Cultured Hippocampal Neurons

    Science.gov (United States)

    Ivenshitz, Miriam; Segal, Menahem; Sapoznik, Stav

    2006-01-01

    A common denominator for the induction of morphological and functional plasticity in cultured hippocampal neurons involves the activation of excitatory synapses. We now demonstrate massive morphological plasticity in mature cultured hippocampal neurons caused by a brief exposure to glutamate. This plasticity involves a slow, 70%-80% increase in…

  7. Electrophysiology of embryonic, adult and aged rat hippocampal neurons in serum-free culture.

    Science.gov (United States)

    Evans, M S; Collings, M A; Brewer, G J

    1998-01-31

    Methods were recently developed for culturing neurons from adult rat hippocampus using the serum-free medium Neurobasal with B27 supplement. To determine whether adult cultured neurons have normal electrical properties, we studied cultures from rats of three age groups: (1) embryonic; (2) 10-11 months old and (3) 35-36 months old. Neurons had a polarized morphology with a large branching apical dendrite and small basal dendrites. Mean resting potentials were similar in the three age groups. All neurons had nonlinear current-voltage relationships, indicating the presence of voltage-sensitive ion channels. Most neurons had a voltage-sensitive inward current followed by a sustained voltage-sensitive outward current. Tetrodotoxin blocked the inward current, which is likely to be a sodium current. The sustained outward current, which is likely to be a potassium current, reversed at -71 mV. Most neurons exhibited anomalous rectification. Calcium currents were present in both embryonic and adult neurons. Embryonic neurons would sometimes fire multiple action potentials but adult neurons fired only single action potentials. Our results indicate that both embryonic and adult cultured neurons retain a clearly neuronal electrophysiological phenotype in Neurobasal/B27 serum-free medium.

  8. Susceptibility of primary culture neurons from rats of different ages to encephalomyocarditis (EMC) virus infection.

    Science.gov (United States)

    Su, Weiping; Ikegami, Hisashi; Nakayama, Yumi; Suzuki, Kazuhiko; Katayama, Kei-ichi; Nakayama, Hiroyuki; Doi, Kunio

    2003-10-01

    The changes in susceptibility of neurons to the D variant of EMC virus (EMC-D) (10(6) PFU/well) were investigated in developing hippocampal primary cultures from postnatal days of 1, 7, and 56 Fischer 344 rats (P1, P7, and P56) for up to 12 h after infection (12 HAI). The virus titer of primary culture neurons increased at 1 HAI, decreased at 2 HAI, increased at 3 HAI, peaked at 8 HAI, and decreased at 12 HAI in all age groups. The titers at 1 and 8 HAI were lowest in P56 cultures. The virus titer of neurons was always higher than that of culture media, especially at 1 HAI, in P1 cultures, whereas the former was lower than the latter from 2 to 3 HAI in P7 cultures and from 2 to 4 HAI in P56 cultures, respectively. Signals of viral RNA detected by in situ hybridization were first observed in the peripheral cytoplasm of neurons at 1 HAI in P1 and P7 cultures and at 4 HAI in P56 cultures, respectively. The signals spread to a large or whole area of cytoplasm and also to processes thereafter. The number of viral RNA-positive neurons and the amount of signals decreased with age. The present results indicated that the susceptibility of primary culture neurons to EMC-D decreased with age but viral replication still occurred in P56 cultures.

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

    Science.gov (United States)

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

    1999-12-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    Science.gov (United States)

    Mattson, M P; Kater, S B

    1988-01-01

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

  12. Encoding of High Frequencies Improves with Maturation of Action Potential Generation in Cultured Neocortical Neurons

    Science.gov (United States)

    Nikitin, Evgeny S.; Bal, Natalia V.; Malyshev, Aleksey; Ierusalimsky, Victor N.; Spivak, Yulia; Balaban, Pavel M.; Volgushev, Maxim

    2017-01-01

    The ability of neocortical neurons to detect and encode rapid changes at their inputs is crucial for basic neuronal computations, such as coincidence detection, precise synchronization of activity and spike-timing dependent plasticity. Indeed, populations of cortical neurons can respond to subtle changes of the input very fast, on a millisecond time scale. Theoretical studies and model simulations linked the encoding abilities of neuronal populations to the fast onset dynamics of action potentials (APs). Experimental results support this idea, however mechanisms of fast onset of APs in cortical neurons remain elusive. Studies in neuronal cultures, that are allowing for accurate control over conditions of growth and microenvironment during the development of neurons and provide better access to the spike initiation zone, may help to shed light on mechanisms of AP generation and encoding. Here we characterize properties of AP encoding in neocortical neurons grown for 11–25 days in culture. We show that encoding of high frequencies improves upon culture maturation, which is accompanied by the development of passive electrophysiological properties and AP generation. The onset of APs becomes faster with culture maturation. Statistical analysis using correlations and linear model approaches identified the onset dynamics of APs as a major predictor of age-dependent changes of encoding. Encoding of high frequencies strongly correlated also with the input resistance of neurons. Finally, we show that maturation of encoding properties of neurons in cultures is similar to the maturation of encoding in neurons studied in slices. These results show that maturation of AP generators and encoding is, to a large extent, determined genetically and takes place even without normal micro-environment and activity of the whole brain in vivo. This establishes neuronal cultures as a valid experimental model for studying mechanisms of AP generation and encoding, and their maturation. PMID

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

    Science.gov (United States)

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

    2015-06-07

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

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

    Directory of Open Access Journals (Sweden)

    Chin Wai Hui

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

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

    Directory of Open Access Journals (Sweden)

    Chao-Yin Chen

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

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

    Science.gov (United States)

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

    2014-06-01

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

  17. Increased hypothalamic serotonin turnover in inflammation-induced anorexia

    OpenAIRE

    Dwarkasing, J.T.; Witkamp, R F; Boekschoten, M.V.; Laak, ter, H.J.; Heins, M.S.; Norren, van, K.

    2016-01-01

    Background 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....

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    1994-09-01

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

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

    Directory of Open Access Journals (Sweden)

    Belrose Jillian C

    2012-04-01

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

  1. Network dynamics of cultured hippocampal neurons in a multi-electrode array

    Science.gov (United States)

    Taguchi, Takahisa; Kudoh, Suguru N.

    2005-02-01

    The neurons in dissociation culture autonomously re-organized their functional neuronal networks, after the process for elongating neurites and establishing synaptic connections. The spatio-temporal patterns of activity in the networks might be a reflection of functional neuron assemblies. The functional connections were dynamically modified by synaptic potentiation and the process may be required for reorganization of the functional group of neurons. Such neuron assemblies are critical for information processing in brain. To visualize the functional connections between neurons, we have analyzed the autonomous activity of synaptically induced action potentials in the living neuronal networks on a multi-electrode array, using "connection map analysis" that we developed for this purpose. Moreover, we designed aan original wide area covering electrode array and succeeded in recording spontaneous action potentials from wider area than commercial multi electrode arrays.

  2. Simulation of Code Spectrum and Code Flow of Cultured Neuronal Networks.

    Science.gov (United States)

    Tamura, Shinichi; Nishitani, Yoshi; Hosokawa, Chie; Miyoshi, Tomomitsu; Sawai, Hajime

    2016-01-01

    It has been shown that, in cultured neuronal networks on a multielectrode, pseudorandom-like sequences (codes) are detected, and they flow with some spatial decay constant. Each cultured neuronal network is characterized by a specific spectrum curve. That is, we may consider the spectrum curve as a "signature" of its associated neuronal network that is dependent on the characteristics of neurons and network configuration, including the weight distribution. In the present study, we used an integrate-and-fire model of neurons with intrinsic and instantaneous fluctuations of characteristics for performing a simulation of a code spectrum from multielectrodes on a 2D mesh neural network. We showed that it is possible to estimate the characteristics of neurons such as the distribution of number of neurons around each electrode and their refractory periods. Although this process is a reverse problem and theoretically the solutions are not sufficiently guaranteed, the parameters seem to be consistent with those of neurons. That is, the proposed neural network model may adequately reflect the behavior of a cultured neuronal network. Furthermore, such prospect is discussed that code analysis will provide a base of communication within a neural network that will also create a base of natural intelligence.

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  4. Human Neuron Cultures: Micropatterning Facilitates the Long-Term Growth and Analysis of iPSC-Derived Individual Human Neurons and Neuronal Networks (Adv. Healthcare Mater. 15/2016).

    Science.gov (United States)

    Burbulla, Lena F; Beaumont, Kristin G; Mrksich, Milan; Krainc, Dimitri

    2016-08-01

    Dimitri Krainc, Milan Mrksich, and co-workers demonstrate the utility of microcontact printing technology for culturing of human neurons in defined patterns over extended periods of time on page 1894. This approach facilitates studies of neuronal development, cellular trafficking, and related mechanisms that require assessment of individual neurons and neuronal networks.

  5. 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...

  6. Caspase-Mediated Apoptosis in Sensory Neurons of Cultured Dorsal Root Ganglia in Adult Mouse

    Directory of Open Access Journals (Sweden)

    Hamid Reza Momeni

    2013-01-01

    Full Text Available Objective: Sensory neurons in dorsal root ganglia (DRG undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG.Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342 and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM and immunohistochemistry for activated caspase-3 were used.Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours.Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

  7. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  9. 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....

  10. Effects of GSM 1800 MHz on dendritic development of cultured hippo-campal neurons

    Institute of Scientific and Technical Information of China (English)

    Wei NING; Shu-jun XU; Huai CHIANG; Zheng-ping XU; Su-ya ZHOU; Wei YANG; Jian-hong LUO

    2007-01-01

    Aim: To evaluate the effects of global system for mobile communications (GSM)1800 MHz microwaves on dendritic filopodia, dendritic arborization, and spine maturation during development in cultured hippocampal neurons in rats. Methods: The cultured hippocampal neurons were exposed to GSM 1800 MHz microwaves with 2.4 and 0.8 W/kg, respectively, for 15 min each day from 6 days in vitro (DIV6) to DIV14. The subtle structures of dendrites were displayed by transfection with farnesylated enhanced green fluorescent protein (F-GFP) and GFP-actin on DIV5 into the hippocampal neurons. Results: There was a significant decrease in the density and mobility of dendritic filopodia at DIV8 and in the density of mature spines at DIV14 in the neurons exposed to GSM 1800 MHz microwaves with 2.4 W/kg. In addition, the average length of dendrites per neuron at DIV10 and DIV14 was decreased, while the dendritic arborization was unaltered in these neurons. However, there were no significant changes found in the neurons ex- posed to the GSM 1800 MHz microwaves with 0.8 W/kg. Conclusion: These data indicate that the chronic exposure to 2.4 W/kg GSM 1800 MHz micro- waves during the early developmental stage may affect dendritic development and the formation of excitatory synapses of hippocampal neurons in culture.

  11. Activity of protease-activated receptors in primary cultured human myenteric neurons

    Directory of Open Access Journals (Sweden)

    Eva Maria Kugler

    2012-09-01

    Full Text Available Activity of the four known protease-activated receptors (PARs has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (AP. Application of the PAR1-AP (TFLLR or PAR4-AP (GYPGQV evoked spike discharge in 79% or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs of PAR1/PAR2 in 51%, PAR1/PAR4 in 43% and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.

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

    CERN Document Server

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

    2014-01-01

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

  13. Firing patterns of long-term cultured neuronal network on multi-electrode array

    Institute of Scientific and Technical Information of China (English)

    LI Xiangning; ZHOU Wei; LIU Man; ZENG Shaoqun; LUO Qingming

    2006-01-01

    Spontaneous neuronal activity plays an important role in the development and plasticity of brain. To explore the developmental changes in the firing pattern of the neuronal networks in vitro, the hippocampal neurons were cultured on the multi-microelectrode array dish for over 14 weeks and the spontaneous activity was recorded. The results showed that random firing was observed in the 1st week and transformed into synchronized activity after two weeks, then tightly synchronized activity appeared in week 2 to 7 and finally the activities transformed into the random firing pattern. These results suggested three stages in the long-term development of neuronal network in vitro: the stage for connection, the stage of synchronized activity and the mature stage. Synchronized firing shown by spontaneous activity was an important phenomenon in high density cultured neuronal network and transformed patterns during development.

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

    Science.gov (United States)

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

    1995-09-01

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

  15. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Jing Wang; Yanjun Zhang; Shuai Du; Mixia Zhang

    2011-01-01

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

  17. 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.

  18. 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.

  19. 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.

  20. Androgen receptors and estrogen receptors are colocalized in male rat hypothalamic and limbic neurons that express Fos immunoreactivity induced by mating.

    Science.gov (United States)

    Gréco, B; Edwards, D A; Michael, R P; Clancy, A N

    1998-01-01

    Conversion of testosterone into estradiol is important for male rat sexual behavior, and both steroids probably contribute to mating. The distributions of neurons containing androgen receptors (AR) and estrogen receptors (ER) overlap, and many AR-immunoreactive (AR-ir) neurons express Fos immunoreactivity (Fos-ir) induced by mating. Because mating-induced Fos-ir in the male rat occurs mainly in AR-ir neurons, and because both steroids are important for mating, we hypothesized that (i) AR-ir and ER-ir are colocalized and that (ii) some of these neurons are activated during mating. We examined, in adjacent sections from the medial preoptic area (MPN) through the central tegmental field (CTF), the expression of ER-ir in: (i) AR-ir-containing neurons, and (ii) Fos-ir-expressive neurons. PG21 anti-AR, OA-11-824 anti-c-fos, H222 or 1D5 anti-ER primary antibodies were visualized, respectively, with cyanine-conjugated, fluorescein- or cyanine-conjugated, and fluorescein-conjugated secondary antibodies in male rats which were killed 1 h after ejaculating with a receptive female. In MPN, bed nucleus of the stria terminalis (BNST), and medial amygdala (MEA), 80-90% of ER-ir labeling occurred in AR-ir-positive neurons but only about 30% of AR-ir neurons were ER-ir-positive. No ER-ir was found in the CTF. This suggests the presence of three types of brain neurons sensitive to gonadal steroid hormones: neurons sensitive to androgens only, neurons sensitive to both androgens and estrogens, and neurons sensitive to estrogens only. About 50% of ER-ir labeling occurred in cells expressing mating-induced Fos-ir but only about 30% of Fos-ir neurons were ER-ir-positive. These findings suggest that, in the MPN, at least two different neuronal populations are activated during mating: the first contains AR-ir only and the second contains AR-ir and ER-ir. In the BNST and MEA, at least three hormonally sensitive populations are activated during mating: the two described above plus a third

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2016-04-06

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  4. Single pulse responses in cultured neuronal networks to describe connectivity

    NARCIS (Netherlands)

    Feber, le Joost; Corner, Michael

    2011-01-01

    Synaptic connections between neurons play a crucial role in cognitive processes like learning and memory. In recent work we developed a method, using conditional firing probability (CFP analysis), to estimate functional connectivity in terms of strength and latency, and here we further explored on t

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Hubbard Kyle S

    2012-10-01

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

  7. Age-related alterations in hypothalamic kisspeptin, neurokinin B, and dynorphin neurons and in pulsatile LH release in female and male rats.

    Science.gov (United States)

    Kunimura, Yuyu; Iwata, Kinuyo; Ishigami, Akihito; Ozawa, Hitoshi

    2017-02-01

    Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) decreases during aging. Kisspeptin (encoded by Kiss1) neurons in the arcuate nucleus coexpress neurokinin B (Tac3) and dynorphin (Pdyn) and are critical for regulating the GnRH/LH pulse. We therefore examined kisspeptin neurons by histochemistry and pulsatile LH release in rats aged 2-3 (Young), 12-13 (Young-Middle), 19-22 (Late-Middle), and 24-26 (Old) months. Total LH concentrations, sampled for 3 hours, decreased in both sexes with aging. In females, numbers of Tac3 and Pdyn neurons were significantly reduced in all aging rats, and numbers of Kiss1 neurons were significantly reduced in Late-Middle and Old rats. In males, numbers of all 3 neuron-types were significantly decreased in all aging rats. GnRH agonist induced LH release in all animals; however, the increased LH concentration in all aging rats was less than that in Young rats. These results suggest that expression of each gene in kisspeptin neurons may be controlled individually during aging, and that reduction of their expression or change in pituitary responsiveness may cause attenuated pulsatile LH secretion.

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

    Science.gov (United States)

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

    2016-01-01

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

  9. Effect of polybrominated diphenyl ether on development of cultured hippocampal neuron

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Polybrominated diphenyl ether (PBDE) is a persistently environmental pollutant ubiquitously found in wildlife and humans. Although concern on PBDE's toxic effects is steadily increasing, its action on the central nervous system (CNS) remains largely unknown. To address this issue, the present study examined the development inhibition of PBDE in neurons. The primary cultured hippocampal neurons of rat were exposed to the commercial decabromodiphenyl ether (deca-BDE), and the neurite length, bifurcation, and synapse formation and maturation were evaluated, based on the confocal microscope imaging. The results showed that the development inhibition in neurons occurred at 15 μmol/L, indicating that PBDE is a potent neurotoxicant and it might obviously inhibit the development of cultured neurons.

  10. Effect of polybrominated diphenyl ether on development of cultured hippocampal neuron

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Polybrominated diphenyl ether (PBDE) is a persistently environmental pollutant ubiquitously found in wildlife and humans. Although concern on PBDE’s toxic effects is steadily increasing, its action on the central nervous system (CNS) remains largely unknown. To address this issue, the present study ex- amined the development inhibition of PBDE in neurons. The primary cultured hippocampal neurons of rat were exposed to the commercial decabromodiphenyl ether (deca-BDE), and the neurite length, bi- furcation, and synapse formation and maturation were evaluated, based on the confocal microscope imaging. The results showed that the development inhibition in neurons occurred at 15 μmol/L, indi- cating that PBDE is a potent neurotoxicant and it might obviously inhibit the development of cultured neurons.

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

    Science.gov (United States)

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

    2003-01-01

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

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

    Science.gov (United States)

    Stachniak, Tevye J E; Bourque, Charles W

    2006-07-01

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

  13. [Hypothalamic dysfunction in obesity].

    Science.gov (United States)

    van de Sande-Lee, Simone; Velloso, Licio A

    2012-08-01

    Obesity, defined as abnormal or excessive fat accumulation that may impair life quality, is one of the major public health problems worldwide. It results from an imbalance between food intake and energy expenditure. The control of energy balance in animals and humans is performed by the central nervous system (CNS) by means of neuroendocrine connections, in which circulating peripheral hormones, such as leptin and insulin, provide signals to specialized neurons of the hypothalamus reflecting body fat stores, and induce appropriate responses to maintain the stability of these stores. The majority of obesity cases are associated with central resistance to both leptin and insulin actions. In experimental animals, high-fat diets can induce an inflammatory process in the hypothalamus, which impairs leptin and insulin intracellular signaling pathways, and results in hyperphagia, decreased energy expenditure and, ultimately, obesity. Recent evidence obtained from neuroimaging studies and assessment of inflammatory markers in the cerebrospinal fluid of obese subjects suggests that similar alterations may be also present in humans. In this review, we briefly present the mechanisms involved with the loss of homeostatic control of energy balance in animal models of obesity, and the current evidence of hypothalamic dysfunction in obese humans.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2016-09-06

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

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

    Science.gov (United States)

    Kern, M; Audesirk, T; Audesirk, G

    1993-01-01

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

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

    Science.gov (United States)

    Nishikawa, Shin; Hirashima, Naohide; Tanaka, Masahiko

    2014-09-01

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

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

    Science.gov (United States)

    Nadeau, H.; Lester, H. A.

    2002-01-01

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

  19. 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.

  20. Identification and dynamics of spontaneous burst initiation zones in unidimensional neuronal cultures.

    Science.gov (United States)

    Feinerman, Ofer; Segal, Menahem; Moses, Elisha

    2007-04-01

    Spontaneous activity is typical of in vitro neural networks, often in the form of large population bursts. The origins of this activity are attributed to intrinsically bursting neurons and to noisy backgrounds as well as to recurrent network connections. Spontaneous activity is often observed to emanate from localized sources or initiation zones, propagating from there to excite large populations of neurons. In this study, we use unidimensional cultures to overcome experimental difficulties in identifying initiation zones in vivo and in dissociated two-dimensional cultures. We found that spontaneous activity in these cultures is initiated exclusively in localized zones that are characterized by high neuronal density but also by recurrent and inhibitory network connections. We demonstrate that initiation zones compete in driving network activity in a winner-takes-most scenario.

  1. 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

    Although intrinsic neuronal properties in invertebrates are well known to undergo specific adaptive changes in culture, long-term adaptation of similar properties in mature vertebrate neurons remain poorly understood. To investigate this, we used an organotypic slice preparation from the spinal...... 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...... receptor antagonists or by high extracellular Mg2+ concentrations. However, in contrast with results from motoneurons in the acute slice, NMDA-induced oscillations in culture did not depend on CaV1.3 channel activation as they still remained after nifedipine application. Other CaV1.3 channel...

  2. Effects of hypothalamic neurodegeneration on energy balance.

    Directory of Open Access Journals (Sweden)

    Allison Wanting Xu

    2005-12-01

    Full Text Available Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp or proopiomelanocortin (Pomc, neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.

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

    Science.gov (United States)

    Strasser, U; Fischer, G

    1995-04-01

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

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

    Science.gov (United States)

    Saijilafu; Zhou, Feng-Quan

    2012-08-17

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

  5. Hypothalamic neurosecretory and circadian vasopressinergic neuronal systems in the blind cone-rod homeobox knock out mouse (Crx(-/-) ) and the 129sv wild type mouse

    DEFF Research Database (Denmark)

    Rovsing, Louise; Rath, Martin Fredensborg; Møller, Morten

    2013-01-01

    Vasopressin (AVP) is both a neuroendocrine hormone located in magnocellular neurosecretory neurons of the hypothalamus of mammals but also a neurotransmitter/neuromodulator in the parvocellular suprachiasmatic nucleus (SCN). The SCN is the endogenous clock of the brain and exhibits a prominent...... magnocellular and parvocellular vasopressinergic systems in both genotypes. We here present a detailed mapping of all classical hypothalamo-pituitary and accessory magnocellular nuclei and neurons in the hypothalamus by use of immunohistochemistry and in situ hybridization in both genotypes. Semiquantitative...... at late day time and nadir during the dark in both the Crx(-/-) and the wild type mouse. None of the magnocellular neurosecretory neurons exhibited a diurnal vasopressin expression. Light stimulation of both genotypes during the dark period did not change the Avp-expression in the SCN. This shows that Avp...

  6. 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.

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

    Science.gov (United States)

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

    2015-07-01

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

  8. Concentration-Dependent Dual Role of Thrombin In Protection of Cultured Rat Cortical Neurons

    Science.gov (United States)

    García, Paul S.; Ciavatta, Vincent T.; Fidler, Jonathan A.; Woodbury, Anna; Levy, Jerrold H.; Tyor, William R.

    2015-01-01

    Background Thrombin’s role in the nervous system is not well understood. Under conditions of blood-brain barrier compromise (e.g., neurosurgery or stroke), thrombin can result in neuroapoptosis and the formation of glial scars. Despite this, preconditioning with thrombin has been found to be neuroprotective in models of cerebral ischemia and intracerebral hemorrhage. Methods We investigated the effects of physiologically relevant concentrations of thrombin on cortical neurons using two culture-based assays. We examined thrombin’s effect on neurites by quantitative analysis of fluorescently labeled neurons. To characterize thrombin’s effects on neuron survival, we spectrophotometrically measured changes in enzymatic activity. Using receptor agonists and thrombin inhibitors, we separately examined the role of thrombin and its receptor in neuroprotection. Results We found that low concentrations of thrombin (1 nM) enhances neurite growth and branching, neuron viability, and protects against excitotoxic damage. In contrast, higher concentrations of thrombin (100 nM) are potentially detrimental to neuronal health as evidenced by inhibition of neurite growth. Lower concentrations of thrombin resulted in equivalent neuroprotection as the antifibrinolytic, aprotinin, and the direct thrombin inhibitor, argatroban. Interestingly, exogenous application of the species-specific thrombin inhibitor, antithrombin III, was detrimental to neuronal health; suggesting that some endogenous thrombin is necessary for optimal neuron health in our culture system. Activation of the thrombin receptor, protease-activated receptor - 1 (PAR-1), via micromolar concentrations of the thrombin receptor agonist peptide, TRAP, did not adversely affect neuronal viability. Conclusions An optimal concentration of thrombin exists to enhance neuronal health. Neurotoxic effects of thrombin do not involve activation of PAR receptors and thus separate pharmacologic manipulation of thrombin’s receptor

  9. 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.

  10. Pharmacodynamics of potassium channel openers in cultured neuronal networks.

    Science.gov (United States)

    Wu, Calvin; V Gopal, Kamakshi; Lukas, Thomas J; Gross, Guenter W; Moore, Ernest J

    2014-06-01

    A novel class of drugs - potassium (K(+)) channel openers or activators - has recently been shown to cause anticonvulsive and neuroprotective effects by activating hyperpolarizing K(+) currents, and therefore, may show efficacy for treating tinnitus. This study presents measurements of the modulatory effects of four K(+) channel openers on the spontaneous activity and action potential waveforms of neuronal networks. The networks were derived from mouse embryonic auditory cortices and grown on microelectrode arrays. Pentylenetetrazol was used to create hyperactivity states in the neuronal networks as a first approximation for mimicking tinnitus or tinnitus-like activity. We then compared the pharmacodynamics of the four channel activators, retigabine and flupirtine (voltage-gated K(+) channel KV7 activators), NS1619 and isopimaric acid ("big potassium" BK channel activators). The EC50 of retigabine, flupirtine, NS1619, and isopimaric acid were 8.0, 4.0, 5.8, and 7.8µM, respectively. The reduction of hyperactivity compared to the reference activity was significant. The present results highlight the notion of re-purposing the K(+) channel activators for reducing hyperactivity of spontaneously active auditory networks, serving as a platform for these drugs to show efficacy toward target identification, prevention, as well as treatment of tinnitus.

  11. 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.

  12. 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.

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

    Science.gov (United States)

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

    2015-11-13

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

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

    Science.gov (United States)

    Meyers, D E; Cooke, I M

    1997-02-01

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

  15. Crustacean peptidergic neurons in culture show immediate outgrowth in simple medium.

    Science.gov (United States)

    Cooke, I; Graf, R; Grau, S; Haylett, B; Meyers, D; Ruben, P

    1989-01-01

    The survival and outgrowth of neurons in culture has usually required conditioning factors. We now report that crustacean neurons, taken from the peptidergic neurosecretory system of the eyestalk of crabs (Cardisoma carnifex) and lobsters (Panulirus marginatus), show immediate outgrowth, sustained for a week or more, in defined medium as simple as physiological saline with glucose and glutamine. The neurons show peptide hormone immunoreactivity that is prominent at growth cones, exhibit differences in form correlated with their immunoreactivity, release peptides to the medium, and have voltage-dependent currents, including a well-sustained Ca current. Cd blocks secretion, growth, and the Ca current. Peptidergic secretory neurons may be able to utilize existing membrane from their store of granules and already active synthetic, transport, and secretory mechanisms for immediate outgrowth.

  16. Batroxobin Against Anoxic Damage of Rat Hippocampal Neurons in Culture: Morphological Changes and Hsp70 Expression

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    @@Batroxobin,the thrombin-like enzyme,is used for therapeutic defibrination. We have found that batroxobin has good therapeutic effect in ischemic reperfusion rats and clinical practices in vivo. But we have not studied the neuroprotective effect of batroxobin on anoxic hippocampal neurons in vitro. The purpose of this study was to obtain further information on the mechanism of the batroxobin-induced neuroprotection and examine the neuroprotective effect on neurons exposed to anoxia. The effect of batroxobin on anoxic damages in cultured hippocampal neurons of neonatal rats was investigated by using morphological changes and heat shock protein 70Kd (Hsp70) immunoreactive expression as indicators. The results indicate that batroxobin, besides its defibrination, may have a direct neuroprotective effect on anoxic damage of hippocampal neurons.

  17. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Directory of Open Access Journals (Sweden)

    Rafael Rodríguez-Muñoz

    Full Text Available The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f, during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV. By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60% and multipolar Glutamatergic (≤40% neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC: dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively, in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

  18. Novel Nuclear Protein Complexes of Dystrophin 71 Isoforms in Rat Cultured Hippocampal GABAergic and Glutamatergic Neurons.

    Science.gov (United States)

    Rodríguez-Muñoz, Rafael; Cárdenas-Aguayo, María Del Carmen; Alemán, Víctor; Osorio, Beatriz; Chávez-González, Oscar; Rendon, Alvaro; Martínez-Rojas, Dalila; Meraz-Ríos, Marco Antonio

    2015-01-01

    The precise functional role of the dystrophin 71 in neurons is still elusive. Previously, we reported that dystrophin 71d and dystrophin 71f are present in nuclei from cultured neurons. In the present work, we performed a detailed analysis of the intranuclear distribution of dystrophin 71 isoforms (Dp71d and Dp71f), during the temporal course of 7-day postnatal rats hippocampal neurons culture for 1h, 2, 4, 10, 15 and 21 days in vitro (DIV). By immunofluorescence assays, we detected the highest level of nuclear expression of both dystrophin Dp71 isoforms at 10 DIV, during the temporal course of primary culture. Dp71d and Dp71f were detected mainly in bipolar GABAergic (≥60%) and multipolar Glutamatergic (≤40%) neurons, respectively. We also characterized the existence of two nuclear dystrophin-associated protein complexes (DAPC): dystrophin 71d or dystrophin 71f bound to β-dystroglycan, α1-, β-, α2-dystrobrevins, α-syntrophin, and syntrophin-associated protein nNOS (Dp71d-DAPC or Dp71f-DAPC, respectively), in the hippocampal neurons. Furthermore, both complexes were localized in interchromatin granule cluster structures (nuclear speckles) of neuronal nucleoskeleton preparations. The present study evinces that each Dp71's complexes differ slightly in dystrobrevins composition. The results demonstrated that Dp71d-DAPC was mainly localized in bipolar GABAergic and Dp71f-DAPC in multipolar Glutamatergic hippocampal neurons. Taken together, our results show that dystrophin 71d, dystrophin 71f and DAP integrate protein complexes, and both complexes were associated to nuclear speckles structures.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2014-10-01

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

  1. 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

  2. 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

  3. 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...

  4. 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…

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

    Directory of Open Access Journals (Sweden)

    Michelle M Garred

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

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

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    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

  7. Slow-pressor angiotensin II hypertension and concomitant dendritic NMDA receptor trafficking in estrogen receptor β-containing neurons of the mouse hypothalamic paraventricular nucleus are sex and age dependent.

    Science.gov (United States)

    Marques-Lopes, Jose; Van Kempen, Tracey; Waters, Elizabeth M; Pickel, Virginia M; Iadecola, Costantino; Milner, Teresa A

    2014-09-01

    The incidence of hypertension increases after menopause. Similar to humans, "slow-pressor" doses of angiotensin II (AngII) increase blood pressure in young males, but not in young female mice. However, AngII increases blood pressure in aged female mice, paralleling reproductive hormonal changes. These changes could influence receptor trafficking in central cardiovascular circuits and contribute to hypertension. Increased postsynaptic N-methyl-D-aspartate (NMDA) receptor activity in the hypothalamic paraventricular nucleus (PVN) is crucial for the sympathoexcitation driving AngII hypertension. Estrogen receptors β (ERβs) are present in PVN neurons. We tested the hypothesis that changes in ovarian hormones with age promote susceptibility to AngII hypertension, and influence NMDA receptor NR1 subunit trafficking in ERβ-containing PVN neurons. Transgenic mice expressing enhanced green fluorescent protein (EGFP) in ERβ-containing cells were implanted with osmotic minipumps delivering AngII (600 ng/kg/min) or saline for 2 weeks. AngII increased blood pressure in 2-month-old males and 18-month-old females, but not in 2-month-old females. By electron microscopy, NR1-silver-intensified immunogold (SIG) was mainly in ERβ-EGFP dendrites. At baseline, NR1-SIG density was greater in 2-month-old females than in 2-month-old males or 18-month-old females. After AngII infusion, NR1-SIG density was decreased in 2-month-old females, but increased in 2-month-old males and 18-month-old females. These findings suggest that, in young female mice, NR1 density is decreased in ERβ-PVN dendrites thus reducing NMDA receptor activity and preventing hypertension. Conversely, in young males and aged females, NR1 density is upregulated in ERβ-PVN dendrites and ultimately leads to the neurohumoral dysfunction driving hypertension.

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Daniel de Santos-Sierra

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

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

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

  13. Organotypic human neuronal culture derived from traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    David Riascos

    2009-11-01

    Full Text Available Introducción: El trauma craneoencefálico (TEC es un problema de salud global que puede generar en los pacientes que lo padecen, muerte, discapacidad y/o alteraciones psiquiátricas con gran impacto sobre su desempeño posterior y sobre su ámbito familiar. En los últimos años se ha avanzado en el conocimiento de los mecanismos fisiopatológicos que subyacen al TCE. Sin embargo, esto no está completamente entendido, como tampoco hay claridad sobre los mecanismos de neuroprotección. Por esta razón cada vez más se buscan modelos que permitan aproximarse al estudio de este síndrome y de esta manera aproximarse a la neuroprotección. Objetivo: Caracterizar un modelo de cultivo organotípico de neuronas corticales humanas obtenidas de personas que sufrieron TCE y a las cuales se les practicó remoción de la contusión. Metodología: Se utilizó tejido cortical humano procedente de 4 individuos que sufrieron TCE y a los cuales se les removió la contusión. Se obtuvieron tajadas de corteza cerebral de 1,500-2,000 mm, las cuales se mantuvieron en un flujo continuo de LCRa a 2 ml/min y una mezcla gaseosa de O2 al 95% y CO2 al 5% con burbujeo permanente durante 2, 8 y 14 horas. Se tomó como tiempo cero el momento de obtención de la muestra. Después de cada tiempo se tomaron las tajadas, se cortaron en un vibrátomo de medio líquido a 50 mm y se procesaron inmunohistoquímicamente con los marcadores neuronales de degeneración NeuN y MAP2. Resultados: Los resultados indican que las muestras de corteza cerebral se pudieron mantener con cierto grado de integridad celular y laminar hasta las 2 horas de cultivo. Se observó que a partir de este tiempo se inicia un proceso de alteración de la citoarquitectura neuronal y laminar, determinada por la pérdida y alteración de la inmunorreactividad a los marcadores NeuN y MAP2. Además se encontró que hay vulnerabilidad celular que compromete en mayor medida a las neuronas localizadas en las l

  14. 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

  15. Direct evidence for the co-expression of URP and GnRH in a sub-population of rat hypothalamic neurones: anatomical and functional correlation.

    Directory of Open Access Journals (Sweden)

    Johann-Günther Egginger

    Full Text Available Urotensin-II-related peptide (URP is an eight amino-acid neuropeptide recently isolated from rat brain and considered as the endogenous ligand for the GPR14 receptor. Using single and double immunohistochemical labelling, in situ hybridization and ultrastructural immunocytochemistry, we explored the cellular and subcellular localization of URP in the male rat brain. URP peptide was detected in numerous varicose fibres of the median eminence (ME and organum vasculosum laminae terminalis (OVLT as well as in neuronal cell bodies of the medial septal nucleus and diagonal band of Broca where corresponding mRNA were also detected. Combining in situ hybridization with immunohistochemistry, we showed that cell bodies of the rat anterior hypothalamus contained both URP mRNA and GnRH peptide. In addition, double ultrastructural immunodetection of URP and GnRH peptides clearly revealed, in the median eminence, the co-localization of both peptides in the same neuronal processes in the vicinity of fenestrated portal vessels. This remarkable cellular and subcellular distribution led us to test the effect of URP on the GnRH-induced gonadotrophins release in the anterior pituitary, and to discuss its putative role at the level of the median eminence.

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

    Science.gov (United States)

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

    2014-11-01

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

  17. 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.

  18. Fluorescent diamond nanoparticle as a probe of intracellular traffic in primary neurons in culture

    Science.gov (United States)

    Le, Xuan Loc; Lepagnol-Bestel, Aude-Marie; Adam, Marie-Pierre; Thomas, Alice; Dantelle, Géraldine; Chang, Cheng-Chun; Mohan, Nitin; Chang, Huan-Cheng; Treussart, François; Simonneau, Michel

    2012-03-01

    Neurons display dendritic spines plasticity and morphology anomalies in numerous psychiatric and neurodegenerative diseases. These changes are associated to abnormal dendritic traffic that can be evidenced by fluorescence microscopy. As a fluorescent probe we propose to use fluorescent diamond nanoparticles with size of < 50 nm. Color centers embedded inside the diamond nanoparticles are perfectly photostable emitters allowing for long-term tracking. Nanodiamond carbon surface is also well suited for biomolecule functionalization to target specific cellular compartments. We show that fluorescent nanodiamonds can be spontaneously internalized in neurons in culture and imaged by confocal and Total Internal Reflection (TIRF) microscopy with a high signal over background ratio.

  19. 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.

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

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

    2014-09-01

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

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

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    Natalya Shulyakova

    2010-09-01

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

  2. Hypothalamic neuropeptides and the regulation of appetite.

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    Parker, Jennifer A; Bloom, Stephen R

    2012-07-01

    Neuropeptides released by hypothalamic neurons play a major role in the regulation of feeding, acting both within the hypothalamus, and at other appetite regulating centres throughout the brain. Where classical neurotransmitters signal only within synapses, neuropeptides diffuse over greater distances affecting both nearby and distant neurons expressing the relevant receptors, which are often extrasynaptic. As well as triggering a behavioural output, neuropeptides also act as neuromodulators: altering the response of neurons to both neurotransmitters and circulating signals of nutrient status. The mechanisms of action of hypothalamic neuropeptides with established roles in feeding, including melanin-concentrating hormone (MCH), the orexins, α-melanocyte stimulating hormone (α-MSH), agouti-gene related protein (AgRP), neuropeptide Y, and oxytocin, are reviewed in this article, with emphasis laid on both their effects on appetite regulating centres throughout the brain, and on examining the evidence for their physiological roles. In addition, evidence for the involvement of several putative appetite regulating hypothalamic neuropeptides is assessed including, ghrelin, cocaine and amphetamine-regulated transcript (CART), neuropeptide W and the galanin-like peptides. This article is part of a Special Issue entitled 'Central control of Food Intake'.

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

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

    2008-01-01

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

  4. Dorsal Root Ganglia Sensory Neuronal Cultures: a tool for drug discovery for peripheral neuropathies

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    Melli, Giorgia; Höke, Ahmet

    2010-01-01

    Background Peripheral neuropathies affect many people worldwide and are caused by or associated with a wide range of conditions, both genetic and acquired. Current therapies are directed at symptomatic control because no effective regenerative treatment exists. Primary challenge is that mechanisms that lead to distal axonal degeneration, a common feature of all peripheral neuropathies, are largely unknown. Objective/Methods To address the role and specific characteristics of dorsal root ganglia (DRG) derived sensory neuron culture system as a useful model in evaluating the pathogenic mechanisms of peripheral neuropathies and examination and validation of potential therapeutic compounds. A thorough review of the recent literature was completed and select examples of the use of DRG neurons in different peripheral neuropathy models were chosen to highlight the utility of these cultures. Conclusion Many useful models of different peripheral neuropathies have been developed using DRG neuronal culture and potential therapeutic targets have been examined, but so far none of the potential therapeutic compounds have succeeded in clinical trials. In recent years, focus has changed to evaluation of axon degeneration as the primary outcome measure advocating a drug development strategy starting with phenotypic drug screening, followed by validation in primary complex co-cultures and animal models. PMID:20657751

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

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

    2008-01-03

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

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

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

    2012-01-15

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

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

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    Zheyan Chen

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

  8. Automated detection of soma location and morphology in neuronal network cultures.

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    Burcin Ozcan

    Full Text Available Automated identification of the primary components of a neuron and extraction of its sub-cellular features are essential steps in many quantitative studies of neuronal networks. The focus of this paper is the development of an algorithm for the automated detection of the location and morphology of somas in confocal images of neuronal network cultures. This problem is motivated by applications in high-content screenings (HCS, where the extraction of multiple morphological features of neurons on large data sets is required. Existing algorithms are not very efficient when applied to the analysis of confocal image stacks of neuronal cultures. In addition to the usual difficulties associated with the processing of fluorescent images, these types of stacks contain a small number of images so that only a small number of pixels are available along the z-direction and it is challenging to apply conventional 3D filters. The algorithm we present in this paper applies a number of innovative ideas from the theory of directional multiscale representations and involves the following steps: (i image segmentation based on support vector machines with specially designed multiscale filters; (ii soma extraction and separation of contiguous somas, using a combination of level set method and directional multiscale filters. We also present an approach to extract the soma's surface morphology using the 3D shearlet transform. Extensive numerical experiments show that our algorithms are computationally efficient and highly accurate in segmenting the somas and separating contiguous ones. The algorithms presented in this paper will facilitate the development of a high-throughput quantitative platform for the study of neuronal networks for HCS applications.

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

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2009-05-05

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

  11. Visualizing sensory transmission between dorsal root ganglion and dorsal horn neurons in co-culture with calcium imaging.

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    Ohshiro, Hiroyuki; Ogawa, Shinji; Shinjo, Katsuhiro

    2007-09-15

    Sensory information is conveyed to the central nervous system by primary afferent neurons within dorsal root ganglia (DRG), which synapse onto neurons of the dorsal horn of the spinal cord. This synaptic connection is central to the processing of both sensory and pain stimuli. Here, we describe a model system to monitor synaptic transmission between DRG neurons and dorsal horn neurons that is compatible with high-throughput screening. This co-culture preparation comprises DRG and dorsal horn neurons and utilizes Ca(2+) imaging with the indicator dye Fura-2 to visualize synaptic transmission. Addition of capsaicin to co-cultures stimulated DRG neurons and led to activation of dorsal horn neurons as well as increased intracellular Ca(2+) concentrations. This effect was dose-dependent and absent when DRG neurons were omitted from the culture. NMDA receptors are a critical component of synapses between DRG and dorsal horn neurons as MK-801, a use-dependent non-competitive antagonist, prevented activation of dorsal horn neurons following capsaicin treatment. This model system allows for rapid and efficient analysis of noxious stimulus-evoked Ca(2+) signal transmission and provides a new approach both for investigating synaptic transmission in the spinal cord and for screening potential analgesic compounds.

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

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

    2016-10-19

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

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

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    Katherine A Hosie

    2012-02-01

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

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

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    Thirukumaran T eKanagasabapathi

    2011-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Ying Liu; Jiangbao Zhou

    2006-01-01

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

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

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    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.

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

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    Shinichi Takahashi

    2014-09-01

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

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

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

    1989-09-01

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

  19. Developmental Expression of Kv Potassium Channels at the Axon Initial Segment of Cultured Hippocampal Neurons

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    Sánchez-Ponce, Diana; DeFelipe, Javier; Garrido, Juan José; Muñoz, Alberto

    2012-01-01

    Axonal outgrowth and the formation of the axon initial segment (AIS) are early events in the acquisition of neuronal polarity. The AIS is characterized by a high concentration of voltage-dependent sodium and potassium channels. However, the specific ion channel subunits present and their precise localization in this axonal subdomain vary both during development and among the types of neurons, probably determining their firing characteristics in response to stimulation. Here, we characterize the developmental expression of different subfamilies of voltage-gated potassium channels in the AISs of cultured mouse hippocampal neurons, including subunits Kv1.2, Kv2.2 and Kv7.2. In contrast to the early appearance of voltage-gated sodium channels and the Kv7.2 subunit at the AIS, Kv1.2 and Kv2.2 subunits were tethered at the AIS only after 10 days in vitro. Interestingly, we observed different patterns of Kv1.2 and Kv2.2 subunit expression, with each confined to distinct neuronal populations. The accumulation of Kv1.2 and Kv2.2 subunits at the AIS was dependent on ankyrin G tethering, it was not affected by disruption of the actin cytoskeleton and it was resistant to detergent extraction, as described previously for other AIS proteins. This distribution of potassium channels in the AIS further emphasizes the heterogeneity of this structure in different neuronal populations, as proposed previously, and suggests corresponding differences in action potential regulation. PMID:23119056

  20. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2010-07-15

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

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

    Science.gov (United States)

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

    2012-04-05

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

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

    Directory of Open Access Journals (Sweden)

    Junko Kimura-Kuroda

    2016-10-01

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

  6. Hypothalamic inflammation and gliosis in obesity.

    Science.gov (United States)

    Dorfman, Mauricio D; Thaler, Joshua P

    2015-10-01

    Hypothalamic inflammation and gliosis are recently discovered mechanisms that may contribute to obesity pathogenesis. Current research in this area suggests that investigation of these central nervous system responses may provide opportunities to develop new weight loss treatments. In rodents, hypothalamic inflammation and gliosis occur rapidly with high-fat diet consumption prior to significant weight gain. In addition, sensitivity or resistance to diet-induced obesity in rodents generally correlates with the presence or absence of hypothalamic inflammation and reactive gliosis (brain response to injury). Moreover, functional interventions that increase or decrease inflammation in neurons and glia correspondingly alter diet-associated weight gain. However, some conflicting data have recently emerged that question the contribution of hypothalamic inflammation to obesity pathogenesis. Nevertheless, several studies have detected gliosis and disrupted connectivity in obese humans, highlighting the potential translational importance of this mechanism. There is growing evidence that obesity is associated with brain inflammation in humans, particularly in the hypothalamus where its presence may disrupt body weight control and glucose homeostasis. More work is needed to determine whether this response is common in human obesity and to what extent it can be manipulated for therapeutic benefit.

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

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

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

  8. Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions.

    Science.gov (United States)

    López, F J; Merchenthaler, I; Ching, M; Wisniewski, M G; Negro-Vilar, A

    1991-01-01

    Galanin (GAL) is widely distributed in the peripheral and the central nervous systems. In the brain, the highest GAL concentrations are observed within the hypothalamus and, particularly, in nerve terminals of the median eminence. This location, as well as GAL actions on prolactin, growth hormone, luteinizing hormone (LH), and LH-releasing hormone (LHRH) secretion, suggest the possibility that GAL may act as a putative hypothalamic-hypophysiotropic hormone. To establish this, GAL and LHRH levels were measured in hypophyseal portal plasma samples using specific radioimmunoassays. Rat galanin (rGAL) concentrations in portal blood were approximately 7-fold higher than those observed in peripheral plasma in male and female (estrus, diestrus) rats, indicating an active secretory process of rGAL into the portal vasculature. Frequent (10 min) sampling revealed that rGAL and LHRH were secreted into the portal circulation in a pulsatile manner with a pulse frequency of one pulse per hour. Interestingly, both hormone series depicted a high degree of coincident episodes. In fact, the probability of random coincidence, calculated by the algorithm HYPERGEO, was less than 0.01. Moreover, the retrograde tracer Fluoro-Gold, when given systemically, was taken up by GAL neurons in the hypothalamus, including a subset of neurons expressing rGAL and LHRH, strengthening the notion of the existence of a GAL neuronal system connected to the hypophyseal portal circulation. These observations reinforce the concept that GAL regulates pituitary hormone secretion. To analyze this in further detail, the effects of rGAL on LH secretion were evaluated under basal and stimulated conditions. rGAL induced a small but dose-dependent increase in LH secretion from cultured, dispersed pituitary cells. Interestingly, rGAL enhanced the ability of LHRH to stimulate LH release. The tight link between GAL and LHRH neuronal systems is strengthened by the observation that during the estrous cycle of the rat

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2011-01-01

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

  11. 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.

  12. Bone morphogenetic protein-5 (BMP-5 promotes dendritic growth in cultured sympathetic neurons

    Directory of Open Access Journals (Sweden)

    Higgins Dennis

    2001-09-01

    Full Text Available Abstract Background BMP-5 is expressed in the nervous system throughout development and into adulthood. However its effects on neural tissues are not well defined. BMP-5 is a member of the 60A subgroup of BMPs, other members of which have been shown to stimulate dendritic growth in central and peripheral neurons. We therefore examined the possibility that BMP-5 similarly enhances dendritic growth in cultured sympathetic neurons. Results Sympathetic neurons cultured in the absence of serum or glial cells do not form dendrites; however, addition of BMP-5 causes these neurons to extend multiple dendritic processes, which is preceded by an increase in phosphorylation of the Smad-1 transcription factor. The dendrite-promoting activity of BMP-5 is significantly inhibited by the BMP antagonists noggin and follistatin and by a BMPR-IA-Fc chimeric protein. RT-PCR and immunocytochemical analyses indicate that BMP-5 mRNA and protein are expressed in the superior cervical ganglia (SCG during times of initial growth and rapid expansion of the dendritic arbor. Conclusions These data suggest a role for BMP-5 in regulating dendritic growth in sympathetic neurons. The signaling pathway that mediates the dendrite-promoting activity of BMP-5 may involve binding to BMPR-IA and activation of Smad-1, and relative levels of BMP antagonists such as noggin and follistatin may modulate BMP-5 signaling. Since BMP-5 is expressed at relatively high levels not only in the developing but also the adult nervous system, these findings suggest the possibility that BMP-5 regulates dendritic morphology not only in the developing, but also the adult nervous system.

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

    Science.gov (United States)

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

    2012-04-01

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

  14. The effects of triethyl lead on the development of hippocampal neurons in culture.

    Science.gov (United States)

    Audesirk, T; Shugarts, D; Cabell-Kluch, L; Wardle, K

    1995-02-01

    Triethyl lead is the major metabolite of tetraethyl lead, which is used in industrial processes and as an antiknock additive to gasoline. We tested the hypothesis that low levels of triethyl lead (0.1 nmol/L to 5 mumol/L) interfere with the normal development of cultured E18 rat hippocampal neurons, possibly through increases in intracellular free calcium ion concentration, [Ca2+]in. The study assessed survival and differentiation using morphometric analysis of individual neurons. We also looked at short-term (up to 3.75-h) changes in intracellular calcium using the calcium-sensitive dye fura-2. Survival of neurons was significantly reduced at 5 mumol/L, and overall production of neurites was reduced at > or = 2 mumol/L. The length of axons and the number of axons and dendrites were reduced at > or = 1 mumol/L. Neurite branching was inhibited at 10 nmol/L for dendrites and 100 nmol/L for axons. Increases in intracellular calcium were observed during a 3.75-h exposure of newly plated neurons to 5 mumol/L triethyl lead. These increases were prevented by BAPTA-AM; which clamps [Ca2+]in at about 100 nmol/L. Culturing neurons with BAPTA-AM and 5 mumol/L triethyl lead did not reverse the effects of triethyl lead, suggesting that elevation of [Ca2+]in is not responsible for decreases in survival and neurite production. Triethyl lead has been shown to disrupt cytoskeletal elements, particularly neurofilaments, at very low levels, suggesting a possible mechanism for its inhibition of neurite branching at nanomolar concentrations.

  15. 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 (CHCl3) 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, CHCl3 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, CHCl3 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/CHCl3 subfraction and EtOAc fraction promote neurite development by up or downregulating several key proteins.

  16. 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.

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

    Science.gov (United States)

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

    2013-04-01

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

  18. S-Nitrosoglutathione and glutathione act as NMDA receptor agonists in cultured hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Ting-yu CHIN; Sheau-huei CHUEH; Pao-luh TAO

    2006-01-01

    Aim: To characterize the effect of combined pre- and postnatal morphine exposure on Af-methyl-D-aspartate receptor (NMDA) receptor signaling in hippocampal neurons of the offspring of morphine-addicted female rats. Methods: Cultured hippocampal neurons and synaptosomes were prepared from neonatal and 2-week-old offspring, respectively, of control or morphine-addicted female rats. The increase in the cytosolic Ca2+ concentration ([Ca2+]i) of cultured cells was measured using Fura-2, and glutamate release from synaptosomes was measured enzymatically. Results: Both glutamate and NMDA caused a dose-dependent increase in the [Ca2+]i. The nitric oxide (NO) donor, S-nitrosoglutathione (GSNO), but not 3-morpholinosydnonimine, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine, also induced a [Ca2+]i increase. GSNO and glutathione caused a dose-dependent increase in the [Ca2+]i with respective EC50 values of 56 and 414 μmol/L. Both effects were inhibited by Mg2+ or an NMDA receptor antagonist and were unaffected by the presence of a glutamate scavenger. The other glutathione derivatives, oxidized glutathione, S-methylglutathione, S-ethylglutathione, S-propylglutathione, and S-butylglutathione, the dipeptides, Glu-Cys and Cys-Gly, and the antioxidants, dithiothreitol and mercaptoethanol, failed to induce a [Ca2+]i increase. In addition, glutathione caused a dose-dependent increase in glutamate release from synaptosomes. The maximal responses and the EC50 values for the glutamate-, NMDA-, GSNO-, and glutathione-induced [Ca2+]i increases and the glutathione-induced glutamate release were indistinguishable in the neurons of the offspring from control and morphine-addicted female rats. Conclusion: GSNO and glutathione act as NMDA receptor agonists and, in contrast to hippocampal brain slice, combined pre- and postnatal morphine exposure does not modulate NMDA receptor signaling in the cultured hippocampal neurons.

  19. 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...

  20. Fluorescence imaging of local membrane electric fields during the excitation of single neurons in culture.

    Science.gov (United States)

    Gogan, P; Schmiedel-Jakob, I; Chitti, Y; Tyc-Dumont, S

    1995-01-01

    The spatial distribution of depolarized patches of membrane during the excitation of single neurons in culture has been recorded with a high spatial resolution (1 micron2/pixel) imaging system based on a liquid-nitrogen-cooled astronomical camera mounted on an inverted microscope. Images were captured from rat nodose neurons stained with the voltage-sensitive dye RH237. Conventional intracellular microelectrode recordings were made in synchrony with the images. During an action potential the fluorescence changes occurred in localized, unevenly distributed membrane areas, which formed clusters of depolarized sites of different sizes and intensities. When fast conductances were blocked by the addition of tetrodotoxin, a reduction in the number and the intensities of the depolarized sites was observed. The blockade by tetrodotoxin of voltage-clamped neurons also reduced the number of depolarized sites, although the same depolarizing voltage step was applied. Similarly, when a voltage-clamped neuron was depolarized by a constant-amplitude voltage step, the number of depolarized sites varied according to the degree of activation of the voltage-sensitive channels, which was modified by changing the holding potential. These results suggest that the spatial patterns of depolarization observed during excitation are related to the operations of ionic channels in the membrane. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:8527643

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

    Science.gov (United States)

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

    2017-08-26

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

  2. A transient, RCK4-like K+ current in cultured Xenopus olfactory bulb neurons.

    Science.gov (United States)

    Engel, J; Rabba, J; Schild, D

    1996-09-01

    A transient K+ current in cultured olfactory bulb neurons of Xenopus tadpoles was studied using the whole-cell patch-clamp technique. The current, which was resistant to 80 mM tetraethylammoniumchloride (TEA) and 10 nM charybdotoxin but blocked by 5 mM 4-aminopyridine (4-AP), activated between -60 and -40 mV and showed time- and voltage-dependent inactivation. Its peak amplitude was nearly independent of the extracellular K+ concentration ([K+]o) in the range of 0.05 to 10 mM, indicating that its conductance increased upon increasing [K+]o. The transient K+ current showed a slow recovery from inactivation with the time for half-maximum recovery from a conditioning pulse to 80 mV for 1 s varying from 100 ms to 500 ms. Complete recovery required as much as 5-10 s at -80 mV, but could be speeded up at hyperpolarized potentials. The current resembles the RCK4 (Kv1.4) current of rat neurons except that its recovery from inactivation was independent of [K+]o. High-frequency stimulation (20-67 Hz) of the neurons with short (5 ms) voltage pulses resulted in a frequency-dependent, progressive inactivation of the transient K+ current. This suggests that, during phasic responses of olfactory bulb neurons, inactivation of the transient K+ current occurs and may lead to lengthening of action potentials and facilitation of synaptic transmission.

  3. Stimulation of glutamate receptors in cultured hippocampal neurons causes Ca2+-dependent mitochondrial contraction.

    Science.gov (United States)

    Brustovetsky, Tatiana; Li, Viacheslav; Brustovetsky, Nickolay

    2009-07-01

    Cultured hippocampal neurons expressing mitochondrially-targeted enhanced yellow fluorescent protein (mito-eYFP) were used to quantitatively examine mitochondrial remodelling in response to excitotoxic glutamate. Mitochondrial morphology was evaluated using laser spinning-disk confocal microscopy followed by calibrated image processing and 3D image rendering. Glutamate triggered an increase in cytosolic Ca(2+) and mitochondrial depolarization accompanied by Ca(2+)-dependent morphological transformation of neuronal mitochondria from "thread-like" to rounded structures. The quantitative analysis of the mitochondrial remodelling revealed that exposure to glutamate resulted in a decrease in mitochondrial volume and surface area concurrent with an increase in sphericity of the organelles. NIM811, an inhibitor of the mitochondrial permeability transition, attenuated the glutamate-induced sustained increase in cytosolic Ca(2+) and suppressed mitochondrial remodelling in the majority of affected neurons, but it did not rescue mitochondrial membrane potential. Shortening, fragmentation, and formation of circular mitochondria with decreased volume and surface area accompanied mitochondrial depolarization with FCCP. However, FCCP-induced morphological alterations appeared to be distinctly different from mitochondrial remodelling caused by glutamate. Moreover, FCCP prevented glutamate-induced mitochondrial remodelling suggesting an important role of Ca(2+) influx into mitochondria in the morphological alterations. Consistent with this, in saponin-permeabilized neurons, Ca(2+) caused mitochondrial remodelling which could be prevented by Ru(360).

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

    Directory of Open Access Journals (Sweden)

    T.T. Logan

    2015-11-01

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

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

    Science.gov (United States)

    Yoon, So-Young; Oh, Young J

    2015-09-01

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

  6. Nanoparticle Targeting to Neurons in a Rat Hippocampal Slice Culture Model

    Directory of Open Access Journals (Sweden)

    Ryan Walters

    2012-09-01

    Full Text Available We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots coated with PEG [poly(ethylene glycol]-appended DHLA (dihydrolipoic acid can bind AcWG(PalVKIKKP9GGH6 (Palm1 through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry, demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.

  7. Deoxyschisandrin modulates synchronized Ca2+ oscillations and spontaneous synaptic transmission of cultured hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Min FU; Zhao-hui SUN; Min ZONG; Xiang-ping HE; Huan-cong ZUO; Zuo-ping XIE

    2008-01-01

    Aim: Deoxyschisandrin is one of the most effective composites of Schisandra chinensis, a famous Chinese medicine widely used as an antistress, anti-aging, and neurological performance-improving herb. In this study, we examined its spe- cific mechanisms of action on cultured hippocampal neurons. Methods: Hippoc- ampal neurons, primarily cultured for 9-11 d in vitro, were used for this study. DS were dissolved in DMSO and applied to calcium imaging and whole-cell patch clamp. Results: The application of 3 mg/L DS decreased the frequency of sponta- neous and synchronous oscillations of intracellular Ca2+ to 72%±2% (mean±SEM), and the spontaneous inhibitory postsynaptic currents to 60%±3% (mean±SEM). The inhibitory concentraton 50% (IC50) for the effect of DS on calcium oscillations was 3.8 mg/L. DS also depressed the high voltage-gated Ca2+ channel and the voltage-gated Na+ channel currents at the same time point. It had no effect, however, on voltage-gated K+ and spontaneous excitatory postsynaptic currents. Conclusion: DS inhibited the spontaneous and synchronous oscillations of intra- cellular Ca2+ through the depression of influx of extracellular calcium and the initiation of action potential. By repressing the spontaneous neurotransmitter release, DS modulated the neuronal network activities.

  8. Nanoparticle targeting to neurons in a rat hippocampal slice culture model

    Directory of Open Access Journals (Sweden)

    Richard P Kraig

    2012-10-01

    Full Text Available We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots coated with PEG [poly(ethylene glycol]-appended DHLA (dihydrolipoic acid can bind AcWG(PalVKIKKP9GGH6 (Palm1 through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry, demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.

  9. 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.

  10. Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons.

    Science.gov (United States)

    Xu, Shangcheng; Zhou, Zhou; Zhang, Lei; Yu, Zhengping; Zhang, Wei; Wang, Yuan; Wang, Xubu; Li, Maoquan; Chen, Yang; Chen, Chunhai; He, Mindi; Zhang, Guangbin; Zhong, Min

    2010-01-22

    Increasing evidence indicates that oxidative stress may be involved in the adverse effects of radiofrequency (RF) radiation on the brain. Because mitochondrial DNA (mtDNA) defects are closely associated with various nervous system diseases and mtDNA is particularly susceptible to oxidative stress, the purpose of this study was to determine whether radiofrequency radiation can cause oxidative damage to mtDNA. In this study, we exposed primary cultured cortical neurons to pulsed RF electromagnetic fields at a frequency of 1800 MHz modulated by 217 Hz at an average special absorption rate (SAR) of 2 W/kg. At 24 h after exposure, we found that RF radiation induced a significant increase in the levels of 8-hydroxyguanine (8-OHdG), a common biomarker of DNA oxidative damage, in the mitochondria of neurons. Concomitant with this finding, the copy number of mtDNA and the levels of mitochondrial RNA (mtRNA) transcripts showed an obvious reduction after RF exposure. Each of these mtDNA disturbances could be reversed by pretreatment with melatonin, which is known to be an efficient antioxidant in the brain. Together, these results suggested that 1800 MHz RF radiation could cause oxidative damage to mtDNA in primary cultured neurons. Oxidative damage to mtDNA may account for the neurotoxicity of RF radiation in the brain.

  11. Long-term live imaging of neuronal circuits in organotypic hippocampal slice cultures.

    Science.gov (United States)

    Gogolla, Nadine; Galimberti, Ivan; DePaola, Vincenzo; Caroni, Pico

    2006-01-01

    This protocol details a method for imaging organotypic slice cultures from the mouse hippocampus. The cultures are based on the interface method, which does not require special equipment, is easy to execute, and yields slice cultures that can be imaged repeatedly after they are isolated on postnatal day 6-9 and for up to 6 months in vitro. The preserved tissue architecture facilitates the analysis of defined hippocampal synapses, cells and entire projections. Time-lapse imaging is based on transgenes expressed in the mice, or on constructs introduced through transfection or viral vectors; it can reveal processes that develop over time periods ranging from seconds to months. Imaging can be repeated at least eight times without detectable morphological damage to neurons. Subsequent to imaging, the slices can be processed for immunocytochemistry or electron microscopy, to collect further information about the structures that have been imaged. This protocol can be completed in 35 min.

  12. CHARACTERIZING CALCIUM INFLUX VIA VOLTAGE- AND LIGAND-GATED CALCIUM CHANNELS IN EMBRYONIC ALLIGATOR NEURONS IN CULTURE

    Science.gov (United States)

    Ju, Weina; Wu, Jiang; Pritz, Michael B.; Khanna, Rajesh

    2013-01-01

    Vertebrate brains share many features in common. Early in development, both the hindbrain and diencephalon are built similarly. Only later in time do differences in morphology occur. Factors that could potentially influence such changes include certain physiological properties of neurons. As an initial step to investigate this problem, embryonic Alligator brain neurons were cultured and calcium responses were characterized. The present report is the first to document culture of Alligator brain neurons in artificial cerebrospinal fluid (ACSF) as well as in standard mammalian tissue culture medium supplemented with growth factors. Alligator brain neuron cultures were viable for at least 1 week with unipolar neurites emerging by 24 hours. Employing Fura-2 AM, robust depolarization-induced calcium influx, was observed in these neurons. Using selective blockers of the voltage-gated calcium channels, the contributions of N-, P/Q-, R-, T-, and L-type channels in these neurons were assessed and their presence documented. Lastly, Alligator brain neurons were challenged with an excitotoxic stimulus (glutamate + glycine) where delayed calcium deregulation could be prevented by a classical NMDA receptor antagonist. PMID:24260711

  13. L-carnitine protects neurons from 1-methyl-4-phenylpyridinium-induced neuronal apoptosis in rat forebrain culture.

    Science.gov (United States)

    Wang, C; Sadovova, N; Ali, H K; Duhart, H M; Fu, X; Zou, X; Patterson, T A; Binienda, Z K; Virmani, A; Paule, M G; Slikker, W; Ali, S F

    2007-01-05

    1-Methyl-4-phenylpyridinium ion (MPP+), an inhibitor of mitochondrial complex I, has been widely used as a neurotoxin because it elicits a severe Parkinson's disease-like syndrome with an elevation of intracellular reactive oxygen species (ROS) and apoptosis. L-carnitine plays an integral role in attenuating the brain injury associated with mitochondrial neurodegenerative disorders. The present study investigates the effects of L-carnitine against the toxicity of MPP+ in rat forebrain primary cultures. Cells in culture were treated for 24 h with 100, 250, 500 and 1000 microM MPP+ alone or co-incubated with L-carnitine. MPP+ produced a dose-related increase in DNA fragmentation as measured by cell death ELISA (enzyme-linked immunosorbent assay), an increase in the number of TUNEL (terminal dUTP nick-end labeling)-positive cells and a reduction in the mitochondrial metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). No significant effect was observed with the release of lactate dehydrogenase (LDH), indicating that cell death presumably occurred via apoptotic mechanisms. Co-incubation of MPP+ with L-carnitine significantly reduced MPP+-induced apoptosis. Western blot analyses showed that neurotoxic concentrations of MPP+ decreased the ratio of BCL-X(L) to Bax and decreased the protein levels of polysialic acid neural cell adhesion molecules (PSA-NCAM), a neuron specific marker. L-carnitine blocked these effects of MPP+ suggesting its potential therapeutic utility in degenerative disorders such as Parkinson's disease, Alzheimer's disease, ornithine transcarbamylase deficiency and other mitochondrial diseases.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Xueneng Guan; Fuling Yan

    2008-01-01

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

  16. Buffer capacity of rat cortical tissue as well as of cultured neurons and astrocytes.

    Science.gov (United States)

    Katsura, K; Mellergård, P; Theander, S; Ouyang, Y B; Siesjö, B K

    1993-08-06

    The primary objective of this work was to assess the intrinsic nonbicarbonate buffer capacity (beta i) of cultured neurons and astrocytes and to compare the beta i values obtained to those of neocortical tissue. A second objective was to determine the pH dependence of beta i. Titration of homogenates of whole-brain cortical tissue and cultured neurons with NaOH and HCl gave beta i values of 25-30 mmol.l-1 x pH-1. The buffer capacity was essentially constant in the pH range of 6-7. Astrocytes showed a higher buffer capacity and a clear relationship between beta i and pH. However, beta i decreased when pH was reduced from 7 to 6. The beta i values derived from microspectrofluorometric studies on neurons and astrocytes were surprisingly variable, ranging from 10 to 50 mmol.l-1 x pH-1. The ammonia "step method" suggested that beta i increased dramatically when pH was lowered from 7 to 6 but the propionic "step method" failed to reveal such a pH dependence. Some techniques obviously give erroneous values for beta i, presumably because changes in buffer base concentration (due to transmembrane fluxes of H+, HCO3-, NH4+ or anions of weak acids) violate the principles upon which the calculations are based. From the results obtained by direct titration and with the propionate technique, we tentatively conclude that beta i in neurons and astrocytes are approximately 20 and 30 mmol.l-1 x pH-1, respectively. We further suggest that the term "intrinsic buffer capacity", as commonly used, is redefined.

  17. 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.

  18. 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.

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

    Institute of Scientific and Technical Information of China (English)

    杨立业; 郑佳坤; 刘相名; 惠国桢; 郭礼和

    2004-01-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  1. Inhibition of calcium currents in cultured rat dorsal root ganglion neurones by (-)-baclofen.

    OpenAIRE

    Dolphin, A C; Scott, R.H.

    1986-01-01

    Voltage-dependent inward calcium currents (ICa) activated in cultured rat dorsal root ganglion neurones were reversibly reduced in a dose-dependent manner by (-)-baclofen (10 microM to 100 microM). Baclofen (100 microM) reduced the calcium-dependent slow outward potassium current (IK(Ca)). This current was abolished in calcium-free medium and by 300 microM cadmium chloride. The action of baclofen on IK(Ca) was reduced when the calcium concentration in the medium was increased from 5 mM to 30 ...

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

    Science.gov (United States)

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

    2015-12-01

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

  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

    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...

  4. 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.

  5. 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) stud

  6. 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.

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

    Science.gov (United States)

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

    2008-01-01

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

  8. 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.

  9. 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

  10. 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.

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

    Science.gov (United States)

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

    1989-12-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

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    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

  14. A case for hypothalamic acromegaly: a clinicopathological study of six patients with hypothalamic gangliocytomas producing growth hormone-releasing factor.

    Science.gov (United States)

    Asa, S L; Scheithauer, B W; Bilbao, J M; Horvath, E; Ryan, N; Kovacs, K; Randall, R V; Laws, E R; Singer, W; Linfoot, J A

    1984-05-01

    We report the histological, ultrastructural, and immunocytochemical features of six hypothalamic gangliocytomas associated with pituitary GH cell adenomas and/or acromegaly. In four patients, the gangliocytoma was intrasellar, and no hypothalamic investigation was performed; in two patients, autopsy confirmed hypothalamic involvement. Four patients had a gangliocytoma associated with pituitary GH cell adenoma and acromegaly; electron microscopy demonstrated an intimate association between neurons and adenomatous GH cells. One patient had a gangliocytoma and a GH cell adenoma but no clinical evidence of acromegaly. In the sixth patient, clinical and biochemical acromegaly was manifest, but no pituitary adenoma was demonstrated. Using immunocytochemistry, human pancreatic tumor GRF (hptGRF-40) was localized in the majority of neurons of all six gangliocytomas. The pituitary adenomas and nontumorous adenohypophyses were negative for hptGRF-40. In addition, somatostatin, glucagon, and GnRH were demonstrated within some neurons of several tumors; insulin and gastrin stains were equivocal. These findings confirm previous proposals of production of a GRF by such gangliocytomas. While the significance of other peptides found in some of the tumors is uncertain, the presence of hptGRF-40 in neurons of these gangliocytomas supports the theory that GRF excess is the mechanism responsible for over-production of GH and provides evidence for a syndrome of hypothalamic acromegaly.

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

    Science.gov (United States)

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

    2010-09-01

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

  16. The presence of cortical neurons in striatal-cortical co-cultures alters the effects of dopamine and BDNF on Medium Spiny Neuron dendritic development

    Directory of Open Access Journals (Sweden)

    Rachel D Penrod

    2015-07-01

    Full Text Available Medium spiny neurons (MSNs are the major striatal neuron and receive synaptic input from both glutamatergic and dopaminergic afferents. These synapses are made on MSN dendritic spines, which undergo density and morphology changes in association with numerous disease and experience-dependent states. Despite wide interest in the structure and function of mature MSNs, relatively little is known about MSN development. Furthermore, most in vitro studies of MSN development have been done in simple striatal cultures that lack any type of non-autologous synaptic input, leaving open the question of how MSN development is affected by a complex environment that includes other types of neurons, glia, and accompanying secreted and cell-associated cues. Here we characterize the development of MSNs in striatal-cortical co-culture, including quantitative morphological analysis of dendritic arborization and spine development, describing progressive changes in density and morphology of developing spines. Overall, MSN growth is much more robust in the striatal-cortical co-culture compared to striatal mono-culture. Inclusion of dopamine in the co-culture further enhances MSN dendritic arborization and spine density, but the effects of dopamine on dendritic branching are only significant at later times in development. In contrast, exogenous Brain Derived Neurotrophic Factor (BDNF has only a minimal effect on MSN development in the co-culture, but significantly enhances MSN dendritic arborization in striatal mono-culture. Importantly, inhibition of NMDA receptors in the co-culture significantly enhances the effect of exogenous BDNF, suggesting that the efficacy of BDNF depends on the cellular environment. Combined, these studies identify specific periods of MSN development that may be particularly sensitive to perturbation by external factors and demonstrate the importance of studying MSN development in a complex signaling environment.

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

    Science.gov (United States)

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

    2007-02-01

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

  18. Acute and chronic effects of clozapine on cholinergic transmission in cultured mouse superior cervical ganglion neurons.

    Science.gov (United States)

    Saur, Taixiang; Cohen, Bruce M; Ma, Qi; Babb, Suzann M; Buttner, Edgar A; Yao, Wei-Dong

    Cholinergic dysfunction contributes to cognitive deficits in schizophrenia. The atypical antipsychotic clozapine improves cognition in patients with schizophrenia, possibly through modulation of the cholinergic system. However, little is known about specific underlying mechanisms. We investigated the acute and chronic effects of clozapine on cholinergic synaptic transmission in cultured superior cervical ganglion (SCG) neurons. Spontaneous excitatory postsynaptic currents (sEPSCs) were detected and were reversibly inhibited by the nicotinic receptor antagonist d-tubocurarine, confirming that the synaptic responses were primarily mediated by nicotinic receptors. Bath application of clozapine at therapeutic concentrations rapidly and reversely inhibited both the amplitude and frequency of sEPSCs in a concentration-dependent manner, without changing either rise or decay time, suggesting that clozapine effects have both presynaptic and postsynaptic origins. The acute effects of clozapine on sEPSCs were recapitulated by chronic treatment of SCG cultures with similar concentrations of clozapine, as clozapine treatment for 4 d reduced the frequency and amplitude of sEPSCs without affecting their kinetics. Cell survival analysis indicated that SCG neuron cell counts after chronic clozapine treatment were comparable to the control group. These results demonstrate that therapeutic concentrations of clozapine suppress nicotinic synaptic transmission in SCG cholinergic synapses, a simple in vitro preparation of cholinergic transmission.

  19. PACAP enhances axon outgrowth in cultured hippocampal neurons to a comparable extent as BDNF.

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    Katsuya Ogata

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP exerts neurotrophic activities including modulation of synaptic plasticity and memory, hippocampal neurogenesis, and neuroprotection, most of which are shared with brain-derived neurotrophic factor (BDNF. Therefore, the aim of this study was to compare morphological effects of PACAP and BDNF on primary cultured hippocampal neurons. At days in vitro (DIV 3, PACAP increased neurite length and number to similar levels by BDNF, but vasoactive intestinal polypeptide showed much lower effects. In addition, PACAP increased axon, but not dendrite, length, and soma size at DIV 3 similarly to BDNF. The PACAP antagonist PACAP6-38 completely blocked the PACAP-induced increase in axon, but not dendrite, length. Interestingly, the BDNF-induced increase in axon length was also inhibited by PACAP6-38, suggesting a mechanism involving PACAP signaling. K252a, a TrkB receptor inhibitor, inhibited axon outgrowth induced by PACAP and BDNF without affecting dendrite length. These results indicate that in primary cultured hippocampal neurons, PACAP shows morphological actions via its cognate receptor PAC1, stimulating neurite length and number, and soma size to a comparable extent as BDNF, and that the increase in total neurite length is ascribed to axon outgrowth.

  20. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

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    Samuel D Robinson

    2015-10-01

    Full Text Available NMDA receptors (NMDARs play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM but not high (50 μM concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-AP. Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and RAP, a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

  1. Mechanism underlying blockade of voltage-gated calcium channels by agmatine in cultured rat hippocampal neurons

    Institute of Scientific and Technical Information of China (English)

    Jian-quan ZHENG; Xie-chuan WENG; Xiao-dan GAI; Jin LI; Wen-bin XIAO

    2004-01-01

    AIM: To investigate whether agmatine could selectively block a given type of the voltage-gated calcium channels (VGCC) and whether related receptors are involved in the blocking effect of agmatine on VGCC. METHODS: The whole-cell patch recording technique was performed to record VGCC currents in the cultured neonatal rat hippocampal neurons. RESULTS: Verapamil (100 μmol/L), a selective blocker of L-type calcium channel, significantly inhibited VGCC current by 80 %± 7 %. Agmatine (100 μmol/L) could further depress the remained currents by 25 %±6 %. The α2-adrenoceptor antagonist yohimbine (10 μmol/L) and the I2 imidazoline receptor antagonist idazoxon (10 and 40 μmol/L) had no significant effect on VGCC currents when used respectively. When the mixture of yohimbine and agmatine was applied, VGCC currents were still depressed remarkably. However, the blocking effect of agmatine was decreased by 29 %± 8 % in the presence of idazoxon (10 μmol/L). The effect of idazoxon did not increase at a higher concentration (40 μmol/L). CONCLUSION: Agmatine could block the L- and other types of VGCC currents in the cultured rat hippocampal neurons. Blocking effect of agmatine on VGCC was partially related to I2 imidazoline receptor and had no relationship with α2-adrenoceptors.

  2. Glutamine as an energy substrate in cultured neurons during glucose deprivation.

    Science.gov (United States)

    Peng, Liang; Gu, Li; Zhang, Hongliang; Huang, Xueshi; Hertz, Elna; Hertz, Leif

    2007-11-15

    During glucose deprivation an increase in aspartate formation from glutamine has been observed in different brain preparations, including synaptosomes and cultured astrocytes. To what extent this reaction, which provides a substantial amount of energy, occurs in different types of neurons is unknown. The present study shows that (14)CO(2) formation from [U-(14)C]glutamine in cerebellar granule neurons, a glutamatergic preparation, increased by 60% during glucose deprivation, indicating enhanced aspartate formation or increased complete oxidative degradation of glutamine. In primary cultures of cerebrocortical interneurons, a GABAergic preparation, the rate of (14)CO(2) production from [U-(14) C] glutamine was four times lower and not stimulated by glucose deprivation. During incubation with glutamine (0.8 mM) as the only metabolic substrate, cerebellar granule cells maintained an oxygen consumption rate of 12 nmol/min/mg protein, corresponding to an aspartate formation of 8 nmol/min/mg protein (three oxidations occur between glutamine and aspartate) or to a total oxidative degradation of 3 nmol/min/mg protein. During glucose deprivation, the rate of aspartate formation increased, and during a 20-min incubation in phosphate-buffered saline it amounted to 3.3 nmol/min/mg protein at 0.2 mM glutamine, which might have been more if measured at 0.8 mM glutamine. These values are consistent with the rate of glutamine utilization calculated based on oxygen consumption and leaves open the possibility that some glutamine is completely degraded oxidatively, as has been shown by other authors based on pyruvate recycling and labeling of lactate from aspartate in cerebellar granule neurons.

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

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    Sanjana, Neville E; Fuller, Sawyer B

    2004-07-30

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

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

    Science.gov (United States)

    Ferguson, C A; Audesirk, G

    1990-01-01

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

  5. The potential of apolipoprotein E4 to act as a substrate for primary cultures of hippocampal neurons.

    Science.gov (United States)

    Kim, Kwang-Min; Vicenty, Janice; Palmore, G Tayhas R

    2013-04-01

    The E4 isoform of apolipoprotein (apoE4) is known to be a major risk factor for Alzheimer's Disease (AD). Previous in vitro studies have shown apoE4 to have a negative effect on neuronal outgrowth when incubated with lipids. The effect of apoE4 itself on the development of neurons from the central nervous system (CNS), however, has not been well characterized. Consequently, apoE4 alone has not been pursued as a substrate for neuronal cultures. In this study, the effect of surface-bound apoE4 on developmental features of rat hippocampal neurons was examined. We show that apoE4 substrates elicit significantly enhanced values in all developmental features at day 2 of culture when compared to laminin (LN) substrates, which is the current substrate-of-choice for neuronal cultures. Interestingly, the adhesion of hippocampal neurons was found to be significantly lower on LN substrates than on glass substrates, but the axon lengths on both substrates were similar. In addition, this study demonstrates that the adhesion- and growth-enhancing effects of apoE4 substrates are not mediated by heparan sulfate proteoglycans (HSPGs), proteins that have been indicated to function as receptors or co-receptors for apoE4. In the absence of lipids, apoE4 appears to use an unknown pathway for up-regulating neuronal adhesion and neurite outgrowth. Our results indicate that apoE4 is better than LN as a substrate for primary cultures of CNS neurons and should be considered in the design of tissue engineered CNS.

  6. 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...

  7. 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...

  8. Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons.

    Science.gov (United States)

    Audesirk, G; Cabell, L; Kern, M

    1997-09-20

    The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein

  9. Protective effects of aloperine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion.

    Science.gov (United States)

    Ma, Ning-Tian; Zhou, Ru; Chang, Ren-Yuan; Hao, Yin-Ju; Ma, Lin; Jin, Shao-Ju; Du, Juan; Zheng, Jie; Zhao, Cheng-Jun; Niu, Yang; Sun, Tao; Li, Wei; Koike, Kazuo; Yu, Jian-Qiang; Li, Yu-Xiang

    2015-10-01

    Aloperine (ALO), one of the alkaloids isolated from Sophora alopecuroides L., is traditionally used for various diseases including neuronal disorders. This study investigated the protective effects of ALO on neonatal rat primary-cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion (OGD/RP). Treatment with ALO (25, 50, and 100 mg/l) attenuated neuronal damage (p oxygen species and malondialdehyde production and enhanced the antioxidant enzymatic activities of catalase, superoxide dismutase, glutathione peroxidase and the total antioxidant capacity. The results suggested that ALO has significant neuroprotective effects that can be attributed to anti-oxidative stress.

  10. Low cost production of 3D-printed devices and electrostimulation chambers for the culture of primary neurons.

    Science.gov (United States)

    Wardyn, Joanna D; Sanderson, Chris; Swan, Laura E; Stagi, Massimiliano

    2015-08-15

    The analysis of primary neurons is a basic requirement for many areas of neurobiology. However, the range of commercial systems available for culturing primary neurons is functionally limiting, and the expense of these devices is a barrier to both exploratory and large-scale studies. This is especially relevant as primary neurons often require unusual geometries and specialised coatings for optimum growth. Fortunately, the recent revolution in 3D printing offers the possibility to generate customised devices, which can support neuronal growth and constrain neurons in defined paths, thereby enabling many aspects of neuronal physiology to be studied with relative ease. In this article, we provide a detailed description of the system hardware and software required to produce affordable 3D-printed culture devices, which are also compatible with live-cell imaging. In addition, we also describe how to use these devices to grow and stimulate neurons within geometrically constrained compartments and provide examples to illustrate the practical utility and potential that these protocols offer for many aspects of experimental neurobiology.

  11. Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: effects of cytisine and its bromo derivatives.

    Science.gov (United States)

    Varas, Rodrigo; Valdés, Viviana; Iturriaga-Vásquez, Patricio; Cassels, Bruce K; Iturriaga, Rodrigo; Alcayaga, Julio

    2006-02-09

    Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although alpha4 and alpha7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5-10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine > acetylcholine approximately = cytisine > 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

  12. Effects of brain-derived neurotrophic factor on synapsin expression in rat spinal cord anterior horn neurons cultured in vitro

    Institute of Scientific and Technical Information of China (English)

    Zhifei Wang; Daguang Liao; Changqi Li

    2010-01-01

    Brain-derived neurotrophic factor(BDNF)promotes synaptic formation and functional maturation by upregulating synapsin expression in cortical and hippocampal neurons.However,it remains controversial whether BDNF affects synapsin expression in spinal cord anterior horn neurons.Wistar rat spinal cord anterior hom neurons were cultured in serum-supplemented medium containing BDNF,BDNF antibody,and Hank's solution for 3 days,and then synapsin I and synaptophysin protein and mRNA expression was detected.Under serum-supplemented conditions,the number of surviving neurons in the spinal cord anterior horn was similar among BDNF,anti-BDNF,and control groups(P > 0.05).Synapsin I and synaptophysin protein and mRNA expressions were increased in BDNF-treated neurons,but decreased in BDNF antibody-treated neurons(P< 0.01).These results indicated that BDNF significantly promotes synapsin I and synaptophysin expression in in vitro-cultured rat spinal cord anterior horn neurons.

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

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  14. Dexamethasone enhances glutamine synthetase activity and reduces N-methyl-D-aspartate neurotoxicity in mixed cultures of neurons and astrocytes

    Directory of Open Access Journals (Sweden)

    Edith Debroas

    2015-05-01

    Full Text Available Astrocytes are claimed to protect neurons against excitotoxicity by clearing glutamate from the extracellular space and rapidly converting it into glutamine. Glutamine, is then released into the extracellular medium, taken up by neurons and transformed back into glutamate which is then stored into synaptic vesicles. Glutamine synthetase (GS, the key enzyme that governs this glutamate/glutamine cycle, is known to be upregulated by glucocorticoids. In the present work we have thus studied in parallel the effects of dexamethasone on glutamine synthetase activity and NMDA-induced neuronal death in cultures derived from the brain cortex of murine embryos. We showed that dexamethasone was able to markedly enhance GS activity in cultures of astrocytes but not in near pure neuronal cultures. The pharmacological characteristics of the dexamethasone action strongly suggest that it corresponds to a typical receptor-mediated effect. We also observed that long lasting incubation (72 h of mixed astrocyte-neuron cultures in the presence of 100 nM dexamethasone significantly reduced the toxicity of NMDA treatment. Furthermore we demonstrated that methionine sulfoximine, a selective inhibitor of GS, abolished the dexamethasone-induced increase in GS activity and also markedly potentiated NMDA toxicity. Altogether these results suggest that dexamethasone may promote neuroprotection through a stimulation of astrocyte glutamine synthetase.

  15. 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

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

    Institute of Scientific and Technical Information of China (English)

    Nailong Yang; Lili Xu; Fen Yang

    2008-01-01

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

  17. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures.

    Science.gov (United States)

    Valtcheva, Manouela V; Copits, Bryan A; Davidson, Steve; Sheahan, Tayler D; Pullen, Melanie Y; McCall, Jordan G; Dikranian, Krikor; Gereau, Robert W

    2016-10-01

    Primary cultures of rodent sensory neurons are widely used to investigate the cellular and molecular mechanisms involved in pain, itch, nerve injury and regeneration. However, translation of these preclinical findings may be greatly improved by direct validation in human tissues. We have developed an approach to extract and culture human sensory neurons in collaboration with a local organ procurement organization (OPO). Here we describe the surgical procedure for extraction of human dorsal root ganglia (hDRG) and the necessary modifications to existing culture techniques to prepare viable adult human sensory neurons for functional studies. Dissociated sensory neurons can be maintained in culture for >10 d, and they are amenable to electrophysiological recording, calcium imaging and viral gene transfer. The entire process of extraction and culturing can be completed in <7 h, and it can be performed by trained graduate students. This approach can be applied at any institution with access to organ donors consenting to tissue donation for research, and is an invaluable resource for improving translational research.

  18. Dynamic localization of glucokinase and its regulatory protein in hypothalamic tanycytes.

    Directory of Open Access Journals (Sweden)

    Magdiel Salgado

    Full Text Available Glucokinase (GK, the hexokinase involved in glucose sensing in pancreatic β cells, is also expressed in hypothalamic tanycytes, which cover the ventricular walls of the basal hypothalamus and are implicated in an indirect control of neuronal activity by glucose. Previously, we demonstrated that GK was preferentially localized in tanycyte nuclei in euglycemic rats, which has been reported in hepatocytes and is suggestive of the presence of the GK regulatory protein, GKRP. In the present study, GK intracellular localization in hypothalamic and hepatic tissues of the same rats under several glycemic conditions was compared using confocal microscopy and Western blot analysis. In the hypothalamus, increased GK nuclear localization was observed in hyperglycemic conditions; however, it was primarily localized in the cytoplasm in hepatic tissue under the same conditions. Both GK and GKRP were next cloned from primary cultures of tanycytes. Expression of GK by Escherichia coli revealed a functional cooperative protein with a S0.5 of 10 mM. GKRP, expressed in Saccharomyces cerevisiae, inhibited GK activity in vitro with a Ki 0.2 µM. We also demonstrated increased nuclear reactivity of both GK and GKRP in response to high glucose concentrations in tanycyte cultures. These data were confirmed using Western blot analysis of nuclear extracts. Results indicate that GK undergoes short-term regulation by nuclear compartmentalization. Thus, in tanycytes, GK can act as a molecular switch to arrest cellular responses to increased glucose.

  19. 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.

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

    Science.gov (United States)

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

    2015-05-11

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

  1. 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.

  2. Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture.

    Science.gov (United States)

    López, E; Arce, C; Oset-Gasque, M J; Cañadas, S; González, M P

    2006-03-15

    Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.

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

    Science.gov (United States)

    Vianney, John-Mary; Spitsbergen, John M

    2011-05-16

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

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

    Science.gov (United States)

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

    2001-01-01

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

  5. Iridium oxide microelectrode arrays for in vitro stimulation of individual rat neurons from dissociated cultures.

    Science.gov (United States)

    Eick, Stefan; Wallys, Jens; Hofmann, Boris; van Ooyen, André; Schnakenberg, Uwe; Ingebrandt, Sven; Offenhäusser, Andreas

    2009-01-01

    We present the first in vitro extracellular stimulation of individual neurons from dissociated cultures with iridium oxide (IrO(x)) electrodes. Microelectrode arrays with sputtered IrO(x) films (SIROF) were developed for electrophysiological investigations with electrogenic cells. The microelectrodes were characterized with scanning electron and atomic force microscopy, revealing rough and porous electrodes with enlarged surface areas. As shown by cyclic voltammetry and electrochemical impedance spectroscopy, the large surface area in combination with the good electrochemical properties of SIROF resulted in high charge storage capacity and low electrode impedance. Thus, we could transfer the good properties of IrO(x) as material for in vivo stimulation electrodes to multi-electrode arrays with electrode diameters as small as 10 mum for in vitro applications. Single rat cortical neurons from dissociated cultures were successfully stimulated to fire action potentials using single or trains of biphasic rectangular voltage-controlled stimulation pulses. The stimulated cell's membrane potential was simultaneously monitored using whole-cell current-clamp recordings. This experimental configuration allowed direct evaluation of the influence of pulse phase sequence, amplitude, and number on the stimulation success ratio and action potential latency. Negative phase first pulses were more effective for extracellular stimulation and caused reduced latency in comparison to positive phase first pulses. Increasing the pulse amplitude also improved stimulation reliability. However, in order to prevent cell or electrode damage, the pulse amplitude is limited to voltages below the threshold for irreversible electrochemical reactions at the electrode. As an alternative to increasing the amplitude, a higher number of stimulation pulses was also shown to increase stimulation success.

  6. Iridium oxide microelectrode arrays for in vitro stimulation of individual rat neurons from dissociated cultures

    Directory of Open Access Journals (Sweden)

    Stefan Eick

    2009-11-01

    Full Text Available We present the first in-vitro extracellular stimulation of individual neurons from dissociated cultures with iridium oxide (IrOx electrodes. Microelectrode arrays with sputtered IrOx films (SIROF were developed for electrophysiological investigations with electrogenic cells. The microelectrodes were characterized with scanning electron and atomic force microscopy, revealing rough and porous electrodes with enlarged surface areas. As shown by cyclic voltammetry and electrochemical impedance spectroscopy, the large surface area in combination with the good electrochemical properties of SIROF resulted in high charge storage capacity and low electrode impedance. Thus, we could transfer the good properties of IrOx as material for in-vivo stimulation electrodes to MEAs with electrode diameters as small as 10 µm for in-vitro applications. Single rat cortical neurons from dissociated cultures were successfully stimulated to fire action potentials using single or trains of biphasic rectangular voltage-controlled stimulation pulses. The stimulated cell’s membrane potential was simultaneously monitored using whole-cell current-clamp recordings. This experimental configuration allowed direct evaluation of the influence of pulse phase sequence, amplitude, and number on the stimulation success ratio and action potential latency. Negative phase first pulses were more effective for extracellular stimulation and caused reduced latency in comparison to positive phase first pulses. Increasing the pulse amplitude also improved stimulation reliability. However, in order to prevent cell or electrode damage, the pulse amplitude is limited to voltages below the threshold for irreversible electrochemical reactions at the electrode. As an alternative to increasing the amplitude, a higher number of stimulation pulses was also shown to increase stimulation success.

  7. Methamphetamine modulates glutamatergic synaptic transmission in rat primary cultured hippocampal neurons.

    Science.gov (United States)

    Zhang, Shuzhuo; Jin, Yuelei; Liu, Xiaoyan; Yang, Lujia; Ge, Zhi juan; Wang, Hui; Li, Jin; Zheng, Jianquan

    2014-09-25

    Methamphetamine (METH) is a psychostimulant drug. Abuse of METH produces long-term behavioral changes including behavioral, sensitization, tolerance, and dependence. It induces neurotoxic effects in several areas of the brain via enhancing dopamine (DA) level abnormally, which may cause a secondary release of glutamate (GLU). However, repeated administration of METH still increases release of GLU even when dopamine content in tissue is significantly depleted. It implies that some other mechanisms are likely to involve in METH-induced GLU release. The goal of this study was to observe METH affected glutamatergic synaptic transmission in rat primary cultured hippocampal neurons and to explore the mechanism of METH modulated GLU release. Using whole-cell patch-clamp recordings, we found that METH (0.1-50.0μM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). However, METH decreased the frequency of sEPSCs and mEPSCs at high concentration of 100μM. The postsynaptic NMDA receptor currents and P/Q-type calcium channel were not affected by the use of METH (10,100μM). METH did not present visible effect on N-type Ca(2+) channel current at the concentration lower than 50.0μM, but it was inhibited by use of METH at a 100μM. The effect of METH on glutamatergic synaptic transmission was not revered by pretreated with DA receptor antagonist SCH23390. These results suggest that METH directly modulated presynaptic GLU release at a different concentration, while dopaminergic system was not involved in METH modulated release of GLU in rat primary cultured hippocampal neurons.

  8. Full Length Bid is sufficient to induce apoptosis of cultured rat hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Ward Manus W

    2007-02-01

    Full Text Available Abstract Background Bcl-2 homology domain (BH 3-only proteins are pro-apoptotic proteins of the Bcl-2 family that couple stress signals to the mitochondrial cell death pathways. The BH3-only protein Bid can be activated in response to death receptor activation via caspase 8-mediated cleavage into a truncated protein (tBid, which subsequently translocates to mitochondria and induces the release of cytochrome-C. Using a single-cell imaging approach of Bid cleavage and translocation during apoptosis, we have recently demonstrated that, in contrast to death receptor-induced apoptosis, caspase-independent excitotoxic apoptosis involves a translocation of full length Bid (FL-Bid from the cytosol to mitochondria. We induced a delayed excitotoxic cell death in cultured rat hippocampal neurons by a 5-min exposure to the glutamate receptor agonist N-methyl-D-aspartate (NMDA; 300 μM. Results Western blot experiments confirmed a translocation of FL-Bid to the mitochondria during excitotoxic apoptosis that was associated with the release of cytochrome-C from mitochondria. These results were confirmed by immunofluorescence analysis of Bid translocation during excitotoxic cell death using an antibody raised against the amino acids 1–58 of mouse Bid that is not able to detect tBid. Finally, inducible overexpression of FL-Bid or a Bid mutant that can not be cleaved by caspase-8 was sufficient to induce apoptosis in the hippocampal neuron cultures. Conclusion Our data suggest that translocation of FL-Bid is sufficient for the activation of mitochondrial cell death pathways in response to glutamate receptor overactivation.

  9. Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorylation.

    Science.gov (United States)

    Kern, M; Audesirk, G

    1995-09-01

    Inorganic lead inhibits neurite initiation in cultured rat hippocampal neurons at concentrations as low as 100 nM. Conflicting reports suggest that Pb2+ may stimulate or inhibit protein kinase C, adenylyl cyclase, phosphodiesterase, and calmodulin, or increase intracellular free Ca2+ concentrations. Therefore, Pb2+ may alter the activities of Ca2+/calmodulin-dependent protein kinase (CaM kinase) or protein kinases C or A. We cultured rat hippocampal neurons in 100 nM PbCI2 alone or in combination with kinase or calmodulin inhibitors. Inhibiting protein kinase C with calphostin C exacerbated the inhibition of neurite initiation caused by PbCI2, but inhibiting protein kinase A with KT5720, CaM kinase with KN62, or calmodulin with calmidazolium completely reversed the effects of PbCI2. These results indicate that Pb2+ may inhibit neurite initiation by inappropriately stimulating protein phosphorylation by CaM kinase or cyclic AMP-dependent protein kinase (PKA), possibly by stimulating calmodulin. This hypothesis is supported by findings that other treatments that should increase protein phosphorylation (okadaic acid, a protein phosphatase inhibitor, and Sp-cAMPS, a PKA activator) also reduced neurite initiation. Whole-cell intracellular free Ca2+ ion concentrations were not significantly altered by 100 nM PbCI2 at 4, 12, 24, or 48 hr. Therefore, the hypothesized stimulatory effects of Pb2+ exposure on calmodulin, CaM kinase, or PKA are probably not caused by increases in whole-cell intracellular free Ca2+, but may be attributable either to intracellular Pb2+ or to localized increases in [Ca2+]in that are not reflected in whole-cell measurements.

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

    Science.gov (United States)

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

    2007-01-22

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

  11. 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.

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

    Science.gov (United States)

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

    2010-09-01

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

  13. Acute neuregulin-1 signaling influences AMPA receptor mediated responses in cultured cerebellar granule neurons.

    Science.gov (United States)

    Fenster, Catherine; Vullhorst, Detlef; Buonanno, Andres

    2012-01-04

    Neuregulin-1 (NRG1) is a trophic and differentiation factor that signals through ErbB receptor tyrosine kinases to regulate nervous system development. Previous studies have demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons of the hippocampus and frontal cortex; however, immunohistochemical and genetic analyses strongly suggest these effects are indirect and mediated via ErbB4 receptors on GABAergic interneurons. Here, we used cultured cerebellar granule cells (CGCs) that express ErbB4 to analyze the cell-autonomous effects of NRG1 stimulation on glutamatergic function. These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We show that acute NRG1 treatment does not affect whole-cell AMPA or NMDA receptor (NMDAR) mediated currents in CGCs at 10-12 days in vitro. NRG1 also does not affect the frequency or amplitude of spontaneous AMPAR or NMDAR mediated miniature excitatory post-synaptic currents (mEPSCs). To further investigate the effects of NRG1 on activity-dependent plasticity of glutamatergic synapses in CGCs, we characterized the effects of high-glyine/0 Mg(2+) (which activates synaptic NMDARs) on AMPAR-mEPSC frequency and amplitude. We show that high-glycine induces a form of chemical long-term potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency but not amplitude. Moreover, NRG1 induces a decrease in AMPAR-mEPSC frequency following chemLTP, but does not affect AMPAR-mEPSC amplitude. CGCs in our cultures conditions express low levels of GluR1, in contrast to dissociated hippocampal cultures, but do express the long isoform of GluR4. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and (2) that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar

  14. 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.

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

    Institute of Scientific and Technical Information of China (English)

    Xianying Wang; Honghai Wu; Gai Xue; Yanning Hou

    2012-01-01

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

  16. Hypothalamic miRNAs: emerging roles in energy balance control.

    Science.gov (United States)

    Schneeberger, Marc; Gomez-Valadés, Alicia G; Ramirez, Sara; Gomis, Ramon; Claret, Marc

    2015-01-01

    The hypothalamus is a crucial central nervous system area controlling appetite, body weight and metabolism. It consists in multiple neuronal types that sense, integrate and generate appropriate responses to hormonal and nutritional signals partly by fine-tuning the expression of specific batteries of genes. However, the mechanisms regulating these neuronal gene programmes in physiology and pathophysiology are not completely understood. MicroRNAs (miRNAs) are key regulators of gene expression that recently emerged as pivotal modulators of systemic metabolism. In this article we will review current evidence indicating that miRNAs in hypothalamic neurons are also implicated in appetite and whole-body energy balance control.

  17. Hypothalamic miRNAs: emerging roles in energy balance control

    Directory of Open Access Journals (Sweden)

    Marc eSchneeberger

    2015-02-01

    Full Text Available The hypothalamus is a crucial central nervous system area controlling appetite, body weight and metabolism. It consists in multiple neuronal types that sense, integrate and generate appropriate responses to hormonal and nutritional signals partly by fine-tuning the expression of specific batteries of genes. However, the mechanisms regulating these neuronal gene programmes in physiology and pathophysiology are not completely understood. MicroRNAs (miRNAs are key regulators of gene expression that recently emerged as pivotal modulators of systemic metabolism. In this article we will review current evidence indicating that miRNAs in hypothalamic neurons are also implicated in appetite and whole-body energy balance control.

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

    Science.gov (United States)

    Pamenter, Matthew E; Ryu, Julie; Hua, Serena T; Perkins, Guy A; Mendiola, Vincent L; Gu, Xiang Q; Ellisman, Mark H; Haddad, Gabriel G

    2012-01-01

    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 prevented stimulus

  19. 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

  20. Dopamine-dependent effects on basal and glutamate stimulated network dynamics in cultured hippocampal neurons.

    Science.gov (United States)

    Li, Yan; Chen, Xin; Dzakpasu, Rhonda; Conant, Katherine

    2017-02-01

    Oscillatory activity occurs in cortical and hippocampal networks with specific frequency ranges thought to be critical to working memory, attention, differentiation of neuronal precursors, and memory trace replay. Synchronized activity within relatively large neuronal populations is influenced by firing and bursting frequency within individual cells, and the latter is modulated by changes in intrinsic membrane excitability and synaptic transmission. Published work suggests that dopamine, a potent modulator of learning and memory, acts on dopamine receptor 1-like dopamine receptors to influence the phosphorylation and trafficking of glutamate receptor subunits, along with long-term potentiation of excitatory synaptic transmission in striatum and prefrontal cortex. Prior studies also suggest that dopamine can influence voltage gated ion channel function and membrane excitability in these regions. Fewer studies have examined dopamine's effect on related endpoints in hippocampus, or potential consequences in terms of network burst dynamics. In this study, we record action potential activity using a microelectrode array system to examine the ability of dopamine to modulate baseline and glutamate-stimulated bursting activity in an in vitro network of cultured murine hippocampal neurons. We show that dopamine stimulates a dopamine type-1 receptor-dependent increase in number of overall bursts within minutes of its application. Notably, however, at the concentration used herein, dopamine did not increase the overall synchrony of bursts between electrodes. Although the number of bursts normalizes by 40 min, bursting in response to a subsequent glutamate challenge is enhanced by dopamine pretreatment. Dopamine-dependent potentiation of glutamate-stimulated bursting was not observed when the two modulators were administered concurrently. In parallel, pretreatment of murine hippocampal cultures with dopamine stimulated lasting increases in the phosphorylation of the

  1. Green Tea Polyphenols Attenuated Glutamate Excitotoxicity via Antioxidative and Antiapoptotic Pathway in the Primary Cultured Cortical Neurons.

    Science.gov (United States)

    Cong, Lin; Cao, Chang; Cheng, Yong; Qin, Xiao-Yan

    2016-01-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Yinzhi Lai

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

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

    DEFF Research Database (Denmark)

    Jensen, J B; Lund, Trine Meldgaard; Timmermann, D 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...

  4. 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

    1996-01-01

    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 r

  5. 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

  6. Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology

    NARCIS (Netherlands)

    Frega, M.; Tedesco, M.; Massobrio, P.; Pesce, M.; Martinoia, S.

    2014-01-01

    Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These

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

    OpenAIRE

    Georg Welzel; Daniel Seitz; Stefan Schuster

    2015-01-01

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

  8. 5-HT7 Receptors Are Not Involved in Neuropeptide Release in Primary Cultured Rat Trigeminal Ganglion Neurons.

    Science.gov (United States)

    Wang, Xiaojuan; Hu, Rong; Liang, Jianbo; Li, Ze; Sun, Weiwen; Pan, Xiaoping

    2016-06-01

    Migraine is a common but complex neurological disorder. Its precise mechanisms are not fully understood. Increasing indirect evidence indicates that 5-HT7 receptors may be involved; however, their role remains unknown. Our previous in vivo study showed that selective blockade of 5-HT7 receptors caused decreased serum levels of calcitonin gene-related peptide (CGRP) in the external jugular vein following electrical stimulation of the trigeminal ganglion (TG) in an animal model of migraine. In the present study, we used an in vitro model of cultured TG cells to further investigate whether 5-HT7 receptors are directly responsible for the release of CGRP and substance P from TG neurons. We stimulated rat primary cultured TG neurons with capsaicin or potassium chloride (KCl) to mimic neurogenic inflammation, resulting in release of CGRP and substance P. 5-HT7 receptors were abundantly expressed in TG neurons. Greater than 93 % of 5-HT7 receptor-positive neurons co-expressed CGRP and 56 % co-expressed substance P. Both the capsaicin- and KCl-induced release of CGRP and substance P were unaffected by pretreatment of cultured TG cells with the selective 5-HT7 receptor agonist AS19 and antagonist SB269970. This study demonstrates for the first time that 5-HT7 receptors are abundantly co-expressed with CGRP and substance P in rat primary TG neurons and suggests that they are not responsible for the release of CGRP and substance P from cultured TG neurons evoked by capsaicin or KCl.

  9. Aged garlic extract and its components protect cultured rat hippocampal neurons from amyloid β—protein—in—duced neuronal death

    Institute of Scientific and Technical Information of China (English)

    ItoY; KosuY

    2002-01-01

    Aged garlic extract and its components such as S-allyl-L-cysteine (SAC) and sllixin have been shown to possess various biological effects including neurotrophic activity.We characterized the neuronal death induced by amyloid β-protein (Aβ),4-hydroxynoenal (HNE),tunicamycin(TM),and trophic factor-deprivation (TFD),and ivestigated whether these garlic compounds could prevent this in cultured PC12 cells and rat hippocampal neurons.Treatment with SAC protected these cells against Aβ- and TM-induced neuronal death.SAC also attenuated the processing of procaspase-12 induced by Aβ25-35 or TM.In contrast,allixin and its analogue,DHP,afforded no protection against Aβ-induced cell death.SAC afforded no protection against HNE- and TFD-induced cell death,which has been shown to be mediated by caspase-3 dependent pathway.These results suggest that SAC protect against the neuronal cell death that is triggered by ER dysfunction.

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

    National Research Council Canada - National Science Library

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

    2015-01-01

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

  11. 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.

  12. Membrane voltage modulates the GABA(A) receptor gating in cultured rat hippocampal neurons.

    Science.gov (United States)

    Pytel, Maria; Mercik, Katarzyna; Mozrzymas, Jerzy W

    2006-02-01

    The kinetics of GABAergic currents in neurons is known to be modulated by the membrane voltage but the underlying mechanisms have not been fully explored. In particular, the impact of membrane potential on the GABA(A) receptor gating has not been elucidated. In the present study, the effect of membrane voltage on current responses elicited by ultrafast GABA applications was studied in cultured hippocampal neurons. The current to voltage relationship (I-V) for responses to saturating [GABA] (10 mM) showed an inward rectification (slope conductance at positive voltages was 0.62 +/- 0.05 of that at negative potentials). On the contrary, I-V for currents evoked by low [GABA] (1 microM) showed an outward rectification. The onset of currents elicited by saturating [GABA] was significantly accelerated at positive potentials. Analysis of currents evoked by prolonged applications of saturating [GABA] revealed that positive voltages significantly increased the rate and extent of desensitization. The onsets of current responses to non-saturating [GABA] were significantly accelerated at positive voltages indicating an enhancement of the binding rate. However, at low [GABA] at which the onset rate is expected to approach an asymptote set by opening/closing and unbinding rates, no significant modification of current onset by voltage was observed. Quantitative analysis based on model simulations indicated that the major effect of membrane depolarization was to increase the rates of binding, desensitization and of opening as well as to slightly reduce the rate of exit from desensitization. In conclusion, we provide evidence that membrane voltage affects the GABA(A) receptor microscopic gating.

  13. High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Thomas eAndreska

    2014-04-01

    Full Text Available In the mammalian brain, the neurotrophin brain-derived neurotrophic factor (BDNF has emerged as a key factor for synaptic refinement, plasticity and learning. Although BDNF-induced signaling cascades are well known, the spatial aspects of the synaptic BDNF localization remained unclear. Recent data provide strong evidence for an exclusive presynaptic location and anterograde secretion of endogenous BDNF at synapses of the hippocampal circuit. In contrast, various studies using BDNF overexpression in cultured hippocampal neurons support the idea that postsynaptic synapses and other dendritic structures are the preferential sites of BDNF localization and release. In this study we used rigorously tested anti-BDNF antibodies and achieved a dense labeling of endogenous BDNF close to synapses. Confocal microscopy showed natural BDNF close to many, but not all glutamatergic synapses, while neither GABAergic synapses nor postsynaptic structures carried a typical synaptic BDNF label. To visualize the BDNF distribution within the fine structure of synapses, we implemented super resolution fluorescence imaging by direct stochastic optical reconstruction microscopy (dSTORM. Two-color dSTORM images of neurites were acquired with a spatial resolution of ~20 nm. At this resolution, the synaptic scaffold proteins Bassoon and Homer exhibit hallmarks of mature synapses and form juxtaposed bars, separated by a synaptic cleft. BDNF imaging signals form granule-like clusters with a mean size of ~60 nm and are preferentially found within the fine structure of the glutamatergic presynapse. Individual glutamatergic presynapses carried up to 90% of the synaptic BDNF immunoreactivity, and only a minor fraction of BDNF molecules was found close to the postsynaptic bars. Our data proof that hippocampal neurons are able to enrich and store high amounts of BDNF in small granules within the mature glutamatergic presynapse, at a principle site of synaptic plasticity.

  14. Pregabalin reduces the release of synaptic vesicles from cultured hippocampal neurons.

    Science.gov (United States)

    Micheva, Kristina D; Taylor, Charles P; Smith, Stephen J

    2006-08-01

    Pregabalin [S-[+]-3-isobutylGABA or (S)-3-(aminomethyl)-5-methylhexanoic acid, Lyrica] is an anticonvulsant and analgesic medication that is both structurally and pharmacologically related to gabapentin (Neurontin; Pfizer Inc., New York, NY). Previous studies have shown that pregabalin reduces the release of neurotransmitters in several in vitro preparations, although the molecular details of these effects are less clear. The present study was performed using living cultured rat hippocampal neurons with the synaptic vesicle fluorescent dye probe FM4-64 to determine details of the action of pregabalin to reduce neurotransmitter release. Our results indicate that pregabalin treatment, at concentrations that are therapeutically relevant, slightly but significantly reduces the emptying of neurotransmitter vesicles from presynaptic sites in living neurons. Dye release is reduced in both glutamic acid decarboxylase (GAD)-immunoreactive and GAD-negative (presumed glutamatergic) synaptic terminals. Furthermore, both calcium-dependent release and hyperosmotic (calcium-independent) dye release are reduced by pregabalin. The effects of pregabalin on dye release are masked in the presence of l-isoleucine, consistent with the fact that both of these compounds have a high binding affinity to the calcium channel alpha(2)-delta protein. The effect of pregabalin is not apparent in the presence of an N-methyl-d-aspartate (NMDA) antagonist [D(-)-2-amino-5-phosphonopentanoic acid], suggesting that pregabalin action depends on NMDA receptor activation. Finally, the action of pregabalin on dye release is most apparent before and early during a train of electrical stimuli when vesicle release preferentially involves the readily releasable pool.

  15. Differential estrogenic effects of the persistent organochlorine pesticides dieldrin, endosulfan, and lindane in primary neuronal cultures.

    Science.gov (United States)

    Briz, Víctor; Molina-Molina, José-Manuel; Sánchez-Redondo, Sara; Fernández, Mariana F; Grimalt, Joan O; Olea, Nicolás; Rodríguez-Farré, Eduard; Suñol, Cristina

    2011-04-01

    The organochlorine chemicals endosulfan, dieldrin, and γ-hexachlorocyclohexane (lindane) are persistent pesticides to which people are exposed mainly via diet. Their antagonism of the γ-aminobutyric acid-A (GABA(A)) receptor makes them convulsants. They are also endocrine disruptors because of their interaction with the estrogen receptor (ER). Here, we study the effects of dieldrin, endosulfan, and lindane on ERs in primary cultures of cortical neurons (CN) and cerebellar granule cells (CGC). All the compounds tested inhibited the binding of [(3)H]-estradiol to the ER in both CN and CGC, with dieldrin in CGC showing the highest affinity. We also determined the effects of the pesticides on protein kinase B (Akt) and extracellular-regulated kinase 1 and 2 (ERK1/2) phosphorylation. Dieldrin and endosulfan increased Akt phosphorylation in CN, which was inhibited by the ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Instead, Akt and ERK1/2 phosphorylation induced by dieldrin in CGC was mediated by multiple activation of ERα, ERβ, and G protein-coupled receptor 30. Lindane did not activate these pathways, but it inhibited estradiol-mediated Akt and ERK1/2 activation. In CN, all the chemicals activated ERK1/2 through a mechanism involving GABA(A) and glutamate receptors. Long-term exposure to these pesticides reduced the levels of ERα, but not of ERβ. Moreover, extracts of CN treated with endosulfan, dieldrin, or lindane induced cell proliferation in MCF-7 human breast cancer-derived cells, whereas only extracts of CGC treated with dieldrin induced MCF-7 cell proliferation. Overall, the observed alterations on ER-mediated signaling and ER levels in neurons might contribute to the neurotoxicity of these organochlorine pesticides.

  16. Quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using FM dyes.

    Science.gov (United States)

    Cheung, Giselle; Cousin, Michael A

    2011-11-11

    obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable(11). Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)(12,13).

  17. Expression of the neuronal nitric oxide synthase in the paraventricular hypothalamic nucleus in rats with metabolic syndrome%代谢综合征大鼠下丘脑室旁核神经元型一氧化氮合酶的表达

    Institute of Scientific and Technical Information of China (English)

    马同军; 吴锋

    2012-01-01

    Objective:To examine the changes of neuronal nitric oxide synthase (nNOS) expression in paraventricular hypothalamic nucleus (PVN) in rats with metabolic syndrome, and explore the mechanisms of nitric oxide(NO) in PVN in inducing the metabolic syndrome. Methods : A high-fat,refined-carbohydrate diet was given to the rats for 24 weeks to induce metabolic syndrome. Immunohistostaining was performed to examine the expression of nNOS in PVN. Results: Compared with the normal control group,the expression of nNOS in the model group were up-regulated significantly(P<0.01). Conclusion :The fact that evidently up-regulated nNOS expression in PVN was seen in rats with metabolic syndrome suggest that the changes of nNOS activities may be involved in variation of the cerebral nemo endocrine.%目的:观察代谢综合征大鼠下丘脑室旁核(paraventricular hypothalamic nucleus,PVN)内神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)表达的变化,探讨PVN内一氧化氮(nitric oxide,NO)在代谢综合征发病中的作用机制.方法:高脂高糖诱导大鼠代谢综合征24周,应用免疫组织化学染色方法观察代谢综合征大鼠PVN内nNOS的表达.结果:与对照组相比较,模型组PVN内nNOS表达明显增加(P<0.01).结论:PVN内nNOS的表达在代谢综合征大鼠中明显升高,nNOS活性改变可能与代谢综合征神经内分泌改变有关.

  18. Neuropeptide exocytosis involving synaptotagmin-4 and oxytocin in hypothalamic programming of body weight and energy balance.

    Science.gov (United States)

    Zhang, Guo; Bai, Hua; Zhang, Hai; Dean, Camin; Wu, Qiang; Li, Juxue; Guariglia, Sara; Meng, Qingyuan; Cai, Dongsheng

    2011-02-10

    Hypothalamic neuropeptides play essential roles in regulating energy and body weight balance. Energy imbalance and obesity have been linked to hypothalamic signaling defects in regulating neuropeptide genes; however, it is unknown whether dysregulation of neuropeptide exocytosis could be critically involved. This study discovered that synaptotagmin-4, an atypical modulator of synaptic exocytosis, is expressed most abundantly in oxytocin neurons of the hypothalamus. Synaptotagmin-4 negatively regulates oxytocin exocytosis, and dietary obesity is associated with increased vesicle binding of synaptotagmin-4 and thus enhanced negative regulation of oxytocin release. Overexpressing synaptotagmin-4 in hypothalamic oxytocin neurons and centrally antagonizing oxytocin in mice are similarly obesogenic. Synaptotagmin-4 inhibition prevents against dietary obesity by normalizing oxytocin release and energy balance under chronic nutritional excess. In conclusion, the negative regulation of synaptotagmin-4 on oxytocin release represents a hypothalamic basis of neuropeptide exocytosis in controlling obesity and related diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

    OpenAIRE

    Saijilafu,; Zhou, Feng-Quan

    2012-01-01

    It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which ...

  20. 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.

  1. Secondary Ion Mass Spectrometry Imaging of Molecular Distributions in Cultured Neurons and Their Processes: Comparative Analysis of Sample Preparation

    Science.gov (United States)

    Tucker, Kevin R.; Li, Zhen; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

    2012-11-01

    Neurons often exhibit a complex chemical distribution and topography; therefore, sample preparation protocols that preserve structures ranging from relatively large cell somata to small neurites and growth cones are important factors in secondary ion mass spectrometry (SIMS) imaging studies. Here, SIMS was used to investigate the subcellular localization of lipids and lipophilic species in neurons from Aplysia californica. Using individual neurons cultured on silicon wafers, we compared and optimized several SIMS sampling approaches. After an initial step to remove the high salt culturing media, formaldehyde, paraformaldehyde, and glycerol, and various combinations thereof, were tested for their ability to achieve cell stabilization during and after the removal of extracellular media. These treatments improved the preservation of cellular morphology as visualized with SIMS imaging. For analytes >250 Da, coating the cell surface with a 3.2 nm-thick gold layer increased the ion intensity; multiple analytes previously not observed or observed at low abundance were detected, including intact cholesterol and vitamin E molecular ions. However, once a sample was coated, many of the lower molecular mass (cell stabilization with glycerol and 4 % paraformaldehyde. The sample preparation methods described here enhance SIMS imaging of processes of individual cultured neurons over a broad mass range with enhanced image contrast.

  2. Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation.

    Science.gov (United States)

    Wiegreffe, Christoph; Feldmann, Svenja; Gaessler, Simeon; Britsch, Stefan

    2017-07-25

    In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

  3. Mitochondria-targeted antioxidant mitotempo protects mitochondrial function against amyloid beta toxicity in primary cultured mouse neurons.

    Science.gov (United States)

    Hu, Hongtao; Li, Mo

    2016-09-01

    Mitochondrial defects including excess reactive oxygen species (ROS) production and compromised ATP generation are featured pathology in Alzheimer's disease (AD). Amyloid beta (Aβ)-mediated mitochondrial ROS overproduction disrupts intra-neuronal Redox balance, in turn exacerbating mitochondrial dysfunction leading to neuronal injury. Previous studies have found the beneficial effects of mitochondria-targeted antioxidants in preventing mitochondrial dysfunction and neuronal injury in AD animal and cell models, suggesting that mitochondrial ROS scavengers hold promise for the treatment of this neurological disorder. In this study, we have determined that mitotempo, a novel mitochondria-targeted antioxidant protects mitochondrial function from the toxicity of Aβ in primary cultured neurons. Our results showed that Aβ-promoted mitochondrial superoxide production and neuronal lipid oxidation were significantly suppressed by the application of mitotempo. Moreover, mitotempo also demonstrated protective effects on mitochondrial bioenergetics evidenced by preserved mitochondrial membrane potential, cytochrome c oxidase activity as well as ATP production. In addition, the Aβ-induced mitochondrial DNA (mtDNA) depletion and decreased expression levels of mtDNA replication-related DNA polymerase gamma (DNA pol γ) and Twinkle were substantially mitigated by mitotempo. Therefore, our study suggests that elimination of excess mitochondrial ROS rescues mitochondrial function in Aβ-insulted neruons; and mitotempo has the potential to be a promising therapeutic agent to protect mitochondrial and neuronal function in AD.

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

    Science.gov (United States)

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

    2001-01-01

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

  5. Medical therapy of hypothalamic diseases

    Energy Technology Data Exchange (ETDEWEB)

    Werder, K. von; Mueller, O.A. (Muenchen Univ. (Germany, F.R.). Medizinische Klinik 1)

    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.

  6. 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.

  7. Peptidergic neurons of the crab, Cardisoma carnifex, in defined culture maintain characteristic morphologies under a variety of conditions.

    Science.gov (United States)

    Grau, S M; Cooke, I M

    1992-11-01

    Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, "superlarge," found occasionally, has a soma diameter greater than 40 microns and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with L-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26 degrees C instead of 22 degrees C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K+]o medium ([K+]o = 15-110 mM; standard = 11 mM) has no long-term effect, but growth is arrested by [K+]o greater than 30 mM. Cultures were also grown in media in which [Ca2+]o ranged from 0.1 microM to 26 mM (standard = 13 mM). Outgrowth occurred from all neuronal types in all [Ca2+]o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.

  8. Hypothalamic proopiomelanocortin processing and the regulation of energy balance

    OpenAIRE

    Wardlaw, Sharon L.

    2011-01-01

    Hypothalamic proopiomelanocortin (POMC) neurons play a key role in regulating energy balance and neuroendocrine function. Much attention has been focused on regulation of POMC gene expression with less emphasis on regulated peptide processing. This is particularly important given the complexity of posttranslational POMC processing which is essential for the generation of biologically active MSH peptides. Mutations that impair POMC sorting and processing are associated with obesity in humans a...

  9. 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.

  10. 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

    in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected......, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted...... 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...

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

    Science.gov (United States)

    Brimson, James M; Tencomnao, Tewin

    2011-07-26

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

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

    Directory of Open Access Journals (Sweden)

    James M. Brimson

    2011-07-01

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

  13. 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.

  14. 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

    2011-07-01

    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.

  15. 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.

  16. Insulin/PI3K signaling protects dentate neurons from oxygen-glucose deprivation in organotypic slice cultures.

    Science.gov (United States)

    Sun, Xiaolu; Yao, Hang; Douglas, Robert M; Gu, Xiang Q; Wang, Juan; Haddad, Gabriel G

    2010-01-01

    It is known that ischemia/reperfusion induces neurodegeneration in the hippocampus in a subregion-dependent manner. This study investigated the mechanism of selective resistance/vulnerability to oxygen-glucose deprivation (OGD) using mouse organotypic hippocampal cultures. Analysis of propidium iodide uptake showed that OGD-induced duration- and subregion-dependent neuronal injury. When compared with the CA1-3 subregions, dentate neuronal survival was more sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling under basal conditions. Dentate neuronal sensitivity to PI3K/Akt signaling activation was inversely related to its vulnerability to OGD-induced injury; insulin/insulin-like growth factor 1 pre-treatment conferred neuroprotection to dentate neurons via activation of PI3K/Akt signaling. In contrast, CA1 and CA3 neurons were less sensitive to disruptions of endogenous PI3K/Akt signaling and protective effects of insulin/insulin-like growth factor 1, but more vulnerable to OGD. OGD-induced injury in CA1 was reduced by inhibition of NMDA receptor or mitogen-activated protein kinase signaling, and was prevented by blocking NMDA receptor in the presence of insulin. The CA2 subregion was distinctive in its response to glutamate, OGD, and insulin, compared with other CA subregions. CA2 neurons were sensitive to the protective effects of insulin against OGD-induced injury, but more resistant to glutamate. Distinctive distribution of insulin receptor beta and basal phospho-Akt was detected in our slice cultures. Our results suggest a role for insulin signaling in subregional resistance/vulnerability to cerebral ischemia.

  17. Serotonin (5-HT) regulates neurite outgrowth through 5-HT1A and 5-HT7 receptors in cultured hippocampal neurons.

    Science.gov (United States)

    Rojas, Paulina S; Neira, David; Muñoz, Mauricio; Lavandero, Sergio; Fiedler, Jenny L

    2014-08-01

    Serotonin (5-HT) production and expression of 5-HT receptors (5-HTRs) occur early during prenatal development. Recent evidence suggests that, in addition to its classical role as a neurotransmitter, 5-HT regulates neuronal connectivity during mammalian development by modulating cell migration and neuronal cytoarchitecture. Given the variety of 5-HTRs, researchers have had difficulty clarifying the specific role of each receptor subtype in brain development. Signalling mediated by the G-protein-coupled 5-HT1A R and 5-HT7 R, however, has been associated with neuronal plasticity. Thus, we hypothesized that 5-HT promotes neurite outgrowth through 5-HT1A R and 5-HT7 R. The involvement of 5-HT1A R and 5-HT7 R in the morphology of rat hippocampal neurons was evaluated by treating primary cultures at 2 days in vitro with 5-HT and specific antagonists for 5-HT1A R and 5-HT7 R (WAY-100635 and SB269970, respectively). The stimulation of hippocampal neurons with 100 nM 5-HT for 24 hr produced no effect on either the number or the length of primary neurites. Nonetheless, after 5HT7 R was blocked, the addition of 5-HT increased the number of primary neurites, suggesting that 5HT7 R could inhibit neuritogenesis. In contrast, 5-HT induced secondary neurite outgrowth, an effect inhibited by 1 μM WAY-100635 or SB269970. These results suggest that both serotonergic receptors participate in secondary neurite outgrowth. We conclude that 5-HT1A R and 5-HT7 R regulate neuronal morphology in primary hippocampal cultures by promoting secondary neurite outgrowth.

  18. Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

    Institute of Scientific and Technical Information of China (English)

    Ye-Nan Chi; Xu Zhang; Jie Cai; Feng-Yu Liu; Guo-Gang Xing; You Wan

    2012-01-01

    Objective Formaldehyde at high concentrations is a contributor to air pollution.It is also an endogenous metabolic product in cells,and when beyond physiological concentrations,has pathological effects on neurons.Formaldehyde induces mis-folding and aggregation of neuronal tau protein,hippocampal neuronal apoptosis,cognitive impairment and loss of memory functions,as well as excitation of peripheral nociceptive neurons in cancer pain models.Intracellular calcium ([Ca2+]i) is an important intracellular messenger,and plays a key role in many pathological processes.The present study aimed to investigate the effect of formaldehyde on [Ca2+]i and the possible involvement of N-methyl-D-aspartate receptors (NMDARs) and T-type Ca2+ channels on the cell membrane.Methods Using primary cultured hippocampal neurons as a model,changes of [Ca2+]i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy.Results Formaldehyde at 1 mmol/L approximately doubled [Ca2+]i.(2R)-amino-5-phosphonopentanoate (AP5,25 μtmol/L,an NMDAR antagonist) and mibefradil (MIB,1 μtmol/L,a T-type Ca2+ channel blocker),given 5 min after formaldehyde perfusion,each partly inhibited the formaldehyde-induced increase of [Ca2+]i,and this inhibitory effect was reinforced by combined application of AP5 and MIB.When applied 3 min before formaldehyde perfusion,AP5 (even at 50 μmol/L) did not inhibit the formaldehyde-induced increase of [Ca2+]i,but MIB (1 μmol/L) significantly inhibited this increase by 70%.Conclusion These results suggest that formaldehyde at a low concentration increases [Ca2+]i in cultured hippocampal neurons; NMDARs and T-type Ca2+ channels may be involved in this process.

  19. 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

    2016-06-18

    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 pro