Science.gov

Sample records for inhibits neuronal cell

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

    PubMed

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

    2016-06-01

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

  2. Tonabersat inhibits trigeminal ganglion neuronal-satellite glial cell signaling.

    PubMed

    Damodaram, Srikanth; Thalakoti, Srikanth; Freeman, Stacy E; Garrett, Filip G; Durham, Paul L

    2009-01-01

    Sensitization and activation of trigeminal neurons are implicated in the underlying pathology of migraine, acute sinusitis, and allergic rhinitis. Cell bodies of trigeminal neurons that provide sensory innervation of the dura and nasal mucosa reside in the trigeminal ganglion in association with satellite glial cells where they communicate via gap junctions. Gap junctions, channels formed by connexins, modulate the excitability state of both neurons and glia under pathological conditions. Tonabersat, a compound being tested as an antimigraine drug, is thought to block gap junction activity. To investigate the cellular events within trigeminal ganglia that may account for the significant comorbidity of migraine and rhinosinusitis and determine the effect of tonabersat on neuron-satellite glia communication. Sprague Dawley rats injected with True Blue were used to localize neuronal cell bodies in the ganglion and study neuron-glia signaling via gap junctions in the trigeminal ganglion. Dye coupling studies were conducted under basal conditions and in response to tumor necrosis factor-alpha injection into the whisker pad and/or capsaicin injection into the eyebrow. Changes in connexin 26 and active p38 levels were determined by immunohistochemistry. In addition, the effect of tonabersat prior to chemical stimulation on gap junction activity and expression of connexins and active p38 was investigated. Injection of tumor necrosis factor-alpha, a cytokine implicated in the pathology of acute sinusitis and allergic rhinitis, into the V2 region was shown to lower the amount of capsaicin required to stimulate neurons located in the V1 region of the ganglion. While injection of tumor necrosis factor-alpha into the whisker pad or capsaicin injection into the eyebrow alone did not cause increased dye movement, the combination of both stimuli greatly increased neuron-satellite glia communication via gap junctions in both V1 and V2 regions. The change in gap junction activity

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

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

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

  4. Humanin Derivatives Inhibit Necrotic Cell Death in Neurons

    PubMed Central

    Cohen, Aviv; Lerner-Yardeni, Jenny; Meridor, David; Kasher, Roni; Nathan, Ilana; Parola, Abraham H

    2015-01-01

    Humanin and its derivatives are peptides known for their protective antiapoptotic effects against Alzheimer’s disease. Herein, we identify a novel function of the humanin-derivative AGA(C8R)-HNG17 (namely, protection against cellular necrosis). Necrosis is one of the main modes of cell death, which was until recently considered an unmoderated process. However, recent findings suggest the opposite. We have found that AGA(C8R)-HNG17 confers protection against necrosis in the neuronal cell lines PC-12 and NSC-34, where necrosis is induced in a glucose-free medium by either chemohypoxia or by a shift from apoptosis to necrosis. Our studies in traumatic brain injury models in mice, where necrosis is the main mode of neuronal cell death, have shown that AGA(C8R)-HNG17 has a protective effect. This result is demonstrated by a decrease in a neuronal severity score and by a reduction in brain edema, as measured by magnetic resonance imaging (MRI). An insight into the peptide’s antinecrotic mechanism was attained through measurements of cellular ATP levels in PC-12 cells under necrotic conditions, showing that the peptide mitigates a necrosis-associated decrease in ATP levels. Further, we demonstrate the peptide’s direct enhancement of the activity of ATP synthase activity, isolated from rat-liver mitochondria, suggesting that AGA(C8R)-HNG17 targets the mitochondria and regulates cellular ATP levels. Thus, AGA(C8R)-HNG17 has potential use for the development of drug therapies for necrosis-related diseases, for example, traumatic brain injury, stroke, myocardial infarction, and other conditions for which no efficient drug-based treatment is currently available. Finally, this study provides new insight into the mechanisms underlying the antinecrotic mode of action of AGA(C8R)-HNG17. PMID:26062019

  5. Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

    PubMed Central

    Ng, KY; Yeung, BHS; Wong, YH; Wise, H

    2013-01-01

    Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

  6. Cell volume changes upon sodium pump inhibition in Helix aspersa neurones.

    PubMed Central

    Alvarez-Leefmans, F J; Gamiño, S M; Reuss, L

    1992-01-01

    1. Identified neurones of the suboesophageal ganglia of Helix aspersa were loaded with tetramethylammonium (TMA+). Experimentally induced changes in cell water volume and membrane potential were measured continuously by monitoring changes in intracellular [TMA+] using ion-sensitive double-barrelled microelectrodes. The technique allowed measurements of cell water volume changes of less than 5%. 2. Exposure to hyperosmotic (up to +24%) or hyposmotic (up to about -10%) solutions caused reversible decreases and increases in cell water volume respectively, which agreed with near-ideal osmometric behaviour. Upon exposure to hyposmotic solutions whose osmolality was decreased by 30-40%, the cell water volume increased to maximum values below those expected for ideal osmometric behaviour and exhibited partial regulatory volume decrease. 3. The sodium pump was inhibited in twenty identified neurones by sustained exposure to 1 mM ouabain. In every case ouabain caused cell membrane depolarization, as expected for inhibition of an electrogenic sodium pump. 4. Upon pump inhibition most cells (n = 14) shrank by up to 13% of their initial water volume. In five of these cells, shrinkage was preceded by one or more short-lived swelling phases. In two other neurones short-lived swelling was followed by cell volume recovery without appreciable shrinkage. In four out of the twenty cells, there were no measurable volume changes. 5. The lack of an initial swelling phase in the cells that shrank, as well as the absence of detectable volume changes in some of the neurones, was not due to loss of ion-selective electrode sensitivity since predictable changes in cell volume elicited by osmotic challenges were monitored in the same cells. 6. It is concluded that neurones can be endowed with ouabain-insensitive mechanisms of volume control, whose activation following Na+ pump inhibition prevents them from short-term swelling and lysis. PMID:1338793

  7. Integration of Purkinje cell inhibition by cerebellar nucleo-olivary neurons.

    PubMed

    Najac, Marion; Raman, Indira M

    2015-01-14

    Neurons in the cerebellar cortex, cerebellar nuclei, and inferior olive (IO) form a trisynaptic loop critical for motor learning. IO neurons excite Purkinje cells via climbing fibers and depress their parallel fiber inputs. Purkinje cells inhibit diverse cells in the cerebellar nuclei, including small GABAergic nucleo-olivary neurons that project to the IO. To investigate how these neurons integrate synaptic signals from Purkinje cells, we retrogradely labeled nucleo-olivary cells in the contralateral interpositus and lateral nuclei with cholera toxin subunit B-Alexa Fluor 488 and recorded their electrophysiological properties in cerebellar slices from weanling mice. Nucleo-olivary cells fired action potentials over a relatively narrow dynamic range (maximal rate, ∼ 70 spikes/s), unlike large cells that project to premotor areas (maximal rate, ∼ 400 spikes/s). GABA(A) receptor-mediated IPSCs evoked by electrical or optogenetic stimulation of Purkinje cells were more than 10-fold slower in nucleo-olivary cells (decay time, ∼ 25 ms) than in large cells (∼ 2 ms), and repetitive stimulation at 20-150 Hz evoked greatly summating IPSCs. Nucleo-olivary firing rates varied inversely with IPSP frequency, and the timing of Purkinje IPSPs and nucleo-olivary spikes was uncorrelated. These attributes contrast with large cells, whose brief IPSCs and rapid firing rates can permit well timed postinhibitory spiking. Thus, the intrinsic and synaptic properties of these two projection neurons from the cerebellar nuclei tailor them for differential integration and transmission of their Purkinje cell input.

  8. Inhibition of telomerase causes vulnerability to endoplasmic reticulum stress-induced neuronal cell death.

    PubMed

    Hosoi, Toru; Nakatsu, Kanako; Shimamoto, Akira; Tahara, Hidetoshi; Ozawa, Koichiro

    2016-08-26

    Endoplasmic reticulum (ER) stress is implicated in several diseases, such as cancer and neurodegenerative diseases. In the present study, we investigated the possible involvement of telomerase in ER stress-induced cell death. ER stress-induced cell death was ameliorated in telomerase reverse transcriptase (TERT) over-expressing MCF7 cells (MCF7-TERT cell). Telomerase specific inhibitor, BIBR1532, reversed the inhibitory effect of TERT on ER stress-induced cell death in MCF7-TERT cells. These findings suggest that BIBR1532 may specifically inhibit telomerase activity, thereby inducing cell death in ER stress-exposed cells. TERT was expressed in the SH-SY5Y neuroblastoma cell line. To analyze the possible involvement of telomerase in ER stress-induced neuronal cell death, we treated SH-SY5Y neuroblastoma cells with BIBR1532 and analyzed ER stress-induced cell death. We found that BIBR1532 significantly enhanced the ER stress-induced neuronal cell death. These findings suggest that inhibition of telomerase activity may enhance vulnerability to neuronal cell death caused by ER stress.

  9. Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae.

    PubMed

    Appelt, Denah M; Roupas, Maria R; Way, Deana S; Bell, Marcus G; Albert, Elizabeth V; Hammond, Christine J; Balin, Brian J

    2008-01-24

    Chlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions. SK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection. At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements. These data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain.

  10. Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae

    PubMed Central

    Appelt, Denah M; Roupas, Maria R; Way, Deana S; Bell, Marcus G; Albert, Elizabeth V; Hammond, Christine J; Balin, Brian J

    2008-01-01

    Background Chlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions. Results SK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection. At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements. Conclusion These data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain. PMID:18218130

  11. Fatty Acid Biosynthesis Inhibition Increases Reduction Potential in Neuronal Cells under Hypoxia

    PubMed Central

    Brose, Stephen A.; Golovko, Svetlana A.; Golovko, Mikhail Y.

    2016-01-01

    Recently, we have reported a novel neuronal specific pathway for adaptation to hypoxia through increased fatty acid (FA) biosynthesis followed by esterification into lipids. However, the biological role of this pathway under hypoxia remains to be elucidated. In the presented study, we have tested our hypothesis that activation of FA synthesis maintains reduction potential and reduces lactoacidosis in neuronal cells under hypoxia. To address this hypothesis, we measured the effect of FA synthesis inhibition on NADH2+/NAD+ and NADPH2+/NADP+ ratios, and lactic acid levels in neuronal SH-SY5Y cells exposed to normoxic and hypoxic conditions. FA synthesis inhibitors, TOFA (inhibits Acetyl-CoA carboxylase) and cerulenin (inhibits FA synthase), increased NADH2+/NAD+ and NADPH2+/NADP+ ratios under hypoxia. Further, FA synthesis inhibition increased lactic acid under both normoxic and hypoxic conditions, and caused cytotoxicity under hypoxia but not normoxia. These results indicate that FA may serve as hydrogen acceptors under hypoxia, thus supporting oxidation reactions including anaerobic glycolysis. These findings may help to identify a radically different approach to attenuate hypoxia related pathophysiology in the nervous system including stroke. PMID:27965531

  12. Tocilizumab inhibits neuronal cell apoptosis and activates STAT3 in cerebral infarction rat model.

    PubMed

    Wang, Shaojun; Zhou, Jun; Kang, Weijie; Dong, Zhaoni; Wang, Hezuo

    2016-01-15

    Cerebral infarction is a severe hypoxic ischemic necrosis with accelerated neuronal cell apoptosis in the brain. As a monoclonal antibody against interleukin 6, tocilizumab (TCZ) is widely used in immune diseases, whose function in cerebral infarction has not been studied. This study aims to reveal the role of TCZ in regulating neuronal cell apoptosis in cerebral infarction. The cerebral infarction rat model was constructed by middle cerebral artery occlusion and treated with TCZ. Cell apoptosis in hippocampus and cortex of the brain was examined with TUNEL method. Rat neuronal cells cultured in oxygen-glucose deprivation (OGD) conditions and treated with TCZ were used to compare cell viability and apoptosis. Apoptosis-related factors including B-cell lymphoma extra large (Bcl-xL) and Caspase 3, as well as the phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in brain cortex were analyzed from the protein level. Results indicated that TCZ treatment could significantly prevent the promoted cell apoptosis caused by cerebral infarction or OGD (P < 0.05 or P < 0.01). In brain cortex of the rat model, TCZ up-regulated Bcl-xL and down-regulated Caspase 3, consistent with the inhibited cell apoptosis. It also promoted tyrosine 705 phosphorylation of STAT3, which might be the potential regulatory mechanism of TCZ in neuronal cells. This study provided evidence for the protective role of TCZ against neuronal cell apoptosis in cerebral infarction. Based on these fundamental data, TCZ is a promising option for treating cerebral infarction, but further investigations on related mechanisms are still necessary.

  13. Tocilizumab inhibits neuronal cell apoptosis and activates STAT3 in cerebral infarction rat model

    PubMed Central

    Wang, Shaojun; Zhou, Jun; Kang, Weijie; Dong, Zhaoni; Wang, Hezuo

    2016-01-01

    Cerebral infarction is a severe hypoxic ischemic necrosis with accelerated neuronal cell apoptosis in the brain. As a monoclonal antibody against interleukin 6, tocilizumab (TCZ) is widely used in immune diseases, whose function in cerebral infarction has not been studied. This study aims to reveal the role of TCZ in regulating neuronal cell apoptosis in cerebral infarction. The cerebral infarction rat model was constructed by middle cerebral artery occlusion and treated with TCZ. Cell apoptosis in hippocampus and cortex of the brain was examined with TUNEL method. Rat neuronal cells cultured in oxygen-glucose deprivation (OGD) conditions and treated with TCZ were used to compare cell viability and apoptosis. Apoptosis-related factors including B-cell lymphoma extra large (Bcl-xL) and Caspase 3, as well as the phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in brain cortex were analyzed from the protein level. Results indicated that TCZ treatment could significantly prevent the promoted cell apoptosis caused by cerebral infarction or OGD (P < 0.05 or P < 0.01). In brain cortex of the rat model, TCZ up-regulated Bcl-xL and down-regulated Caspase 3, consistent with the inhibited cell apoptosis. It also promoted tyrosine 705 phosphorylation of STAT3, which might be the potential regulatory mechanism of TCZ in neuronal cells. This study provided evidence for the protective role of TCZ against neuronal cell apoptosis in cerebral infarction. Based on these fundamental data, TCZ is a promising option for treating cerebral infarction, but further investigations on related mechanisms are still necessary. PMID:26773188

  14. Synergistic effects of CoCl(2) and ROCK inhibition on mesenchymal stem cell differentiation into neuron-like cells.

    PubMed

    Pacary, Emilie; Legros, Hélène; Valable, Samuel; Duchatelle, Pascal; Lecocq, Myriam; Petit, Edwige; Nicole, Olivier; Bernaudin, Myriam

    2006-07-01

    Bone-marrow-derived mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after neurodegenerative diseases. Recently, several studies suggested that oxygen-dependent gene expression is of crucial importance in governing the essential steps of neurogenesis such as cell proliferation, survival and differentiation. In this context, we analysed the effect of the HIF-1 (hypoxia inducible factor-1) activation-mimicking agent CoCl(2) on MSCs. CoCl(2) treatment increased the expression of the anti-proliferative gene BTG2/PC3 and decreased cyclin D1 expression. Expression of HIF-1alpha and its target genes EPO, VEGF and p21 was also upregulated. These changes were followed by inhibition of cell proliferation and morphological changes resulting in neuron-like cells, which had increased neuronal marker expression and responded to neurotransmitters. Echinomycin, a molecule inhibiting HIF-1 DNA-binding activity, blocked the CoCl(2) effect on MSCs. Additionally, by using Y-27632, we demonstrated that Rho kinase (ROCK) inhibition potentiated CoCl(2)-induced MSC differentiation in particular into dopaminergic neuron-like cells as attested by its effect on tyrosine hydroxylase expression. Altogether, these results support the ability of MSCs to differentiate into neuron-like cells in response to CoCl(2), an effect that might act, in part, through HIF-1 activation and cell-cycle arrest, and which is potentiated by inhibition of ROCK.

  15. Tanshinone inhibits neuronal cell apoptosis and inflammatory response in cerebral infarction rat model.

    PubMed

    Zhou, Liang; Zhang, Jie; Wang, Chao; Sun, Qiangsan

    2017-06-01

    We aimed to investigate the effect and mechanisms of tanshinone (TSN) IIA in cerebral infarction. The cerebral infarction rat model was established by middle cerebral artery occlusion (MCAO). After pretreatment with TSN, cerebral infarct volume, cerebral edema, and neurological deficits score were evaluated, as well as cell apoptosis in hippocampus and cortex of the brain was examined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). In addition, rat primary neuronal cells were isolated and cultured in oxygen-glucose deprivation (OGD) conditions. After pretreatment with TSN, cell viability and apoptosis were observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. The expressions of Bax and B-cell lymphoma 2 (Bcl-2) were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. Compared with untreated cerebral infarction rat, TSN treatment significantly reduced cerebral infarct volume, cerebral edema, and neurological deficits score ( P < 0.05). Cell apoptosis as well as the levels of IL-6, TNF-α, and CRP in hippocampus and cortex of cerebral infarction rat were inhibited after pretreatment with TSN ( P < 0.05). Furthermore, TSN remarkably increased cell viability and inhibited cell apoptosis ratio ( P < 0.05) in OGD-induced rat neuronal cells. Besides, TSN significantly downregulated the expression of Bax and upregulated Bcl-2 ( P < 0.05). TSN IIA has a preventive effect on cerebral infarction by inhibiting neuronal cell apoptosis and inflammatory response in vitro and in vivo.

  16. Inhibition among olfactory receptor neurons

    PubMed Central

    Van der Goes van Naters, Wynand

    2013-01-01

    Often assumed to be epiphenomena of a cell’s activity, extracellular currents and resulting potential changes are increasingly recognized to influence the function of other cells in the vicinity. Experimental evidence shows that even small electric fields can modulate spike timing in neurons. Moreover, when neurons are brought close together experimentally or in pathological conditions, activity in one neuron can excite its neighbors. Inhibitory ephaptic mechanisms, however, may depend on more specialized coupling among cells. Recent studies in the Drosophila olfactory system have shown that excitation of a sensory neuron can inhibit its neighbor, and it was speculated that this interaction was ephaptic. Here we give an overview of ephaptic interactions that effect changes in spike timing, excitation or inhibition in diverse systems with potential relevance to human neuroscience. We examine the mechanism of the inhibitory interaction in the Drosophila system and that of the well-studied ephaptic inhibition of the Mauthner cell in more detail. We note that both current towards and current away from the local extracellular environment of a neuron can inhibit it, but the mechanism depends on the specific architecture of each system. PMID:24167484

  17. Nigericin-induced impairment of autophagic flux in neuronal cells is inhibited by overexpression of Bak.

    PubMed

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B H; Yue, Zhenyu; Oh, Young J

    2012-07-06

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K(+)/H(+) ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis.

  18. Nigericin-induced Impairment of Autophagic Flux in Neuronal Cells Is Inhibited by Overexpression of Bak*

    PubMed Central

    Lim, Junghyun; Lee, Yunsu; Kim, Hyun-Wook; Rhyu, Im Joo; Oh, Myung Sook; Youdim, Moussa B. H.; Yue, Zhenyu; Oh, Young J.

    2012-01-01

    Bak is a prototypic pro-apoptotic Bcl-2 family protein expressed in a wide variety of tissues and cells. Recent studies have revealed that Bcl-2 family proteins regulate apoptosis as well as autophagy. To investigate whether and how Bak exerts a regulatory role on autophagy-related events, we treated independent cell lines, including MN9D neuronal cells, with nigericin, a K+/H+ ionophore. Treatment of MN9D cells with nigericin led to an increase of LC3-II and p62 levels with concomitant activation of caspase. Ultrastructural examination revealed accumulation of autophagic vacuoles and swollen vacuoles in nigericin-treated cells. We further found that the LC3-II accumulated as a consequence of impaired autophagic flux and the disrupted degradation of LC3-II in nigericin-treated cells. In this cell death paradigm, both transient and stable overexpression of various forms of Bak exerted a protective role, whereas it did not inhibit the extent of nigericin-mediated activation of caspase-3. Subsequent biochemical and electron microscopic studies revealed that overexpressed Bak maintained autophagic flux and reduced the area occupied by swollen vacuoles in nigericin-treated cells. Similar results were obtained in nigericin-treated non-neuronal cells and another proton ionophore-induced cell death paradigm. Taken together, our study indicates that a protective role for Bak during ionophore-induced cell death may be closely associated with its regulatory effect on maintenance of autophagic flux and vacuole homeostasis. PMID:22493436

  19. Dipyrone Inhibits Neuronal Cell Death and Diminishes Hypoxic/Ischemic Brain Injury

    PubMed Central

    Zhang, Yi; Wang, Xin; Baranov, Sergei V.; Zhu, Shan; Huang, Zhihong; Fellows-Mayle, Wendy; Jiang, Jiying; Day, Arthur L.; Kristal, Bruce S.; Friedlander, Robert M.

    2011-01-01

    Background and Objective Dipyrone is an analgesic and antipyretic drug usually prescribed for patients with inflammatory conditions. We recently identified dipyrone as an anti-apoptotic agent by screening a library of 1040 compounds for their ability to inhibit cytochrome c release from isolated mitochondria. We investigated the potential neuroprotective properties of dipyrone in cerebral ischemia. Methods We evaluated the protective effects of dipyrone in experimental models of neuronal hypoxia/ischemia, including an oxygen/glucose deprivation model in primary cerebrocortical neurons and a focal cerebral ischemia model in mice. Results Dipyrone reduced hypoxia/ischemia injury in both cellular and animal models. Dipyrone inhibited the release of cytochrome c and other mitochondrial apoptogenic factors from mitochondria into the cytoplasm, and attenuated subsequent caspase-9 and caspase-3 activation both in vitro and in vivo. Moreover, dipyrone prevented ischemia-induced changes in Bcl-2 and tBid, and ameliorated OGD-mediated loss of mitochondrial membrane potential. Dipyrone also inhibited ischemia-induced reactive microgliosis. In the cellular models evaluated, dipyrone did not inhibit OGD-induced COX-2 activation. Conclusion This study demonstrates that dipyrone is remarkably neuroprotective in cerebral ischemia, and its COX-independent protective properties are, at least in part, due to the inhibition of mitochondrial cell death cascades. PMID:21552169

  20. Dipyrone inhibits neuronal cell death and diminishes hypoxic/ischemic brain injury.

    PubMed

    Zhang, Yi; Wang, Xin; Baranov, Sergei V; Zhu, Shan; Huang, Zhihong; Fellows-Mayle, Wendy; Jiang, Jiying; Day, Arthur L; Kristal, Bruce S; Friedlander, Robert M

    2011-10-01

    Dipyrone is an analgesic and antipyretic drug usually prescribed for patients with inflammatory conditions. We recently identified dipyrone as an antiapoptotic agent by screening a library of 1040 compounds for their ability to inhibit cytochrome c release from isolated mitochondria. We investigated the potential neuroprotective properties of dipyrone in cerebral ischemia. We evaluated the protective effects of dipyrone in experimental models of neuronal hypoxia/ischemia, including an oxygen/glucose deprivation model in primary cerebrocortical neurons and a focal cerebral ischemia model in mice. Dipyrone reduced hypoxia/ischemia injury in both cellular and animal models. Dipyrone inhibited the release of cytochrome c and other mitochondrial apoptogenic factors from mitochondria into the cytoplasm, and attenuated subsequent caspase-9 and caspase-3 activation both in vitro and in vivo. Moreover, dipyrone prevented ischemia-induced changes in Bcl-2 and tBid, and ameliorated oxygen/glucose deprivation-mediated loss of mitochondrial membrane potential. Dipyrone also inhibited ischemia-induced reactive microgliosis. In the cellular models evaluated, dipyrone did not inhibit oxygen/glucose deprivation-induced cyclooxygenase-2 activation. Dipyrone is remarkably neuroprotective in cerebral ischemia, and its cyclooxygenase-independent protective properties are, at least in part, due to the inhibition of mitochondrial cell death cascades.

  1. Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury.

    PubMed

    Shin, Min Jea; Kim, Dae Won; Lee, Yeom Pyo; Ahn, Eun Hee; Jo, Hyo Sang; Kim, Duk-Soo; Kwon, Oh-Shin; Kang, Tae-Cheon; Cho, Yong-Jun; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2014-02-01

    Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H2O2-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H2O2-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H2O2-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases. © 2013 Elsevier Inc. All rights reserved.

  2. Vitamin B12 offers neuronal cell protection by inhibiting Aβ-42 amyloid fibrillation.

    PubMed

    Alam, Parvez; Siddiqi, Mohammad Khursheed; Chaturvedi, Sumit Kumar; Zaman, Masihuz; Khan, Rizwan Hasan

    2017-03-04

    Protein misfolding and aggregation has been implicated as the cause of more than 20 diseases in humans such as Alzheimer's and Parkinson's and systemic amyloidosis. Retardation of Aβ- 42 aggregation is considered as a promising and challenging strategy for developing effective therapeutics against Alzheimer's disease. Herein, we demonstrated the effect of vitamin B12 (VB) on inhibiting amyloid formation by employing ThT fluorescence assay, circular dichroism, ANS fluorescence assay, dynamic light scattering measurements and transmission electron microscopy and cell viability assay. Our results demonstrate that vitamin B12 (VB), inhibits Aβ- 42 aggregation in a concentration dependent manner. Further VB also provide protection against amyloid induced cytotoxicity in human neuronal cell line. This study points towards a promising strategy to combat Aβ- 42 aggregation and may have broader implication for targeting other neurological disorders whose distinct hallmark is also amyloid formation.

  3. Botulinum neurotoxin dose-dependently inhibits release of neurosecretory vesicle-vargeted luciferase from neuronal cells.

    PubMed

    Pathe-Neuschäfer-Rube, Andrea; Neuschäfer-Rube, Frank; Genz, Lara; Püchel, Gerhard P

    2015-01-01

    Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations.Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by of botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.

  4. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines

    PubMed Central

    Ring, Avi; Strom, Bjorn Oddvar; Turner, Simon R.; Timperley, Christopher M.; Bird, Michael; Green, A. Christopher; Chad, John E.; Worek, Franz; Tattersall, John E. H.

    2015-01-01

    Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase), but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21) and neuronal (SH-SY5Y) cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning. PMID:26274808

  5. miR-455 inhibits neuronal cell death by targeting TRAF3 in cerebral ischemic stroke

    PubMed Central

    Yao, Shengtao; Tang, Bo; Li, Gang; Fan, Ruiming; Cao, Fang

    2016-01-01

    Ischemic stroke is one of the leading causes of brain disease, with high morbidity, disability, and mortality. MicroRNAs (miRNAs) have been identified as vital gene regulators in various types of human diseases. Accumulating evidence has suggested that aberrant expression of miRNAs play critical roles in the pathologies of ischemic stroke. Yet, the precise mechanism by which miRNAs control cerebral ischemic stroke remains unclear. In the present study, we explored whether miR-455 suppresses neuronal death by targeting TRAF3 in cerebral ischemic stroke. The expression levels of miR-455 and TRAF3 were detected by quantitative real-time polymerase chain reaction and Western blot. The role of miR-455 in cell death caused by oxygen–glucose deprivation (OGD) was assessed using Cell Counting Kit-8 (CCK-8) assay. The influence of miR-455 on infarct volume was evaluated in mouse brain after middle cerebral artery occlusion (MCAO). Bioinformatics softwares and luciferase analysis were used to find and confirm the targets of miR-455. The results showed that the expression levels of miR-455 significantly decreased in primary neuronal cells subjected to OGD and mouse brain subjected to MCAO. In addition, forced expression of miR-455 inhibited neuronal death and weakened ischemic brain infarction in focal ischemia-stroked mice. Furthermore, TRAF3 was proved to be a direct target of miR-455, and miR-455 could negatively suppress TRAF3 expression. Biological function analysis showed that TRAF3 silencing displayed the neuroprotective effect in ischemic stroke and could enhance miR-455-induced positive impact on ischemic injury both in vitro and in vivo. Taken together, miR-455 played a vital role in protecting neuronal cells from death by downregulating TRAF3 protein expression. These findings may represent a novel latent therapeutic target for cerebral ischemic stroke. PMID:27980410

  6. Apoptotic neuron-secreted HN12 inhibits cell apoptosis in Hirschsprung’s disease

    PubMed Central

    Du, Chunxia; Xie, Hua; Zang, Rujin; Shen, Ziyang; Li, Hongxing; Chen, Pingfa; Xu, Xiaoqun; Xia, Yankai; Tang, Weibing

    2016-01-01

    Perturbation in apoptosis can lead to Hirschsprung’s disease (HSCR), which is a genetic disorder of neural crest development. It is believed that long noncoding RNAs (lncRNAs) play a role in the progression of HSCR. This study shows that apoptotic neurons can suppress apoptosis of nonapoptotic cells by secreting exosomes that contain high levels of HN12 lncRNA. Elevated exogenous HN12 in nonapoptotic cells effectively inhibited cell apoptosis by maintaining the function of mitochondria, including the production of ATP and the release of cytochrome C. These results demonstrate that secreted lncRNAs may serve as signaling molecules mediating intercellular communication in HSCR. In addition, high HN12 levels in the circulation worked as a biomarker for predicting HSCR, providing a potential, novel, noninvasive diagnostic approach for early screening of HSCR. PMID:27853370

  7. Tat-HSP22 inhibits oxidative stress-induced hippocampal neuronal cell death by regulation of the mitochondrial pathway.

    PubMed

    Jo, Hyo Sang; Kim, Dae Won; Shin, Min Jea; Cho, Su Bin; Park, Jung Hwan; Lee, Chi Hern; Yeo, Eun Ji; Choi, Yeon Joo; Yeo, Hyeon Ji; Sohn, Eun Jeong; Son, Ora; Cho, Sung-Woo; Kim, Duk-Soo; Yu, Yeon Hee; Lee, Keun Wook; Park, Jinseu; Eum, Won Sik; Choi, Soo Young

    2017-01-04

    Oxidative stress plays an important role in the progression of various neuronal diseases including ischemia. Heat shock protein 22 (HSP22) is known to protect cells against oxidative stress. However, the protective effects and mechanisms of HSP22 in hippocampal neuronal cells under oxidative stress remain unknown. In this study, we determined whether HSP22 protects against hydrogen peroxide (H2O2)-induced oxidative stress in HT-22 using Tat-HSP22 fusion protein. We found that Tat-HSP22 transduced into HT-22 cells and that H2O2-induced cell death, oxidative stress, and DNA damage were significantly reduced by Tat-HSP22. In addition, Tat-HSP22 markedly inhibited H2O2-induced mitochondrial membrane potential, cytochrome c release, cleaved caspase-3, and Bax expression levels, while Bcl-2 expression levels were increased in HT-22 cells. Further, we showed that Tat-HSP22 transduced into animal brain and inhibited cleaved-caspase-3 expression levels as well as significantly inhibited hippocampal neuronal cell death in the CA1 region of animals in the ischemic animal model. In the present study, we demonstrated that transduced Tat-HSP22 attenuates oxidative stress-induced hippocampal neuronal cell death through the mitochondrial signaling pathway and plays a crucial role in inhibiting neuronal cell death, suggesting that Tat-HSP22 protein may be used to prevent oxidative stress-related brain diseases including ischemia.

  8. Neuronal Nitric Oxide Synthase in Neural Stem Cells Induces Neuronal Fate Commitment via the Inhibition of Histone Deacetylase 2

    PubMed Central

    Jin, Xing; Yu, Zhang-Feng; Chen, Fang; Lu, Guang-Xian; Ding, Xin-Yuan; Xie, Lin-Jun; Sun, Jian-Tong

    2017-01-01

    Active adult neurogenesis produces new functional neurons, which replace the lost ones and contribute to brain repair. Promoting neurogenesis may offer a therapeutic strategy for human diseases associated with neurodegeneration. Here, we report that endogenous neuronal nitric oxide synthase (nNOS) for neural stem cells (NSCs) or progenitors positively regulates neurogenesis. nNOS repression exhibits significantly decreased neuronal differentiation and nNOS upregulation promotes neurons production from NSCs. Using a quantitative approach, we show that instructive effect, that is instruction of NSCs to adopt a neuronal fate, contributes to the favorable effect of endogenous nNOS on neurogenesis. Furthermore, nNOS-mediated instruction of neuronal fate commitment is predominantly due to the reduction of histone deacetylase 2 (HDAC2) expression and enzymatic activity. Further investigation will be needed to test whether HDAC2 can serve as a new target for therapeutic intervention of neurodegenerative disorders. PMID:28326018

  9. APP and APLP1 are degraded through autophagy in response to proteasome inhibition in neuronal cells.

    PubMed

    Zhou, Fangfang; van Laar, Theo; Huang, Huizhe; Zhang, Long

    2011-05-01

    Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer's disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.

  10. The Ubiquitin Ligase Praja1 Reduces NRAGE Expression and Inhibits Neuronal Differentiation of PC12 Cells

    PubMed Central

    Teuber, Jan; Mueller, Bettina; Fukabori, Ryoji; Lang, Daniel; Albrecht, Anne; Stork, Oliver

    2013-01-01

    Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect. PMID:23717400

  11. Cell-type-specific inhibition of the dendritic plateau potential in striatal spiny projection neurons.

    PubMed

    Du, Kai; Wu, Yu-Wei; Lindroos, Robert; Liu, Yu; Rózsa, Balázs; Katona, Gergely; Ding, Jun B; Kotaleski, Jeanette Hellgren

    2017-09-05

    Striatal spiny projection neurons (SPNs) receive convergent excitatory synaptic inputs from the cortex and thalamus. Activation of spatially clustered and temporally synchronized excitatory inputs at the distal dendrites could trigger plateau potentials in SPNs. Such supralinear synaptic integration is crucial for dendritic computation. However, how plateau potentials interact with subsequent excitatory and inhibitory synaptic inputs remains unknown. By combining computational simulation, two-photon imaging, optogenetics, and dual-color uncaging of glutamate and GABA, we demonstrate that plateau potentials can broaden the spatiotemporal window for integrating excitatory inputs and promote spiking. The temporal window of spiking can be delicately controlled by GABAergic inhibition in a cell-type-specific manner. This subtle inhibitory control of plateau potential depends on the location and kinetics of the GABAergic inputs and is achieved by the balance between relief and reestablishment of NMDA receptor Mg(2+) block. These findings represent a mechanism for controlling spatiotemporal synaptic integration in SPNs.

  12. Cell-Autonomous Inhibition of α7-Containing Nicotinic Acetylcholine Receptors Prevents Death of Parasympathetic Neurons during Development

    PubMed Central

    Hruska, Martin; Nishi, Rae

    2010-01-01

    Neurotrophic molecules are key retrograde influences of cell survival in the developing nervous system, but other influences such as activity are also emerging as important factors. In the avian ciliary ganglion, half the neurons are eliminated between embryonic day 8 (E8) and E14, but it is not known how cell death is initiated. Because systemic application of α7-nicotinic acetylcholine receptor (nAChR) antagonists prevents this cell loss, we examined differences in receptor densities and responses of intracellular calcium to nicotine using the calcium-sensitive dye fura-2. In addition, we determined whether cell-autonomous inhibition of α7 activation in neurons prevented cell death. E8 neurons are heterogeneous with respect to α7-nAChR density, which leads to large increases in [Ca2+]i in some neurons; E8 neurons also exhibit a slower rate of Ca2+decay after nicotinic stimulation than E13 neurons. Expressing α-bungarotoxin that is tethered to the membrane by a glycosylphosphatidylinositol linkage (GPIαbtx) in ciliary ganglion neurons with the retroviral vector RCASBP(A) blocks increases in intracellular calcium induced by nicotine through α7-nAChRs and prevents neurons from dying. Expression of GPIαbtx in surrounding non-neural tissues, but not in neurons, does not prevent cell loss. Furthermore, the GPIαbtx is not efficiently expressed in the accessory oculomotor neurons, eliminating preganglionic inputs as another site for action of the antagonist. These results support the hypothesis that cholinergic inputs facilitate cell death in the developing autonomic nervous system by activating α7-nAChRs, possibly by leading to increases in intracellular calcium that exceed the threshold for cell survival. PMID:17959793

  13. Concomitant inhibition of prolyl hydroxylases and ROCK initiates differentiation of mesenchymal stem cells and PC12 towards the neuronal lineage.

    PubMed

    Pacary, Emilie; Petit, Edwige; Bernaudin, Myriam

    2008-12-12

    This study demonstrates that a prolyl hydroxylase inhibitor, FG-0041, is able, in combination with the ROCK inhibitor, Y-27632, to initiate differentiation of mesenchymal stem cells (MSCs) into neuron-like cells. FG-0041/Y-27632 co-treatment provokes morphological changes into neuron-like cells, increases neuronal marker expression and provokes modifications of cell cycle-related gene expression consistent with a cell cycle arrest of MSC, three events showing the engagement of MSC towards the neuronal lineage. Moreover, as we observed in our previous studies with cobalt chloride and desferroxamine, the activation of HIF-1 by this prolyl hydroxylase inhibitor is potentiated by Y-27632 which could explain at least in part the effect of this co-treatment on MSC neuronal differentiation. In addition, we show that this co-treatment enhances neurite outgrowth and tyrosine hydroxylase expression in PC12 cells. Altogether, these results evidence that concomitant inhibition of prolyl hydroxylases and ROCK represents a relevant protocol to initiate neuronal differentiation.

  14. Genetically modified Schwann cells producing glial cell line-derived neurotrophic factor inhibit neuronal apoptosis in rat spinal cord injury.

    PubMed

    Liu, Guomin; Wang, Xukai; Shao, Guoxi; Liu, Qinyi

    2014-04-01

    Schwann cells (SCs) are the major cells constituting the peripheral nerve structure and function, and also secret a variety of neurotrophic factors. Schwann cell (SC) transplantation has recently emerged as a promising therapeutic strategy for spinal cord injury (SCI). In the present study, the ability of genetically modified SCs producing high levels of glial cell line‑derived neurotrophic factor (GDNF) to promote spinal cord repair was assessed. The GDNF gene was transduced into SCs. The engineered SCs were characterized by their ability to express and secrete biologically active GDNF, which was shown to inhibit apoptosis of primary rat neurons induced by radiation, and upregulate the expression of B‑cell lymphoma 2 (Bcl‑2) and downregulate the expression of Bcl‑2 associated X protein (Bax) in vitro. Following SC implantation into the spinal cord of adult rats with SCI induced by weight‑drop impact, the survival of rats with transplanted SCs, histology of the spinal cord and expression levels of Bcl‑2 and Bax were examined. Transplantation of unmodified and genetically modified SCs producing GDNF attenuated SCI by inhibiting apoptosis via the Bcl‑2/Bax pathways. The genetically modified SCs demonstrated markedly improved recovery of SCI as compared with unmodified SCs. The present study combined the outgrowth‑promoting property of SCs with the neuroprotective effects of overexpressed GDNF and identified this as a potential novel therapeutic strategy for SCI.

  15. Neuronal cell death in the inner retina and the influence of vascular endothelial growth factor inhibition in a diabetic rat model.

    PubMed

    Park, Hae-Young Lopilly; Kim, Jie Hyun; Park, Chan Kee

    2014-06-01

    To inhibit vascular changes in diabetic retinopathy, inhibiting vascular endothelial growth factor (VEGF) has become a mainstay of the treatment of diabetic retinopathy. However, its effects on neuronal cells remain to be elucidated. We aimed to evaluate the effect of VEGF inhibition on neuronal cells in a streptozotocin-induced diabetic rat retina. VEGF inhibition was performed by intravitreal VEGF-A antibody injection. After anti-VEGF treatment, apoptosis in retinal ganglion cells (RGCs) increased, and novel apoptosis in amacrine and bipolar cells of the inner nuclear layer was observed by TUNEL staining. Phosphorylated Akt expression was significantly higher in RGCs but was decreased in neuronal cells of the inner nuclear layer after anti-VEGF treatment by Western blot analysis and immunohistochemical staining. These results demonstrate that VEGF inhibition significantly increased RGC apoptosis and neuronal cell apoptosis in the inner nuclear layer of a diabetic retina, which seems to consist primarily of amacrine and bipolar cells. The phosphorylated Akt pathway, which plays a neuroprotective role via VEGF, was significantly affected by VEGF inhibition in the inner nuclear layer, suggesting that neurotrophic factor deprivation is the main mechanism for neuronal cell death after inhibiting VEGF. The results of this study show that inhibiting VEGF may have detrimental effects on the apoptosis of neuronal cells in the inner layers of the diabetic retina.

  16. Cofilin inhibition restores neuronal cell death in oxygen glucose deprivation model of ischemia

    PubMed Central

    Madineni, Anusha; Alhadidi, Qasim; Shah, Zahoor A.

    2014-01-01

    Ischemia is a condition associated with decreased blood supply to the brain, eventually leading to death of neurons. It is associated with a diverse cascade of responses involving both degenerative and regenerative mechanisms. At the cellular level, the changes are initiated prominently in the neuronal cytoskeleton. Cofilin, a cytoskeletal actin severing protein, is known to be involved in the early stages of apoptotic cell death. Evidence supports its intervention in the progression of disease states like Alzheimer's and ischemic kidney disease. In the present study, we have hypothesized the possible involvement of cofilin in ischemia. Using PC12 cells and mouse primary cultures of cortical neurons, we investigated the potential role of cofilin in ischemia in two different in vitro ischemic models: chemical induced oxidative stress and oxygen-glucose deprivation/reperfusion (OGD/R). The expression profile studies demonstrated a decrease in phosphocofilin levels in all models of ischemia, implying stress-induced cofilin activation. Furthermore, calcineurin and slingshot 1L (SSH) phosphatases were found to be the signaling mediators of the cofilin activation. In primary cultures of cortical neurons, cofilin was found to be significantly activated after 1 h of OGD. To delineate the role of activated cofilin in ischemia, we knocked down cofilin by siRNA technique and tested the impact of cofilin silencing on neuronal viability. Cofilin siRNA-treated neurons showed a significant reduction of cofilin levels in all treatment groups (control, OGD and OGD/R). Additionally, cofilin siRNA reduced cofilin mitochondrial translocation and caspase 3 cleavage, with a concomitant increase in neuronal viability. These results strongly support the active role of cofilin in ischemia-induced neuronal degeneration and apoptosis. We believe that targeting this protein mediator has a potential for therapeutic intervention in ischemic brain injury and stroke. PMID:25526862

  17. Cofilin Inhibition Restores Neuronal Cell Death in Oxygen-Glucose Deprivation Model of Ischemia.

    PubMed

    Madineni, Anusha; Alhadidi, Qasim; Shah, Zahoor A

    2016-03-01

    Ischemia is a condition associated with decreased blood supply to the brain, eventually leading to death of neurons. It is associated with a diverse cascade of responses involving both degenerative and regenerative mechanisms. At the cellular level, the changes are initiated prominently in the neuronal cytoskeleton. Cofilin, a cytoskeletal actin severing protein, is known to be involved in the early stages of apoptotic cell death. Evidence supports its intervention in the progression of disease states like Alzheimer's and ischemic kidney disease. In the present study, we have hypothesized the possible involvement of cofilin in ischemia. Using PC12 cells and mouse primary cultures of cortical neurons, we investigated the potential role of cofilin in ischemia in two different in vitro ischemic models: chemical induced oxidative stress and oxygen-glucose deprivation/reperfusion (OGD/R). The expression profile studies demonstrated a decrease in phosphocofilin levels in all models of ischemia, implying stress-induced cofilin activation. Furthermore, calcineurin and slingshot 1L (SSH) phosphatases were found to be the signaling mediators of the cofilin activation. In primary cultures of cortical neurons, cofilin was found to be significantly activated after 1 h of OGD. To delineate the role of activated cofilin in ischemia, we knocked down cofilin by small interfering RNA (siRNA) technique and tested the impact of cofilin silencing on neuronal viability. Cofilin siRNA-treated neurons showed a significant reduction of cofilin levels in all treatment groups (control, OGD, and OGD/R). Additionally, cofilin siRNA-reduced cofilin mitochondrial translocation and caspase 3 cleavage, with a concomitant increase in neuronal viability. These results strongly support the active role of cofilin in ischemia-induced neuronal degeneration and apoptosis. We believe that targeting this protein mediator has a potential for therapeutic intervention in ischemic brain injury and stroke.

  18. Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

    PubMed Central

    Schwer, Christian I.; Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Strosing, Karl M.; Spassov, Sashko; Erxleben, Anika; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

    2013-01-01

    Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. PMID:24167567

  19. Neuronal Inhibition under the Spotlight.

    PubMed

    Mortensen, Martin; Smart, Trevor G

    2015-12-02

    In this issue of Neuron,Lin et al. (2015) report an optical method to precisely manipulate the activity of GABAA receptors by designing a mutant receptor that binds photosensitive compounds. This allows for studying GABAA receptors in situ and represents a valuable tool to investigate how inhibition affects brain physiology.

  20. Growth inhibition, morphological differentiation and stimulation of survival in neuronal cell type (Neuro-2a) treated with trophic molecules.

    PubMed

    Blanco, V; Lopez Camelo, J; Carri, N G

    2001-01-01

    Trophic molecules are key regulators of survival, growth and differentiation of neural cells. Neuronal cell type Neuro-2a is a good model to study development and molecules modulating this process, and retinoic acid (RA) and neurotrophins (NGF, BDNF, NT-3 and NT-4) have been shown to be active in this modulation. The purpose of the present study was the functional analysis of these trophic molecules in our short-term bioassay of Neuro-2a cells, an immortalised murine neuroblastoma cell line. Through cell counting, image process and arithmetic combination of digital parameters of treated and untreated cultures, we show that RA inhibits growth and induces morphological neuronal phenotype of treated cells. Through DNA labelling with BrdU we also show that NGF, BDNF, and NT-3 increase survival and proliferation of cells, grown in serum-deprived media. From these results we conclude that neurotrophins have manifest trophic effects on cells improving survival, growth and proliferation and we also confirm the growth arrest and differentiation properties of RA on Neuro-2a cells. Copyright 2001 Academic Press.

  1. Second Generation Amphiphilic Poly-Lysine Dendrons Inhibit Glioblastoma Cell Proliferation without Toxicity for Neurons or Astrocytes

    PubMed Central

    Janiszewska, Jolanta; Posadas, Inmaculada; Játiva, Pablo; Bugaj-Zarebska, Marta; Urbanczyk-Lipkowska, Zofia; Ceña, Valentín

    2016-01-01

    Glioblastomas are the most common malignant primary brain tumours in adults and one of the most aggressive and difficult-to-treat cancers. No effective treatment exits actually for this tumour and new therapeutic approaches are needed for this disease. One possible innovative approach involves the nanoparticle-mediated specific delivery of drugs and/or genetic material to glioblastoma cells where they can provide therapeutic benefits. In the present work, we have synthesised and characterised several second generation amphiphilic polylysine dendrons to be used as siRNA carriers. We have found that, in addition to their siRNA binding properties, these new compounds inhibit the proliferation of two glioblastoma cell lines while being nontoxic for non-tumoural central nervous system cells like neurons and glia, cell types that share the anatomical space with glioblastoma cells during the course of the disease. The selective toxicity of these nanoparticles to glioblastoma cells, as compared to neurons and glial cells, involves mitochondrial depolarisation and reactive oxygen species production. This selective toxicity, together with the ability to complex and release siRNA, suggests that these new polylysine dendrons might offer a scaffold in the development of future nanoparticles designed to restrict the proliferation of glioblastoma cells. PMID:27832093

  2. Berberine induces neuronal differentiation through inhibition of cancer stemness and epithelial-mesenchymal transition in neuroblastoma cells.

    PubMed

    Naveen, C R; Gaikwad, Sagar; Agrawal-Rajput, Reena

    2016-06-15

    Berberine, a plant alkaloid, has been used since many years for treatment of gastrointestinal disorders. It also shows promising medicinal use against metabolic disorders, neurodegenerative disorders and cancer; however its efficacy in neuroblastoma (NB) is poorly explored. EMT is important in cancer stemness and metastasis resulting in failure to differentiate; thus targeting EMT and related pathways can have clinical benefits. Potential of berberine was investigated for (i) neuronal differentiation and cancer stemness inhibition, (ii) underlying molecular mechanisms regulating cancer-stemness and (iii) EMT reversal. Using neuro2a (N2a) neuroblastoma cells (NB); we investigated effect of berberine on neuronal differentiation, cancer-stemness, EMT and underlying signalling by immunofluorescence, RT-PCR, Western blot. High glucose-induced TGF-β mediated EMT model was used to test EMT reversal potential by Western blot and RT-PCR. STRING analysis was done to determine and validate functional protein-interaction networks. We demonstrate berberine induces neuronal differentiation accompanying increased neuronal differentiation markers like MAP2, β-III tubulin and NCAM; generated neurons were viable. Berberine attenuated cancer stemness markers CD133, β-catenin, n-myc, sox2, notch2 and nestin. Berberine potentiated G0/G1 cell cycle arrest by inhibiting proliferation, cyclin dependent kinases and cyclins resulting in apoptosis through increased bax/bcl-2 ratio. Restoration of tumor suppressor proteins, p27 and p53, indicate promising anti-cancer property. The induction of NCAM and reduction in its polysialylation indicates anti-migratory potential which is supported by down regulation of MMP-2/9. It increased epithelial marker laminin and smad and increased Hsp70 levels also suggest its protective role. Molecular insights revealed that berberine regulates EMT via downregulation of PI3/Akt and Ras-Raf-ERK signalling and subsequent upregulation of p38-MAPK. TGF

  3. Atoh1 inhibits neuronal differentiation and collaborates with Gli1 to generate medulloblastoma-initiating cells

    PubMed Central

    Ayrault, Olivier; Zhao, Haotian; Zindy, Frederique; Qu, Chunxu; Sherr, Charles J.; Roussel, Martine F.

    2010-01-01

    The morphogen and mitogen Sonic Hedgehog activates a Gli1-dependent transcription program that drives proliferation of granule neuron progenitors (GNPs) within the external germinal layer of the postnatally developing cerebellum. Medulloblastomas with mutations activating the Sonic Hedgehog signaling pathway preferentially arise within the external germinal layer, and the tumor cells closely resemble GNPs. Atoh1/Math1, a basic helix-loop-helix transcription factor essential for GNP histogenesis, does not induce medulloblastomas when expressed in primary mouse GNPs that are explanted from the early postnatal cerebellum and transplanted back into the brains of naïve mice. However, enforced expression of Atoh1 in primary GNPs enhances the oncogenicity of cells overexpressing Gli1 by almost three orders of magnitude. Unlike Gli1, Atoh1 cannot support GNP proliferation in the absence of Sonic Hedgehog signaling and does not govern expression of canonical cell cycle genes. Instead, Atoh1 maintains GNPs in a Sonic Hedgehog-responsive state by regulating genes that trigger neuronal differentiation, including many expressed in response to bone morphogenic protein-4. Therefore, by targeting multiple genes regulating the differentiation state of GNPs, Atoh1 collaborats with the pro-proliferative Gli1-dependent transcriptional program to influence medulloblastoma development. PMID:20516124

  4. Acetaminophen inhibits neuronal inflammation and protects neurons from oxidative stress

    PubMed Central

    Tripathy, Debjani; Grammas, Paula

    2009-01-01

    Background Recent studies have demonstrated a link between the inflammatory response, increased cytokine formation, and neurodegeneration in the brain. The beneficial effects of anti-inflammatory drugs in neurodegenerative diseases, such as Alzheimer's disease (AD), have been documented. Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. The objectives of this study are to determine the effects of acetaminophen on cultured brain neuronal survival and inflammatory factor expression when exposed to oxidative stress. Methods Cerebral cortical cultured neurons are pretreated with acetaminophen and then exposed to the superoxide-generating compound menadione (5 μM). Cell survival is assessed by MTT assay and inflammatory protein (tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES) release quantitated by ELISA. Expression of pro- and anti-apoptotic proteins is assessed by western blots. Results Acetaminophen has pro-survival effects on neurons in culture. Menadione, a superoxide releasing oxidant stressor, causes a significant (p < 0.001) increase in neuronal cell death as well as in the release of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES from cultured neurons. Pretreatment of neuronal cultures with acetaminophen (50 μM) increases neuronal cell survival and inhibits the expression of these cytokines and chemokines. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2 in brain neurons and decreases the menadione-induced elevation of the proapoptotic protein, cleaved caspase 3. We show that blocking acetaminophen-induced expression of Bcl2 reduces the pro-survival effect of the drug. Conclusion These data show that acetaminophen has anti-oxidant and anti-inflammatory effects on neurons and suggest a heretofore unappreciated therapeutic potential for

  5. Curcumin protects neuronal cells against status-epilepticus-induced hippocampal damage through induction of autophagy and inhibition of necroptosis.

    PubMed

    Wang, Jin; Liu, Yuan; Li, Xiao-Hui; Zeng, Xiang-Chang; Li, Jian; Zhou, Jun; Xiao, Bo; Hu, Kai

    2017-05-01

    Status epilepticus, the most severe form of epilepsy, is characterized by progressive functional and structural damage in the hippocampus, ultimately leading to the development and clinical appearance of spontaneous, recurrent seizures. Although the pathogenesis underlying epileptogenesis processes remains unclear, a substantial body of evidence has shown that status epilepticus acts as an important initial factor in triggering epileptogenesis. Notably, besides classical cell death mechanisms such as apoptosis and necrosis, 2 novel regulators of cell fate known as necroptosis and autophagy, are demonstrated to be involved in neuronal damage in various neurodegenerative and neuropsychiatric disorders. However, whether necroptosis and autophagy play a role in post-status-epilepticus rat hippocampus and other epilepsy mechanisms deserves further research effort. In addition, research is needed to determine whether compounds from traditional Chinese herbs possess antiepileptic effects through the modulation of necroptosis and autophagy. In this study, we found that curcumin, a polyphenolic phytochemical extracted from the Curcuma longa plant, protects neuronal cells against status-epilepticus-induced hippocampal neuronal damage in the lithium-pilocarpine-induced status epilepticus rat model through induction of autophagy and inhibition of necroptosis.

  6. WNT/β-catenin pathway activation in Myc immortalised cerebellar progenitor cells inhibits neuronal differentiation and generates tumours resembling medulloblastoma.

    PubMed

    Rogers, H A; Sousa, S; Salto, C; Arenas, E; Coyle, B; Grundy, R G

    2012-09-25

    Medulloblastoma is the most common malignant childhood brain tumour. Aberrant activation of the WNT/β-catenin pathway occurs in approximately 25% of medulloblastomas. However, its role in medulloblastoma pathogenesis is not understood. We have developed a model of WNT/β-catenin pathway-activated medulloblastoma. Pathway activation was induced in a Myc immortalised cerebellar progenitor cell line through stable expression of Wnt1. In vitro and in vivo analysis was undertaken to understand the effect of pathway activation and identify the potential cell of origin. Tumours that histologically resembled classical medulloblastoma formed in vivo using cells overexpressing Wnt1, but not with the control cell line. Wnt1 overexpression inhibited neuronal differentiation in vitro, suggesting WNT/β-catenin pathway activation prevents cells terminally differentiating, maintaining them in a more 'stem-like' state. Analysis of cerebellar progenitor cell markers demonstrated the cell line resembled cells from the cerebellar ventricular zone. We have developed a cell line with the means of orthotopically modelling WNT/β-catenin pathway-activated medulloblastoma. We provide evidence of the role pathway activation is playing in tumour pathogenesis and suggest medulloblastomas can arise from cells other than granule cell progenitors. This cell line is a valuable resource to further understand the role of pathway activation in tumorigenesis and for investigation of targeted therapies.

  7. Trazodone treatment protects neuronal-like cells from inflammatory insult by inhibiting NF-κB, p38 and JNK.

    PubMed

    Daniele, Simona; Da Pozzo, Eleonora; Zappelli, Elisa; Martini, Claudia

    2015-08-01

    inhibited, and the release of interleukin-10 was restored to control levels. Furthermore, the intracellular signalling mechanism regulating TDZ-elicited effects was specifically investigated. TDZ induced extracellular signal-regulated kinase (ERK) phosphorylation and inhibited constitutive p38 activation. Moreover, TDZ counteracted the activation of p38 and c-Jun NH2-terminal kinase (JNK) elicited by LPS-TNF-α, suggesting that the neuro-protective role of TDZ could be mediated by p38 and JNK. Overall, our results demonstrated that the protective effects of TDZ under inflammation in neuronal-like cells function by decreasing pro-inflammatory signalling and by enhancing anti-inflammatory signalling.

  8. IGF-1 protects against Aβ25-35-induced neuronal cell death via inhibition of PUMA expression and Bax activation.

    PubMed

    Hou, Xunyao; Jin, Yan; Chen, Jian; Hong, Yan; Luo, Dingzhen; Yin, Qingqing; Liu, Xueping

    2017-01-10

    Amyloid-β-peptide (Aβ) is considered to be the toxic species in AD and causes cell death in the affected areas of patient's brain. Insulin-like growth factor 1 (IGF-1) has been reported to attenuate Aβ toxicity in neuronal cells. However, the molecular mechanisms involved in the neuroprotective function of IGF-1 remain largely unknown. In the present study, we for the first time demonstrated that IGF-1 protects against Aβ-induced neurotoxicity via inhibition of PUMA expression and Bax activation. We found that IGF-1 could activate Akt, which in turn inhibited Aβ-induced FOXO3a nuclear translocation and thus decreased the binding ability of FOXO3a to PUMA promoter, leading to decreased PUMA expression. In addition, IGF-1 inhibited the translocation of Bax to the mitochondria induced by Aβ. Notably, addition of wortmannin, a specific inhibitor of PI3K, significantly abolished the neuroprotective effect of IGF-1, suggesting that IGF-1 exerts its anti-apoptotic effect depend on PI3K activity. Our findings may provide new insights into molecular mechanisms mediated by IGF-1 in cell survival against Aβ-induced apoptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Inhibition Controls Asynchronous States of Neuronal Networks

    PubMed Central

    Treviño, Mario

    2016-01-01

    Computations in cortical circuits require action potentials from excitatory and inhibitory neurons. In this mini-review, I first provide a quick overview of findings that indicate that GABAergic neurons play a fundamental role in coordinating spikes and generating synchronized network activity. Next, I argue that these observations helped popularize the notion that network oscillations require a high degree of spike correlations among interneurons which, in turn, produce synchronous inhibition of the local microcircuit. The aim of this text is to discuss some recent experimental and computational findings that support a complementary view: one in which interneurons participate actively in producing asynchronous states in cortical networks. This requires a proper mixture of shared excitation and inhibition leading to asynchronous activity between neighboring cells. Such contribution from interneurons would be extremely important because it would tend to reduce the spike correlation between neighboring pyramidal cells, a drop in redundancy that could enhance the information-processing capacity of neural networks. PMID:27274721

  10. MC1568 Inhibits Thimerosal-Induced Apoptotic Cell Death by Preventing HDAC4 Up-Regulation in Neuronal Cells and in Rat Prefrontal Cortex.

    PubMed

    Guida, Natascia; Laudati, Giusy; Mascolo, Luigi; Cuomo, Ornella; Anzilotti, Serenella; Sirabella, Rossana; Santopaolo, Marianna; Galgani, Mario; Montuori, Paolo; Di Renzo, Gianfranco; Canzoniero, Lorella M T; Formisano, Luigi

    2016-12-01

    Ethylmercury thiosalicylate (thimerosal) is an organic mercury-based compound commonly used as an antimicrobial preservative that has been found to be neurotoxic. In contrast, histone deacetylases (HDACs) inhibition has been found to be neuroprotective against several environmental contaminants, such as polychlorinated biphenyls, di-2-ethylhexyl phthalate, and methylmercury. The aim of this study was to investigate the effect of HDAC inhibition on thimerosal-induced neurotoxicity in neuroblastoma cells and cortical neurons. Interestingly, we found that thimerosal, at 0.5 μM in SH-SY5Y cells and at 1 μM in neurons, caused cell death by activation of apoptosis, which was prevented by the HDAC class IIA inhibitor MC1568 but not the class I inhibitor MS275. Furthermore, thimerosal specifically increased HDAC4 protein expression but not that of HDACs 5, 6, 7, and 9. Western blot analysis revealed that MC1568 prevented thimerosal-induced HDAC4 increase. In addition, both HDAC4 knocking-down and MC1568 inhibited thimerosal-induced cell death in SH-SY5Y cells and cortical neurons. Importantly, intramuscular injection of 12 μg/kg thimerosal on postnatal days 7, 9, 11, and 15 increased HDAC4 levels in the prefrontal cortex (PFC), which decreased histone H4 acetylation in infant male rats, in parallel increased motor activity changes. In addition, coadministration of 40 mg/kg MC1568 (intraperitoneal injection) moderated the HDAC4 increase which reduced histone H4 deacetylation and caspase-3 cleavage in the PFC. Finally, open-field testing showed that thimerosal-induced motor activity changes are reduced by MC1568. These findings indicate that HDAC4 regulates thimerosal-induced cell death in neurons and that treatment with MC1568 prevents thimerosal-induced activation of caspase-3 in the rat PFC.

  11. Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos, necroptosis, and apoptosis, but not autophagy.

    PubMed

    Kong, Deyan; Zhu, Juehua; Liu, Qian; Jiang, Yongjun; Xu, Lily; Luo, Ning; Zhao, Zhenqiang; Zhai, Qijin; Zhang, Hao; Zhu, Mingyue; Liu, Xinfeng

    2017-03-01

    Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.

  12. Intact glycosaminoglycans from intervertebral disc-derived notochordal cell-conditioned media inhibit neurite growth while maintaining neuronal cell viability.

    PubMed

    Purmessur, Devina; Cornejo, Marisa C; Cho, Samuel K; Roughley, Peter J; Linhardt, Robert J; Hecht, Andrew C; Iatridis, James C

    2015-05-01

    Painful human intervertebral discs (IVDs) exhibit nerve growth deep into the IVD. Current treatments for discogenic back pain do not address the underlying mechanisms propagating pain and are often highly invasive or only offer temporary symptom relief. The notochord produces factors during development that pattern the spine and inhibit the growth of dorsal root ganglion (DRG) axons into the IVD. We hypothesize that notochordal cell (NC)-conditioned medium (NCCM) includes soluble factors capable of inhibiting neurite growth and may represent a future therapeutic target. To test if NCCM can inhibit neurite growth and determine if NC-derived glycosaminoglycans (GAGs) are necessary candidates for this inhibition. Human neuroblastoma (SH-SY5Y) cells and rat DRG cells were treated with NCCM in two-dimensional culture in vitro, and digestion and mechanistic studies determined if specific GAGs were responsible for inhibitory effects. Notochordal cell-conditioned medium was generated from porcine nucleus pulposus tissue that was cultured in Dulbecco's modified eagle's medium for 4 days. A dose study was performed using SH-SY5Y cells that were seeded in basal medium for 24 hours and neurite outgrowth and cell viability were assessed after treatment with basal media or NCCM (10% and 100%) for 48 hours. Glycosaminoglycans from NCCM were characterized using multiple digestions and liquid chromatography mass spectroscopy (LC-MS). Neurite growth was assessed on both SH-SY5Y and DRG cells after treatment with NCCM with and without GAG digestion. Notochordal cell-conditioned medium significantly inhibited the neurite outgrowth from SH-SY5Y cells compared with basal controls without dose or cytotoxic effects; % of neurite expressing cells were 39.0±2.9%, 27.3±3.6%, and 30.2±2.7% and mean neurite length was 60.3±3.5, 50.8±2.4, 53.2±3.7 μm for basal, 10% NCCM, and 100% NCCM, respectively. Digestions and LC-MS determined that chondroitin-6-sulfate was the major GAG chain in

  13. Cellular form of prion protein inhibits Reelin-mediated shedding of Caspr from the neuronal cell surface to potentiate Caspr-mediated inhibition of neurite outgrowth.

    PubMed

    Devanathan, Vasudharani; Jakovcevski, Igor; Santuccione, Antonella; Li, Shen; Lee, Hyun Joon; Peles, Elior; Leshchyns'ka, Iryna; Sytnyk, Vladimir; Schachner, Melitta

    2010-07-07

    Extension of axonal and dendritic processes in the CNS is tightly regulated by outgrowth-promoting and -inhibitory cues to assure precision of synaptic connections. We identify a novel role for contactin-associated protein (Caspr) as an inhibitory cue that reduces neurite outgrowth from CNS neurons. We show that proteolysis of Caspr at the cell surface is regulated by the cellular form of prion protein (PrP), which directly binds to Caspr. PrP inhibits Reelin-mediated shedding of Caspr from the cell surface, thereby increasing surface levels of Caspr and potentiating the inhibitory effect of Caspr on neurite outgrowth. PrP deficiency results in reduced levels of Caspr at the cell surface, enhanced neurite outgrowth in vitro, and more efficient regeneration of axons in vivo following spinal cord injury. Thus, we reveal a previously unrecognized role for Caspr and PrP in inhibitory modulation of neurite outgrowth in CNS neurons, which is counterbalanced by the proteolytic activity of Reelin.

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

    PubMed

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

    2008-01-28

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

  15. Time course of morphine's effects on adult hippocampal subgranular zone reveals preferential inhibition of cells in S phase of the cell cycle and a subpopulation of immature neurons.

    PubMed

    Arguello, A A; Harburg, G C; Schonborn, J R; Mandyam, C D; Yamaguchi, M; Eisch, A J

    2008-11-11

    Opiates, such as morphine, decrease neurogenesis in the adult hippocampal subgranular zone (SGZ), raising the possibility that decreased neurogenesis contributes to opiate-induced cognitive deficits. However, there is an incomplete understanding of how alterations in cell cycle progression and progenitor maturation contribute to this decrease. The present study examined how morphine regulates progenitor cell cycle, cell death and immature SGZ neurons (experiment 1) as well as the progression of SGZ progenitors through key stages of maturation (experiment 2). In experiment 1, mice received sham or morphine pellets (s.c., 0 and 48 h) and bromodeoxyuridine (BrdU) 2 h prior to sacrifice (24, 72 or 96 h). Morphine decreased both the number of S phase and total cycling cells, as there were fewer cells immunoreactive (IR) for the S phase marker BrdU and the cell cycle marker Ki67. The percentage of Ki67-IR cells that were BrdU-IR was decreased after 24 but not 96 h of morphine, suggesting a disproportionate effect on S phase cells relative to all cycling cells at this time point. Cell death (activated caspase-3 counts) was increased after 24 but not 96 h. In experiment 2, nestin-green fluorescent protein (GFP) mice given BrdU 1 day prior to morphine or sham surgery (0 and 48 h, sacrifice 96 h) had fewer Ki67-IR cells, but no change in BrdU-IR cell number, suggesting that this population of BrdU-IR cells was less sensitive to morphine. Interestingly, examination of key stages of progenitor cell maturation revealed that morphine increased the percent of BrdU-IR cells that were type 2b and decreased the percent that were immature neurons. These data suggest that chronic morphine decreases SGZ neurogenesis by inhibiting dividing cells, particularly those in S phase, and progenitor cell progression to a more mature neuronal stage.

  16. Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

    SciTech Connect

    Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena; Accordi, Benedetta; Basso, Giuseppe; Gaspar, John Antonydas; Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina; Sachinidis, Agapios; Bremer-Hoffmann, Susanne

    2014-10-15

    According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.

  17. Inhibition of neuronal cell mitochondrial complex I with rotenone increases lipid β-oxidation, supporting acetyl-coenzyme A levels.

    PubMed

    Worth, Andrew J; Basu, Sankha S; Snyder, Nathaniel W; Mesaros, Clementina; Blair, Ian A

    2014-09-26

    Rotenone is a naturally occurring mitochondrial complex I inhibitor with a known association with parkinsonian phenotypes in both human populations and rodent models. Despite these findings, a clear mechanistic link between rotenone exposure and neuronal damage remains to be determined. Here, we report alterations to lipid metabolism in SH-SY5Y neuroblastoma cells exposed to rotenone. The absolute levels of acetyl-CoA were found to be maintained despite a significant decrease in glucose-derived acetyl-CoA. Furthermore, palmitoyl-CoA levels were maintained, whereas the levels of many of the medium-chain acyl-CoA species were significantly reduced. Additionally, using isotopologue analysis, we found that β-oxidation of fatty acids with varying chain lengths helped maintain acetyl-CoA levels. Rotenone also induced increased glutamine utilization for lipogenesis, in part through reductive carboxylation, as has been found previously in other cell types. Finally, palmitoylcarnitine levels were increased in response to rotenone, indicating an increase in fatty acid import. Taken together, these findings show that alterations to lipid and glutamine metabolism play an important compensatory role in response to complex I inhibition by rotenone. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Inhibition of Neuronal Cell Mitochondrial Complex I with Rotenone Increases Lipid β-Oxidation, Supporting Acetyl-Coenzyme A Levels*

    PubMed Central

    Worth, Andrew J.; Basu, Sankha S.; Snyder, Nathaniel W.; Mesaros, Clementina; Blair, Ian A.

    2014-01-01

    Rotenone is a naturally occurring mitochondrial complex I inhibitor with a known association with parkinsonian phenotypes in both human populations and rodent models. Despite these findings, a clear mechanistic link between rotenone exposure and neuronal damage remains to be determined. Here, we report alterations to lipid metabolism in SH-SY5Y neuroblastoma cells exposed to rotenone. The absolute levels of acetyl-CoA were found to be maintained despite a significant decrease in glucose-derived acetyl-CoA. Furthermore, palmitoyl-CoA levels were maintained, whereas the levels of many of the medium-chain acyl-CoA species were significantly reduced. Additionally, using isotopologue analysis, we found that β-oxidation of fatty acids with varying chain lengths helped maintain acetyl-CoA levels. Rotenone also induced increased glutamine utilization for lipogenesis, in part through reductive carboxylation, as has been found previously in other cell types. Finally, palmitoylcarnitine levels were increased in response to rotenone, indicating an increase in fatty acid import. Taken together, these findings show that alterations to lipid and glutamine metabolism play an important compensatory role in response to complex I inhibition by rotenone. PMID:25122772

  19. Inhibition of mTOR by Rapamycin Results in Auditory Hair Cell Damage and Decreased Spiral Ganglion Neuron Outgrowth and Neurite Formation In Vitro

    PubMed Central

    Leitmeyer, Katharina; Glutz, Andrea; Radojevic, Vesna; Setz, Cristian; Huerzeler, Nathan; Bumann, Helen; Bodmer, Daniel; Brand, Yves

    2015-01-01

    Rapamycin is an antifungal agent with immunosuppressive properties. Rapamycin inhibits the mammalian target of rapamycin (mTOR) by blocking the mTOR complex 1 (mTORC1). mTOR is an atypical serine/threonine protein kinase, which controls cell growth, cell proliferation, and cell metabolism. However, less is known about the mTOR pathway in the inner ear. First, we evaluated whether or not the two mTOR complexes (mTORC1 and mTORC2, resp.) are present in the mammalian cochlea. Next, tissue explants of 5-day-old rats were treated with increasing concentrations of rapamycin to explore the effects of rapamycin on auditory hair cells and spiral ganglion neurons. Auditory hair cell survival, spiral ganglion neuron number, length of neurites, and neuronal survival were analyzed in vitro. Our data indicates that both mTOR complexes are expressed in the mammalian cochlea. We observed that inhibition of mTOR by rapamycin results in a dose dependent damage of auditory hair cells. Moreover, spiral ganglion neurite number and length of neurites were significantly decreased in all concentrations used compared to control in a dose dependent manner. Our data indicate that the mTOR may play a role in the survival of hair cells and modulates spiral ganglion neuronal outgrowth and neurite formation. PMID:25918725

  20. One cell model establishment to inhibit CaMKIIγ mRNA expression in the dorsal root ganglion neuron by RNA interfere.

    PubMed

    Wen, Xianjie; Li, Xiaohong; Liang, Hua; Yang, Chenxiang; Zhong, Jiying; Wang, Hanbing; Liu, Hongzhen

    2017-09-01

    CaMKIIγ in dorsal root ganglion neurons is closely related to the neuropathic pain, neuron injury induced by local anesthetics. To get great insight into the function of CaMKIIγ in dorsal root ganglion neurons, we need one cell model to specially inhibit the CaMKIIγ mRNA expression. The present study was aimed to establish one cell model to specially inhibit the CaMKIIγ mRNA expression. We designed the CaMKIIγ shRNA sequence and connected with pYr-1.1 plasmid. The ligation product of the CaMKIIγshRNA and pYr-1.1 plasmid was recombined with pAd/PL-DEST vector into pAD-CaMKIIγ-shRNA. adenovirus vector. pAD-CaMKIIγ-shRNA. adenovirus vector infected the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA expression in vitro. The pAD-CaMKIIγ-shRNA adenovirus vector was verified to be correct by the digestion, sequence. And pAD-CaMKIIγ-shRNA. adenovirus vector can infect the DRG cells to inhibit the CaMKIIγ mRNA or protein expression by the real-time polymerase chain reaction (PCR) or western blotting. Those results showed that we successfully constructed one adenovirus vector that can infect the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA and protein expression. That will supply with one cell model for the CaMKIIγ function study.

  1. Combined small-molecule inhibition accelerates the derivation of functional cortical neurons from human pluripotent stem cells.

    PubMed

    Qi, Yuchen; Zhang, Xin-Jun; Renier, Nicolas; Wu, Zhuhao; Atkin, Talia; Sun, Ziyi; Ozair, M Zeeshan; Tchieu, Jason; Zimmer, Bastian; Fattahi, Faranak; Ganat, Yosif; Azevedo, Ricardo; Zeltner, Nadja; Brivanlou, Ali H; Karayiorgou, Maria; Gogos, Joseph; Tomishima, Mark; Tessier-Lavigne, Marc; Shi, Song-Hai; Studer, Lorenz

    2017-02-01

    Considerable progress has been made in converting human pluripotent stem cells (hPSCs) into functional neurons. However, the protracted timing of human neuron specification and functional maturation remains a key challenge that hampers the routine application of hPSC-derived lineages in disease modeling and regenerative medicine. Using a combinatorial small-molecule screen, we previously identified conditions to rapidly differentiate hPSCs into peripheral sensory neurons. Here we generalize the approach to central nervous system (CNS) fates by developing a small-molecule approach for accelerated induction of early-born cortical neurons. Combinatorial application of six pathway inhibitors induces post-mitotic cortical neurons with functional electrophysiological properties by day 16 of differentiation, in the absence of glial cell co-culture. The resulting neurons, transplanted at 8 d of differentiation into the postnatal mouse cortex, are functional and establish long-distance projections, as shown using iDISCO whole-brain imaging. Accelerated differentiation into cortical neuron fates should facilitate hPSC-based strategies for disease modeling and cell therapy in CNS disorders.

  2. Inspiratory neurons that are activated when inspiration is inhibited behaviorally.

    PubMed

    Orem, J

    1987-12-29

    Respiration can be automatic or controlled behaviorally. Behavioral control in the cat occurs, at least in part, through control of the brainstem respiratory neurons that constitute the automatic system. Thus, when inspiration is inhibited behaviorally, inspiratory neurons in the medulla are inactivated. Reported herein are data on inspiratory cells, located in both the dorsal and ventral respiratory groups, that were activated when other inspiratory cells there were inhibited behaviorally. During spontaneous breathing, their activity showed much variability unattributable to the respiratory cycle--indicating that they receive a considerable non-respiratory input. These cells might act as the interface through which behavioral inhibition of inspiration occurs.

  3. Vanillin Protects Dopaminergic Neurons against Inflammation-Mediated Cell Death by Inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway

    PubMed Central

    Yan, Xuan; Liu, Dian-Feng; Zhang, Xiang-Yang; Liu, Dong; Xu, Shi-Yao; Chen, Guang-Xin; Huang, Bing-Xu; Ren, Wen-Zhi; Wang, Wei; Fu, Shou-Peng; Liu, Ju-Xiong

    2017-01-01

    Neuroinflammation plays a very important role in the pathogenesis of Parkinson’s disease (PD). After activation, microglia produce pro-inflammatory mediators that damage surrounding neurons. Consequently, the inhibition of microglial activation might represent a new therapeutic approach of PD. Vanillin has been shown to protect dopaminergic neurons, but the mechanism is still unclear. Herein, we further study the underlying mechanisms in lipopolysaccharide (LPS)-induced PD models. In vivo, we firstly established rat models of PD by unilateral injection of LPS into substantia nigra (SN), and then examined the role of vanillin in motor dysfunction, microglial activation and degeneration of dopaminergic neurons. In vitro, murine microglial BV-2 cells were treated with vanillin prior to the incubation of LPS, and then the inflammatory responses and the related signaling pathways were analyzed. The in vivo results showed that vanillin markedly improved the motor dysfunction, suppressed degeneration of dopaminergic neurons and inhibited microglial over-activation induced by LPS intranigral injection. The in vitro studies demonstrated that vanillin reduces LPS-induced expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and IL-6 through regulating ERK1/2, p38 and NF-κB signaling. Collectively, these data indicated that vanillin has a role in protecting dopaminergic neurons via inhibiting inflammatory activation. PMID:28208679

  4. Vanillin Protects Dopaminergic Neurons against Inflammation-Mediated Cell Death by Inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway.

    PubMed

    Yan, Xuan; Liu, Dian-Feng; Zhang, Xiang-Yang; Liu, Dong; Xu, Shi-Yao; Chen, Guang-Xin; Huang, Bing-Xu; Ren, Wen-Zhi; Wang, Wei; Fu, Shou-Peng; Liu, Ju-Xiong

    2017-02-12

    Neuroinflammation plays a very important role in the pathogenesis of Parkinson's disease (PD). After activation, microglia produce pro-inflammatory mediators that damage surrounding neurons. Consequently, the inhibition of microglial activation might represent a new therapeutic approach of PD. Vanillin has been shown to protect dopaminergic neurons, but the mechanism is still unclear. Herein, we further study the underlying mechanisms in lipopolysaccharide (LPS)-induced PD models. In vivo, we firstly established rat models of PD by unilateral injection of LPS into substantia nigra (SN), and then examined the role of vanillin in motor dysfunction, microglial activation and degeneration of dopaminergic neurons. In vitro, murine microglial BV-2 cells were treated with vanillin prior to the incubation of LPS, and then the inflammatory responses and the related signaling pathways were analyzed. The in vivo results showed that vanillin markedly improved the motor dysfunction, suppressed degeneration of dopaminergic neurons and inhibited microglial over-activation induced by LPS intranigral injection. The in vitro studies demonstrated that vanillin reduces LPS-induced expression of inducible nitric oxide (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and IL-6 through regulating ERK1/2, p38 and NF-κB signaling. Collectively, these data indicated that vanillin has a role in protecting dopaminergic neurons via inhibiting inflammatory activation.

  5. Complex II inhibition by 3-NP causes mitochondrial fragmentation and neuronal cell death via an NMDA- and ROS-dependent pathway

    PubMed Central

    Liot, G; Bossy, B; Lubitz, S; Kushnareva, Y; Sejbuk, N; Bossy-Wetzel, E

    2009-01-01

    Mitochondrial respiratory complex II inhibition plays a central role in Huntington’s disease (HD). Remarkably, 3-NP, a complex II inhibitor, recapitulates HD-like symptoms. Furthermore, decreases in mitochondrial fusion or increases in mitochondrial fission have been implicated in neurodegenerative diseases. However, the relationship between mitochondrial energy defects and mitochondrial dynamics has never been explored in detail. In addition, the mechanism of neuronal cell death by complex II inhibition remains unclear. Here, we tested the temporal and spatial relationship between energy decline, impairment of mitochondrial dynamics, and neuronal cell death in response to 3-NP using quantitative fluorescence time-lapse microscopy and cortical neurons. 3-NP caused an immediate drop in ATP. This event corresponded with a mild rise in reactive oxygen species (ROS), but mitochondrial morphology remained unaltered. Unexpectedly, several hours after this initial phase, a second dramatic rise in ROS occurred, associated with profound mitochondrial fission characterized by the conversion of filamentous to punctate mitochondria and neuronal cell death. Glutamate receptor antagonist AP5 abolishes the second peak in ROS, mitochondrial fission, and cell death. Thus, secondary excitotoxicity, mediated by glutamate receptor activation of the NMDA subtype, and consequent oxidative and nitrosative stress cause mitochondrial fission, rather than energy deficits per se. These results improve our understanding of the cellular mechanisms underlying HD pathogenesis. PMID:19300456

  6. [Astragalus induces human amniotic epithelial cells (WISH) to differentiate toward neurons, inhibits the expression of Notch1 and promotes cell survival].

    PubMed

    Chen, Xu-Dong; Wang, Jian-Guo

    2012-12-25

    The aim of the study was to investigate the effect of astragalus on differentiation of human amniotic epithelial cell line WISH into neurons, the expression of Notch1 gene and cell viability. WISH were randomly divided into astragalus group (4 subgroups), alltransretinoic acid (RA) group and control group. Astragalus group and RA group were induced to differentiate into neurocytes by using chemical inducer RA and astragalus, respectively. The expression of neuron-specific enolase (NSE), microtubule associated protein 2 (MAP-2), Nestin and GFAP of induced cells in three groups were detected using immunocytochemical method. RT- PCR was further used to detect the expression of Oct4, Notch1, Hes1, Nestin and NSE. The cell viability was measured by methyl thiazolyl tetrazolium methods. Under the convert microscope it was observed that WISH cells started to change their shape, and there were several axon or dendrite-like processes out from the cell body induced by astragalus for 24 h or RA for 12 h. The positive cell rates of NSE and MAP-2 in 100 μL/mL astragalus-induced group were less than those in RA-induced group at 48 h (P < 0.05), but higher than those in control group. Cell viability in astragalus group was higher than that of RA group (P < 0.05). While the positive cell rates of Nestin and GFAP in 100 μL/mL astragalus-induced group were higher than those in RA-induced group at 48 h (P < 0.05). The positive cell rates of Nestin in the two induced groups were lower than those in control group. RT-PCR showed that the expressions of Oct4, Notch1 and Hes1 in RA and astragalus (100 μL/mL) groups were less than those in control group, but the expression of NSE was higher than that in control group. These results suggest that astragalus (especially at 100 μL/mL, 48 h) and RA can both induce human amniotic epithelial cell line WISH cells into neuron-like cells, but astragalus induction has a higher cell survival rate than RA induction, and the expression of Notch1

  7. Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons.

    PubMed

    Benavides, Gloria A; Liang, Qiuli; Dodson, Matthew; Darley-Usmar, Victor; Zhang, Jianhua

    2013-12-01

    Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular

  8. Pharmacological inhibition of DNA methyltransferase 1 promotes neuronal differentiation from rodent and human nasal olfactory stem/progenitor cell cultures.

    PubMed

    Franco, I; Ortiz-López, L; Roque-Ramírez, B; Ramírez-Rodríguez, G B; Lamas, M

    2017-05-01

    Nasal olfactory stem and neural progenitor cells (NOS/PCs) are considered possible tools for regenerative stem cell therapies in neurodegenerative diseases. Neurogenesis is a complex process regulated by extrinsic and intrinsic signals that include DNA-methylation and other chromatin modifications that could be experimentally manipulated in order to increase neuronal differentiation. The aim of the present study was the characterization of primary cultures and consecutive passages (P2-P10) of NOS/PCs isolated from male Swiss-Webster (mNOS/PCs) or healthy humans (hNOS/PCs). We evaluated and compared cellular morphology, proliferation rates and the expression pattern of pluripotency-associated markers and DNA methylation-associated gene expression in these cultures. Neuronal differentiation was induced by exposure to all-trans retinoic acid and forskolin for 7 days and evaluated by morphological analysis and immunofluorescence against neuronal markers MAP2, NSE and MAP1B. In response to the inductive cues mNOS/PCs expressed NSE (75.67%) and MAP2 (35.34%); whereas the majority of the hNOS/PCs were immunopositive to MAP1B. Treatment with procainamide, a specific inhibitor of DNA methyltransferase 1 (DNMT1), increases in the number of forskolin'/retinoic acid-induced mature neuronal marker-expressing mNOS/PCs cells and enhances neurite development in hNOS/PCs. Our results indicate that mice and human nasal olfactory stem/progenitors cells share pluripotency-related gene expression suggesting that their application for stem cell therapy is worth pursuing and that DNA methylation inhibitors could be efficient tools to enhance neuronal differentiation from these cells.

  9. Oxygen-glucose deprivation inducing B1 RNA inhibits neuronal cells metabolic activity by NLRP3 and associated proinflammatory cytokines production.

    PubMed

    Wang, Li; Wang, Weihua; Zhang, Lei; Dai, Peng; Wang, Kai; Hui, Hao; Rao, Wei; Peng, Cheng; Yang, Jinghua; Yan, Zhen; Fei, Zhou

    2015-02-19

    Cerebral ischemia occurs when blood flow to part of the brain is obstructed, which can result in oxygen and glucose deprivation (OGD) and neuronal damage. However, the mechanisms remain poorly understood. The present study investigated the production and effects of double-stranded RNA (dsRNA) induced by OGD in neuronal cells. By confocal microscopy, dsRNA containing B1 and B2 RNA, was found accumulating in HT22 cells under OGD treatment. The sequence of B1 RNA was identified and transfected into HT22 cells. Interestingly, B1 RNA induced transcription and expression of NLRP3, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α, which was similar to the effects of OGD treatment. Moreover, HT22 cell growth inhibition and proinflammatory cytokines production induced by OGD and B1 RNA treatment were down-regulated by NLRP3 knock-down. These findings suggest that B1 RNA induced by OGD forms as dsRNA and inhibits neuronal cell metabolic activity by regulating the NLRP3 and associated proinflammatory cytokines production. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Inhibition of autophagy induction delays neuronal cell loss caused by dysfunctional ESCRT-III in frontotemporal dementia.

    PubMed

    Lee, Jin-A; Gao, Fen-Biao

    2009-07-01

    Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in age-dependent neurodegenerative diseases remain largely unknown. Here we show that the autophagy inhibitor 3-methyladenine delays neuronal cell loss caused by dysfunctional endosomal sorting complex required for transport III (ESCRT-III), either through loss of its essential component mSnf7-2 or ectopic expression of the disease protein CHMP2B(Intron5), which is associated with frontotemporal dementia linked to chromosome 3. Neuronal loss was also delayed by reduced activity of the autophagy genes atg5 and atg7. However, the endosomal accumulation of ubiquitinated proteins induced by dysfunctional ESCRT-III was not significantly affected, further confirming the essential contribution of dysregulated autophagy pathway in neurodegeneration. These findings show that autophagic stress by excess accumulation of autophagosomes is detrimental to neuronal survival under certain neurodegenerative conditions.

  11. Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation.

    PubMed

    Kim, Bokyung; Park, Junghyung; Chang, Kyu-Tae; Lee, Dong-Seok

    2016-01-01

    Alzheimer's disease (AD), a neurodegenerative disorder, is caused by amyloid-beta oligomers (AβOs). AβOs induce cell death by triggering oxidative stress and mitochondrial dysfunction. A recent study showed that AβO-induced oxidative stress is associated with extracellular signal-regulated kinase (ERK)-dynamin related protein 1 (Drp1)-mediated mitochondrial fission. Reactive oxygen species (ROS) are regulated by antioxidant enzymes, especially peroxiredoxins (Prxs) that scavenge H2O2. These enzymes inhibit neuronal cell death induced by various neurotoxic reagents. However, it is unclear whether Prx5, which is specifically expressed in neuronal cells, protects these cells from AβO-induced damage. In this study, we found that Prx5 expression was upregulated by AβO-induced oxidative stress and that Prx5 decreased ERK-Drp1-mediated mitochondrial fragmentation and apoptosis of HT-22 neuronal cells. Prx5 expression was affected by AβO, and amelioration of oxidative stress by N-acetyl-L-cysteine decreased AβO-induced Prx5 expression. Prx5 overexpression reduced ROS as well as RNS and apoptotic cell death but Prx5 knockdown did not. In addition, Prx5 overexpression ameliorated ERK-Drp1-mediated mitochondrial fragmentation but Prx5 knockdown did not. These results indicated that inducible Prx5 expression by AβO plays a key role in inhibiting both ERK-Drp1-induced mitochondrial fragmentation and neuronal cell death by regulating oxidative stress. Thus, Prx5 may be a new therapeutic agent for treating AD.

  12. Epigenetic regulation of Dpp6 expression by Dnmt3b and its novel role in the inhibition of RA induced neuronal differentiation of P19 cells.

    PubMed

    Sheikh, Muhammad Abid; Malik, Yousra Saeed; Yu, Huali; Lai, Mingming; Wang, Xingzhi; Zhu, Xiaojuan

    2013-01-01

    DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells. Dnmt3b enriched chromatin library from RA treated P19 cells identified dipeptidyl peptidase 6 (Dpp6) gene as a novel target of Dnmt3b. Further, quantitative ChIP analysis showed that the amount of Dnmt3b recruited on Dpp6 promoter was equal in both RA treated as well as untreated p19 cells. Bisulfite genomic sequencing, COBRA, and methylation specific PCR analysis revealed that Dpp6 promoter was heavily methylated in both RA treated and untreated P19 cells. Dnmt3b was responsible for transcriptional silencing of Dpp6 gene as depletion of Dnmt3b resulted in increased mRNA and protein expression of Dpp6. Consequently, the average methylation of Dpp6 gene promoter was reduced to half in Dnmt3b knockdown cells. In the absence of Dnmt3b, Dnmt3a was associated with Dpp6 gene promoter and regulated its expression and methylation in P19 cells. RA induced neuronal differentiation was inhibited upon ectopic expression of Dpp6 in P19 cells. Taken together, the present study described epigenetic silencing of Dpp6 expression by DNA methylation and established that its ectopic expression can act as negative signal during RA induced neuronal differentiation of P19 cells.

  13. Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

    PubMed

    Vázquez-Gómez, Elizabeth; Arias, Hugo R; Feuerbach, Dominik; Miranda-Morales, Marcela; Mihailescu, Stefan; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof; García-Colunga, Jesús

    2014-10-05

    The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Inhibition of the proliferation and acceleration of migration of vascular endothelial cells by increased cysteine-rich motor neuron 1

    SciTech Connect

    Nakashima, Yukiko; Morimoto, Mayuka; Toda, Ken-ichi; Shinya, Tomohiro; Sato, Keizo; Takahashi, Satoru

    2015-07-03

    Cysteine-rich motor neuron 1 (CRIM1) is upregulated only in extracellular matrix gels by angiogenic factors such as vascular endothelial growth factor (VEGF). It then plays a critical role in the tube formation of endothelial cells. In the present study, we investigated the effects of increased CRIM1 on other endothelial functions such as proliferation and migration. Knock down of CRIM1 had no effect on VEGF-induced proliferation or migration of human umbilical vein endothelial cells (HUVECs), indicating that basal CRIM1 is not involved in the proliferation or migration of endothelial cells. Stable CRIM1-overexpressing endothelial F-2 cells, termed CR1 and CR2, were constructed, because it was difficult to prepare monolayer HUVECs that expressed high levels of CRIM1. Proliferation was reduced and migration was accelerated in both CR1 and CR2 cells, compared with normal F-2 cells. Furthermore, the transient overexpression of CRIM1 resulted in decreased proliferation and increased migration of bovine aortic endothelial cells. In contrast, neither proliferation nor migration of COS-7 cells were changed by the overexpression of CRIM1. These results demonstrate that increased CRIM1 reduces the proliferation and accelerates the migration of endothelial cells. These CRIM1 effects might contribute to tube formation of endothelial cells. CRIM1 induced by angiogenic factors may serve as a regulator in endothelial cells to switch from proliferating cells to morphological differentiation. - Highlights: • CRIM1 was upregulated only in tubular endothelial cells, but not in monolayers. • Increased CRIM1 reduced the proliferation of endothelial cells. • Increased CRIM1 accelerated the migration of endothelial cells. • Increased CRIM1 had no effect on the proliferation or migration of COS-7 cells.

  15. Neuronal Cell Cultures.

    DTIC Science & Technology

    1982-10-01

    AD-A123 120 NEURONAL . CELL CULTURES( U) FEDERATION OF AMERICAN / SOCIETIES FOR EXPER IMENTAL B IOLOGY BETHES DA MD R BUNOF ET AL 01 0C 82 AFOSR-- a...REPRT NUM2. GOVT ACCESSION No. 3. RECIIENT’S CATALOG NUmBER 4. TITLE (end Subtitle) 5, TYPE OF REPORT & PERIOD COVERED Neuronal Cell Cultures FNLRPR...10. PROGRAM ELEMENT. PRZjECT. TASK~ Federation of American Societies for Experi- AREA & WORK UNIT N.jVBERS mental Biology (FASEB), 9650 Rockville

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

    PubMed Central

    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

  17. GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

    PubMed

    Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

    2011-09-22

    The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons.

  18. GDE2 regulates subtype specific motor neuron generation through inhibition of Notch signaling

    PubMed Central

    Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

    2011-01-01

    The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here, we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons. PMID:21943603

  19. Diversity of vestibular nuclei neurons targeted by cerebellar nodulus inhibition

    PubMed Central

    Meng, Hui; Blázquez, Pablo M; Dickman, J David; Angelaki, Dora E

    2014-01-01

    Abstract A functional role of the cerebellar nodulus and ventral uvula (lobules X and IXc,d of the vermis) for vestibular processing has been strongly suggested by direct reciprocal connections with the vestibular nuclei, as well as direct vestibular afferent inputs as mossy fibres. Here we have explored the types of neurons in the macaque vestibular nuclei targeted by nodulus/ventral uvula inhibition using orthodromic identification from the caudal vermis. We found that all nodulus-target neurons are tuned to vestibular stimuli, and most are insensitive to eye movements. Such non-eye-movement neurons are thought to project to vestibulo-spinal and/or thalamo-cortical pathways. Less than 20% of nodulus-target neurons were sensitive to eye movements, suggesting that the caudal vermis can also directly influence vestibulo-ocular pathways. In general, response properties of nodulus-target neurons were diverse, spanning the whole continuum previously described in the vestibular nuclei. Most nodulus-target cells responded to both rotation and translation stimuli and only a few were selectively tuned to translation motion only. Other neurons were sensitive to net linear acceleration, similar to otolith afferents. These results demonstrate that, unlike the flocculus and ventral paraflocculus which target a particular cell group, nodulus/ventral uvula inhibition targets a large diversity of cell types in the vestibular nuclei, consistent with a broad functional significance contributing to vestibulo-ocular, vestibulo-thalamic and vestibulo-spinal pathways. PMID:24127616

  20. Memantine inhibits serotonin-induced rise of cytosolic Ca2+ activity and of cyclic GMP level in a neuronal cell line.

    PubMed

    Reiser, G; Koch, R

    1989-05-11

    Serotonin (5-HT) evoked a rise of cytosolic Ca2+ activity in neuroblastoma X glioma hybrid cells, most probably due to the entry of extracellular Ca2+; cyclic GMP synthesis was also stimulated. The rise of both cytosolic Ca2+ activity and of cyclic GMP level was blocked by memantine (1-amino-3,5-dimethyladamantane). Memantine inhibited the rise of the cyclic GMP level non-competitively (Ki about 50 microM). Thus, memantine suppresses the effects of 5-HT in the neuronal cell line, most likely by blocking Ca2+-permeable ion channels. This interpretation is in line with the previously reported finding that memantine suppressed the 5-HT-induced depolarizing response in the same cell line.

  1. Sensory experience regulates cortical inhibition by inducing IGF1 in VIP neurons.

    PubMed

    Mardinly, A R; Spiegel, I; Patrizi, A; Centofante, E; Bazinet, J E; Tzeng, C P; Mandel-Brehm, C; Harmin, D A; Adesnik, H; Fagiolini, M; Greenberg, M E

    2016-03-17

    Inhibitory neurons regulate the adaptation of neural circuits to sensory experience, but the molecular mechanisms by which experience controls the connectivity between different types of inhibitory neuron to regulate cortical plasticity are largely unknown. Here we show that exposure of dark-housed mice to light induces a gene program in cortical vasoactive intestinal peptide (VIP)-expressing neurons that is markedly distinct from that induced in excitatory neurons and other subtypes of inhibitory neuron. We identify Igf1 as one of several activity-regulated genes that are specific to VIP neurons, and demonstrate that IGF1 functions cell-autonomously in VIP neurons to increase inhibitory synaptic input onto these neurons. Our findings further suggest that in cortical VIP neurons, experience-dependent gene transcription regulates visual acuity by activating the expression of IGF1, thus promoting the inhibition of disinhibitory neurons and affecting inhibition onto cortical pyramidal neurons.

  2. Ventral hippocampal neurons inhibit postprandial energy intake.

    PubMed

    Hannapel, Reilly C; Henderson, Yoko H; Nalloor, Rebecca; Vazdarjanova, Almira; Parent, Marise B

    2017-03-01

    Evidence suggests that the memory of a recently ingested meal limits subsequent intake. Given that ventral hippocampal (vHC) neurons are involved in memory and energy intake, the present experiment tested the hypothesis that vHC neurons contribute to the formation of a memory of a meal and inhibit energy intake during the postprandial period. We tested (1) whether pharmacological inactivation of vHC neurons during the period following a sucrose meal, when the memory of the meal would be undergoing consolidation, accelerates the onset of the next sucrose meal and increases intake and (2) whether sucrose intake increases vHC expression of the synaptic plasticity marker activity-regulated cytoskeletal-associated protein (Arc). Adult male Sprague-Dawley rats were trained to consume a 32% sucrose solution daily at the same time and location. On the experimental day, the rats were given intra-vHC infusions of the GABAA receptor agonist muscimol or vehicle after they finished their first sucrose meal. Compared to vehicle infusions, postmeal intra-vHC muscimol infusions decreased the latency to the next sucrose meal, increased the amount of sucrose consumed during that meal, increased the total number of sucrose meals and the total amount of sucrose ingested. In addition, rats that consumed sucrose had higher levels of Arc expression in both vHC CA1 and CA3 subfields than cage control rats. Collectively, these findings are the first to show that vHC neurons inhibit energy intake during the postprandial period and support the hypothesis that vHC neurons form a memory of a meal and inhibit subsequent intake. © 2016 Wiley Periodicals, Inc.

  3. Neuroprotective effect of Citrus unshiu immature peel and nobiletin inhibiting hydrogen peroxide-induced oxidative stress in HT22 murine hippocampal neuronal cells

    PubMed Central

    Cho, Hyun Woo; Jung, Su Young; Lee, Gyeong Hwan; Cho, Jung Hee; Choi, In Young

    2015-01-01

    Background: Oxidative stress-induced cell damage is common in the etiology of several neurobiological disorders, including Alzheimer's disease and Parkinson's disease. In a case study, nobiletin-rich Citrus reticulata peels could prevent the progression of cognitive impairment in donepezil-preadministered Alzheimer's disease patients. Objective: In this study, we investigated the effects and underlying mechanism of nobiletin and Citrus unshiu immature peel (CUIP) water extract, which contains nobiletin as a major compound, on hydrogen peroxide-induced oxidative stress in HT22 cells, a murine hippocampal neuronal model. Materials and Methods: HT22 cells were treated with hydrogen peroxide in the presence or absence of various concentrations of CUIP and nobiletin. Cytotoxicity and apoptotic protein levels were measured by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and Western blotting. Results: Pretreatment with CUIP and nobiletin inhibited cell death due to hydrogen peroxide. Hydrogen peroxide-induced the expression of phospho-Jun N-terminal kinases (p-JNK) and p-p38 proteins in HT22 cells; however CUIP and nobiletin suppressed p-JNK and p-p38 without changing JNK or p38. Regarding apoptosis, caspase 3, B-cell lymphoma 2 (Bcl-2), and Bax protein expression was determined. CUIP and nobiletin suppressed caspase 3 and Bax expression, but they induced Bcl-2 expression in HT22 cells. Conclusion: These results show that CUIP and nobiletin can protect against hydrogen peroxide-induced cell death in HT22 neurons via mitogen-activated protein kinases and apoptotic pathways. PMID:26664016

  4. Inhibition of neuronal ferroptosis protects hemorrhagic brain.

    PubMed

    Li, Qian; Han, Xiaoning; Lan, Xi; Gao, Yufeng; Wan, Jieru; Durham, Frederick; Cheng, Tian; Yang, Jie; Wang, Zhongyu; Jiang, Chao; Ying, Mingyao; Koehler, Raymond C; Stockwell, Brent R; Wang, Jian

    2017-04-06

    Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell-derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

  5. Inhibition of neuronal ferroptosis protects hemorrhagic brain

    PubMed Central

    Li, Qian; Han, Xiaoning; Lan, Xi; Gao, Yufeng; Wan, Jieru; Durham, Frederick; Cheng, Tian; Yang, Jie; Wang, Zhongyu; Jiang, Chao; Ying, Mingyao; Stockwell, Brent R.

    2017-01-01

    Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell–derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies. PMID:28405617

  6. Ethanol inhibition of N-methyl-D-aspartate-activated current in mouse hippocampal neurones: whole-cell patch-clamp analysis.

    PubMed

    Peoples, R W; White, G; Lovinger, D M; Weight, F F

    1997-11-01

    1. The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. 3. Concentration-response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4. The polyamine spermine (1 microM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. 5. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 7. Mg2+ (10, 100, 500 microM), (5, 20 microM) or ketamine (2, 10 microM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 8. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor-ionophore complex.

  7. Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons.

    PubMed

    Schapira, Anthony H V; Cleeter, Michael W J; Muddle, John R; Workman, Jane M; Cooper, J Mark; King, Rosalind H M

    2006-08-01

    Dysfunction of the ubiquitin-proteasomal system (UPS) has been implicated in the pathogenesis of Parkinson's disease. The systemic administration of UPS inhibitors has been reported to induce nigrostriatal cell death and model Parkinson's disease pathology in rodents. We administered a synthetic, specific UPS inhibitor (PSI) subcutaneously to rats and quantified substantia nigral tyrosine hydroxylase-positive dopaminergic neurons by stereology. PSI caused a 15% decrease in UPS activity at 2 weeks and a 42% reduction in substantia nigra pars compacta tyrosine hydroxylase-positive neurons at 8 weeks. Systemic inhibition of the UPS warrants further evaluation as a means to model Parkinson's disease.

  8. Eugenol inhibits the GABAA current in trigeminal ganglion neurons.

    PubMed

    Lee, Sang Hoon; Moon, Jee Youn; Jung, Sung Jun; Kang, Jin Gu; Choi, Seung Pyo; Jang, Jun Ho

    2015-01-01

    Eugenol has sedative, antioxidant, anti-inflammatory, and analgesic effects, but also serves as an irritant through the regulation of a different set of ion channels. Activation of gamma aminobutyric acid (GABA) receptors on sensory neurons leads to the stabilization of neuronal excitability but contributes to formalin-induced inflammatory pain. In this study, we examined the effect of eugenol on the GABA-induced current in rat trigeminal ganglia (TG) neurons and in human embryonic kidney (HEK) 293 cells expressing the GABAA receptor α1β2γ2 subtype using the whole-cell patch clamp technique. RT-PCR and Western blot analysis were used to confirm the expression of GABAA receptor γ2 subunit mRNA and protein in the TG and hippocampus. Eugenol decreased the amplitude ratio of the GABA-induced current to 27.5 ± 3.2% (p < 0.05) in TG neurons, which recovered after a 3-min washout. In HEK 293 cells expressing the α1β2γ2 subtype, eugenol inhibited GABA-induced currents in a dose-dependent manner. Application of eugenol also decreased the GABA response in the presence of a G-protein blocker. Eugenol pretreatment with different concentrations of GABA resulted in similar inhibition of the GABA-induced current in a non-competitive manner. In conclusion, eugenol inhibits the GABA-induced current in TG neurons and HEK 293 cells expressing the GABAA receptor in a reversible, dose-dependent, and non-competitive manner, but not via the G-protein pathway. We suggest that the GABAA receptor could be a molecular target for eugenol in the modulation of nociceptive information.

  9. Lipid nanocapsules containing the non-ionic surfactant Solutol HS15 inhibit the transport of calcium through hyperforin-activated channels in neuronal cells.

    PubMed

    Chauvet, Sylvain; Barras, Alexandre; Boukherroub, Rabah; Bouron, Alexandre

    2015-12-01

    Hyperforin is described as a natural antidepressant inhibiting the reuptake of neurotransmitters and also activating cation channels. However the blood-brain barrier limits the access to the brain of this biomolecule. To circumvent this problem it was envisaged to encapsulate hyperforin into biomimetic lipid nano-carriers like lipid nanocapsules (LNCs). When testing the safety of 25 nm LNCs it appeared that they strongly blocked hyperforin-activated Ca2+ channels of cultured cortical neurons. This inhibition was due to one of their main component: solutol HS15 (polyoxyethylene-660-12-hydroxy stearate), a non-ionic soluble surfactant. Solutol HS15 rapidly depresses in a concentration-dependent manner the entry of Ca2+ through hyperforin-activated channels without influencing store-operated channels. This effect is mimicked by Brij58 but not by PEG600, indicating that the lipid chain of Solutol HS15 is important in determining its effects on the channels. The inhibition of the Ca2+ fluxes depends on the cellular cholesterol content; it is stronger after depleting cholesterol with methyl-β-cyclodextrin and is nearly absent on cells cultured in a cholesterol-rich medium. When chronically applied for 24 h, Solutol HS15 slightly up-regulates the entry of Ca2+ through hyperforin-activated channels. Similar observations were made when testing 25 nm lipid nanocapsules containing the surfactant Solutol HS15. Altogether, this study shows that Solutol HS15 perturbs in a cholesterol-dependent manner the activity of some neuronal channels. This is the first demonstration that LNCs containing this surfactant can influence cellular calcium signaling in the brain, a finding that can have important clinical implications.

  10. Cell biology of neuronal endocytosis.

    PubMed

    Parton, R G; Dotti, C G

    1993-09-01

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

  11. Diva/BclB regulates differentiation by inhibiting NDPKB/Nm23H2-mediated neuronal differentiation in PC-12 cells

    PubMed Central

    2012-01-01

    Background Diva (death inducer binding to vBcl-2 and Apaf-1)/BclB is a Bcl-2 family member, which is known for its function in apoptosis. Diva/BclB has been shown to interact with NDPKB/Nm23H2, which is involved in cellular differentiation. Thus far, there has been no direct evidence of Diva/BclB having a role in differentiation. In the present study, we investigated the expression of Diva/BclB and NDPKB/Nm23H2 during differentiation in PC-12 cell line. Results Our results show that after differentiation, Diva/BclB expression was decreased and reciprocally, NDPKB/Nm23H2 expression was increased and it translocated into the nucleus. Overexpression of NDPKB/Nm23H2 promoted PC-12 neuronal differentiation by increasing neurite outgrowth and arresting cell cycle progression. There was a concurrent downregulation of Diva/Boo when NDPKB/Nm23H2 was overexpressed, which mirrors the effect of NGF on PC-12 cell differentiation. Overexpression of Diva/BclB did not change the expression level of NDPKB/Nm23H2, but inhibited its nuclear localization. Cells that overexpressed Diva/BclB presented a decreased percentage of differentiated cells and average neurite length was shortened. This was due to an increase in the formation of Diva/BclB and NDPKB/Nm23H2 complexes as well as Diva/BclB and β-tubulin complexes. Concomitantly, there was a decrease in formation of NDPKB/Nm23H2 and β-tubulin complexes. Overexpression of Diva/BclB also resulted in a higher percentage of S-phase cells. Conclusion Our results showed a novel role for Diva/BclB in neuronal differentiation. Its downregulation during neuronal differentiation may be necessary to allow NDPKB/Nm23H2 and β-tubulin interaction that promotes NDPKB/Nm23H2 mediated differentiation. PMID:23057762

  12. Estragole blocks neuronal excitability by direct inhibition of Na+ channels

    PubMed Central

    Silva-Alves, K.S.; Ferreira-da-Silva, F.W.; Peixoto-Neves, D.; Viana-Cardoso, K.V.; Moreira-Júnior, L.; Oquendo, M.B.; Oliveira-Abreu, K.; Albuquerque, A.A.C.; Coelho-de-Souza, A.N.; Leal-Cardoso, J.H.

    2013-01-01

    Estragole is a volatile terpenoid, which occurs naturally as a constituent of the essential oils of many plants. It has several pharmacological and biological activities. The objective of the present study was to investigate the mechanism of action of estragole on neuronal excitability. Intact and dissociated dorsal root ganglion neurons of rats were used to record action potential and Na+ currents with intracellular and patch-clamp techniques, respectively. Estragole blocked the generation of action potentials in cells with or without inflexions on their descendant (repolarization) phase (Ninf and N0 neurons, respectively) in a concentration-dependent manner. The resting potentials and input resistances of Ninf and N0 cells were not altered by estragole (2, 4, and 6 mM). Estragole also inhibited total Na+ current and tetrodotoxin-resistant Na+ current in a concentration-dependent manner (IC50 of 3.2 and 3.6 mM, respectively). Kinetic analysis of Na+ current in the presence of 4 mM estragole showed a statistically significant reduction of fast and slow inactivation time constants, indicating an acceleration of the inactivation process. These data demonstrate that estragole blocks neuronal excitability by direct inhibition of Na+ channel conductance activation. This action of estragole is likely to be relevant to the understanding of the mechanisms of several pharmacological effects of this substance. PMID:24345915

  13. Synaptic Inhibition in Avian Interaural Level Difference Sound Localizing Neurons

    PubMed Central

    2016-01-01

    Abstract Synaptic inhibition plays a fundamental role in the neural computation of the interaural level difference (ILD), an important cue for the localization of high-frequency sound. Here, we studied the inhibitory synaptic currents in the chicken posterior portion of the dorsal nucleus of the lateral lemniscus (LLDp), the first binaural level difference encoder of the avian auditory pathway. Using whole-cell recordings in brain slices, we provide the first evidence confirming a monosynaptic inhibition driven by direct electrical and chemical stimulation of the contralateral LLDp, establishing the reciprocal inhibitory connection between the two LLDps, a long-standing assumption in the field. This inhibition was largely mediated by GABAA receptors; however, functional glycine receptors were also identified. The reversal potential for the Cl− channels measured with gramicidin-perforated patch recordings was hyperpolarizing (−88 mV), corresponding to a low intracellular Cl− concentration (5.2 mm). Pharmacological manipulations of KCC2 (outwardly Cl− transporter) activity demonstrate that LLDp neurons can maintain a low intracellular Cl− concentration under a high Cl− load, allowing for the maintenance of hyperpolarizing inhibition. We further demonstrate that hyperpolarizing inhibition was more effective at regulating cellular excitability than depolarizing inhibition in LLDp neurons. PMID:28032116

  14. Sumatriptan Inhibits TRPV1 Channels in Trigeminal Neurons

    PubMed Central

    Evans, M. Steven; Cheng, Xiangying; Jeffry, Joseph A.; Disney, Kimberly E.; Premkumar, Louis S.

    2011-01-01

    Objective To understand a possible role for transient potential receptor vanilloid 1 (TRPV1) ion channels in sumatriptan relief of pain mediated by trigeminal nociceptors. Background TRPV1 channels are expressed in small nociceptive sensory neurons. In dorsal root ganglia (DRG), TRPV1-containing nociceptors mediate certain types of inflammatory pain. Neurogenic inflammation of cerebral dura and blood vessels in the trigeminal nociceptive system is thought to be important in migraine pain, but the ion channels important in transducing migraine pain are not known. Sumatriptan is an agent effective in treatment of migraine and cluster headache. We hypothesized that sumatriptan might modulate activity of TRPV1 channels found in the trigeminal nociceptive system. Methods We used immunohistochemistry to detect the presence of TRPV1 channel protein, whole cell recording in acutely dissociated trigeminal ganglia (TG) to detect functionality of TRPV1 channels, and whole cell recording in trigeminal nucleus caudalis (TNC) to detect effects on release of neurotransmitters from trigeminal neurons onto second order sensory neurons. Effects specifically on TG neurons that project to cerebral dura were assessed by labeling dural nociceptors with DiI. Results Immunohistochemistry demonstrated that TRPV1 channels are present in cerebral dura, trigeminal ganglion, and in the trigeminal nucleus caudalis. Capsaicin, a TRPV1 agonist, produced depolarization and repetitive action potential firing in current clamp recordings and large inward currents in voltage clamp recordings from acutely dissociated TG neurons, demonstrating that TRPV1 channels are functional in trigeminal neurons. Capsaicin increased spontaneous excitatory postsynaptic currents (sEPSCs) in neurons of layer II in TNC slices, showing that these channels have a physiological effect on central synaptic transmission. Sumatriptan (10 μM), a selective anti-migraine drug inhibited TRPV1-mediated inward currents in TG. and

  15. Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons

    PubMed Central

    Hu, Tao; Liu, Nana; Lv, Minhua; Ma, Longxian; Peng, Huizhen; Peng, Sicong

    2016-01-01

    BACKGROUND: Lidocaine, which blocks voltage-gated sodium channels, is widely used in surgical anesthesia and pain management. Recently, it has been proposed that the hyperpolarization-activated cyclic nucleotide (HCN) channel is one of the other novel targets of lidocaine. Substantia gelatinosa in the spinal dorsal horn, which plays key roles in modulating nociceptive information from primary afferents, comprises heterogeneous interneurons that can be electrophysiologically categorized by firing pattern. Our previous study demonstrated that a substantial proportion of substantia gelatinosa neurons reveal the presence of HCN current (Ih); however, the roles of lidocaine and HCN channel expression in different types of substantia gelatinosa neurons remain unclear. METHODS: By using the whole-cell patch-clamp technique, we investigated the effect of lidocaine on Ih in rat substantia gelatinosa neurons of acute dissociated spinal cord slices. RESULTS: We found that lidocaine rapidly decreased the peak Ih amplitude with an IC50 of 80 μM. The inhibition rate on Ih was not significantly different with a second application of lidocaine in the same neuron. Tetrodotoxin, a sodium channel blocker, did not affect lidocaine’s effect on Ih. In addition, lidocaine shifted the half-activation potential of Ih from −109.7 to −114.9 mV and slowed activation. Moreover, the reversal potential of Ih was shifted by −7.5 mV by lidocaine. In the current clamp, lidocaine decreased the resting membrane potential, increased membrane resistance, delayed rebound depolarization latency, and reduced the rebound spike frequency. We further found that approximately 58% of substantia gelatinosa neurons examined expressed Ih, in which most of them were tonically firing. CONCLUSIONS: Our studies demonstrate that lidocaine strongly inhibits Ih in a reversible and concentration-dependent manner in substantia gelatinosa neurons, independent of tetrodotoxin-sensitive sodium channels. Thus, our

  16. Preceding Inhibition Silences Layer 6 Neurons in Auditory Cortex

    PubMed Central

    Zhou, Yi; Liu, Bao-hua; Wu, Guangying K.; Kim, Young; Xiao, Zhongju; Tao, Huizhong W.; Zhang, Li I.

    2010-01-01

    Summary A canonical feedforward circuit is proposed to underlie sensory cortical responses with balanced excitation and inhibition in layer 4 (L4). However, in another input layer, L6, sensory responses and the underlying synaptic circuits remain largely unclear. Here, cell-attached recordings in rat primary auditory cortex revealed that for the majority of L6 excitatory neurons, tonal stimuli did not drive spike responses, but suppressed spontaneous firings. Whole-cell recordings further revealed that the silencing resulted from tone-evoked strong inhibition arriving earlier than excitation. This pattern of inputs can be attributed to a parallel feedforward circuit with both excitatory and inhibitory inputs disynaptically relayed. In contrast, in the other neurons directly driven by thalamic input, stimuli evoked excitation preceding relatively weak inhibition, resulting in robust spike responses. Thus, the dichotomy of L6 response properties arises from two distinct patterns of excitatory-inhibitory interplay. The parallel circuit module generating preceding inhibition may provide a gating mechanism for conditional corticothalamic feedback. PMID:20223205

  17. Defective neuronal migration and inhibition of bipolar to multipolar transition of migrating neural cells by Mesoderm-Specific Transcript, Mest, in the developing mouse neocortex.

    PubMed

    Ji, Liting; Bishayee, Kausik; Sadra, Ali; Choi, Seunghyuk; Choi, Wooyul; Moon, Sungho; Jho, Eek-Hoon; Huh, Sung-Oh

    2017-07-04

    Brain developmental disorders such as lissencephaly can result from faulty neuronal migration and differentiation during the formation of the mammalian neocortex. The cerebral cortex is a modular structure, where developmentally, newborn neurons are generated as a neuro-epithelial sheet and subsequently differentiate, migrate and organize into their final positions in the cerebral cortical plate via a process involving both tangential and radial migration. The specific role of Mest, an imprinted gene, in neuronal migration has not been previously studied. In this work, we reduced expression of Mest with in utero electroporation of neuronal progenitors in the developing embryonic mouse neocortex. Reduction of Mest levels by shRNA significantly reduced the number of neurons migrating to the cortical plate. Also, Mest-knockdown disrupted the transition of bipolar neurons into multipolar neurons migrating out of the sub-ventricular zone region. The migrating neurons also adopted a more tangential migration pattern upon knockdown of the Mest message, losing their potential to attach to radial glia cells, required for radial migration. The differentiation and migration properties of neurons via Wnt-Akt signaling were affected by Mest changes. In addition, miR-335, encoded in a Mest gene intron, was identified as being responsible for blocking the default tangential migration of the neurons. Our results suggest that Mest and its intron product, miR-335, play important roles in neuronal migration with Mest regulating the morphological transition of primary neurons required in the formation of the mammalian neocortex. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Anisomycin uses multiple mechanisms to stimulate mitogen-activated protein kinases and gene expression and to inhibit neuronal differentiation in PC12 phaeochromocytoma cells.

    PubMed

    Törocsik, B; Szeberényi, J

    2000-02-01

    Treatment of PC12 cells with nerve growth factor (NGF) stimulates extracellular signal-regulated kinases (ERKs), as well as stress-activated c-Jun N-terminal kinases (JNKs) and p38 kinase, and induces neuronal differentiation. While the pivotal role of ERKs in NGF-induced morphological differentiation is well established, the contribution of JNK- and p38-pathways is less clear. The role of the JNK- and p38-pathway in PC12 cells was analysed by using anisomycin, a protein synthesis inhibitor that activates JNKs and p38. Non-toxic concentrations of anisomycin were found to stimulate these enzyme activities as well as the expression of the early response genes c-jun, c-fos and zif268, and to inhibit NGF-induced neurite formation. These effects of anisomycin appear to be mediated by the generation of reactive oxygen species (ROS), which in turn act through both TrkA/Ras-dependent and -independent signalling pathways. In addition, cross-talk between the p38- and ERK-pathways appears to play a role in the action of anisomycin.

  19. All optical experimental design for neuron excitation, inhibition, and action potential detection

    NASA Astrophysics Data System (ADS)

    Walsh, Alex J.; Tolstykh, Gleb; Martens, Stacey; Sedelnikova, Anna; Ibey, Bennett L.; Beier, Hope T.

    2016-03-01

    Recently, infrared light has been shown to both stimulate and inhibit excitatory cells. However, studies of infrared light for excitatory cell inhibition have been constrained by the use of invasive and cumbersome electrodes for cell excitation and action potential recording. Here, we present an all optical experimental design for neuronal excitation, inhibition, and action potential detection. Primary rat neurons were transfected with plasmids containing the light sensitive ion channel CheRiff. CheRiff has a peak excitation around 450 nm, allowing excitation of transfected neurons with pulsed blue light. Additionally, primary neurons were transfected with QuasAr2, a fast and sensitive fluorescent voltage indicator. QuasAr2 is excited with yellow or red light and therefore does not spectrally overlap CheRiff, enabling imaging and action potential activation, simultaneously. Using an optic fiber, neurons were exposed to blue light sequentially to generate controlled action potentials. A second optic fiber delivered a single pulse of 1869nm light to the neuron causing inhibition of the evoked action potentials (by the blue light). When used in concert, these optical techniques enable electrode free neuron excitation, inhibition, and action potential recording, allowing research into neuronal behaviors with high spatial fidelity.

  20. Inhibition of adenylyl cyclase by neuronal P2Y receptors

    PubMed Central

    Unterberger, Ursula; Moskvina, Eugenia; Scholze, Thomas; Freissmuth, Michael; Boehm, Stefan

    2002-01-01

    P2Y receptors inhibiting adenylyl cyclase have been found in blood platelets, glioma cells, and endothelial cells. In platelets and glioma cells, these receptors were identified as P2Y12. Here, we have used PC12 cells to search for adenylyl cyclase inhibiting P2Y receptors in a neuronal cellular environment.ADP and ATP (0.1 – 100 μM) left basal cyclic AMP accumulation unaltered, but reduced cyclic AMP synthesis stimulated by activation of endogenous A2A or recombinant β2 receptors. Forskolin-dependent cyclic AMP production was reduced by ⩽1 μM and enhanced by 10 – 100 μM ADP; this latter effect was turned into an inhibition when A2A receptors were blocked.The nucleotide inhibition of cyclic AMP synthesis was not altered when P2X receptors were blocked, but abolished by pertussis toxin.The rank order of agonist potencies for the reduction of cyclic AMP was (IC50 values): 2-methylthio-ADP (0.12 nM)=2-methylthio-ATP (0.13 nM)>ADPβS (71 nM)>ATP (164 nM)=ADP (244 nM). The inhibition by ADP was not antagonized by suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid, or adenosine-3′-phosphate-5′-phosphate, but attenuated by reactive blue 2, ATPαS, and 2-methylthio-AMP.RT – PCR demonstrated the expression of P2Y2, P2Y4, P2Y6, and P2Y12, but not P2Y1, receptors in PC12 cells. In Northern blots, only P2Y2 and P2Y12 were detectable. Differentiation with NGF did not alter these hybridization signals and left the nucleotide inhibition of adenylyl cyclase unchanged.We conclude that P2Y12 receptors are expressed in neuronal cells and inhibit adenylyl cyclase activity. PMID:11834615

  1. UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons.

    PubMed

    Edwards, Stacey L; Morrison, Logan M; Yorks, Rosalina M; Hoover, Christopher M; Boominathan, Soorajnath; Miller, Kenneth G

    2015-09-01

    The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(-) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(-) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(-) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function. Copyright © 2015 by the Genetics Society of America.

  2. Reflex inhibition of cutaneous and muscle vasoconstrictor neurons during stimulation of cutaneous and muscle nociceptors.

    PubMed

    Kirillova-Woytke, Irina; Baron, Ralf; Jänig, Wilfrid

    2014-05-01

    Cutaneous (CVC) and muscle (MVC) vasoconstrictor neurons exhibit typical reflex patterns to physiological stimulation of somatic and visceral afferent neurons. Here we tested the hypothesis that CVC neurons are inhibited by stimulation of cutaneous nociceptors but not of muscle nociceptors and that MVC neurons are inhibited by stimulation of muscle nociceptors but not of cutaneous nociceptors. Activity in the vasoconstrictor neurons was recorded from postganglionic axons isolated from the sural nerve or the lateral gastrocnemius-soleus nerve in anesthetized rats. The nociceptive afferents were excited by mechanical stimulation of the toes of the ipsilateral hindpaw (skin), by hypertonic saline injected into the ipsi- or contralateral gastrocnemius-soleus muscle, or by heat or noxious cold stimuli applied to the axons in the common peroneal nerve or tibial nerve. The results show that CVC neurons are inhibited by noxious stimulation of skin but not by noxious stimulation of skeletal muscle and that MVC neurons are inhibited by noxious stimulation of skeletal muscle but not by noxious stimulation of skin. These inhibitory reflexes are mostly lateralized and are most likely organized in the spinal cord. Stimulation of nociceptive cold-sensitive afferents does not elicit inhibitory or excitatory reflexes in CVC or MVC neurons. The reflex inhibition of activity in CVC or MVC neurons generated by stimulation of nociceptive cutaneous or muscle afferents during tissue injury leads to local increase of blood flow, resulting in an increase of transport of immunocompetent cells, proteins, and oxygen to the site of injury and enhancing the processes of healing.

  3. (±)3,4-methylenedioxyamphetamine inhibits the TEA-sensitive K⁺ current in the hippocampal neuron and the Kv2.1 current expressed in H1355 cells.

    PubMed

    Lin, Chia-Hsien; Yang, Chin-Tsang; Tsai, Ming-Cheng; Wu, Ya-Ting; MacDonald, Iona; Wang, Mei-Ling; Wu, Chien-Hua; Leung, Yuk-Man; Chen, Yi-Hung

    2015-02-01

    The whole-cell patch clamp method was used to study the effects of (±)3,4-methylenedioxyamphetamine (MDA) in hippocampal CA1 neurons from neonatal rats and in lung epithelial H1355 cells expressing Kv2.1. Extracellular application of MDA (30 μM) induced bursts of action potentials in hippocampal CA1 neurons exhibiting single spike action potentials without a bursting firing pattern, and promoted action potential bursts in hippocampal CA1 neurons exhibiting bursting firing of action potentials. Whereas MDA (30 and 100 μM) markedly decreased the delayed outward current in hippocampal CA1 neurons, MDA (100 μM) only slightly decreased the fast-inactivating K(+) current (I(A)) current. Furthermore, MDA (100 μM) substantially decreased the delayed outward current in the presence of 4-aminopyridine (4-AP; 3 mM), but did not significantly decrease the delayed outward current in the presence of tetraethylammonium (TEA; 30 mM). MDA (100 μM) also inhibited the current in H1355 cells expressing Kv2.1. Our results have shown that MDA inhibits the TEA-sensitive K(+) current in the hippocampus and the Kv2.1 current expressed in H1355 cells. These effects may contribute to the pharmacological and toxicological effects of MDA.

  4. Neurovascular coupling protects neurons against hypoxic injury via inhibition of potassium currents by generation of nitric oxide in direct neuron and endothelium cocultures.

    PubMed

    Wu, Kun-Wei; Kou, Zeng-Wei; Mo, Jia-Lin; Deng, Xu-Xu; Sun, Feng-Yan

    2016-10-15

    This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (IA) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (IK) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced IA inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the IA. These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal IA by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury.

  5. Neuronal cell cycle: the neuron itself and its circumstances.

    PubMed

    Frade, José M; Ovejero-Benito, María C

    2015-01-01

    Neurons are usually regarded as postmitotic cells that undergo apoptosis in response to cell cycle reactivation. Nevertheless, recent evidence indicates the existence of a defined developmental program that induces DNA replication in specific populations of neurons, which remain in a tetraploid state for the rest of their adult life. Similarly, de novo neuronal tetraploidization has also been described in the adult brain as an early hallmark of neurodegeneration. The aim of this review is to integrate these recent developments in the context of cell cycle regulation and apoptotic cell death in neurons. We conclude that a variety of mechanisms exists in neuronal cells for G1/S and G2/M checkpoint regulation. These mechanisms, which are connected with the apoptotic machinery, can be modulated by environmental signals and the neuronal phenotype itself, thus resulting in a variety of outcomes ranging from cell death at the G1/S checkpoint to full proliferation of differentiated neurons.

  6. Neuroprotector effect of stem cells from human exfoliated deciduous teeth transplanted after traumatic spinal cord injury involves inhibition of early neuronal apoptosis.

    PubMed

    Nicola, Fabrício do Couto; Marques, Marília Rossato; Odorcyk, Felipe; Arcego, Danusa Mar; Petenuzzo, Letícia; Aristimunha, Dirceu; Vizuete, Adriana; Sanches, Eduardo Farias; Pereira, Daniela Pavulack; Maurmann, Natasha; Dalmaz, Carla; Pranke, Patricia; Netto, Carlos A

    2017-05-15

    Stem cells from human exfoliated deciduous teeth (SHED) transplants have been investigated as a possible treatment strategy for spinal cord injuries (SCI) due to their potential for promoting functional recovery. The aim of present study was to investigate the effects of SHED on neuronal death after an experimental model of SCI. Wistar rats were spinalized using NYU impactor®. Animals were randomly distributed into 4 groups: Control (Naive) or Surgical control, Sham (laminectomy with no SCI); SCI (laminectomy followed by SCI, treated with vehicle); SHED (SCI treated with intraspinal transplantation of 3×10(5) SHED, 1h after SCI). Functional evaluations and morphological analysis were performed to confirm the spinal injury and the benefit of SHED transplantation on behavior, tissue protection and motor neuron survival. Flow cytometry of neurons, astrocytes, macrophages/microglia and T cells of spinal cord tissue were run at six, twenty-four, forty-eight and seventy-two hours after lesion. Six hours after SCI, ELISA and Western Blot were run to assess pro- and anti-apoptotic factors. The SHED group showed a significant functional improvement in comparison to the SCI animals, as from the first week until the end of the experiment. This behavioral protection was associated with less tissue impairment and greater motor neuron preservation. SHED reduced neuronal loss over time, as well as the overexpression of pro-apoptotic factor TNF-α, while maintained basal levels of the anti-apoptotic BCL-XL six hours after lesion. Data here presented show that SHED transplantation one hour after SCI interferes with the balance between pro- and anti-apoptotic factors and reduces early neuronal apoptosis, what contributes to tissue and motor neuron preservation and hind limbs functional recovery. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. L-carnosine inhibits neuronal cell apoptosis through signal transducer and activator of transcription 3 signaling pathway after acute focal cerebral ischemia.

    PubMed

    Wang, Jian-Ping; Yang, Zhi-Tang; Liu, Cong; He, Yuan-Hong; Zhao, Shan-Shan

    2013-04-24

    Considerable studies have showed that L-carnosine provides anti-oxidative and anti-apoptotic roles in the animal models of global or focal cerebral ischemia. However, the anti-apoptotic mechanisms of L-carnosine in the focal cerebral ischemia model have yet to be elucidated. To investigate the molecular mechanisms, rat models of permanent middle cerebral artery occlusion (pMCAO) and sham operation were first established and then pMCAO and sham-operated rats were treated with L-carnosine or vehicle alone. After this treatment, neurological deficits were evaluated at 12, 24 and 72 h after operation and the infarct volume was measured at 72 h after treatment. In addition, we also detected the mRNA expression of signal transducer and activator of transcription 3 (STAT3) and Pim-1 and the protein expression of phosphorylated STAT3, Pim-1, bcl-2 and cleaved caspase-3 at 12, 24 and 72 h post-pMCAO. Our results showed that the L-carnosine-treated rats had milder neurological deficits and smaller infarct volume and showed up-regulated expression in mRNA levels of STAT3 and Pim-1 than vehicle-treated rats at 72 h after treatment. Meanwhile, compared with vehicle-treated rats, the L-carnosine-treated rats exhibited higher protein expressions of pSTAT3, Pim-1 and bcl-2 but lower expression of cleaved caspase-3 protein at 72 h following operation. These results indicate that L-carnosine plays an important role in inhibiting neuronal cell apoptosis through STAT3 signaling pathway after acute cerebral ischemia.

  8. (-)-Deprenyl reduces neuronal apoptosis and facilitates neuronal outgrowth by altering protein synthesis without inhibiting monoamine oxidase.

    PubMed

    Tatton, W G; Wadia, J S; Ju, W Y; Chalmers-Redman, R M; Tatton, N A

    1996-01-01

    (-)-Deprenyl stereospecifically reduces neuronal death even after neurons have sustained seemingly lethal damage at concentrations too small to cause monoamine oxidase-B (MAO-B) inhibition. (-)-Deprenyl can also influence the process growth of some glial and neuronal populations and can reduce the concentrations of oxidative radicals in damaged cells at concentrations too small to inhibit MAO. In accord with the earlier work of others, we showed that (-)-deprenyl alters the expression of a number mRNAs or proteins in nerve and glial cells and that the alterations in gene expression/protein synthesis are the result of a selective action on transcription. The alterations in gene expression/protein synthesis are accompanied by a decrease in DNA fragmentation characteristic of apoptosis and the death of responsive cells. The onco-proteins Bcl-2 and Bax and the scavenger proteins Cu/Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) are among the 40-50 proteins whose synthesis is altered by (-)-deprenyl. Since mitochondrial ATP production depends on mitochondrial membrane potential (MMP) and mitochondrial failure has been shown to be one of the earliest events in apoptosis, we used confocal laser imaging techniques in living cells to show that the transcriptional changes induced by (-)-deprenyl are accompanied by a maintenance of mitochondrial membrane potential, a decrease in intramitochondrial calcium and a decrease in cytoplasmic oxidative radical levels. We therefore propose that (-)-deprenyl acts on gene expression to maintain mitochondrial function and to decrease cytoplasmic oxidative radical levels and thereby to reduce apoptosis. An understanding of the molecular steps by which (-)-deprenyl selectively alters transcription may contribute to the development of new therapies for neurodegenerative diseases.

  9. Human prion protein-induced autophagy flux governs neuron cell damage in primary neuron cells.

    PubMed

    Moon, Ji-Hong; Lee, Ju-Hee; Nazim, Uddin Md; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-05-24

    An unusual molecular structure of the prion protein, PrPsc is found only in mammals with transmissible prion diseases. Prion protein stands for either the infectious pathogen itself or a main component of it. Recent studies suggest that autophagy is one of the major functions that keep cells alive and has a protective effect against the neurodegeneration. In this study, we investigated that the effect of human prion protein on autophagy-lysosomal system of primary neuronal cells. The treatment of human prion protein induced primary neuron cell death and decreased both LC3-II and p62 protein amount indicating autophagy flux activation. Electron microscope pictures confirmed the autophagic flux activation in neuron cells treated with prion protein. Inhibition of autophagy flux using pharmacological and genetic tools prevented neuron cell death induced by human prion protein. Autophagy flux induced by prion protein is more activated in prpc expressing cells than in prpc silencing cells. These data demonstrated that prion protein-induced autophagy flux is involved in neuron cell death in prion disease and suggest that autophagy flux might play a critical role in neurodegenerative diseases including prion disease.

  10. Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens

    PubMed Central

    Zhang, YueMei; Bhavnani, Bhagu R

    2006-01-01

    Background Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22. Results Glutamate-induced apoptotic cell death in neuronal cells was associated with characteristic DNA fragmentation, morphological changes, activation of calpain and caspase-3 as well as the upregulation and/or translocation of AIF from mitochondria into cytosol and nuclei. Our results reveal that primary cortical cells and HT22 cells display different patterns of regulation of these genes/proteins. In primary cortical cells, glutamate induces activation of calpain, caspase-3 and translocation of AIF from mitochondria to cytosol and nuclei. In contrast, in HT22 cells, only the activation of calpain and upregulation and translocation of AIF occurred. In both cell types, these processes were inhibited/reversed by 17β-estradiol and Δ8,17β-estradiol with the latter being more potent. Conclusion Depending upon the neuronal cell type, at least two mechanisms are involved in glutamate-induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpain and AIF. Since HT22 cells lack caspase-3, glutamate-induced apoptosis is mediated via the caspase-independent pathway in this cell line. Kinetics of this apoptotic pathway further indicate that calpain rather than caspase-3, plays a critical role in the glutamate-induced apoptosis. Our studies further indicate that glutamate- induced changes

  11. Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation.

    PubMed

    Liu, Jie; Huang, Dongping; Xu, Jing; Tong, Jiabin; Wang, Zishan; Huang, Li; Yang, Yufang; Bai, Xiaochen; Wang, Pan; Suo, Haiyun; Ma, Yuanyuan; Yu, Mei; Fei, Jian; Huang, Fang

    2015-10-26

    Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson's disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD.

  12. Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation

    PubMed Central

    Liu, Jie; Huang, Dongping; Xu, Jing; Tong, Jiabin; Wang, Zishan; Huang, Li; Yang, Yufang; Bai, Xiaochen; Wang, Pan; Suo, Haiyun; Ma, Yuanyuan; Yu, Mei; Fei, Jian; Huang, Fang

    2015-01-01

    Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson’s disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD. PMID:26499517

  13. Salidroside protects cortical neurons against glutamate-induced cytotoxicity by inhibiting autophagy.

    PubMed

    Yin, Wei-Yong; Ye, Qiang; Huang, Huan-Jie; Xia, Nian-Ge; Chen, Yan-Yan; Zhang, Yi; Qu, Qiu-Min

    2016-08-01

    Recent evidence suggests that glutamate-induced cytotoxicity contributes to autophagic neuron death and is partially mediated by increased oxidative stress. Salidroside has been demonstrated to have neuroprotective effects in glutamate-induced neuronal damage. The precise mechanism of its regulatory role in neuronal autophagy is, however, poorly understood. This study aimed to probe the effects and mechanisms of salidroside in glutamate-induced autophagy activation in cultured rat cortical neurons. Cell viability assay, Western blotting, coimmunoprecipitation, and small interfering RNA were performed to analyze autophagy activities during glutamate-evoked oxidative injury. We found that salidroside protected neonatal neurons from glutamate-induced apoptotic cell death. Salidroside significantly attenuated the LC3-II/LC3-I ratio and expression of Beclin-1, but increased (SQSTM1)/p62 expression under glutamate exposure. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, decreased LC3-II/LC3-I ratio, attenuated glutamate-induced cell injury, and mimicked some of the protective effects of salidroside against glutamate-induced cell injury. Molecular analysis demonstrated that salidroside inhibited cortical neuron autophagy in response to glutamate exposure through p53 signaling by increasing the accumulation of cytoplasmic p53. Salidroside inhibited the glutamate-induced dissociation of the Bcl-2-Beclin-1 complex with minor affects on the PI3K/Akt/mTOR signaling pathways. These data demonstrate that the inhibition of autophagy could be responsible for the neuroprotective effects of salidroside on glutamate-induced neuronal injury.

  14. Post-synaptic inhibition of bulbar inspiratory neurones in the cat.

    PubMed Central

    Ballantyne, D; Richter, D W

    1984-01-01

    Stable intracellular recordings from thirty-six bulbar inspiratory neurones revealed three centrally originating, rhythmic patterns of synaptic inhibition (i.p.s.p.s). (i) A declining pattern of i.p.s.p.s accompanying the early stages of inspiration (early inspiratory inhibition) was identified in a total of twenty neurones representing examples of each of the functional classes of bulbar neurones examined, i.e. six R alpha- and two R beta-neurones of the dorsal respiratory group and twelve R alpha-neurones of the ventral respiratory group. (ii) A transient pattern of i.p.s.p.s just preceding or coinciding with the cessation of inspiration (late inspiratory inhibition) was present in the remaining sixteen neurones which were tested, representing six R alpha-neurones and three R beta-neurones of the dorsal respiratory group and seven R alpha-neurones of the ventral respiratory group. (iii) An augmenting pattern of expiratory i.p.s.p.s was present in all thirty-six neurones. Late inspiratory and expiratory i.p.s.p.s in the same neurones showed a similar time course of reversal when chloride was injected or allowed to diffuse into the cells and were associated with similar increases in input conductance. Both patterns of i.p.s.p.s appear to arise at or close to the cell soma. Early inspiratory i.p.s.p.s required a relatively longer period of chloride injection for reversal to be accomplished. Input conductance changes were either absent or smaller than those associated with late inspiratory or expiratory inhibition. These i.p.s.p.s appear to arise at more distal dendritic sites. These patterns of i.p.s.p.s are discussed in relation to the mechanisms shaping the growth of central inspiratory activity, bringing this activity to an end, and suppressing its redevelopment during expiration. PMID:6716297

  15. Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity.

    PubMed

    Bal-Price, A; Brown, G C

    2001-09-01

    Glia undergo inflammatory activation in most CNS pathologies and are capable of killing cocultured neurons. We investigated the mechanisms of this inflammatory neurodegeneration using a mixed culture of neurons, microglia, and astrocytes, either when the astrocytes were activated directly with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or LPS/IFN-gamma-activated microglia were added to mixed neuronal cultures. In either case, activated glia caused 75-100% necrotic cell death within 48 hr, which was completely prevented by inhibitors of inducible nitric oxide synthase (iNOS) (aminoguanidine or 1400W). Activated astrocytes or microglia produced nitric oxide (NO) (steady-state level approximately 0.5 microm), which immediately inhibited the cellular respiration of cocultured neurons, as did authentic NO. NO donors also decreased ATP levels and stimulated lactate production by neurons, consistent with NO-induced respiratory inhibition. NO donors or a specific respiratory inhibitor caused rapid (<1 min) release of glutamate from neuronal and neuronal-astrocytic cultures and subsequent neuronal death that was blocked by an antagonist of NMDA receptor (MK-801). MK-801 also blocked neuronal death induced by activated glia. High oxygen also prevented NO-induced neuronal death, consistent with death being induced by NO inhibition of cytochrome c oxidation in competition with oxygen. Thus activated glia kill neurons via NO from iNOS, which inhibits neuronal respiration resulting in glutamate release and subsequent excitotoxicity. This may contribute to neuronal cell death in inflammatory, infectious, ischemic, and neurodegenerative diseases.

  16. Nerve growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons

    PubMed Central

    1992-01-01

    Previous studies have shown that in neuronal cells the developmental phenomenon of programmed cell death is an active process, requiring synthesis of both RNA and protein. This presumably reflects a requirement for novel gene products to effect cell death. It is shown here that the death of nerve growth factor-deprived neuronal PC12 cells occurs at the same rate as that of rat sympathetic neurons and, like rat sympathetic neurons, involves new transcription and translation. In nerve growth factor-deprived neuronal PC12 cells, a decline in metabolic activity, assessed by uptake of [3H]2-deoxyglucose, precedes the decline in cell number, assessed by counts of trypan blue-excluding cells. Both declines are prevented by actinomycin D and anisomycin. In contrast, the death of nonneuronal (chromaffin-like) PC12 cells is not inhibited by transcription or translation inhibitors and thus does not require new protein synthesis. DNA fragmentation by internucleosomal cleavage does not appear to be a consistent or significant aspect of cell death in sympathetic neurons, neuronal PC12 cells, or nonneuronal PC12 cells, notwithstanding that the putative nuclease inhibitor aurintricarboxylic acid protects sympathetic neurons, as well as neuronal and nonneuronal PC12 cells, from death induced by trophic factor removal. Both phenotypic classes of PC12 cells respond to aurintricarboxylic acid with similar dose-response characteristics. Our results indicate that programmed cell death in neuronal PC12 cells, but not in nonneuronal PC12 cells, resembles programmed cell death in sympathetic neurons in significant mechanistic aspects: time course, role of new protein synthesis, and lack of a significant degree of DNA fragmentation. PMID:1469055

  17. Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat

    PubMed Central

    Choi, Won-Seok; Kruse, Shane E.; Palmiter, Richard D.; Xia, Zhengui

    2008-01-01

    Inhibition of mitochondrial complex I is one of the leading hypotheses for dopaminergic neuron death associated with Parkinson's disease (PD). To test this hypothesis genetically, we used a mouse strain lacking functional Ndufs4, a gene encoding a subunit required for complete assembly and function of complex I. Deletion of the Ndufs4 gene abolished complex I activity in midbrain mesencephalic neurons cultured from embryonic day (E) 14 mice, but did not affect the survival of dopaminergic neurons in culture. Although dopaminergic neurons were more sensitive than other neurons in these cultures to cell death induced by rotenone, MPP+, or paraquat treatments, the absence of complex I activity did not protect the dopaminergic neurons, as would be expected if these compounds act by inhibiting complex 1. In fact, the dopaminergic neurons were more sensitive to rotenone. These data suggest that dopaminergic neuron death induced by treatment with rotenone, MPP+, or paraquat is independent of complex I inhibition. PMID:18812510

  18. Inhibiting the inhibition: a neuronal network for sound localization in reverberant environments.

    PubMed

    Pecka, Michael; Zahn, Thomas P; Saunier-Rebori, Bernadette; Siveke, Ida; Felmy, Felix; Wiegrebe, Lutz; Klug, Achim; Pollak, George D; Grothe, Benedikt

    2007-02-14

    The precedence effect describes the phenomenon whereby echoes are spatially fused to the location of an initial sound by selectively suppressing the directional information of lagging sounds (echo suppression). Echo suppression is a prerequisite for faithful sound localization in natural environments but can break down depending on the behavioral context. To date, the neural mechanisms that suppress echo directional information without suppressing the perception of echoes themselves are not understood. We performed in vivo recordings in Mongolian gerbils of neurons of the dorsal nucleus of the lateral lemniscus (DNLL), a GABAergic brainstem nucleus that targets the auditory midbrain, and show that these DNLL neurons exhibit inhibition that persists tens of milliseconds beyond the stimulus offset, so-called persistent inhibition (PI). Using in vitro recordings, we demonstrate that PI stems from GABAergic projections from the opposite DNLL. Furthermore, these recordings show that PI is attributable to intrinsic features of this GABAergic innervation. Implementation of these physiological findings into a neuronal model of the auditory brainstem demonstrates that, on a circuit level, PI creates an enhancement of responsiveness to lagging sounds in auditory midbrain cells. Moreover, the model revealed that such response enhancement is a sufficient cue for an ideal observer to identify echoes and to exhibit echo suppression, which agrees closely with the percepts of human subjects.

  19. CB₂ cannabinoid receptors inhibit synaptic transmission when expressed in cultured autaptic neurons.

    PubMed

    Atwood, Brady K; Straiker, Alex; Mackie, Ken

    2012-09-01

    The role of CB₂ in the central nervous system, particularly in neurons, has generated much controversy. Fueling the controversy are imperfect tools, which have made conclusive identification of CB₂ expressing neurons problematic. Imprecise localization of CB₂ has made it difficult to determine its function in neurons. Here we avoid the localization controversy and directly address the question if CB₂ can modulate neurotransmission. CB₂ was expressed in excitatory hippocampal autaptic neurons obtained from CB₁ null mice. Whole-cell patch clamp recordings were made from these neurons to determine the effects of CB₂ on short-term synaptic plasticity. CB₂ expression restored depolarization induced suppression of excitation to these neurons, which was lost following genetic ablation of CB₁. The endocannabinoid 2-arachidonylglycerol (2-AG) mimicked the effects of depolarization in CB₂ expressing neurons. Interestingly, ongoing basal production of 2-AG resulted in constitutive activation of CB₂, causing a tonic inhibition of neurotransmission that was relieved by the CB₂ antagonist AM630 or the diacylglycerol lipase inhibitor RHC80267. Through immunocytochemistry and analysis of spontaneous EPSCs, paired pulse ratios and coefficients of variation we determined that CB₂ exerts its function at a presynaptic site of action, likely through inhibition of voltage gated calcium channels. Therefore CB₂ expressed in neurons effectively mimics the actions of CB₁. Thus neuronal CB₂ is well suited to integrate into conventional neuronal endocannabinoid signaling processes, with its specific role determined by its unique and highly inducible expression profile.

  20. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  1. Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neurons.

    PubMed

    Santiago, Ammy M; Clegg, Deborah J; Routh, Vanessa H

    2016-10-01

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

  2. Proteasome inhibition increases DNA and RNA oxidation in astrocyte and neuron cultures.

    PubMed

    Ding, Qunxing; Dimayuga, Edgardo; Markesbery, William R; Keller, Jeffrey N

    2004-12-01

    Increased levels of nucleic acid oxidation have been described as part of normal brain aging and have been demonstrated to occur in multiple neurological disorders. The basis for increased nucleic acid oxidation in each of these conditions is presently unknown. Proteasome inhibition occurs in a host of neurodegenerative conditions and likely contributes to increased levels of oxidative damage and neurotoxicity. In the present study we demonstrate for the first time the ability of proteasome inhibition to increase the level of nucleic acid oxidation in primary neuron and astrocyte cultures. Administration of proteasome inhibitors (MG262, MG115) at concentrations that do not induce neuron death in the first 24 h of treatment, dramatically increase the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8OHG) immunoreactivity in both cell types. Neurons underwent larger increases in nucleic acid oxidation compared to astrocyte cultures. While both DNA and RNA oxidation were observed following proteasome inhibition, RNA appeared to undergo a greater degree of oxidation than DNA. Both 18S and 28S ribosomal RNA were dramatically decreased following proteasome inhibition. Interestingly, an accumulation of unprocessed and/or cross-linked RNA species was observed following proteasome inhibition. Taken together, these data indicate the ability of proteasome inhibition to increase the levels of nucleic acid oxidation in both neurons and astrocytes, and suggest that proteasome inhibition may have deleterious effects on transcription and translation in both neurons and glia.

  3. Attenuation of Magnesium Sulfate on CoCl₂-Induced Cell Death by Activating ERK1/2/MAPK and Inhibiting HIF-1α via Mitochondrial Apoptotic Signaling Suppression in a Neuronal Cell Line.

    PubMed

    Huang, Chih-Yang; Hsieh, You-Liang; Ju, Da-Tong; Lin, Chien-Chung; Kuo, Chia-Hua; Liou, Yi-Fan; Ho, Tsung-Jung; Tsai, Chang-Hai; Tsai, Fuu-Jen; Lin, Jing-Ying

    2015-08-31

    Magnesium sulfate (MgSO₄) ameliorates hypoxia/ischemia-induced neuronal apoptosis in a rat model. This study aimed to investigate the mechanisms governing the anti-apoptotic effect of MgSO₄ on cobalt chloride (CoCl₂)-exposed NB41A3 mouse neuroblastoma cells. MgSO₄ increased the viability of NB41A3 cells treated with CoCl₂ in a dose-dependent manner. MgSO₄ treatment was shown to lead to an increase in the anti-apoptotic Bcl-2 family proteins, with a concomitant decrease in the pro-apoptotic proteins. MgSO₄ also attenuated the CoCl₂-induced disruption of mitochondrial membrane potential (ΔΨ(m)) and reduced the release of cytochrome c form the mitochondria to the cytosol. Furthermore, exposure to CoCl₂ caused activation of the hypoxia-inducible factor 1α (HIF-1α). On the other hand, MgSO₄ markedly reduced CoCl₂-induced HIF-1α activation and suppressed HIF-1α downstream protein BNIP3. MgSO₄ treatment induced ERK1/2 activation and attenuated CoCl₂-induced activation of p38 and JNK. Addition of the ERK1/2 inhibitor U0126 significantly reduced the ability of MgSO₄ to protect neurons from CoCl₂-induced mitochondrial apoptotic events. However, incubation of cultures with the p38 and JNK inhibitors did not significantly affect MgSO₄-mediated neuroprotection. MgSO₄ appears to suppress CoCl₂-induced NB41A3 cell death by activating ERK1/2/ MAPK pathways, which further modulates the role of Bcl-2 family proteins and mitochondria in NB41A3 cells. Our data suggest that MgSO₄ may act as a survival factor that preserves mitochondrial integrity and inhibits apoptotic pathways.

  4. Tumor suppressor p53 induces miR-15a processing to inhibit neuronal apoptosis inhibitory protein (NAIP) in the apoptotic response DNA damage in breast cancer cell

    PubMed Central

    Yang, Li; Zhao, Wei; Wei, Ping; Zuo, Wenshu; Zhu, Shouhui

    2017-01-01

    This study was aimed to investigate the functional role of miR-15a in breast cancer cells in response to DNA damage and to illustrate the possible potential underlying molecular mechanism(s). Human breast cancer cell lines MCF-7 cells and/or MDA-MB-231 cells were pre-treated with or without bleomycin. Cells were transfected with corresponding vectors. qRT-PCR was used to detect the expression of mRNA or miRNA, and immunoprecipitation and immunoblot analysis were performed to explore the status of protein association. Cell apoptosis was analyzed with flow cytometry. The results showed that neuronal apoptosis inhibitory protein (NAIP) was negatively regulated by p53 in MCF-7 cells, and NAIP expression was still high in bleomycin-treated MCF-7 cells. In addition, we observed that miR-15a expression was regulated by p53, and the effects of miR-15a on DNA damage was also mediated by p53. Furthermore, the results revealed that the cell apoptosis was mediated by miR-15a. Taken together, this study reveals that p53 negatively regulates NAIP expression by targeting miR-15a processing from primary into precursor miRNA in breast cancer. PMID:28337296

  5. Inhibition of Aminoacylase 3 Protects Rat Brain Cortex Neuronal Cells from the Toxicity of 4-Hydroxy-2-nonenal Mercapturate and 4-Hydroxy-2-nonenal

    PubMed Central

    Tsirulnikov, Kirill; Abuladze, Natalia; Bragin, Anatol; Faull, Kym; Cascio, Duilio; Damoiseaux, Robert; Schibler, Matthew J.; Pushkin, Alexander

    2017-01-01

    4-Hydroxy-2-nonenal (HNE) and acrolein are highly reactive neurotoxic products of lipid peroxidation that accumulate in Alzheimer’s and Parkinson’s disease brain and are implicated in the pathogenesis and progression of these neurological diseases. Conjugation with glutathione (GSH) initiates the HNE and acrolein detoxification pathway, which generates the mercapturates of HNE and acrolein that can be excreted. Prior work has shown that the efficiency of the GSH-dependent renal detoxification of haloalkene derived mercapturates is significantly decreased upon their deacetylation. Moreover the deacetylated products are rapidly transformed via β-lyases into toxic compounds responsible for the nephrotoxicity of the mercapturates. Because the enzymes of the GSH-conjugation pathway and β-lyases are expressed in brain, we hypothesized that a similar toxicity mechanism may be initiated in brain by the deacetylation of HNE and acrolein mercapturates. The present study was performed to identify an enzyme(s) involved in HNE and acrolein mercapturate deacetylation, characterize the brain expression of this enzyme and determine whether its inhibition decreases HNE and HNE mercapturate toxicity in neurons. We demonstrated that of two candidate enzymes known to deacetylate mercapturic acids, aminoacylases 1 (AA1) and 3 (AA3), only AA3 efficiently deacetylates both HNE and acrolein mercapturates. AA3 was further localized to neurons and blood vessels. Using a small molecule screen we further generated high-affinity AA3 inhibitors. Two of them completely protected rat brain cortex neurons expressing AA3 from the toxicity of HNE mercapturate. The results suggest that AA3 mediated deacetylation of HNE mercapturate may be involved in the neurotoxicity of HNE. PMID:22819785

  6. Aberrant Neuronal Differentiation and Inhibition of Dendrite Outgrowth Resulting from Endoplasmic Reticulum Stress

    PubMed Central

    Kawada, Koichi; Iekumo, Takaaki; Saito, Ryo; Kaneko, Masayuki; Mimori, Seisuke; Nomura, Yasuyuki; Okuma, Yasunobu

    2014-01-01

    Neural stem cells (NSCs) play an essential role in development of the central nervous system. Endoplasmic reticulum (ER) stress induces neuronal death. After neuronal death, neurogenesis is generally enhanced to repair the damaged regions. However, it is unclear whether ER stress directly affects neurogenesis-related processes such as neuronal differentiation and dendrite outgrowth. We evaluated whether neuronal differentiation and dendrite outgrowth were regulated by HRD1, a ubiquitin ligase that was induced under mild conditions of tunicamycin-induced ER stress. Neurons were differentiated from mouse embryonic carcinoma P19 cells by using retinoic acid. The differentiated cells were cultured for 8 days with or without tunicamycin and HRD1 knockdown. The ER stressor led to markedly increased levels of ER stress. ER stress increased the expression levels of neuronal marker βIII-tubulin in 8-day-differentiated cells. However, the neurites of dendrite marker microtubule-associated protein-2 (MAP-2)-positive cells appeared to retract in response to ER stress. Moreover, ER stress markedly reduced the dendrite length and MAP-2 expression levels, whereas it did not affect the number of surviving mature neurons. In contrast, HRD1 knockdown abolished the changes in expression of proteins such as βIII-tubulin and MAP-2. These results suggested that ER stress caused aberrant neuronal differentiation from NSCs followed by the inhibition of neurite outgrowth. These events may be mediated by increased HRD1 expression. PMID:24723324

  7. Adenosine Inhibits the Excitatory Synaptic Inputs to Basal Forebrain Cholinergic, GABAergic, and Parvalbumin Neurons in Mice

    PubMed Central

    Yang, Chun; Franciosi, Serena; Brown, Ritchie E.

    2013-01-01

    Coffee and tea contain the stimulants caffeine and theophylline. These compounds act as antagonists of adenosine receptors. Adenosine promotes sleep and its extracellular concentration rises in association with prolonged wakefulness, particularly in the basal forebrain (BF) region involved in activating the cerebral cortex. However, the effect of adenosine on identified BF neurons, especially non-cholinergic neurons, is incompletely understood. Here we used whole-cell patch-clamp recordings in mouse brain slices prepared from two validated transgenic mouse lines with fluorescent proteins expressed in GABAergic or parvalbumin (PV) neurons to determine the effect of adenosine. Whole-cell recordings were made from BF cholinergic neurons and from BF GABAergic and PV neurons with the size (>20 μm) and intrinsic membrane properties (prominent H-currents) corresponding to cortically projecting neurons. A brief (2 min) bath application of adenosine (100 μM) decreased the frequency but not the amplitude of spontaneous excitatory postsynaptic currents (EPSCs) in all groups of BF cholinergic, GABAergic, and PV neurons we recorded. In addition, adenosine decreased the frequency of miniature EPSCs in BF cholinergic neurons. Adenosine had no effect on the frequency of spontaneous inhibitory postsynaptic currents in cholinergic neurons or GABAergic neurons with large H-currents but reduced them in a group of GABAergic neurons with smaller H-currents. All effects of adenosine were blocked by a selective, adenosine A1 receptor antagonist, cyclopentyltheophylline (CPT, 1 μM). Adenosine had no postsynaptic effects. Taken together, our work suggests that adenosine promotes sleep by an A1 receptor-mediated inhibition of glutamatergic inputs to cortically projecting cholinergic and GABA/PV neurons. Conversely, caffeine and theophylline promote attentive wakefulness by inhibiting these A1 receptors in BF thereby promoting the high-frequency oscillations in the cortex required

  8. A Molecular Mechanism for Ibuprofen-Mediated RhoA Inhibition in Neurons

    PubMed Central

    Dill, John; Patel, Ankur R.; Yang, Xiao-Li; Bachoo, Robert; Powell, Craig M.; Li, Shuxin

    2014-01-01

    Ibuprofen is a nonsteroidal anti-inflammatory drug widely used to relieve pain and inflammation in many disorders via inhibition of cyclooxygenases. Recently, we have demonstrated that ibuprofen inhibits intracellular signaling of RhoA and promotes significant axonal growth and functional recovery following spinal cord lesions in rodents. In addition, another study suggests that ibuprofen reduces generation of amyloid-β42 peptide via inactivation of RhoA signaling, although it may also regulate amyloid-β42 formation by direct inhibition of the γ-secretase complex. The molecular mechanisms by which ibuprofen inhibits the RhoA signal in neurons, however, remain unclear. Here, we report that the transcription factor peroxisome proliferator-activated receptorγ (PPARγ) is essential for coupling ibuprofen to RhoA inhibition and subsequent neurite growth promotion in neurons. Ibuprofen activates PPARγ in neuron-like PC12 and B104 cells. Activation of PPARγ with traditional agonists mimics the RhoA-inhibiting properties of ibuprofen in PC12 cells and, like ibuprofen, promotes neurite elongation in primary cultured neurons exposed to axonal growth inhibitors. Protein knockdown with small interfering RNA specific for PPARγ blocks RhoA suppression of PPARγ agonists in PC12 cells. Moreover, the effect of ibuprofen on RhoA activity and neurite growth in neuronal cultures is prevented by selective PPARγ inhibition. These findings support that PPARγ plays an essential role in mediating the RhoA-inhibiting effect of ibuprofen. Elucidation of the novel molecular mechanisms linking ibuprofen to RhoA inhibition may provide additional therapeutic targets to the disorders characterized by RhoA activation, including spinal cord injuries and Alzheimer’s disease. PMID:20089905

  9. Calcineurin inhibition enhances motor neuron survival following injury

    PubMed Central

    Hui, Kelvin KW; Liadis, Nicole; Robertson, Jennifer; Kanungo, Anish; Henderson, Jeffrey T

    2010-01-01

    Abstract The immunosuppressive agents cyclosporin A (CsA) and FK-506 have previously been shown to exhibit neurotrophic and neuroprotective properties in vivo. Given that significant clinical expertise exists for both drugs, they represent an attractive starting point for treatment of acute neural injuries. One putative mechanism for neuroprotection by these drugs relates to inhibition of calcineurin activity. However each drug–immunophilin complex can potentially influence additional signal transduction pathways. Furthermore, several non-immunosuppressive immunophilin ligands have been described as possessing neuroprotective properties, suggesting that neuroprotection may be separable from calcineurin inhibition. In the present study, we examined the mechanism of this neuroprotection in facial motor neurons following axotomy-induced injury. Similar to previous studies in rats, CsA and FK-506 enhanced motor neuron survival in mice following acute injury. To examine the mechanism responsible for neuroprotection by these agents, pharmacologic inhibitors of several potential alternate signalling pathways (17-(allylamino)-17-demethoxygeldanamycin, rapamycin, cypermethrin) were evaluated with respect to neuroprotection. Of these, only cypermethrin, a direct calcineurin inhibitor not previously associated with neuronal survival properties, was observed to significantly enhance motor neuron survival following injury. The results demonstrate for the first time that direct inhibition of calcineurin is neuroprotective in vivo. These data support a model in which calcineurin inhibition promotes neuronal survival, distinct from effects upon neurite outgrowth. PMID:19243469

  10. Inhibition of acid-sensing ion channel currents by propofol in rat dorsal root ganglion neurons.

    PubMed

    Lei, Zhen; Li, Xiaoyu; Wang, Guizhi; Fei, Jianchun; Meng, Tao; Zhang, Xinyu; Yu, Jingya; Yu, Jingui; Li, Jingxin

    2014-04-01

    Acid-sensing ion channels (ASICs), part of the epithelial sodium channel/degenerin family, are activated by extracellular protons. The ASICs play a significant role in the acidosis-mediated perception of pain. The anaesthetic agent propofol also exerts antinociceptive effects, but the underlying mechanisms for this effect are not clear. We used whole-cell patch clamping to investigate the effect of propofol on proton-gated currents in: (i) rat dorsal root ganglion (DRG) neurons; and (ii) HEK293 cells transfected with either ASIC1a or ASIC3. Propofol inhibited the amplitude of proton-gated currents in DRG neurons, but did not change the sensitivity of ASICs to H(+). Notably, propofol altered acid-evoked excitability of rat DRG neurons and decreased the number of action potentials induced by acid stimuli. In addition, we demonstrated that propofol inhibited ASICs by directly binding with these channels in HEK293 cells. These results suggest that propofol inhibits proton-gated currents in DRG neurons and that inhibition of proton-gated currents explains, in part, the antinociceptive effects of propofol in primary afferent neurons.

  11. Curcumin inhibits apoptosis by regulating intracellular calcium release, reactive oxygen species and mitochondrial depolarization levels in SH-SY5Y neuronal cells.

    PubMed

    Uğuz, Abdülhadi Cihangir; Öz, Ahmi; Nazıroğlu, Mustafa

    2016-08-01

    Neurological diseases such as Alzheimer's and Parkinson's diseases are incurable progressive neurological disorders caused by the degeneration of neuronal cells and characterized by motor and non-motor symptoms. Curcumin, a turmeric product, is an anti-inflammatory agent and an effective reactive oxygen and nitrogen species scavenging molecule. Hydrogen peroxide (H2O2) is the main source of oxidative stress, which is claimed to be the major source of neurological disorders. Hence, in this study we aimed to investigate the effect of curcumin on Ca(2+) signaling, oxidative stress parameters, mitochondrial depolarization levels and caspase-3 and -9 activities that are induced by the H2O2 model of oxidative stress in SH-SY5Y neuronal cells. SH-SY5Y neuronal cells were divided into four groups namely, the control, curcumin, H2O2, and curcumin + H2O2 groups. The dose and duration of curcumin and H2O2 were determined from published data. The cells in the curcumin, H2O2, and curcumin + H2O2 groups were incubated for 24 h with 5 µM curcumin and 100 µM H2O2. Lipid peroxidation and cytosolic free Ca(2+) concentrations were higher in the H2O2 group than in the control group; however, their levels were lower in the curcumin and curcumin + H2O2 groups than in the H2O2 group alone. Reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) values were lower in the H2O2 group although they were higher in the curcumin and curcumin + H2O2 groups than in the H2O2 group. Caspase-3 activity was lower in the curcumin group than in the H2O2 group. In conclusion, curcumin strongly induced modulator effects on oxidative stress, intracellular Ca(2+) levels, and the caspase-3 and -9 values in an experimental oxidative stress model in SH-SY5Y cells.

  12. Intrinsically Active and Pacemaker Neurons in Pluripotent Stem Cell-Derived Neuronal Populations

    PubMed Central

    Illes, Sebastian; Jakab, Martin; Beyer, Felix; Gelfert, Renate; Couillard-Despres, Sébastien; Schnitzler, Alfons; Ritter, Markus; Aigner, Ludwig

    2014-01-01

    Summary Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks. PMID:24672755

  13. Inhibition of aminoacylase 3 protects rat brain cortex neuronal cells from the toxicity of 4-hydroxy-2-nonenal mercapturate and 4-hydroxy-2-nonenal

    SciTech Connect

    Tsirulnikov, Kirill; Abuladze, Natalia; Bragin, Anatol; Faull, Kym; Cascio, Duilio; Damoiseaux, Robert; Schibler, Matthew J.; Pushkin, Alexander

    2012-09-15

    4-Hydroxy-2-nonenal (4HNE) and acrolein (ACR) are highly reactive neurotoxic products of lipid peroxidation that are implicated in the pathogenesis and progression of Alzheimer's and Parkinson's diseases. Conjugation with glutathione (GSH) initiates the 4HNE and ACR detoxification pathway, which generates the mercapturates of 4HNE and ACR that can be excreted. Prior work has shown that the efficiency of the GSH-dependent renal detoxification of haloalkene derived mercapturates is significantly decreased upon their deacetylation because of rapid transformation of the deacetylated products into toxic compounds mediated by β-lyase. The enzymes of the GSH-conjugation pathway and β-lyases are expressed in the brain, and we hypothesized that a similar toxicity mechanism may be initiated in the brain by the deacetylation of 4HNE- and ACR-mercapturate. The present study was performed to identify an enzyme(s) involved in 4HNE- and ACR-mercapturate deacetylation, characterize the brain expression of this enzyme and determine whether its inhibition decreases 4HNE and 4HNE-mercapturate neurotoxicity. We demonstrated that of two candidate deacetylases, aminoacylases 1 (AA1) and 3 (AA3), only AA3 efficiently deacetylates both 4HNE- and ACR-mercapturate. AA3 was further localized to neurons and blood vessels. Using a small molecule screen we generated high-affinity AA3 inhibitors. Two of them completely protected rat brain cortex neurons expressing AA3 from the toxicity of 4HNE-mercapturate. 4HNE-cysteine (4HNE-Cys) was also neurotoxic and its toxicity was mostly prevented by a β-lyase inhibitor, aminooxyacetate. The results suggest that the AA3 mediated deacetylation of 4HNE-mercapturate may be involved in the neurotoxicity of 4HNE.

  14. Inhibition of aminoacylase 3 protects rat brain cortex neuronal cells from the toxicity of 4-hydroxy-2-nonenal mercapturate and 4-hydroxy-2-nonenal.

    PubMed

    Tsirulnikov, Kirill; Abuladze, Natalia; Bragin, Anatol; Faull, Kym; Cascio, Duilio; Damoiseaux, Robert; Schibler, Matthew J; Pushkin, Alexander

    2012-09-15

    4-Hydroxy-2-nonenal (4HNE) and acrolein (ACR) are highly reactive neurotoxic products of lipid peroxidation that are implicated in the pathogenesis and progression of Alzheimer's and Parkinson's diseases. Conjugation with glutathione (GSH) initiates the 4HNE and ACR detoxification pathway, which generates the mercapturates of 4HNE and ACR that can be excreted. Prior work has shown that the efficiency of the GSH-dependent renal detoxification of haloalkene derived mercapturates is significantly decreased upon their deacetylation because of rapid transformation of the deacetylated products into toxic compounds mediated by β-lyase. The enzymes of the GSH-conjugation pathway and β-lyases are expressed in the brain, and we hypothesized that a similar toxicity mechanism may be initiated in the brain by the deacetylation of 4HNE- and ACR-mercapturate. The present study was performed to identify an enzyme(s) involved in 4HNE- and ACR-mercapturate deacetylation, characterize the brain expression of this enzyme and determine whether its inhibition decreases 4HNE and 4HNE-mercapturate neurotoxicity. We demonstrated that of two candidate deacetylases, aminoacylases 1 (AA1) and 3 (AA3), only AA3 efficiently deacetylates both 4HNE- and ACR-mercapturate. AA3 was further localized to neurons and blood vessels. Using a small molecule screen we generated high-affinity AA3 inhibitors. Two of them completely protected rat brain cortex neurons expressing AA3 from the toxicity of 4HNE-mercapturate. 4HNE-cysteine (4HNE-Cys) was also neurotoxic and its toxicity was mostly prevented by a β-lyase inhibitor, aminooxyacetate. The results suggest that the AA3 mediated deacetylation of 4HNE-mercapturate may be involved in the neurotoxicity of 4HNE.

  15. Flavonoids, flavonoid metabolites, and phenolic acids inhibit oxidative stress in the neuronal cell line HT-22 monitored by ECIS and MTT assay: a comparative study.

    PubMed

    Kling, Beata; Bücherl, Daniel; Palatzky, Peter; Matysik, Frank-Michael; Decker, Michael; Wegener, Joachim; Heilmann, Jörg

    2014-03-28

    A real-time and label-free in vitro assay based on electric cell-substrate impedance sensing (ECIS) was established, validated, and compared to an end-point MTT assay within an experimental trial addressing the cytoprotective effects of 19 different flavonoids, flavonoid metabolites, and phenolic acids and their methyl esters on the HT-22 neuronal cell line, after induction of oxidative stress with tert-butyl hydroperoxide. Among the flavonoids under study, only those with a catechol unit and an additional 4-keto group provided cytoprotection. The presence of a 2,3-double bond was not a structural prerequisite for a neuroprotective effect. In the case of the phenolics, catechol substitution was the only structural requirement for activity. The flavonoids and other phenolics with a ferulic acid substitution or a single hydroxy group showed no activity. Electrochemical characterization of all compounds via square-wave voltammetry provided a rather specific correlation between cytoprotective activity and redox potential for the active flavonoids, but not for the active phenolics with a low molecular weight. Moreover this study was used to compare label-free ECIS recordings with results of the established MTT assay. Whereas the former provides time-resolved and thus entirely unbiased information on changes of cell morphology that are unequivocally associated with cell death, the latter requires predefined exposure times and a strict causality between metabolic activity and cell death. However, MTT assays are based on standard lab equipment and provide a more economic way to higher throughput.

  16. Calcium imaging in neuron cell death.

    PubMed

    Calvo, María; Villalobos, Carlos; Núñez, Lucía

    2015-01-01

    Intracellular Ca2+ is involved in control of a large variety of cell functions including apoptosis and neuron cell death. For example, intracellular Ca2+ overload is critical in neuron cell death induced by excitotoxicity. Thus, single cell monitoring of intracellular Ca2+ concentration ([Ca2+]cyt ) in neurons concurrently with apoptosis and neuron cell death is widely required. Procedures for culture and preparation of primary cultures of hippocampal rat neurons and fluorescence imaging of cytosolic Ca2+ concentration in Fura2/AM -loaded neurons are described. We also describe a method for apoptosis detection by immunofluorescence imaging. Finally, a simple method for concurrent measurements of [Ca2+]cyt and apoptosis in the same neurons is described.

  17. Modafinil inhibits rat midbrain dopaminergic neurons through D2-like receptors.

    PubMed

    Korotkova, T M; Klyuch, B P; Ponomarenko, A A; Lin, J S; Haas, H L; Sergeeva, O A

    2007-02-01

    Modafinil is a well-tolerated medication for excessive sleepiness, attention-deficit disorder, cocaine dependence and as an adjunct to antidepressants with low propensity for abuse. We investigated the modafinil action on identified dopaminergic and GABAergic neurons in the ventral tegmental area (VTA) and substantia nigra (SN) of rat brain slices. Modafinil (20 microM) inhibited the firing of dopaminergic, but not GABAergic neurons. This inhibition was maintained in the presence of tetrodotoxin and was accompanied by hyperpolarization. Sulpiride (10 microM), a D2-receptor antagonist, but not prazosine (20 microM, an alpha1-adrenoreceptor blocker) abolished the modafinil action. Inhibition of dopamine reuptake with a low dose of nomifensine (1 microM) reduced the firing of DA neurons in a sulpiride-dependent manner and blunted the effect of modafinil. On acutely isolated neurons, modafinil evoked D2-receptor-mediated outward currents in tyrosine-hydroxylase positive cells, identified by single-cell RT-PCR, which reversed polarity near the K(+) equilibrium potential and were unchanged in the presence of nomifensine. Thus modafinil directly inhibits DA neurons through D2 receptors.

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

    PubMed

    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.

  19. [Death of neurons and glial cells, induced by a photodynamic injury: signaling processes and neurone-glial interactions].

    PubMed

    Uzdenskiĭ, A B; Kolosov, M S; Lobanov, A V

    2007-01-01

    The mechanisms of photodynamic (PD) injury of neurons and glial cells are reviewed. Neuron responses: firing stimulation at high photosensitizer concentrations and inhibition at low concentrations (< 10(-7) M) that were followed by necrosis, are described. Glial cells died from both necrosis and apoptosis. Local laser inactivation of a neuron enhanced PD-induced apoptosis of glial cells, thus indicating that neuron maintained the survival of glia. Inter- and intracellular signaling mediated photodamage of these cells. Using inhibitors or activators of signaling proteins, the involvement of Ca(2+)-, adenylate cyclase- and tyrosine kinase-mediated signaling pathways in responses of neurons and glial cells to photosensitization was shown. Their pharmacological modulation can change selectivity of PD injury of neuronal and glial cells and efficiency of PD therapy.

  20. Molecular basis of vitamin E action. Tocotrienol potently inhibits glutamate-induced pp60(c-Src) kinase activation and death of HT4 neuronal cells.

    PubMed

    Sen, C K; Khanna, S; Roy, S; Packer, L

    2000-04-28

    HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than alpha-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of alpha-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60(c-Src) kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of alpha-tocotrienol, but not alpha-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. alpha-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4-10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.

  1. Dipeptidyl peptidase-IV inhibition prevents blood-retinal barrier breakdown, inflammation and neuronal cell death in the retina of type 1 diabetic rats.

    PubMed

    Gonçalves, Andreia; Marques, Catarina; Leal, Ermelindo; Ribeiro, Carlos F; Reis, Flávio; Ambrósio, António F; Fernandes, Rosa

    2014-09-01

    Diabetic retinopathy, a leading cause of vision loss in working-age population, is often associated with inflammation and apoptosis. We have previously reported that sitagliptin, a DPP-IV inhibitor, exerts beneficial effects in the retina of type 2 diabetic animals. The present study aimed to evaluate whether sitagliptin can exert protective effects in the retina of type 1 diabetic animals by a mechanism independent of insulin secretion and glycemia normalization. Streptozotocin-induced diabetic rats were treated orally with sitagliptin (5mg/kg/day) for the last two weeks of 4 weeks of diabetes. Sitagliptin treatment did not change the weight and glucose, HbA1c or insulin levels. However, it prevented the diabetes-induced increase in DPP-IV/CD26 activity and levels in serum and retina. Sitagliptin also prevented the increase in blood-retinal barrier (BRB) permeability and inhibited the changes in immunoreactivity and endothelial subcellular distribution of occludin, claudin-5 and ZO-1 proteins induced by diabetes. Furthermore, sitagliptin decreased the retinal inflammatory state and neuronal apoptosis. Sitagliptin inhibited the BRB breakdown in a type 1 diabetic animal model, by a mechanism independent of normalization of glycemia, by preventing changes in tight junctions (TJs) organization. Sitagliptin also exerted protective effects against inflammation and pro-apoptotic state in the retina of diabetic rats. Altogether, these results suggest that sitagliptin might be envisaged to be used to prevent or delay some of the alterations associated with the development of diabetic retinopathy. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Astroglial U87 Cells Protect Neuronal SH-SY5Y Cells from Indirect Effect of Radiation by Reducing DNA Damage and Inhibiting Fas Mediated Apoptotic Pathway in Coculture System.

    PubMed

    Saeed, Yasmeen; Rehman, Abdul; Xie, Bingjie; Xu, Jin; Hong, Ma; Hong, Qing; Deng, Yulin

    2015-08-01

    Recent studies provide the evidence that indirect effects of radiation could lead to neuronal cells death but underlying mechanism is not completely understood. On the other hand astroglial cells are known to protect neuronal cells against stress conditions in vivo and invitro. Yet, the fate of neuronal cells and the neuroprotective effect of coculture system (with glial cells) in response to indirect radiation exposure remain rarely discussed. Here, we purpose that the indirect effect of radiation may induce DNA damage by cell cycle arrest and receptor mediated apoptotic cascade which lead to apoptotic death of neuronal SH-SY5Y cells. We also hypothesized that coculture (with glial U87) may relieved the neuronal SH-SY5Y cells from toxicity of indirect effects radiation by reducing DNA damage and expression of apoptotic proteins in vitro. In the present study irradiated cell conditioned medium (ICCM) was used as source of indirect effect of radiation. Neuronal SH-SY5Y cells were exposed to ICCM with and without coculture with (glial U87) in transwell coculture system respectively. Various endpoints such as, cell survival number assay, Annexin V/PI assay, cell cycle analysis by flow cytometer, mRNA level of Fas receptor by q RT-PCR, expression of key apoptotic proteins by western blot and estimation of neurotrophic factors by ELISA method were analyzed into neuronal SH-SY5Y cells with and without co culture after ICCM exposure respectively. We found that ICCM induced DNA damage in neuronal SH-SY5Y cells by significant increase in cell cycle arrest at S-phase (***P < 0.001) which was further supported by over expression of P53 protein (**P < 0.01). While coculture (with glial U87), significantly reduced the ICCM induced cell cycle arrest and expression of P53 ((###) P < 0.001) neuronal SH-SY5Y cells. Further investigation of the underlying apoptotic mechanism revealed that in coculture system; ICCM induced elevated level of FAS mRNA level was significantly reduced

  3. Exercise inhibits neuronal apoptosis and improves cerebral function following rat traumatic brain injury.

    PubMed

    Itoh, Tatsuki; Imano, Motohiro; Nishida, Shozo; Tsubaki, Masahiro; Hashimoto, Shigeo; Ito, Akihiko; Satou, Takao

    2011-09-01

    Exercise is reported to inhibit neuronal apoptotic cell death in the hippocampus and improve learning and memory. However, the effect of exercise on inhibition of neuronal apoptosis surrounding the area of damage after traumatic brain injury (TBI) and the improvement of cerebral dysfunction following TBI are unknown. Here, we investigate the effect of exercise on morphology and cerebral function following TBI in rats. Wistar rats received TBI by a pneumatic controlled injury device were randomly divided into two groups: (1) non-exercise group and (2) exercise group. The exercise group ran on a treadmill for 30 min/day at 22 m/min for seven consecutive days. Immunohistochemical and behavioral studies were performed following TBI. The number of single-stranded DNA (ssDNA)-positive cells around the damaged area early after TBI was significantly reduced in the exercise group compared with the non-exercise group (P < 0.05). Furthermore, most ssDNA-positive cells in the non-exercise group co-localized with neuronal cells. However, in the exercise group, a few ssDNA-positive cells co-localized with neurons. In addition, there was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the exercise group compared with the non-exercise group (P < 0.05). These results indicate that exercise following TBI inhibits neuronal degeneration and apoptotic cell death around the damaged area, which results in improvement of cerebral dysfunction. In summary, treadmill running improved cerebral dysfunction following TBI, indicating its potential as an effective clinical therapy. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction.

  4. Apelin-13 Enhances Arcuate POMC Neuron Activity via Inhibiting M-Current

    PubMed Central

    Lee, Dong Kun; Jeong, Jae Hoon; Oh, Seunghoon; Jo, Young-Hwan

    2015-01-01

    The hypothalamus is a key element of the neural circuits that control energy homeostasis. Specific neuronal populations within the hypothalamus are sensitive to a variety of homeostatic indicators such as circulating nutrient levels and hormones that signal circulating glucose and body fat content. Central injection of apelin secreted by adipose tissues regulates feeding and glucose homeostasis. However, the precise neuronal populations and cellular mechanisms involved in these physiological processes remain unclear. Here we examine the electrophysiological impact of apelin-13 on proopiomelanocortin (POMC) neuron activity. Approximately half of POMC neurons examined respond to apelin-13. Apelin-13 causes a dose-dependent depolarization. This effect is abolished by the apelin (APJ) receptor antagonist. POMC neurons from animals pre-treated with pertussis toxin still respond to apelin, whereas the Gβγ signaling inhibitor gallein blocks apelin-mediated depolarization. In addition, the effect of apelin is inhibited by the phospholipase C and protein kinase inhibitors. Furthermore, single-cell qPCR analysis shows that POMC neurons express the APJ receptor, PLC-β isoforms, and KCNQ subunits (2, 3 and 5) which contribute to M-type current. Apelin-13 inhibits M-current that is blocked by the KCNQ channel inhibitor. Therefore, our present data indicate that apelin activates APJ receptors, and the resultant dissociation of the Gαq heterotrimer triggers a Gβγ-dependent activation of PLC-β signaling that inhibits M-current. PMID:25782002

  5. Neurons on Parafilm: versatile elastic substrates for neuronal cell cultures.

    PubMed

    Yoo, Sang Jin; Nam, Yoonkey

    2012-02-15

    A variety of materials has been applied to neuronal cell culture substrates to improve the efficiency of the culture and to provide pertinent cell growth environment. Here we report the application of Parafilm(®) M ('Parafilm') as a novel substrate for neuronal culture and patterning. Cell culture results show that elastic Parafilm had effects on cell viability, length and number of neurites, and soma spreading. Parafilm was also an effective substrate to obtain patterned neuronal cultures using a conventional micro-contract printing (μCP) technique. Polylysine micropatterns in line or grid forms were readily transferred from PDMS stamp to bare Parafilm surfaces and spatially confined neuronal cultures were successfully maintained for over three weeks. We also demonstrate that batch-processing cell culture substrates can be easily fabricated using a piece of Parafilm. The softness, plasticity, and hydrophobicity were main features that made it attractive for Parafilm to be considered as a practical cell culture platform. The results can be extended to develop an inexpensive and practical neuronal culture substrates in tissue engineering and biochip applications.

  6. GABA-mediated oxytocinergic inhibition in dorsal horn neurons by hypothalamic paraventricular nucleus stimulation.

    PubMed

    Rojas-Piloni, Gerardo; López-Hidalgo, Mónica; Martínez-Lorenzana, Guadalupe; Rodríguez-Jiménez, Javier; Condés-Lara, Miguel

    2007-03-16

    In anaesthetized rats, we tested whether the unit activity of dorsal horn neurons that receive nociceptive input is modulated by electrical stimulation of the hypothalamic paraventricular nucleus (PVN). An electrophysiological mapping of dorsal horn neurons at L3-L4 let us choose cells responding to a receptive field located in the toes region of the left hindpaw. Dorsal horn neurons were classified according to their response properties to peripheral stimulation. Wide Dynamic Range (WDR) cells responding to electrical stimulation of the peripheral receptive field and presenting synaptic input of Adelta, Abeta, and C-fibers were studied. Suspected interneurons that are typically silent and lack peripheral receptive field responses were also analyzed. PVN electrical stimulation inhibits Adelta (-55.0+/-10.2%), C-fiber (-73.1+/-6.7%), and post-discharge (-75.0+/-8.9%) peripheral activation in WDR cells, and silent interneurons were activated. So, this last type of interneuron was called a PVN-ON cell. In WDR cells, the inhibition of peripheral responses caused by PVN stimulation was blocked by intrathecal administration of a specific oxytocin antagonist or bicuculline. However, PVN-ON cell activation was blocked by the same specific oxytocin antagonist, but not by bicuculline. Our results suggest that PVN stimulation inhibits nociceptive peripheral-evoked responses in WDR neurons by a descending oxytocinergic pathway mediated by GABAergic PVN-ON cells. We discuss our observation that the PVN electrical stimulation selectively inhibits Adelta and C-fiber activity without affecting Abeta fibers. We conclude that Adelta and C-fibers receive a presynaptic inhibition mediated by GABA.

  7. miR-525-5p inhibits ADAMTS13 and is correlated with Ischemia/reperfusion injury-induced neuronal cell death.

    PubMed

    Zhao, Liyan; Hua, Cong; Li, Yunqian; Sun, Qingqing; Wu, Wei

    2015-01-01

    The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. In this study, miR-525-5p is clearly reduced in the ischemic brain after oxygen-glucose deprivation (OGD). Using TargetScan, MicroCosm Targets version 5, and microRNA.org databases, we identified miR-525-5p as a possible regulator of the ADAMTS13. We validated that ADAMTS13 is a target for miR-525-5p with a luciferase reporter activity assay. Moreover, adult rats subjected to focal cerebral ischemia exhibited a substantial reduction of miR-525-5p expression, which was inversely upregulated by ADAMTS13 expression. In vivo treatment with miR-525-5p agomir effectively decreased ADAMTS13 mRNA and protein levels in the ischemic region. Furthermore, knockdown of cerebral miR-525-5p reduced cell death and infarct size. In addition, the knockdown of ADAMTS13 by ADAMTS13 siRNA apparently abrogated the protective effect of miR-525-5p antagomir on OGD-induced cell death. Our data demonstrate that miR-525-5p is an endogenous regulator of ADAMTS13 that improves ischemia/reperfusion (I/R)-induced brain injury and dysfunction.

  8. CaMKII inhibition promotes neuronal apoptosis by transcriptionally upregulating Bim expression.

    PubMed

    Zhao, Yiwei; Zhu, Lin; Yu, Shaojun; Zhu, Jing; Wang, Chong

    2016-09-28

    The effects of Ca/calmodulin-dependent protein kinase II (CaMKII) on neuronal apoptosis are complex and contradictory, and the underlying mechanisms remain unclear. Bcl-2-interacting mediator of cell death (Bim) is an important proapoptotic protein under many physiological and pathophysiological conditions. However, there is no evidence that CaMKII and Bim are mechanistically linked in neuronal apoptosis. In this study, we showed that CaMKII inhibition by the inhibitors KN-62 and myristoylated autocamtide-2-related inhibitory peptide promoted apoptosis in cerebellar granule neurons in a dose-dependent manner. CaMKII inhibition increased Bim protein and messenger RNA levels. The expression of early growth response factor-1, a transcription factor of Bim, was also induced by CaMKII inhibitors. These data suggested that CaMKII repressed the transcriptional expression of Bim. Moreover, knockdown of Bim using small interfering RNAs attenuated the proapoptotic effects of CaMKII inhibition. Taken together, this is the first report to show that CaMKII inhibition transcriptionally upregulates Bim expression to promote neuronal apoptosis, providing new insights into the proapoptotic mechanism of CaMKII inhibition.

  9. Habenula-Induced Inhibition of Midbrain Dopamine Neurons Is Diminished by Lesions of the Rostromedial Tegmental Nucleus.

    PubMed

    Brown, P Leon; Palacorolla, Heather; Brady, Dana; Riegger, Katelyn; Elmer, Greg I; Shepard, Paul D

    2017-01-04

    Neurons in the lateral habenula (LHb) are transiently activated by aversive events and have been implicated in associative learning. Functional changes associated with tonic and phasic activation of the LHb are often attributed to a corresponding inhibition of midbrain dopamine (DA) neurons. Activation of GABAergic neurons in the rostromedial tegmental nucleus (RMTg), a region that receives dense projections from the LHb and projects strongly to midbrain monoaminergic nuclei, is believed to underlie the transient inhibition of DA neurons attributed to activation of the LHb. To test this premise, the effects of axon-sparing lesions of the RMTg were assessed on LHb-induced inhibition of midbrain DA cell firing in anesthetized rats. Quinolinic acid lesions decreased the number of NeuN-positive neurons in the RMTg significantly while largely sparing cells in neighboring regions. Lesions of the RMTg reduced both the number of DA neurons inhibited by, and the duration of inhibition resulting from, LHb stimulation. Although the firing rate was not altered, the regularity of DA cell firing was increased in RMTg-lesioned rats. Locomotor activity in an open field was also elevated. These results are the first to show that RMTg neurons contribute directly to LHb-induced inhibition of DA cell activity and support the widely held proposition that GABAergic neurons in the mesopontine tegmentum are an important component of a pathway that enables midbrain DA neurons to encode the negative valence associated with failed expectations and aversive stimuli. Phasic changes in the activity of midbrain dopamine cells motivate and guide future behavior. Activation of the lateral habenula by aversive events inhibits dopamine neurons transiently, providing a neurobiological representation of learning models that incorporate negative reward prediction errors. Anatomical evidence suggests that this inhibition occurs via the rostromedial tegmental nucleus, but this hypothesis has yet to be

  10. Autaptic self-inhibition of cortical GABAergic neurons: synaptic narcissism or useful introspection?

    PubMed

    Deleuze, Charlotte; Pazienti, Antonio; Bacci, Alberto

    2014-06-01

    Fast synaptic inhibition sculpts all forms of cortical activity by means of a specialized connectivity pattern between highly heterogeneous inhibitory interneurons and principal excitatory cells. Importantly, inhibitory neurons connect also to each other extensively, following a detailed blueprint, and, indeed, specific forms of disinhibition affect important behavioral functions. Here we discuss a peculiar form of cortical disinhibition: the massive autaptic self-inhibition of parvalbumin-(PV) positive basket cells. Despite being described long ago, autaptic inhibition onto PV basket cells is rarely included in cortical circuit diagrams, perhaps because of its still elusive function. We propose here a potential dual role of autaptic feedback inhibition in temporally coordinating PV basket cells during cortical network activity.

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

    PubMed Central

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

    2013-01-01

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

  12. Inhibition of mitochondrial calcium uptake 1 in Drosophila neurons.

    PubMed

    M'Angale, P G; Staveley, B E

    2017-02-08

    The mitochondrial calcium uptake 1 (MICU1) is a regulatory subunit of the mitochondrial calcium uniporter that plays an important role in calcium sensing. It contains two EF-hand domains that are well conserved across diverse species from protozoa to plants and metazoans. The loss of MICU1 function in mammals is attributed to several neurological disorders that involve movement dysfunction. The CG4495 gene in Drosophila melanogaster was identified as a putative homolog of MICU1 in the HomoloGene database of the National Centre for Biotechnology Information (NCBI). In agreement with previous studies that have shown the development of neurological disorders and movement defects in MICU1 loss-of-function organisms, we attempted to identify the function of CG4495/MICU1 in Drosophila neurons. We analyzed survival and locomotor ability of these flies and additionally performed biometric analysis of the Drosophila developing eye. The inducible RNA interference-mediated inhibition of CG4495/MICU1 in the Ddc-Gal4-expressing neurons of Drosophila presented with reduction in survival coupled with a precocious loss of locomotor ability. Since the pro-survival Bcl-2 family genes have been shown to be protective towards mitochondria, and CG4495/MICU1 has a mitochondrial targeting sequence, we attempted to rescue the phenotypes resulting from the inhibition of CG4495/MICU1 by overexpressing Buffy, the sole Bcl-2 homologue in Drosophila. The co-expression of CG4495/MICU1-RNAi along with Buffy resulted in the suppression of the phenotypes induced by the inhibition of CG4495/MICU1. Subsequently, the inhibition of CG4495/MICU1 in the Drosophila developing eye, a neuron-rich organ, resulted in reduced number of ommatidia and a highly fused ommatidial array. These developmental eye defects were rescued by the overexpression of Buffy. Our study suggests an important role for MICU1 in the normal function of neurons in Drosophila.

  13. Ca2+-induced uncoupling of Aplysia bag cell neurons.

    PubMed

    Dargaei, Zahra; Standage, Dominic; Groten, Christopher J; Blohm, Gunnar; Magoski, Neil S

    2015-02-01

    Electrical transmission is a dynamically regulated form of communication and key to synchronizing neuronal activity. The bag cell neurons of Aplysia are a group of electrically coupled neuroendocrine cells that initiate ovulation by secreting egg-laying hormone during a prolonged period of synchronous firing called the afterdischarge. Accompanying the afterdischarge is an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). We used whole cell recording from paired cultured bag cell neurons to demonstrate that electrical coupling is regulated by both Ca2+ and PKC. Elevating Ca2+ with a train of voltage steps, mimicking the onset of the afterdischarge, decreased junctional current for up to 30 min. Inhibition was most effective when Ca2+ entry occurred in both neurons. Depletion of Ca2+ from the mitochondria, but not the endoplasmic reticulum, also attenuated the electrical synapse. Buffering Ca2+ with high intracellular EGTA or inhibiting calmodulin kinase prevented uncoupling. Furthermore, activating PKC produced a small but clear decrease in junctional current, while triggering both Ca2+ influx and PKC inhibited the electrical synapse to a greater extent than Ca2+ alone. Finally, the amplitude and time course of the postsynaptic electrotonic response were attenuated after Ca2+ influx. A mathematical model of electrically connected neurons showed that excessive coupling reduced recruitment of the cells to fire, whereas less coupling led to spiking of essentially all neurons. Thus a decrease in electrical synapses could promote the afterdischarge by ensuring prompt recovery of electrotonic potentials or making the neurons more responsive to current spreading through the network.

  14. ROLES OF INHIBITION IN COMPLEX AUDITORY RESPONSES IN THE INFERIOR COLLICULUS: INHIBITED COMBINATION-SENSITIVE NEURONS

    PubMed Central

    Nataraj, Kiran; Wenstrup, Jeffrey J.

    2006-01-01

    We studied the functional properties and underlying neural mechanisms associated with inhibitory combination-sensitive neurons in the mustached bat’s inferior colliculus (IC). In these neurons, the excitatory response to best frequency tones was suppressed by lower frequency signals (usually in the range 12-30 kHz) in a time-dependant manner. Of 143 inhibitory units, the majority (71%) were type I, in which low frequency sounds evoked inhibition only. In the remainder, however, the low frequency inhibitory signal also evoked excitation. Of these, excitation preceded the inhibition in type E/I units (16%), while in type I/E units (13%) excitation followed the inhibition. Type E/I and I/E units were distinct in the tuning and threshold sensitivity of low frequency responses, while type I units overlapped the other types in these features. In 71 neurons, antagonists to receptors for glycine (strychnine, STRY) or γ-aminobutyric acid (GABA) (bicuculline, BIC) were applied micro-iontophoretically. These antagonists failed to eliminate combination-sensitive inhibition in 92% (STRY), 93% (BIC), and 87% (BIC+STRY) of the type I units tested. However, inhibition was reduced in many neurons. Results were similar for type E/I and I/E inhibitory neurons. The results indicate that there are distinct populations of combination-sensitive inhibited neurons in the IC, and that these populations are at least partly independent of glycine or GABAA receptors in the IC. We propose that these populations originate in different brainstem auditory nuclei, that they may be modified by interactions within the IC, and that they may perform different spectrotemporal analyses of vocal signals. PMID:16371455

  15. Yokukansan Inhibits Neuronal Death during ER Stress by Regulating the Unfolded Protein Response

    PubMed Central

    Miyata, Shingo; Kinoshita, Mitsuhiro; Kakehi, Kazuaki; Nishida, Shinji; Katayama, Taiichi; Tohyama, Masaya

    2010-01-01

    Background Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death. Methods We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein. Results Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs. Conclusions Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD. PMID:20967273

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

    PubMed

    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, P<0.001) and 41±24% (n=8, P<0.05), respectively. GABA or neurotensin receptor blockade prevented leptin's effect on glucose sensitivity. Fasting increased activation of LHA orexin-GI neurons by decreased glucose, as would be predicted by these hormonal effects. We also evaluated putative VTA-DA neurons in a novel horizontal slice preparation containing the LHA and VTA. Decreased glucose increased the frequency of spontaneous excitatory post-synaptic currents (sEPSCs; 125 ± 40%, n=9, 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

  17. The influences of somatic and dendritic inhibition on bursting patterns in a neuronal circuit model.

    PubMed

    Yang, Keun-Hang; Franaszczuk, Piotr J; Bergey, Gregory K

    2003-10-01

    The balance between inhibition and excitation plays a crucial role in the generation of synchronous bursting activity in neuronal circuits. In human and animal models of epilepsy, changes in both excitatory and inhibitory synaptic inputs are known to occur. Locations and distribution of these excitatory and inhibitory synaptic inputs on pyramidal cells play a role in the integrative properties of neuronal activity, e.g., epileptiform activity. Thus the location and distribution of the inputs onto pyramidal cells are important parameters that influence neuronal activity in epilepsy. However, the location and distribution of inhibitory synapses converging onto pyramidal cells have not been fully studied. The objectives of this study are to investigate the roles of the relative location of inhibitory synapses on the dendritic tree and soma in the generation of bursting activity. We investigate influences of somatic and dendritic inhibition on bursting activity patterns in several paradigms of potential connections using a simplified multicompartmental model. We also investigate the effects of distribution of fast and slow components of GABAergic inhibition in pyramidal cells. Interspike interval (ISI) analysis is used for examination of bursting patterns. Simulations show that the inhibitory interneuron regulates neuronal bursting activity. Bursting behavior patterns depend on the synaptic weight and delay of the inhibitory connection as well as the location of the synapse. When the inhibitory interneuron synapses on the pyramidal neuron, inhibitory action is stronger if the inhibitory synapse is close to the soma. Alterations of synaptic weight of the interneuron can be compensatory for changes in the location of synaptic input. The relative changes in these parameters exert a considerable influence on whether synchronous bursting activity is facilitated or reduced. Additional simulations show that the slow GABAergic inhibitory component is more effective than the

  18. Activation and inhibition of tph2 serotonergic neurons operate in tandem to influence larval zebrafish preference for light over darkness.

    PubMed

    Cheng, Ruey-Kuang; Krishnan, Seetha; Jesuthasan, Suresh

    2016-02-12

    Serotonergic neurons have been implicated in a broad range of processes, but the principles underlying their effects remain a puzzle. Here, we ask how these neurons influence the tendency of larval zebrafish to swim in the light and avoid regions of darkness. Pharmacological inhibition of serotonin synthesis reduces dark avoidance, indicating an involvement of this neuromodulator. Calcium imaging of tph2-expressing cells demonstrates that a rostral subset of dorsal raphe serotonergic neurons fire continuously while the animal is in darkness, but are inhibited in the light. Optogenetic manipulation of tph2 neurons by channelrhodopsin or halorhodopsin expression modifies preference, confirming a role for these neurons. In particular, these results suggest that fish prefer swimming in conditions that elicits lower activity in tph2 serotonergic neurons in the rostral raphe.

  19. Activation and inhibition of tph2 serotonergic neurons operate in tandem to influence larval zebrafish preference for light over darkness

    PubMed Central

    Cheng, Ruey-Kuang; Krishnan, Seetha; Jesuthasan, Suresh

    2016-01-01

    Serotonergic neurons have been implicated in a broad range of processes, but the principles underlying their effects remain a puzzle. Here, we ask how these neurons influence the tendency of larval zebrafish to swim in the light and avoid regions of darkness. Pharmacological inhibition of serotonin synthesis reduces dark avoidance, indicating an involvement of this neuromodulator. Calcium imaging of tph2-expressing cells demonstrates that a rostral subset of dorsal raphe serotonergic neurons fire continuously while the animal is in darkness, but are inhibited in the light. Optogenetic manipulation of tph2 neurons by channelrhodopsin or halorhodopsin expression modifies preference, confirming a role for these neurons. In particular, these results suggest that fish prefer swimming in conditions that elicits lower activity in tph2 serotonergic neurons in the rostral raphe. PMID:26868164

  20. [ERK activation effects on GABA secretion inhibition induced by SDF-1 in hippocampal neurons of rats].

    PubMed

    Zhang, Zi-juan; Guo, Mei-xia; Xing, Ying

    2015-09-01

    To investigate the effect of extracellular regulating kinase (ERK) signaling pathway on the secretion of gamma-aminobutyric acid (GABA) in cultured rat hippocampal neurons induced by stromal cell derived factor-1 (SDF-1). The hippocampal neurons of newborn SD rats were cultured and identified in vitro; the phosphorylation level of ERK1/2 was examined by Western blot; ELISA was used to detect the effect of PD98059, a ERK1/2 specific blocker on GABA secretion of cultured hippocampal neurons and Western blot were adopted to measure the protein expression levels of glutamate decarboxylase (GAD65/67) and gamma aminobutyric acid transporter (GAT); after blocking ERK1/2 signaling pathway with PD98059; RT-PCR was used to detect the mRNA expression levels of GAT-1 and GAD65 after treated with PD98059. The levels of ERKl/2 phosphorylation were increased significantly by SDF1 acting on hippocampal neurons, and CX-CR4 receptor blocker AMD3100, could inhibit SDF-1 induced ERK1/2 activation; SDF-1 could inhibit the secretion of GABA in cultured hippocampal neurons, and ERK1/2 specific inhibitor PD98059, could partly reverse the inhibition of GABA secretion by SDF-1. The effects of SDF-1 on cultured hippocampal neurons was to decrease the mRNA genesis of glutamic acid decarboxylase GAD65 and GABA transporter GAT-1, besides, ERK inhibitor PD98059 could effectively flip the effect of SDF-1. The results of Western blot showed that SDF-1 could inhibit the protein expression of GAT-1 and GAD65/67 in hippocampal neurons and the inhibition of GAT-1 and GAD65/67 protein expression could be partially restored by ERK1/2 blocker. SDF-1 acts on the CXCR4 of hippocampal neurons in vitro, and inhibits the expression of GAD by activating the ERK1/2 signaling pathway, and this may represent one possible pathway of GABA secretion inhibition.

  1. Hypoxia and hypercapnia inhibit hypothalamic orexin neurons in rats.

    PubMed

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

    2016-11-01

    Evidence of impaired function of orexin neurons has been found in individuals with cardiorespiratory disorders, such as obstructive sleep apnea (OSA) and sudden infant death syndrome (SIDS), but the mechanisms responsible are unknown. Individuals with OSA and SIDS experience repetitive breathing cessations and/or rebreathing of expired air, resulting in hypoxia/hypercapnia (H/H). In this study, we examined the responses of fluorescently identified rat orexin neurons in the lateral hypothalamus to acute H/H to test if and how these neurons alter their activity and function during this challenge. Experiments were conducted in an in vitro slice preparation using voltage-clamp and current-clamp configurations. H/H (10 min) induced hyperpolarization, accompanied by rapid depression, and finally, cessation of firing activity in orexin neurons. Hypoxia alone had similar but less potent effects. H/H did not alter the frequency of inhibitory glycinergic postsynaptic currents. The frequency of GABAergic currents was diminished but only at 8-10 min of H/H. In contrast, the frequency of excitatory glutamatergic postsynaptic events was diminished as early as 2-4 min of H/H. In the presence of glutamatergic receptor blockers, the inhibitory effects of H/H on the firing activity and membrane potential of orexin neurons persisted but to a lesser extent. In conclusion, both direct alteration of postsynaptic membrane properties and diminished glutamatergic neurotransmission likely contribute to the inhibition of orexin neurons by H/H. These mechanisms could be responsible for the decreased function of orexin in individuals at risk for OSA and SIDS. Copyright © 2016 the American Physiological Society.

  2. NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

    PubMed

    Mao, Xianrong; Phanavanh, Bounleut; Hamdan, Hamdan; Moerman-Herzog, Andréa M; Barger, Steven W

    2016-04-01

    The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions. No component of the NFκB pathway was permissive for endogenous NFκB activity, and none stimulated the activity of exogenous RelA. Surprisingly, however, the latter was inhibited by cotransfection of NFκB-inducing kinase (NIK). Fluorescence imaging of RelA indicated that co-expression of NIK sequestered RelA in the cytoplasm, similar to the effect of IκBα. NIK-knockout mice showed elevated expression of an NFκB-reporter construct in neurons in vivo. Cortical neurons cultured from NIK-knockout mice showed elevated expression of an NFκB-reporter transgene. Consistent with data from other cell types, a C-terminal fragment of NIK suppressed RelA activity in astrocytes as well as neurons. Therefore, the inhibitory ability of the NIK C-terminus was unbiased with regard to cell type. However, inhibition of NFκB by full-length NIK is a novel outcome that appears to be specific to CNS neurons. This has implications for unique aspects of transcription in the CNS, perhaps relevant to aspects of development, neuroplasticity, and neuroinflammation. Full-length NIK was found to inhibit (down arrow) transcriptional activation of NFκB in neurons, while it elevated (up arrow) activity in astrocytes. Deletion constructs corresponding to the N-terminus or C-terminus also inhibited NFκB in neurons, while only the C-terminus did so in astrocytes. One possible explanation is that the inhibition in neurons occurs via two different

  3. Inhibition of glycine receptor function of native neurons by aliphatic n-alcohols

    PubMed Central

    Tao, Liang; Ye, Jiang Hong

    2002-01-01

    The inhibitory effects of n-alcohols (methanol to dodecanol) on glycine-activated currents were studied in neurons freshly dissociated from the ventral tegmental area of neonatal rats using whole-cell patch-clamp recording technique.Ethanol enhanced and depressed glycine-activated currents in 35% and 45%, respectively, of neurons of ventral tegmental area of neonatal rats. In this report, we extended our focus of ethanol-induced inhibition of glycine currents to other straight-chain alcohols.Aliphatic n-alcohols, which have carbon numbers less than nine, suppressed glycine currents in 45% (71/158) of the neurons. All results from this study are obtained from the 45% of cells displaying inhibition; the other 55% of the neurons were not studied.Alcohol potency increased as the number of carbon atoms increased from one to five, and was at a maximal plateau from five to nine; alcohols with 10 or more carbons did not inhibit glycine-activated currents. Thus, a ‘cutoff' point in their potency for inhibition of glycine receptor function occurred at about decanol.A coapplication of dodecanol with ethanol eliminated the inhibition resulting from ethanol. Thus, dodecanol may bind to the receptor silently and compete with ethanol.These observations indicate that straight-chain n-alcohols exhibit a ‘cutoff' point in their potency for inhibition of the glycine receptor function between nine and 10 carbon atoms. The inability of longer alcohols to change the activation properties of the receptors may contribute to the cutoff effect. PMID:12055142

  4. Merkel cells and neurons keep in touch

    PubMed Central

    Woo, Seung-Hyun; Lumpkin, Ellen A.; Patapoutian, Ardem

    2014-01-01

    The Merkel cell-neurite complex is a unique vertebrate touch receptor comprising two distinct cell types in the skin. Its presence in touch-sensitive skin areas was recognized more than a century ago, but the functions of each cell type in sensory transduction have been unclear. Three recent studies demonstrate that Merkel cells are mechanosensitive cells that function in touch transduction via Piezo2. One study concludes that Merkel cells rather than sensory neurons are principal sites of mechanotransduction, whereas the other two studies report that both Merkel cells and neurons encode mechanical inputs. Together, these studies settle a longstanding debate on whether Merkel cells are mechanosensory cells, and enable future investigations of how these skin cells communicate with neurons. PMID:25480024

  5. Merkel cells and neurons keep in touch.

    PubMed

    Woo, Seung-Hyun; Lumpkin, Ellen A; Patapoutian, Ardem

    2015-02-01

    The Merkel cell-neurite complex is a unique vertebrate touch receptor comprising two distinct cell types in the skin. Its presence in touch-sensitive skin areas was recognized more than a century ago, but the functions of each cell type in sensory transduction have been unclear. Three recent studies demonstrate that Merkel cells are mechanosensitive cells that function in touch transduction via Piezo2. One study concludes that Merkel cells, rather than sensory neurons, are principal sites of mechanotransduction, whereas two other studies report that both Merkel cells and neurons encode mechanical inputs. Together, these studies settle a long-standing debate on whether or not Merkel cells are mechanosensory cells, and enable future investigations of how these skin cells communicate with neurons. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Aspirin prevention of NMDA-induced neuronal death by direct protein kinase Czeta inhibition.

    PubMed

    Crisanti, P; Leon, A; Lim, D M; Omri, B

    2005-06-01

    Abstract Aspirin has been shown to protect against glutamate neurotoxicity via the nuclear factor kappaB pathway. Some studies have implicated the atypical protein kinase C (PKC) zeta (zeta) isoform in cell protection, but the mechanism involved remains unclear. We show here that aspirin exerts at least some of its effects through PKCzeta, decreasing the NMDA-induced activation, cleavage and nuclear translocation of this molecule. Aspirin (acetylsalicylic acid) directly inhibited the protein kinase activity of PKCzeta, whereas salicylic acid did not. This direct effect of aspirin on purified human PKCzeta is consistent with PKCzeta inhibition preventing the NMDA-induced death of cortical neurones. Caspase-3 inhibition blocked the cleavage and nuclear translocation of PKCzeta, whereas caspase-1-inhibition did not. Thus, PKCzeta (protein kinase Mzeta) regulates nuclear events essential for the initiation of the apoptotic pathway. Aspirin protects cells against NMDA-induced apoptosis by means of a novel mechanism targeting PKCzeta, a key molecule in inflammatory responses and neurodegeneration.

  7. Methamphetamine inhibits the glucose uptake by human neurons and astrocytes: stabilization by acetyl-L-carnitine.

    PubMed

    Abdul Muneer, P M; Alikunju, Saleena; Szlachetka, Adam M; Haorah, James

    2011-04-27

    Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 µM increased the uptake while 200 µM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers.

  8. Neurons Are Host Cells for Mycobacterium tuberculosis

    PubMed Central

    Randall, Philippa J.; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M.; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston

    2014-01-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system. PMID:24566619

  9. Neurons are host cells for Mycobacterium tuberculosis.

    PubMed

    Randall, Philippa J; Hsu, Nai-Jen; Lang, Dirk; Cooper, Susan; Sebesho, Boipelo; Allie, Nasiema; Keeton, Roanne; Francisco, Ngiambudulu M; Salie, Sumayah; Labuschagné, Antoinette; Quesniaux, Valerie; Ryffel, Bernhard; Kellaway, Lauriston; Jacobs, Muazzam

    2014-05-01

    Mycobacterium tuberculosis infection of the central nervous system is thought to be initiated once the bacilli have breached the blood brain barrier and are phagocytosed, primarily by microglial cells. In this study, the interactions of M. tuberculosis with neurons in vitro and in vivo were investigated. The data obtained demonstrate that neurons can act as host cells for M. tuberculosis. M. tuberculosis bacilli were internalized by murine neuronal cultured cells in a time-dependent manner after exposure, with superior uptake by HT22 cells compared to Neuro-2a cells (17.7% versus 9.8%). Internalization of M. tuberculosis bacilli by human SK-N-SH cultured neurons suggested the clinical relevance of the findings. Moreover, primary murine hippocampus-derived neuronal cultures could similarly internalize M. tuberculosis. Internalized M. tuberculosis bacilli represented a productive infection with retention of bacterial viability and replicative potential, increasing 2- to 4-fold within 48 h. M. tuberculosis bacillus infection of neurons was confirmed in vivo in the brains of C57BL/6 mice after intracerebral challenge. This study, therefore, demonstrates neurons as potential new target cells for M. tuberculosis within the central nervous system.

  10. Interneuron-mediated inhibition synchronizes neuronal activity during slow oscillation

    PubMed Central

    Chen, Jen-Yung; Chauvette, Sylvain; Skorheim, Steven; Timofeev, Igor; Bazhenov, Maxim

    2012-01-01

    The signature of slow-wave sleep in the electroencephalogram (EEG) is large-amplitude fluctuation of the field potential, which reflects synchronous alternation of activity and silence across cortical neurons. While initiation of the active cortical states during sleep slow oscillation has been intensively studied, the biological mechanisms which drive the network transition from an active state to silence remain poorly understood. In the current study, using a combination of in vivo electrophysiology and thalamocortical network simulation, we explored the impact of intrinsic and synaptic inhibition on state transition during sleep slow oscillation. We found that in normal physiological conditions, synaptic inhibition controls the duration and the synchrony of active state termination. The decline of interneuron-mediated inhibition led to asynchronous downward transition across the cortical network and broke the regular slow oscillation pattern. Furthermore, in both in vivo experiment and computational modelling, we revealed that when the level of synaptic inhibition was reduced significantly, it led to a recovery of synchronized oscillations in the form of seizure-like bursting activity. In this condition, the fast active state termination was mediated by intrinsic hyperpolarizing conductances. Our study highlights the significance of both intrinsic and synaptic inhibition in manipulating sleep slow rhythms. PMID:22641778

  11. MLKL inhibition attenuates hypoxia-ischemia induced neuronal damage in developing brain.

    PubMed

    Qu, Yi; Shi, Jing; Tang, Ying; Zhao, Fengyan; Li, Shiping; Meng, Junjie; Tang, Jun; Lin, Xuemei; Peng, Xiaodong; Mu, Dezhi

    2016-05-01

    Mixed lineage kinase domain-like protein (MLKL) is a critical molecule mediating cell necroptosis. However, its role in brain injury remains obscure. We first investigated the functions and mechanisms of MLKL in mediating neuronal damage in developing brain after hypoxia-ischemia. Neuronal necroptosis was induced by oxygen-glucose deprivation (OGD) plus caspase inhibitor zVAD treatment (OGD/zVAD). We found that two important necroptosis related proteins, receptor-interacting protein 1 and 3 (RIP1, RIP3) were upregulated. Furthermore, the interaction of RIP1-RIP3 with MLKL increased. Inhibition of MLKL through siRNA diminished RIP1-RIP3-MLKL interaction and attenuated neuronal death induced by OGD/zVAD. The translocation of oligomerized MLKL to the neuronal membrane leading to the injury of cellular membrane is the possible new mechanism of neuronal necroptosis. Animal experiment with neonatal rats further proved that MLKL inhibition attenuated brain damage induced by hypoxia-ischemia. These findings suggest that MLKL is a target to attenuate brain damage in developing brain.

  12. In vivo inhibition of the mitochondrial H+-ATP synthase in neurons promotes metabolic preconditioning.

    PubMed

    Formentini, Laura; Pereira, Marta P; Sánchez-Cenizo, Laura; Santacatterina, Fulvio; Lucas, José J; Navarro, Carmen; Martínez-Serrano, Alberto; Cuezva, José M

    2014-04-01

    A key transducer in energy conservation and signaling cell death is the mitochondrial H(+)-ATP synthase. The expression of the ATPase inhibitory factor 1 (IF1) is a strategy used by cancer cells to inhibit the activity of the H(+)-ATP synthase to generate a ROS signal that switches on cellular programs of survival. We have generated a mouse model expressing a mutant of human IF1 in brain neurons to assess the role of the H(+)-ATP synthase in cell death in vivo. The expression of hIF1 inhibits the activity of oxidative phosphorylation and mediates the shift of neurons to an enhanced aerobic glycolysis. Metabolic reprogramming induces brain preconditioning affording protection against quinolinic acid-induced excitotoxicity. Mechanistically, preconditioning involves the activation of the Akt/p70S6K and PARP repair pathways and Bcl-xL protection from cell death. Overall, our findings provide the first in vivo evidence highlighting the H(+)-ATP synthase as a target to prevent neuronal cell death.

  13. Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage.

    PubMed

    Morel, Gustavo R; Carón, Rubén W; Cónsole, Gloria M; Soaje, Marta; Sosa, Yolanda E; Rodríguez, Silvia S; Jahn, Graciela A; Goya, Rodolfo G

    2009-12-16

    Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.

  14. Imbalance between excitation and inhibition among synaptic connections of CA3 pyramidal neurons in cultured hippocampal slices.

    PubMed

    Cruz-Martín, Alberto; Schweizer, Felix E

    2008-03-01

    A fundamental property of small neuronal ensembles is their ability to be selectively activated by distinct stimuli. One cellular mechanism by which neurons achieve this input selectivity is by modulating the temporal dynamics of excitation and inhibition. We explored the interplay of excitation and inhibition in synapses between pyramidal neurons of cornu ammonis field 3 of the hippocampal formation (CA3) in cultured rat hippocampal slices, where activation of a single excitatory cell can readily recruit local interneurons. Simultaneous whole-cell recordings from pairs of CA3 pyramidal neurons revealed that the strength of connections was neither uniform nor balanced. Rather, stimulation of presynaptic neurons elicited distinct combinations of excitatory postsynaptic current-inhibitory postsynaptic current (EPSC-IPSC) amplitudes in the postsynaptic neurons. EPSC-IPSC sequences with small EPSCs had large IPSCs and sequences that contained large EPSCs had small IPSCs. In addition to differences in the amplitudes of the responses, the kinetics of the EPSCs were also different, creating distinct temporal dynamics of excitation and inhibition. Weaker EPSCs had significantly slower kinetics and were efficiently occluded by IPSCs, thereby further limiting their contribution to depolarizing the postsynaptic membrane. Our data suggest that hippocampal pyramidal cells may use an imbalance between excitation and inhibition as a filter to enhance selectivity toward preferential excitatory connections.

  15. Activin A Inhibits MPTP and LPS-Induced Increases in Inflammatory Cell Populations and Loss of Dopamine Neurons in the Mouse Midbrain In Vivo

    PubMed Central

    Stayte, Sandy; Rentsch, Peggy; Tröscher, Anna R.; Bamberger, Maximilian; Li, Kong M.; Vissel, Bryce

    2017-01-01

    Parkinson’s disease is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta region and a subsequent loss of dopamine within the striatum. A promising avenue of research has been the administration of growth factors to promote the survival of remaining midbrain neurons, although the mechanism by which they provide neuroprotection is not understood. Activin A, a member of the transforming growth factor β superfamily, has been shown to be a potent anti-inflammatory following acute brain injury and has been demonstrated to play a role in the neuroprotection of midbrain neurons against MPP+-induced degeneration in vitro. We hypothesized that activin A may offer similar anti-inflammatory and neuroprotective effects in in vivo mouse models of Parkinson’s disease. We found that activin A significantly attenuated the inflammatory response induced by both MPTP and intranigral administration of lipopolysaccharide in C57BL/6 mice. We found that administration of activin A promoted survival of dopaminergic and total neuron populations in the pars compacta region both 8 days and 8 weeks after MPTP-induced degeneration. Surprisingly, no corresponding protection of striatal dopamine levels was found. Furthermore, activin A failed to protect against loss of striatal dopamine transporter expression in the striatum, suggesting the neuroprotective action of activin A may be localized to the substantia nigra. Together, these results provide the first evidence that activin A exerts potent neuroprotection and anti-inflammatory effects in the MPTP and lipopolysaccharide mouse models of Parkinson’s disease. PMID:28121982

  16. Non-associative Potentiation of Perisomatic Inhibition Alters the Temporal Coding of Neocortical Layer 5 Pyramidal Neurons

    PubMed Central

    Lourenço, Joana; Pacioni, Simone; Rebola, Nelson; van Woerden, Geeske M.; Marinelli, Silvia; DiGregorio, David; Bacci, Alberto

    2014-01-01

    In the neocortex, the coexistence of temporally locked excitation and inhibition governs complex network activity underlying cognitive functions, and is believed to be altered in several brain diseases. Here we show that this equilibrium can be unlocked by increased activity of layer 5 pyramidal neurons of the mouse neocortex. Somatic depolarization or short bursts of action potentials of layer 5 pyramidal neurons induced a selective long-term potentiation of GABAergic synapses (LTPi) without affecting glutamatergic inputs. Remarkably, LTPi was selective for perisomatic inhibition from parvalbumin basket cells, leaving dendritic inhibition intact. It relied on retrograde signaling of nitric oxide, which persistently altered presynaptic GABA release and diffused to inhibitory synapses impinging on adjacent pyramidal neurons. LTPi reduced the time window of synaptic summation and increased the temporal precision of spike generation. Thus, increases in single cortical pyramidal neuron activity can induce an interneuron-selective GABAergic plasticity effectively altering the computation of temporally coded information. PMID:25003184

  17. Epigenetic regulation of motor neuron cell death through DNA methylation.

    PubMed

    Chestnut, Barry A; Chang, Qing; Price, Ann; Lesuisse, Catherine; Wong, Margaret; Martin, Lee J

    2011-11-16

    DNA methylation is an epigenetic mechanism for gene silencing engaged by DNA methyltransferase (Dnmt)-catalyzed methyl group transfer to cytosine residues in gene-regulatory regions. It is unknown whether aberrant DNA methylation can cause neurodegeneration. We tested the hypothesis that Dnmts can mediate neuronal cell death. Enforced expression of Dnmt3a induced degeneration of cultured NSC34 cells. During apoptosis of NSC34 cells induced by camptothecin, levels of Dnmt1 and Dnmt3a increased fivefold and twofold, respectively, and 5-methylcytosine accumulated in nuclei. Truncation mutation of the Dnmt3a catalytic domain and Dnmt3a RNAi blocked apoptosis of cultured neurons. Inhibition of Dnmt catalytic activity with RG108 and procainamide protected cultured neurons from excessive DNA methylation and apoptosis. In vivo, Dnmt1 and Dnmt3a are expressed differentially during mouse brain and spinal cord maturation and in adulthood when Dnmt3a is abundant in synapses and mitochondria. Dnmt1 and Dnmt3a are expressed in motor neurons of adult mouse spinal cord, and, during their apoptosis induced by sciatic nerve avulsion, nuclear and cytoplasmic 5-methylcytosine immunoreactivity, Dnmt3a protein levels and Dnmt enzyme activity increased preapoptotically. Inhibition of Dnmts with RG108 blocked completely the increase in 5-methycytosine and the apoptosis of motor neurons in mice. In human amyotrophic lateral sclerosis (ALS), motor neurons showed changes in Dnmt1, Dnmt3a, and 5-methylcytosine similar to experimental models. Thus, motor neurons can engage epigenetic mechanisms to drive apoptosis, involving Dnmt upregulation and increased DNA methylation. These cellular mechanisms could be relevant to human ALS pathobiology and disease treatment.

  18. Leptin-inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance.

    PubMed

    Flak, Jonathan N; Patterson, Christa M; Garfield, Alastair S; D'Agostino, Giuseppe; Goforth, Paulette B; Sutton, Amy K; Malec, Paige A; Wong, Jenny-Marie T; Germani, Mark; Jones, Justin C; Rajala, Michael; Satin, Leslie; Rhodes, Christopher J; Olson, David P; Kennedy, Robert T; Heisler, Lora K; Myers, Martin G

    2014-12-01

    Hypoglycemia initiates the counter-regulatory response (CRR), in which the sympathetic nervous system, glucagon and glucocorticoids restore glucose to appropriate concentrations. During starvation, low leptin levels restrain energy utilization, enhancing long-term survival. To ensure short-term survival during hypoglycemia in fasted animals, the CRR must overcome this energy-sparing program and nutrient depletion. Here we identify in mice a previously unrecognized role for leptin and a population of leptin-regulated neurons that modulate the CRR to meet these challenges. Hypoglycemia activates neurons of the parabrachial nucleus (PBN) that coexpress leptin receptor (LepRb) and cholecystokinin (CCK) (PBN LepRb(CCK) neurons), which project to the ventromedial hypothalamic nucleus. Leptin inhibits these cells, and Cck(cre)-mediated ablation of LepRb enhances the CRR. Inhibition of PBN LepRb cells blunts the CRR, whereas their activation mimics the CRR in a CCK-dependent manner. PBN LepRb(CCK) neurons are a crucial component of the CRR system and may be a therapeutic target in hypoglycemia.

  19. Phosphodiesterase 7 Inhibition Preserves Dopaminergic Neurons in Cellular and Rodent Models of Parkinson Disease

    PubMed Central

    Morales-Garcia, Jose A.; Redondo, Miriam; Alonso-Gil, Sandra; Gil, Carmen; Perez, Concepción; Martinez, Ana; Santos, Angel; Perez-Castillo, Ana

    2011-01-01

    Background Phosphodiesterase 7 plays a major role in down-regulation of protein kinase A activity by hydrolyzing cAMP in many cell types. This cyclic nucleotide plays a key role in signal transduction in a wide variety of cellular responses. In the brain, cAMP has been implicated in learning, memory processes and other brain functions. Methodology/Principal Findings Here we show a novel function of phosphodiesterase 7 inhibition on nigrostriatal dopaminergic neuronal death. We found that S14, a heterocyclic small molecule inhibitor of phosphodiesterase 7, conferred significant neuronal protection against different insults both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures. S14 treatment also reduced microglial activation, protected dopaminergic neurons and improved motor function in the lipopolysaccharide rat model of Parkinson disease. Finally, S14 neuroprotective effects were reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase A. Conclusions/Significance Our findings demonstrate that phosphodiesterase 7 inhibition can protect dopaminergic neurons against different insults, and they provide support for the therapeutic potential of phosphodiesterase 7 inhibitors in the treatment of neurodegenerative disorders, particularly Parkinson disease. PMID:21390306

  20. Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment.

    PubMed

    Matusica, Dusan; Alfonsi, Fabienne; Turner, Bradley J; Butler, Tim J; Shepheard, Stephanie R; Rogers, Mary-Louise; Skeldal, Sune; Underwood, Clare K; Mangelsdorf, Marie; Coulson, Elizabeth J

    2016-02-01

    The p75 neurotrophin receptor (p75(NTR); also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75(NTR)-derived trophic cell-permeable peptide, c29, to inhibit p75(NTR)-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1(G93A) mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75(NTR) cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75(NTR) in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75(NTR) cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1(G93A) mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo. © 2016. Published by The Company of Biologists Ltd.

  1. Autophagy Inhibition Favors Survival of Rubrospinal Neurons After Spinal Cord Hemisection.

    PubMed

    Bisicchia, Elisa; Latini, Laura; Cavallucci, Virve; Sasso, Valeria; Nicolin, Vanessa; Molinari, Marco; D'Amelio, Marcello; Viscomi, Maria Teresa

    2016-08-11

    Spinal cord injuries (SCIs) are devastating conditions of the central nervous system (CNS) for which there are no restorative therapies. Neuronal death at the primary lesion site and in remote regions that are functionally connected to it is one of the major contributors to neurological deficits following SCI.Disruption of autophagic flux induces neuronal death in many CNS injuries, but its mechanism and relationship with remote cell death after SCI are unknown. We examined the function and effects of the modulation of autophagy on the fate of axotomized rubrospinal neurons in a rat model of spinal cord dorsal hemisection (SCH) at the cervical level. Following SCH, we observed an accumulation of LC3-positive autophagosomes (APs) in the axotomized neurons 1 and 5 days after injury. Furthermore, this accumulation was not attributed to greater initiation of autophagy but was caused by a decrease in AP clearance, as demonstrated by the build-up of p62, a widely used marker of the induction of autophagy. In axotomized rubrospinal neurons, the disruption of autophagic flux correlated strongly with remote neuronal death and worse functional recovery. Inhibition of AP biogenesis by 3-methyladenine (3-MA) significantly attenuated remote degeneration and improved spontaneous functional recovery, consistent with the detrimental effects of autophagy in remote damage after SCH. Collectively, our results demonstrate that autophagic flux is blocked in axotomized neurons on SCI and that the inhibition of AP formation improves their survival. Thus, autophagy is a promising target for the development of therapeutic interventions in the treatment of SCIs.

  2. Neurites regrowth of cortical neurons by GSK3beta inhibition independently of Nogo receptor 1.

    PubMed

    Seira, Oscar; Gavín, Rosalina; Gil, Vanessa; Llorens, Franc; Rangel, Alejandra; Soriano, Eduardo; del Río, José Antonio

    2010-06-01

    Lesioned axons do not regenerate in the adult mammalian CNS, owing to the over-expression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3beta (GSK3beta) and extracellular-related kinase (ERK) 1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: (i) cerebellar granule cells and (ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Finally, these regenerative effects were corroborated in the lesioned entorhino-hippocampal pathway in NgR1-/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

  3. Role of platinum DNA damage-induced transcriptional inhibition in chemotherapy-induced neuronal atrophy and peripheral neurotoxicity.

    PubMed

    Yan, Fang; Liu, Johnson J; Ip, Virginia; Jamieson, Stephen M F; McKeage, Mark J

    2015-12-01

    Platinum-based anticancer drugs cause peripheral neurotoxicity by damaging sensory neurons within the dorsal root ganglia (DRG), but the mechanisms are incompletely understood. The roles of platinum DNA binding, transcription inhibition and altered cell size were investigated in primary cultures of rat DRG cells. Click chemistry quantitative fluorescence imaging of RNA-incorporated 5-ethynyluridine showed high, but wide ranging, global levels of transcription in individual neurons that correlated with their cell body size. Treatment with platinum drugs reduced neuronal transcription and cell body size to an extent that corresponded to the amount of preceding platinum DNA binding, but without any loss of neuronal cells. The effects of platinum drugs on neuronal transcription and cell body size were inhibited by blocking platinum DNA binding with sodium thiosulfate, and mimicked by treatment with a model transcriptional inhibitor, actinomycin D. In vivo oxaliplatin treatment depleted the total RNA content of DRG tissue concurrently with altering DRG neuronal size. These findings point to a mechanism of chemotherapy-induced peripheral neurotoxicity, whereby platinum DNA damage induces global transcriptional arrest leading in turn to neuronal atrophy. DRG neurons may be particularly vulnerable to this mechanism of toxicity because of their requirements for high basal levels of global transcriptional activity. Findings point to a new stepwise mechanism of chemotherapy-induced peripheral neurotoxicity, whereby platinum DNA damage induces global transcriptional arrest leading in turn to neuronal atrophy. Dorsal root ganglion neurons may be particularly vulnerable to this neurotoxicity because of their high global transcriptional outputs, demonstrated in this study by click chemistry quantitative fluorescence imaging.

  4. Molecular characterization of Thy1 expressing fear-inhibiting neurons within the basolateral amygdala

    PubMed Central

    McCullough, Kenneth M.; Choi, Dennis; Guo, Jidong; Zimmerman, Kelsey; Walton, Jordan; Rainnie, Donald G.; Ressler, Kerry J.

    2016-01-01

    Molecular characterization of neuron populations, particularly those controlling threat responses, is essential for understanding the cellular basis of behaviour and identifying pharmacological agents acting selectively on fear-controlling circuitry. Here we demonstrate a comprehensive workflow for identification of pharmacologically tractable markers of behaviourally characterized cell populations. Thy1-eNpHR-, Thy1-Cre- and Thy1-eYFP-labelled neurons of the BLA consistently act as fear inhibiting or ‘Fear-Off' neurons during behaviour. We use cell-type-specific optogenetics and chemogenetics (DREADDs) to modulate activity in this population during behaviour to block or enhance fear extinction. Dissociated Thy1-eYFP neurons are isolated using FACS. RNA sequencing identifies genes strongly upregulated in RNA of this population, including Ntsr2, Dkk3, Rspo2 and Wnt7a. Pharmacological manipulation of neurotensin receptor 2 confirms behavioural effects observed in optogenetic and chemogenetic experiments. These experiments identify and validate Ntsr2-expressing neurons within the BLA, as a putative ‘Fear-Off' population. PMID:27767183

  5. Dopamine Inhibition Differentially Controls Excitability of Substantia Nigra Dopamine Neuron Subpopulations through T-Type Calcium Channels.

    PubMed

    Evans, Rebekah C; Zhu, Manhua; Khaliq, Zayd M

    2017-03-29

    While there is growing appreciation for diversity among ventral tegmental area dopamine neurons, much less is known regarding functional heterogeneity among the substantia nigra pars compacta (SNc) neurons. Here, we show that calbindin-positive dorsal tier and calbindin-negative ventral tier SNc dopaminergic neurons in mice comprise functionally distinct subpopulations distinguished by their dendritic calcium signaling, rebound excitation, and physiological responses to dopamine D2-receptor (D2) autoinhibition. While dopamine is known to inhibit action potential backpropagation, our experiments revealed an unexpected enhancement of excitatory responses and dendritic calcium signals in the presence of D2-receptor inhibition. Specifically, dopamine inhibition and direct hyperpolarization enabled the generation of low-threshold depolarizations that occurred in an all-or-none or graded manner, due to recruitment of T-type calcium channels. Interestingly, these effects occurred selectively in calbindin-negative dopaminergic neurons within the SNc. Thus, calbindin-positive and calbindin-negative SNc neurons differ substantially in their calcium channel composition and efficacy of excitatory inputs in the presence of dopamine inhibition.SIGNIFICANCE STATEMENT Substantia nigra dopaminergic neurons can be divided into two populations: the calbindin-negative ventral tier, which is vulnerable to neurodegeneration in Parkinson's disease, and the calbindin-positive dorsal tier, which is relatively resilient. Although tonic firing is similar in these subpopulations, we find that their responses to dopamine-mediated inhibition are strikingly different. During inhibition, calbindin-negative neurons exhibit increased sensitivity to excitatory inputs, which can then trigger large dendritic calcium transients due to strong expression of T-type calcium channels. Therefore, SNc neurons differ substantially in their calcium channel composition, which may contribute to their differential

  6. Sigma receptor activation inhibits voltage-gated sodium channels in rat intracardiac ganglion neurons

    PubMed Central

    Zhang, Hongling; Katnik, Christopher; Cuevas, Javier

    2010-01-01

    Sigma (σ) receptors have been shown to regulate multiple ion channel types in intracardiac ganglion neurons, including voltage-gated calcium and potassium channels. However, the inhibition of these channels alone cannot fully account for σ receptor-induced changes in neuronal excitability previously reported. Whole-cell patch clamp experiments were conducted under current-clamp mode in isolated intracardiac neurons from neonatal rats to assess the effects of σ receptor activation on the active membrane properties of these cells. Bath application of the pan-selective σ receptor agonist, 1,3-Di-o-tolylguanidine (DTG), and the σ-1-selective agonist, (+)-pentazocine, significantly increased the action potential latency and decreased action potential overshoot in response to depolarizing current ramps, which suggests inhibition of voltage-gated sodium channels. Whole-cell voltage clamp experiments showed that these σ agonists reversibly decrease depolarization-activated Na+ currents in these cells at all potentials tested. The peak currents generated by membrane depolarizations were decreased in a dose dependent manner with IC50 values for DTG and (+)-pentazocine of 32 μM and 49 μM, respectively. The σ-1 receptor-selective antagonist, BD 1063 (100 nM), inhibited DTG (30 μM) block of Na+ currents by ∼ 50%, suggesting that the effects are mediated by activation of σ-1 receptors. DTG also shifted the steady-state inactivation curve of Na+ channels to more negative potentials, with the membrane potential of half-activation shifting from -49 mV to -63 mV in the absence and presence of 30 μM DTG, respectively. Taken together, these results suggest that σ-1 receptor activation decreases intracardiac ganglion neuron excitability by modulating voltage-gated Na+ channels. PMID:21383893

  7. Muscarine inhibits high-threshold calcium currents with two distinct modes in rat embryonic hippocampal neurons.

    PubMed Central

    Toselli, M; Taglietti, V

    1995-01-01

    1. Ca2+ channel modulation by muscarine was investigated in primary cultured embryonic rat hippocampal neurons using the whole-cell variant of the patch-clamp technique. 2. Muscarine produced a reversible and concentration-dependent decrease in the Ba2+ current amplitude. In 65% of neurons sensitive to the agonist, current inhibition was time and voltage dependent, being maximal between -20 and 0 mV and decreasing at depolarizing potentials. In the remaining 35% of neurons, the effects of muscarine were voltage independent, inhibition being constant in a wide potential range between -20 and +80 mV. 3. Different receptors might be involved in the two modes of modulation. Muscarine-induced voltage-dependent inhibition of Ba2+ current was best suppressed by the muscarinic receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine methiodide (81% suppression), while voltage-independent inhibition was best suppressed by AFDX116 (75% suppression). 4. In cells treated with omega-conotoxin (omega-CgTX), the voltage-independent mode of inhibition was strongly prevented, suggesting that the two modulatory mechanisms (voltage dependent and voltage independent) operate on separate classes of high-voltage-activated (HVA) Ca2+ channels. 5. A pertussis toxin-sensitive G-protein is involved in both modes of action of muscarine, since both modes were prevented by pretreatment of the cells with 50 ng ml-1 pertussis toxin. 6. Both modes of modulation were mimicked in different cells by intracellular application of GTP-gamma-S. However, the onset of voltage-independent inhibition was about 5 times slower than that of voltage-dependent inhibition, suggesting involvement of a more complex metabolic pathway for the former mode of channel modulation. 7. Relief of the voltage-dependent inhibition was obtained by depolarizing voltage prepulses and occurred with kinetics that depended on agonist concentration. 8. The voltage-dependent inhibition could be simulated by a kinetic model in which

  8. Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration

    PubMed Central

    Zheng, Xinde; Boyer, Leah; Jin, Mingji; Kim, Yongsung; Fan, Weiwei; Bardy, Cedric; Berggren, Travis; Evans, Ronald M; Gage, Fred H; Hunter, Tony

    2016-01-01

    mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here, we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels, particularly when oxidative phosphorylation is impaired, such as in neurons treated with mitochondrial inhibitors, or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly, in mitochondrially defective neurons, but not neuroprogenitor cells, ribosomal S6 and S6 kinase phosphorylation increased over time, despite activation of AMPK, which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis, a major energy-consuming process, may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration. DOI: http://dx.doi.org/10.7554/eLife.13378.001 PMID:27008180

  9. Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

    PubMed

    Zhang, Hai-Tao; Xue, Jing-Hui; Zhang, Zhi-Wen; Kong, Hai-Bo; Liu, Ai-Jun; Li, Shou-Chun; Xu, Dong-Gang

    2015-10-05

    Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

  10. Ghrelin inhibits LPS-induced release of IL-6 from mouse dopaminergic neurones

    PubMed Central

    2013-01-01

    Background Ghrelin is an orexigenic stomach hormone that acts centrally to increase mid-brain dopamine neurone activity, amplify dopamine signaling and protect against neurotoxin-induced dopamine cell death in the mouse substantia nigra pars compacta (SNpc). In addition, ghrelin inhibits the lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines from peripheral macrophages, T-cells and from LPS stimulated microglia. Here we sought to determine whether ghrelin attenuates pro-inflammatory cytokine release from dopaminergic neurones. Findings The dopaminergic SN4741 cell-line, which derives from the mouse substantia nigra (SN) and expresses the ghrelin-receptor (growth hormone secretagogue receptor (GHS-R)) and the ghrelin-O-acyl transferase (GOAT) enzyme, was used to determine the neuro-immunomodulatory action of ghrelin. We induced innate immune activation via LPS challenge (1 μg/ml) of SN4741 neurones that had been pre-cultured in the presence or absence of ghrelin (1, 10, 100 nM) for 4 h. After 24 h supernatants were collected for detection of IL-1 beta (IL-1β ), TNF alpha (TNF-α) and IL-6 cytokines via enzyme linked immunosorbent assay (ELISA) analysis. Nuclear translocation of the transcription factor nuclear factor kappa B (NF-κB) was analyzed by Western blotting, and to determine viability of treatments a cell viability assay and caspase-3 immunohistochemistry were performed. We provide evidence that while IL-1β and TNF-α were not detectable under any conditions, SN4741 neurones constitutively released IL-6 under basal conditions and treatment with LPS significantly increased IL-6 secretion. Pre-treatment of neurones with ghrelin attenuated LPS-mediated IL-6 release at 24 h, an affect that was inhibited by the GHS-R antagonist [D-Lys3]-GHRP-6. However, while ghrelin pre-treatment attenuated the LPS-mediated increase in NF-κB, there was no alteration in its nuclear translocation. Cell viability assay and caspase-3 immunocytochemistry

  11. Ghrelin inhibits LPS-induced release of IL-6 from mouse dopaminergic neurones.

    PubMed

    Beynon, Amy L; Brown, M Rowan; Wright, Rhiannon; Rees, Mark I; Sheldon, I Martin; Davies, Jeffrey S

    2013-03-19

    Ghrelin is an orexigenic stomach hormone that acts centrally to increase mid-brain dopamine neurone activity, amplify dopamine signaling and protect against neurotoxin-induced dopamine cell death in the mouse substantia nigra pars compacta (SNpc). In addition, ghrelin inhibits the lipopolysaccharide (LPS)-induced release of pro-inflammatory cytokines from peripheral macrophages, T-cells and from LPS stimulated microglia. Here we sought to determine whether ghrelin attenuates pro-inflammatory cytokine release from dopaminergic neurones. The dopaminergic SN4741 cell-line, which derives from the mouse substantia nigra (SN) and expresses the ghrelin-receptor (growth hormone secretagogue receptor (GHS-R)) and the ghrelin-O-acyl transferase (GOAT) enzyme, was used to determine the neuro-immunomodulatory action of ghrelin. We induced innate immune activation via LPS challenge (1 μg/ml) of SN4741 neurones that had been pre-cultured in the presence or absence of ghrelin (1, 10, 100 nM) for 4 h. After 24 h supernatants were collected for detection of IL-1 beta (IL-1β ), TNF alpha (TNF-α) and IL-6 cytokines via enzyme linked immunosorbent assay (ELISA) analysis. Nuclear translocation of the transcription factor nuclear factor kappa B (NF-κB) was analyzed by Western blotting, and to determine viability of treatments a cell viability assay and caspase-3 immunohistochemistry were performed.We provide evidence that while IL-1β and TNF-α were not detectable under any conditions, SN4741 neurones constitutively released IL-6 under basal conditions and treatment with LPS significantly increased IL-6 secretion. Pre-treatment of neurones with ghrelin attenuated LPS-mediated IL-6 release at 24 h, an affect that was inhibited by the GHS-R antagonist [D-Lys3]-GHRP-6. However, while ghrelin pre-treatment attenuated the LPS-mediated increase in NF-κB, there was no alteration in its nuclear translocation. Cell viability assay and caspase-3 immunocytochemistry demonstrated that the

  12. The actions of Pasteurella multocida toxin on neuronal cells.

    PubMed

    Surguy, Susan M; Duricki, Denise A; Reilly, Joanne M; Lax, Alistair J; Robbins, Jon

    2014-02-01

    Pasteurella multocida toxin (PMT) activates the G-proteins Gαi(₁₋₃), Gα(q), Gα₁₁, Gα₁₂ and Gα₁₃ by deamidation of specific glutamine residues. A number of these alpha subunits have signalling roles in neurones. Hence we studied the action of this toxin on rat superior cervical ganglion (SCG) neurones and NG108-15 neuronal cells. Both Gα(q) and Gα₁₁ could be identified in SCGs with immunocytochemistry. PMT had no direct action on Kv7 or Cav2 channels in SCGs. However PMT treatment enhanced muscarinic receptor mediated inhibition of M-current (Kv7.2 + 7. 3) as measured by a 19-fold leftward shift in the oxotremorine-M concentration-inhibition curve. Agonists of other receptors, such as bradykinin or angiotensin, that inhibit M-current did not produce this effect. However the amount of PIP₂ hydrolysis could be enhanced by PMT for all three agonists. In a transduction system in SCGs that is unlikely to be affected by PMT, Go mediated inhibition of calcium current, PMT was ineffective whereas the response was blocked by pertussis toxin as expected. M1 muscarinic receptor evoked calcium mobilisation in transformed NG108-15 cells was enhanced by PMT. The calcium rises evoked by uridine triphosphate acting on endogenous P2Y₂ receptors in NG108-15 cells were enhanced by PMT. The time and concentration dependence of the PMT effect was different for the resting calcium compared to the calcium rise produced by activation of P2Y₂ receptors. PMT's action on these neuronal cells would suggest that if it got into the brain, symptoms of a hyperexcitable nature would be seen, such as seizures.

  13. Cell cycle inhibition and retinoblastoma protein overexpression prevent Purkinje cell death in organotypic slice cultures.

    PubMed

    Padmanabhan, Jaya; Brown, Kristy; Shelanski, Michael L

    2007-05-01

    Purkinje cells are vulnerable to a number of physical, chemical, and genetic insults during development and maturity. Normal development of these cells depends on the cell-cell interactions between granule and astroglial cell populations. Apoptotic death in Purkinje neurons had been shown to be associated with cell cycle activation, and new DNA synthesis is associated with Purkinje cell death in staggerer and lurcher mutant mice. Here using an in vitro organotypic slice culture model from 9 (P9) and 4 days (P4) old postnatal rats we show that the cyclin dependent kinase (cdk) inhibitors (roscovitine, olomoucine, and flavopiridol) protect the Purkinje cells from cell death. The results are more pronounced in the cerebellar sections from P4 rats. Analysis of Purkinje neurons in sections from P4 rats after 1 week of culturing showed that while there were very limited calbindin positive neurons in the untreated sections the cdk inhibitor treated sections had a notably higher number. Although treatment with cdk inhibitors inhibited Purkinje cell loss significantly, the morphology of these neurons was abnormal, with stunted dendrites and axons. Since the retinoblastoma protein (Rb) is the major pocket protein involved in determining the differentiated state of neurons we examined the effect of over-expressing Rb in the organotypic cultures. Rb overexpression significantly inhibited the Purkinje cell death and these neurons maintained their normal morphology. Thus our studies show that the cell death in Purkinje neurons observed in organotypic cultures is cell cycle dependent and the optimal survival requires Rb.

  14. Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease.

    PubMed

    Moruno-Manchon, Jose F; Uzor, Ndidi-Ese; Blasco-Conesa, Maria P; Mannuru, Sishira; Putluri, Nagireddy; Furr-Stimming, Erin E; Tsvetkov, Andrey S

    2017-04-01

    Huntington disease (HD) is the most common inherited neurodegenerative disorder. It has no cure. The protein huntingtin causes HD, and mutations to it confer toxic functions to the protein that lead to neurodegeneration. Thus, identifying modifiers of mutant huntingtin-mediated neurotoxicity might be a therapeutic strategy for HD. Sphingosine kinases 1 (SK1) and 2 (SK2) synthesize sphingosine-1-phosphate (S1P), a bioactive lipid messenger critically involved in many vital cellular processes, such as cell survival. In the nucleus, SK2 binds to and inhibits histone deacetylases 1 and 2 (HDAC1/2). Inhibiting both HDACs has been suggested as a potential therapy in HD. Here, we found that SK2 is nuclear in primary neurons and, unexpectedly, overexpressed SK2 is neurotoxic in a dose-dependent manner. SK2 promotes DNA double-strand breaks in cultured primary neurons. We also found that SK2 is hyperphosphorylated in the brain samples from a model of HD, the BACHD mice. These data suggest that the SK2 pathway may be a part of a pathogenic pathway in HD. ABC294640, an inhibitor of SK2, reduces DNA damage in neurons and increases survival in two neuron models of HD. Our results identify a novel regulator of mutant huntingtin-mediated neurotoxicity and provide a new target for developing therapies for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Molecular mechanisms of subtype-specific inhibition of neuronal T-type calcium channels by ascorbate.

    PubMed

    Nelson, Michael T; Joksovic, Pavle M; Su, Peihan; Kang, Ho-Won; Van Deusen, Amy; Baumgart, Joel P; David, Laurence S; Snutch, Terrance P; Barrett, Paula Q; Lee, Jung-Ha; Zorumski, Charles F; Perez-Reyes, Edward; Todorovic, Slobodan M

    2007-11-14

    T-type Ca2+ channels (T-channels) are involved in the control of neuronal excitability and their gating can be modulated by a variety of redox agents. Ascorbate is an endogenous redox agent that can function as both an anti- and pro-oxidant. Here, we show that ascorbate selectively inhibits native Ca(v)3.2 T-channels in peripheral and central neurons, as well as recombinant Ca(v)3.2 channels heterologously expressed in human embryonic kidney 293 cells, by initiating the metal-catalyzed oxidation of a specific, metal-binding histidine residue in domain 1 of the channel. Our biophysical experiments indicate that ascorbate reduces the availability of Ca(v)3.2 channels over a wide range of membrane potentials, and inhibits Ca(v)3.2-dependent low-threshold-Ca2+ spikes as well as burst-firing in reticular thalamic neurons at physiologically relevant concentrations. This study represents the first mechanistic demonstration of ion channel modulation by ascorbate, and suggests that ascorbate may function as an endogenous modulator of neuronal excitability.

  16. An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo

    PubMed Central

    Wietek, Jonas; Beltramo, Riccardo; Scanziani, Massimo; Hegemann, Peter; Oertner, Thomas G.; Simon Wiegert, J.

    2015-01-01

    Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source. PMID:26443033

  17. An improved chloride-conducting channelrhodopsin for light-induced inhibition of neuronal activity in vivo.

    PubMed

    Wietek, Jonas; Beltramo, Riccardo; Scanziani, Massimo; Hegemann, Peter; Oertner, Thomas G; Wiegert, J Simon

    2015-10-07

    Channelrhodopsins are light-gated cation channels that have been widely used for optogenetic stimulation of electrically excitable cells. Replacement of a glutamic acid in the central gate with a positively charged amino acid residue reverses the ion selectivity and produces chloride-conducting ChRs (ChloCs). Expressed in neurons, published ChloCs produced a strong shunting effect but also a small, yet significant depolarization from the resting potential. Depending on the state of the neuron, the net result of illumination might therefore be inhibitory or excitatory with respect to action potential generation. Here we report two additional amino acid substitutions that significantly shift the reversal potential of improved ChloC (iChloC) to the reversal potential of endogenous GABAA receptors. As a result, light-evoked membrane depolarization was strongly reduced and spike initiation after current injection or synaptic stimulation was reliably inhibited in iChloC-transfected neurons in vitro. In the primary visual cortex of anesthetized mice, activation of iChloC suppressed spiking activity evoked by visual stimulation. Due to its high operational light sensitivity, iChloC makes it possible to inhibit neurons in a large volume of brain tissue from a small, point-like light source.

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

    PubMed

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

    2009-07-01

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

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

    PubMed

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

    2007-01-01

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

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

    PubMed

    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.

  1. Light Activates Output from Evening Neurons and Inhibits Output from Morning Neurons in the Drosophila Circadian Clock

    PubMed Central

    Picot, Marie; Cusumano, Paola; Klarsfeld, André; Ueda, Ryu; Rouyer, François

    2007-01-01

    Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons. PMID:18044989

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

    PubMed

    Sakai, Tetsuro; Xu, Yan

    2012-01-01

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

  3. SLO BK Potassium Channels Couple Gap Junctions to Inhibition of Calcium Signaling in Olfactory Neuron Diversification

    PubMed Central

    Schumacher, Jennifer A.; Wang, Xiaohong; Merrill, Sean A.; Millington, Grethel; Bayne, Brittany; Jorgensen, Erik M.; Chuang, Chiou-Fen

    2016-01-01

    The C. elegans AWC olfactory neuron pair communicates to specify asymmetric subtypes AWCOFF and AWCON in a stochastic manner. Intercellular communication between AWC and other neurons in a transient NSY-5 gap junction network antagonizes voltage-activated calcium channels, UNC-2 (CaV2) and EGL-19 (CaV1), in the AWCON cell, but how calcium signaling is downregulated by NSY-5 is only partly understood. Here, we show that voltage- and calcium-activated SLO BK potassium channels mediate gap junction signaling to inhibit calcium pathways for asymmetric AWC differentiation. Activation of vertebrate SLO-1 channels causes transient membrane hyperpolarization, which makes it an important negative feedback system for calcium entry through voltage-activated calcium channels. Consistent with the physiological roles of SLO-1, our genetic results suggest that slo-1 BK channels act downstream of NSY-5 gap junctions to inhibit calcium channel-mediated signaling in the specification of AWCON. We also show for the first time that slo-2 BK channels are important for AWC asymmetry and act redundantly with slo-1 to inhibit calcium signaling. In addition, nsy-5-dependent asymmetric expression of slo-1 and slo-2 in the AWCON neuron is necessary and sufficient for AWC asymmetry. SLO-1 and SLO-2 localize close to UNC-2 and EGL-19 in AWC, suggesting a role of possible functional coupling between SLO BK channels and voltage-activated calcium channels in AWC asymmetry. Furthermore, slo-1 and slo-2 regulate the localization of synaptic markers, UNC-2 and RAB-3, in AWC neurons to control AWC asymmetry. We also identify the requirement of bkip-1, which encodes a previously identified auxiliary subunit of SLO-1, for slo-1 and slo-2 function in AWC asymmetry. Together, these results provide an unprecedented molecular link between gap junctions and calcium pathways for terminal differentiation of olfactory neurons. PMID:26771544

  4. Optical control of neuronal excitation and inhibition using a single opsin protein, ChR2

    NASA Astrophysics Data System (ADS)

    Liske, Holly; Qian, Xiang; Anikeeva, Polina; Deisseroth, Karl; Delp, Scott

    2013-10-01

    The effect of electrical stimulation on neuronal membrane potential is frequency dependent. Low frequency electrical stimulation can evoke action potentials, whereas high frequency stimulation can inhibit action potential transmission. Optical stimulation of channelrhodopsin-2 (ChR2) expressed in neuronal membranes can also excite action potentials. However, it is unknown whether optical stimulation of ChR2-expressing neurons produces a transition from excitation to inhibition with increasing light pulse frequencies. Here we report optical inhibition of motor neuron and muscle activity in vivo in the cooled sciatic nerves of Thy1-ChR2-EYFP mice. We also demonstrate all-optical single-wavelength control of neuronal excitation and inhibition without co-expression of inhibitory and excitatory opsins. This all-optical system is free from stimulation-induced electrical artifacts and thus provides a new approach to investigate mechanisms of high frequency inhibition in neuronal circuits in vivo and in vitro.

  5. Bee Venom Protects against Rotenone-Induced Cell Death in NSC34 Motor Neuron Cells.

    PubMed

    Jung, So Young; Lee, Kang-Woo; Choi, Sun-Mi; Yang, Eun Jin

    2015-09-21

    Rotenone, an inhibitor of mitochondrial complex I of the mitochondrial respiratory chain, is known to elevate mitochondrial reactive oxygen species and induce apoptosis via activation of the caspase-3 pathway. Bee venom (BV) extracted from honey bees has been widely used in oriental medicine and contains melittin, apamin, adolapin, mast cell-degranulating peptide, and phospholipase A₂. In this study, we tested the effects of BV on neuronal cell death by examining rotenone-induced mitochondrial dysfunction. NSC34 motor neuron cells were pretreated with 2.5 μg/mL BV and stimulated with 10 μM rotenone to induce cell toxicity. We assessed cell death by Western blotting using specific antibodies, such as phospho-ERK1/2, phospho-JNK, and cleaved capase-3 and performed an MTT assay for evaluation of cell death and mitochondria staining. Pretreatment with 2.5 μg/mL BV had a neuroprotective effect against 10 μM rotenone-induced cell death in NSC34 motor neuron cells. Pre-treatment with BV significantly enhanced cell viability and ameliorated mitochondrial impairment in rotenone-treated cellular model. Moreover, BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. Taken together, we suggest that BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death.

  6. Bee Venom Protects against Rotenone-Induced Cell Death in NSC34 Motor Neuron Cells

    PubMed Central

    Jung, So Young; Lee, Kang-Woo; Choi, Sun-Mi; Yang, Eun Jin

    2015-01-01

    Rotenone, an inhibitor of mitochondrial complex I of the mitochondrial respiratory chain, is known to elevate mitochondrial reactive oxygen species and induce apoptosis via activation of the caspase-3 pathway. Bee venom (BV) extracted from honey bees has been widely used in oriental medicine and contains melittin, apamin, adolapin, mast cell-degranulating peptide, and phospholipase A2. In this study, we tested the effects of BV on neuronal cell death by examining rotenone-induced mitochondrial dysfunction. NSC34 motor neuron cells were pretreated with 2.5 μg/mL BV and stimulated with 10 μM rotenone to induce cell toxicity. We assessed cell death by Western blotting using specific antibodies, such as phospho-ERK1/2, phospho-JNK, and cleaved capase-3 and performed an MTT assay for evaluation of cell death and mitochondria staining. Pretreatment with 2.5 μg/mL BV had a neuroprotective effect against 10 μM rotenone-induced cell death in NSC34 motor neuron cells. Pre-treatment with BV significantly enhanced cell viability and ameliorated mitochondrial impairment in rotenone-treated cellular model. Moreover, BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. Taken together, we suggest that BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death. PMID:26402700

  7. Caspase Inhibition in Select Olfactory Neurons Restores Innate Attraction Behavior in Aged Drosophila

    PubMed Central

    Chihara, Takahiro; Takeuchi, Ken-ichi; Masuyama, Kaoru; Tonoki, Ayako; Davis, Ronald L.; Wang, Jing W.; Miura, Masayuki

    2014-01-01

    Sensory and cognitive performance decline with age. Neural dysfunction caused by nerve death in senile dementia and neurodegenerative disease has been intensively studied; however, functional changes in neural circuits during the normal aging process are not well understood. Caspases are key regulators of cell death, a hallmark of age-related neurodegeneration. Using a genetic probe for caspase-3-like activity (DEVDase activity), we have mapped age-dependent neuronal changes in the adult brain throughout the lifespan of Drosophila. Spatio-temporally restricted caspase activation was observed in the antennal lobe and ellipsoid body, brain structures required for olfaction and visual place memory, respectively. We also found that caspase was activated in an age-dependent manner in specific subsets of Drosophila olfactory receptor neurons (ORNs), Or42b and Or92a neurons. These neurons are essential for mediating innate attraction to food-related odors. Furthermore, age-induced impairments of neural transmission and attraction behavior could be reversed by specific inhibition of caspase in these ORNs, indicating that caspase activation in Or42b and Or92a neurons is responsible for altering animal behavior during normal aging. PMID:24967585

  8. Schwann cells induce neuronal differentiation of bone marrow stromal cells.

    PubMed

    Zurita, Mercedes; Vaquero, Jesús; Oya, Santiago; Miguel, Miriam

    2005-04-04

    Bone marrow stromal cells are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. Recently, bone marrow stromal cells have been shown to have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. We now describe how bone marrow stromal cells can be induced to differentiate into neuron-like cells when they are co-cultured with Schwann cells. When compared with chemical differentiation, expression of neuronal differentiation markers begins later, but one week after beginning co-culture, most bone marrow stromal cells showed a typical neuronal morphology. Our present findings support the transdifferentiation of bone marrow stromal cells, and the potential utility of these cells for the treatment of degenerative and acquired disorders of the nervous system.

  9. Hydroxysafflor Yellow A Protects Neurons From Excitotoxic Death through Inhibition of NMDARs

    PubMed Central

    Wang, Xingtao; Ma, Zhiyuan; Fu, Zhongxiao; Gao, Su; Yang, Liu; Jin, Yan; Sun, Hui; Wang, Chaoyun; Fan, Weiming; Chen, Lin; Zheng, Qing-Yin; Bi, Guoqiang

    2016-01-01

    Excessive glutamate release causes overactivation of N-methyl d-aspartate receptors (NMDARs), leading to excitatory neuronal damage in cerebral ischemia. Hydroxysafflor yellow A (HSYA), a compound extracted from Carthamus tinctorius L., has been reported to exert a neuroprotective effect in many pathological conditions, including brain ischemia. However, the underlying mechanism of HSYA's effect on neurons remains elusive. In the present study, we conducted experiments using patch-clamp recording of mouse hippocampal slices. In addition, we performed Ca2+ imaging, Western blots, as well as mitochondrial-targeted circularly permuted yellow fluorescent protein transfection into cultured hippocampal neurons in order to decipher the physiological mechanism underlying HSYA's neuroprotective effect. Through the electrophysiology experiments, we found that HSYA inhibited NMDAR-mediated excitatory postsynaptic currents without affecting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor and γ-aminobutyric acid A-type receptor-mediated currents. This inhibitory effect of HSYA on NMDARs was concentration dependent. HSYA did not show any preferential inhibition of either N-methyl d-aspartate receptor subtype 2A- or N-methyl d-aspartate receptor subtype 2B- subunit-containing NMDARs. Additionally, HSYA exhibits a facilitatory effect on paired NMDAR-mediated excitatory postsynaptic currents. Furthermore, HSYA reduced the magnitude of NMDAR-mediated membrane depolarization currents evoked by oxygen-glucose deprivation, and suppressed oxygen-glucose deprivation–induced and NMDAR-dependent ischemic long-term potentiation, which is believed to cause severe reperfusion damage after ischemia. Through the molecular biology experiments, we found that HSYA inhibited the NMDA-induced and NMDAR-mediated intracellular Ca2+ concentration increase in hippocampal cultures, reduced apoptotic and necrotic cell deaths, and prevented mitochondrial damage. Together, our data

  10. The Interglomerular Circuit Potently Inhibits Olfactory Bulb Output Neurons by Both Direct and Indirect Pathways

    PubMed Central

    Puche, Adam C.; Shipley, Michael T.

    2016-01-01

    Sensory processing shapes our perception. In mammals, odor information is encoded by combinatorial activity patterns of olfactory bulb (OB) glomeruli. Glomeruli are richly interconnected by short axon cells (SACs), which form the interglomerular circuit (IGC). It is unclear how the IGC impacts OB output to downstream neural circuits. We combined in vitro and in vivo electrophysiology with optogenetics in mice and found the following: (1) the IGC potently and monosynaptically inhibits the OB output neurons mitral/tufted cells (MTCs) by GABA release from SACs: (2) gap junction-mediated electrical coupling is strong for the SAC→MTC synapse, but negligible for the SAC→ETC synapse; (3) brief IGC-mediated inhibition is temporally prolonged by the intrinsic properties of MTCs; and (4) sniff frequency IGC activation in vivo generates persistent MTC inhibition. These findings suggest that the temporal sequence of glomerular activation by sensory input determines which stimulus features are transmitted to downstream olfactory networks and those filtered by lateral inhibition. SIGNIFICANCE STATEMENT Odor identity is encoded by combinatorial patterns of activated glomeruli, the initial signal transformation site of the olfactory system. Lateral circuit processing among activated glomeruli modulates olfactory signal transformation before transmission to higher brain centers. Using a combination of in vitro and in vivo optogenetics, this work demonstrates that interglomerular circuitry produces potent inhibition of olfactory bulb output neurons via direct chemical and electrical synapses as well as by indirect pathways. The direct inhibitory synaptic input engages mitral cell intrinsic membrane properties to generate inhibition that outlasts the initial synaptic action. PMID:27629712

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

    PubMed

    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.

  12. Inhibition of T-type calcium current in rat thalamocortical neurons by isoflurane

    PubMed Central

    Eckle, Veit-Simon; DiGruccio, Michael R.; Uebele, Victor N.; Renger, John J.; Todorovic, Slobodan M.

    2012-01-01

    Thalamocortical (TC) neurons provide the major sensory input to the mammalian somatosensory cortex. Decreased activity of these cells may be pivotal in the ability of general anesthetics to induce loss of consciousness and promote sleep (hypnosis). T-type voltage-gated calcium currents (T-currents) have a key function regulating the cellular excitability of TC neurons and previous studies have indicated that volatile general anesthetics may alter the excitability of these neurons. Using a patch-clamp technique, we investigated the mechanisms whereby isoflurane, a common volatile anesthetic, modulates isolated T-currents and T-current-dependent excitability of native TC neurons in acute brain slices of the rat. In voltage-clamp experiments, we found that isoflurane strongly inhibited peak amplitude of T-current, yielding an IC50 of 1.1% at physiological membrane potentials. Ensuing biophysical studies demonstrated that inhibition was more prominent at depolarized membrane potentials as evidenced by hyperpolarizing shifts in channel availability curves. In current-clamp experiments we found that isoflurane decreased the rate of depolarization of low-threshold-calcium spikes (LTCSs) and consequently increased the latency of rebound spike firing at the same concentrations that inhibited isolated T-currents. This effect was mimicked by a novel selective T-channel blocker 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2). In contrast, isoflurane and TTA-P2 had minimal effect on resting membrane potential and cell input resistance. We propose that depression of thalamic T-currents may contribute to some clinical properties of isoflurane. PMID:22491022

  13. Radial Glial Cell-Neuron Interaction Directs Axon Formation at the Opposite Side of the Neuron from the Contact Site.

    PubMed

    Xu, Chundi; Funahashi, Yasuhiro; Watanabe, Takashi; Takano, Tetsuya; Nakamuta, Shinichi; Namba, Takashi; Kaibuchi, Kozo

    2015-10-28

    How extracellular cues direct axon-dendrite polarization in mouse developing neurons is not fully understood. Here, we report that the radial glial cell (RGC)-cortical neuron interaction directs axon formation at the opposite side of the neuron from the contact site. N-cadherin accumulates at the contact site between the RGC and cortical neuron. Inhibition of the N-cadherin-mediated adhesion decreases this oriented axon formation in vitro, and disrupts the axon-dendrite polarization in vivo. Furthermore, the RGC-neuron interaction induces the polarized distribution of active RhoA at the contacting neurite and active Rac1 at the opposite neurite. Inhibition of Rho-Rho-kinase signaling in a neuron impairs the oriented axon formation in vitro, and prevents axon-dendrite polarization in vivo. Collectively, these results suggest that the N-cadherin-mediated radial glia-neuron interaction determines the contacting neurite as the leading process for radial glia-guided neuronal migration and directs axon formation to the opposite side acting through the Rho family GTPases.

  14. Distinct forms of synaptic inhibition and neuromodulation regulate calretinin-positive neuron excitability in the spinal cord dorsal horn.

    PubMed

    Smith, K M; Boyle, K A; Mustapa, M; Jobling, P; Callister, R J; Hughes, D I; Graham, B A

    2016-06-21

    The dorsal horn (DH) of the spinal cord contains a heterogenous population of neurons that process incoming sensory signals before information ascends to the brain. We have recently characterized calretinin-expressing (CR+) neurons in the DH and shown that they can be divided into excitatory and inhibitory subpopulations. The excitatory population receives high-frequency excitatory synaptic input and expresses delayed firing action potential discharge, whereas the inhibitory population receives weak excitatory drive and exhibits tonic or initial bursting discharge. Here, we characterize inhibitory synaptic input and neuromodulation in the two CR+ populations, in order to determine how each is regulated. We show that excitatory CR+ neurons receive mixed inhibition from GABAergic and glycinergic sources, whereas inhibitory CR+ neurons receive inhibition, which is dominated by glycine. Noradrenaline and serotonin produced robust outward currents in excitatory CR+ neurons, predicting an inhibitory action on these neurons, but neither neuromodulator produced a response in CR+ inhibitory neurons. In contrast, enkephalin (along with selective mu and delta opioid receptor agonists) produced outward currents in inhibitory CR+ neurons, consistent with an inhibitory action but did not affect the excitatory CR+ population. Our findings show that the pharmacology of inhibitory inputs and neuromodulator actions on CR+ cells, along with their excitatory inputs can define these two subpopulations further, and this could be exploited to modulate discrete aspects of sensory processing selectively in the DH.

  15. Neuronal uptake of pesticides disrupts chemosensory cells of nematodes.

    PubMed

    Winter, M D; McPherson, M J; Atkinson, H J

    2002-12-01

    Low doses of the acetylcholinesterase-inhibiting carbamate nematicides disrupt chemoreception in plant-parasitic nematodes. Fluorescein isothiocyanate (FITC)/dextran conjugates up to 12 kDa are taken up from the external medium by certain chemosensory neurons in Caenorhabditis elegans. Similar chemoreceptive neurons of the non-feeding infective stage of Heterodera glycines (soybean cyst nematode) fill with FITC and the nuclei of their cell bodies selectively stain with bisbenzimide. The widely used nematicide aldicarb disrupts the chemoreceptive response of H. glycines with 50% inhibition at very low concentrations (ca 1 pM), some 10(-6)-fold lower than required to affect locomotion. Similarly, the anthelmintic levamisole had this effect at 1 nM. Peptides selected as mimetics of aldicarb and levamisole also disrupt chemoreception in H. glycines and Globodera pallida at 10(-3)-fold or lower concentration than required to inhibit locomotion. We propose an uptake pathway for aldicarb, levamisole, peptide mimetics and other soluble molecules by retrograde transport along dendrites of chemoreceptive neurons to the cell bodies and synapses where they act. This may prove to be a general mechanism for the low-dose effects of some nematicides and anthelmintics.

  16. Maintenance of postmitotic neuronal cell identity

    PubMed Central

    Deneris, Evan S.; Hobert, Oliver

    2015-01-01

    The identity of specific cell types in the nervous system is defined by the expression of neuron type–specific gene batteries. How the expression of such batteries is initiated during nervous system development has been under intensive study over the past few decades. However, comparatively little is known about how gene batteries that define the terminally differentiated state of a neuron type are maintained throughout the life of an animal. We provide here an overview of studies in invertebrate and vertebrate model systems that have carved out the general and not commonly appreciated principle that neuronal identity is maintained in postmitotic neurons by the sustained, and often autoregulated expression of the same transcription factors that have initiated terminal differentiation in a developing organism. Disruption of postmitotic maintenance mechanisms may result in neuropsychiatric and neurodegenerative conditions. PMID:24929660

  17. Maintenance of postmitotic neuronal cell identity.

    PubMed

    Deneris, Evan S; Hobert, Oliver

    2014-07-01

    The identity of specific cell types in the nervous system is defined by the expression of neuron type-specific gene batteries. How the expression of such batteries is initiated during nervous system development has been under intensive study over the past few decades. However, comparatively little is known about how gene batteries that define the terminally differentiated state of a neuron type are maintained throughout the life of an animal. Here we provide an overview of studies in invertebrate and vertebrate model systems that have carved out the general and not commonly appreciated principle that neuronal identity is maintained in postmitotic neurons by the sustained, and often autoregulated, expression of the same transcription factors that initiate terminal differentiation in a developing organism. Disruption of postmitotic maintenance mechanisms may result in neuropsychiatric and neurodegenerative conditions.

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

    PubMed

    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. Detection of Temperature Difference in Neuronal Cells.

    PubMed

    Tanimoto, Ryuichi; Hiraiwa, Takumi; Nakai, Yuichiro; Shindo, Yutaka; Oka, Kotaro; Hiroi, Noriko; Funahashi, Akira

    2016-03-01

    For a better understanding of the mechanisms behind cellular functions, quantification of the heterogeneity in an organism or cells is essential. Recently, the importance of quantifying temperature has been highlighted, as it correlates with biochemical reaction rates. Several methods for detecting intracellular temperature have recently been established. Here we develop a novel method for sensing temperature in living cells based on the imaging technique of fluorescence of quantum dots. We apply the method to quantify the temperature difference in a human derived neuronal cell line, SH-SY5Y. Our results show that temperatures in the cell body and neurites are different and thus suggest that inhomogeneous heat production and dissipation happen in a cell. We estimate that heterogeneous heat dissipation results from the characteristic shape of neuronal cells, which consist of several compartments formed with different surface-volume ratios. Inhomogeneous heat production is attributable to the localization of specific organelles as the heat source.

  20. Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase.

    PubMed

    Shinozaki, Y; Sato, Y; Koizumi, S; Ohno, Y; Nagao, T; Inoue, K

    2007-06-15

    Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.

  1. Neuronal cell death in hepatic encephalopathy.

    PubMed

    Butterworth, Roger F

    2007-12-01

    It is generally assumed that neuronal cell death is minimal in liver failure and is insufficient to account for the neuropsychiatric symptoms characteristic of hepatic encephalopathy. However, contrary to this assumption, neuronal cell damage and death are well documented in liver failure patients, taking the form of several distinct clinical entities namely acquired (non-Wilsonian) hepatocerebral degeneration, cirrhosis-related Parkinsonism, post-shunt myelopathy and cerebellar degeneration. In addition, there is evidence to suggest that liver failure contributes to the severity of neuronal loss in Wernicke's encephalopathy. The long-standing nature of the thalamic and cerebellar lesions, over 80% of which are missed by routine clinical evaluation, together with the probability that they are nutritional in origin, underscores the need for careful nutritional management (adequate dietary protein, Vitamin B(1)) in liver failure patients. Mechanisms identified with the potential to cause neuronal cell death in liver failure include NMDA receptor-mediated excitotoxicity, lactic acidosis, oxidative/nitrosative stress and the presence of pro-inflammatory cytokines. The extent of neuronal damage in liver failure may be attenuated by compensatory mechanisms that include down-regulation of NMDA receptors, hypothermia and the presence of neuroprotective steroids such as allopregnanolone. These findings suggest that some of the purported "sequelae" of liver transplantation (gait ataxia, memory loss, confusion) could reflect preexisting neuropathology.

  2. Gut-neuron interaction via Hh signaling regulates intestinal progenitor cell differentiation in Drosophila.

    PubMed

    Han, Hui; Pan, Chenyu; Liu, Chunying; Lv, Xiangdong; Yang, Xiaofeng; Xiong, Yue; Lu, Yi; Wu, Wenqing; Han, Junhai; Zhou, Zhaocai; Jiang, Hai; Zhang, Lei; Zhao, Yun

    2015-01-01

    Intestinal homeostasis is maintained by intestinal stem cells (ISCs) and their progenies. A complex autonomic nervous system spreads over posterior intestine. However, whether and how neurons regulate posterior intestinal homeostasis is largely unknown. Here we report that neurons regulate Drosophila posterior intestinal homeostasis. Specifically, downregulation of neuronal Hedgehog (Hh) signaling inhibits the differentiation of ISCs toward enterocytes (ECs), whereas upregulated neuronal Hh signaling promotes such process. We demonstrate that, among multiple sources of Hh ligand, those secreted by ECs induces similar phenotypes as does neuronal Hh. In addition, intestinal JAK/STAT signaling responds to activated neuronal Hh signaling, suggesting that JAK/STAT signaling acts downstream of neuronal Hh signaling in intestine. Collectively, our results indicate that neuronal Hh signaling is essential for the determination of ISC fate.

  3. Gut–neuron interaction via Hh signaling regulates intestinal progenitor cell differentiation in Drosophila

    PubMed Central

    Han, Hui; Pan, Chenyu; Liu, Chunying; Lv, Xiangdong; Yang, Xiaofeng; Xiong, Yue; Lu, Yi; Wu, Wenqing; Han, Junhai; Zhou, Zhaocai; Jiang, Hai; Zhang, Lei; Zhao, Yun

    2015-01-01

    Intestinal homeostasis is maintained by intestinal stem cells (ISCs) and their progenies. A complex autonomic nervous system spreads over posterior intestine. However, whether and how neurons regulate posterior intestinal homeostasis is largely unknown. Here we report that neurons regulate Drosophila posterior intestinal homeostasis. Specifically, downregulation of neuronal Hedgehog (Hh) signaling inhibits the differentiation of ISCs toward enterocytes (ECs), whereas upregulated neuronal Hh signaling promotes such process. We demonstrate that, among multiple sources of Hh ligand, those secreted by ECs induces similar phenotypes as does neuronal Hh. In addition, intestinal JAK/STAT signaling responds to activated neuronal Hh signaling, suggesting that JAK/STAT signaling acts downstream of neuronal Hh signaling in intestine. Collectively, our results indicate that neuronal Hh signaling is essential for the determination of ISC fate. PMID:27462407

  4. Phasic, suprathreshold excitation and sustained inhibition underlie neuronal selectivity for short-duration sounds

    PubMed Central

    Rose, Gary J.; Hanson, Jessica L.; Leary, Christopher J.; Vasquez-Opazo, Gustavo A.; Graham, Jalina A.; Wilkerson, Jeremy

    2016-01-01

    Sound duration is important in acoustic communication, including speech recognition in humans. Although duration-selective auditory neurons have been found, the underlying mechanisms are unclear. To investigate these mechanisms we combined in vivo whole-cell patch recordings from midbrain neurons, extraction of excitatory and inhibitory conductances, and focal pharmacological manipulations. We show that selectivity for short-duration stimuli results from integration of short-latency, sustained inhibition with delayed, phasic excitation; active membrane properties appeared to amplify responses to effective stimuli. Blocking GABAA receptors attenuated stimulus-related inhibition, revealed suprathreshold excitation at all stimulus durations, and decreased short-pass selectivity without changing resting potentials. Blocking AMPA and NMDA receptors to attenuate excitation confirmed that inhibition tracks stimulus duration and revealed no evidence of postinhibitory rebound depolarization inherent to coincidence models of duration selectivity. These results strongly support an anticoincidence mechanism of short-pass selectivity, wherein inhibition and suprathreshold excitation show greatest temporal overlap for long duration stimuli. PMID:26976602

  5. Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

    PubMed Central

    Takeda, Yuji S.; Xu, Qiaobing

    2015-01-01

    Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC. PMID:26248331

  6. Neuroprotective effect of acute melatonin treatment on hippocampal neurons against irradiation by inhibition of caspase-3

    PubMed Central

    LI, JIANGUO; ZHANG, GUOWEI; MENG, ZHUANGZHI; WANG, LINGZHAN; LIU, HAIYING; LIU, QIANG; BUREN, BATU

    2016-01-01

    Neuronal cell apoptosis is associated with various factors that induce neurological damage, including radiation exposure. When administered prior to exposure to radiation, a protective agent may prevent cellular and molecular injury. The present study aimed to investigate whether melatonin exerts a neuroprotective effect by inhibiting the caspase cell death pathway. Male Sprague-Dawley rats were administered melatonin (100 mg/kg body weight) 30 min prior to radiation exposure in red light during the evening. In order to elucidate whether melatonin has a neuroprotective role, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling, Nissl staining, reverse transcription-quantitative polymerase chain reaction, reactive oxygen species analysis and western blotting were performed. At 24 h post-melatonin treatment, caspase-3 mRNA and protein expression levels were significantly decreased. These results demonstrated that melatonin may protect hippocampal neurons via the inhibition of caspase-3 when exposed to irradiation. Therefore, caspase-3 inhibition serves a neuroprotective and antioxidant role in the interventional treatment of melatonin. The results of the present study suggested that melatonin may have a potential therapeutic effect against irradiation; however, further studies are required in order to elucidate the underlying antioxidant mechanisms. PMID:27313671

  7. Neuronal cell sheet of cortical motor neuron phenotype derived from human iPS cells.

    PubMed

    Suzuki, Noboru; Arimitsu, Nagisa; Shimizu, Jun; Takai, Kenji; Hirotsu, Chieko; Takada, Erika; Ueda, Yuji; Wakisaka, Sueshige; Fujiwara, Naruyoshi; Suzuki, Tomoko

    2017-03-17

    Transplantation of stem cells which differentiate into more mature neural cells brings about functional improvement in pre-clinical studies of stroke. Previous transplant approaches in diseased brain have utilized injection of the cells in a cell suspension. In addition, neural stem cells were preferentially used as graft. However, these cells had no specific relationship to the damaged tissue of stroke patients and brain injury. The injection of cells in a suspension destroyed the cell-cell interactions that are suggested to be important for promoting functional integrity as cortical motor neurons.

    In order to obtain suitable cell types for grafting patients with stroke and brain damage, we have modified a protocol for differentiating human iPS cells to cells phenotypically related to cortical motor neurons. Moreover, we applied cell sheet technology to neural cell transplantation due to the idea in which keeping cell-cell communications was regarded as important for the repair of host brain architecture.

    Accordingly, we developed neuronal cell sheets being positive for FEZ family zinc finger 2 (Fezf2), COUP-TF-interacting protein 2 (CTIP2), insulin-like growth factor-binding protein 4 (Igfbp4), cysteine-rich motor neuron 1 protein precursor (CRIM1) and forkhead box p2 (Foxp2). These markers are associated with cortical motoneuron which is appropriate for the transplant location in the lesions. The sheets allowed preservation of cell-cell interactions shown by synapsin1 staining after transplantation to damaged mouse brain. The sheet transplantation brought about structural restoration partly and improvement of motor functions in hemiplegic mice.

    Collectively, the cell sheets were transplanted to damaged motor cortex in a way of a novel neuronal cell sheet that maintained cell-cell interactions and improved motor functions of the hemiplegic model mice. The motoneuron cell sheets are possibly applicable for stroke patients and patients with

  8. Tuberculoventral neurons project to the multipolar cell area but not to the octopus cell area of the posteroventral cochlear nucleus.

    PubMed

    Wickesberg, R E; Whitlon, D; Oertel, D

    1991-11-15

    Tuberculoventral neurons in the deep layer of the dorsal cochlear nucleus (DCN) provide frequency-specific inhibition to neurons in the anteroventral cochlear nucleus (AVCN) of the mouse (Wickesberg and Oertel, '88, '90). The present experiments examine the projection from the deep DCN to the posteroventral cochlear nucleus (PVCN). Horseradish peroxidase (HRP) injections into the PVCN reveal that the multipolar cell area, but not the octopus cell area, is innervated by neurons in the deep layer of the DCN. Injections into the multipolar cell area, in the rostral and ventral PVCN, labeled neurons across the entire rostrocaudal extent of the deep DCN. The labeled tuberculoventral neurons generally lay within the band of labeled auditory nerve terminals in the DCN. Injections of HRP into the octopus cell area, in the dorsal caudal PVCN, labeled almost no cells within the band of auditory nerve fiber terminals that were labeled by the same injection. The inhibition from tuberculoventral neurons onto ventral cochlear nucleus (VCN) neurons is likely to be mediated by glycine (Wickesberg and Oertel, '90). Slices of the cochlear nuclear complex were immunolabeled by an antibody against glycine conjugated with glutaraldehyde to bovine serum albumin (Wenthold et al., '87). Glycine-like immunoreactivity was found throughout the DCN, the AVCN and the multipolar cell area, but there was little labeling in the octopus cell area. This finding provides independent evidence that tuberculoventral neurons do not innervate the octopus cell area and indicates that the octopus cell area is anatomically and functionally distinct.

  9. Frequency tuning of synaptic inhibition underlying duration-tuned neurons in the mammalian inferior colliculus.

    PubMed

    Valdizón-Rodríguez, Roberto; Faure, Paul A

    2017-04-01

    Inhibition plays an important role in creating the temporal response properties of duration-tuned neurons (DTNs) in the mammalian inferior colliculus (IC). Neurophysiological and computational studies indicate that duration selectivity in the IC is created through the convergence of excitatory and inhibitory synaptic inputs offset in time. We used paired-tone stimulation and extracellular recording to measure the frequency tuning of the inhibition acting on DTNs in the IC of the big brown bat (Eptesicus fuscus). We stimulated DTNs with pairs of tones differing in duration, onset time, and frequency. The onset time of a short, best-duration (BD), probe tone set to the best excitatory frequency (BEF) was varied relative to the onset of a longer-duration, nonexcitatory (NE) tone whose frequency was varied. When the NE tone frequency was near or within the cell's excitatory bandwidth (eBW), BD tone-evoked spikes were suppressed by an onset-evoked inhibition. The onset of the spike suppression was independent of stimulus frequency, but both the offset and duration of the suppression decreased as the NE tone frequency departed from the BEF. We measured the inhibitory frequency response area, best inhibitory frequency (BIF), and inhibitory bandwidth (iBW) of each cell. We found that the BIF closely matched the BEF, but the iBW was broader and usually overlapped the eBW measured from the same cell. These data suggest that temporal selectivity of midbrain DTNs is created and preserved by having cells receive an onset-evoked, constant-latency, broadband inhibition that largely overlaps the cell's excitatory receptive field. We conclude by discussing possible neural sources of the inhibition.NEW & NOTEWORTHY Duration-tuned neurons (DTNs) arise from temporally offset excitatory and inhibitory synaptic inputs. We used single-unit recording and paired-tone stimulation to measure the spectral tuning of the inhibitory inputs to DTNs. The onset of inhibition was independent of

  10. Human pluripotent stem cell differentiation into authentic striatal projection neurons.

    PubMed

    Delli Carri, Alessia; Onorati, Marco; Castiglioni, Valentina; Faedo, Andrea; Camnasio, Stefano; Toselli, Mauro; Biella, Gerardo; Cattaneo, Elena

    2013-08-01

    Here we present the principles and steps of a protocol that we have recently developed for the differentiation of hES/iPS cells into the authentic human striatal projection medium spiny neurons (MSNs) that die in Huntington's Disease (HD). Authenticity is judged by the convergence of multiple features within individual cells. Our procedure lasts 80 days and couples neural induction via BMP/TGF-β inhibition with exposure to the developmental factors sonic hedgehog (SHH) and dickkopf1 (DKK-1) to drive ventral telencephalic specification, followed by terminal differentiation [1]. Authenticity of the resulting neuronal population is monitored by the appearance of FOXG1(+)/GSX2(+) progenitor cells of the lateral ganglionic eminence (LGE) at day 15-25 of differentiation, followed by appearance of CTIP2-, FOXP1- and FOXP2-positive cells at day 45. These precursor cells then mature into MAP2(+)/GABA(+) neurons with 20 % of them ultimately co-expressing the DARPP-32 and CTIP2 diagnostic markers and carrying electrophysiological properties expected for fully functional MSNs.The protocol is characterized by its replicability in at least three human pluripotent cell lines. Altogether this protocol defines a useful platform for in vitro developmental neurobiology studies, drug screening, and regenerative medicine approaches.

  11. Unique processing during a period of high excitation/inhibition balance in adult-born neurons.

    PubMed

    Marín-Burgin, Antonia; Mongiat, Lucas A; Pardi, M Belén; Schinder, Alejandro F

    2012-03-09

    The adult dentate gyrus generates new granule cells (GCs) that develop over several weeks and integrate into the preexisting network. Although adult hippocampal neurogenesis has been implicated in learning and memory, the specific role of new GCs remains unclear. We examined whether immature adult-born neurons contribute to information encoding. By combining calcium imaging and electrophysiology in acute slices, we found that weak afferent activity recruits few mature GCs while activating a substantial proportion of the immature neurons. These different activation thresholds are dictated by an enhanced excitation/inhibition balance transiently expressed in immature GCs. Immature GCs exhibit low input specificity that switches with time toward a highly specific responsiveness. Therefore, activity patterns entering the dentate gyrus can undergo differential decoding by a heterogeneous population of GCs originated at different times.

  12. Ethanol inhibits histaminergic neurons in mouse tuberomammillary nucleus slices via potentiating GABAergic transmission onto the neurons at both pre- and postsynaptic sites

    PubMed Central

    Sun, Yu; Jiang, Shi-yu; Ni, Jian; Luo, Yan-jia; Chen, Chang-rui; Hong, Zong-yuan; Yanagawa, Yuchio; Qu, Wei-min; Wang, Lu; Huang, Zhi-li

    2016-01-01

    Aim: Ethanol, one of the most frequently used and abused substances in our society, has a profound impact on sedation. However, the neuronal mechanisms underlying its sedative effect remain unclear. In this study, we investigated the effects of ethanol on histaminergic neurons in the tuberomammillary nucleus (TMN), a brain region thought to be critical for wakefulness. Methods: Coronal brain slices (250 μm thick) containing the TMN were prepared from GAD67-GFP knock-in mice. GAD67-GFP was used to identify histaminergic neurons in the TMN. The spontaneous firing and membrane potential of histaminergic neurons, and GABAergic transmission onto these neurons were recorded using whole-cell patch-clamp recordings. Drugs were applied through superfusion. Results: Histaminergic and GAD67-expressing neurons in the TMN of GAD67-GFP mice were highly co-localized. TMN GFP-positive neurons exhibited a regular spontaneous discharge at a rate of 2–4 Hz without burst firing. Brief superfusion of ethanol (64, 190, and 560 mmol/L) dose-dependently and reversibly suppressed the spontaneous firing of the neurons in the TMN; when synaptic transmission was blocked by tetrodotoxin (1 μmol/L), ethanol caused hyperpolarization of the membrane potential. Furthermore, superfusion of ethanol markedly increased the frequency and amplitude of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs), which were abolished in the presence of the GABAA receptor antagonist bicuculline (20 μmol/L). Finally, ethanol-mediated enhancement of sIPSCs and mIPSCs was significantly attenuated when the slices were pretreated with the GABAB agonist baclofen (30 μmol/L). Conclusion: Ethanol inhibits the excitability of histaminergic neurons in mouse TMN slices, possibly via potentiating GABAergic transmission onto the neurons at both pre- and postsynaptic sites. PMID:27498778

  13. The tricyclic antidepressant desipramine inhibits T3 import into primary neurons.

    PubMed

    Roth, Stephan; Kinne, Anita; Schweizer, Ulrich

    2010-06-30

    Transport of thyroid hormones across the plasma membrane is required for binding to their nuclear receptors. Monocarboxylate transporter 8 (MCT8) is a plasma membrane thyroid hormone transport protein, which has recently gained much attention, since mutations in MCT8 are associated with severe mental retardation in patients afflicted with the Allan-Herndon-Dudley syndrome. MCT8 is expressed along the blood-brain-barrier and on central neurons. We have found that desipramine (DMI), a tricyclic antidepressant, acts as an inhibitor of thyroid hormone transport by MCT8. Uptake of 3,5,3'-triiodo-L-thyronine (T(3)) into primary cortical neurons could be blocked with desipramine as well as with the known, but unspecific, inhibitor bromosulphtalein (BSP). T(3) uptake by neurons derived from Mct8-deficient cells was not further decreased by DMI. In a heterologous expression system, both human MCT8 and its close homolog, MCT10, were sensitive to inhibition by DMI. Kinetic experiments demonstrated a non-competitive mode of inhibition. Numerous interactions between thyroid hormones, depressive symptoms, and antidepressant treatments have been reported in the literature. Our findings add to the evidence that antidepressant drugs may affect CNS thyroid hormone function.

  14. Two PTP receptors mediate CSPG inhibition by convergent and divergent signaling pathways in neurons

    PubMed Central

    Ohtake, Yosuke; Wong, Daniella; Abdul-Muneer, P. M.; Selzer, Michael E.; Li, Shuxin

    2016-01-01

    Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways (RhoA, Akt and Erk), but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures in vitro. Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors. PMID:27849007

  15. Insulin inhibits AMPA-induced neuronal damage via stimulation of protein kinase B (Akt).

    PubMed

    Kim, S-J; Han, Y

    2005-02-01

    We designed a series of experiments to explore the neuroprotective effects of insulin. Insulin significantly inhibited the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced neuronal cell damage as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. However, insulin had little affect on the AMPA-induced glial cell damage. To determine whether insulin inhibits AMPA-induced excitotoxicity, we performed grease-gap recording assays using rat brain slices. In these experiments, insulin also significantly inhibited AMPA-induced depolarization. Flow cytometry and DNA fragmentation assays showed that insulin inhibits AMPA-induced apoptosis and DNA fragmentation, respectively. Insulin stimulated protein kinase B (Akt) activity, whereas AMPA pretreatment did not alter the insulin-stimulated Akt activity. On the contrary, insulin blocked induction of SAPK/JNK, which AMPA stimulated. Taken together, these results suggest that insulin exerts neuroprotective effects by inhibiting AMPA-induced excitotoxicity and apoptosis, possibly by activating Akt and blocking SAPK/JNK.

  16. Starvation and inhibition of lysosomal function increased tau secretion by primary cortical neurons.

    PubMed

    Mohamed, Nguyen-Vi; Plouffe, Vanessa; Rémillard-Labrosse, Gaudeline; Planel, Emmanuel; Leclerc, Nicole

    2014-07-17

    Recent studies have demonstrated that human tau can be secreted by neurons and non-neuronal cells, an event linked to the propagation of tau pathology in the brain. In the present study, we confirmed that under physiological conditions, one tau-positive band was detected in the culture medium with an anti-tau antibody recognizing total tau and the Tau-1 antibody directed against unphosphorylated tau. We then examined whether tau secretion was modified upon insults. Tau secretion was increased by starvation [Earle's Balanced Salt Solution (EBSS)], inhibition of lysosomal function (leupeptin) and when both of these conditions were superimposed, this combined treatment having the most important effects on tau secretion. Interestingly, the pattern of tau secretion was distinct from that of control neurons when neurons were treated either with EBSS alone or EBSS + leupeptin. In these conditions, three tau-positive bands were detected in the culture medium. Two of these three bands were immunoreactive to Tau-1 antibody revealing that at least two tau species were released upon these treatments. Collectively, our results indicate that insults such as nutrient deprivation and lysosomal dysfunction observed in neurodegenerative diseases could result in an increase of tau secretion and propagation of tau pathology in the brain.

  17. Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels

    PubMed Central

    Bautista, Diana M; Sigal, Yaron M; Milstein, Aaron D; Garrison, Jennifer L; Zorn, Julie A; Tsuruda, Pamela R; Nicoll, Roger A; Julius, David

    2011-01-01

    In traditional folk medicine, Xanthoxylum plants are referred to as ‘toothache trees’ because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-α-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-α-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-α-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience. PMID:18568022

  18. Pungent agents from Szechuan peppers excite sensory neurons by inhibiting two-pore potassium channels.

    PubMed

    Bautista, Diana M; Sigal, Yaron M; Milstein, Aaron D; Garrison, Jennifer L; Zorn, Julie A; Tsuruda, Pamela R; Nicoll, Roger A; Julius, David

    2008-07-01

    In traditional folk medicine, Xanthoxylum plants are referred to as 'toothache trees' because their anesthetic or counter-irritant properties render them useful in the treatment of pain. Psychophysical studies have identified hydroxy-alpha-sanshool as the compound most responsible for the unique tingling and buzzing sensations produced by Szechuan peppercorns or other Xanthoxylum preparations. Although it is generally agreed that sanshool elicits its effects by activating somatosensory neurons, the underlying cellular and molecular mechanisms remain a matter of debate. Here we show that hydroxy-alpha-sanshool excites two types of sensory neurons, including small-diameter unmyelinated cells that respond to capsaicin (but not mustard oil) as well as large-diameter myelinated neurons that express the neurotrophin receptor TrkC. We found that hydroxy-alpha-sanshool excites neurons through a unique mechanism involving inhibition of pH- and anesthetic-sensitive two-pore potassium channels (KCNK3, KCNK9 and KCNK18), providing a framework for understanding the unique and complex psychophysical sensations associated with the Szechuan pepper experience.

  19. Estradiol Rapidly Attenuates ORL-1 Receptor-Mediated Inhibition of Proopiomelanocortin Neurons via Gq-Coupled, Membrane-Initiated Signaling.

    PubMed

    Conde, Kristie; Meza, Cecilia; Kelly, Martin J; Sinchak, Kevin; Wagner, Edward J

    2016-01-01

    Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiological recordings were generated in ARH neurons held at or near -60 mV, and neuronal phenotype was determined post hoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via G protein-gated, inwardly rectifying K+ (GIRK) channels that were attenuated by pretreatment with either 17-β estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and mimicked by the Gq-coupled membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

  20. Estradiol rapidly attenuates ORL-1 receptor-mediated inhibition of proopiomelanocortin neurons via Gq-coupled, membrane-initiated signaling

    PubMed Central

    Conde, Kristie; Meza, Cecilia; Kelly, Martin J.; Sinchak, Kevin; Wagner, Edward J.

    2016-01-01

    Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiologic recordings were generated in ARH neurons held at or near −60 mV, and neuronal phenotype was determined posthoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via GIRK channels that were attenuated by pretreatment with either 17-β estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780, and mimicked by the Gq-coupled, membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-coupled mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS. PMID:26765570

  1. Melanocortin 4 receptor constitutive activity inhibits L-type voltage-gated calcium channels in neurons.

    PubMed

    Agosti, F; Cordisco Gonzalez, S; Martinez Damonte, V; Tolosa, M J; Di Siervi, N; Schioth, H B; Davio, C; Perello, M; Raingo, J

    2017-03-27

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain nuclei playing a crucial role in the regulation of energy balance controlling the homeostasis of the organism. It displays both agonist-evoked and constitutive activity, and moreover, it can couple to different G proteins. Most of the research on MC4R has been focused on agonist-induced activity, while the molecular and cellular basis of MC4R constitutive activity remains scarcely studied. We have previously shown that neuronal N-type voltage-gated calcium channels (CaV2.2) are inhibited by MC4R agonist-dependent activation, while the CaV subtypes that carry L- and P/Q-type current are not. Here, we tested the hypothesis that MC4R constitutive activity can affect CaV, with focus on the channel subtypes that can control transcriptional activity coupled to depolarization (L-type, CaV1.2/1.3) and neurotransmitter release (N- and P/Q-type, CaV2.2 and CaV2.1). We found that MC4R constitutive activity inhibits specifically CaV1.2/1.3 and CaV2.1 subtypes of CaV. We also explored the signaling pathways mediating this inhibition, and thus propose that agonist-dependent and basal MC4R activation modes signal differentially through Gs and Gi/o pathways to impact on different CaV subtypes. In addition, we found that chronic incubation with MC4R endogenous inverse agonist, agouti and agouti-related peptide (AgRP), occludes CaV inhibition in a cell line and in amygdaloid complex cultured neurons as well. Thus, we define new mechanisms of control of the main mediators of depolarization-induced calcium entry into neurons by a GPCR that displays constitutive activity.

  2. Reactive nucleolar and Cajal body responses to proteasome inhibition in sensory ganglion neurons.

    PubMed

    Palanca, Ana; Casafont, Iñigo; Berciano, María T; Lafarga, Miguel

    2014-06-01

    The dysfunction of the ubiquitin proteasome system has been related to a broad array of neurodegenerative disorders in which the accumulation of misfolded protein aggregates causes proteotoxicity. The ability of proteasome inhibitors to induce cell cycle arrest and apoptosis has emerged as a powerful strategy for cancer therapy. Bortezomib is a proteasome inhibitor used as an antineoplastic drug, although its neurotoxicity frequently causes a severe sensory peripheral neuropathy. In this study we used a rat model of bortezomib treatment to study the nucleolar and Cajal body responses to the proteasome inhibition in sensory ganglion neurons that are major targets of bortezomib-induced neurotoxicity. Treatment with bortezomib induced dose-dependent dissociation of protein synthesis machinery (chromatolysis) and nuclear retention of poly(A) RNA granules resulting in neuronal dysfunction. However, as a compensatory response to the proteotoxic stress, both nucleoli and Cajal bodies exhibited reactive changes. These include an increase in the number and size of nucleoli, strong nucleolar incorporation of the RNA precursor 5'-fluorouridine, and increased expression of both 45S rRNA and genes encoding nucleolar proteins UBF, fibrillarin and B23. Taken together, these findings appear to reflect the activation of the nucleolar transcription in response to proteotoxic stress Furthermore, the number of Cajal bodies, a parameter related to transcriptional activity, increases upon proteasome inhibition. We propose that nucleoli and Cajal bodies are important targets in the signaling pathways that are activated by the proteotoxic stress response to proteasome inhibition. The coordinating activity of these two organelles in the production of snRNA, snoRNA and rRNA may contribute to neuronal survival after proteasome inhibition. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

  3. PIP₂ hydrolysis is responsible for voltage independent inhibition of CaV2.2 channels in sympathetic neurons.

    PubMed

    Vivas, Oscar; Castro, Hector; Arenas, Isabel; Elías-Viñas, David; García, David E

    2013-03-08

    GPCRs regulate Ca(V)2.2 channels through both voltage dependent and independent inhibition pathways. The aim of the present work was to assess the phosphatidylinositol-4,5-bisphosphate (PIP2) as the molecule underlying the voltage independent inhibition of Ca(V)2.2 channels in SCG neurons. We used a double pulse protocol to study the voltage independent inhibition and changed the PIP(2) concentration by means of blocking the enzyme PLC, filling the cell with a PIP(2) analogue and preventing the PIP(2) resynthesis with wortmannin. We found that voltage independent inhibition requires the activation of PLC and can be hampered by internal dialysis of exogenous PIP(2). In addition, the recovery from voltage independent inhibition is blocked by inhibition of the enzymes involved in the resynthesis of PIP(2). These results support that the hydrolysis of PIP(2) is responsible for the voltage independent inhibition of Ca(V)2.2 channels.

  4. Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease

    PubMed Central

    Sánchez-Pernaute, Rosario; Ferree, Andrew; Cooper, Oliver; Yu, Meixiang; Brownell, Anna-Liisa; Isacson, Ole

    2004-01-01

    Several lines of evidence point to a significant role of neuroinflammation in Parkinson's disease (PD) and other neurodegenerative disorders. In the present study we examined the protective effect of celecoxib, a selective inhibitor of the inducible form of cyclooxygenase (COX-2), on dopamine (DA) cell loss in a rat model of PD. We used the intrastriatal administration of 6-hydroxydopamine (6-OHDA) that induces a retrograde neuronal damage and death, which progresses over weeks. Animals were randomized to receive celecoxib (20 mg/kg/day) or vehicle starting 1 hour before the intrastriatal administration of 6-OHDA. Evaluation was performed in vivo using micro PET and selective radiotracers for DA terminals and microglia. Post mortem analysis included stereological quantification of tyrosine hydroxylase, astrocytes and microglia. 12 days after the 6-OHDA lesion there were no differences in DA cell or fiber loss between groups, although the microglial cell density and activation was markedly reduced in animals receiving celecoxib (p < 0.01). COX-2 inhibition did not reduce the typical astroglial response in the striatum at any stage. Between 12 and 21 days, there was a significant progression of DA cell loss in the vehicle group (from 40 to 65%) that was prevented by celecoxib. Therefore, inhibition of COX-2 by celecoxib appears to be able, either directly or through inhibition of microglia activation to prevent or slow down DA cell degeneration. PMID:15285796

  5. Effect of GABAergic inhibition on odorant concentration coding in mushroom body intrinsic neurons of the honeybee.

    PubMed

    Froese, Anja; Szyszka, Paul; Menzel, Randolf

    2014-03-01

    Kenyon cells, the intrinsic neurons of the insect mushroom body, have the intriguing property of responding in a sparse way to odorants. Sparse neuronal codes are often invariant to changes in stimulus intensity and duration, and sparse coding often depends on global inhibition. We tested if this is the case for honeybees' Kenyon cells, too, and used in vivo Ca²⁺ imaging to record their responses to different odorant concentrations. Kenyon cells responded not only to the onset of odorant stimuli (ON responses), but also to their termination (OFF responses). Both, ON and OFF responses increased with increasing odorant concentration. ON responses were phasic and invariant to the duration of odorant stimuli, while OFF responses increased with increasing odorant duration. Pharmacological blocking of GABA receptors in the brain revealed that ionotropic GABA(A) and metabotropic GABA(B) receptors attenuate Kenyon cells' ON responses without changing their OFF responses. Ionotropic GABA(A) receptors attenuated Kenyon cell ON responses more strongly than metabotropic GABA(B) receptors. However, the response dynamic, temporal resolution and paired-pulse depression did not depend on GABA(A) transmission. These data are discussed in the context of mechanisms leading to sparse coding in Kenyon cells.

  6. Thrombospondin-4 Promotes Neuronal Differentiation of NG2 Cells via the ERK/MAPK Pathway.

    PubMed

    Yang, Hai Jie; Ma, Shuang Ping; Ju, Fei; Zhang, Ya Ping; Li, Zhi Chao; Zhang, Bin Bin; Lian, Jun Jiang; Wang, Lei; Cheng, Bin Feng; Wang, Mian; Feng, Zhi Wei

    2016-12-01

    NG2-expressing neural progenitors can produce neurons in the central nervous system, providing a potential cell resource of therapy for neurological disorders. However, the mechanism underlying neuronal differentiation of NG2 cells remains largely unknown. In this report, we found that a thrombospondin (TSP) family member, TSP4, is involved in the neuronal differentiation of NG2 cells. When TSP4 was overexpressed, NG2 cells underwent spontaneous neuronal differentiation, as demonstrated by the induction of various neuronal differentiation markers such as NeuN, Tuj1, and NF200, at the messenger RNA and protein levels. In contrast, TSP4 silencing had an opposite effect on the expression of neuronal differentiation markers in NG2 cells. Next, the signaling pathway responsible for TSP4-mediated NG2 cell differentiation was investigated. We found that ERK but not p38 and AKT signaling was affected by TSP4 overexpression. Furthermore, when ERK signaling was blocked by the inhibitor U0126, the neuronal marker expression of NG2 cells was substantially increased. Together, these findings suggested that TSP4 promoted neuronal differentiation of NG2 cells by inhibiting ERK/MAPK signaling, revealing a novel role of TSP4 in cell fate specification of NG2 cells.

  7. Inhibition of propofol on single neuron and neuronal ensemble activity in prefrontal cortex of rats during working memory task.

    PubMed

    Xu, Xinyu; Tian, Yu; Wang, Guolin; Tian, Xin

    2014-08-15

    Working memory (WM) refers to the temporary storage and manipulation of information necessary for performance of complex cognitive tasks. There is a growing interest in whether and how propofol anesthesia inhibits WM function. The aim of this study is to investigate the possible inhibition mechanism of propofol anesthesia from the view of single neuron and neuronal ensemble activities. Adult SD rats were randomly divided into two groups: propofol group (0.9 mg kg(-1)min(-1), 2h via a tail vein catheter) and control group. All the rats were tested for working memory performances in a Y-maze-rewarded alternation task (a task of delayed non-matched-to-sample) at 24, 48, 72 h after propofol anesthesia, and the behavior results of WM tasks were recorded at the same time. Spatio-temporal trains of action potentials were obtained from the original signals. Single neuron activity was characterized by peri-event time histograms analysis and neuron ensemble activities were characterized by Granger causality to describe the interactions within the neuron ensemble. The results show that: comparing with the control group, the percentage of neurons excited and related to WM was significantly decreased (p<0.01 in 24h, p<0.05 in 48 h); the interactions within neuron ensemble were significantly weakened (p<0.01 in 24h, p<0.05 in 48 h), whereas no significant difference in 72 h (p>0.05), which were consistent with the behavior results. These findings could lead to improved understanding of the mechanism of anesthesia inhibition on WM functions from the view of single neuron activity and neuron ensemble interactions.

  8. Murine Mueller cells are progenitor cells for neuronal cells and fibrous tissue cells

    SciTech Connect

    Florian, Christian; Langmann, Thomas; Weber, Bernhard H.F.; Morsczeck, Christian

    2008-09-19

    Mammalian Mueller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Mueller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Mueller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Mueller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Mueller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.

  9. WNT3 Inhibits Cerebellar Granule Neuron Progenitor Proliferation and Medulloblastoma Formation via MAPK Activation

    PubMed Central

    Ayrault, Olivier; Kim, Jee Hae; Zhu, Xiaodong; Murphy, David A.; Van Aelst, Linda; Roussel, Martine F.; Hatten, Mary E.

    2013-01-01

    During normal cerebellar development, the remarkable expansion of granule cell progenitors (GCPs) generates a population of granule neurons that outnumbers the total neuronal population of the cerebral cortex, and provides a model for identifying signaling pathways that may be defective in medulloblastoma. While many studies focus on identifying pathways that promote growth of GCPs, a critical unanswered question concerns the identification of signaling pathways that block mitogenic stimulation and induce early steps in differentiation. Here we identify WNT3 as a novel suppressor of GCP proliferation during cerebellar development and an inhibitor of medulloblastoma growth in mice. WNT3, produced in early postnatal cerebellum, inhibits GCP proliferation by down-regulating pro-proliferative target genes of the mitogen Sonic Hedgehog (SHH) and the bHLH transcription factor Atoh1. WNT3 suppresses GCP growth through a non-canonical Wnt signaling pathway, activating prototypic mitogen-activated protein kinases (MAPKs), the Ras-dependent extracellular-signal-regulated kinases 1/2 (ERK1/2) and ERK5, instead of the classical β-catenin pathway. Inhibition of MAPK activity using a MAPK kinase (MEK) inhibitor reversed the inhibitory effect of WNT3 on GCP proliferation. Importantly, WNT3 inhibits proliferation of medulloblastoma tumor growth in mouse models by a similar mechanism. Thus, the present study suggests a novel role for WNT3 as a regulator of neurogenesis and repressor of neural tumors. PMID:24303070

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

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    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-aminopyridine (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 receptor-associated protein (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. PMID:26500501

  11. Cholesterol Inhibits M-type K+ Channels via Protein Kinase C-dependent Phosphorylation in Sympathetic Neurons*

    PubMed Central

    Lee, Seul-Yi; Choi, Hyun-Kyung; Kim, Seong-Tae; Chung, Sungkwon; Park, Myoung Kyu; Cho, Jung-Hwa; Ho, Won-Kyung; Cho, Hana

    2010-01-01

    M-type (KCNQ) potassium channels play an important role in regulating the action potential firing in neurons. Here, we investigated the effect of cholesterol on M current in superior cervical ganglion (SCG) sympathetic neurons, using the patch clamp technique. M current was inhibited in a dose-dependent manner by cholesterol loading with a methyl-β-cyclodextrin-cholesterol complex. This effect was prevented when membrane cholesterol level was restored by including empty methyl-β-cyclodextrin in the pipette solution. Dialysis of cells with AMP-PNP instead of ATP prevented cholesterol action on M currents. Protein kinase C (PKC) inhibitor, calphostin C, abolished cholesterol-induced inhibition whereas the PKC activator, PDBu, mimicked the inhibition of M currents by cholesterol. The in vitro kinase assay showed that KCNQ2 subunits of M channel can be phosphorylated by PKC. A KCNQ2 mutant that is defective in phosphorylation by PKC failed to show current inhibition not only by PDBu but also by cholesterol. These results indicate that cholesterol-induced inhibition of M currents is mediated by PKC phosphorylation. The inhibition of M currents by PDBu and cholesterol was completely blocked by PIP2 loading, indicating that the decrease in PIP2-channel interaction underlies M channel inhibition by PKC-mediated phosphorylation. We conclude that cholesterol specifically regulates M currents in SCG neurons via PKC activation. PMID:20123983

  12. Inhibition by morphine and its analogs of action potentials in adult rat dorsal root ganglion neurons.

    PubMed

    Mizuta, Kotaro; Fujita, Tsugumi; Kumamoto, Eiichi

    2012-09-01

    Although opioids inhibit action potential (AP) conduction in primary-afferent fibers, this has not yet been fully examined. We investigated by using the sharp glass microelectrode technique how opioids (morphine, codeine, and ethylmorphine) affect APs recorded from adult rat dorsal root ganglion (DRG) neurons in response to sciatic nerve stimulation. The DRG neurons were classified into three types, Aα/β, Aδ, and C, according to AP characteristics, including the fiber conduction velocity (CV) of the neuron. AP of the Aα/β neuron was reduced in peak amplitude by each of the opioids in a reversible and concentration-dependent manner. The potency sequence was ethylmorphine > codeine = morphine (IC(50) = 0.70, 2.5, and 2.9 mM, respectively), indicating that this AP inhibition is related to the chemical structure of the opioid. Each of the Aδ and C neuron APs was also inhibited by the opioids; ethylmorphine had a tendency to inhibit APs more effectively than codeine and morphine. This inhibition was variable in extent among neurons and was either comparable to or greater than that of the Aα/β neuron AP. The opioid-induced AP inhibitions were unaffected by nonspecific opioid-receptor antagonist naloxone; opioid-receptor agonists did not affect APs. In conclusion, the opioids inhibited APs in DRG neurons without opioid-receptor activation; this inhibition was different among neurons having different primary-afferent fiber CVs and also among the three kinds of opioid. The inhibition by opioid of primary-afferent fiber AP conduction is suggested to be distinct in extent among fibers conveying distinct types of nociceptive information. Copyright © 2012 Wiley Periodicals, Inc.

  13. Varicella-zoster virus (VZV) infection of neurons derived from human embryonic stem cells: direct demonstration of axonal infection, transport of VZV, and productive neuronal infection.

    PubMed

    Markus, Amos; Grigoryan, Sergei; Sloutskin, Anna; Yee, Michael B; Zhu, Hua; Yang, In Hong; Thakor, Nitish V; Sarid, Ronit; Kinchington, Paul R; Goldstein, Ronald S

    2011-07-01

    Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons.

  14. Varicella-Zoster Virus (VZV) Infection of Neurons Derived from Human Embryonic Stem Cells: Direct Demonstration of Axonal Infection, Transport of VZV, and Productive Neuronal Infection▿

    PubMed Central

    Markus, Amos; Grigoryan, Sergei; Sloutskin, Anna; Yee, Michael B.; Zhu, Hua; Yang, In Hong; Thakor, Nitish V.; Sarid, Ronit; Kinchington, Paul R.; Goldstein, Ronald S.

    2011-01-01

    Study of the human neurotrophic herpesvirus varicella-zoster virus (VZV) and of its ability to infect neurons has been severely limited by strict viral human tropism and limited availability of human neurons for experimentation. Human embryonic stem cells (hESC) can be differentiated to all the cell types of the body including neurons and are therefore a potentially unlimited source of human neurons to study their interactions with human neurotropic viruses. We report here reproducible infection of hESC-derived neurons by cell-associated green fluorescent protein (GFP)-expressing VZV. hESC-derived neurons expressed GFP within 2 days after incubation with mitotically inhibited MeWo cells infected with recombinant VZV expressing GFP as GFP fusions to VZV proteins or under an independent promoter. VZV infection was confirmed by immunostaining for immediate-early and viral capsid proteins. Infection of hESC-derived neurons was productive, resulting in release into the medium of infectious virions that appeared fully assembled when observed by electron microscopy. We also demonstrated, for the first time, VZV infection of axons and retrograde transport from axons to neuronal cell bodies using compartmented microfluidic chambers. The use of hESC-derived human neurons in conjunction with fluorescently tagged VZV shows great promise for the study of VZV neuronal infection and axonal transport and has potential for the establishment of a model for VZV latency in human neurons. PMID:21525353

  15. Electric impedance sensing during the inhibition of cell-cell adhesion.

    PubMed

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

    2008-01-01

    Electric cell impedance sensing (ECIS) was used to monitor the change of in vitro neuron-neuron adhesion in response to the blocking of N-Cam, N-Cadherin and L1. ECIS is a method in which cell morphology and cell mobility can be indirectly measured by changes in intercellular resistance. Antibodies and soluble extracellular domains of the cell adhesion molecules N-Cam, N-Cadherin and L1 were used as blockers of these adhesion molecules on the cell surface. In a 96 hour aggregation assay on a low adhesive substrate, the effect of mentioned blockers on the aggregation was investigated. The N-Cadherin antibody showed effective in aggregation inhibition at concentrations of 3 and 10 micrograms/ml. Up to 96 hours no aggregation occurred. A similar effect was achieved by the N-Cadherin protein, although less distinct. Blocking of N-CAM and L1 revealed no inhibition of aggregation. Results from impedance measurements correspond to those of the aggregation assays. The neuron-neuron adhesion in monolayers was inhibited by blocking of cell adhesion molecules and monitored by ECIS. Impedances of neuron covered electrodes were significantly lower in the presence of N-Cadherin antibody and protein at concentrations of 1, 3 and 10 micrograms/ml, indicating a less profound binding between adjacent neuron.The results from both the aggregation assays and the impedance measurements demonstrate the applicability of CAM blocking for the regulation of culture topography.

  16. The effects of short-term JNK inhibition on the survival and growth of aged sympathetic neurons.

    PubMed

    Guha, Isa; Slamova, Ivana; Chun, Soyon; Clegg, Arthur; Golos, Michal; Thrasivoulou, Chris; Simons, J Paul; Al-Shawi, Raya

    2016-10-01

    During the course of normal aging, certain populations of nerve growth factor (NGF)-responsive neurons become selectively vulnerable to cell death. Studies using dissociated neurons isolated from neonates have shown that c-Jun N-terminal kinases (JNKs) are important in regulating the survival and neurite outgrowth of NGF-responsive sympathetic neurons. Unlike neonatal neurons, adult sympathetic neurons are not dependent on NGF for their survival. Moreover, the NGF precursor, proNGF, is neurotoxic for aging but not young adult NGF-responsive neurons. Because of these age-related differences, the effects of JNK inhibition on the survival and growth of sympathetic neurons isolated from aged mice were studied. Aged neurons, as well as glia, were found to be dependent on JNK for their growth but not their survival. Conversely, proNGF neurotoxicity was JNK-dependent and mediated by the p75-interacting protein NRAGE, whereas neurite outgrowth was independent of NRAGE. These results have implications for the potential use of JNK inhibitors as therapies for ameliorating age-related neurodegenerative disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Pterostilbene attenuates lipopolysaccharide-induced learning and memory impairment possibly via inhibiting microglia activation and protecting neuronal injury in mice.

    PubMed

    Hou, Yue; Xie, Guanbo; Miao, Fengrong; Ding, Lingling; Mou, Yanhua; Wang, Lihui; Su, Guangyue; Chen, Guoliang; Yang, Jingyu; Wu, Chunfu

    2014-10-03

    The present study aims to evaluate the effects of pterostilbene on lipopolysaccharide (LPS)-induced learning and memory impairment as well as the possible changes of microglia and neurons. Firstly, learning and memory function was investigated by behavioral tests. Pterostilbene attenuated LPS-induced learning and memory impairment tested by Y-maze and Morris water maze. Secondly, immunohistochemical method was used to study the changes of microglia and neurons. The results showed that pterostilbene produced a significant decrease in the number of Iba-1 and Doublecortin (DCX) positive cells and a significant increase in neuronal nuclear antigen (NeuN)-stained area of neurons in mouse hippocampal compared to the LPS group. Finally, an in vitro study was performed to further confirm the inhibitory effect on microglia activation and protective effect on neurons exerted by pterostilbene. The results demonstrated that pterostilbene significantly inhibited microglia activation, showing the obvious decrease of LPS-induced production of NO, TNF-α and IL-6 in N9 microglial cells. In addition, the viability of SH-SY5Y cells decreased by conditioned media of LPS-activated N9 microglial cells was remarkably recovered by pterostilbene. In summary, the present study demonstrated for the first time that pterostilbene attenuated LPS-induced learning and memory impairment, which may be associated with its inhibitory effect on microglia activation and protective effect on neuronal injury.

  18. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines

    PubMed Central

    Gibson, Gary E.; Xu, Hui; Chen, Huan-Lian; Chen, Wei; Denton, Travis; Zhang, Sheng

    2015-01-01

    Reversible post-translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins are unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α-ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans-succinylase that mediates succinylation in an α-ketoglutarate-dependent manner. Inhibition of KGDHC reduced suc-cinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid (TCA) cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl CoA suggests that the catalysis due to the E2k suc-cinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC. PMID:25772995

  19. Interferon alpha inhibits spinal cord synaptic and nociceptive transmission via neuronal-glial interactions

    PubMed Central

    Liu, Chien-Cheng; Gao, Yong-Jing; Luo, Hao; Berta, Temugin; Xu, Zhen-Zhong; Ji, Ru-Rong; Tan, Ping-Heng

    2016-01-01

    It is well known that interferons (IFNs), such as type-I IFN (IFN-α) and type-II IFN (IFN-γ) are produced by immune cells to elicit antiviral effects. IFNs are also produced by glial cells in the CNS to regulate brain functions. As a proinflammatory cytokine, IFN-γ drives neuropathic pain by inducing microglial activation in the spinal cord. However, little is known about the role of IFN-α in regulating pain sensitivity and synaptic transmission. Strikingly, we found that IFN-α/β receptor (type-I IFN receptor) was expressed by primary afferent terminals in the superficial dorsal horn that co-expressed the neuropeptide CGRP. In the spinal cord IFN-α was primarily expressed by astrocytes. Perfusion of spinal cord slices with IFN-α suppressed excitatory synaptic transmission by reducing the frequency of spontaneous excitatory postsynaptic current (sEPSCs). IFN-α also inhibited nociceptive transmission by reducing capsaicin-induced internalization of NK-1 and phosphorylation of extracellular signal-regulated kinase (ERK) in superficial dorsal horn neurons. Finally, spinal (intrathecal) administration of IFN-α reduced inflammatory pain and increased pain threshold in naïve rats, whereas removal of endogenous IFN-α by a neutralizing antibody induced hyperalgesia. Our findings suggest a new form of neuronal-glial interaction by which IFN-α, produced by astrocytes, inhibits nociceptive transmission in the spinal cord. PMID:27670299

  20. Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons

    SciTech Connect

    Vaccarino, F.; Guidotti, A.; Costa, E.

    1987-12-01

    In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-..beta..-(/sup 3/H)phorbol 12,13-dibutyrate /(/sup 3/H)-P(BtO)/sub 2// binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of /sup 32/P into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg/sup 2 +/ sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca/sup 2 +/-channel-blocker) but is abolished by the removal of Ca/sup 2 +/ from the incubation medium, suggesting that glutamate-mediated Ca/sup 2 +/ influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) of monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site of with the (/sup 3/H)P(BtO)/sub 2/ binding, nor do they affect the Ca/sup 2 +/ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine.

  1. Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

    PubMed

    Agosti, Francina; López Soto, Eduardo J; Cabral, Agustina; Castrogiovanni, Daniel; Schioth, Helgi B; Perelló, Mario; Raingo, Jesica

    2014-09-01

    The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

  2. Indomethacin inhibits tetrodotoxin-resistant Na(+) channels at acidic pH in rat nociceptive neurons.

    PubMed

    Nakamura, Michiko; Jang, Il-Sung

    2016-06-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) are well-known inhibitors of cyclooxygenases (COXs) and are widely used for the treatment of inflammatory pain; however several NSAIDs display COX-independent analgesic action including the inhibition of voltage-gated Na(+) channels expressed in primary afferent neurons. In the present study, we examined whether NSAIDs modulate tetrodotoxin-resistant (TTX-R) Na(+) channels and if this modulation depends on the extracellular pH. The TTX-R Na(+) currents were recorded from small-sized trigeminal ganglion neurons by using a whole-cell patch clamp technique. Among eight NSAIDs tested in this study, several drugs, including aspirin and ibuprofen, did not affect TTX-R Na(+) channels either at pH 7.4 or at pH 6.0. However, we found that indomethacin, and, to a lesser extent, ibuprofen and naproxen potently inhibited the peak amplitude of TTX-R Na(+) currents at pH 6.0. The indomethacin-induced inhibition of TTX-R Na(+) channels was more potent at depolarized membrane potentials. Indomethacin significantly shifted both the voltage-activation and voltage-inactivation relationships to depolarizing potentials at pH 6.0. Indomethacin accelerated the development of inactivation and retarded the recovery from inactivation of TTX-R Na(+) channels at pH 6.0. Given that indomethacin and several other NSAIDs could further suppress local nociceptive signals by inhibiting TTX-R Na(+) channels at an acidic pH in addition to the classical COX inhibition, these drugs could be particularly useful for the treatment of inflammatory pain. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Inhibition of the neuronal NFκB pathway attenuates bortezomib-induced neuropathy in a mouse model.

    PubMed

    Alé, Albert; Bruna, Jordi; Calls, Aina; Karamita, Maria; Haralambous, Sylva; Probert, Lesley; Navarro, Xavier; Udina, Esther

    2016-07-01

    Bortezomib is a proteasome inhibitor with a remarkable antitumor activity, used in the clinic as first line treatment for multiple myeloma. One hallmark of bortezomib mechanism of action in neoplastic cells is the inhibition of nuclear factor kappa B (NFκB), a transcription factor involved in cell survival and proliferation. Bortezomib-induced peripheral neuropathy is a dose-limiting toxicity that often requires adjustment of treatment and affects patient's prognosis and quality of life. Since disruption of NFκB pathway can also affect neuronal survival, we assessed the role of NFκB in bortezomib-induced neuropathy by using a transgenic mouse that selectively provides blockage of the NFκB pathway in neurons. Interestingly, we observed that animals with impaired NFκB activation developed significantly less severe neuropathy than wild type animals, with particular preservation of large myelinated fibers, thus suggesting that neuronal NFκB activation plays a positive role in bortezomib induced neuropathy and that bortezomib treatment might induce neuropathy by inhibiting NFκΒ in non-neuronal cell types or by targeting other signaling pathways. Therefore, inhibition of NFκB might be a promising strategy for the cotreatment of cancer and neuropathy.

  4. Transition in subicular burst firing neurons from epileptiform activity to suppressed state by feedforward inhibition.

    PubMed

    Sah, Nirnath; Sikdar, Sujit K

    2013-08-01

    The subiculum, a para-hippocampal structure positioned between the cornu ammonis 1 subfield and the entorhinal cortex, has been implicated in temporal lobe epilepsy in human patients and in animal models of epilepsy. The structure is characterized by the presence of a significant population of burst firing neurons that has been shown previously to lead epileptiform activity locally. Phase transitions in epileptiform activity in neurons following a prolonged challenge with an epileptogenic stimulus has been shown in other brain structures, but not in the subiculum. Considering the importance of the subicular burst firing neurons in the propagation of epileptiform activity to the entorhinal cortex, we have explored the phenomenon of phase transitions in the burst firing neurons of the subiculum in an in vitro rat brain slice model of epileptogenesis. Whole-cell patch-clamp and extracellular field recordings revealed a distinct phenomenon in the subiculum wherein an early hyperexcitable state was followed by a late suppressed state upon continuous perfusion with epileptogenic 4-aminopyridine and magnesium-free medium. The suppressed state was characterized by inhibitory post-synaptic potentials in pyramidal excitatory neurons and bursting activity in local fast-spiking interneurons at a frequency of 0.1-0.8 Hz. The inhibitory post-synaptic potentials were mediated by GABAA receptors that coincided with excitatory synaptic inputs to attenuate action potential discharge. These inhibitory post-synaptic potentials ceased following a cut between the cornu ammonis 1 and subiculum. The suppression of epileptiform activity in the subiculum thus represents a homeostatic response towards the induced hyperexcitability. Our results suggest the importance of feedforward inhibition in exerting this homeostatic control.

  5. Inhibition of neuronal nicotinic acetylcholine receptors by the abused solvent, toluene

    PubMed Central

    Bale, Ambuja S; Smothers, Corigan T; Woodward, John J

    2002-01-01

    Toluene is a representative example of a class of industrial solvents that are voluntarily inhaled as drugs of abuse. Previous data from this lab and others has shown that toluene modulates the function of N-methyl-D-aspartate (NMDA), γ-aminobutyric acid (GABA) and glycine receptors at concentrations that do not affect non-NMDA receptors. We utilized two-electrode voltage-clamp and whole cell patch-clamp techniques to assess the effects of toluene on neuronal nicotinic acetylcholine receptors expressed in oocytes and cultured hippocampal neurons. Toluene (50 μM to 10 mM) produced a reversible, concentration-dependent inhibition of acetylcholine-induced current in Xenopus oocytes expressing various nicotinic receptor subtypes. The α4β2 and α3β2 subunit combinations were significantly more sensitive to toluene inhibition than the α4β4, α3β4 and α7 receptors. Receptors composed of α4 and β2(V253F) subunits showed α4β4-like toluene sensitivity while those containing α4 and β4(F255V) subunits showed α4β2-like sensitivity. In hippocampal neurons, toluene (50 μM to 10 mM) dose-dependently inhibited ACh-mediated responses with an IC50 of 1.1 mM. Taken together, these results suggest that nicotinic receptors, like NMDA receptors, show a subunit-dependent sensitivity to toluene and may represent an important site of action for some of the neurobehavioural effects of toluene. PMID:12237258

  6. Inhibition of glial hemichannels by boldine treatment reduces neuronal suffering in a murine model of Alzheimer's disease.

    PubMed

    Yi, Chenju; Ezan, Pascal; Fernández, Paola; Schmitt, Julien; Sáez, Juan C; Giaume, Christian; Koulakoff, Annette

    2017-10-01

    The contribution of reactive gliosis to the pathological phenotype of Alzheimer's disease (AD) opened the way for therapeutic strategies targeting glial cells instead of neurons. In such context, connexin hemichannels were proposed recently as potential targets since neuronal suffering is alleviated when connexin expression is genetically suppressed in astrocytes of a murine model of AD. Here, we show that boldine, an alkaloid from the boldo tree, inhibited hemichannel activity in astrocytes and microglia without affecting gap junctional communication in culture and acute hippocampal slices. Long-term oral administration of boldine in AD mice prevented the increase in glial hemichannel activity, astrocytic Ca(2+) signal, ATP and glutamate release and alleviated hippocampal neuronal suffering. These findings highlight the important pathological role of hemichannels in AD mice. The neuroprotective effect of boldine treatment might provide the basis for future pharmacological strategies that target glial hemichannels to reduce neuronal damage in neurodegenerative diseases. © 2017 Wiley Periodicals, Inc.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

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

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

    PubMed

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

    2014-05-16

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

  9. Optical inhibition of striatal neurons promotes focal neurogenesis and neurobehavioral recovery in mice after middle cerebral artery occlusion.

    PubMed

    He, Xiaosong; Lu, Yifan; Lin, Xiaojie; Jiang, Lu; Tang, Yaohui; Tang, Guanghui; Chen, Xiaoyan; Zhang, Zhijun; Wang, Yongting; Yang, Guo-Yuan

    2017-03-01

    Striatal neurons regulate the activity of neural progenitor cells in the subventricular zone, but the effect of striatal neuronal activity on neurogenesis after ischemic stroke is unclear. In this study, we used optogenetic tools to investigate the impact of striatal neuronal activity on the neurogenesis and functional recovery after cerebral ischemia. We transfected striatal neurons with channelrhodopsin-2 or halorhodopsin from Natronomonas so that they can be excited by 473 nm laser or inhibited by 594 nm laser, respectively. Neural inhibition but not excitation at 4-7 days after middle cerebral artery occlusion resulted in reduced atrophy volume (6.8 ± 0.7 vs 8.5 ± 1.2 mm(3), p < 0.05) and better performance represented by longer sustaining time on rotarod (99.3 ± 9 vs 80.1 ± 11 s, p < 0.01) and faster moving speed (7.7 ± 2 vs 5.7 ± 1.1 cm/s, p < 0.05) in open field tests. Furthermore, neural inhibition increased the number of nestin(+), BrdU(+)/doublecortin(+) and BrdU(+)/NeuN(+) cells ( p < 0.001) in the subventricular zone and peri-focal region, and the expression level of axon guidance factor Netrin-1 (0.39 ± 0.16 vs 0.16 ± 0.02, p < 0.05) in the peri-focal region. These data suggest that striatal neuronal activity plays an important role in regulating neurogenesis and neural-behavioral outcomes, and that inhibiting striatal neurons by optogenetics promotes the recovery after ischemic stroke in mice.

  10. Basal Forebrain Cholinergic Neurons Primarily Contribute to Inhibition of Electroencephalogram Delta Activity, Rather Than Inducing Behavioral Wakefulness in Mice.

    PubMed

    Chen, Li; Yin, Dou; Wang, Tian-Xiao; Guo, Wei; Dong, Hui; Xu, Qi; Luo, Yan-Jia; Cherasse, Yoan; Lazarus, Michael; Qiu, Zi-Long; Lu, Jun; Qu, Wei-Min; Huang, Zhi-Li

    2016-07-01

    The basal forebrain (BF) cholinergic neurons have long been thought to be involved in behavioral wakefulness and cortical activation. However, owing to the heterogeneity of BF neurons and poor selectivity of traditional methods, the precise role of BF cholinergic neurons in regulating the sleep-wake cycle remains unclear. We investigated the effects of cell-selective manipulation of BF cholinergic neurons on the sleep-wake behavior and electroencephalogram (EEG) power spectrum using the pharmacogenetic technique, the 'designer receptors exclusively activated by designer drugs (DREADD)' approach, and ChAT-IRES-Cre mice. Our results showed that activation of BF cholinergic neurons expressing hM3Dq receptors significantly and lastingly decreased the EEG delta power spectrum, produced low-delta non-rapid eye movement sleep, and slightly increased wakefulness in both light and dark phases, whereas inhibition of BF cholinergic neurons expressing hM4Di receptors significantly increased EEG delta power spectrum and slightly decreased wakefulness. Next, the projections of BF cholinergic neurons were traced by humanized Renilla green fluorescent protein (hrGFP). Abundant and highly dense hrGFP-positive fibers were observed in the secondary motor cortex and cingulate cortex, and sparse hrGFP-positive fibers were observed in the ventrolateral preoptic nucleus, a known sleep-related structure. Finally, we found that activation of BF cholinergic neurons significantly increased c-Fos expression in the secondary motor cortex and cingulate cortex, but decreased c-Fos expression in the ventrolateral preoptic nucleus. Taken together, these findings reveal that the primary function of BF cholinergic neurons is to inhibit EEG delta activity through the activation of cerebral cortex, rather than to induce behavioral wakefulness.

  11. Cerium oxide nanoparticles inhibit differentiation of neural stem cells.

    PubMed

    Gliga, Anda R; Edoff, Karin; Caputo, Fanny; Källman, Thomas; Blom, Hans; Karlsson, Hanna L; Ghibelli, Lina; Traversa, Enrico; Ceccatelli, Sandra; Fadeel, Bengt

    2017-08-24

    Cerium oxide nanoparticles (nanoceria) display antioxidant properties and have shown cytoprotective effects both in vitro and in vivo. Here, we explored the effects of nanoceria on neural progenitor cells using the C17.2 murine cell line as a model. First, we assessed the effects of nanoceria versus samarium (Sm) doped nanoceria on cell viability in the presence of the prooxidant, DMNQ. Both particles were taken up by cells and nanoceria, but not Sm-doped nanoceria, elicited a temporary cytoprotective effect upon exposure to DMNQ. Next, we employed RNA sequencing to explore the transcriptional responses induced by nanoceria or Sm-doped nanoceria during neuronal differentiation. Detailed computational analyses showed that nanoceria altered pathways and networks relevant for neuronal development, leading us to hypothesize that nanoceria inhibits neuronal differentiation, and that nanoceria and Sm-doped nanoceria both interfere with cytoskeletal organization. We confirmed that nanoceria reduced neuron specific β3-tubulin expression, a marker of neuronal differentiation, and GFAP, a neuroglial marker. Furthermore, using super-resolution microscopy approaches, we could show that both particles interfered with cytoskeletal organization and altered the structure of neural growth cones. Taken together, these results reveal that nanoceria may impact on neuronal differentiation, suggesting that nanoceria could pose a developmental neurotoxicity hazard.

  12. AgRP Neurons Can Increase Food Intake during Conditions of Appetite Suppression and Inhibit Anorexigenic Parabrachial Neurons.

    PubMed

    Essner, Rachel A; Smith, Alison G; Jamnik, Adam A; Ryba, Anna R; Trutner, Zoe D; Carter, Matthew E

    2017-09-06

    To maintain energy homeostasis, orexigenic (appetite-inducing) and anorexigenic (appetite suppressing) brain systems functionally interact to regulate food intake. Within the hypothalamus, neurons that express agouti-related protein (AgRP) sense orexigenic factors and orchestrate an increase in food-seeking behavior. In contrast, calcitonin gene-related peptide (CGRP)-expressing neurons in the parabrachial nucleus (PBN) suppress feeding. PBN CGRP neurons become active in response to anorexigenic hormones released following a meal, including amylin, secreted by the pancreas, and cholecystokinin (CCK), secreted by the small intestine. Additionally, exogenous compounds, such as lithium chloride (LiCl), a salt that creates gastric discomfort, and lipopolysaccharide (LPS), a bacterial cell wall component that induces inflammation, exert appetite-suppressing effects and activate PBN CGRP neurons. The effects of increasing the homeostatic drive to eat on feeding behavior during appetite suppressing conditions are unknown. Here, we show in mice that food deprivation or optogenetic activation of AgRP neurons induces feeding to overcome the appetite suppressing effects of amylin, CCK, and LiCl, but not LPS. AgRP neuron photostimulation can also increase feeding during chemogenetic-mediated stimulation of PBN CGRP neurons. AgRP neuron stimulation reduces Fos expression in PBN CGRP neurons across all conditions. Finally, stimulation of projections from AgRP neurons to the PBN increases feeding following administration of amylin, CCK, and LiCl, but not LPS. These results demonstrate that AgRP neurons are sufficient to increase feeding during noninflammatory-based appetite suppression and to decrease activity in anorexigenic PBN CGRP neurons, thereby increasing food intake during homeostatic need.SIGNIFICANCE STATEMENT The motivation to eat depends on the relative balance of activity in distinct brain regions that induce or suppress appetite. An abnormal amount of activity in

  13. Pyrethroids inhibit K2P channels and activate sensory neurons: basis of insecticide-induced paraesthesias.

    PubMed

    Castellanos, Aida; Andres, Alba; Bernal, Laura; Callejo, Gerard; Comes, Nuria; Gual, Arcadi; Giblin, Jonathan P; Roza, Carolina; Gasull, Xavier

    2017-09-25

    Pyrethroid insecticides are widely used for pest control, in agriculture or in human public health commonly as a topical treatment for scabies and head lice. Exposure to pyrethroids such as permethrin or tetramethrin (TM) causes sensory alterations such as transient pain, burning, stinging sensations and paraesthesias. Despite the well-known effects of pyrethroids on sodium channels, actions on other channels that control sensory neuron excitability are less studied. Given the role of two-pore domain potassium (K2P) channels in modulating sensory neuron excitability and firing, both in physiological and pathological conditions, we examined the effect of pyrethroids on K2P channels mainly expressed in sensory neurons. Through electrophysiological and calcium imaging experiments, we show that a high percentage of TM-responding neurons were nociceptors, which were also activated by TRPA1 and/or TRPV1 agonists. This pyrethroid also activated and enhanced the excitability of peripheral saphenous nerve fibers. Pyrethroids produced a significant inhibition of native TRESK, TRAAK, TREK-1 and TREK-2 currents. Similar effects were found in transfected HEK293 cells. At the behavioral level, intradermal TM injection in the mouse paw produced nocifensive responses and caused mechanical allodynia, demonstrating that the effects seen on nociceptors in culture lead to pain-associated behaviors in vivo. In TRESK knockout mice, pain-associated behaviors elicited by TM were enhanced, providing further evidence for a role of this channel in preventing excessive neuronal activation. Our results indicate that inhibition of K2P channels facilitates sensory neuron activation and increases their excitability. These effects contribute to the generation of paraesthesias and pain after pyrethroid exposure.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and

  14. Environmental CO2 inhibits Caenorhabditis elegans egg-laying by modulating olfactory neurons and evokes widespread changes in neural activity

    PubMed Central

    Fenk, Lorenz A.; de Bono, Mario

    2015-01-01

    Carbon dioxide (CO2) gradients are ubiquitous and provide animals with information about their environment, such as the potential presence of prey or predators. The nematode Caenorhabditis elegans avoids elevated CO2, and previous work identified three neuron pairs called “BAG,” “AFD,” and “ASE” that respond to CO2 stimuli. Using in vivo Ca2+ imaging and behavioral analysis, we show that C. elegans can detect CO2 independently of these sensory pathways. Many of the C. elegans sensory neurons we examined, including the AWC olfactory neurons, the ASJ and ASK gustatory neurons, and the ASH and ADL nociceptors, respond to a rise in CO2 with a rise in Ca2+. In contrast, glial sheath cells harboring the sensory endings of C. elegans’ major chemosensory neurons exhibit strong and sustained decreases in Ca2+ in response to high CO2. Some of these CO2 responses appear to be cell intrinsic. Worms therefore may couple detection of CO2 to that of other cues at the earliest stages of sensory processing. We show that C. elegans persistently suppresses oviposition at high CO2. Hermaphrodite-specific neurons (HSNs), the executive neurons driving egg-laying, are tonically inhibited when CO2 is elevated. CO2 modulates the egg-laying system partly through the AWC olfactory neurons: High CO2 tonically activates AWC by a cGMP-dependent mechanism, and AWC output inhibits the HSNs. Our work shows that CO2 is a more complex sensory cue for C. elegans than previously thought, both in terms of behavior and neural circuitry. PMID:26100886

  15. Phenotype-dependent inhibition of glutamatergic transmission on nucleus accumbens medium spiny neurons by the abused inhalant toluene

    PubMed Central

    Beckley, Jacob T.; Randall, Patrick K.; Smith, Rachel J.; Hughes, Benjamin A.; Kalivas, Peter W.; Woodward, John J.

    2015-01-01

    Abused inhalants are voluntary inhaled at high concentrations to produce intoxicating effects. Results from animal studies show that the abused inhalant toluene triggers behaviors, such as self-administration and conditioned place preference that are commonly associated with addictive drugs. Little is known however about how toluene affects neurons within the nucleus accumbens (NAc), a brain region within the basal ganglia that mediates goal-directed behaviors and is implicated in the development and maintenance of addictive behaviors. Here, we report that toluene inhibits a component of the after-hyperpolarization potential (AHP), and dose-dependently inhibits NMDA-mediated currents in rat NAc medium spiny neurons (MSN). Moreover, using the multivariate statistical technique, partial least squares discriminative analysis (PLS-DA) to analyze electrophysiological measures from rat NAc MSNs, we show that toluene induces a persistent depression of AMPA-mediated currents in one subtype of NAc medium spiny neurons, and that the electrophysiological features of MSN neurons predicts their sensitivity to toluene. The CB1 receptor antagonist AM281 blocked the toluene-induced long-term depression of AMPA currents, indicating that this process is dependent on endocannabinoid signaling. The neuronal identity of recorded cells was examined using dual histochemistry and shows that toluene-sensitive NAc neurons are dopamine D2 MSNs that express preproenkephalin mRNA. Overall, the results from these studies indicate that physiological characteristics obtained from NAc MSNs during whole-cell patch clamp recordings reliably predict neuronal phenotype, and that the abused inhalant toluene differentially depresses excitatory neurotransmission in NAc neuronal subtypes. PMID:25752326

  16. Heat shock protein 70 protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus via inhibition of nuclear factor-κB activation-induced nitric oxide synthase II expression.

    PubMed

    Chang, Chiung-Chih; Chen, Shang-Der; Lin, Tsu-Kung; Chang, Wen-Neng; Liou, Chia-Wei; Chang, Alice Y W; Chan, Samuel H H; Chuang, Yao-Chung

    2014-02-01

    Status epilepticus induces subcellular changes that may eventually lead to neuronal cell death in the hippocampus. Based on an animal model of status epilepticus, our laboratory showed previously that sustained hippocampal seizure activity activates nuclear factor-κB (NF-κB) and upregulates nitric oxide synthase (NOS) II gene expression, leading to apoptotic neuronal cell death in the hippocampus. The present study examined the potential modulatory role of heat shock protein 70 (HSP70) on NF-κB signaling in the hippocampus following experimental status epilepticus. In Sprague-Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Expression of HSP70 was elevated as early as 1h after the elicitation of sustained seizure activity, followed by a progressive elevation that peaked at 24h. Pretreatment with an antisense oligonucleotide against hsp70 decreased the HSP70 expression, and significantly augmented IκB kinase (IKK) activity and phosphorylation of IκBα, alongside enhanced nuclear translocation and DNA binding activity of NF-κB in the hippocampal CA3 neurons and glial cells. These cellular events were followed by enhanced upregulation of NOS II and peroxynitrite expression 3h after sustained seizure activity that led to an increase of caspase-3 and DNA fragmentation in the hippocampal CA3 neurons 7days after experimental status epilepticus. We concluded that HSP70 protects against apoptotic cell death induced by NF-κB activation and NOS II-peroxynitrite signaling cascade in the hippocampal CA3 and glial cells following experimental status epilepticus via suppression of IKK activity and deactivation of IκBα.

  17. Reinnervation of Hair Cells by Auditory Neurons after Selective Removal of Spiral Ganglion Neurons

    PubMed Central

    Martinez-Monedero, Rodrigo; Corrales, C. Eduardo; Cuajungco, Math P.; Heller, Stefan; Edge, Albert S.B.

    2007-01-01

    Hearing loss can be caused by primary degeneration of spiral ganglion neurons or by secondary degeneration of these neurons after hair cell loss. The replacement of auditory neurons would be an important step in any attempt to restore auditory function in patients with damaged inner ear neurons or hair cells. Application of β-bungarotoxin, a toxin derived from snake venom, to an explant of the cochlea eradicates spiral ganglion neurons while sparing the other cochlear cell types. The toxin was found to bind to the neurons and to cause apoptotic cell death without affecting hair cells or other inner ear cell types as indicated by TUNEL staining, and, thus, the toxin provides a highly specific means of deafferentation of hair cells. We therefore used the denervated organ of Corti for the study of neuronal regeneration and synaptogenesis with hair cells and found that spiral ganglion neurons obtained from the cochlea of an untreated newborn mouse reinnervated hair cells in the toxin-treated organ of Corti and expressed synaptic vesicle markers at points of contact with hair cells. These findings suggest that it may be possible to replace degenerated neurons by grafting new cells into the organ of Corti. PMID:16408287

  18. Chemotherapy Agents and the Inhibition of Neuronal Birthing in the Brain - The Cause of Chemo Brain

    DTIC Science & Technology

    2006-11-01

    of Neuronal Birthing in the Brain – The Cause of “Chemo Brain” PRINCIPAL INVESTIGATOR: Robert A Gross, M.D., Ph.D...Chemotherapy Agents and the Inhibition of Neuronal Birthing in the Brain – The 5a. CONTRACT NUMBER Cause of “Chemo Brain” 5b. GRANT NUMBER DAMD17-03-1...agents that do not (Adriamycin and Taxol) with respect to their ability to impair the birthing of new neurons in the hippocampus of adult mice. By

  19. Over-expressed copper/zinc superoxide dismutase localizes to mitochondria in neurons inhibiting the angiotensin II-mediated increase in mitochondrial superoxide.

    PubMed

    Li, Shumin; Case, Adam J; Yang, Rui-Fang; Schultz, Harold D; Zimmerman, Matthew C

    2013-01-01

    Angiotensin II (AngII) is the main effector peptide of the renin-angiotensin system (RAS), and contributes to the pathogenesis of cardiovascular disease by exerting its effects on an array of different cell types, including central neurons. AngII intra-neuronal signaling is mediated, at least in part, by reactive oxygen species, particularly superoxide (O2 (•-)). Recently, it has been discovered that mitochondria are a major subcellular source of AngII-induced O2 (•-). We have previously reported that over-expression of manganese superoxide dismutase (MnSOD), a mitochondrial matrix-localized O2 (•-) scavenging enzyme, inhibits AngII intra-neuronal signaling. Interestingly, over-expression of copper/zinc superoxide dismutase (CuZnSOD), which is believed to be primarily localized to the cytoplasm, similarly inhibits AngII intra-neuronal signaling and provides protection against AngII-mediated neurogenic hypertension. Herein, we tested the hypothesis that CuZnSOD over-expression in central neurons localizes to mitochondria and inhibits AngII intra-neuronal signaling by scavenging mitochondrial O2 (•-). Using a neuronal cell culture model (CATH.a neurons), we demonstrate that both endogenous and adenovirus-mediated over-expressed CuZnSOD (AdCuZnSOD) are present in mitochondria. Furthermore, we show that over-expression of CuZnSOD attenuates the AngII-mediated increase in mitochondrial O2 (•-) levels and the AngII-induced inhibition of neuronal potassium current. Taken together, these data clearly show that over-expressed CuZnSOD in neurons localizes in mitochondria, scavenges AngII-induced mitochondrial O2 (•-), and inhibits AngII intra-neuronal signaling.

  20. [Cold inducible RNA-binding protein inhibits hippocampal neuronal apoptosis under hypothermia by regulating redox system].

    PubMed

    Li, Jing-Hui; Zhang, Xue; Meng, Yu; Li, Chang-Sheng; Ji, Hong; Yang, Huan-Min; Li, Shi-Ze

    2015-08-25

    In this study, we intend to confirm our hypothesis that cold inducible RNA-binding protein (CIRP) can inhibit neuronal apoptosis through suppressing the formation of oxygen free radicals under hypothermia. Primary rat hippocampal neurons were isolated and cultured in vitro, and were divided into five groups: (1) normal control group (37 °C), (2) cells infected by empty viral vector group, (3) CIRP over-expressed group, (4) CIRP knock-down group, and (5) hypothermia control group. Cells in groups 2-5 were cultured under 32 °C, 5% CO2. Apoptosis of hippocampal neurons were detected by Annexin V-FITC/PI staining and flow cytometry; Expression of CIRP was determined by Western blot; Redox-related parameters (T-AOC, GSH-Px, SOD, MDA) were detected by ELISA kits. Results showed that CIRP expression levels were significantly increased (P < 0.01) and the apoptotic rates were significantly decreased (P < 0.01) in hypothermia control group and CIRP over-expressed group when compared with normal control group. On the other hand, the apoptotic rate was significantly increased (P < 0.05) in CIRP knock-down group compared with that in hypothermia control group. The levels of redox parameters in hypothermia control group and CIRP over-expressed group were significantly changed in comparison with those in normal control group, CIRP knock-down group and empty viral vector infected group, respectively (P < 0.05 or P < 0.01). These results suggest that up-regulation of CIRP by hypothermia treatment can protect the neuron from apoptosis through suppressing the formation of oxygen free radicals.

  1. Heat pulse excitability of vestibular hair cells and afferent neurons

    PubMed Central

    Brichta, Alan M.; Tabatabaee, Hessam; Boutros, Peter J.; Ahn, JoongHo; Della Santina, Charles C.; Poppi, Lauren A.; Lim, Rebecca

    2016-01-01

    In the present study we combined electrophysiology with optical heat pulse stimuli to examine thermodynamics of membrane electrical excitability in mammalian vestibular hair cells and afferent neurons. We recorded whole cell currents in mammalian type II vestibular hair cells using an excised preparation (mouse) and action potentials (APs) in afferent neurons in vivo (chinchilla) in response to optical heat pulses applied to the crista (ΔT ≈ 0.25°C per pulse). Afferent spike trains evoked by heat pulse stimuli were diverse and included asynchronous inhibition, asynchronous excitation, and/or phase-locked APs synchronized to each infrared heat pulse. Thermal responses of membrane currents responsible for APs in ganglion neurons were strictly excitatory, with Q10 ≈ 2. In contrast, hair cells responded with a mix of excitatory and inhibitory currents. Excitatory hair cell membrane currents included a thermoelectric capacitive current proportional to the rate of temperature rise (dT/dt) and an inward conduction current driven by ΔT. An iberiotoxin-sensitive inhibitory conduction current was also evoked by ΔT, rising in <3 ms and decaying with a time constant of ∼24 ms. The inhibitory component dominated whole cell currents in 50% of hair cells at −68 mV and in 67% of hair cells at −60 mV. Responses were quantified and described on the basis of first principles of thermodynamics. Results identify key molecular targets underlying heat pulse excitability in vestibular sensory organs and provide quantitative methods for rational application of optical heat pulses to examine protein biophysics and manipulate cellular excitability. PMID:27226448

  2. Agomelatine and duloxetine synergistically modulates apoptotic pathway by inhibiting oxidative stress triggered intracellular calcium entry in neuronal PC12 cells: role of TRPM2 and voltage-gated calcium channels.

    PubMed

    Akpinar, Abdullah; Uğuz, Abdülhadi Cihangir; Nazıroğlu, Mustafa

    2014-05-01

    Calcium ion (Ca(2+)) is one of the universal second messengers, which acts in a wide range of cellular processes. Results of recent studies indicated that ROS generated by depression leads to loss of endoplasmic reticulum-Ca(2+) homeostasis, oxidative stress, and apoptosis. Agomelatine and duloxetine are novel antidepressant and antioxidant drugs and may reduce oxidative stress, apoptosis, and Ca(2+) entry through TRPM2 and voltage-gated calcium channels. We tested the effects of agomelatine, duloxetine, and their combination on oxidative stress, Ca(2+) influx, mitochondrial depolarization, apoptosis, and caspase values in the PC-12 neuronal cells. PC-12 neuronal cells were exposed in cell culture and exposed to appropriate non-toxic concentrations and incubation times for agomelatine were determined in the neurons by assessing cell viability. Then PC-12 cells were incubated with agomelatine and duloxetine for 24 h. Treatment of cultured PC-12 cells with agomelatine, duloxetine, and their combination results in a protection on apoptosis, caspase-3, caspase-9, mitochondrial membrane depolarization, cytosolic ROS production, glutathione peroxidase, reduced glutathione, and lipid peroxidation, values. Ca(2+) entry through non-specific TRPM2 channel blocker (2-APB) and voltage-gated Ca(2+) channel blockers (verapamil and diltiazem) was modulated by agomelatine and duloxetine. However, effects of duloxetine on the Ca(2+) entry through TRPM2 channels were higher than in agomelatine. Results of current study suggest that the agomelatine and duloxetine are useful against apoptotic cell death and oxidative stress in PC-12 cells, which seem to be dependent on mitochondrial damage and increased levels of intracellular Ca(2+) through activation of TRPM2 and voltage-gated Ca(2+) channels.

  3. Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization

    PubMed Central

    Auffenberg, Eva; Jurik, Angela; Mattusch, Corinna; Stoffel, Rainer; Genewsky, Andreas; Namendorf, Christian; Schmid, Roland M.; Rammes, Gerhard; Biel, Martin; Uhr, Manfred; Moosmang, Sven; Michalakis, Stylianos; Wotjak, Carsten T.; Thoeringer, Christoph K.

    2016-01-01

    Manipulating the function of neurons and circuits that translate electrical and chemical signals into behavior represents a major challenges in neuroscience. In addition to optogenetic methods using light-activatable channels, pharmacogenetic methods with ligand induced modulation of cell signaling and excitability have been developed. However, they are largely based on ectopic expression of exogenous or chimera proteins. Now, we describe the remote and reversible expression of a Kir2.1 type potassium channel using the chemogenetic technique of small molecule induced protein stabilization. Based on shield1-mediated shedding of a destabilizing domain fused to a protein of interest and inhibition of protein degradation, this principle has been adopted for biomedicine, but not in neuroscience so far. Here, we apply this chemogenetic approach in brain research for the first time in order to control a potassium channel in a remote and reversible manner. We could show that shield1-mediated ectopic Kir2.1 stabilization induces neuronal silencing in vitro and in vivo in the mouse brain. We also validated this novel pharmacogenetic method in different neurobehavioral paradigms.The DD-Kir2.1 may complement the existing portfolio of pharmaco- and optogenetic techniques for specific neuron manipulation, but it may also provide an example for future applications of this principle in neuroscience research. PMID:26757616

  4. Changing numbers of neuronal and non-neuronal cells underlie postnatal brain growth in the rat

    PubMed Central

    Bandeira, Fabiana; Lent, Roberto; Herculano-Houzel, Suzana

    2009-01-01

    The rat brain increases >6× in mass from birth to adulthood, presumably through the addition of glial cells and increasing neuronal size, without the addition of neurons. To test this hypothesis, here we investigate quantitatively the postnatal changes in the total number of neuronal and non-neuronal cells in the developing rat brain, and examine how these changes correlate with brain growth. Total numbers of cells were determined with the isotropic fractionator in the brains of 53 Wistar rats, from birth to young adulthood. We find that at birth, >90% of the cells in the rat brain are neurons. Following a dormant period of ≈3 days after birth, the net number of neurons in the cerebral cortex, hippocampus, and remaining tissue (excluding cerebellum and olfactory bulb) doubles during the first week, then is reduced by 70% during the second postnatal week, concurrently with net gliogenesis. A second round of net addition of 6 million neurons is observed in the cerebral cortex over the following 2 weeks. During the first postnatal week, brain growth relates mainly to increased numbers of neurons of larger average size. In the second and third weeks, it correlates with increased numbers of non-neuronal cells that are smaller in size than the preexisting neurons. Postnatal rat brain development is thus characterized by dramatic changes in the cellular composition of the brain, whose growth is governed by different combinations of cell addition and loss, and changes in average cell size during the first months after birth. PMID:19666520

  5. Apolipoprotein A-IV inhibits AgRP/NPY neurons and activates POMC neurons in the arcuate nucleus

    PubMed Central

    Xu, Yuanzhong; Shu, Gang; Wang, Chunmei; Yang, Yongjie; Saito, Kenji; Xu, Pingwen; Hinton, Antentor Othrell; Yan, Xiaofeng; Yu, Likai; Wu, Qi; Tso, Patrick; Tong, Qingchun; Xu, Yong

    2015-01-01

    Background/Aims Apolipoprotein A-IV (apoA-IV) in the brain potently suppresses food intake. However the mechanisms underlying its anorexigenic effects remain to be identified. Methods We first examined the effects of apoA-IV on cellular activities in hypothalamic neurons that co-express agouti-related peptide (AgRP) and neuropeptide Y (NPY) and in neurons that express pro-opiomelanocortin (POMC). We then compared anorexigenic effects of apoA-IV in wild type mice and in mutant mice lacking melanocortin 4 receptors (MC4Rs, the receptors of AgRP and the POMC gene product). Finally, we examined expression of apoA-IV in mouse hypothalamus and quantified its protein levels at fed vs. fasted states. Results We demonstrate that apoA-IV inhibited the firing rate of AgRP/NPY neurons. The decreased firing was associated with hyperpolarized membrane potential and decreased miniature excitatory postsynaptic current. We further used c-fos immunoreactivity to show that intracerebroventricular (i.c.v.) injections of apoA-IV abolished the fasting-induced activation of AgRP/NPY neurons in mice. Further, we found that apoA-IV depolarized POMC neurons and increased their firing rate. In addition, genetic deletion of MC4Rs blocked anorexigenic effects of i.c.v. apoA-IV. Finally, we detected endogenous apoA-IV in multiple neural populations in mouse hypothalamus, including AgRP/NPY neurons, and food deprivation suppresses hypothalamic apoA-IV protein levels. Conclusion Our findings support a model where central apoA-IV inhibits AgRP/NPY neurons and activates POMC neurons to activate MC4Rs, which in turn suppresses food intake. PMID:26337236

  6. Substance P Depolarizes Lamprey Spinal Cord Neurons by Inhibiting Background Potassium Channels

    PubMed Central

    Thörn Pérez, Carolina; Hill, Russell H.; Grillner, Sten

    2015-01-01

    Substance P is endogenously released in the adult lamprey spinal cord and accelerates the burst frequency of fictive locomotion. This is achieved by multiple effects on interneurons and motoneurons, including an attenuation of calcium currents, potentiation of NMDA currents and reduction of the reciprocal inhibition. While substance P also depolarizes spinal cord neurons, the underlying mechanism has not been resolved. Here we show that effects of substance P on background K+ channels are the main source for this depolarization. Hyperpolarizing steps induced inward currents during whole-cell voltage clamp that were reduced by substance P. These background K+ channels are pH sensitive and are selectively blocked by anandamide and AVE1231. These blockers counteracted the effect of substance P on these channels and the resting membrane potential depolarization in spinal cord neurons. Thus, we have shown now that substance P inhibits background K+ channels that in turn induce depolarization, which is likely to contribute to the frequency increase observed with substance P during fictive locomotion. PMID:26197458

  7. Monocarboxylate transporter 8 in neuronal cell growth.

    PubMed

    James, S R; Franklyn, J A; Reaves, B J; Smith, V E; Chan, S Y; Barrett, T G; Kilby, M D; McCabe, C J

    2009-04-01

    Thyroid hormones are essential for the normal growth and development of the fetus, and even small alterations in maternal thyroid hormone status during early pregnancy may be associated with neurodevelopmental abnormalities in childhood. Mutations in the novel and specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) have been associated with severe neurodevelopmental impairment. However, the mechanism by which MCT8 influences neural development remains poorly defined. We have therefore investigated the effect of wild-type (WT) MCT8, and the previously reported L471P mutant, on the growth and function of human neuronal precursor NT2 cells as well as MCT8-null JEG-3 cells. HA-tagged WT MCT8 correctly localized to the plasma membrane in NT2 cells and increased T(3) uptake in both cell types. In contrast, L471P MCT8 was largely retained in the endoplasmic reticulum and displayed no T(3) transport activity. Transient overexpression of WT and mutant MCT8 proteins failed to induce endoplasmic reticular stress or apoptosis. However, MCT8 overexpression significantly repressed cell proliferation in each cell type in both the presence and absence of the active thyroid hormone T(3) and in a dose-dependent manner. In contrast, L471P MCT8 showed no such influence. Finally, small interfering RNA depletion of endogenous MCT8 resulted in increased cell survival and decreased T(3) uptake. Given that T(3) stimulated proliferation in embryonic neuronal NT2 cells, whereas MCT8 repressed cell growth, these data suggest an entirely novel role for MCT8 in addition to T(3) transport, mediated through the modulation of cell proliferation in the developing brain.

  8. Target neurons of floccular middle zone inhibition in medial vestibular nucleus.

    PubMed

    Sato, Y; Kanda, K; Kawasaki, T

    1988-04-19

    Unitary activities of 288 neurons were recorded extracellularly in the medial vestibular nucleus (MV) in anesthetized cats. In 19 neurons, located in the rostral part of the MV adjacent to the stria acustica, floccular middle zone stimulation resulted in cessation of spontaneous discharges. Systematic microstimulation in the brainstem during recording of 16 of 19 target neurons of floccular middle zone inhibition revealed that the target neurons projected to the ipsilateral abducens nucleus (ABN), and not to the contralateral ABN nor the oculomotor nucleus. The conjugate ipsilateral horizontal eye movement elicited by middle zone stimulation may be mediated by this pathway to motoneurons and internuclear neurons in the ipsilateral ABN. In additional experiments, the MV neurons responding antidromically to ipsilateral ABN stimulation and orthodromically to ipsilateral 8 nerve stimulation were recorded extracellularly. In only 7 of 36 recorded neurons, middle zone stimulation depressed the orthodromic and spontaneous activities. Many neurons were free of floccular inhibition. As to the route of floccular inhibitory control over the vestibulo-ocular reflex (VOR) during visual-vestibular stimulation, we propose that the interaction of target and VOR relay neurons takes place at the ipsilateral ABN and modulates the VOR, in addition to well known Ito's proposal that the interaction of the floccular output and the VOR takes place at secondary vestibular neurons and modulates the VOR.

  9. Rhubarb extract has a protective role against radiation-induced brain injury and neuronal cell apoptosis.

    PubMed

    Lu, Kui; Zhang, Cheng; Wu, Wenjun; Zhou, Min; Tang, Yamei; Peng, Ying

    2015-08-01

    Oxidative stress caused by ionizing radiation is involved in neuronal damage in a number of disorders, including trauma, stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Ionizing radiation can lead to the formation of free radicals, which cause neuronal apoptosis and have important roles in the development of some types of chronic brain disease. The present study evaluated the effects of varying concentrations (2, 5 and 10 µg/ml) of ethanolic rhubarb extract on the neuronal damage caused by irradiation in primary neuronal cultures obtained from the cortices of rat embryos aged 20 days. Brain damage was induced with a single dose of γ-irradiation that induced DNA fragmentation, increased lactate dehydrogenase release in neuronal cells and acted as a trigger for microglial cell proliferation. Treatment with rhubarb extract significantly decreased radiation-induced lactate dehydrogenase release and DNA fragmentation, which are important in the process of cell apoptosis. The rhubarb extract exhibited dose-dependent inhibition of lactate dehydrogenase release and neuronal cell apoptosis that were induced by the administration of ionizing radiation. The effect of a 10 µg/ml dose of rhubarb extract on the generation of reactive oxygen species (ROS) induced by radiation was also investigated. This dose led to significant inhibition of ROS generation. In conclusion, the present study showed a protective role of rhubarb extract against irradiation-induced apoptotic neuronal cell death and ROS generation.

  10. Neuronal somatic ATP release triggers neuron-satellite glial cell communication in dorsal root ganglia.

    PubMed

    Zhang, X; Chen, Y; Wang, C; Huang, L-Y M

    2007-06-05

    It has been generally assumed that the cell body (soma) of a neuron, which contains the nucleus, is mainly responsible for synthesis of macromolecules and has a limited role in cell-to-cell communication. Using sniffer patch recordings, we show here that electrical stimulation of dorsal root ganglion (DRG) neurons elicits robust vesicular ATP release from their somata. The rate of release events increases with the frequency of nerve stimulation; external Ca(2+) entry is required for the release. FM1-43 photoconversion analysis further reveals that small clear vesicles participate in exocytosis. In addition, the released ATP activates P2X7 receptors in satellite cells that enwrap each DRG neuron and triggers the communication between neuronal somata and glial cells. Blocking L-type Ca(2+) channels completely eliminates the neuron-glia communication. We further show that activation of P2X7 receptors can lead to the release of tumor necrosis factor-alpha (TNFalpha) from satellite cells. TNFalpha in turn potentiates the P2X3 receptor-mediated responses and increases the excitability of DRG neurons. This study provides strong evidence that somata of DRG neurons actively release transmitters and play a crucial role in bidirectional communication between neurons and surrounding satellite glial cells. These results also suggest that, contrary to the conventional view, neuronal somata have a significant role in cell-cell signaling.

  11. Ethanol Inhibition of Up-States in Prefrontal Cortical Neurons Expressing the Genetically Encoded Calcium Indicator GCaMP3

    PubMed Central

    Woodward, John J; Pava, Matthew

    2011-01-01

    Background The prefrontal cortex (PFC) is critically involved in working memory, cognition and decision-making; processes significantly affected by ethanol. During quiet restfulness or sleep, prefrontal cortical neurons show synaptically-evoked oscillations in membrane potential between hyperpolarized down-states and depolarized up-states. Previous studies from this laboratory used whole-cell electrophysiology and demonstrated that in individual neurons, ethanol inhibited PFC up-states at concentrations associated with behavioral impairment. While those studies monitored activity in one or two neurons at a time, it is likely that in vivo, larger networks of neurons participate in the complex functions of the prefrontal cortex. In the present study, we used imaging and a genetically encoded calcium sensor to examine the effects of ethanol on the activity of multiple neurons simultaneously during up-states. Methods Slice cultures of mouse prefrontal cortex were infected with an AAV virus encoding the calcium indicator GCaMP3 whose expression was driven by the neuron-specific synapsin promoter. After 2–3 weeks in culture, a fast CCD-camera imaging system was used to capture changes in GCaMP3 fluorescence before, during and after exposure to ethanol. Results PFC neurons displayed robust and reproducible changes in GCaMP3 fluorescence during evoked and spontaneous up-states. Simultaneous whole-cell patch-clamp recording and GCaMP3 imaging verified that neurons transitioned into and out of up-states together. Acute application of ethanol reliably depressed up-state calcium signals with lower doses having a greater effect on up-state duration than amplitude. These effects of ethanol on up-state parameters were reversed during washout. Conclusions The results of the present study indicate that ethanol has profound effects on upstate activity in prefrontal neurons and suggest that this action may underlie some of the cognitive impairment associated with acute alcohol

  12. bcl-2 transgene expression can protect neurons against developmental and induced cell death.

    PubMed Central

    Farlie, P G; Dringen, R; Rees, S M; Kannourakis, G; Bernard, O

    1995-01-01

    The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life. Images Fig. 1 Fig. 2 Fig. 4 PMID:7753817

  13. Anesthetic activation of central respiratory chemoreceptor neurons involves inhibition of a THIK-1-like background K+ current

    PubMed Central

    Lazarenko, Roman M.; Fortuna, Michal G.; Shi, Yingtang; Mulkey, Daniel K.; Takakura, Ana C.; Moreira, Thiago S.; Guyenet, Patrice G.; Bayliss, Douglas A.

    2010-01-01

    At surgical depths of anesthesia, inhalational anesthetics cause a loss of motor response to painful stimuli (i.e., immobilization) that is characterized by profound inhibition of spinal motor circuits. Yet, although clearly depressed, the respiratory motor system continues to provide adequate ventilation under these same conditions. Here, we show that isoflurane causes robust activation of CO2/pH-sensitive, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) of the rodent brainstem, in vitro and in vivo. In brainstem slices from Phox2b-eGFP mice, the firing of pH-sensitive RTN neurons was strongly increased by isoflurane, independent of prevailing pH conditions. At least two ionic mechanisms contributed to anesthetic activation of RTN neurons: activation of a Na+-dependent cationic current and inhibition of a background K+ current. Single cell RT-PCR analysis of dissociated GFP-labeled RTN neurons revealed expression of THIK-1 (K2P13.1), a channel that shares key properties with the native RTN current (i.e., suppression by inhalational anesthetics, weak rectification, inhibition by extracellular Na+, and pH-insensitivity). Isoflurane also increased firing rate of RTN chemosensitive neurons in urethane-anesthetized rats, again independent of CO2 levels. In these animals, isoflurane transiently enhanced activity of the respiratory system, an effect that was most prominent at low levels of respiratory drive and mediated largely by an increase in respiratory frequency. These data indicate that inhalational anesthetics cause activation of RTN neurons, which serve an important integrative role in respiratory control; the increased drive provided by enhanced RTN neuronal activity may contribute, in part, to maintaining respiratory motor activity under immobilizing anesthetic conditions. PMID:20610767

  14. Cyclin D1 is an essential mediator of apoptotic neuronal cell death.

    PubMed Central

    Kranenburg, O; van der Eb, A J; Zantema, A

    1996-01-01

    Many neurons in the developing nervous system undergo programmed cell death, or apoptosis. However, the molecular mechanism underlying this phenomenon is largely unknown. In the present report, we present evidence that the cell cycle regulator cyclin D1 is involved in the regulation of neuronal cell death. During neuronal apoptosis, cyclin D1-dependent kinase activity is stimulated, due to an increase in cyclin D1 levels. Moreover, artificial elevation of cyclin D1 levels is sufficient to induce apoptosis, even in non-neural cell types. Cyclin D1-induced apoptosis, like neuronal apoptosis, can be inhibited by 21 kDa E1B, Bcl2 and pRb, but not by 55 kDa E1B. Most importantly, however, overexpression of the cyclin D-dependent kinase inhibitor p16INK4 protects neurons from apoptotic cell death, demonstrating that activation of endogenous cyclin D1-dependent kinases is essential during neuronal apoptosis. These data support a model in which neuronal apoptosis results from an aborted attempt to activate the cell cycle in terminally differentiated neurons. Images PMID:8598205

  15. TETRAMETHRIN AND DDT INHIBIT SPONTANEOUS FIRING IN CORTICAL NEURONAL NETWORKS

    EPA Science Inventory

    The insecticidal and neurotoxic effects of pyrethroids result from prolonged sodium channel inactivation, which causes alterations in neuronal firing and communication. Previously, we determined the relative potencies of 11 type I and type II pyrethroid insecticides using microel...

  16. TETRAMETHRIN AND DDT INHIBIT SPONTANEOUS FIRING IN CORTICAL NEURONAL NETWORKS

    EPA Science Inventory

    The insecticidal and neurotoxic effects of pyrethroids result from prolonged sodium channel inactivation, which causes alterations in neuronal firing and communication. Previously, we determined the relative potencies of 11 type I and type II pyrethroid insecticides using microel...

  17. Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia.

    PubMed

    Satoh, Kunio; Niwa, Masayuki; Goda, Wael; Binh, Nguyen Huy; Nakashima, Masaya; Takamatsu, Manabu; Hara, Akira

    2011-03-25

    The ischemic damage in the hippocampal CA1 sector following transient ischemia, delayed neuronal death, is a typical apoptosis, but the mechanism underlying the delayed neuronal death is still far from fully understood. Galectin-3 is a β-galactosidase-binding lectin which is important in cell proliferation and apoptotic regulation. Galectin-3 is expressed by microglial cells in experimental models of adult stroke. It has been reported that activated microglial cells are widely observed in the brain, including in the hippocampal CA1 region after transient ischemic insult. In the present study, time course expression of galectin-3 following transient forebrain ischemia in gerbils was examined by immunohistochemistry, combined with Iba-1 immunostaining (a specific microglial cell marker), hematoxylin and eosin staining (for morphological observation), and in situ terminal dUTP-biotin nick end labeling of DNA fragments method (for determination of cell death). Following transient ischemia, we observed a transient increase of galectin-3 expression in CA1 region, which was maximal 96h after reperfusion. Galectin-3 expression was predominately localized within CA1 region and observed only in cells which expressed Iba-1. The galectin-3-positive microglial cells emerge after the onset of neuronal cell damage. Expressions of galectin-3 and Iba-1 were strongly reduced by hypothermia during ischemic insult. Prevention of galectin-3 and Iba-1 expression in microglia by hypothermia has led us to propose that hypothermia either inhibits microglial activation or prevents delayed neuronal death itself. Our results indicate that galectin-3 might exert its effect by modulating the neuronal damage in delayed neuronal death.

  18. Neuropeptides released from trigeminal neurons promote the stratification of human corneal epithelial cells.

    PubMed

    Ko, Ji-Ae; Mizuno, Yukari; Ohki, Chihiro; Chikama, Tai-ichiro; Sonoda, Koh-Hei; Kiuchi, Yoshiaki

    2014-01-07

    To examine the effects of neural cells on the stratification of and junctional protein expression by corneal epithelial cells with a coculture system. PC12 cells induced to undergo neuronal differentiation or rat trigeminal nerve cells were cultured together with simian virus 40-transformed human corneal epithelial (HCE) cells on opposite sides of a collagen vitrigel membrane. Stratification of HCE cells was examined by immunofluorescence analysis with antibodies to zonula occludens-1. Expression of junctional proteins in HCE cells was assessed by RT-PCR and immunoblot analyses. The presence of neural cells (PC12 cells or trigeminal neurons) markedly promoted the stratification of HCE cells as well as increased the amounts of N-cadherin mRNA and protein in these cells. These effects of the neural cells were mimicked by conditioned medium prepared from differentiating PC12 cells or by the neuropeptides substance P and calcitonin gene-related peptide (CGRP). Furthermore, the stimulatory effects of trigeminal neurons on the stratification of and N-cadherin expression by HCE cells were inhibited by antagonists of substance P or of CGRP. These results suggest that trigeminal neurons play an important role in the differentiation of corneal epithelial cells. Neuropeptides released from these neurons may thus regulate adhesion between corneal epithelial cells and thereby contribute to the establishment and maintenance of corneal structure and function.

  19. A novel role for PTEN in the inhibition of neurite outgrowth by Myelin-associated glycoprotein in cortical neurons

    PubMed Central

    Perdigoto, Ana Luisa; Chaudhry, Nagarathnamma; Barnes, Gregory N.; Filbin, Marie T.; Carter, Bruce D.

    2010-01-01

    Axonal regeneration in the central nervous system is prevented, in part, by inhibitory proteins expressed by myelin, including Myelin-associated glycoprotein (MAG). Although injury to the corticospinal tract can result in permanent disability, little is known regarding the mechanisms by which MAG affects cortical neurons. Here, we demonstrate that cortical neurons plated on MAG expressing CHO cells, exhibit a striking reduction in process outgrowth. Interestingly, none of the receptors previously implicated in MAG signaling, including the p75 neurotrophin receptor or gangliosides, contributed significantly to MAG-mediated inhibition. However, blocking the small GTPase Rho or its downstream effector kinase, ROCK, partially reversed the effects of MAG on the neurons. In addition, we identified the lipid phosphatase PTEN as a mediator of MAG’s inhibitory effects on neurite outgrowth. Knockdown or gene deletion of PTEN or over expression of activated AKT in cortical neurons resulted in significant, although partial, rescue of neurite outgrowth on MAG-CHO cells. Moreover, MAG decreased the levels of phospho-Akt, suggesting that it activates PTEN in the neurons. Taken together, these results suggest a novel pathway activated by MAG in cortical neurons involving the PTEN/PI3K/AKT axis. PMID:20869442

  20. Enhanced Sensitivity to Hyperpolarizing Inhibition in Mesoaccumbal Relative to Nigrostriatal Dopamine Neuron Subpopulations

    PubMed Central

    2017-01-01

    Midbrain dopamine neurons recorded in vivo pause their firing in response to reward omission and aversive stimuli. While the initiation of pauses typically involves synaptic or modulatory input, intrinsic membrane properties may also enhance or limit hyperpolarization, raising the question of how intrinsic conductances shape pauses in dopamine neurons. Using retrograde labeling and electrophysiological techniques combined with computational modeling, we examined the intrinsic conductances that shape pauses evoked by current injections and synaptic stimulation in subpopulations of dopamine neurons grouped according to their axonal projections to the nucleus accumbens or dorsal striatum in mice. Testing across a range of conditions and pulse durations, we found that mesoaccumbal and nigrostriatal neurons differ substantially in rebound properties with mesoaccumbal neurons displaying significantly longer delays to spiking following hyperpolarization. The underlying mechanism involves an inactivating potassium (IA) current with decay time constants of up to 225 ms, and small-amplitude hyperpolarization-activated currents (IH), characteristics that were most often observed in mesoaccumbal neurons. Pharmacological block of IA completely abolished rebound delays and, importantly, shortened synaptically evoked inhibitory pauses, thereby demonstrating the involvement of A-type potassium channels in prolonging pauses evoked by GABAergic inhibition. Therefore, these results show that mesoaccumbal and nigrostriatal neurons display differential responses to hyperpolarizing inhibitory stimuli that favors a higher sensitivity to inhibition in mesoaccumbal neurons. These findings may explain, in part, observations from in vivo experiments that ventral tegmental area neurons tend to exhibit longer aversive pauses relative to SNc neurons. SIGNIFICANCE STATEMENT Our study examines rebound, postburst, and synaptically evoked inhibitory pauses in subpopulations of midbrain dopamine

  1. BET bromodomain inhibition promotes neurogenesis while inhibiting gliogenesis in neural progenitor cells.

    PubMed

    Li, Jingjun; Ma, Jing; Meng, Guofeng; Lin, Hong; Wu, Sharon; Wang, Jamie; Luo, Jie; Xu, Xiaohong; Tough, David; Lindon, Matthew; Rioja, Inmaculada; Zhao, Jing; Mei, Hongkang; Prinjha, Rab; Zhong, Zhong

    2016-09-01

    Neural stem cells and progenitor cells (NPCs) are increasingly appreciated to hold great promise for regenerative medicine to treat CNS injuries and neurodegenerative diseases. However, evidence for effective stimulation of neuronal production from endogenous or transplanted NPCs for neuron replacement with small molecules remains limited. To identify novel chemical entities/targets for neurogenesis, we had established a NPC phenotypic screen assay and validated it using known small-molecule neurogenesis inducers. Through screening small molecule libraries with annotated targets, we identified BET bromodomain inhibition as a novel mechanism for enhancing neurogenesis. BET bromodomain proteins, Brd2, Brd3, and Brd4 were found to be downregulated in NPCs upon differentiation, while their levels remain unaltered in proliferating NPCs. Consistent with the pharmacological study using bromodomain selective inhibitor (+)-JQ-1, knockdown of each BET protein resulted in an increase in the number of neurons with simultaneous reduction in both astrocytes and oligodendrocytes. Gene expression profiling analysis demonstrated that BET bromodomain inhibition induced a broad but specific transcription program enhancing directed differentiation of NPCs into neurons while suppressing cell cycle progression and gliogenesis. Together, these results highlight a crucial role of BET proteins as epigenetic regulators in NPC development and suggest a therapeutic potential of BET inhibitors in treating brain injuries and neurodegenerative diseases. Copyright © 2016 Helmholtz Zentrum München. Published by Elsevier B.V. All rights reserved.

  2. Manganese inhibits the ability of astrocytes to promote neuronal differentiation

    SciTech Connect

    Giordano, Gennaro; Pizzurro, Daniella; VanDeMark, Kathryn; Guizzetti, Marina; Costa, Lucio G.

    2009-10-15

    Manganese (Mn) is a known neurotoxicant and developmental neurotoxicant. As Mn has been shown to accumulate in astrocytes, we sought to investigate whether Mn would alter astrocyte-neuronal interactions, specifically the ability of astrocytes to promote differentiation of neurons. We found that exposure of rat cortical astrocytes to Mn (50-500 {mu}M) impaired their ability to promote axonal and neurite outgrowth in hippocampal neurons. This effect of Mn appeared to be mediated by oxidative stress, as it was reversed by antioxidants (melatonin and PBN) and by increasing glutathione levels, while it was potentiated by glutathione depletion in astrocytes. As the extracellular matrix protein fibronectin plays an important role in astrocyte-mediated neuronal neurite outgrowth, we also investigated the effect of Mn on fibronectin. Mn caused a concentration-dependent decrease of fibronectin protein and mRNA in astrocytes lysate and of fibronectin protein in astrocyte medium; these effects were also antagonized by antioxidants. Exposure of astrocytes to two oxidants, H{sub 2}O{sub 2} and DMNQ, similarly impaired their neuritogenic action, and led to a decreased expression of fibronectin. Mn had no inhibitory effect on neurite outgrowth when applied directly onto hippocampal neurons, where it actually caused a small increase in neuritogenesis. These results indicate that Mn, by targeting astrocytes, affects their ability to promote neuronal differentiation by a mechanism which is likely to involve oxidative stress.

  3. Inhibition of Catechol-O-methyl Transferase (COMT) by Tolcapone Restores Reductions in Microtubule-associated Protein 2 (MAP2) and Synaptophysin (SYP) Following Exposure of Neuronal Cells to Neurotropic HIV

    PubMed Central

    Lee, Ting Ting; Chana, Gursharan; Gorry, Paul R.; Ellett, Anne; Bousman, Chad A.; Churchill, Melissa J.; Gray, Lachlan R.; Everall, Ian P.

    2015-01-01

    This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by Tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40% lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of Tolcapone for 6 days. RNA was extracted and qPCR was performed using Qiagen RT2-custom-array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the mRNA expression of COMT while reduced the expression of microtubule-associated protein 2 (MAP2) (p=0.0015) and synaptophysin (SYP) (p=0.012) compared to control. A concomitant exposure of Tolcapone ameliorated the perturbed expression of MAP2 (p=0.009) and COMT (p=0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV, and that concomitant exposure of Tolcapone increased SYP (p=0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND. PMID:26037113

  4. Inhibition of catechol-O-methyl transferase (COMT) by tolcapone restores reductions in microtubule-associated protein 2 (MAP2) and synaptophysin (SYP) following exposure of neuronal cells to neurotropic HIV.

    PubMed

    Lee, Ting Ting; Chana, Gursharan; Gorry, Paul R; Ellett, Anne; Bousman, Chad A; Churchill, Melissa J; Gray, Lachlan R; Everall, Ian P

    2015-10-01

    This investigation aimed to assess whether inhibition of cathecol-O-methyl transferase (COMT) by tolcapone could provide neuroprotection against HIV-associated neurodegenerative effects. This study was conducted based on a previous work, which showed that a single nucleotide polymorphism (SNP) at position 158 (val158met) in COMT, resulted in 40 % lower COMT activity. Importantly, this reduction confers a protective effect against HIV-associated neurocognitive disorders (HAND), which have been linked to HIV-associated brain changes. SH-SY5Y-differentiated neurons were exposed to macrophage-propagated HIV (neurotropic MACS2-Br strain) in the presence or absence of tolcapone for 6 days. RNA was extracted, and qPCR was performed using Qiagen RT2 custom array consisting of genes for neuronal and synaptic integrity, COMT and pro-inflammatory markers. Immunofluorescence was conducted to validate the gene expression changes at the protein level. Our findings demonstrated that HIV significantly increased the messenger RNA (mRNA) expression of COMT while reducing the expression of microtubule-associated protein 2 (MAP2) (p = 0.0015) and synaptophysin (SYP) (p = 0.012) compared to control. A concomitant exposure of tolcapone ameliorated the perturbed expression of MAP2 (p = 0.009) and COMT (p = 0.024) associated with HIV. Immunofluorescence revealed a trend reduction of SYP and MAP2 with exposure to HIV and that concomitant exposure of tolcapone increased SYP (p = 0.016) compared to HIV alone. Our findings demonstrated in vitro that inhibition of COMT can ameliorate HIV-associated neurodegenerative changes that resulted in the decreased expression of the structural and synaptic components MAP2 and SYP. As HIV-associated dendritic and synaptic damage are contributors to HAND, inhibition of COMT may represent a potential strategy for attenuating or preventing some of the symptoms of HAND.

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

    PubMed

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

    2011-06-01

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

  6. Mitochondrial impairment induced by 3-nitropropionic acid is enhanced by endogenous metalloprotease activity inhibition in cultured rat striatal neurons.

    PubMed

    de Oca Balderas, Pavel Montes; Ospina, Gabriel Gutiérrez; Del Ángel, Abel Santamaría

    2013-06-24

    Metalloproteases from the metzincin family mediate molecule processing at the cell membrane termed ectodomain shedding (ES). This mechanism enables the generation of intracellular and extracellular fragments from cell membrane molecules that exert additional functions involved in cell processes including cell death, beyond those of full length molecules. Micotoxin 3-nitropropionic acid (3-NP) induces striatal neuronal degeneration in vivo and in vitro through mitochondrial complex II inhibition. In this study, we hypothesized that metalloproteases regulate mitochondrial activity in cultured rat striatal neurons undergoing degeneration. To test this idea, striatal neuronal cultures characterized by NeuN and GAD-67 expression were treated with 3-NP together with the metalloprotease inhibitor GM6001 and their mitochondrial activity was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Our results showed that metalloprotease inhibition potentiated mitochondrial activity impairment induced by 3-NP whereas the inhibitor alone had no effect. These results indicate that metalloproteases regulate and promote mitochondrial functionality in striatal neurons undergoing degeneration induced by 3-NP. Since NMDA receptor is involved in the excitotoxic neuronal death triggered by 3-NP and is known to undergo ES, we analyzed NMDAR subunit NR1 phenotypic distribution by immunofluorescence. 3-NP and GM6001 induced abnormal perinuclear NR1 accumulation that was not observed with 3-NP or GM6001 alone. This observation suggests that metalloproteases are involved in NR1 cellular reorganization induced by 3-NP, and that their inhibition results in abnormal NR1 distribution. Together results indicate that endogenous metalloproteases are activated during striatal neurodegeneration induced by 3-NP eliciting an adaptative or compensatory response that protects mitochondrial functionality.

  7. Inhibition of the Rho/ROCK pathway prevents neuronal degeneration in vitro and in vivo following methylmercury exposure

    SciTech Connect

    Fujimura, Masatake; Usuki, Fusako; Kawamura, Miwako; Izumo, Shuji

    2011-01-01

    Methylmercury (MeHg) is an environmental neurotoxicant which induces neuropathological changes in both the central nervous and peripheral sensory nervous systems. Our recent study demonstrated that down-regulation of Ras-related C3 botulinum toxin substrate 1 (Rac1), which is known to promote neuritic extension, preceded MeHg-induced damage in cultured cortical neurons, suggesting that MeHg-mediated axonal degeneration is due to the disturbance of neuritic extension. Therefore we hypothesized that MeHg-induced axonal degeneration might be caused by neuritic extension/retraction incoordination. This idea brought our attention to the Ras homolog gene (Rho)/Rho-associated coiled coil-forming protein kinase (ROCK) pathway because it has been known to be associated with the development of axon and apoptotic neuronal cell death. Here we show that inhibition of the Rho/ROCK pathway prevents MeHg-intoxication both in vitro and in vivo. A Rho inhibitor, C3 toxin, and 2 ROCK inhibitors, Fasudil and Y-27632, significantly protected against MeHg-induced axonal degeneration and apoptotic neuronal cell death in cultured cortical neuronal cells exposed to 100 nM MeHg for 3 days. Furthermore, Fasudil partially prevented the loss of large pale neurons in dorsal root ganglia, axonal degeneration in dorsal spinal root nerves, and vacuolar degeneration in the dorsal columns of the spinal cord in MeHg-intoxicated model rats (20 ppm MeHg in drinking water for 28 days). Hind limb crossing sign, a characteristic MeHg-intoxicated sign, was significantly suppressed in this model. The results suggest that inhibition of the Rho/ROCK pathway rescues MeHg-mediated neuritic extension/retraction incoordination and is effective for the prevention of MeHg-induced axonal degeneration and apoptotic neuronal cell death.

  8. Voltage-Independent Inhibition of the Tetrodotoxin-Sensitive Sodium Currents by Oxotremorine and Angiotensin II in Rat Sympathetic Neurons.

    PubMed

    Puente, Erika I; De la Cruz, Lizbeth; Arenas, Isabel; Elias-Viñas, David; Garcia, David E

    2016-04-01

    Tetrodotoxin-sensitive Na(+) currents have been extensively studied because they play a major role in neuronal firing and bursting. In this study, we showed that voltage-dependent Na(+) currents are regulated in a slow manner by oxotremorine (oxo-M) and angiotensin II in rat sympathetic neurons. We found that these currents can be readily inhibited through a signaling pathway mediated by G proteins and phospholipase C (PLC) β1. This inhibition is slowly established, pertussis toxin-insensitive, partially reversed within tens of seconds after oxo-M washout, and not relieved by a strong depolarization, suggesting a voltage-insensitive mechanism of inhibition. Specificity of the M1 receptor was tested by the MT-7 toxin. Activation and inactivation curves showed no shift in the voltage dependency under the inhibition by oxo-M. This inhibition is blocked by a PLC inhibitor (U73122, 1-(6-{[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione), and recovery from inhibition is prevented by wortmannin, a PI3/4 kinase inhibitor. Hence, the pathway involves Gq/11 and is mediated by a diffusible second messenger. Oxo-M inhibition is occluded by screening phosphatidylinositol 4,5-bisphosphate (PIP2)-negative charges with poly-l-lysine and prevented by intracellular dialysis with a PIP2 analog. In addition, bisindolylmaleimide I, a specific ATP-competitive protein kinase C (PKC) inhibitor, rules out that this inhibition may be mediated by this protein kinase. Furthermore, oxo-M-induced suppression of Na(+) currents remains unchanged when neurons are treated with calphostin C, a PKC inhibitor that targets the diacylglycerol-binding site of the kinase. These results support a general mechanism of Na(+) current inhibition that is widely present in excitable cells through modulation of ion channels by specific G protein-coupled receptors. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  9. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    PubMed

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

  10. Generation of motor neurons from pluripotent stem cells.

    PubMed

    Chipman, Peter H; Toma, Jeremy S; Rafuse, Victor F

    2012-01-01

    Alpha motor neurons (also known as lower or skeletal motor neurons) have been studied extensively for over 100 years. Motor neurons control the contraction of skeletal muscles and thus are the final common pathway in the nervous system responsible for motor behavior. Muscles become paralyzed when their innervating motor neurons die because of injury or disease. Motor neuron diseases (MNDs), such as Amyotrophic Lateral Sclerosis, progressively destroy motor neurons until those inflicted succumb to the illness due to respiratory failure. One strategy being explored to study and treat muscle paralysis due to motor neuron loss involves deriving surrogate motor neurons from pluripotent stem cells. Guided by decades of research on the development of the spinal cord, recent advances in neurobiology have shown that functional motor neurons can be derived from mouse and human embryonic stem (ES) cells. Furthermore, ES cell-derived motor neurons restore motor behavior when transplanted into animal models of motor dysfunction. The recent discovery that mouse and human motor neurons can be derived from induced pluripotent stem (iPS) cells (i.e., somatic cells converted to pluripotency) has set the stage for the development of patient-specific therapies designed to treat movement disorders. Indeed, there is now hope within the scientific community that motor neurons derived from pluripotent stem cells will be used to treat MNDs through cell transplantation and/or to screen molecules that will prevent motor neuron death. In this chapter, we review the journey that led to the generation of motor neurons from ES and iPS cells, how stem cell-derived motor neurons have been used to treat/study motor dysfunction, and where the technology will likely lead to in the future.

  11. ASIC channel inhibition enhances excitotoxic neuronal death in an in vitro model of spinal cord injury.

    PubMed

    Mazzone, Graciela L; Veeraraghavan, Priyadharishini; Gonzalez-Inchauspe, Carlota; Nistri, Andrea; Uchitel, Osvaldo D

    2017-02-20

    In the spinal cord high extracellular glutamate evokes excitotoxic damage with neuronal loss and severe locomotor impairment. During the cell dysfunction process, extracellular pH becomes acid and may activate acid-sensing ion channels (ASICs) which could be important contributors to neurodegenerative pathologies. Our previous studies have shown that transient application of the glutamate analog kainate (KA) evokes delayed excitotoxic death of spinal neurons, while white matter is mainly spared. The present goal was to enquire if ASIC channels modulated KA damage in relation to locomotor network function and cell death. Mouse spinal cord slices were treated with KA (0.01 or 0.1mM) for 1h, and then washed out for 24h prior to analysis. RT-PCR results showed that KA (at 0.01mM concentration that is near-threshold for damage) increased mRNA expression of ASIC1a, ASIC1b, ASIC2 and ASIC3, an effect reversed by the ASIC inhibitor 4',6-diamidino-2-phenylindole (DAPI). A KA neurotoxic dose (0.1mM) reduced ASIC1a and ASIC2 expression. Cell viability assays demonstrated KA-induced large damage in spinal slices from mice with ASIC1a gene ablation. Likewise, immunohistochemistry indicated significant neuronal loss when KA was followed by the ASIC inhibitors DAPI or amiloride. Electrophysiological recording from ventral roots of isolated spinal cords showed that alternating oscillatory cycles were slowed down by 0.01mMKA, and intensely inhibited by subsequently applied DAPI or amiloride. Our data suggest that early rise in ASIC expression and function counteracted deleterious effects on spinal networks by raising the excitotoxicity threshold, a result with potential implications for improving neuroprotection. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Somatostatin modulates mast cell-induced responses in murine spinal neurons and satellite cells.

    PubMed

    Van Op den bosch, Joeri; Van Nassauw, Luc; Van Marck, Eric; Timmermans, Jean-Pierre

    2009-08-01

    The course of intestinal inflammatory responses is tightly coordinated by the extensive communication between the immune system and the enteric nervous system, among which the bidirectional mast cell-neuron interaction within the intestinal wall plays a prominent role. Recent research suggests that somatostatin (SOM) is able to inhibit this self-reinforcing network by simultaneously suppressing the inflammatory activities of both neurons and mast cells. Therefore, we assessed the modulatory effects of SOM on both the short-term and long-term effects induced by the main mast cell mediators histamine (HIS) and 5-HT on spinal sensory neurons. Short-term incubation of dorsal root ganglion cultures with HIS and 5-HT induced neuronal CGRP-release and calcium-mediated activation of both neurons and nonneuronal cells, both of which effects were significantly reduced by SOM. In addition, SOM was also able to suppress the increased neuronal expression of pro- and anti-inflammatory peptides induced by long-term exposure to HIS and 5-HT. Immunocytochemical and molecular-biological experiments revealed the possible involvement of somatostatin receptor 1 (SSTR1) and SSTR2A in these profound SOM-dependent effects. These data, combined with the increased expression of pro- and anti-inflammatory peptides and several SSTRs in murine dorsal root ganglia following intestinal inflammation, reveal that intestinal inflammation not only induces the onset of proinflammatory cascades but simultaneously triggers endogenous systems destined to prevent excessive tissue damage. Moreover, these data provide for the first time functional evidence that SOM is able to directly modulate intestinal inflammatory responses by interference with the coordinating mast cell-neuron communication.

  13. Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

    PubMed

    Rao, Mala V; Campbell, Jabbar; Palaniappan, Arti; Kumar, Asok; Nixon, Ralph A

    2016-04-01

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly, CAST

  14. Phenotype-dependent inhibition of glutamatergic transmission on nucleus accumbens medium spiny neurons by the abused inhalant toluene.

    PubMed

    Beckley, Jacob T; Randall, Patrick K; Smith, Rachel J; Hughes, Benjamin A; Kalivas, Peter W; Woodward, John J

    2016-05-01

    Abused inhalants are voluntarily inhaled at high concentrations to produce intoxicating effects. Results from animal studies show that the abused inhalant toluene triggers behaviors, such as self-administration and conditioned place preference, which are commonly associated with addictive drugs. However, little is known about how toluene affects neurons within the nucleus accumbens (NAc), a brain region within the basal ganglia that mediates goal-directed behaviors and is implicated in the development and maintenance of addictive behaviors. Here we report that toluene inhibits a component of the after-hyperpolarization potential, and dose-dependently inhibits N-methyl-D-aspartate (NMDA)-mediated currents in rat NAc medium spiny neurons (MSN). Moreover, using the multivariate statistical technique, partial least squares discriminative analysis to analyze electrophysiological measures from rat NAc MSNs, we show that toluene induces a persistent depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated currents in one subtype of NAc MSNs, and that the electrophysiological features of MSN neurons predicts their sensitivity to toluene. The CB1 receptor antagonist AM281 blocked the toluene-induced long-term depression of AMPA currents, indicating that this process is dependent on endocannabinoid signaling. The neuronal identity of recorded cells was examined using dual histochemistry and shows that toluene-sensitive NAc neurons are dopamine D2 MSNs that express preproenkephalin mRNA. Overall, the results from these studies indicate that physiological characteristics obtained from NAc MSNs during whole-cell patch-clamp recordings reliably predict neuronal phenotype, and that the abused inhalant toluene differentially depresses excitatory neurotransmission in NAc neuronal subtypes.

  15. Weak Noise in Neurons May Powerfully Inhibit the Generation of Repetitive Spiking but Not Its Propagation

    PubMed Central

    Tuckwell, Henry C.; Jost, Jürgen

    2010-01-01

    Many neurons have epochs in which they fire action potentials in an approximately periodic fashion. To see what effects noise of relatively small amplitude has on such repetitive activity we recently examined the response of the Hodgkin-Huxley (HH) space-clamped system to such noise as the mean and variance of the applied current vary, near the bifurcation to periodic firing. This article is concerned with a more realistic neuron model which includes spatial extent. Employing the Hodgkin-Huxley partial differential equation system, the deterministic component of the input current is restricted to a small segment whereas the stochastic component extends over a region which may or may not overlap the deterministic component. For mean values below, near and above the critical values for repetitive spiking, the effects of weak noise of increasing strength is ascertained by simulation. As in the point model, small amplitude noise near the critical value dampens the spiking activity and leads to a minimum as noise level increases. This was the case for both additive noise and conductance-based noise. Uniform noise along the whole neuron is only marginally more effective in silencing the cell than noise which occurs near the region of excitation. In fact it is found that if signal and noise overlap in spatial extent, then weak noise may inhibit spiking. If, however, signal and noise are applied on disjoint intervals, then the noise has no effect on the spiking activity, no matter how large its region of application, though the trajectories are naturally altered slightly by noise. Such effects could not be discerned in a point model and are important for real neuron behavior. Interference with the spike train does nevertheless occur when the noise amplitude is larger, even when noise and signal do not overlap, being due to the instigation of secondary noise-induced wave phenomena rather than switching the system from one attractor (firing regularly) to another (a stable

  16. In vivo vulnerability of dopamine neurons to inhibition of energy metabolism.

    PubMed

    Zeevalk, G D; Manzino, L; Hoppe, J; Sonsalla, P

    1997-02-12

    In vitro studies indicate that mesencephalic dopamine neurons are more vulnerable than other neurons to impairment of energy metabolism. Such findings may have bearing on the loss of dopamine neurons in Parkinson's disease, in which mitochondrial deficiencies have been identified, but would only be relevant if the selective vulnerability were maintained in vivo. To examine this, rats were stereotaxically administered various concentrations of the succinate dehydrogenase inhibitor, malonate (0.25-4 mumol), either into the left substantia nigra or striatum. One week following injection, dopamine and gamma-aminobutyric acid (GABA) levels in the mesencephalon and striatum were measured. Intranigral injection of malonate caused nigral dopamine and GABA to be comparably reduced at all doses tested. The 50% dose level for malonate vs. dopamine and GABA loss was 0.39 and 0.42 mumol, respectively. Tyrosine hydroxylase immunocytochemistry of the midbrains of rats which received an intranigral injection of malonate showed normal staining with 0.25 mumol malonate, but almost complete loss of tyrosine hydroxylase positive nigral pars compacta cells with 1 mumol malonate. Intrastriatal injection of malonate produced a loss of both tyrosine hydroxylase activity and dopamine. In contrast to what was seen in substantia nigra, there was a greater loss of dopamine than GABA in striatal regions nearest the injection site. In striatal regions most distal to the injection site, and which received the lowest concentration of malonate due to diffusion, dopamine levels were significantly reduced with all doses of malonate (0.5-4 mumol), whereas GABA levels were unaffected. Intrastriatal coinfusion of succinate along with malonate completely prevented the loss of dopamine and GABA indicating that succinate dehydrogenase inhibition was the cause of toxicity. These findings indicate that dopamine terminals in the striatum of adult rats are selectively more vulnerable than are the GABA neurons

  17. Weak noise in neurons may powerfully inhibit the generation of repetitive spiking but not its propagation.

    PubMed

    Tuckwell, Henry C; Jost, Jürgen

    2010-05-27

    Many neurons have epochs in which they fire action potentials in an approximately periodic fashion. To see what effects noise of relatively small amplitude has on such repetitive activity we recently examined the response of the Hodgkin-Huxley (HH) space-clamped system to such noise as the mean and variance of the applied current vary, near the bifurcation to periodic firing. This article is concerned with a more realistic neuron model which includes spatial extent. Employing the Hodgkin-Huxley partial differential equation system, the deterministic component of the input current is restricted to a small segment whereas the stochastic component extends over a region which may or may not overlap the deterministic component. For mean values below, near and above the critical values for repetitive spiking, the effects of weak noise of increasing strength is ascertained by simulation. As in the point model, small amplitude noise near the critical value dampens the spiking activity and leads to a minimum as noise level increases. This was the case for both additive noise and conductance-based noise. Uniform noise along the whole neuron is only marginally more effective in silencing the cell than noise which occurs near the region of excitation. In fact it is found that if signal and noise overlap in spatial extent, then weak noise may inhibit spiking. If, however, signal and noise are applied on disjoint intervals, then the noise has no effect on the spiking activity, no matter how large its region of application, though the trajectories are naturally altered slightly by noise. Such effects could not be discerned in a point model and are important for real neuron behavior. Interference with the spike train does nevertheless occur when the noise amplitude is larger, even when noise and signal do not overlap, being due to the instigation of secondary noise-induced wave phenomena rather than switching the system from one attractor (firing regularly) to another (a stable

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

    NASA Technical Reports Server (NTRS)

    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.

  19. Mitochondria modify exercise-induced development of stem cell-derived neurons in the adult brain.

    PubMed

    Steib, Kathrin; Schäffner, Iris; Jagasia, Ravi; Ebert, Birgit; Lie, D Chichung

    2014-05-07

    Neural stem cells in the adult mammalian hippocampus continuously generate new functional neurons, which modify the hippocampal network and significantly contribute to cognitive processes and mood regulation. Here, we show that the development of new neurons from stem cells in adult mice is paralleled by extensive changes to mitochondrial mass, distribution, and shape. Moreover, exercise-a strong modifier of adult hippocampal neurogenesis-accelerates neuronal maturation and induces a profound increase in mitochondrial content and the presence of mitochondria in dendritic segments. Genetic inhibition of the activity of the mitochondrial fission factor dynamin-related protein 1 (Drp1) inhibits neurogenesis under basal and exercise conditions. Conversely, enhanced Drp1 activity furthers exercise-induced acceleration of neuronal maturation. Collectively, these results indicate that adult hippocampal neurogenesis requires adaptation of the mitochondrial compartment and suggest that mitochondria are targets for enhancing neurogenesis-dependent hippocampal plasticity.

  20. TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain

    PubMed Central

    Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A.; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

    2014-01-01

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology. PMID:24381309

  1. TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

    PubMed

    Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

    2014-05-15

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

  2. Astrocytic αVβ3 Integrin Inhibits Neurite Outgrowth and Promotes Retraction of Neuronal Processes by Clustering Thy-1

    PubMed Central

    Herrera-Molina, Rodrigo; Frischknecht, Renato; Maldonado, Horacio; Seidenbecher, Constanze I.; Gundelfinger, Eckart D.; Hetz, Claudio; Aylwin, María de la Luz; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2012-01-01

    Thy-1 is a membrane glycoprotein suggested to stabilize or inhibit growth of neuronal processes. However, its precise function has remained obscure, because its endogenous ligand is unknown. We previously showed that Thy-1 binds directly to αVβ3 integrin in trans eliciting responses in astrocytes. Nonetheless, whether αVβ3 integrin might also serve as a Thy-1-ligand triggering a neuronal response has not been explored. Thus, utilizing primary neurons and a neuron-derived cell line CAD, Thy-1-mediated effects of αVβ3 integrin on growth and retraction of neuronal processes were tested. In astrocyte-neuron co-cultures, endogenous αVβ3 integrin restricted neurite outgrowth. Likewise, αVβ3-Fc was sufficient to suppress neurite extension in Thy-1(+), but not in Thy-1(−) CAD cells. In differentiating primary neurons exposed to αVβ3-Fc, fewer and shorter dendrites were detected. This effect was abolished by cleavage of Thy-1 from the neuronal surface using phosphoinositide-specific phospholipase C (PI-PLC). Moreover, αVβ3-Fc also induced retraction of already extended Thy-1(+)-axon-like neurites in differentiated CAD cells as well as of axonal terminals in differentiated primary neurons. Axonal retraction occurred when redistribution and clustering of Thy-1 molecules in the plasma membrane was induced by αVβ3 integrin. Binding of αVβ3-Fc was detected in Thy-1 clusters during axon retraction of primary neurons. Moreover, αVβ3-Fc-induced Thy-1 clustering correlated in time and space with redistribution and inactivation of Src kinase. Thus, our data indicates that αVβ3 integrin is a ligand for Thy-1 that upon binding not only restricts the growth of neurites, but also induces retraction of already existing processes by inducing Thy-1 clustering. We propose that these events participate in bi-directional astrocyte-neuron communication relevant to axonal repair after neuronal damage. PMID:22479590

  3. Distinct cognitive effects and underlying transcriptome changes upon inhibition of individual miRNAs in hippocampal neurons

    PubMed Central

    Malmevik, Josephine; Petri, Rebecca; Knauff, Pina; Brattås, Per Ludvik; Åkerblom, Malin; Jakobsson, Johan

    2016-01-01

    MicroRNAs (miRNA) are small, non-coding RNAs mediating post-transcriptional regulation of gene expression. miRNAs have recently been implicated in hippocampus-dependent functions such as learning and memory, although the roles of individual miRNAs in these processes remain largely unknown. Here, we achieved stable inhibition using AAV-delivered miRNA sponges of individual, highly expressed and brain-enriched miRNAs; miR-124, miR-9 and miR-34, in hippocampal neurons. Molecular and cognitive studies revealed a role for miR-124 in learning and memory. Inhibition of miR-124 resulted in an enhanced spatial learning and working memory capacity, potentially through altered levels of genes linked to synaptic plasticity and neuronal transmission. In contrast, inhibition of miR-9 or miR-34 led to a decreased capacity of spatial learning and of reference memory, respectively. On a molecular level, miR-9 inhibition resulted in altered expression of genes related to cell adhesion, endocytosis and cell death, while miR-34 inhibition caused transcriptome changes linked to neuroactive ligand-receptor transduction and cell communication. In summary, this study establishes distinct roles for individual miRNAs in hippocampal function. PMID:26813637

  4. Molecular misreading in non-neuronal cells.

    PubMed

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

    2000-08-01

    +1 Frame-shifted proteins such as amyloid precursor protein(+1) and ubiquitin-B(+1) have been identified in the neuropathological hallmarks of Alzheimer's disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed 'molecular misreading'. A key question is whether this process is confined to neurons or whether it could also occur in non-neuronal cells. A transgenic mouse line (MV-B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV-LTR promotor was used to screen non-neuronal tissues for molecular misreading by means of detection of the rat vasopressin(+1) protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non-neural tissues too, could be affected by cellular derangements caused by molecular misreading.

  5. Ghrelin Inhibits Visceral Afferent Activation of Catecholamine Neurons in the Solitary Tract Nucleus

    PubMed Central

    Cui, Ran Ji; Li, Xiaojun; Appleyard, Suzanne M.

    2011-01-01

    Brainstem A2/C2 catecholamine (CA) neurons in the solitary tract nucleus (NTS) are thought to play an important role in the control of food intake and other homeostatic functions. We have previously demonstrated that these neurons, which send extensive projections to brain regions involved in the regulation of appetite, are strongly and directly activated by solitary tract (ST) visceral afferents. Ghrelin, a potent orexigenic peptide released from the stomach, is proposed to act in part through modulating NTS CA neurons but the underlying cellular mechanisms are unknown. Here we identified CA neurons using transgenic mice that express enhanced green florescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). We then determined how ghrelin modulates TH-EGFP neurons using patch clamp techniques in a horizontal brain slice preparation. Ghrelin inhibited the frequency of spontaneous glutamate inputs (sEPSCs) onto TH-EGFP neurons, including cholecystokinin-sensitive neurons, an effect blocked by the GHSR1 antagonist, D-Lys-3-GHRP-6. This resulted in a decrease in the basal firing rate of NTS TH-EGFP neurons, an effect blocked by the glutamate antagonist NBQX. Ghrelin also dose-dependently inhibited the amplitude of ST afferent evoked EPSCs (ST-EPSCs) in TH-EGFP NTS neurons, decreasing the success rate for ST-evoked action potentials. In addition, ghrelin decreased the frequency of mini-EPSCs suggesting its actions are pre-synaptic to reduce glutamate release. Lastly, ghrelin’s inhibition of the ST-EPSCs was significantly increased by an 18 hour fast. These results demonstrate a potential mechanism by which ghrelin inhibits NTS TH neurons through a pathway whose responsiveness is increased during fasting. PMID:21368060

  6. Opioids inhibit visceral afferent activation of catecholamine neurons in the solitary tract nucleus.

    PubMed

    Cui, R J; Roberts, B L; Zhao, H; Andresen, M C; Appleyard, S M

    2012-10-11

    Brainstem A2/C2 catecholamine (CA) neurons within the solitary tract nucleus (NTS) influence many homeostatic functions, including food intake, stress, respiratory and cardiovascular reflexes. They also play a role in both opioid reward and withdrawal. Injections of opioids into the NTS modulate many autonomic functions influenced by catecholamine neurons including food intake and cardiac function. We recently showed that NTS-CA neurons are directly activated by incoming visceral afferent inputs. Here we determined whether opioid agonists modulate afferent activation of NTS-CA neurons using transgenic mice with EGFP expressed under the control of the tyrosine hydroxylase promoter (TH-EGFP) to identify catecholamine neurons. The opioid agonist Met-enkephalin (Met-Enk) significantly attenuated solitary tract-evoked excitatory postsynaptic currents (ST-EPSCs) in NTS TH-EGFP neurons by 80%, an effect reversed by wash or the mu opioid receptor-specific antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP). Met-Enk had a significantly greater effect to inhibit afferent inputs onto TH-EGFP-positive neurons than EGFP-negative neurons, which were only inhibited by 50%. The mu agonist, DAMGO, also inhibited the ST-EPSC in TH-EGFP neurons in a dose-dependent manner. In contrast, neither the delta agonist DPDPE, nor the kappa agonist, U69,593, consistently inhibited the ST-EPSC amplitude. Met-Enk and DAMGO increased the paired pulse ratio, decreased the frequency, but not amplitude, of mini-EPSCs and had no effect on holding current, input resistance or current-voltage relationships in TH-EGFP neurons, suggesting a presynaptic mechanism of action on afferent terminals. Met-Enk significantly reduced both the basal firing rate of NTS TH-EGFP neurons and the ability of afferent stimulation to evoke an action potential. These results suggest that opioids inhibit NTS-CA neurons by reducing an excitatory afferent drive onto these neurons through presynaptic inhibition of

  7. Intraganglionic interactions between satellite cells and adult sensory neurons.

    PubMed

    Christie, Kimberly; Koshy, Dilip; Cheng, Chu; Guo, GuiFang; Martinez, Jose A; Duraikannu, Arul; Zochodne, Douglas W

    2015-07-01

    Perineuronal satellite cells have an intimate anatomical relationship with sensory neurons that suggests close functional collaboration and mutual support. We examined several facets of this relationship in adult sensory dorsal root ganglia (DRG). Collaboration included the support of process outgrowth by clustering of satellite cells, induction of distal branching behavior by soma signaling, the capacity of satellite cells to respond to distal axon injury of its neighboring neurons, and evidence of direct neuron-satellite cell exchange. In vitro, closely adherent coharvested satellite cells routinely clustered around new outgrowing processes and groups of satellite cells attracted neurite processes. Similar clustering was encountered in the pseudounipolar processes of intact sensory neurons within intact DRG in vivo. While short term exposure of distal growth cones of unselected adult sensory neurons to transient gradients of a PTEN inhibitor had negligible impacts on their behavior, exposure of the soma induced early and substantial growth of their distant neurites and branches, an example of local soma signaling. In turn, satellite cells sensed when distal neuronal axons were injured by enlarging and proliferating. We also observed that satellite cells were capable of internalizing and expressing a neuron fluorochrome label, diamidino yellow, applied remotely to distal injured axons of the neuron and retrogradely transported to dorsal root ganglia sensory neurons. The findings illustrate a robust interaction between intranganglionic neurons and glial cells that involve two way signals, features that may be critical for both regenerative responses and ongoing maintenance.

  8. Hyperbaric oxygenation alleviates chronic constriction injury (CCI)-induced neuropathic pain and inhibits GABAergic neuron apoptosis in the spinal cord.

    PubMed

    Fu, Huiqun; Li, Fenghua; Thomas, Sebastian; Yang, Zhongjin

    2017-09-15

    Dysfunction of GABAergic inhibitory controls contributes to the development of neuropathic pain. We examined our hypotheses that (1) chronic constriction injury (CCI)-induced neuropathic pain is associated with increased spinal GABAergic neuron apoptosis, and (2) hyperbaric oxygen therapy (HBO) alleviates CCI-induced neuropathic pain by inhibiting GABAergic neuron apoptosis. Male rats were randomized into 3 groups: CCI, CCI+HBO and the control group (SHAM). Mechanical allodynia was tested daily following CCI procedure. HBO rats were treated at 2.4 atmospheres absolute (ATA) for 60min once per day. The rats were euthanized and the spinal cord harvested on day 8 and 14 post-CCI. Detection of GABAergic cells and apoptosis was performed. The percentages of double positive stained cells (NeuN/GABA), cleaved caspase-3 or Cytochrome C in total GABAergic cells or in total NeuN positive cells were calculated. HBO significantly alleviated mechanical allodynia. CCI-induced neuropathic pain was associated with significantly increased spinal apoptotic GABA-positive neurons. HBO considerably decreased these spinal apoptotic cells. Cytochrome-C-positive neurons and cleaved caspase-3-positive neurons were also significantly higher in CCI rats. HBO significantly decreased these positive cells. Caspase-3 mRNA was also significantly higher in CCI rats. HBO reduced mRNA expression of caspase-3. CCI-induced neuropathic pain was associated with increased apoptotic GABAergic neurons induced by activation of key proteins of mitochondrial apoptotic pathways in the dorsal horn of the spinal cord. HBO alleviated CCI-induced neuropathic pain and reduced GABAergic neuron apoptosis. The beneficial effect of HBO may be via its inhibitory role in CCI-induced GABAergic neuron apoptosis by suppressing mitochondrial apoptotic pathways in the spinal cord. Increased apoptotic GABAergic neurons induced by activation of key proteins of mitochondrial apoptotic pathways in the dorsal horn of the spinal

  9. RARβ regulates neuronal cell death and differentiation in the avian ciliary ganglion

    PubMed Central

    Boerries, Melanie; Busch, Hauke

    2015-01-01

    ABSTRACT Programmed cell death during chicken ciliary ganglion (CG) development is mostly discussed as an extrinsically regulated process, guided either by the establishment of a functional balance between preganglionic and postganglionic activity or the availability of target‐derived neurotrophic factors. We found that the expression of the gene coding for the nuclear retinoic acid receptor β (RARB) is transiently upregulated prior to and during the execution phase of cell death in the CG. Using retroviral vectors, the expression of RARB was knocked down during embryonic development in ovo. The knockdown led to a significant increase in CG neuron number after the cell death phase. BrdU injections and active caspase‐3 staining revealed that this increase in neuron number was due to an inhibition of apoptosis during the normal cell death phase. Furthermore, apoptotic neuron numbers were significantly increased at a stage when cell death is normally completed. While the cholinergic phenotype of the neurons remained unchanged after RARB knockdown, the expression of the proneural gene Cash1 was increased, but somatostatin‐like immunoreactivity, a hallmark of the mature choroid neuron population, was decreased. Taken together, these results point toward a delay in neuronal differentiation as well as cell death. The availability of nuclear retinoic acid receptor β (RARβ) and RARβ‐induced transcription of genes could therefore be a new intrinsic cue for the maturation of CG neurons and their predisposition to undergo cell death. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1204–1218, 2015 PMID:25663354

  10. Antioxidant gene therapy against neuronal cell death

    PubMed Central

    Navarro-Yepes, Juliana; Zavala-Flores, Laura; Annadurai, Anandhan; Wang, Fang; Skotak, Maciej; Chandra, Namas; Li, Ming; Pappa, Aglaia; Martinez-Fong, Daniel; Razo, Luz Maria Del; Quintanilla-Vega, Betzabet; Franco, Rodrigo

    2014-01-01

    Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy. PMID:24333264

  11. Synchronized Inhibition Boosts Information Transfer in Entrained Neurons

    NASA Astrophysics Data System (ADS)

    Tiesinga, Paul; Fellous, Jean-Marc; Jose, Jorge; Sejnowski, Terrence

    2001-03-01

    We investigated the ability of a single neuron to transduce the information content of a synchronized inhibitory synaptic drive, generated by an interneuron network, into an information-rich output of neuron spike times. The neuron was entrained to the periodic network drive when the jitter in the input spike times is sufficiently small (i.e. high precision), and the number of presynaptic spikes during one drive cycle is sufficiently large. The Shannon entropy of the output of spike times was reduced sharply during entrainment. Surprisingly, however, the amount of transduced information as measured by the mutual information was significantly increased during entrainment. This increase was due to the reduced contribution of the internal correlations to the output variability. These theoretical predictions were confirmed in experimental recordings from the rat neocortex and hippocampus.

  12. μ-Opioid receptor activation and noradrenaline transport inhibition by tapentadol in rat single locus coeruleus neurons.

    PubMed

    Sadeghi, Mahsa; Tzschentke, Thomas M; Christie, MacDonald J

    2015-01-01

    Tapentadol is a novel analgesic that combines moderate μ-opioid receptor agonism and noradrenaline reuptake inhibition in a single molecule. Both mechanisms of action are involved in producing analgesia; however, the potency and efficacy of tapentadol in individual neurons has not been characterized. Whole-cell patch-clamp recordings of G-protein-coupled inwardly rectifying K(+) (KIR 3.x) currents were made from rat locus coeruleus neurons in brain slices to investigate the potency and relative efficacy of tapentadol and compare its intrinsic activity with other clinically used opioids. Tapentadol showed agonist activity at μ receptors and was approximately six times less potent than morphine with respect to KIR 3.x current modulation. The intrinsic activity of tapentadol was lower than [Met]enkephalin, morphine and oxycodone, but higher than buprenorphine and pentazocine. Tapentadol inhibited the noradrenaline transporter (NAT) with potency similar to that at μ receptors. The interaction between these two mechanisms of action was additive in individual LC neurons. Tapentadol displays similar potency for both µ receptor activation and NAT inhibition in functioning neurons. The intrinsic activity of tapentadol at the μ receptor lies between that of buprenorphine and oxycodone, potentially explaining the favourable profile of side effects, related to μ receptors. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2013 The British Pharmacological Society.

  13. IPP5 inhibits neurite growth in primary sensory neurons by maintaining TGF-β/Smad signaling.

    PubMed

    Han, Qing-Jian; Gao, Nan-Nan; Guo-QiangMa; Zhang, Zhen-Ning; Yu, Wen-Hui; Pan, Jing; Wang, Qiong; Zhang, Xu; Bao, Lan

    2013-01-15

    During nerve regeneration, neurite growth is regulated by both intrinsic molecules and extracellular factors. Here, we found that inhibitor 5 of protein phosphatase 1 (IPP5), a newly identified inhibitory subunit of protein phosphatase 1 (PP1), inhibited neurite growth in primary sensory neurons as an intrinsic regulator. IPP5 was highly expressed in the primary sensory neurons of rat dorsal root ganglion (DRG) and was downregulated after sciatic nerve axotomy. Knocking down IPP5 with specific shRNA increased the length of the longest neurite, the total neurite length and the number of neurite ends in cultured rat DRG neurons. Mutation of the PP1-docking motif K(8)IQF(11) or the PP1-inhibiting motif at Thr(34) eliminated the IPP5-induced inhibition of neurite growth. Furthermore, biochemical experiments showed that IPP5 interacted with type I transforming growth factor-β receptor (TβRI) and PP1 and enhanced transforming growth factor-β (TGF-β)/Smad signaling in a PP1-dependent manner. Overexpressing IPP5 in DRG neurons aggravated TGF-β-induced inhibition of neurite growth, which was abolished by blocking PP1 or IPP5 binding to PP1. Blockage of TGF-β signaling with the TβRI inhibitor SB431542 or Smad2 shRNA attenuated the IPP5-induced inhibition of neurite growth. Thus, these data indicate that selectively expressed IPP5 inhibits neurite growth by maintaining TGF-β signaling in primary sensory neurons.

  14. Notch signaling alters sensory or neuronal cell fate specification of inner ear stem cells.

    PubMed

    Jeon, Sang-Jun; Fujioka, Masato; Kim, Shi-Chan; Edge, Albert S B

    2011-06-08

    Multipotent progenitor cells in the otic placode give rise to the specialized cell types of the inner ear, including neurons, supporting cells, and hair cells. The mechanisms governing acquisition of specific fates by the cells that form the cochleovestibular organs remain poorly characterized. Here we show that whereas blocking Notch signaling with a γ-secretase inhibitor increased the conversion of inner ear stem cells to hair cells by a mechanism that involved the upregulation of bHLH transcription factor, Math1 (mouse Atoh1), differentiation to a neuronal lineage was increased by expression of the Notch intracellular domain. The shift to a neuronal lineage could be attributed in part to continued cell proliferation in cells that did not undergo sensory cell differentiation due to the high Notch signaling, but also involved upregulation of Ngn1. The Notch intracellular domain influenced Ngn1 indirectly by upregulation of Sox2, a transcription factor expressed in many neural progenitor cells, and directly by an interaction with an RBP-J binding site in the Ngn1 promoter/enhancer. The induction of Ngn1 was blocked partially by mutation of the RBP-J site and nearly completely when the mutation was combined with inhibition of Sox2 expression. Thus, Notch signaling had a significant role in the fate specification of neurons and hair cells from inner ear stem cells, and decisions about cell fate were mediated in part by a differential effect of combinatorial signaling by Notch and Sox2 on the expression of bHLH transcription factors.

  15. NOTCH SIGNALING ALTERS SENSORY OR NEURONAL CELL FATE SPECIFICATION OF INNER EAR STEM CELLS

    PubMed Central

    Jeon, Sang-Jun; Fujioka, Masato; Kim, Shi-Chan; Edge, Albert S.B.

    2011-01-01

    Multipotent progenitor cells in the otic placode give rise to the specialized cell types of the inner ear, including neurons, supporting cells and hair cells. The mechanisms governing acquisition of specific fates by the cells that form the cochleovestibular organs remain poorly characterized. Here we show that whereas blocking Notch signaling with a γ-secretase inhibitor increased the conversion of inner ear stem cells to hair cells by a mechanism that involved the upregulation of bHLH transcription factor, Math1 (mouse Atoh1), differentiation to a neuronal lineage was increased by expression of the Notch intracellular domain. The shift to a neuronal lineage could be attributed in part to the continued cell proliferation in cells that did not undergo sensory cell differentiation due to the high Notch signaling, but also involved upregulation of Ngn1. The Notch intracellular domain influenced Ngn1 indirectly by upregulation of Sox2, a transcription factor expressed in many neural progenitor cells, and directly by an interaction with an RBP-J binding site in the Ngn1 promoter/enhancer. The induction of Ngn1 was blocked partially by mutation of the RBP-J site and nearly completely when the mutation was combined with inhibition of Sox2 expression. Thus Notch signaling had a significant role in the fate specification of neurons and hair cells from inner ear stem cells, and decisions about cell fate were mediated in part by a differential effect of combinatorial signaling by Notch and Sox2 on the expression of bHLH transcription factors. PMID:21653840

  16. Inhibition of primate spinothalamic tract neurons by spinal glycine and GABA is modulated by guanosine 3',5'-cyclic monophosphate.

    PubMed

    Lin, Q; Wu, J; Peng, Y B; Cui, M; Willis, W D

    1999-03-01

    Our recent work has suggested that the nitric oxide/guanosine 3', 5'-cyclic monophosphate (NO/cGMP) signal transduction system contributes to central sensitization of spinothalamic tract (STT) neurons in part by influencing the descending inhibition of nociception resulting from stimulation in the periaqueductal gray. This study was designed to examine further whether activation of the NO/cGMP cascade reduces the inhibition of the activity of STT neurons mediated by spinal inhibitory amino acid (IAA) receptors. Responses of STT cells to noxious cutaneous stimuli were inhibited by iontophoresis of glycine and GABA agonists in anesthetized monkeys. Administration of 8-bromoguanosine-3',5'-cyclophosphate sodium (8-bromo-cGMP), a membrane permeable analogue of cGMP, either by microdialysis or by iontophoresis reduced significantly the IAA-induced inhibition of wide dynamic range (WDR) STT cells in the deep layers of the dorsal horn. The reduction in inhibition lasted for up to 1-1.5 h after the cessation of drug infusion. In contrast, IAA-induced inhibition of WDR STT cells in the superficial dorsal horn and high-threshold (HT) cells in superficial or deep layers was not significantly changed during 8-bromo-cGMP infusion. Iontophoresis of 8-bromo-cGMP onto STT cells produced the same actions as produced by microdialysis of this agent, but the effect was not as long-lasting nor as potent. Finally, an attenuation of the IAA receptor-mediated inhibition of STT cells produced by iontophoretic release of a NO donor, 3-morpholinosydnonimine, could be blocked by pretreatment of the spinal cord with a guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. These results suggest that an increased spinal cGMP level contributes to the sensitization of WDR STT neurons in the deep dorsal horn in part by down-regulating spinal IAA receptors. However, no evidence is provided in this study that the NO/cGMP cascade regulates IAA receptors on HT and superficial WDR

  17. [Morphological transformation of sensory ganglion neurons and satellite cells].

    PubMed

    Matsuda, S; Kobayashi, N; Mominoki, K; Wakisaka, H; Mori, M; Murakami, S

    1998-12-01

    Sensory ganglion neurons in higher vertebrates are unique in that they are pseudounipolar with a single stem process that divides at some distance from the cell body into central and peripheral processes. In the early stages of development, these neurons are bipolar but later they became pseudounipolar. This developmental process of sensory ganglion neurons with satellite cells was examined by scanning electron microscopy following removal of connective tissue. This pseudo-unipolarization began earlier but proceeded at a slower rate in chick than in rat embryos. This difference may due to the difference found in the extent and intimacy of satellite cell investments in these two animals, which was due to the fact that sensory neurons undergo pseudo-unipolarization only in the presence of satellite cells in vitro. The neuronal perikaryal projections were observed by scanning electron microscopy after removal of connective tissue and satellite cells. Morphometric analysis reveal that perikaryal projections were more numerous on the surface of mature pseudounipolar neurons than on the surface of premature bipolar neurons, and that the number of projections increased as the neuronal cell bodies grew larger. This may support the hypothesis that perikaryal projections are structural devices for increasing the neuron-satellite interface and for improving the efficiency of metabolic exchange between these two cell types. These results suggest that satellite cells play an important role in neuronal maturation.

  18. Prospects for Replacement of Auditory Neurons by Stem Cells

    PubMed Central

    Shi, Fuxin; Edge, Albert S.B.

    2013-01-01

    Sensorineural hearing loss is caused by degeneration of hair cells or auditory neurons. Spiral ganglion cells, the primary afferent neurons of the auditory system, are patterned during development and send out projections to hair cells and to the brainstem under the control of largely unknown guidance molecules. The neurons do not regenerate after loss and even damage to their projections tends to be permanent. The genesis of spiral ganglion neurons and their synapses forms a basis for regenerative approaches. In this review we critically present the current experimental findings on auditory neuron replacement. We discuss the latest advances with a focus on (a) exogenous stem cell transplantation into the cochlea for neural replacement, (b) expression of local guidance signals in the cochlea after loss of auditory neurons, (c) the possibility of neural replacement from an endogenous cell source, and (d) functional changes from cell engraftment. PMID:23370457

  19. Prospects for replacement of auditory neurons by stem cells.

    PubMed

    Shi, Fuxin; Edge, Albert S B

    2013-03-01

    Sensorineural hearing loss is caused by degeneration of hair cells or auditory neurons. Spiral ganglion cells, the primary afferent neurons of the auditory system, are patterned during development and send out projections to hair cells and to the brainstem under the control of largely unknown guidance molecules. The neurons do not regenerate after loss and even damage to their projections tends to be permanent. The genesis of spiral ganglion neurons and their synapses forms a basis for regenerative approaches. In this review we critically present the current experimental findings on auditory neuron replacement. We discuss the latest advances with a focus on (a) exogenous stem cell transplantation into the cochlea for neural replacement, (b) expression of local guidance signals in the cochlea after loss of auditory neurons, (c) the possibility of neural replacement from an endogenous cell source, and (d) functional changes from cell engraftment. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Intracisternal injection of palmitoylethanolamide inhibits the peripheral nociceptive evoked responses of dorsal horn wide dynamic range neurons.

    PubMed

    González-Hernández, Abimael; Martínez-Lorenzana, Guadalupe; Rodríguez-Jiménez, Javier; Rojas-Piloni, Gerardo; Condés-Lara, Miguel

    2015-03-01

    Endogenous palmitoylethanolamide (PEA) has a key role in pain modulation. Central or peripheral PEA can reduce nociceptive behavior, but no study has yet reported a descending inhibitory effect on the neuronal nociceptive activity of Aδ- and C-fibers. This study shows that intracisternal PEA inhibits the peripheral nociceptive responses of dorsal horn wide dynamic range cells (i.e., inhibition of Aδ- and C-fibers), an effect blocked by spinal methiothepin. These results suggest that a descending analgesic mechanism mediated by the serotonergic system could be activated by central PEA.

  1. GABAergic inhibition sharpens the frequency tuning and enhances phase locking in chicken nucleus magnocellularis neurons.

    PubMed

    Fukui, Iwao; Burger, R Michael; Ohmori, Harunori; Rubel, Edwin W

    2010-09-08

    GABAergic modulation of activity in avian cochlear nucleus neurons has been studied extensively in vitro. However, how this modulation actually influences processing in vivo is not known. We investigated responses of chicken nucleus magnocellularis (NM) neurons to sound while pharmacologically manipulating the inhibitory input from the superior olivary nucleus (SON). SON receives excitatory inputs from nucleus angularis (NA) and nucleus laminaris (NL), and provides GABAergic inputs to NM, NA, NL, and putatively to the contralateral SON. Results from single-unit extracellular recordings from 2 to 4 weeks posthatch chickens show that firing rates of auditory nerve fibers increased monotonically with sound intensity, while that of NM neurons saturated or even decreased at moderate or loud sound levels. Blocking GABAergic input with local application of TTX into the SON induced an increase in firing rate of ipsilateral NM, while that of the contralateral NM decreased at high sound levels. Moreover, local application of bicuculline to NM also increased the firing rate of NM neurons at high sound levels, reduced phase locking, and broadened the frequency-tuning properties of NM neurons. Following application of DNQX, clear evidence of inhibition was observed. Furthermore, the inhibition was tuned to a broader frequency range than the excitatory response areas. We conclude that GABAergic inhibition from SON has at least three physiological influences on the activity of NM neurons: it regulates the firing activity of NM units in a sound-level-dependent manner; it improves phase selectivity; and it sharpens frequency tuning of NM neuronal responses.

  2. GABAergic inhibition sharpens the frequency tuning and enhances phase locking in chicken nucleus magnocellularis neurons

    PubMed Central

    Fukui, Iwao; Burger, R Michael; Ohmori, Harunori; Rubel, Edwin W

    2010-01-01

    GABAergic modulation of activity in avian cochlear nucleus neurons has been studied extensively in vitro. However, how this modulation actually influences processing in vivo is not known. We investigated responses of chicken nucleus magnocellularis (NM) neurons to sound while pharmacologically manipulating the inhibitory input from the superior olivary nucleus (SON). SON receives excitatory inputs from nucleus angularis (NA) and nucleus laminaris (NL), and provides GABAergic inputs to NM, NA, NL, and putatively to the contralateral SON. Results from single unit extracellular recordings from 2–4 wks posthatch chickens show that firing rates of auditory nerve fibers (ANFs) increased monotonically with sound intensity, while that of NM neurons saturated or even decreased at moderate or loud sound levels. Blocking GABAergic input with local application of TTX into the SON induced an increase in firing rate of ipsilateral NM while that of the contralateral NM decreased at high sound levels. Moreover, local application of bicuculline to NM also increased the firing rate of NM neurons at high sound levels, reduced phase-locking, and broadened the frequency tuning properties of NM neurons. Following application of DNQX, clear evidence of inhibition was observed. Furthermore, the inhibition was tuned to a broader frequency range than the excitatory response areas. We conclude that GABAergic inhibition from SON has at least three physiological influences on the activity of NM neurons: it regulates the firing activity of NM units in a sound-level dependent manner; it improves phase selectivity; and it sharpens frequency tuning of NM neuronal responses. PMID:20826670

  3. Paraneoplastic cerebellar degeneration with a circulating antibody against neurons and non-neuronal cells.

    PubMed

    Tomimoto, H; Brengman, J M; Yanagihara, T

    1993-01-01

    We describe a woman with paraneoplastic cerebellar degeneration associated with para-ovarian adenocarcinoma, who had a circulating antibody with a corresponding antigen not only in cerebellar Purkinje cells but also in neurons located in the molecular layer of the human and rat cerebellum. The antigen was also present in neurons in the cerebral cortex, brain stem, anterior horn cells, dorsal root ganglia, intestinal autonomic neurons, retinal ganglion cells, Schwann cells of the peripheral nerve and epithelial cells of the renal glomerulus in rats. Immunoelectron microscopy revealed immunoprecipitates in the smooth and rough endoplasmic reticulum and polyribosomes in human and rat cerebellar Purkinje cells and other neuronal cell bodies as well as Schwann cells of the peripheral nerve. Even though patients with this disorder manifest primarily with cerebellar and some extracerebellar signs, the antigen also exists in many neurons other than cerebellar Purkinje cells and even in non-neuronal cells. The clinicopathologic significance of the observed immunologic reaction in diverse neurons remains to be determined.

  4. mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy.

    PubMed

    Srivastava, Isha N; Shperdheja, Jona; Baybis, Marianna; Ferguson, Tanya; Crino, Peter B

    2016-01-01

    Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP. Mouse pups (P6) were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified. Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in

  5. Cannabinoids inhibit network-driven synapse loss between hippocampal neurons in culture

    PubMed Central

    Kim, Hee Jung; Waataja, Jonathan J.; Thayer, Stanley A.

    2008-01-01

    Dendritic pruning and loss of synaptic contacts are early events in many neurodegenerative diseases. These effects are dynamic and appear to differ mechanistically from the cell death process. Cannabinoids modulate synaptic activity and afford protection in some neurotoxicity models. We investigated the effects of cannabinoids on activity-induced changes in the number of synapses between rat hippocampal neurons in culture. Morphology and synapses were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 fused to enhanced green fluorescent protein (PSD95-GFP). Reducing the extracellular Mg2+ concentration to 0.1 mM for 4 hr induced intense synaptic activity that decreased the number of PSD95-GFP puncta by 45 ± 13 %. Synapse loss was an early event, required activation of NMDA receptors and was mediated by the ubiquitin-proteasome pathway. The cannabinoid receptor full agonist (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate (WIN55,212-2; EC50=2.5±0.5 nM) and the partial agonist Δ9-tetrahydrocannabinol (THC; EC50=9±3 nM) inhibited PSD loss in a manner reversed by the CB1 receptor antagonist rimonabant. The protection was mimicked by inhibition of presynaptic Ca2+ channels and WIN55,212-2 did not prevent PSD loss elicited by direct application of glutamate, suggesting a presynaptic mechanism. Prolonged exposure to WIN55,212-2, but not THC, desensitized the protective effect. Treating cells that had undergone PSD loss with WIN55,212-2 reversed the loss and enabled recovery of a full compliment of synapses. The modulation of synaptic number by acute and prolonged exposure to cannabinoids may account for some of the effects of these drugs on the plasticity, survival and function of neural networks. PMID:18310474

  6. JNK inhibition of VMAT2 contributes to rotenone-induced oxidative stress and dopamine neuron death

    PubMed Central

    Choi, Won-Seok; Kim, Hyung-Wook; Xia, Zhengui

    2014-01-01

    Treatment with rotenone, both in vitro and in vivo, is widely used to model dopamine neuron death in Parkinson’s disease upon exposure to environmental neurotoxicants and pesticides. Mechanisms underlying rotenone neurotoxicity are still being defined. Our recent studies suggest that rotenone-induced dopamine neuron death involves microtubule destabilization, which leads to accumulation of cytosolic dopamine and consequently reactive oxygen species (ROS). Furthermore, the c-Jun N-terminal protein kinase (JNK) is required for rotenone-induced dopamine neuron death. Here we report that the neural specific JNK3 isoform of the JNKs, but not JNK1 or JNK2, is responsible for this neuron death in primary cultured dopamine neurons. Treatment with taxol, a microtubule stabilizing agent, attenuates rotenone-induced phosphorylation and presumably activation of JNK. This suggests that JNK is activated by microtubule destabilization upon rotenone exposure. Moreover, rotenone inhibits VMAT2 activity but not VMAT2 protein levels. Significantly, treatment with SP600125, a pharmacological inhibitor of JNKs, attenuates rotenone inhibition of VMAT2. Furthermore, decreased VMAT2 activity following in vitro incubation of recombinant JNK3 protein with purified mesencephalic synaptic vesicles suggests that JNK3 can inhibit VMAT2 activity. Together with our previous findings, these results suggest that rotenone induces dopamine neuron death through a series of sequential events including microtubule destabilization, JNK3 activation, VMAT2 inhibition, accumulation of cytosolic dopamine, and generation of ROS. Our data identify JNK3 as a novel regulator of VMAT2 activity. PMID:25496994

  7. Human recombinant interleukin-1 beta inhibits nicotinic transmission in neurons of guinea pig pelvic plexus ganglia.

    PubMed

    Lin, J; Krier, J

    1995-12-01

    The actions of human recombinant interleukin-1 beta (hrIL-1 beta) were tested on guinea pig pelvic plexus ganglion neurons using intracellular electrophysiological methods in vitro. hrIL-1 beta caused membrane depolarization associated with a decreased input resistance or inward currents in 54% of neurons tested. hrIL-1 beta caused a hyperpolarization associated with an increase in input resistance or outward currents in 30% of neurons tested. hrIL-1 beta-evoked responses were not altered by hexamethonium (100 microM), atropine (0.5 microM), yohimbine (0.3 microM), or naloxone (1 microM), indicating that cholinergic, alpha 2-adrenergic, or opioid receptors were not involved. Drugs that inhibit Na+, Ca2+, or K+ channels did not change hrIL-1 beta-evoked responses. Stimulation of synaptic inputs to pelvic ganglion neurons evoked nicotinic cholinergic fast excitatory postsynaptic potentials (fEPSPs). hrIL-1 beta inhibited fEPSPs in 44% of neurons tested but had no effect on acetylcholine-induced depolarizations. An IL-1 beta receptor antagonist blocked all actions of hrIL-1 beta. In summary, hrIL-1 beta has excitatory and inhibitory actions on pelvic ganglion neurons. Inhibition of fEPSPs by hrIL-1 beta may be due to presynaptic inhibition of acetylcholine release.

  8. Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

    PubMed Central

    de Haas, A. H.; van Weering, H. R. J.; de Jong, E. K.; Boddeke, H. W. G. M.

    2007-01-01

    Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron–astrocyte, neuron–microglia, and neuron–neuron interaction. PMID:17952658

  9. The extracellular protease stl functions to inhibit migration of v'ch1 sensory neuron during Drosophila embryogenesis.

    PubMed

    Lhamo, Tashi; Ismat, Afshan

    2015-08-01

    Proper migration of cells through the dense and complex extracellular matrix (ECM) requires constant restructuring of the ECM to allow cells to move forward in a smooth manner. This restructuring can occur through the action of extracellular enzymes. Among these extracellular enzymes is the ADAMTS (A Disintegrin And Metalloprotease with ThromboSpondin repeats) family of secreted extracellular proteases. Drosophila stl encodes an ADAMTS protease expressed in and around the peripheral nervous system (PNS) during embryogenesis. The absence of stl displayed one specific neuron, the v'ch1 sensory neuron, migrating to its target sooner than in wild type. During normal development, the v'ch1 sensory neuron migrates dorsally at the same time it is extending an axon ventrally toward the CNS. Surprisingly, in the absence of stl, the v'ch1 neuron migrated further dorsally as compared to the wild type at stage 15, but did not migrate past its correct target at stage 16, suggesting a novel role for this extracellular protease in inhibiting migration of this neuron past a certain point.

  10. The Polyphenol Altenusin Inhibits in Vitro Fibrillization of Tau and Reduces Induced Tau Pathology in Primary Neurons.

    PubMed

    Chua, Sook Wern; Cornejo, Alberto; van Eersel, Janet; Stevens, Claire H; Vaca, Inmaculada; Cueto, Mercedes; Kassiou, Michael; Gladbach, Amadeus; Macmillan, Alex; Lewis, Lev; Whan, Renee; Ittner, Lars M

    2017-04-19

    In Alzheimer's disease, the microtubule-associated protein tau forms intracellular neurofibrillary tangles (NFTs). A critical step in the formation of NFTs is the conversion of soluble tau into insoluble filaments. Accordingly, a current therapeutic strategy in clinical trials is aimed at preventing tau aggregation. Here, we assessed altenusin, a bioactive polyphenolic compound, for its potential to inhibit tau aggregation. Altenusin inhibits aggregation of tau protein into paired helical filaments in vitro. This was associated with stabilization of tau dimers and other oligomers into globular structures as revealed by atomic force microscopy. Moreover, altenusin reduced tau phosphorylation in cells expressing pathogenic tau, and prevented neuritic tau pathology induced by incubation of primary neurons with tau fibrils. However, treatment of tau transgenic mice did not improve neuropathology and functional deficits. Taken together, altenusin prevents tau fibrillization in vitro and induced tau pathology in neurons.

  11. Alternative functions of core cell cycle regulators in neuronal migration, neuronal maturation, and synaptic plasticity

    PubMed Central

    Frank, Christopher L.; Tsai, Li-Huei

    2009-01-01

    Recent studies have demonstrated that boundaries separating a cycling cell from a post-mitotic neuron are not as concrete as expected. Novel and unique physiological functions in neurons have been ascribed for proteins fundamentally required for cell cycle progression and control. These “core” cell cycle regulators serve diverse post-mitotic functions that span various developmental stages of a neuron, including neuronal migration, axonal elongation, axon pruning, dendrite morphogenesis, and synaptic maturation and plasticity. In this review, we detail the non-proliferative post-mitotic roles that these cell cycle proteins have recently been reported to play, the significance of their expression in neurons, mechanistic insight when available, and future prospects. PMID:19447088

  12. Orientation selectivity in inhibition-dominated networks of spiking neurons: effect of single neuron properties and network dynamics.

    PubMed

    Sadeh, Sadra; Rotter, Stefan

    2015-01-01

    The neuronal mechanisms underlying the emergence of orientation selectivity in the primary visual cortex of mammals are still elusive. In rodents, visual neurons show highly selective responses to oriented stimuli, but neighboring neurons do not necessarily have similar preferences. Instead of a smooth map, one observes a salt-and-pepper organization of orientation selectivity. Modeling studies have recently confirmed that balanced random networks are indeed capable of amplifying weakly tuned inputs and generating highly selective output responses, even in absence of feature-selective recurrent connectivity. Here we seek to elucidate the neuronal mechanisms underlying this phenomenon by resorting to networks of integrate-and-fire neurons, which are amenable to analytic treatment. Specifically, in networks of perfect integrate-and-fire neurons, we observe that highly selective and contrast invariant output responses emerge, very similar to networks of leaky integrate-and-fire neurons. We then demonstrate that a theory based on mean firing rates and the detailed network topology predicts the output responses, and explains the mechanisms underlying the suppression of the common-mode, amplification of modulation, and contrast invariance. Increasing inhibition dominance in our networks makes the rectifying nonlinearity more prominent, which in turn adds some distortions to the otherwise essentially linear prediction. An extension of the linear theory can account for all the distortions, enabling us to compute the exact shape of every individual tuning curve in our networks. We show that this simple form of nonlinearity adds two important properties to orientation selectivity in the network, namely sharpening of tuning curves and extra suppression of the modulation. The theory can be further extended to account for the nonlinearity of the leaky model by replacing the rectifier by the appropriate smooth input-output transfer function. These results are robust and do not

  13. Orientation Selectivity in Inhibition-Dominated Networks of Spiking Neurons: Effect of Single Neuron Properties and Network Dynamics

    PubMed Central

    Sadeh, Sadra; Rotter, Stefan

    2015-01-01

    The neuronal mechanisms underlying the emergence of orientation selectivity in the primary visual cortex of mammals are still elusive. In rodents, visual neurons show highly selective responses to oriented stimuli, but neighboring neurons do not necessarily have similar preferences. Instead of a smooth map, one observes a salt-and-pepper organization of orientation selectivity. Modeling studies have recently confirmed that balanced random networks are indeed capable of amplifying weakly tuned inputs and generating highly selective output responses, even in absence of feature-selective recurrent connectivity. Here we seek to elucidate the neuronal mechanisms underlying this phenomenon by resorting to networks of integrate-and-fire neurons, which are amenable to analytic treatment. Specifically, in networks of perfect integrate-and-fire neurons, we observe that highly selective and contrast invariant output responses emerge, very similar to networks of leaky integrate-and-fire neurons. We then demonstrate that a theory based on mean firing rates and the detailed network topology predicts the output responses, and explains the mechanisms underlying the suppression of the common-mode, amplification of modulation, and contrast invariance. Increasing inhibition dominance in our networks makes the rectifying nonlinearity more prominent, which in turn adds some distortions to the otherwise essentially linear prediction. An extension of the linear theory can account for all the distortions, enabling us to compute the exact shape of every individual tuning curve in our networks. We show that this simple form of nonlinearity adds two important properties to orientation selectivity in the network, namely sharpening of tuning curves and extra suppression of the modulation. The theory can be further extended to account for the nonlinearity of the leaky model by replacing the rectifier by the appropriate smooth input-output transfer function. These results are robust and do not

  14. Cortical cell and neuron density estimates in one chimpanzee hemisphere.

    PubMed

    Collins, Christine E; Turner, Emily C; Sawyer, Eva Kille; Reed, Jamie L; Young, Nicole A; Flaherty, David K; Kaas, Jon H

    2016-01-19

    The density of cells and neurons in the neocortex of many mammals varies across cortical areas and regions. This variability is, perhaps, most pronounced in primates. Nonuniformity in the composition of cortex suggests regions of the cortex have different specializations. Specifically, regions with densely packed neurons contain smaller neurons that are activated by relatively few inputs, thereby preserving information, whereas regions that are less densely packed have larger neurons that have more integrative functions. Here we present the numbers of cells and neurons for 742 discrete locations across the neocortex in a chimpanzee. Using isotropic fractionation and flow fractionation methods for cell and neuron counts, we estimate that neocortex of one hemisphere contains 9.5 billion cells and 3.7 billion neurons. Primary visual cortex occupies 35 cm(2) of surface, 10% of the total, and contains 737 million densely packed neurons, 20% of the total neurons contained within the hemisphere. Other areas of high neuron packing include secondary visual areas, somatosensory cortex, and prefrontal granular cortex. Areas of low levels of neuron packing density include motor and premotor cortex. These values reflect those obtained from more limited samples of cortex in humans and other primates.

  15. Electrophysiological Properties of Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Risner-Janiczek, Jessica R.; Ungless, Mark A.; Li, Meng

    2011-01-01

    In vitro generation of functional neurons from embryonic stem (ES) cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP) and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP+ neurons in culture display functional neuronal properties even at early stages of differentiation. PMID:21887381

  16. Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons.

    PubMed

    Pepponi, Rita; Ferrante, Antonella; Ferretti, Roberta; Martire, Alberto; Popoli, Patrizia

    2009-09-01

    Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Adenosine A(2A) receptors regulate extracellular glutamate levels by acting on both the release and the uptake of glutamate. The aim of this study was to evaluate whether the inhibition of the effects of glutamate uptake blockers by adenosine A(2A) receptor antagonists resulted in neuroprotection. In cortical and striatal neuronal cultures, the application of l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), induced a dose-dependent increase in lactate dehydrogenase (LDH) levels, an index of cytotoxicity. Such an effect of PDC was significantly reduced by pre-treatment with the adenosine A(2A) receptor antagonist ZM 241385 (50 nM) in striatal, but not cortical, cultures. The protective effects of ZM 241385 were specifically due to a counteraction of PDC effects, since ZM 241385 was totally ineffective in preventing the cytotoxicity induced by direct application of glutamate to cultures. These results indicate that adenosine A(2A) receptor antagonists prevent the toxic effects induced by a transportable competitive inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity.

  17. Dopamine and baclofen inhibit the hyperpolarization-activated cation current in rat ventral tegmental neurones.

    PubMed Central

    Jiang, Z G; Pessia, M; North, R A

    1993-01-01

    1. Whole-cell patch-clamp recordings were made from dopamine-containing ventral tegmental area neurones in slices of rat midbrain. An inward current (Ih) was activated by hyperpolarization from -60 mV. 2. Dopamine (30 microM) reduced the amplitude of Ih by 10-30% at potentials from -70 to -120 mV. The effect was concentration dependent, mimicked by the D2 agonist quinpirole, and prevented by the D2 antagonist (-)-sulpiride. Baclofen (0.3-3 microM) also inhibited Ih; this action was antagonized by 2-hydroxysaclofen but not by (-)-sulpiride. The decrease in Ih resulted from a reduction in the maximal current with no change in the voltage dependence. 3. The action of dopamine was unaffected by cadmium (200 microM), forskolin (10 microM), the adenylyl cyclase inhibitor 2',3'-dideoxyadenosine (100 microM), or by intracellular solution containing cyclic AMP (2 mM). 4. Ih was progressively reduced during the first 5-10 min of recording with electrodes containing guanosine 5'-O-(3-thiotriphosphate); after this time, dopamine had no further effect. 5. It is concluded that agonists acting at D2 receptors and GABAB receptors reduce Ih in ventral tegmental neurones. PMID:8392580

  18. Dehydration-induced modulation of kappa-opioid inhibition of vasopressin neurone activity.

    PubMed

    Scott, Victoria; Bishop, Valerie R; Leng, Gareth; Brown, Colin H

    2009-12-01

    Dehydration increases vasopressin (antidiuretic hormone) secretion from the posterior pituitary gland to reduce water loss in the urine. Vasopressin secretion is determined by action potential firing in vasopressin neurones, which can exhibit continuous, phasic (alternating periods of activity and silence), or irregular activity. Autocrine kappa-opioid inhibition contributes to the generation of activity patterning of vasopressin neurones under basal conditions and so we used in vivo extracellular single unit recording to test the hypothesis that changes in autocrine kappa-opioid inhibition drive changes in activity patterning of vasopressin neurones during dehydration. Dehydration increased the firing rate of rat vasopressin neurones displaying continuous activity (from 7.1 +/- 0.5 to 9.0 +/- 0.6 spikes s(1)) and phasic activity (from 4.2 +/- 0.7 to 7.8 +/- 0.9 spikes s(1)), but not those displaying irregular activity. The dehydration-induced increase in phasic activity was via an increase in intraburst firing rate. The selective -opioid receptor antagonist nor-binaltorphimine increased the firing rate of phasic neurones in non-dehydrated rats (from 3.4 +/- 0.8 to 5.3 +/- 0.6 spikes s(1)) and dehydrated rats (from 6.4 +/- 0.5 to 9.1 +/- 1.2 spikes s(1)), indicating that kappa-opioid feedback inhibition of phasic bursts is maintained during dehydration. In a separate series of experiments, prodynorphin mRNA expression was increased in vasopressin neurones of hyperosmotic rats, compared to hypo-osmotic rats. Hence, it appears that dynorphin expression in vasopressin neurones undergoes dynamic changes in proportion to the required secretion of vasopressin so that, even under stimulated conditions, autocrine feedback inhibition of vasopressin neurones prevents over-excitation.

  19. Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

    PubMed Central

    Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

    2015-01-01

    Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

  20. DJ-1 mediates paraquat-induced dopaminergic neuronal cell death.

    PubMed

    Kwon, Hyun Joo; Heo, Jun Young; Shim, Jung Hee; Park, Ji Hoon; Seo, Kang Sik; Ryu, Min Jeong; Han, Jeong Su; Shong, Minho; Son, Jin H; Kweon, Gi Ryang

    2011-04-25

    There are two causes of Parkinson's disease (PD): environmental insults and genetic mutations of PD-associated genes. Environmental insults and genetic mutations lead to mitochondrial dysfunction, and a combination of mitochondrial dysfunction and increased oxidative stress in dopaminergic neurons is thought to contribute to the pathogenesis of PD. Among the PD-associated genes, DJ-1 acts as a redox sensor for oxidative stress and has been also proposed to maintain mitochondrial complex I activity. To understand molecular functions of DJ-1 in the cell, we have generated DJ-1 null cells from the DJ-1(-/-) mouse embryos. Using these null cells, we investigated the susceptibility to an environmental toxin, paraquat, which is known to inhibit mitochondrial complex I. Interestingly, we found that DJ-1 null cells showed a resistance to paraquat-induced apoptosis, including reduced poly (ADP-ribose) polymerase and procaspase-3. Also DJ-1 null cells generated less superoxide than SN4741 cells by paraquat treatment. Consistent with the reduced paraquat sensitivity, DJ-1 null cells showed reduced complex I activity, which was partially rescued by ectopic DJ-I expression. In summary, our results suggest that DJ-1 is critical to maintain mitochondrial complex I and complex I could be a key target in interaction of paraquat toxicity and DJ-1 for giving rise to PD.

  1. Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion

    PubMed Central

    LaMotte, Robert H.; Chao, MA

    2009-01-01

    The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG (“CCD” model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accompanied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword. PMID:18958366

  2. Hyperexcitable neurons and altered non-neuronal cells in the compressed spinal ganglion.

    PubMed

    LaMotte, Robert H; Ma, Chao

    2008-10-25

    The cell body or soma in the dosal root ganglion (DRG) is normally excitable and this excitability can increase and persist after an injury of peripheral sensory neurons. In a rat model of radicular pain, an intraforaminal implantation of a rod that chronically compressed the lumbar DRG ("CCD" model) resulted in neuronal somal hyperexcitability and spontaneous activity that was accompanied by hyperalgesia in the ipsilateral hind paw. By the 5th day after onset of CCD, there was a novel upregulation in neuronal expression of the chemokine, monocyte chemoattractant protein-1 (MCP-1 or CCL2) and also its receptor, CCR2. The neurons developed, in response to topically applied MCP-1, an excitatory response that they normally do not have. CCD also activated non-neuronal cells including, for example, the endothelial cells as evidenced by angiogenesis in the form of an increased number of capillaries in the DRG after 7 days. A working hypothesis is that the CCD induced changes in neurons and non-neuronal cells that may act together to promote the survival of the injured tissue. The release of ligands such as CCL2, in addition to possibly activating nociceptive neurons (maintaining the pain), may also act to preserve injured cells in the face of ischemia and hypoxia, for example, by promoting angiogenesis. Thus, somal hyperexcitability, as often said of inflammation, may represent a double edged sword.

  3. Acupuncture inhibits GABA neuron activity in the ventral tegmental area and reduces ethanol self-administration.

    PubMed

    Yang, Chae Ha; Yoon, Seong Shoon; Hansen, David M; Wilcox, Jeffrey D; Blumell, Bryan R; Park, Jung Jae; Steffensen, Scott C

    2010-12-01

    Withdrawal from chronic ethanol enhances ventral tegmental area (VTA) GABA neuron excitability and reduces mesolimbic dopamine (DA) neurotransmission, which is suppressed by acupuncture at Shenmen (HT7) points (Zhao et al., 2006). The aim of this study was to evaluate the effects of HT7 acupuncture on VTA GABA neuron excitability, ethanol inhibition of VTA GABA neuron firing rate, and ethanol self-administration. A role for opioid receptors (ORs) in ethanol and acupuncture effects is also explored. Using electrophysiological methods in mature rats, we evaluated the effects of HT7 stimulation and opioid antagonists on VTA GABA neuron firing rate. Using behavioral paradigms in rats, we evaluated the effects of HT7 stimulation and opioid antagonists on ethanol self-administration using a modification of the sucrose-fading procedure. HT7 stimulation produced a biphasic modulation of VTA GABA neuron firing rate characterized by transient enhancement followed by inhibition and subsequent recovery in 5 minutes. HT7 inhibition of VTA GABA neuron firing rate was blocked by systemic administration of the nonselective μ-opioid receptor antagonist naloxone. HT7 stimulation significantly reduced ethanol suppression of VTA GABA neuron firing rate, which was also blocked by naloxone. HT7 acupuncture reduced ethanol self-administration without affecting sucrose consumption. Systemic administration of the δ-opioid receptor (DOR) antagonist naltrindole blocked ethanol suppression of VTA GABA neuron firing rate and significantly reduced ethanol self-administration without affecting sucrose consumption. These findings suggest that DOR-mediated opioid modulation of VTA GABA neurons may mediate acupuncture's role in modulating mesolimbic DA release and suppressing the reinforcing effects of ethanol. Copyright © 2010 by the Research Society on Alcoholism.

  4. Interplay between low threshold voltage-gated K(+) channels and synaptic inhibition in neurons of the chicken nucleus laminaris along its frequency axis.

    PubMed

    Hamlet, William R; Liu, Yu-Wei; Tang, Zheng-Quan; Lu, Yong

    2014-01-01

    Central auditory neurons that localize sound in horizontal space have specialized intrinsic and synaptic cellular mechanisms to tightly control the threshold and timing for action potential generation. However, the critical interplay between intrinsic voltage-gated conductances and extrinsic synaptic conductances in determining neuronal output are not well understood. In chicken, neurons in the nucleus laminaris (NL) encode sound location using interaural time difference (ITD) as a cue. Along the tonotopic axis of NL, there exist robust differences among low, middle, and high frequency (LF, MF, and HF, respectively) neurons in a variety of neuronal properties such as low threshold voltage-gated K(+) (LTK) channels and depolarizing inhibition. This establishes NL as an ideal model to examine the interactions between LTK currents and synaptic inhibition across the tonotopic axis. Using whole-cell patch clamp recordings prepared from chicken embryos (E17-E18), we found that LTK currents were larger in MF and HF neurons than in LF neurons. Kinetic analysis revealed that LTK currents in MF neurons activated at lower voltages than in LF and HF neurons, whereas the inactivation of the currents was similar across the tonotopic axis. Surprisingly, blockade of LTK currents using dendrotoxin-I (DTX) tended to broaden the duration and increase the amplitude of the depolarizing inhibitory postsynaptic potentials (IPSPs) in NL neurons without dependence on coding frequency regions. Analyses of the effects of DTX on inhibitory postsynaptic currents led us to interpret this unexpected observation as a result of primarily postsynaptic effects of LTK currents on MF and HF neurons, and combined presynaptic and postsynaptic effects in LF neurons. Furthermore, DTX transferred subthreshold IPSPs to spikes. Taken together, the results suggest a critical role for LTK currents in regulating inhibitory synaptic strength in ITD-coding neurons at various frequencies.

  5. Interplay between low threshold voltage-gated K+ channels and synaptic inhibition in neurons of the chicken nucleus laminaris along its frequency axis

    PubMed Central

    Hamlet, William R.; Liu, Yu-Wei; Tang, Zheng-Quan; Lu, Yong

    2014-01-01

    Central auditory neurons that localize sound in horizontal space have specialized intrinsic and synaptic cellular mechanisms to tightly control the threshold and timing for action potential generation. However, the critical interplay between intrinsic voltage-gated conductances and extrinsic synaptic conductances in determining neuronal output are not well understood. In chicken, neurons in the nucleus laminaris (NL) encode sound location using interaural time difference (ITD) as a cue. Along the tonotopic axis of NL, there exist robust differences among low, middle, and high frequency (LF, MF, and HF, respectively) neurons in a variety of neuronal properties such as low threshold voltage-gated K+ (LTK) channels and depolarizing inhibition. This establishes NL as an ideal model to examine the interactions between LTK currents and synaptic inhibition across the tonotopic axis. Using whole-cell patch clamp recordings prepared from chicken embryos (E17–E18), we found that LTK currents were larger in MF and HF neurons than in LF neurons. Kinetic analysis revealed that LTK currents in MF neurons activated at lower voltages than in LF and HF neurons, whereas the inactivation of the currents was similar across the tonotopic axis. Surprisingly, blockade of LTK currents using dendrotoxin-I (DTX) tended to broaden the duration and increase the amplitude of the depolarizing inhibitory postsynaptic potentials (IPSPs) in NL neurons without dependence on coding frequency regions. Analyses of the effects of DTX on inhibitory postsynaptic currents led us to interpret this unexpected observation as a result of primarily postsynaptic effects of LTK currents on MF and HF neurons, and combined presynaptic and postsynaptic effects in LF neurons. Furthermore, DTX transferred subthreshold IPSPs to spikes. Taken together, the results suggest a critical role for LTK currents in regulating inhibitory synaptic strength in ITD-coding neurons at various frequencies. PMID:24904297

  6. Accelerated neuronal differentiation toward motor neuron lineage from human embryonic stem cell line (H9).

    PubMed

    Lu, David; Chen, Eric Y T; Lee, Philip; Wang, Yung-Chen; Ching, Wendy; Markey, Christopher; Gulstrom, Chase; Chen, Li-Ching; Nguyen, Thien; Chin, Wei-Chun

    2015-03-01

    Motor neurons loss plays a pivotal role in the pathoetiology of various debilitating diseases such as, but not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy. However, advancement in motor neuron replacement therapy has been significantly constrained by the difficulties in large-scale production at a cost-effective manner. Current methods to derive motor neuron heavily rely on biochemical stimulation, chemical biological screening, and complex physical cues. These existing methods are seriously challenged by extensive time requirements and poor yields. An innovative approach that overcomes prior hurdles and enhances the rate of successful motor neuron transplantation in patients is of critical demand. Iron, a trace element, is indispensable for the normal development and function of the central nervous system. Whether ferric ions promote neuronal differentiation and subsequently promote motor neuron lineage has never been considered. Here, we demonstrate that elevated iron concentration can drastically accelerate the differentiation of human embryonic stem cells (hESCs) toward motor neuron lineage potentially via a transferrin mediated pathway. HB9 expression in 500 nM iron-treated hESCs is approximately twofold higher than the control. Moreover, iron treatment generated more matured and functional motor neuron-like cells that are ∼1.5 times more sensitive to depolarization when compared to the control. Our methodology renders an expedited approach to harvest motor neuron-like cells for disease, traumatic injury regeneration, and drug screening.

  7. Patterned activity in stratum lacunosum moleculare inhibits CA1 pyramidal neuron firing.

    PubMed

    Dvorak-Carbone, H; Schuman, E M

    1999-12-01

    CA1 pyramidal cells are the primary output neurons of the hippocampus, carrying information about the result of hippocampal network processing to the subiculum and entorhinal cortex (EC) and thence out to the rest of the brain. The primary excitatory drive to the CA1 pyramidal cells comes via the Schaffer collateral (SC) projection from area CA3. There is also a direct projection from EC to stratum lacunosum-moleculare (SLM) of CA1, an input well positioned to modulate information flow through the hippocampus. High-frequency stimulation in SLM evokes an inhibition sufficiently strong to prevent CA1 pyramidal cells from spiking in response to SC input, a phenomenon we refer to as spike-blocking. We characterized the spike-blocking efficacy of burst stimulation (10 stimuli at 100 Hz) in SLM and found that it is greatest at approximately 300-600 ms after the burst, consistent with the time course of the slow GABA(B) signaling pathway. Spike-blocking efficacy increases in potency with the number of SLM stimuli in a burst, but also decreases with repeated presentations of SLM bursts. Spike-blocking was eliminated in the presence of GABA(B) antagonists. We have identified a candidate population of interneurons in SLM and distal stratum radiatum (SR) that may mediate this spike-blocking effect. We conclude that the output of CA1 pyramidal cells, and hence the hippocampus, is modulated in an input pattern-dependent manner by activation of the direct pathway from EC.

  8. Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network.

    PubMed

    Tarczyluk, Marta A; Nagel, David A; O'Neil, John D; Parri, H Rheinallt; Tse, Erin H Y; Coleman, Michael D; Hill, Eric J

    2013-09-01

    The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture.

  9. Inhibition of Acyl-CoA: cholesterol acyltransferase (ACAT), overexpression of cholesterol transporter gene, and protection of amyloid β (Aβ) oligomers-induced neuronal cell death by tricyclic pyrone molecules.

    PubMed

    Pokhrel, Laxman; Maezawa, Izumi; Nguyen, Thi D T; Chang, Kyeong-Ok; Jin, Lee-Way; Hua, Duy H

    2012-10-25

    A major effort in Alzheimer's disease therapeutic development has targeted Aβ and downstream events. We have synthesized a small library of tricyclic pyrone compounds. Their protective action in MC65 cells and inhibition of ACAT along with the upregulation of cholesterol transporter gene were investigated. Five active compounds exhibited potencies in the nanomolar ranges. The multiple effects of the compounds on Aβ and cellular cholesterol pathways could be potential mechanisms underlying the protective effects in vivo.

  10. Fasting enhances the response of arcuate neuropeptide Y-glucose-inhibited neurons to decreased extracellular glucose

    PubMed Central

    Murphy, Beth Ann; Fioramonti, Xavier; Jochnowitz, Nina; Fakira, Kurt; Gagen, Karen; Contie, Sylvain; Lorsignol, Anne; Penicaud, Luc; Martin, William J.; Routh, Vanessa H.

    2009-01-01

    Fasting increases neuropeptide Y (NPY) expression, peptide levels, and the excitability of NPY-expressing neurons in the hypothalamic arcuate (ARC) nucleus. A subpopulation of ARC-NPY neurons (∼40%) are glucose-inhibited (GI)-type glucose-sensing neurons. Hence, they depolarize in response to decreased glucose. Because fasting enhances NPY neurotransmission, we propose that during fasting, GI neurons depolarize in response to smaller decreases in glucose. This increased excitation in response to glucose decreases would increase NPY-GI neuronal excitability and enhance NPY neurotransmission. Using an in vitro hypothalamic explant system, we show that fasting enhances NPY release in response to decreased glucose concentration. By measuring relative changes in membrane potential using a membrane potential-sensitive dye, we demonstrate that during fasting, a smaller decrease in glucose depolarizes NPY-GI neurons. Furthermore, incubation in low (0.7 mM) glucose enhanced while leptin (10 nM) blocked depolarization of GI neurons in response to decreased glucose. Fasting, leptin, and glucose-induced changes in NPY-GI neuron glucose sensing were mediated by 5′-AMP-activated protein kinase (AMPK). We conclude that during energy sufficiency, leptin reduces the ability of NPY-GI neurons to sense decreased glucose. However, after a fast, decreased leptin and glucose activate AMPK in NPY-GI neurons. As a result, NPY-GI neurons become depolarized in response to smaller glucose fluctuations. Increased excitation of NPY-GI neurons enhances NPY release. NPY, in turn, shifts energy homeostasis toward increased food intake and decreased energy expenditure to restore energy balance. PMID:19211911

  11. Leucokinin mimetic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron.

    PubMed

    Kwon, Hyeogsun; Ali Agha, Moutaz; Smith, Ryan C; Nachman, Ronald J; Marion-Poll, Frédéric; Pietrantonio, Patricia V

    2016-06-21

    Insect kinins (leucokinins) are multifunctional peptides acting as neurohormones and neurotransmitters. In females of the mosquito vector Aedes aegypti (L.), aedeskinins are known to stimulate fluid secretion from the renal organs (Malpighian tubules) and hindgut contractions by activating a G protein-coupled kinin receptor designated "Aedae-KR." We used protease-resistant kinin analogs 1728, 1729, and 1460 to evaluate their effects on sucrose perception and feeding behavior. In no-choice feeding bioassays (capillary feeder and plate assays), the analog 1728, which contains α-amino isobutyric acid, inhibited females from feeding on sucrose. It further induced quick fly-away or walk-away behavior following contact with the tarsi and the mouthparts. Electrophysiological recordings from single long labellar sensilla of the proboscis demonstrated that mixing the analog 1728 at 1 mM with sucrose almost completely inhibited the detection of sucrose. Aedae-KR was immunolocalized in contact chemosensory neurons in prothoracic tarsi and in sensory neurons and accessory cells of long labellar sensilla in the distal labellum. Silencing Aedae-KR by RNAi significantly reduced gene expression and eliminated the feeding-aversion behavior resulting from contact with the analog 1728, thus directly implicating the Aedae-KR in the aversion response. To our knowledge, this is the first report that kinin analogs modulate sucrose perception in any insect. The aversion to feeding elicited by analog 1728 suggests that synthetic molecules targeting the mosquito Aedae-KR in the labellum and tarsi should be investigated for the potential to discover novel feeding deterrents of mosquito vectors.

  12. Leucokinin mimetic elicits aversive behavior in mosquito Aedes aegypti (L.) and inhibits the sugar taste neuron

    PubMed Central

    Kwon, Hyeogsun; Ali Agha, Moutaz; Smith, Ryan C.; Nachman, Ronald J.; Marion-Poll, Frédéric; Pietrantonio, Patricia V.

    2016-01-01

    Insect kinins (leucokinins) are multifunctional peptides acting as neurohormones and neurotransmitters. In females of the mosquito vector Aedes aegypti (L.), aedeskinins are known to stimulate fluid secretion from the renal organs (Malpighian tubules) and hindgut contractions by activating a G protein-coupled kinin receptor designated “Aedae-KR.” We used protease-resistant kinin analogs 1728, 1729, and 1460 to evaluate their effects on sucrose perception and feeding behavior. In no-choice feeding bioassays (capillary feeder and plate assays), the analog 1728, which contains α-amino isobutyric acid, inhibited females from feeding on sucrose. It further induced quick fly-away or walk-away behavior following contact with the tarsi and the mouthparts. Electrophysiological recordings from single long labellar sensilla of the proboscis demonstrated that mixing the analog 1728 at 1 mM with sucrose almost completely inhibited the detection of sucrose. Aedae-KR was immunolocalized in contact chemosensory neurons in prothoracic tarsi and in sensory neurons and accessory cells of long labellar sensilla in the distal labellum. Silencing Aedae-KR by RNAi significantly reduced gene expression and eliminated the feeding-aversion behavior resulting from contact with the analog 1728, thus directly implicating the Aedae-KR in the aversion response. To our knowledge, this is the first report that kinin analogs modulate sucrose perception in any insect. The aversion to feeding elicited by analog 1728 suggests that synthetic molecules targeting the mosquito Aedae-KR in the labellum and tarsi should be investigated for the potential to discover novel feeding deterrents of mosquito vectors. PMID:27274056

  13. Current status of neuronal cell xenotransplantation.

    PubMed

    Vadori, Marta; Aron Badin, Romina; Hantraye, Philippe; Cozzi, Emanuele

    2015-11-01

    Neural cell transplantation has long been considered as an option for the treatment of neurodegenerative disorders. To date, several patients with Parkinson's and Huntington's diseases have been treated with human fetal-derived neurons with disparate results. However, the limited efficacy to date combined with the scarce availability of human fetal tissues and ethical concerns render this procedure inapplicable to a wide population scale. With a view to overcoming these shortcomings, transplantation of pig-derived cell precursors has been proposed and applied in preclinical and clinical trials. Recently long-term survival (more than 18 months) associated with clinical efficacy has been reported following transplantation of genetically engineered porcine neural precursors in fully immunosuppressed primate recipients. Despite the promising results obtained to date, several questions remain unanswered. In particular, the ideal xenogeneic cell-products to transplant, the extent of the immune response against the implanted xenograft and the most suitable therapeutic strategies to improve engraftment are all issues that still need to be thoroughly addressed. The present review describes the current knowledge in the pig-to-primate xenotransplantation field. In this context, recent data on human-to-nonhuman primate xenogeneic stem cell-based treatments for neurological disorders are discussed. Copyright © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

  14. The inhibition of neuronal calcium ion channels by trace levels of yttrium released from carbon nanotubes

    PubMed Central

    Jakubek, Lorin; Marangoudakis, Spiro; Raingo, Jessica; Liu, Xinyuan; Lipscombe, Diane; Hurt, Robert

    2009-01-01

    Carbon nanotubes (CNTs) are used with increasing frequency in neuroengineering applications. CNT scaffolds are used to transmit electrical stimulation to cultured neurons and to control outgrowth and branching patterns of neurites. CNTs have been reported to disrupt normal neuronal function including alterations in endocytotic capability and inhibition of ion channels. Calcium ion channels regulate numerous neuronal and cellular functions including endo and exocytosis, neurite outgrowth, and gene expression. Strong CNT interactions with neuronal calcium ion channels would have profound biological implications. Here we show that physiological solutions containing CNTs inhibit neuronal voltage-gated calcium-ion channels in a dose dependent and CNT-sample-dependent manner with IC50 as low as 1.2 ug/ml. Importantly, we demonstrate that the inhibitory activity does not involve tubular graphene as previously reported, but rather very low concentrations of soluble yttrium released from the nanotube growth catalyst. Cationic yttrium potently inhibits calcium ion channel function with an inhibitory efficacy, IC50, of 0.07 ppm w/w. Because of this potency, unpurified and even some reportedly “purified” CNT samples contain sufficient bioavailable yttrium to inhibit channel function. Our results have important implications for emerging nano-neurotechnology and highlight the critical role that trace components can play in the biological response to complex nanomaterials. PMID:19698989

  15. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons.

    PubMed

    Machado, Carolina Barcellos; Kanning, Kevin C; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-02-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

  16. Reconstruction of phrenic neuron identity in embryonic stem cell-derived motor neurons

    PubMed Central

    Machado, Carolina Barcellos; Kanning, Kevin C.; Kreis, Patricia; Stevenson, Danielle; Crossley, Martin; Nowak, Magdalena; Iacovino, Michelina; Kyba, Michael; Chambers, David; Blanc, Eric; Lieberam, Ivo

    2014-01-01

    Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations. PMID:24496616

  17. Ca(2+)-sensitive inhibition by Pb(2+) of alpha7-containing nicotinic acetylcholine receptors in hippocampal neurons.

    PubMed

    Mike, A; Pereira, E F; Albuquerque, E X

    2000-08-04

    In the present study the patch-clamp technique was applied to cultured hippocampal neurons to determine the kinetics as well as the agonist concentration- and Ca(2+)-dependence of Pb(2+)-induced inhibition of alpha7 nicotinic receptors (nAChRs). Evidence is provided that more than two-thirds of the inhibition by Pb(2+) (3-30 microM) of alpha7 nAChR-mediated whole-cell currents (referred to as type IA currents) develops rapidly and is fully reversible upon washing. The estimated values for tau(onset) and tau(recovery) were 165 and 240 ms, respectively. The magnitude of the effect of Pb(2+) was the same regardless of whether acetylcholine or choline was the agonist. Pre-exposure of the neurons for 800 ms to Pb(2+) (30 microM) decreased the amplitude and accelerated the decay phase of currents evoked by moderate to high agonist concentrations. In contrast, only the amplitude of currents evoked by low agonist concentrations was reduced when the neurons were exposed simultaneously to Pb(2+) and the agonists. Taken together with the findings that Pb(2+) reduces the frequency of opening and the mean open time of alpha7 nAChR channels, these data suggest that Pb(2+) accelerates the rate of receptor desensitization. An additional reduction of type IA current amplitudes occurred after 2-min exposure of the neurons to Pb(2+). This effect was not reversible upon washing of the neurons and was most likely due to an intracellular action of Pb(2+). Pb(2+)-induced inhibition of alpha7 nAChRs, which was hindered by the enhancement of extracellular Ca(2+) concentrations, may contribute to the neurotoxicity of the heavy metal.

  18. Synaptobrevin I mediates exocytosis of CGRP from sensory neurons and inhibition by botulinum toxins reflects their anti-nociceptive potential.

    PubMed

    Meng, Jianghui; Wang, Jiafu; Lawrence, Gary; Dolly, J Oliver

    2007-08-15

    Calcitonin-gene-related peptide (CGRP), a potent vasodilator that mediates inflammatory pain, is elevated in migraine; nevertheless, little is known about its release from sensory neurons. In this study, CGRP was found to occur in the majority of neurons from rat trigeminal ganglia, together with the three exocytotic SNAREs [SNAP25, syntaxin 1 and the synaptobrevin (Sbr, also known as VAMP) isoforms] and synaptotagmin. Ca(2+)-dependent CGRP release was evoked with K(+)-depolarisation and, to lower levels, by capsaicin or bradykinin from neurons that contain the vanilloid receptor 1 and/or bradykinin receptor 2. Botulinum neurotoxin (BoNT) type A cleaved SNAP25 and inhibited release triggered by K(+) > bradykinin > capsaicin. Unlike BoNT type D, BoNT type B did not affect exocytosis, even though the neurons possess its receptor and Sbr II and Sbr III got proteolysed (I is resistant in rat) but, in mouse neurons, it additionally cleaved Sbr I and blocked transmitter release. Sbr I and II were found in CGRP-containing vesicles, and each was shown to separately form a SNARE complex. These new findings, together with punctate staining of Sbr I and CGRP in neurites, implicate isoform Sbr I in exocytosis from large dense-core vesicles together with SNAP25 (also, probably, syntaxin 1 because BoNT type C1 caused partial cleavage and inhibition); this differs from Sbr-II-dependent release of transmitters from small synaptic vesicles. Such use of particular Sbr isoform(s) by different neurons raises the functional implications for other cells previously unrecognised.

  19. Glycinergic tonic inhibition of hippocampal neurons with depolarizing GABAergic transmission elicits histopathological signs of temporal lobe epilepsy

    PubMed Central

    Eichler, Sabrina A; Kirischuk, Sergei; Jüttner, René; Schafermeier, Philipp K; Legendre, Pascal; Lehmann, Thomas-Nicolas; Gloveli, Tengis; Grantyn, Rosemarie; Meier, Jochen C

    2008-01-01

    An increasing number of epilepsy patients are afflicted with drug-resistant temporal lobe epilepsy (TLE) and require alternative therapeutic approaches. High-affinity glycine receptors (haGlyRs) are functionally adapted to tonic inhibition due to their response to hippocampal ambient glycine, and their synthesis is activity-dependent. Therefore, in our study, we scanned TLE hippocampectomies for expression of haGlyRs and characterized the effects mediated by these receptors using primary hippocampal neurons. Increased haGlyR expression occurred in TLE hippocampi obtained from patients with a severe course of disease. Furthermore, in TLE patients, haGlyR and potassium chloride cotransporter 2 (KCC2) expressions were inversely regulated. To examine this potential causal relationship with respect to TLE histopathology, we established a hippocampal cell culture system utilising tonic inhibition mediated by haGlyRs in response to hippocam-pal ambient glycine and in the context of a high Cl equilibrium potential, as is the case in TLE hippocampal neurons. We showed that hypoactive neurons increase their ratio between glutamatergic and GABAergic synapses, reduce their dendrite length and finally undergo excitotoxicity. Pharmacological dissection of the underlying processes revealed ionotropic glutamate and TrkB receptors as critical mediators between neuronal hypoactivity and the emergence of these TLE-characteristic histopathological signs. Moreover, our results indicate a beneficial role for KCC2, because decreasing the Cl− equilibrium potential by KCC2 expression also rescued hypoactive hippocampal neurons. Thus, our data support a causal relationship between increased haGlyR expression and the emergence of histopathological TLE-characteristic signs, and they establish a pathophysiological role for neuronal hypoactivity in the context of a high Cl− equilibrium potential. PMID:19210758

  20. Activation and inhibition of rat neuronal nicotinic receptors by ABT-418

    PubMed Central

    Papke, Roger L; Thinschmidt, Jeffrey S; Moulton, Becky A; Meyer, Edwin M; Poirier, Amy

    1997-01-01

    ABT-418 appeared to function as a relatively broad spectrum activator of neuronal nicotinic receptors, expressed in Xenopus oocytes, with little cross reactivity to the mammalian muscle receptor subtype. However, the relative potencies of ABT-418 at the various subtypes differed from those acetylcholine (ACh). For example, ACh was most potent at α3β2 (EC50≈30 μM) and least potent at α2β2 (EC50≈500 μM). ABT-418 was most potent at α4β2 and α2β2 (EC50≈6 μM and 11 μM, respectively) and least potent at α3β4 (EC50≈188 μM).In addition to activating neuronal receptors, ABT-418 exhibited complex properties, including the inhibition of ACh responses.The current responses elicited by relatively high concentrations of ABT-418 on the α4β2 receptor subtype were protracted beyond the application interval. The coapplication of ABT-418 with either of the use-dependent inhibitors bis(1,2,2,6,6-tetramethyl-4-pipendimyl)sebacate (BTMPS) or tetramethyl-pipenidine (TMP) eliminated the late protracted phase of the currents with only small effects on the initial activation phase. When the reversible inhibitor TMP was washed from the bath, the previously inhibited late current reappeared, suggesting that the observed mixed agonist-antagonist effects of ABT-418 and (±)-epibatidine on α4β2 were due to a concentration-dependent noncompetitive inhibition, an effect similar to that obtained for (−)-nicotine.The inhibition of α4β2 receptors by ABT-418 was voltage-dependent. When high concentrations of ABT-418 were applied under depolarizing conditions, additional late currents could be observed under conditions which suggested that a build up of ABT-418 in an unstirred layer over the surface of the oocyte was occurring. This may have been due to the dissociation of the drug from channel blocking sites on the receptors themselves, or alternatively, from the plasma membrane of the cells. PMID:9031746

  1. Inhibition of the Aplysia sensory neuron calcium current with dopamine and serotonin.

    PubMed

    Dunn, Tyler W; Sossin, Wayne S

    2013-11-01

    The inhibition of Aplysia pleural mechanosensory neuron synapses by dopamine and serotonin through activation of endogenous dopaminergic and expressed 5-HT1Apl(a)/b receptors, respectively, involves a reduction in action potential-associated calcium influx. We show that the inhibition of synaptic efficacy is downstream of the readily releasable pool, suggesting that inhibition is at the level of calcium secretion coupling, likely a result of the changes in the calcium current. Indeed, the inhibitory responses directly reduce a CaV2-like calcium current in isolated sensory neurons. The inhibition of the calcium current is voltage independent as it is not affected by a strong depolarizing prepulse, consistent with other invertebrate CaV2 calcium currents. Similar to voltage-independent inhibition of vertebrate nociceptors, inhibition was blocked with Src tyrosine kinase inhibitors. The data suggest a conserved mechanism by which G protein-coupled receptor activation can inhibit the CaV2 calcium current in nociceptive neurons.

  2. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons.

    PubMed

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A

    2014-10-17

    During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia-NCAMs) modulate cell-cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia-NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb's to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell-cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  3. Müllerian inhibiting substance is anterogradely transported and does not attenuate avulsion-induced death of hypoglossal motor neurons

    PubMed Central

    Clarkson, Andrew N.; Talbot, Caroline L.; Wang, Pei-Yu; MacLaughlin, David T.; Donahoe, Patricia K.; McLennan, Ian S.

    2013-01-01

    Müllerian Inhibiting Substance (MIS, Anti-Müllerian hormone) is a gonadal hormone that contributes to the subtle sex-biases in the nervous system. Mature neurons of both sexes also produce MIS, suggesting that MIS may be a paracrine regulator of adult neural networks. We report here that murine hypoglossal motor neurons produce MIS and its receptors, MISRII and bone morphogenetic protein receptor 1A (BMPR1A, ALK3), but differentially transport them, with only MIS being detectable in axons. The production of MIS and its receptors were rapidly down regulated after axonal damage, which is a characteristic of genes involved in mature neuronal function. MIS is a survival factor for embryonic spinal motor neurons, but the rate of cell loss after hypoglossal nerve avulsion was normal in Mis−/− mice and was not attenuated by intraventricular administration of MIS. These observations suggest that MIS may be involved in anterograde rather than autocrine or retrograde regulation of neurons. PMID:21195071

  4. Estrogens stimulate serotonin neurons to inhibit binge-like eating in mice

    PubMed Central

    Cao, Xuehong; Xu, Pingwen; Oyola, Mario G.; Xia, Yan; Yan, Xiaofeng; Saito, Kenji; Zou, Fang; Wang, Chunmei; Yang, Yongjie; Hinton, Antentor; Yan, Chunling; Ding, Hongfang; Zhu, Liangru; Yu, Likai; Yang, Bin; Feng, Yuxin; Clegg, Deborah J.; Khan, Sohaib; DiMarchi, Richard; Mani, Shaila K.; Tong, Qingchun; Xu, Yong

    2014-01-01

    Binge eating afflicts approximately 5% of US adults, though effective treatments are limited. Here, we showed that estrogen replacement substantially suppresses binge-like eating behavior in ovariectomized female mice. Estrogen-dependent inhibition of binge-like eating was blocked in female mice specifically lacking estrogen receptor-α (ERα) in serotonin (5-HT) neurons in the dorsal raphe nuclei (DRN). Administration of a recently developed glucagon-like peptide-1–estrogen (GLP-1–estrogen) conjugate designed to deliver estrogen to GLP1 receptor–enhanced regions effectively targeted bioactive estrogens to the DRN and substantially suppressed binge-like eating in ovariectomized female mice. Administration of GLP-1 alone reduced binge-like eating, but not to the same extent as the GLP-1–estrogen conjugate. Administration of ERα-selective agonist propylpyrazole triol (PPT) to murine DRN 5-HT neurons activated these neurons in an ERα-dependent manner. PPT also inhibited a small conductance Ca2+-activated K+ (SK) current; blockade of the SK current prevented PPT-induced activation of DRN 5-HT neurons. Furthermore, local inhibition of the SK current in the DRN markedly suppressed binge-like eating in female mice. Together, our data indicate that estrogens act upon ERα to inhibit the SK current in DRN 5-HT neurons, thereby activating these neurons to suppress binge-like eating behavior and suggest ERα and/or SK current in DRN 5-HT neurons as potential targets for anti-binge therapies. PMID:25157819

  5. Phenylethanoid glycosides from Cistanches salsa inhibit apoptosis induced by 1-methyl-4-phenylpyridinium ion in neurons.

    PubMed

    Tian, Xue-Fei; Pu, Xiao-Ping

    2005-02-10

    In our study we investigated the neuroprotective effects of phenylethanoid glycosides (PhGs) from Cistanches salsa on 1-methyl-4-phenylpyridinium ion (MPP(+))-induced apoptosis in cerebellar granule neurons (CGNs). CGNs were treated with 100 microM MPP(+) for 24h to induce apoptosis, simultaneously CGNs were incubated with PhGs at 10, 20 and 40 microg/ml, respectively. In addition CGNs were pretreated with PhGs at 20 microg/ml for 6, 12, 24 h, respectively, and then treated with 100 microM MPP(+) for 24 h. 3-(4,5-Dimethylthiazol-2-ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the treatment of CGNs with PhGs inhibited the decrease of cell viability induced by MPP(+). The activation of caspase-3 and caspase-8 was induced by MPP(+) in apoptosis. The caspase-3 and caspase-8 fluorogenic assays showed that the treatments of CGNs with PhGs efficiently suppressed the activation of caspase-3 and caspase-8 induced by MPP(+). It is concluded that PhGs can prevent the MPP(+)-induced apoptosis in CGNs and exert its anti-apoptosis effect by inhibiting caspase-3 and caspase-8 activities.

  6. Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease

    PubMed Central

    Zhai, Jinbin; Zhang, Lei; Mojsilovic-Petrovic, Jelena; Jian, Xiaoying; Thomas, Jeffrey; Homma, Kengo; Schmitz, Anton; Famulok, Michael; Ichijo, Hidenori; Argon, Yair; Randazzo, Paul A.

    2015-01-01

    Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS. PMID:26085633

  7. Microinfusion of bupropion inhibits putative GABAergic neuronal activity of the ventral tegmental area.

    PubMed

    Amirabadi, Sanaz; Pakdel, Firouz Ghaderi; Shahabi, Parviz; Naderi, Somayyeh; Osalou, Mostafa Ashrafi; Cankurt, Ulker

    2014-01-01

    The most common interpretation for the mechanisms of antidepression is the increase of the brain monoamine levels such as dopamine (DA). The increase of DA can reduce depression but it can also decrease the monoamine release because of autoreceptor inhibition. Although bupropion can decrease the dopamine release, there is evidence about stimulatory effects of chronic application of bupropion on ventral tegmental area (VTA) neurons. In this study, the intra-VTA acute microinfusion of bupropion on putative VTA non-Dopaminergic (VTA-nonDA) neuronal firing rates was evaluated by a single neuron recording technique. Animals were divided into 7 groups (sham, and 6 bupropion-microinfused groups with 1, 10(-1), 10(-2), 10(-3), 10(-4), and 10(-5) mol, 1 µl/3 min, intra-VTA). A single neuron recording technique was done according to the stereotaxic coordination. After 10 min baseline recording, ACSF or bupropion was microinfused. The recording continued to recovery period in the treated groups. The prestimulus time (PST) and interspike interval (ISI) histograms were calculated for every single unit. The assessment of the drug effect was carried out by one-way analysis of variance (ANOVA) and Post-hoc test. 126 non-DA neurons were separated. Bupropion could inhibit 116 neurons and 11 neurons had no significant response. Maximum inhibition was 79.1% of baseline firing rate with 44.3 min duration. The inhibitory effect of bupropion was dose-dependent. The acute inhibitory effects of bupropion on VTA-nonDA neurons can explain the fast inhibitory effects of bupropion and other antidepressants on the VTA. These data can explain some side effects of antidepressants.

  8. Microinfusion of Bupropion Inhibits Putative GABAergic Neuronal Activity of the Ventral Tegmental Area

    PubMed Central

    Amirabadi, Sanaz; Pakdel, Firouz Ghaderi; Shahabi, Parviz; Naderi, Somayyeh; Osalou, Mostafa Ashrafi; Cankurt, Ulker

    2014-01-01

    Introduction The most common interpretation for the mechanisms of antidepression is the increase of the brain monoamine levels such as dopamine (DA). The increase of DA can reduce depression but it can also decrease the monoamine release because of autoreceptor inhibition. Although bupropion can decrease the dopamine release, there is evidence about stimulatory effects of chronic application of bupropion on ventral tegmental area (VTA) neurons. In this study, the intra-VTA acute microinfusion of bupropion on putative VTA non-Dopaminergic (VTA-nonDA) neuronal firing rates was evaluated by a single neuron recording technique. Methods Animals were divided into 7 groups (sham, and 6 bupropion-microinfused groups with 1, 10-1, 10-2, 10-3, 10-4, and 10-5 mol, 1 µl/3 min, intra-VTA). A single neuron recording technique was done according to the stereotaxic coordination. After 10 min baseline recording, ACSF or bupropion was microinfused. The recording continued to recovery period in the treated groups. The prestimulus time (PST) and interspike interval (ISI) histograms were calculated for every single unit. The assessment of the drug effect was carried out by one-way analysis of variance (ANOVA) and Post-hoc test. Results 126 non-DA neurons were separated. Bupropion could inhibit 116 neurons and 11 neurons had no significant response. Maximum inhibition was 79.1% of baseline firing rate with 44.3 min duration. The inhibitory effect of bupropion was dose-dependent. Discussion The acute inhibitory effects of bupropion on VTA-nonDA neurons can explain the fast inhibitory effects of bupropion and other antidepressants on the VTA. These data can explain some side effects of antidepressants. PMID:25337378

  9. Excitation and inhibition onto central courtship neurons biases Drosophila mate choice

    PubMed Central

    Kallman, Benjamin R; Kim, Heesoo; Scott, Kristin

    2015-01-01

    The ability to distinguish males from females is essential for productive mate selection and species propagation. Recent studies in Drosophila have identified different classes of contact chemosensory neurons that detect female or male pheromones and influence courtship decisions. Here, we examine central neural pathways in the male brain that process female and male pheromones using anatomical, calcium imaging, optogenetic, and behavioral studies. We find that sensory neurons that detect female pheromones, but not male pheromones, activate a novel class of neurons in the ventral nerve cord to cause activation of P1 neurons, male-specific command neurons that trigger courtship. In addition, sensory neurons that detect male pheromones, as well as those that detect female pheromones, activate central mAL neurons to inhibit P1. These studies demonstrate that the balance of excitatory and inhibitory drives onto central courtship-promoting neurons controls mating decisions. DOI: http://dx.doi.org/10.7554/eLife.11188.001 PMID:26568316

  10. MARK2 Rescues Nogo-66-Induced Inhibition of Neurite Outgrowth via Regulating Microtubule-Associated Proteins in Neurons In Vitro.

    PubMed

    Zuo, Yu-Chao; Xiong, Nan-Xiang; Shen, Jian-Ying; Yu, Hua; Huang, Yi-Zhi; Zhao, Hong-Yang

    2016-11-01

    The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

  11. Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons

    SciTech Connect

    Park, Kyoung Ho; Yeo, Sang Won; Troy, Frederic A.

    2014-10-17

    Highlights: • PolySia expressed on neurons primarily during early stages of neuronal development. • PolySia–NCAM is expressed on neural stem cells from adult guinea pig spiral ganglion. • PolySia is a biomarker that modulates neuronal differentiation in inner ear stem cells. - Abstract: During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

  12. Brain-derived neurotrophic factor-dependent cdk1 inhibition prevents G2/M progression in differentiating tetraploid neurons.

    PubMed

    Ovejero-Benito, María C; Frade, José M

    2013-01-01

    Neurodegeneration is often associated with DNA synthesis in neurons, the latter usually remaining for a long time as tetraploid cells before dying by apoptosis. The molecular mechanism preventing G2/M transition in these neurons remains unknown, but it may be reminiscent of the mechanism that maintains tetraploid retinal ganglion cells (RGCs) in a G2-like state during normal development, thus preventing their death. Here we show that this latter process, known to depend on brain-derived neurotrophic factor (BDNF), requires the inhibition of cdk1 by TrkB. We demonstrate that a subpopulation of chick RGCs previously shown to become tetraploid co-expresses TrkB and cdk1 in vivo. By using an in vitro system that recapitulates differentiation and cell cycle re-entry of chick retinal neurons we show that BDNF, employed at concentrations specific for the TrkB receptor, reduces the expression of cdk1 in TrkB-positive, differentiating neurons. In this system, BDNF also inhibits the activity of both endogenous cdk1 and exogenously-expressed cdk1/cyclin B1 complex. This inhibition correlates with the phosphorylation of cdk1 at Tyr15, an effect that can be prevented with K252a, a tyrosine kinase inhibitor commonly used to prevent the activity of neurotrophins through their Trk receptors. The effect of BDNF on cdk1 activity is Tyr15-specific since BDNF cannot prevent the activity of a constitutively active form of cdk1 (Tyr15Phe) when expressed in differentiating retinal neurons. We also show that BDNF-dependent phosphorylation of cdk1 at Tyr15 could not be blocked with MK-1775, a Wee1-selective inhibitor, indicating that Tyr15 phosphorylation in cdk1 does not seem to occur through the canonical mechanism observed in proliferating cells. We conclude that the inhibition of both expression and activity of cdk1 through a BDNF-dependent mechanism contributes to the maintenance of tetraploid RGCs in a G2-like state.

  13. Cell specific electrodes for neuronal network reconstruction and monitoring.

    PubMed

    Bendali, Amel; Bouguelia, Sihem; Roupioz, Yoann; Forster, Valérie; Mailley, Pascal; Benosman, Ryad; Livache, Thierry; Sahel, José-Alain; Picaud, Serge

    2014-07-07

    Direct interfacing of neurons with electronic devices has been investigated for both prosthetic and neuro-computing applications. In vitro neuronal networks provide great tools not only for improving neuroprostheses but also to take advantage of their computing abilities. However, it is often difficult to organize neuronal networks according to specific cell distributions. Our aim was to develop a cell-type specific immobilization of neurons on individual electrodes to produce organized in vitro neuronal networks on multi-electrode arrays (MEAs). We demonstrate the selective capture of retinal neurons on antibody functionalized surfaces following the formation of self-assembled monolayers from protein-thiol conjugates by simple contact and protein-polypyrrole deposits by electrochemical functionalization. This neuronal selection was achieved on gold for either cone photoreceptors or retinal ganglion neurons using a PNA lectin or a Thy1 antibody, respectively. Anti-fouling of un-functionalized gold surfaces was optimized to increase the capture efficiencies. The technique was extended to electrode arrays by addressing electropolymerization of pyrrole monomers and pyrrole-protein conjugates to active electrodes. Retinal ganglion cell recording on the array further demonstrated the integrity of these neurons following their selection on polypyrrole-coated electrodes. Therefore, this protein-polypyrrole electrodeposition could provide a new approach to generate organized in vitro neuronal networks.

  14. Inhibiting cholesterol degradation induces neuronal sclerosis and epileptic activity in mouse hippocampus.

    PubMed

    Chali,