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Sample records for cerebellar granule cell

  1. Synaptic representation of locomotion in single cerebellar granule cells

    PubMed Central

    Powell, Kate; Mathy, Alexandre; Duguid, Ian; Häusser, Michael

    2015-01-01

    The cerebellum plays a crucial role in the regulation of locomotion, but how movement is represented at the synaptic level is not known. Here, we use in vivo patch-clamp recordings to show that locomotion can be directly read out from mossy fiber synaptic input and spike output in single granule cells. The increase in granule cell spiking during locomotion is enhanced by glutamate spillover currents recruited during movement. Surprisingly, the entire step sequence can be predicted from input EPSCs and output spikes of a single granule cell, suggesting that a robust gait code is present already at the cerebellar input layer and transmitted via the granule cell pathway to downstream Purkinje cells. Thus, synaptic input delivers remarkably rich information to single neurons during locomotion. DOI: http://dx.doi.org/10.7554/eLife.07290.001 PMID:26083712

  2. Multimodal sensory integration in single cerebellar granule cells in vivo

    PubMed Central

    Ishikawa, Taro; Shimuta, Misa; Häusser, Michael

    2015-01-01

    The mammalian cerebellum is a highly multimodal structure, receiving inputs from multiple sensory modalities and integrating them during complex sensorimotor coordination tasks. Previously, using cell-type-specific anatomical projection mapping, it was shown that multimodal pathways converge onto individual cerebellar granule cells (Huang et al., 2013). Here we directly measure synaptic currents using in vivo patch-clamp recordings and confirm that a subset of single granule cells receive convergent functional multimodal (somatosensory, auditory, and visual) inputs via separate mossy fibers. Furthermore, we show that the integration of multimodal signals by granule cells can enhance action potential output. These recordings directly demonstrate functional convergence of multimodal signals onto single granule cells. DOI: http://dx.doi.org/10.7554/eLife.12916.001 PMID:26714108

  3. Inhibition of Cerebellar Granule Cell Turning by Alcohol

    PubMed Central

    Kumada, Tatsuro; Komuro, Yutaro; Li, Ying; Hu, Taofang; Wang, Zhe; Littner, Yoav; Komuro, Hitoshi

    2010-01-01

    Ectopic neurons are often found in the brains of fetal alcohol spectrum disorders (FASD) and fetal alcohol syndrome (FAS) patients, suggesting that alcohol exposure impairs neuronal cell migration. Although it has been reported that alcohol decreases the speed of neuronal cell migration, little is known about whether alcohol also affects the turning of neurons. Here we show that ethanol exposure inhibits the turning of cerebellar granule cells in vivo and in vitro. First, in vivo studies using P10 mice demonstrated that a single i.p. injection of ethanol not only reduces the number of turning granule cells but also alters the mode of turning at the EGL-ML border of the cerebellum. Second, in vitro analysis using microexplant cultures of P0-P3 mouse cerebella revealed that ethanol directly reduces the frequency of spontaneous granule cell turning in a dose-dependent manner. Third, the action of ethanol on the frequency of granule cell turning was significantly ameliorated by stimulating Ca2+ and cGMP signaling or by inhibiting cAMP signaling. Taken together, these results indicate that ethanol affects the frequency and mode of cerebellar granule cell turning through alteration of the Ca2+ and cyclic nucleotide signaling pathways, suggesting that the abnormal allocation of neurons found in the brains of FASD and FSA patients results, at least in part, from impaired turning of immature neurons by alcohol. PMID:20691765

  4. Extracellular potassium concentration regulates proliferation of immature cerebellar granule cells.

    PubMed

    Borodinsky, L N; Fiszman, M L

    1998-04-17

    The present study examines the effect of depolarizing potassium concentrations on the proliferation of immature rat cerebellar neurons. Cells inoculated in serum free medium and 5 mM KCl (5 K) showed a high degree of 3H-thymidine incorporation that decreased 24-48 h after plating as differentiation began. During the first 24 h after inoculation, cells grown in high potassium (25 K), showed a 34 +/- 3% increase (mean +/- S.E.M., n = 12) in 3H-thymidine incorporation as compared with the values observed in 5 K. After 24 h in vitro, cells grown in 25 K showed 23 +/- 3% (mean +/- S.E.M., n = 3) less DNA synthesis than those inoculated in 5 K. The increase in DNA synthesis due to 25 K was blocked by MgCl2 and nifedipine, but not by omega-conotoxin GVIA, suggesting that it is mediated by a Ca2+ influx via voltage-gated calcium channels (VGCC) of the L-subtype. High potassium-induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor (PD98059, 75 microM). The number of neurons counted after 48 h in vitro in 25 K was 35-100% above of the number obtained with 5 K and this increase also was blocked by MgCl2 and nifedipine. These data support the hypothesis that depolarizing activity during neurogenesis plays a role in the modulation of cerebellar granule cells proliferation. PMID:9602050

  5. AMPA receptors in cerebellar granule cells during development in culture.

    PubMed

    Hack, N J; Sluiter, A A; Balázs, R

    1995-06-27

    The survival and maturation of differentiating cerebellar granule cells in culture are known to be promoted by excitatory amino acids (EAAs) which, however, compromise the survival of mature cells. In contrast to the trophic effect, the toxic effect of alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropiate (AMPA) could only be elicited when the desensitisation of AMPA receptors was blocked, cyclothiazide being used in this study. Nevertheless, even under these conditions, toxicity induced by AMPA in contrast to kainate was, at 9 DIV, only half of the maximal toxicity attained by 13-16 DIV. Since cellular responses to AMPA depend so dramatically on the maturational stage of granule cells, we examined here whether this characteristic is related to developmental changes in AMPA receptor properties, which may result from changes in the subunit composition of the receptor. In contrast to toxicity, AMPA-induced 45Ca2+ influx (determined in the presence of cyclothiazide and the NMDA receptor blocker MK-801) reached a maximum already at 9 DIV. This also applied to a fraction of the 45Ca2+ uptake which persisted either after Cd2+ application or under Na(+)-free conditions and therefore presumably was mediated directly through AMPA receptor channels. Quantitative analysis of Western blots showed that the amounts of GluR4 and to a lesser extent GluR2/3/4c are substantial already at 2 DIV, remaining fairly constant until 9 DIV, followed by an increase by 16 DIV. However GluR1, which is hardly detectable in granule cells in vivo and is also low early in vitro, increased almost linearly with cultivation time.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7554232

  6. Calcium transients in cerebellar granule cell presynaptic terminals.

    PubMed Central

    Regehr, W G; Atluri, P P

    1995-01-01

    Calcium ions act presynaptically to modulate synaptic strength and to trigger neurotransmitter release. Here we detect stimulus-evoked changes in residual free calcium ([Ca2+]i) in rat cerebellar granule cell presynaptic terminals. Granule cell axons, known as parallel fibers, and their associated boutons, were labeled with several calcium indicators. When parallel fibers were extracellularly activated with stimulus trains, calcium accumulated in the terminals, producing changes in the fluorescence of the indicators. During the stimulus train, the fluorescence change per pulse became progressively smaller with the high affinity indicators Fura-2 and calcium green-2 but remained constant with the low affinity dyes BTC and furaptra. In addition, fluorescence transients of high affinity dyes were slower than those of low affinity indicators, which appear to accurately report the time course of calcium transients. Simulations show that differences in the observed transients can be explained by the different affinities and off rates of the fluorophores. The return of [Ca2+]i to resting levels can be approximated by an exponential decay with a time constant of 150 ms. On the basis of the degree of saturation in the response of high affinity dyes observed during trains, we estimate that each action potential increases [Ca2+]i in the terminal by several hundred nanomolar. These findings indicate that in these terminals [Ca2+]i transients are much larger and faster than those observed in larger boutons, such as those at the neuromuscular junction. Such rapid [Ca2+]i dynamics may be found in many of the terminals in the mammalian brain that are similar in size to parallel fiber boutons. Images FIGURE 1 PMID:7612860

  7. Neuroligin-2 accelerates GABAergic synapse maturation in cerebellar granule cells.

    PubMed

    Fu, Zhanyan; Vicini, Stefano

    2009-09-01

    Neuroligins (NLGs) are postsynaptic cell adhesion molecules that are thought to function in synaptogenesis. To investigate the role of NLGs on synaptic transmission once the synapse is formed, we transfected neuroligin-2 (NLG-2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABA(A) (gamma-aminobutyric acid) receptor mediated miniature postsynaptic currents (mIPSCs). NLG-2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV7-8). Down-regulation of NLG-2 by the isoform specific shRNA-NLG-2 resulted in an opposite effect. We and others have shown that the switch of alpha subunits of GABA(A)Rs from alpha2/3 to alpha1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG-2 is due to the recruitment of more alpha1 containing GABA(A)Rs at the synapses, we examined the prolongation of current decay by the Zolpidem, which has been shown to preferentially enhance the activity of alpha1 subunit-containing GABA channel. The application of Zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG-2 over-expressing cells than control cells, suggesting that NLG-2 over-expression accelerates synapse maturation by promoting incorporation of the alpha1 subunit-containing GABA(A)Rs at postsynaptic sites in immature cells. In addition, the effect of NLG-2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from alpha1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG-2 could be also due to the impacts of NLG-2 on the GABA transient in synaptic cleft, we measured the sensitivity of mIPSCs to the fast-off competitive antagonists TPMPA. We found that TPMPA similarly inhibits mIPSCs in control and NLG-2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of

  8. Alcohol enhances GABAergic transmission to cerebellar granule cells via an increase in Golgi cell excitability.

    PubMed

    Carta, Mario; Mameli, Manuel; Valenzuela, C Fernando

    2004-04-14

    Alcohol intoxication alters coordination and motor skills, and this is responsible for a significant number of traffic accident-related deaths around the world. Although the precise mechanism of action of ethanol (EtOH) is presently unknown, studies suggest that it acts, in part, by interfering with normal cerebellar functioning. An important component of cerebellar circuits is the granule cell. The excitability of these abundantly expressed neurons is controlled by the Golgi cell, a subtype of GABAergic interneuron. Granule cells receive GABAergic input in the form of phasic and tonic currents that are mediated by synaptic and extrasynaptic receptors, respectively. Using the acute cerebellar slice preparation and patch-clamp electrophysiological techniques, we found that ethanol induces a parallel increase in both the frequency of spontaneous IPSCs and the magnitude of the tonic current. EtOH (50 mm) did not produce this effect when spontaneous action potentials were blocked with tetrodotoxin. Recordings in the loose-patch cell-attached configuration demonstrated that ethanol increases the frequency of spontaneous action potentials in Golgi cells. Taken together, these findings indicate that ethanol enhances GABAergic inhibition of granule cells via a presynaptic mechanism that involves an increase in action potential-dependent GABA release from Golgi cells. This effect is likely to have an impact on the flow of information through the cerebellar cortex and may contribute to the mechanism by which acute ingestion of alcoholic beverages induces motor impairment. PMID:15084654

  9. Ex vivo imaging of postnatal cerebellar granule cell migration using confocal macroscopy.

    PubMed

    Bénard, Magalie; Lebon, Alexis; Komuro, Hitoshi; Vaudry, David; Galas, Ludovic

    2015-01-01

    During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration. PMID:25992599

  10. Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

    PubMed Central

    Bénard, Magalie; Lebon, Alexis; Komuro, Hitoshi; Vaudry, David; Galas, Ludovic

    2015-01-01

    During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 °C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration. PMID:25992599

  11. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    SciTech Connect

    Schousboe, A.; Frandsen, A.; Drejer, J. )

    1989-09-01

    Evoked release of ({sup 3}H)-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and (3H)-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP.

  12. Synaptic action of ethanol on cerebellar auditory granule cells reveals acute tolerance

    SciTech Connect

    Huang, C.M.; Liu, G.; Huang, R.H. )

    1991-03-11

    The cerebellum is very sensitive to acute intoxication by ethanol. The authors have recorded electrophysiological responses of granule cells to auditory stimulation from the posterior cerebellar vermis of cats before and after a relatively low dose of ethanol. Auditory responses of granule cells were severely inhibited by ethanol at a transient, peak ethanol concentration of 15-18 mM in the cerebrospinal fluid (CSF). Thereafter, the clearance of ethanol from CSF followed an exponential time course, with 50% of the CSF ethanol being cleared with every passing hour. Auditory responses of granule cells returned to control levels within 60-90 minutes, despite the presence of a DSF ethanol concentration at 8-10mM, indicating acute tolerance. Moreover, a second, identical dose of ethanol, delivered two hours after the first dose produced an attenuated inhibition in the auditory response of cerebellar granule cells. The inhibition took a longer time to be evident but a shorter time to recover than that followed by the first dose of ethanol.

  13. Mitotic Events in Cerebellar Granule Progenitor Cells that Expand Cerebellar Surface Area Are Critical for Normal Cerebellar Cortical Lamination in Mice

    PubMed Central

    Chang, Joshua C.; Leung, Mark; Gokozan, Hamza Numan; Gygli, Patrick Edwin; Catacutan, Fay Patsy; Czeisler, Catherine; Otero, José Javier

    2015-01-01

    Late embryonic and postnatal cerebellar folial surface area expansion promotes cerebellar cortical cytoarchitectural lamination. We developed a streamlined sampling scheme to generate unbiased estimates of murine cerebellar surface area and volume using stereological principles. We demonstrate that during the proliferative phase of the external granule layer (EGL) and folial surface area expansion, EGL thickness does not change and thus is a topological proxy for progenitor self-renewal. The topological constraints indicate that during proliferative phases, migration out of the EGL is balanced by self-renewal. Progenitor self-renewal must, therefore, include mitotic events yielding either 2 cells in the same layer to increase surface area (β-events) and mitotic events yielding 2 cells, with 1 cell in a superficial layer and 1 cell in a deeper layer (α-events). As the cerebellum grows, therefore, β-events lie upstream of α-events. Using a mathematical model constrained by the measurements of volume and surface area, we could quantify inter-mitotic times for β-events on a per-cell basis in post-natal mouse cerebellum. Furthermore, we found that loss of CCNA2, which decreases EGL proliferation and secondarily induces cerebellar cortical dyslamination, shows preserved α-type events. Thus, CCNA2-null cerebellar granule progenitor cells are capable of self-renewal of the EGL stem cell niche; this is concordant with prior findings of extensive apoptosis in CCNA2-null mice. Similar methodologies may provide another layer of depth to the interpretation of results from stereological studies. PMID:25668568

  14. Rapid uncoupling of oxidative phosphorylation accompanies glutamate toxicity in rat cerebellar granule cells.

    PubMed

    Atlante, A; Gagliardi, S; Minervini, G M; Marra, E; Passarella, S; Calissano, P

    1996-11-01

    A 100 microM glutamate pulse administered to rat cerebellar granule cells causes a very rapid and progressive decrease in both cell and mitochondrial oxygen consumption caused by glucose and succinate addition, respectively. The respiratory control ratio, which reflects the ability of mitochondria to produce ATP, is reduced by 50% within the first 30 min after glutamate addition. Subsequent to glutamate exposure, a progressive decrease of respiratory control ratio to almost 1 was found within the following 3-5 h. The addition of extra calcium had no effect per se on oxygen consumption by cell homogenate. PMID:8981415

  15. Procaspase-activating compound 1 induces a caspase-3-dependent cell death in cerebellar granule neurons

    SciTech Connect

    Aziz, Gulzeb; Akselsen, Oyvind W.; Hansen, Trond V.; Paulsen, Ragnhild E.

    2010-09-15

    Procaspase-activating compound 1, PAC-1, has been introduced as a direct activator of procaspase-3 and has been suggested as a therapeutic agent against cancer. Its activation of procaspase-3 is dependent on the chelation of zinc. We have tested PAC-1 and an analogue of PAC-1 as zinc chelators in vitro as well as their ability to activate caspase-3 and induce cell death in chicken cerebellar granule neuron cultures. These neurons are non-dividing, primary cells with normal caspase-3. The results reported herein show that PAC-1 chelates zinc, activates procaspase-3, and leads to caspase-3-dependent cell death in neurons, as the specific caspase-3-inhibitor Ac-DEVD-cmk inhibited both the caspase-3 activity and cell death. Thus, chicken cerebellar granule neurons is a suitable model to study mechanisms of interference with apoptosis of PAC-1 and similar compounds. Furthermore, the present study also raises concern about potential neurotoxicity of PAC-1 if used in cancer therapy.

  16. A Reinforcing Circuit Action of Extrasynaptic GABAA Receptor Modulators on Cerebellar Granule Cell Inhibition

    PubMed Central

    Santhakumar, Vijayalakshmi; Otis, Thomas S.

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  17. A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

    PubMed

    Santhakumar, Vijayalakshmi; Meera, Pratap; Karakossian, Movses H; Otis, Thomas S

    2013-01-01

    GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants. PMID:23977374

  18. Glutamate-induced protein phosphorylation in cerebellar granule cells: role of protein kinase C.

    PubMed

    Eboli, M L; Mercanti, D; Ciotti, M T; Aquino, A; Castellani, L

    1994-10-01

    Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells. 32P-labelled cells have been stimulated with 100 microM glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells. PMID:7891841

  19. Model cerebellar granule cells can faithfully transmit modulated firing rate signals

    PubMed Central

    Rössert, Christian; Solinas, Sergio; D'Angelo, Egidio; Dean, Paul; Porrill, John

    2014-01-01

    A crucial assumption of many high-level system models of the cerebellum is that information in the granular layer is encoded in a linear manner. However, granule cells are known for their non-linear and resonant synaptic and intrinsic properties that could potentially impede linear signal transmission. In this modeling study we analyse how electrophysiological granule cell properties and spike sampling influence information coded by firing rate modulation, assuming no signal-related, i.e., uncorrelated inhibitory feedback (open-loop mode). A detailed one-compartment granule cell model was excited in simulation by either direct current or mossy-fiber synaptic inputs. Vestibular signals were represented as tonic inputs to the flocculus modulated at frequencies up to 20 Hz (approximate upper frequency limit of vestibular-ocular reflex, VOR). Model outputs were assessed using estimates of both the transfer function, and the fidelity of input-signal reconstruction measured as variance-accounted-for. The detailed granule cell model with realistic mossy-fiber synaptic inputs could transmit information faithfully and linearly in the frequency range of the vestibular-ocular reflex. This was achieved most simply if the model neurons had a firing rate at least twice the highest required frequency of modulation, but lower rates were also adequate provided a population of neurons was utilized, especially in combination with push-pull coding. The exact number of neurons required for faithful transmission depended on the precise values of firing rate and noise. The model neurons were also able to combine excitatory and inhibitory signals linearly, and could be replaced by a simpler (modified) integrate-and-fire neuron in the case of high tonic firing rates. These findings suggest that granule cells can in principle code modulated firing-rate inputs in a linear manner, and are thus consistent with the high-level adaptive-filter model of the cerebellar microcircuit. PMID:25352777

  20. Characterization of metabotropic glutamate receptor-stimulated phosphoinositide hydrolysis in rat cultured cerebellar granule cells.

    PubMed Central

    Toms, N. J.; Jane, D. E.; Tse, H. W.; Roberts, P. J.

    1995-01-01

    1. The pharmacology of excitatory amino acid (EAA)-stimulated phosphoinositide (PI) hydrolysis, monitored via [3H]-inositol monophosphate accumulation, was investigated in primary cultures of rat cerebellar granule cells. 2. EAA-stimulated PI hydrolysis peaked after 4-5 days in vitro and subsequently declined. 3. The agonist order of potency was found to be (EC50): L-quisqualic acid (Quis) (2 microM) >> L-glutamate (50 microM) > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) (102 microM). L-Glutamate (Emax = 873% of basal activity) elicited the largest stimulation of PI hydrolysis, whereas Quis (Emax = 603%) and (1S,3R)-ACPD (Emax = 306%) produced somewhat lower stimulations. 4. Several phenylglycine derivatives were found to be active in inhibiting 2 microM Quis-stimulated PI hydrolysis, in order of potency (IC50): (S)-4-carboxy-3-hydroxyphenylglycine (41 microM) > or = (S)-4-carboxyphenylglycine (51 microM) >> (+)-alpha-methyl-4-carboxyphenylglycine (243 microM). 5. Cultured cerebellar granule cells of the rat appear to have Group I mGluR pharmacology similar to that reported for cloned mGluR1 and provide an ideal system for investigating novel mGluR1 ligands in a native environment. PMID:8680712

  1. Antiphospholipid antibodies bind to rat cerebellar granule cells: the role of N-methyl-D-aspartate receptors.

    PubMed

    Riccio, A; Andreassi, C; Eboli, M L

    1998-11-27

    IgGs from sera containing antiphospholipid antibodies (aPL), detected as antibodies to cardiolipin, or control sera were incubated with rat cerebellar granule cells in primary culture. Using a mitochondrial dehydrogenase activity assay (MTT test), aPL IgGs were shown to decrease MTT metabolism after 24 h incubation with the cells, and to cause non-toxic amounts of glutamate to become neurotoxic when added to the cells for 45 min. Acute and chronic aPL toxicity were prevented by MK-801. Sera containing aPL bound to intact cerebellar neurons, as revealed by an immunofluorescent technique. These results suggest that antiphospholipid antibodies interfere with excitatory pathways in glutamatergic cerebellar granule cells by a mechanism involving overactivation of the NMDA glutamate receptor. PMID:9865941

  2. Multiple extra-synaptic spillover mechanisms regulate prolonged activity in cerebellar Golgi cell–granule cell loops

    PubMed Central

    Holtzman, Tahl; Sivam, Vanessa; Zhao, Tian; Frey, Oivier; van der Wal, Peter Dow; de Rooij, Nico F; Dalley, Jeffrey W; Edgley, Steve A

    2011-01-01

    Abstract Despite a wealth of in vitro and modelling studies it remains unclear how neuronal populations in the cerebellum interact in vivo. We address the issue of how the cerebellar input layer processes sensory information, with particular focus on the granule cells (input relays) and their counterpart inhibitory interneurones, Golgi cells. Based on the textbook view, granule cells excite Golgi cells via glutamate forming a negative feedback loop. However, Golgi cells express inhibitory mGluR2 receptors suggesting an inhibitory role for glutamate. We set out to test this glutamatergic paradox in Golgi cells. Here we show that granule cells and Golgi cells interact through extra-synaptic signalling mechanisms during sensory information processing, as well as synaptic mechanisms. We demonstrate that such interactions depend on granule cell-derived glutamate acting via inhibitory mGluR2 receptors leading causally to the suppression of Golgi cell activity for several hundreds of milliseconds. We further show that granule cell-derived inhibition of Golgi cell activity is regulated by GABA-dependent extra-synaptic Golgi cell inhibition of granule cells, identifying a regulatory loop in which glutamate and GABA may be critical regulators of Golgi cell–granule cell functional activity. Thus, granule cells may promote their own prolonged activity via paradoxical feed-forward inhibition of Golgi cells, thereby enabling information processing over long timescales. PMID:21669981

  3. GABA induces functionally active low-affinity GABA receptors on cultured cerebellar granule cells.

    PubMed

    Meier, E; Drejer, J; Schousboe, A

    1984-12-01

    The effect of gamma-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 microM GABA in the culture media led to the induction of low-affinity GABA receptors (KD 546 +/- 117 nM) in addition to the high-affinity receptors (KD 7 +/- 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED50 10 +/- 3 microM) only when the cells had been cultured in the presence of 50 microM GABA, 50 microM muscimol, or 150 microM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 microM bicuculline and mimicked by 50 microM muscimol or 150 microM THIP whereas 150 microM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABAA receptors. PMID:6149269

  4. Simulating Spinal Border Cells and Cerebellar Granule Cells under Locomotion – A Case Study of Spinocerebellar Information Processing

    PubMed Central

    Spanne, Anton; Geborek, Pontus; Bengtsson, Fredrik; Jörntell, Henrik

    2014-01-01

    The spinocerebellar systems are essential for the brain in the performance of coordinated movements, but our knowledge about the spinocerebellar interactions is very limited. Recently, several crucial pieces of information have been acquired for the spinal border cell (SBC) component of the ventral spinocerebellar tract (VSCT), as well as the effects of SBC mossy fiber activation in granule cells of the cerebellar cortex. SBCs receive monosynaptic input from the reticulospinal tract (RST), which is an important driving system under locomotion, and disynaptic inhibition from Ib muscle afferents. The patterns of activity of RST neurons and Ib afferents under locomotion are known. The activity of VSCT neurons under fictive locomotion, i.e. without sensory feedback, is also known, but there is little information on how these neurons behave under actual locomotion and for cerebellar granule cells receiving SBC input this is completely unknown. But the available information makes it possible to simulate the interactions between the spinal and cerebellar neuronal circuitries with a relatively large set of biological constraints. Using a model of the various neuronal elements and the network they compose, we simulated the modulation of the SBCs and their target granule cells under locomotion and hence generated testable predictions of their general pattern of modulation under this condition. This particular system offers a unique opportunity to simulate these interactions with a limited number of assumptions, which helps making the model biologically plausible. Similar principles of information processing may be expected to apply to all spinocerebellar systems. PMID:25226298

  5. Protective Effect of Edaravone in Primary Cerebellar Granule Neurons against Iodoacetic Acid-Induced Cell Injury

    PubMed Central

    Zhou, Xinhua; Zhu, Longjun; Wang, Liang; Guo, Baojian; Zhang, Gaoxiao; Sun, Yewei; Zhang, Zaijun; Lee, Simon Ming-Yuen; Yu, Pei; Wang, Yuqiang

    2015-01-01

    Edaravone (EDA) is clinically used for treatment of acute ischemic stroke in Japan and China due to its potent free radical-scavenging effect. However, it has yet to be determined whether EDA can attenuate iodoacetic acid- (IAA-) induced neuronal death in vitro. In the present study, we investigated the effect of EDA on damage of IAA-induced primary cerebellar granule neurons (CGNs) and its possible underlying mechanisms. We found that EDA attenuated IAA-induced cell injury in CGNs. Moreover, EDA significantly reduced intracellular reactive oxidative stress production, loss of mitochondrial membrane potential, and caspase 3 activity induced by IAA. Taken together, EDA protected CGNs against IAA-induced neuronal damage, which may be attributed to its antiapoptotic and antioxidative activities. PMID:26557222

  6. Early release and subsequent caspase-mediated degradation of cytochrome c in apoptotic cerebellar granule cells.

    PubMed

    Bobba, A; Atlante, A; Giannattasio, S; Sgaramella, G; Calissano, P; Marra, E

    1999-08-20

    Cytochrome c (cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt c is released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylchetone (z-VAD-fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt c may be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5-soluble cyt c-mediated fashion. PMID:10486578

  7. Dendritic patch-clamp recordings from cerebellar granule cells demonstrate electrotonic compactness

    PubMed Central

    Delvendahl, Igor; Straub, Isabelle; Hallermann, Stefan

    2015-01-01

    Cerebellar granule cells (GCs), the smallest neurons in the brain, have on average four short dendrites that receive high-frequency mossy fiber inputs conveying sensory information. The short length of the dendrites suggests that GCs are electrotonically compact allowing unfiltered integration of dendritic inputs. The small average diameter of the dendrites (~0.7 µm), however, argues for dendritic filtering. Previous studies based on somatic recordings and modeling indicated that GCs are electrotonically extremely compact. Here, we performed patch-clamp recordings from GC dendrites in acute brain slices of mice to directly analyze the electrotonic properties of GCs. Strikingly, the input resistance did not differ significantly between dendrites and somata of GCs. Furthermore, spontaneous excitatory postsynaptic potentials (EPSP) were similar in amplitude at dendritic and somatic recording sites. From the dendritic and somatic input resistances we determined parameters characterizing the electrotonic compactness of GCs. These data directly demonstrate that cerebellar GCs are electrotonically compact and thus ideally suited for efficient high-frequency information transfer. PMID:25852483

  8. The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration.

    PubMed

    Mogensen, H S; Hack, N; Balázs, R; Jørgensen, O S

    1994-08-01

    The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K+ (5 or 10 mM), as well as in the presence of 'high' K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K(+)-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K(+)-induced membrane depolarization. PMID:7529458

  9. Generation and Characterization of an Nse-CreERT2 Transgenic Line Suitable for Inducible Gene Manipulation in Cerebellar Granule Cells

    PubMed Central

    Pohlkamp, Theresa; Steller, Laura; May, Petra; Günther, Thomas; Schüle, Roland; Frotscher, Michael

    2014-01-01

    We created an Nse-CreERT2 mouse line expressing the tamoxifen-inducible CreERT2 recombinase under the control of the neuron-specific enolase (Nse) promoter. By using Cre reporter lines we could show that this Nse-CreERT2 line has recombination activity in the granule cells of all cerebellar lobules as well as in postmitotic granule cell precursors in the external granular layer of the developing cerebellum. A few hippocampal dentate gyrus granule cells showed Cre-mediated recombination as well. Cre activity could be induced in both the developing and adult mouse brain. The established mouse line constitutes a valuable tool to study the function of genes expressed by cerebellar granule cells in the developing and adult brain. In combination with reporter lines it is a useful model to analyze the development and maintenance of the cerebellar architecture including granule cell distribution, migration, and the extension of granule cell fibers in vivo. PMID:24950299

  10. 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

  11. LXR agonist rescued the deficit in the proliferation of the cerebellar granule cells induced by dexamethasone.

    PubMed

    Bian, Xuting; Zhong, Hongyu; Li, Fen; Cai, Yulong; Li, Xin; Wang, Lian; Fan, Xiaotang

    2016-09-01

    Dexamethasone (DEX) exposure during early postnatal life produces permanent neuromotor and intellectual deficits and stunts cerebellar growth. The liver X receptor (LXR) plays important roles in CNS development. However, the effects of LXR on the DEX-mediated impairment of cerebellar development remain undetermined. Thus, mice were pretreated with LXR agonist TO901317 (TO) and were later exposed to DEX to evaluate its protective effects on DEX-mediated deficit during cerebellar development. The results showed that an acute exposure of DEX on postnatal day 7 resulted in a significant impairment in cerebellar development and decreased the proliferation of granule neuron precursors in the external granule layer of cerebellum. This effect was attenuated by pretreatment with TO. We further found that the decrease in the proliferation caused by DEX occurred via up-regulation of glucocorticoid receptor and p27kip1, which could be partially prevented by LXR agonist pretreatment. Overall, our results suggest that LXR agonist pretreatment could protect against DEX-induced deficits in cerebellar development in postnatal mice and may thus be perspective recruited to counteract such GC side effects. PMID:27369072

  12. A sensitive method to assay the xanthine oxidase activity in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Valenti, D; Gagliardi, S; Passarella, S

    2000-11-01

    Since xanthine oxidase (XO, Xanthine:oxidoreductase, E.C.1.2.3.22) is a key enzyme in reactive oxygen specie formation which plays a major role in cell oxidative stress, the availability of a sensitive and simple assay useful to detect its activity in monolayer cell cultures is worthwhile. In order to achieve this, we developed a method in which the conversion of pterine into isoxanthopterin is monitored fluorimetrically. Temperature assay was 50 degrees C. The activity of XO was detected in cerebellar granule cells exposed to glutamate. Since XO is formed from protease-dependent xanthine dehydrogenase processing, its activity appearance was found to be prevented by the protease inhibitor, leupeptin, as well as the glutamate NMDA-receptor inhibitor, MK-801, and the Ca(++) complexing agent, EGTA. The reported novel protocol, at variance with a conventional method, is shown to be a simple, fast, sensitive and relatively cheap method to assay XO activity. In addition, the reported assay can be applied to any cell type in culture. PMID:11086257

  13. Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis.

    PubMed

    Bobba, A; Casalino, E; Petragallo, V A; Atlante, A

    2014-10-01

    The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs. PMID:25055978

  14. Raising cytosolic Cl− in cerebellar granule cells affects their excitability and vestibulo-ocular learning

    PubMed Central

    Seja, Patricia; Schonewille, Martijn; Spitzmaul, Guillermo; Badura, Aleksandra; Klein, Ilse; Rudhard, York; Wisden, William; Hübner, Christian A; De Zeeuw, Chris I; Jentsch, Thomas J

    2012-01-01

    Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABAA-receptor Cl− channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl−]i roughly two-fold. The reduced Cl− gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning. PMID:22252133

  15. Protective effect of fangchinoline on cyanide-induced neurotoxicity in cultured rat cerebellar granule cells.

    PubMed

    Cho, Soon Ok; Seong, Yeon Hee

    2002-06-01

    The present study was performed to examine the effect of fangchinoline, a bis- benzylisoquinoline alkaloid, which exhibits the characteristics of a Ca2+ channel blocker, on cyanide-induced neurotoxicity using cultured rat cerebellar granule neurons. NaCN produced a concentration-dependent reduction of cell viability, which was blocked by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, L-type Ca2+ channel blocker, and L-NAME, a nitric oxide synthase inhibitor. Pretreatment with fangchinoline over a concentration range of 0.1 to 10 microM significantly decreased the NaCN-induced neuronal cell death, glutamate release into medium, and elevation of [Ca2+]i and oxidants generation. These results suggest that fangchinoline may mitigate the harmful effects of cyanide-induced neuronal cell death by interfering with [Ca2+]i influx, due to its function as a Ca2+ channel blocker, and then by inhibiting glutamate release and oxidants generation. PMID:12135109

  16. Detection of reactive oxygen species in primary cultures of cerebellar granule cells.

    PubMed

    Atlante, A; Passarella, S

    1999-12-01

    The aim of this work was to develop a novel procedure useful to detect the formation of two reactive oxygen species, i.e. superoxide and singlet oxygen, in neuron monolayer primary cultures, thus, making possible the investigation of the effect of certain compounds on reactive oxygen species formation. Thus, use was made of two reactive oxygen species detecting systems consisting of ferricytochrome c (Fe-cyt c) and imidazole-RNO (N, N-dimethyl-4-nitrosoaniline) which allow for the photometric detection of superoxide anion and singlet oxygen, respectively. Both of them were used to assess the formation of reactive oxygen species in cerebellar granule cells exposed to glutamate: both superoxide anion and singlet oxygen proved to be generated in glutamate neurotoxicity in a way sensitive to glutamate NMDA-receptor inhibitor, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a, d)cyclohepten-5,10-imine hydrogen maleate), to Ca(2+) complexing agent, EGTA, and to certain antioxidants. In principle, the reported protocol can be applied to any cell type in culture. PMID:10592334

  17. Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells.

    PubMed

    Wang, Dong; She, Liang; Sui, Ya-nan; Yuan, Xiao-bing; Wen, Yunqing; Poo, Mu-ming

    2012-12-18

    Directional flow of membrane components has been detected at the leading front of fibroblasts and the growth cone of neuronal processes, but whether there exists global directional flow of plasma membrane components over the entire migrating neuron remains largely unknown. By analyzing the trajectories of antibody-coated single quantum dots (QDs) bound to two membrane proteins, overexpressed myc-tagged synaptic vesicle-associated membrane protein VAMP2 and endogenous neurotrophin receptor TrkB, we found that these two proteins exhibited net forward transport, which is superimposed upon Brownian motion, in both leading and trailing processes of migrating cerebellar granule cells in culture. Furthermore, no net directional transport of membrane proteins was observed in nonmigrating cells with either growing or stalling leading processes. Analysis of the correlation of motion direction between two QDs on the same process in migrating neurons also showed a higher frequency of correlated forward than rearward movements. Such correlated QD movements were markedly reduced in the presence of myosin II inhibitor blebbistatin,suggesting the involvement of myosin II-dependent active transport processes. Thus, a net forward transport of plasma membrane proteins exists in the leading and trailing processes of migrating neurons, in line with the translocation of the soma. PMID:23213239

  18. Tactile responses in the granule cell layer of cerebellar folium crus IIa of freely behaving rats

    NASA Technical Reports Server (NTRS)

    Hartmann, M. J.; Bower, J. M.

    2001-01-01

    We recorded activity from the granule cell layer (GCL) of cerebellar folium Crus IIa as freely moving rats engaged in a variety of natural behaviors, including grooming, eating, and free tactile exploration. Multiunit responses in the 1000-4500 Hz range were found to be strongly correlated with tactile stimulation of lip and whisker (perioral) regions. These responses occurred regardless of whether the stimulus was externally or self-generated and during both active and passive touch. In contrast, perioral movements that did not tactually stimulate this region of the face (e.g., chewing) produced no detectable increases in GCL activity. In addition, GCL responses were not correlated with movement extremes. When rats used their lips actively for palpation and exploration, the tactile responses in the GCL were not detectably modulated by ongoing jaw movements. However, active palpation and exploratory behaviors did result in the largest and most continuous bursts of GCL activity: responses were on average 10% larger and 50% longer during palpation and exploration than during grooming or passive stimulation. Although activity levels differed between behaviors, the position and spatial extent of the peripheral receptive field was similar over all behaviors that resulted in tactile input. Overall, our data suggest that the 1000-4500 Hz multiunit responses in the Crus IIa GCL of awake rats are correlated with tactile input rather than with movement or any movement parameter and that these responses are likely to be of particular importance during the acquisition of sensory information by perioral structures.

  19. Bax inactivation in lurcher mutants rescues cerebellar granule cells but not purkinje cells or inferior olivary neurons.

    PubMed

    Selimi, F; Vogel, M W; Mariani, J

    2000-07-15

    Lurcher is a gain-of-function mutation in the delta2 glutamate receptor gene (Grid2) that turns the receptor into a leaky ion channel. The expression of the Lurcher gene in heterozygous (Grid2(Lc/+)) mutants induces the death of almost all Purkinje cells starting from the second postnatal week. Ninety percent of the granule cells and 60-75% of the inferior olivary neurons die because of the loss of their target neurons, the Purkinje cells. The apoptotic nature of the neurodegeneration has been demonstrated previously by the presence of activated caspase-3 and DNA fragmentation. Bax, a pro-apoptotic gene of the Bcl-2 family, has been shown to be involved in developmental neuronal death. To study the role of Bax in Grid2(Lc/+) neurodegeneration, double mutants with Grid2(Lc/)+ mice and Bax knock-out mice (Bax-/-) were generated. Bax deletion had no effect on the death of Purkinje cells and inferior olivary neurons, although a temporary rescue of some Purkinje cells could be detected in P15 Grid2(Lc/)+;Bax-/- animals. From postnatal day 15 (P15) to P60, the number of granule cells in Grid2(Lc/)+;Bax-/-mice did not significantly change and was significantly increased compared with the number found in Grid2(Lc/)+;Bax+/+ mice. Granule cell number in P60 Grid2(Lc/)+;Bax-/- mice corresponded to 70% of the number found in wild-type mice. Our results show that Bax inactivation in Grid2(Lc/+) mice does not rescue intrinsic Purkinje cell death or the target-related cell death of olivary neurons, but Bax inactivation does inhibit persistently target-related cell death in cerebellar granule cells. PMID:10884318

  20. Pharmacological characterization of mGlu1 receptors in cerebellar granule cells reveals biased agonism

    PubMed Central

    Hathaway, Hannah A.; Pshenichkin, Sergey; Grajkowska, Ewa; Gelb, Tara; Emery, Andrew C.; Wolfe, Barry B.; Wroblewski, Jarda T.

    2015-01-01

    The majority of existing research on the function of metabotropic glutamate (mGlu) receptor 1 focuses on G protein-mediated outcomes. However, similar to other G protein-coupled receptors (GPCR), it is becoming apparent that mGlu1 receptor signaling is multi-dimensional and does not always involve G protein activation. Previously, in transfected CHO cells, we showed that mGlu1 receptors activate a G protein-independent, β-arrestin-dependent signal transduction mechanism and that some mGlu1 receptor ligands were incapable of stimulating this response. Here we set out to investigate the physiological relevance of these findings in a native system using primary cultures of cerebellar granule cells. We tested the ability of a panel of compounds to stimulate two mGlu1 receptor-mediated outcomes: (1) protection from decreased cell viability after withdrawal of trophic support and (2) G protein-mediated phosphoinositide (PI) hydrolysis. We report that the commonly used mGlu1 receptor ligands quisqualate, DHPG, and ACPD are completely biased towards PI hydrolysis and do not induce mGlu1 receptor-stimulated neuroprotection. On the other hand, endogenous compounds including glutamate, aspartate, cysteic acid, cysteine sulfinic acid, and homocysteic acid stimulate both responses. These results show that some commonly used mGlu1 receptor ligands are biased agonists, stimulating only a fraction of mGlu1 receptor-mediated responses in neurons. This emphasizes the importance of utilizing multiple agonists and assays when studying GPCR function. PMID:25700650

  1. Reversible suppression of glutamatergic neurotransmission of cerebellar granule cells in vivo by genetically manipulated expression of tetanus neurotoxin light chain.

    PubMed

    Yamamoto, Mutsuya; Wada, Norio; Kitabatake, Yasuji; Watanabe, Dai; Anzai, Masayuki; Yokoyama, Minesuke; Teranishi, Yutaka; Nakanishi, Shigetada

    2003-07-30

    We developed a novel technique that allowed reversible suppression of glutamatergic neurotransmission in the cerebellar network. We generated two lines of transgenic mice termed Tet and TeNT mice and crossed the two transgenic lines to produce the Tet/TeNT double transgenic mice. In the Tet mice, the tetracycline-controlled reverse activator (rtTA) was expressed selectively in cerebellar granule cells by the promoter function of the GABA(A) receptor alpha6 subunit gene. In the TeNT mice, the fusion gene of tetanus neurotoxin light chain (TeNT) and enhanced green fluorescent protein (EGFP) was designed to be induced by the interaction of doxycycline (DOX)-activated rtTA with the tetracycline-responsive promoter. The Tet/TeNT mice grew normally even after DOX treatment and exhibited a restricted DOX-dependent expression of TeNT in cerebellar granule cells. Along with this expression, TeNT proteolytically cleaved the synaptic vesicle protein VAMP2 (also termed synaptobrevin2) and reduced glutamate release from granule cells. Both cleavage of VAMP2/synaptobrevin2 and reduction of glutamate release were reversed by removal of DOX. Among the four genotypes generated by heterozygous crossing of Tet and TeNT mice, only Tet/TeNT mice showed DOX-dependent reversible motor impairments as analyzed with fixed bar and rota-rod tests. Reversible suppression of glutamatergic neurotransmission thus can be manipulated with spatiotemporal accuracy by DOX treatment and removal. These transgenic mice will serve as an animal model to study the cerebellar function in motor coordination and learning. PMID:12890769

  2. Methylmercury differentially affects GABAA receptor-mediated spontaneous IPSCs in Purkinje and granule cells of rat cerebellar slices

    PubMed Central

    Yuan, Yukun; Atchison, William D

    2003-01-01

    Using whole-cell recording techniques we compared effects of the environmental cerebellar neurotoxicant methylmercury (MeHg) on spontaneous IPSCs (sIPSCs) of both Purkinje and granule cells in cerebellar slices of the rat. In Purkinje cells, bath application of 10, 20 or 100 μM MeHg initially increased then suppressed the frequency of sIPSCs to zero. In granule cells, the initial increase in frequency was not observed in ≈50 % of cells examined, but suppression of sIPSCs by MeHg occurred in every cell tested. For both cells, time to onset of effects of MeHg was inversely related to the concentration; moreover, the pattern of changes in mIPSCs induced by MeHg in the presence of tetrodotoxin was similar to that in sIPSCs. For each concentration of MeHg, it took 2–3 times longer to block sIPSCs in Purkinje cells than it did in granule cells. MeHg also initially increased then decreased amplitudes of sIPSCs to block in both cells; again the response was more variable in granule cells. In most Purkinje and some granule cells, MeHg induced a giant, slow inward current during the late stages of exposure. Appearance of this current appeared to be MeHg concentration dependent, and the direction of current flow was reversed by changing the holding potentials. Reduction of the [Cl−] in the internal solution caused inwardly directed, but not outwardly directed giant currents to disappear, suggesting that this current is a Cl−-mediated response. However, bicuculline and picrotoxin failed to block it. MeHg apparently acts at both presynaptic and postsynaptic sites to alter GABAA receptor-mediated inhibitory synaptic transmission. GABAA receptors in granule cells appear to be more sensitive to block by MeHg than are those in Purkinje cells, although the general patterns of effects on the two cells are similar. PMID:12879869

  3. Proneurotrophin-3 promotes cell cycle withdrawal of developing cerebellar granule cell progenitors via the p75 neurotrophin receptor

    PubMed Central

    Zanin, Juan Pablo; Abercrombie, Elizabeth; Friedman, Wilma J

    2016-01-01

    Cerebellar granule cell progenitors (GCP) proliferate extensively in the external granule layer (EGL) of the developing cerebellum prior to differentiating and migrating. Mechanisms that regulate the appropriate timing of cell cycle withdrawal of these neuronal progenitors during brain development are not well defined. The p75 neurotrophin receptor (p75NTR) is highly expressed in the proliferating GCPs, but is downregulated once the cells leave the cell cycle. This receptor has primarily been characterized as a death receptor for its ability to induce neuronal apoptosis following injury. Here we demonstrate a novel function for p75NTR in regulating proper cell cycle exit of neuronal progenitors in the developing rat and mouse EGL, which is stimulated by proNT3. In the absence of p75NTR, GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits. DOI: http://dx.doi.org/10.7554/eLife.16654.001 PMID:27434667

  4. Cell Division Mode Change Mediates the Regulation of Cerebellar Granule Neurogenesis Controlled by the Sonic Hedgehog Signaling

    PubMed Central

    Yang, Rong; Wang, Minglei; Wang, Jia; Huang, Xingxu; Yang, Ru; Gao, Wei-Qiang

    2015-01-01

    Summary Symmetric and asymmetric divisions are important for self-renewal and differentiation of stem cells during neurogenesis. Although cerebellar granule neurogenesis is controlled by sonic hedgehog (SHH) signaling, whether and how this process is mediated by regulation of cell division modes have not been determined. Here, using time-lapse imaging and cell culture from neuronal progenitor-specific and differentiated neuron-specific reporter mouse lines (Math1-GFP and Dcx-DsRed) and Patched+/− mice in which SHH signaling is activated, we find evidence for the existence of symmetric and asymmetric divisions that are closely associated with progenitor proliferation and differentiation. While activation of the SHH pathway enhances symmetric progenitor cell divisions, blockade of the SHH pathway reverses the cell division mode change in Math1-GFP;Dcx-DsRed;Patched+/− mice by promoting asymmetric divisions or terminal neuronal symmetric divisions. Thus, cell division mode change mediates the regulation of cerebellar granule neurogenesis controlled by SHH signaling. PMID:26527387

  5. The effects of neurotrophin-3 and brain-derived neurotrophic factor on cerebellar granule cell movement and neurite extension in vitro.

    PubMed

    Tanaka, S; Sekino, Y; Shirao, T

    2000-01-01

    Migration of the granule cells is a major stage of cerebellar maturation. Granule cells express neurotrophins and their receptors; however, their role in cell migration has not been defined. In this study we investigated the effects of exogenous neurotrophins on the movement and neurite extension of granule cells from glial-free cerebellar cell reaggregates in vitro. Our results provide direct evidence that neurotrophin-3 and brain-derived neurotrophic factor differentially affect the granule cells. Neurotrophin-3 significantly affected granule cell movements by decreasing the migration index (the ratio of the number of cells that moved further than half the neurite length) and the speed of cell soma movement, but did not affect neurite length or growth cone migration. In contrast, brain-derived neurotrophic factor and neurotrophin-4 acted on growing neurites and growth cones by significantly increasing neurite length and the speed of growth cone migration, but had no effect either on the migration index or on the speed of the cell soma movement. The results suggest that neurotrophins differentially affect neurite extension and the movements of cerebellar granule cells. PMID:10842017

  6. Glutamate neurotoxicity in rat cerebellar granule cells: a major role for xanthine oxidase in oxygen radical formation.

    PubMed

    Atlante, A; Gagliardi, S; Minervini, G M; Ciotti, M T; Marra, E; Calissano, P

    1997-05-01

    To gain insight into the mechanism through which the neurotransmitter glutamate causally participates in several neurological diseases, in vitro cultured cerebellar granule cells were exposed to glutamate and oxygen radical production was investigated. To this aim, a novel procedure was developed to detect oxygen radicals; the fluorescent dye 2',7'-dichlorofluorescein was used to detect production of peroxides, and a specific search for the possible conversion of the enzyme xanthine dehydrogenase into xanthine oxidase after the excitotoxic glutamate pulse was undertaken. A 100 microM glutamate pulse administered to 7-day-old cerebellar granule cells is accompanied by the onset of neuronal death, the appearance of xanthine oxidase, and production of oxygen radicals. Xanthine oxidase activation and superoxide (O2.-) production are completely inhibited by concomitant incubation of glutamate with MK-801, a specific NMDA receptor antagonist, or by chelation of external calcium with EGTA. Partial inhibition of both cell death and parallel production of reactive oxygen species is achieved with allopurinol, a xanthine oxidase inhibitor, leupeptin, a protease inhibitor, reducing agents such as glutathione or dithiothreitol, antioxidants such as vitamin E and vitamin C, and externally added superoxide dismutase. It is concluded that glutamate-triggered, NMDA-mediated, massive Ca2+ influx induces rapid conversion of xanthine dehydrogenase into xanthine oxidase with subsequent production of reactive oxygen species that most probably have a causal involvement in the initial steps of the series of intracellular events leading to neuronal degeneration and death. PMID:9109530

  7. Distinct expression patterns of inwardly rectifying potassium currents in developing cerebellar granule cells of the hemispheres and the vermis.

    PubMed

    Brandalise, Federico; Lujan, Rafael; Leone, Roberta; Lodola, Francesco; Cesaroni, Valentina; Romano, Chiara; Gerber, Urs; Rossi, Paola

    2016-06-01

    G-protein-coupled inwardly rectifying potassium (GIRK) channels play a crucial role during the migration and maturation of cerebellar granule cells (GCs) in the vermis. In the cerebellar hemispheres, however, only minor effects on the development of GCs are observed in mice with GIRK channel impairment. This regional difference may reflect distinct ontogenetic expression patterns of GIRK channels. Therefore, inwardly rectifying responses in mice were characterized at different stages of development in the vermis and the hemispheres. In the vermis, GCs in the premigratory zone (PMZ) at P7-P15 exhibit GIRK current but not constitutive inwardly rectifying potassium (CIRK) current, and are relatively depolarized at rest. In contrast, premigratory GCs in the hemispheres express only CIRK channels, which accounts for their more hyperpolarized resting membrane potential. Furthermore, the pattern of voltage-dependent inward currents in the PMZ GCs of cerebellar hemispheres is consistent with a more mature stage of development than the corresponding GCs in the vermis, resulting in robust firing properties mediated by sodium channels. Later in development (P21-P22), CIRK current is then observed in the majority of vermis GCs. This developmental pattern, revealed by electrophysiological recordings, was confirmed by immunohistological experiments that showed greater reactivity for GIRK2 in the PMZ of the vermis than in the hemispheres during development (P7-P15). These findings suggest that regional differences in development are responsible for the differential expression of inwardly rectifying potassium channels in the vermis and in the hemispheres. PMID:26921581

  8. Persistent Nav1.6 current at axon initial segments tunes spike timing of cerebellar granule cells

    PubMed Central

    Osorio, Nancy; Cathala, Laurence; Meisler, Miriam H; Crest, Marcel; Magistretti, Jacopo; Delmas, Patrick

    2010-01-01

    Cerebellar granule (CG) cells generate high-frequency action potentials that have been proposed to depend on the unique properties of their voltage-gated ion channels. To address the in vivo function of Nav1.6 channels in developing and mature CG cells, we combined the study of the developmental expression of Nav subunits with recording of acute cerebellar slices from young and adult granule-specific Scn8a KO mice. Nav1.2 accumulated rapidly at early-formed axon initial segments (AISs). In contrast, Nav1.6 was absent at early postnatal stages but accumulated at AISs of CG cells from P21 to P40. By P40–P65, both Nav1.6 and Nav1.2 co-localized at CG cell AISs. By comparing Na+ currents in mature CG cells (P66–P74) from wild-type and CG-specific Scn8a KO mice, we found that transient and resurgent Na+ currents were not modified in the absence of Nav1.6 whereas persistent Na+ current was strongly reduced. Action potentials in conditional Scn8a KO CG cells showed no alteration in threshold and overshoot, but had a faster repolarization phase and larger post-spike hyperpolarization. In addition, although Scn8a KO CG cells kept their ability to fire action potentials at very high frequency, they displayed increased interspike-interval variability and firing irregularity in response to sustained depolarization. We conclude that Nav1.6 channels at axon initial segments contribute to persistent Na+ current and ensure a high degree of temporal precision in repetitive firing of CG cells. PMID:20173079

  9. Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells.

    PubMed

    Riccio, A; Esposito, E; Eboli, M L

    1996-04-29

    The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells. PMID:8773779

  10. Balanced effect of PACAP and FasL on granule cell death during cerebellar development: a morphological, functional and behavioural characterization.

    PubMed

    Allais, Aurélie; Burel, Delphine; Roy, Vincent; Arthaud, Sébastien; Galas, Ludovic; Isaac, Emma Rachel; Desfeux, Arnaud; Parent, Bénédicte; Fournier, Alain; Chapillon, Pierre; Sherwood, Nancy McKeown; Vaudry, Hubert; Gonzalez, Bruno José

    2010-04-01

    It is now established that the development of the CNS requires equilibrium between cell survival and apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts a powerful protective effect on cerebellar granule cells by inhibiting the caspase 3. In contrast, Fas ligand (FasL) plays an essential role during ontogenesis in eliminating supernumerary neurons by apoptosis. To determine if PACAP and FasL interact during cerebellar development, we characterized the effects of these factors on cerebellar morphogenesis and caspase 3 activity in PACAP+/+ and PACAP-/- mice. First, we demonstrated in vivo that PACAP is able to reverse the diminution of internal granule cell layer thickness induced by FasL in PACAP+/+ and PACAP-/- mice. Second, ex vivo and immunohistochemical studies revealed that interaction between FasL and PACAP occurs through the caspase 3 activity. Third, behavioural study showed a significant difference for the PACAP + FasL group in the righting reflex test at P8 which does not persist at P60. Finally, a time course study revealed that the pro-apoptotic effect of FasL characterized at P8 was followed by a progressive compensatory mechanism in caspase 3 activity and bromodeoxyuridine incorporation. These data suggest that PACAP and FasL interact during cerebellar development to control apoptosis of granule cells and may affect some motor cerebellar functions. PMID:20050979

  11. Endothelin-1 stimulates the release of preloaded ( sup 3 H)D-aspartate from cultured cerebellar granule cells

    SciTech Connect

    Lin, W.W.; Lee, C.Y.; Chuang, D.M. )

    1990-03-16

    We have recently reported that endothelin-1 (ET) induces phosphoinositide hydrolysis in primary cultures of rat cerebellar granule cells. Here we found that ET in a dose-dependent manner (1-30 nM) stimulated the release of preloaded ({sup 3}H)D-aspartate from granule cells. The ET-induced aspartate release was completely blocked in the absence of extracellular Ca{sup 2+}, but was unaffected by 1 mM Co{sup 2+} or 1 microM dihydropyridine derivatives (nisoldipine and nimodipine). At higher concentration (10 microM) of nisoldipine and nimodipine, the release was partially inhibited. Short-term pretreatment of cells with phorbol 12,13-dibutyrate (PDBu) potentiated the ET-induced aspartate release, while long-term pretreatment with PDBu attenuated the release. Long-term exposure of cells to pertussis toxin (PTX), on the other hand, potentiated the ET-induced effects. Our results suggest that ET has a neuromodulatory function in the central nervous system.

  12. NMDA receptors amplify mossy fiber synaptic inputs at frequencies up to at least 750 Hz in cerebellar granule cells.

    PubMed

    Baade, Carolin; Byczkowicz, Niklas; Hallermann, Stefan

    2016-07-01

    Neuronal integration of high-frequency signals is important for rapid information processing. Cerebellar mossy fiber axons (MFs) can fire action potentials (APs) at frequencies of more than one kilohertz. However, it is unclear whether and how the postsynaptic cerebellar granule cells (GCs) are able to process these high-frequency MF inputs. Here, we measured AP firing in GCs during high-frequency MF stimulation and show that GC firing frequency increased non-linearly when MF stimulation frequency was increased from 100 to 750 Hz. To investigate the mechanisms enabling such high-frequency signaling, we analyzed the role of N-methyl-d-aspartate receptors (NMDARs), which have been implicated in synaptic signaling at lower frequencies. Application of D-2-amino-5-phosphonopentanoic acid (APV), a potent inhibitor of NMDARs, strongly impaired the GC firing frequency during high-frequency MF stimulation. APV had no significant effect on single excitatory postsynaptic potentials (EPSPs) or currents (EPSCs) evoked at 1 Hz at resting membrane potentials. However, the time course of EPSCs evoked at 1 Hz at depolarized potentials or following high-frequency MF stimulation was accelerated by APV. Thus, our results show that NMDAR-mediated currents amplify high-frequency MF inputs by prolonging the time courses of synaptic inputs, thereby causing greater synaptic summation of inputs. Hence, NMDARs support the integration of MF synaptic input at frequencies up to at least 750 Hz. Synapse 70:269-276, 2016. © 2016 Wiley Periodicals, Inc. PMID:26887562

  13. Hydroxylated polychlorinated biphenyls increase reactive oxygen species formation and induce cell death in cultured cerebellar granule cells

    SciTech Connect

    Dreiem, Anne Rykken, Sidsel; Lehmler, Hans-Joachim; Robertson, Larry W.; Fonnum, Frode

    2009-10-15

    Polychlorinated biphenyls (PCBs) are persistent organic pollutants that bioaccumulate in the body, however, they can be metabolized to more water-soluble products. Although they are more readily excreted than the parent compounds, some of the metabolites are still hydrophobic and may be more available to target tissues, such as the brain. They can also cross the placenta and reach a developing foetus. Much less is known about the toxicity of PCB metabolites than about the parent compounds. In the present study, we have investigated the effects of eight hydroxylated (OH) PCB congeners (2'-OH PCB 3, 4-OH PCB 14, 4-OH PCB 34, 4'-OH PCB 35, 4-OH PCB 36, 4'-OH PCB 36, 4-OH PCB 39, and 4'-OH PCB 68) on reactive oxygen species (ROS) formation and cell viability in rat cerebellar granule cells. We found that, similar to their parent compounds, OH-PCBs are potent ROS inducers with potency 4-OH PCB 14 < 4-OH PCB 36 < 4-OH PCB 34 < 4'-OH PCB 36 < 4'-OH PCB 68 < 4-OH PCB 39 < 4'-OH PCB 35. 4-OH PCB 36 was the most potent cell death inducer, and caused apoptotic or necrotic morphology depending on concentration. Inhibition of ERK1/2 kinase with U0126 reduced both cell death and ROS formation, suggesting that ERK1/2 activation is involved in OH-PCB toxicity. The results indicate that the hydroxylation of PCBs may not constitute a detoxification reaction. Since OH-PCBs like their parent compounds are retained in the body and may be more widely distributed to sensitive tissues, it is important that not only the levels of the parent compounds but also the levels of their metabolites are taken into account during risk assessment of PCBs and related compounds.

  14. Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

    PubMed Central

    Diaz, Marvin R.; Wadleigh, Aya; Hughes, Benjamin A.; Woodward, John J.; Valenzuela, C. Fernando

    2012-01-01

    The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs. PMID:22275879

  15. Expression and traffic of cellular prolyl oligopeptidase are regulated during cerebellar granule cell differentiation, maturation, and aging.

    PubMed

    Moreno-Baylach, M J; Felipo, V; Männistö, P T; García-Horsman, J A

    2008-10-15

    Prolyl oligopeptidase (POP) is an endopeptidase which cleaves short proline-containing neuropeptides, and it is involved in memory and learning. POP also has an intercellular function mediated through the inositol pathway, and has been involved in cell death. POP has been early considered as a housekeeping enzyme, but the recent research indicates that POP expression is regulated across tissues and intracellularly. In the brain, POP is exclusively expressed in neurons and most abundantly in pyramidal neurons of cerebral cortex, in the CA1 field neurons of hippocampus and in cerebellar Purkinje's cells. Intracellularly, POP is mainly present in the cytoplasm and some in intracellular membranes, like rough endoplasmic reticulum and Golgi apparatus. In this paper, we systematically studied the levels of expression of POP along the life of cerebellar granule cells (CGC) in culture and the distribution of POP within different intracellular compartments. We used the tight-binding inhibitor JTP-4819 covalently coupled with fluorescein (FJTP) as a tool to study the changes on expression and localization of POP protein. Our results indicate that POP activity levels are regulated during the life of the neurons. POP was found mainly in cytoplasm and neuronal projections, but at an early developmental phase significant amounts were found also in nuclei. Along the life of the neurons, POP activity fluctuated in 7-day cycles. In young neurons, the cytosolic POP activity was low but increased by maturation so that the activity peak coincided with full differentiation. Over aging, cytoplasmic POP was concentrated around nucleus, but the activity decreased with time. POP was also present in vesicles across the neuron. No major changes were seen in the nuclear or membrane bound POP over aging until activity disappeared upon neuronal death. This is the first time when POP was found in the nuclei of human neuronal cells. PMID:18718510

  16. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish.

    PubMed

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-10-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  17. Type IV Collagen Controls the Axogenesis of Cerebellar Granule Cells by Regulating Basement Membrane Integrity in Zebrafish

    PubMed Central

    Takeuchi, Miki; Yamaguchi, Shingo; Yonemura, Shigenobu; Kakiguchi, Kisa; Sato, Yoshikatsu; Higashiyama, Tetsuya; Shimizu, Takashi; Hibi, Masahiko

    2015-01-01

    Granule cells (GCs) are the major glutamatergic neurons in the cerebellum, and GC axon formation is an initial step in establishing functional cerebellar circuits. In the zebrafish cerebellum, GCs can be classified into rostromedial and caudolateral groups, according to the locations of their somata in the corresponding cerebellar lobes. The axons of the GCs in the caudolateral lobes terminate on crest cells in the dorsal hindbrain, as well as forming en passant synapses with Purkinje cells in the cerebellum. In the zebrafish mutant shiomaneki, the caudolateral GCs extend aberrant axons. Positional cloning revealed that the shiomaneki (sio) gene locus encodes Col4a6, a subunit of type IV collagen, which, in a complex with Col4a5, is a basement membrane (BM) component. Both col4a5 and col4a6 mutants displayed similar abnormalities in the axogenesis of GCs and retinal ganglion cells (RGCs). Although type IV collagen is reported to control axon targeting by regulating the concentration gradient of an axonal guidance molecule Slit, Slit overexpression did not affect the GC axons. The structure of the BM surrounding the tectum and dorsal hindbrain was disorganized in the col4a5 and col4a6 mutants. Moreover, the abnormal axogenesis of the caudolateral GCs and the RGCs was coupled with aberrant BM structures in the type IV collagen mutants. The regrowth of GC axons after experimental ablation revealed that the original and newly formed axons displayed similar branching and extension abnormalities in the col4a6 mutants. These results collectively suggest that type IV collagen controls GC axon formation by regulating the integrity of the BM, which provides axons with the correct path to their targets. PMID:26451951

  18. Glutamate neurotoxicity in rat cerebellar granule cells involves cytochrome c release from mitochondria and mitochondrial shuttle impairment.

    PubMed

    Atlante, A; Gagliardi, S; Marra, E; Calissano, P; Passarella, S

    1999-07-01

    To gain some insight into the mechanism by which glutamate neurotoxicity takes place in cerebellar granule cells, two steps of glucose oxidation were investigated: the electron flow via respiratory chain from certain substrates to oxygen and the transfer of extramitochondrial reducing equivalents via the mitochondrial shuttles. However, cytochrome c release from intact mitochondria was found to occur in glutamate-treated cells as detected photometrically in the supernatant of the cell homogenate suspension. As a result of cytochrome c release, an increase of the oxidation of externally added NADH was found, probably occurring via the NADH-b5 oxidoreductase of the outer mitochondrial membrane. When the two mitochondrial shuttles glycerol 3-phosphate/dihydroxyacetone phosphate and malate/oxaloacetate, devoted to oxidizing externally added NADH, were reconstructed, both were found to be impaired under glutamate neurotoxicity. Consistent early activation in two NADH oxidizing mechanisms, i.e., lactate production and plasma membrane NADH oxidoreductase activity, was found in glutamate-treated cells. In spite of this, the increase in the cell NADH fluorescence was found to be time-dependent, an index of the progressive damage of the cell. PMID:10386976

  19. Relationship between lipophilicity of C6-10 hydrocarbon solvents and their ROS-inducing potency in rat cerebellar granule cells.

    PubMed

    Dreiem, A; Myhre, O; Fonnum, F

    2002-12-01

    We have studied the effects of aliphatic, alicyclic, and aromatic C6-10 solvents on the formation of reactive oxygen species (ROS) in rat cerebellar granule cell cultures. ROS formation was assessed by monitoring oxidation of 2',7'-dichlorofluorescin (DCFH) to the fluorescent compound 2',7'-dichlorofluorescein (DCF). We found that aromatic solvents with C > 7, and aliphatic and alicyclic solvents with C > or = 7 induce ROS formation in rat cerebellar granule cells in vitro. The response increased with increasing solvent concentration. The potency of the compounds within each homologous group seemed to be correlated to their octanol water partition-coefficients. The aromatic solvents were generally less efficient in inducing ROS formation than the aliphatic and the alicyclic compounds. PMID:12520760

  20. AMPA receptors serum-dependently mediate GABAA receptor alpha1 and alpha6 subunit down-regulation in cultured mouse cerebellar granule cells.

    PubMed

    Uusi-Oukari, Mikko; Kontturi, Leena-Stiina; Kallinen, Sampsa A; Salonen, Virpi

    2010-04-01

    Depolarization of cultured mouse cerebellar granule cells with potassium or kainate results in developmentally arrested state that includes down-regulation of GABA(A) receptor alpha1, alpha6 and beta2 subunit expression. These subunits are normally strongly expressed in cerebellar granule cells from second postnatal week throughout the adulthood. In the present study we demonstrate that selective activation of AMPA subtype of glutamate receptors down-regulates alpha1 and alpha6 subunit mRNA expression. Removal of AMPA agonist from culture medium restores expression of these subunits indicating reversibility of the down-regulation. In serum-free culture medium AMPA receptor activation did not down-regulate alpha1 or alpha6 subunit expression. Furthermore, the down-regulation was strongly attenuated when the cells were cultured in the presence of dialysed fetal calf serum. The results indicate that down-regulation of GABA(A) receptor alpha1 and alpha6 subunits by AMPA receptor activation is dependent on the presence of low molecular weight compounds present in fetal calf serum. In order to study mouse cerebellar granule cell maturation and/or regulation of GABA(A) receptor subunit expression in culture, the experiments should be performed in the absence of fetal calf serum. PMID:20170697

  1. Excitatory amino acid stimulation of the survival of rat cerebellar granule cells in culture is associated with an increase in SMN, the spinal muscular atrophy disease gene product.

    PubMed

    Andreassi, C; Patrizi, A L; Brahe, C; Eboli, M L

    2000-01-01

    Excitatory amino acids which promote the survival of cerebellar granule cells in culture, also promote the expression of the survival of motor neuron (SMN) protein. Immunolocalization studies using SMN monoclonal antibody showed that SMN is decreased in cultures grown in low K+ or chemically defined medium with respect to cultures grown in high K+ medium and that an increase of SMN can be induced by treatment of low K+ cultures with glutamate or N-methyl-D-aspartate. PMID:10901626

  2. The Etv1 transcription factor activity-dependently downregulates a set of genes controlling cell growth and differentiation in maturing cerebellar granule cells.

    PubMed

    Okazawa, Makoto; Abe, Haruka; Nakanishi, Shigetada

    2016-05-13

    In the early postnatal period, cerebellar granule cells exhibit an activity-dependent downregulation of a set of immaturation genes involved in cell growth and migration and are shifted to establishment of a mature network formation. Through the use of a granule cell culture and both pharmacological and RNA interference (siRNA) analyses, the present investigation revealed that the downregulation of these immaturation genes is controlled by strikingly unified signaling mechanisms that operate sequentially through the stimulation of AMPA and NMDA receptors, tetrodotoxin-sensitive Na(+) channels and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). This signaling cascade induces the Etv1 transcription factor, and knockdown of Etv1 by a siRNA technique prevented this activity-dependent downregulation of immaturation genes. Thus, taken into consideration the mechanism that controls the upregulation of maturation genes involved in synaptic formation, these results indicate that Etv1 orchestrates the activity-dependent regulation of both maturation and immaturation genes in developing granule cells and plays a key role in specifying the identity of mature granule cells in the cerebellum. PMID:27059140

  3. Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells.

    PubMed

    Kim, S D; Oh, S K; Kim, H S; Seong, Y H

    2001-04-01

    Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, a bis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 microM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 microM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 microM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 microM), which was measured by HPLC. And fangchinoline (5 microM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions. PMID:11339637

  4. Cell Signaling and Neurotoxicity: 3H-Arachidonic acid release (Phospholipase A2) in cerebellar granule neurons

    EPA Science Inventory

    Cell signaling is a complex process which controls basic cellular activities and coordinates actions to maintain normal cellular homeostasis. Alterations in signaling processes have been associated with neurological diseases such as Alzheimer's and cerebellar ataxia, as well as, ...

  5. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

    PubMed Central

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  6. Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels.

    PubMed

    Basille-Dugay, Magali; Vaudry, Hubert; Fournier, Alain; Gonzalez, Bruno; Vaudry, David

    2013-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca(2+)]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca(2+) response. Preincubation of granule cells with the Ca(2+) ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca(2+)]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca(2+) channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca(2+)]i increase. Preincubation of granule neurons with the N-type Ca(2+) channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca(2+)]i response, whereas the L-type Ca(2+) channel blocker, nifedipine, and the P- and Q-type Ca(2+) channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca(2+)]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca(2+) channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes. PMID:23675369

  7. Mefenamic acid bi-directionally modulates the transient outward K{sup +} current in rat cerebellar granule cells

    SciTech Connect

    Zhang Man; Shi Wenjie; Fei Xiaowei; Liu Yarong; Zeng Ximin; Mei Yanai

    2008-02-01

    The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K{sup +} current (I{sub A}) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 {mu}M to 100 {mu}M, mefenamic acid reversibly inhibited I{sub A} in a dose-dependent manner. However, mefenamic acid at a concentration of 1 {mu}M significantly increased the amplitude of I{sub A} to 113 {+-} 1.5% of the control. At more than 10 {mu}M, mefenamic acid inhibited the amplitude of I{sub A} without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I{sub A}, but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I{sub A} channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.

  8. The natural scorpion peptide, BmK NT1 activates voltage-gated sodium channels and produces neurotoxicity in primary cultured cerebellar granule cells.

    PubMed

    Zou, Xiaohan; He, Yuwei; Qiao, Jinping; Zhang, Chunlei; Cao, Zhengyu

    2016-01-01

    The scorpion Buthus martensii Karsch has been used in Traditional Chinese Medicine to treat neuronal diseases such as neuropathic pain, paralysis and epilepsy for thousands of years. Studies have demonstrated that scorpion venom is the primary active component. Although scorpion venom can effectively attenuate pain in the clinic, it also produces neurotoxic response. In this study, toxicity guided purification led to identify a mammalian toxin termed BmK NT1 comprising of 65 amino acid residues and an amidated C-terminus, a mature peptide encoded by the nucleotide sequence (GenBank No. AF464898). In contract to the recombinant product of the same nucleotide sequence, BmK AGAP, which displayed analgesic and anti-tumor effect, intravenous injection (i.v.) of BmK NT1 produced acute toxicity in mice with an LD50 value of 1.36 mg/kg. In primary cultured cerebellar granule cells, BmK NT1 produced a concentration-dependent cell death with an IC50 value of 0.65 μM (0.41-1.03 μM, 95% Confidence Intervals, 95% CI) which was abolished by TTX, a voltage-gated sodium channel (VGSC) blocker. We also demonstrated that BmK NT1 produced modest sodium influx in cerebellar granule cell cultures with an EC50 value of 2.19 μM (0.76-6.40 μM, 95% CI), an effect similar to VGSC agonist, veratridine. The sodium influx response was abolished by TTX suggesting that BmK NT1-induced sodium influx is solely through activation of VGSC. Considered these data together, we demonstrated that BmK NT1 activated VGSC and produced neurotoxicity in cerebellar granule cell cultures. PMID:26598793

  9. ONTOGENY OF PROTEINS ASSOCIATED WITH NEURITE GROWTH AND SYNAPTOGENESIS IN CEREBELLAR GRANULE CELLS IN VITRO.

    EPA Science Inventory

    In vitro techniques may be useful in screening for effects of developmental neurotoxicants. Previously, we characterized changes in biochemical markers associated with neuronal development in a PC12 cell model of differentiation and growth. The current research extended these stu...

  10. Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca2+-dependent interactions via its EF-hand 5 domain

    PubMed Central

    Arendt, Oliver; Schwaller, Beat; Brown, Edward B; Eilers, Jens; Schmidt, Hartmut

    2013-01-01

    Ca2+-binding proteins (CaBPs) are important regulators of neuronal Ca2+ signalling, acting either as buffers that shape Ca2+ transients and Ca2+ diffusion and/or as Ca2+ sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching. We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca2+ transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca2+-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca2+ buffer and point towards a Ca2+ sensor function of CR. PMID:23732647

  11. COMPARATIVE EFFECTS OF TWO POLYCHLORINATED BIPHENYL CONGENERS ON CALCIUM HOMEOSTASIS IN RAT CEREBELLAR GRANULE CELLS

    EPA Science Inventory

    Some polychlorinated biphenyls (PCBs) have been reported to alter locomotor activity and decrease brain dopamine function in laboratory animals. CBs with orth- and/or para-chlorine substitutions are reportedly most potent in decreasing cell dopamine content in vitro and were dete...

  12. Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

    SciTech Connect

    Vendrell, Iolanda; Carrascal, Montserrat; Abian, Joaquin

    2010-01-01

    Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 +- 199, 345 +- 47, and 243 +- 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 +- 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

  13. Recombinant human insulin-like growth factor I exerts a trophic action and confers glutamate sensitivity on glutamate-resistant cerebellar granule cells.

    PubMed Central

    Calissano, P; Ciotti, M T; Battistini, L; Zona, C; Angelini, A; Merlo, D; Mercanti, D

    1993-01-01

    Cerebellar granule cells grown in the presence of a serum complex differentiate but are resistant to the lethal action of excitatory amino acids. When these cells are grown also in the presence of insulin-like growth factor I (IGF-I) they become fully susceptible to the toxic, lethal action of glutamate. The glutamate-sensitizing action of IGF-I is dependent on concentration (half-maximal effect at 2-4 ng/ml) and time (half-maximal effect at 2-4 days in vitro) and is paralleled by the appearance of functionally active, glutamate-activated, Ca2+ channels and of voltage-gated Na+ and late K+ channels. IGF-I-induced glutamate sensitivity is rapidly reversible (t1/2 = 30-60 min) after removal of this somatomedin. The action of IGF-I is not mimicked by IGF-II, nerve growth factor, basic or acidic fibroblast growth factor, platelet-derived growth factor, or tumor necrosis factor alpha. We postulate that the constitutive phenotype of cerebellar granule cells is glutamate-resistant and becomes responsive to excitatory amino acids under the action of epigenetic cues among which IGF-I may be one of those operative in vivo. Images Fig. 1 PMID:8104340

  14. YB-1 is elevated in medulloblastoma and drives proliferation in Sonic hedgehog-dependent cerebellar granule neuron progenitor cells and medulloblastoma cells.

    PubMed

    Dey, A; Robitaille, M; Remke, M; Maier, C; Malhotra, A; Gregorieff, A; Wrana, J L; Taylor, M D; Angers, S; Kenney, A M

    2016-08-11

    Postnatal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells of origin for the SHH-associated subgroup of medulloblastoma, is driven by Sonic hedgehog (Shh) and insulin-like growth factor (IGF) in the developing cerebellum. Shh induces the oncogene Yes-associated protein (YAP), which drives IGF2 expression in CGNPs and mouse Shh-associated medulloblastomas. To determine how IGF2 expression is regulated downstream of YAP, we carried out an unbiased screen for transcriptional regulators bound to IGF2 promoters. We report that Y-box binding protein-1 (YB-1), an onco-protein regulating transcription and translation, binds to IGF2 promoter P3. We observed that YB-1 is upregulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh subgroup of medulloblastoma in mice, we show for the first time that YB-1 is induced by Shh in CGNPs. Its expression is YAP-dependent and it is required for IGF2 expression in CGNPs. Finally, both gain-of function and loss-of-function experiments reveal that YB-1 activity is required for sustaining CGNP and medulloblastoma cell (MBC) proliferation. Collectively, our findings describe a novel role for YB-1 in driving proliferation in the developing cerebellum and MBCs and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas. PMID:26725322

  15. Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development

    PubMed Central

    Zhang, Peter; Ha, Thomas; Larouche, Matt; Swanson, Douglas; Goldowitz, Dan

    2015-01-01

    Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene’s function in neuronal development. PMID:26226504

  16. Mice deficient in carbonic anhydrase type 8 exhibit motor dysfunctions and abnormal calcium dynamics in the somatic region of cerebellar granule cells.

    PubMed

    Lamont, Matthew G; Weber, John T

    2015-06-01

    The waddles (wdl) mouse is characterized by a namesake "side-to-side" waddling gait due to a homozygous mutation of the Car8 gene. This mutation results in non-functional copies of the protein carbonic anhydrase type 8. Rota-rod testing was conducted to characterize the wdl mutations' effect on motor output. Results indicated that younger homozygotes outperformed their older cohorts, an effect not seen in previous studies. Heterozygotes, which were thought to be free of motor impairment, displayed motor learning deficiencies when compared with wild type performance. Acute cerebellar slices were then utilized for fluorescent calcium imaging experiments, which revealed significant alterations in cerebellar granule cell somatic calcium signaling when exposed to glutamate. The contribution of GABAergic signaling to these alterations was also verified using bath application of bicuculline. Changes in somatic calcium signals were found to be applicable to an in vivo scenario by comparing group responses to electrical stimulation of afferent mossy fiber projections. Finally, intracellular calcium store function was also found to be altered by the wdl mutation when slices were treated with thapsigargin. These findings, taken together with previous work on the wdl mouse, indicate a widespread disruption in cerebellar circuitry hampering proper neuronal communication. PMID:25721739

  17. Peroxisome proliferator-activated receptor gamma agonists protect cerebellar granule cells from cytokine-induced apoptotic cell death by inhibition of inducible nitric oxide synthase.

    PubMed

    Heneka, M T; Feinstein, D L; Galea, E; Gleichmann, M; Wüllner, U; Klockgether, T

    1999-12-01

    Cerebellar granule cells (CGCs) can express the inducible isoform of nitric oxide synthase (iNOS) in response to inflammatory stimuli. We demonstrate that induction of iNOS in CGCs by bacterial lipopolysaccharide and pro-inflammatory cytokines results in cell death that was potentiated by excess L-arginine and inhibited by the selective iNOS inhibitor, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. The NO-mediated cell death was accompanied by increased caspase-3-like activity, DNA fragmentation and positive terminal transferase dUTP nick end labeling (TUNEL), suggesting that apoptosis mediates CGC cell death. Incubation of CGCs with the non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen or indomethacin, or with 15-deoxy-delta12,14 prostaglandin J2 (PGJ2) downregulates iNOS expression and reduces subsequent cell death. Since in other cell types, both NSAIDs and PGJ2 can activate the peroxisome proliferator-activated receptor-gamma (PPARgamma) and downregulate cytokine levels and iNOS expression, and since CGCs express PPARgamma in vivo and in vitro, our data suggest that activation of CGC PPARgamma mediates iNOS suppression and reduced cell death. Because PPARgamma is expressed in brains of Alzheimer's Disease (AD) patients, in which neuronal iNOS expression and apoptotic cell death have been described, these results may help explain the basis for the beneficial effects of NSAIDs in AD. PMID:10695726

  18. Methylmercury-Dependent Increases in Fluo4 Fluorescence in Neonatal Rat Cerebellar Slices Depend on Granule Cell Migrational Stage and GABAA Receptor Modulation.

    PubMed

    Bradford, Aaron B; Mancini, Jayme D; Atchison, William D

    2016-01-01

    Methylmercury (MeHg) disrupts cerebellar function, especially during development. Cerebellar granule cells (CGC), which are particularly susceptible to MeHg by unknown mechanisms, migrate during this process. Transient changes in intracellular Ca(2+) (Ca(2+) i) are crucial to proper migration, and MeHg is well known to disrupt CGC Ca(2+) i regulation. Acutely prepared slices of neonatal rat cerebellum in conjunction with confocal microscopy and fluo4 epifluorescence were used to track changes induced by MeHg in CGC Ca(2+) i regulation in the external (EGL) and internal granule cell layers (IGL) as well as the molecular layer (ML). MeHg caused no cytotoxicity but did cause a time-dependent increase in fluo4 fluorescence that depended on the stage of CGC development. CGCs in the EGL were most susceptible to MeHg-induced increases in fluo4 fluorescence. MeHg increased fluorescence in CGC processes but only diffusely; Purkinje cells rarely fluoresced in these slices. Neither muscimol nor bicuculline alone altered baseline fluo4 fluorescence in any CGC layer, but each delayed the onset and reduced the magnitude of effect of MeHg on fluo4 fluorescence in the EGL and ML. In the IGL, both muscimol and bicuculline delayed the onset of MeHg-induced increases in fluo4 fluorescence but did not affect fluorescence magnitude. Thus, acute exposure to MeHg causes developmental stage-dependent increases in Ca(2+) i in CGCs. Effects are most prominent in CGCs during development or early stages of migration. GABAA receptors participate in an as yet unclear manner to MeHg-induced Ca(2+) i dysregulation of CGCs. PMID:26514794

  19. Analysis of hedgehog signaling in cerebellar granule cell precursors in a conditional Nsdhl allele demonstrates an essential role for cholesterol in postnatal CNS development.

    PubMed

    Cunningham, David; DeBarber, Andrea E; Bir, Natalie; Binkley, Laura; Merkens, Louise S; Steiner, Robert D; Herman, Gail E

    2015-05-15

    NSDHL is a 3β-hydroxysterol dehydrogenase that is involved in the removal of two C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the gene encoding the enzyme are responsible for the X-linked, male lethal mouse mutations bare patches and striated, as well as most cases of human CHILD syndrome. Rare, hypomorphic NSDHL mutations are also associated with X-linked intellectual disability in males with CK syndrome. Since hemizygous male mice with Nsdhl mutations die by midgestation, we generated a conditional targeted Nsdhl mutation (Nsdhl(tm1.1Hrm)) to investigate the essential role of cholesterol in the early postnatal CNS. Ablation of Nsdhl in radial glia using GFAP-cre resulted in live-born, normal appearing affected male pups. However, the pups develop overt ataxia by postnatal day 8-10 and die shortly thereafter. Histological abnormalities include progressive loss of cortical and hippocampal neurons, as well as deficits in the proliferation and migration of cerebellar granule precursors and subsequent massive apoptosis of the cerebellar cortex. We replicated the granule cell precursor proliferation defect in vitro and demonstrate that it results from defective signaling by SHH. Furthermore, this defect is almost completely rescued by supplementation of the culture media with exogenous cholesterol, while methylsterol accumulation above the enzymatic block appears to be associated with increased cell death. These data support the absolute requirement for cholesterol synthesis in situ once the blood-brain-barrier forms and cholesterol transport to the fetus is abolished. They further emphasize the complex ramifications of cholesterogenic enzyme deficiency on cellular metabolism. PMID:25652406

  20. Analysis of hedgehog signaling in cerebellar granule cell precursors in a conditional Nsdhl allele demonstrates an essential role for cholesterol in postnatal CNS development

    PubMed Central

    Cunningham, David; DeBarber, Andrea E.; Bir, Natalie; Binkley, Laura; Merkens, Louise S.; Steiner, Robert D.; Herman, Gail E.

    2015-01-01

    NSDHL is a 3β-hydroxysterol dehydrogenase that is involved in the removal of two C-4 methyl groups in one of the later steps of cholesterol biosynthesis. Mutations in the gene encoding the enzyme are responsible for the X-linked, male lethal mouse mutations bare patches and striated, as well as most cases of human CHILD syndrome. Rare, hypomorphic NSDHL mutations are also associated with X-linked intellectual disability in males with CK syndrome. Since hemizygous male mice with Nsdhl mutations die by midgestation, we generated a conditional targeted Nsdhl mutation (Nsdhltm1.1Hrm) to investigate the essential role of cholesterol in the early postnatal CNS. Ablation of Nsdhl in radial glia using GFAP-cre resulted in live-born, normal appearing affected male pups. However, the pups develop overt ataxia by postnatal day 8–10 and die shortly thereafter. Histological abnormalities include progressive loss of cortical and hippocampal neurons, as well as deficits in the proliferation and migration of cerebellar granule precursors and subsequent massive apoptosis of the cerebellar cortex. We replicated the granule cell precursor proliferation defect in vitro and demonstrate that it results from defective signaling by SHH. Furthermore, this defect is almost completely rescued by supplementation of the culture media with exogenous cholesterol, while methylsterol accumulation above the enzymatic block appears to be associated with increased cell death. These data support the absolute requirement for cholesterol synthesis in situ once the blood-brain-barrier forms and cholesterol transport to the fetus is abolished. They further emphasize the complex ramifications of cholesterogenic enzyme deficiency on cellular metabolism. PMID:25652406

  1. Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling study

    PubMed Central

    Magistretti, Jacopo; Castelli, Loretta; Forti, Lia; D'Angelo, Egidio

    2006-01-01

    Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ∼−40 mV, showed half-maximal activation at ∼−55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at −35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially

  2. Progressive multifocal leukoencephalopathy with bilateral middle cerebellar peduncle lesions confirmed by repeated CSF-JC virus tests and coexistence of JC virus granule cell neuronopathy. Report of a case.

    PubMed

    Ito, Daisuke; Yasui, Keizo; Hasegawa, Yasuhiro; Nakamichi, Kazuo; Katsuno, Masahisa; Takahashi, Akira

    2016-07-28

    A 65 year-old woman with small lymphocytic leukemia presented with subacute cerebellar ataxia. Six months after rituximab chemotherapy, a cranial MRI revealed lesions in the bilateral middle cerebellar peduncles. Both cerebrospinal fluid (CSF) JC virus (JCV)-DNA PCR test on three occasions and brain biopsy were negative. CSF tests were repeated. The fourth test performed 6 months after the onset showed positive JCV-DNA, and a definite diagnosis of progressive multifocal leukoencephalopathy (PML) was made. Neuroimaging of cerebellar atrophy was considered to be coexistence of granule cell neuronopathy. Medication with mirtazapine and mefloquine was temporarily effective for several months. Little are known solitary bilateral MRI lesions of the middle cerebellar peduncle in PML. JCV-PCR test of CSF may be negative at an earlier stage of PML. Repeated CSF tests should be essential to confirming the diagnosis in such cases. PMID:27356732

  3. Simultaneous determination of purine nucleotides, their metabolites and beta-nicotinamide adenine dinucleotide in cerebellar granule cells by ion-pair high performance liquid chromatography.

    PubMed

    Giannattasio, Sergio; Gagliardi, Sara; Samaja, Michele; Marra, Ersilia

    2003-02-01

    The method described here allows the quantitative simultaneous determination of adenosine 5'-triphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine, inosine 5'-monophosphate, inosine, uric acid, xanthine, hypoxanthine and beta-nicotinamide adenine dinucleotide by ion-pair high performance liquid chromatography. The chromatographic analysis requires 26 min per sample and allows the separation of the mentioned metabolites in a time as short as 16 min. Primary cultures of rat cerebellar granule cells were incubated in serum-free medium containing 25 mM KCl for 1.5-48 h and their acid extracts were injected onto column. Uric acid, inosine 5'-monophosphate, inosine, beta-nicotinamide adenine dinucleotide, adenosine, adenosine 5'-monophosphate, guanosine 5'-diphosphate, adenosine 5'-diphosphate, guanosine 5'-triphosphate and adenosine 5'-triphosphate were identified and quantified, while hypoxanthine, xanthine and guanosine were below the detection limit. This method makes use of a single-step sample pre-treatment procedure which allows a greater than 91% recovery of the compounds of interest and provides the assay of the metabolites of interest in little amounts of cell extracts. Therefore, this method is suitable to evaluate the energetic state in a variety of cell types, both under normal and dismetabolic conditions, such as after the induction of apoptosis or necrosis. PMID:12565687

  4. Protective effects of fangchinoline and tetrandrine on hydrogen peroxide-induced oxidative neuronal cell damage in cultured rat cerebellar granule cells.

    PubMed

    Koh, Sang Bum; Ban, Ju Yeon; Lee, Bo Young; Seong, Yeon Hee

    2003-06-01

    The present study was performed to examine the neuroprotective effects of fangchinoline (FAN) and tetrandrine (TET), bis-benzylisoquinoline alkaloids, which exhibit the characteristics of Ca 2+ channel blockers, on H2O2 -induced neurotoxicity using cultured rat cerebellar granule neurons. H2O2 produced a concentration-dependent reduction of cell viability, which was blocked by (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,10-imine (MK-801), an N-methyl- D-aspartate (NMDA) receptor antagonist, verapamil, an L-type Ca 2+ channel blocker, and NG-nitro- L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Pretreatment with FAN and TET over a concentration range of 0.1 to 10 microM significantly decreased the H2O2 -induced neuronal cell death as assessed by a trypan blue exclusion test, a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and the number of apoptotic nuclei. In addition, FAN and TET inhibited the H2O2 -induced elevation of glutamate release into the medium, elevation of the cytosolic free Ca 2+ concentration ([Ca 2+] c ), and generation of reactive oxygen species (ROS). These results suggest that FAN and TET may mitigate the harmful effects of H2O2 -induced neuronal cell death by interfering with the increase of [Ca 2+] c, and then by inhibiting glutamate release and generation of ROS. Abbreviations. AP5:D(-)-2-amino-5-phosphonopentanoic acid DMSO:dimethyl sulfoxide FAN:fangchinoline H 2 DCF-DA:2',7'-dichlorodihydrofluorescin diacetate MK-801:(5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,d]cyclohepten-5,20-imine MTT:3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide L-NAME: NG-Nitro- L-arginine methyl ester NMDA: N-methyl- D-aspartate TET:tetrandrine PMID:12865967

  5. Weaker control of the electrical properties of cerebellar granule cells by tonically active GABAA receptors in the Ts65Dn mouse model of Down’s syndrome

    PubMed Central

    2013-01-01

    Background Down’s syndrome (DS) is caused by triplication of all or part of human chromosome 21 and is characterized by a decrease in the overall size of the brain. One of the brain regions most affected is the cerebellum, in which the number of granule cells (GCs) is markedly decreased. GCs process sensory information entering the cerebellum via mossy fibres and pass it on to Purkinje cells and inhibitory interneurons. How GCs transform incoming signals depends on their input–output relationship, which is adjusted by tonically active GABAA receptor channels. Results We report that in the Ts65Dn mouse model of DS, in which cerebellar volume and GC number are decreased as in DS, the tonic GABAA receptor current in GCs is smaller than in wild-type mice and is less effective in moderating input resistance and raising the minimum current required for action potential firing. We also find that tonically active GABAA receptors curb the height and broaden the width of action potentials in wild-type GCs but not in Ts65Dn GCs. Single-cell real-time quantitative PCR reveals that these electrical differences are accompanied by decreased expression of the gene encoding the GABAA receptor β3 subunit but not genes coding for some of the other GABAA receptor subunits expressed in GCs (α1, α6, β2 and δ). Conclusions Weaker moderation of excitability and action potential waveform in GCs of the Ts65Dn mouse by tonically active GABAA receptors is likely to contribute to atypical transfer of information through the cerebellum. Similar changes may occur in DS. PMID:23870245

  6. In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

    PubMed Central

    2012-01-01

    Background The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes. Methods Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D3-Creatine (D3Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (CT1), L-arginine:glycine amidinotransferase (AGAT), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) gene expression was assessed in the same cells by real time PCR. Results D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, AGAT, GAMT and SLC6A8 were expressed in both kind of cells. Conclusions Our results

  7. Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis

    PubMed Central

    Galli, Cinzia; Piccini, Alessandra; Ciotti, Maria Teresa; Castellani, Loriana; Calissano, Pietro; Zaccheo, Damiano; Tabaton, Massimo

    1998-01-01

    Some clues suggest that neuronal damage induces a secondary change of amyloid β protein (Aβ) metabolism. We investigated this possibility by analyzing the secretion of Aβ and processing of its precursor protein (amyloid precursor protein, APP) in an in vitro model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [35S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Aβ, full-length APP, α- and β-APP secreted forms. Immunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer. Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa Aβ were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the β-APPs/α-APPs ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Aβ sequence showed that the Aβ signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from β-secretase cleavage and suggest that an overproduction of Aβ may be the consequence of neuronal damage from various causes. PMID:9448317

  8. Effects of depolarization and NMDA antagonists on the role survival of cerebellar granule cells: a pivotal role for protein kinase C isoforms.

    PubMed

    Lin, W W; Wang, C W; Chuang, D M

    1997-06-01

    Primary cultures of cerebellar granule cells (CGCs) grown in high-K+ (25 mM; K25) medium progressively differentiate in vitro. Differentiation is noticeable after 3-4 days in vitro (DIV) and reach a mature stage after 8 DIV. Longer cultivation of CGCs (>13 DIV) triggers the processes of spontaneous cell death. However, if cultured in normal physiological K concentration (5 mM; K5), a significant proportion of the cells dies by the end of the first week in culture. To address the role of protein kinase C (PKC) in the development of CGCs, we measured the kinase activity as well as the protein level of the kinase isoforms. As the K25 CGC culture proceeded, the PKC activity time-dependently increased by 3.2-fold, reaching a steady state at 8 DIV. Western blot analysis using PKC isoform-specific antibodies revealed an increase in levels of PKC alpha, gamma, mu, lambda, and iota from 2 to 8 DIV. A slight increase or decrease at 4 DIV was observed for PKC epsilon and betaII, respectively, whereas no significant change was observed for betaI. The isoforms of delta, theta, eta, and zeta were not detected. Comparing the 14 DIV cultures with the 10 DIV cultures, the immunoreactivities of PKC iota and epsilon were decreased, those of PKC alpha, betaI, betaII, gamma, and lambda were unchanged, whereas that of PKC mu was still increased. In K5 cultures, the immunoreactivity of each PKC isoform at 2-4 DIV was similar to that observed in K25 cells, although no remarkable differentiation features were observed. Coordinated with the appearance of cell death at 8 DIV in low-K+ cultures, levels of PKC alpha, mu, lambda, and iota, but not the others, were markedly decreased. The NMDA receptor antagonists MK-801 and 2-amino-5-phosphopentanoic acid markedly prevented the age-induced apoptosis of CGCs, and the cells survived >18 DIV under these conditions. The cytoprotective effect of MK-801 was concomitant with the increases in levels of PKC gamma, lambda, iota, and mu at 10 and 14 DIV

  9. COMPARISON OF NEUROSCREEN-1 AND CEREBELLAR GRANULE CELL CULTURES FOR EVALUATING NEURITE OUTGROWTH USING THE ARRAYSCAN HIGH CONTENT ANALYSIS SYSTEM

    EPA Science Inventory

    A major challenge facing the Environmental Protection Agency is the development of high-throughput screening assays amendable to resource-efficient developmental neurotoxicity for chemical screening and toxicity prioritization. One approach uses in vitro, cell-based assays which...

  10. Comparison of PC12 and Cerebellar Granule Cell Cultures for Evaluating Neurite Outgrowth Using High Content Screening

    EPA Science Inventory

    Development of high-throughput assays for chemical screening and hazard identification is a pressing priority worldwide. One approach uses in vitro, cell-based assays which recapitulate biological events observed in vivo. Neurite outgrowth is one such critical cellular process un...

  11. Aryl hydrocarbon receptor deletion in cerebellar granule neuron precursors impairs neurogenesis.

    PubMed

    Dever, Daniel P; Adham, Zachariah O; Thompson, Bryan; Genestine, Matthieu; Cherry, Jonathan; Olschowka, John A; DiCicco-Bloom, Emanuel; Opanashuk, Lisa A

    2016-05-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated member of the basic-helix-loop-helix/PER-ARNT-SIM(PAS) transcription factor superfamily that also mediates the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increasing evidence suggests that AhR influences the development of many tissues, including the central nervous system. Our previous studies suggest that sustained AhR activation by TCDD and/or AhR deletion disrupts cerebellar granule neuron precursor (GNP) development. In the current study, to determine whether endogenous AhR controls GNP development in a cell-autonomous manner, we created a GNP-specific AhR deletion mouse, AhR(fx/fx) /Math1(CRE/+) (AhR CKO). Selective AhR deletion in GNPs produced abnormalities in proliferation and differentiation. Specifically, fewer GNPs were engaged in S-phase, as demonstrated by ∼25% reductions in thymidine (in vitro) and Bromodeoxyuridine (in vivo) incorporation. Furthermore, total granule neuron numbers in the internal granule layer at PND21 and PND60 were diminished in AhR conditional knockout (CKO) mice compared with controls. Conversely, differentiation was enhanced, including ∼40% increase in neurite outgrowth and 50% increase in GABARα6 receptor expression in deletion mutants. Our results suggest that AhR activity plays a role in regulating granule neuron number and differentiation, possibly by coordinating this GNP developmental transition. These studies provide novel insights for understanding the normal roles of AhR signaling during cerebellar granule cell neurogenesis and may have important implications for the effects of environmental factors in cerebellar dysgenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 533-550, 2016. PMID:26243376

  12. The absence of pleiotrophin modulates gene expression in the hippocampus in vivo and in cerebellar granule cells in vitro.

    PubMed

    González-Castillo, Celia; Ortuño-Sahagún, Daniel; Guzmán-Brambila, Carolina; Márquez-Aguirre, Ana Laura; Raisman-Vozari, Rita; Pallás, Mercé; Rojas-Mayorquín, Argelia E

    2016-09-01

    Pleiotrophin (PTN) is a secreted growth factor recently proposed to act as a neuromodulatory peptide in the Central Nervous System. PTN appears to be involved in neurodegenerative diseases and neural disorders, and it has also been implicated in learning and memory. Specifically, PTN-deficient mice exhibit a lower threshold for LTP induction in the hippocampus, which is attenuated in mice overexpressing PTN. However, there is little information about the signaling systems recruited by PTN to modulate neural activity. To address this issue, the gene expression profile in hippocampus of mice lacking PTN was analyzed using microarrays of 22,000 genes. In addition, we corroborated the effect of the absence of PTN on the expression of these genes by silencing this growth factor in primary neuronal cultures in vitro. The microarray analysis identified 102 genes that are differentially expressed (z-score>3.0) in PTN null mice, and the expression of eight of those modified in the hippocampus of KO mice was also modified in vitro after silencing PTN in cultured neurons with siRNAs. The data obtained indicate that the absence of PTN affects AKT pathway response and modulates the expression of genes related with neuroprotection (Mgst3 and Estrogen receptor 1, Ers 1) and cell differentiation (Caspase 6, Nestin, and Odz4), both in vivo and in vitro. PMID:27468976

  13. Interactive effects of environmentally relevant polychlorinated biphenyls and dioxins on [3H]phorbol ester binding in rat cerebellar granule cells.

    PubMed Central

    Kodavanti, P R; Ward, T R

    1998-01-01

    Polychlorinated biphenyls (PCBs) are persistent contaminants that exist as complex mixtures in the environment. One problem faced by risk assessors is that the possible interactive effects of specific PCB congeners and related chemicals found in environmental and biological samples have not been systematically investigated. Some PCBs perturb Ca2+ homeostasis and cause protein kinase C (PKC) translocation in neuronal cell cultures and in brain homogenate preparations at concentrations where no cytotoxicity is observed, and these systems are necessary for the growth and normal functioning of neurons. The changes in second messenger systems appear to be associated with the extent of noncoplanarity of the PCB molecule. We studied the interactive effects of selected PCB congeners, a PCB metabolite, and a dioxin on PKC translocation, as determined by [3H]phorbol ester binding in cerebellar granule cells. The binary combinations included coplanar and noncoplanar PCB congeners or PCB congeners with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/PCB metabolite. In addition, we tested the interactive effects of several PCB congeners (three or more) found in environmental samples such as human milk and blood, contaminated fish, and brain samples from PCB-treated animals. The results indicated that 1) the coplanar congener [3,3',4, 4'-tetrachlorobiphenyl (TeCB)] did not alter the in vitro activity of the noncoplanar (2,2',5,5'-TeCB) or coplanar [4, 4'-dichlorobiphenyl (DCB)] congeners; 2) binary mixtures of active PCB congeners (2,2',4,4'-TeCB and 2,2'-DCB; 2,2'-DCB and 3,5-DCB; 2,2',3,5',6-PeCB and 2,2',4,4',5-PeCB) interact in a dose-additive manner; 3) TCDD did not alter the activity of either coplanar (3,3', 4,4'-TeCB) or noncoplanar (2,2',5,5'-TeCB) congeners; 4) the interaction between the parent PCB congener and hydroxy metabolite of PCB is additive; 5) PCB congener mixtures at the ratios found in environmental samples are biologically active; and 6) there was no indication

  14. PACT/RAX regulates the migration of cerebellar granule neurons in the developing cerebellum.

    PubMed

    Yong, Yue; Meng, Ya; Ding, Hanqing; Fan, Zhiqin; Tang, Yifen; Zhou, Chenghua; Luo, Jia; Ke, Zun-Ji

    2015-01-01

    PACT and its murine ortholog RAX were originally identified as a protein activator for the dsRNA-dependent, interferon-inducible protein kinase PKR. Recent studies indicated that RAX played a role in embryogenesis and neuronal development. In this study, we investigated the expression of RAX during the postnatal development of the mouse cerebellum and its role in the migration of cerebellar granule neurons (CGNs). High expression of RAX was observed in the cerebellum from postnatal day (PD) 4 to PD9, a period when the CGNs migrate from the external granule layer (EGL) to the internal granule layer (IGL). The migration of the EGL progenitor cells in vivo was inhibited by RAX knockdown on PD4. This finding was confirmed by in vitro studies showing that RAX knockdown impaired the migration of CGNs in cerebellar microexplants. PACT/RAX-regulated migration required its third motif and was independent of PKR. PACT/RAX interacted with focal adhesion kinase (FAK) and PACT/RAX knockdown disturbed the FAK phosphorylation in CGNs. These findings demonstrated a novel function of PACT/RAX in the regulation of neuronal migration. PMID:25609658

  15. GDF15 regulates Kv2.1-mediated outward K+ current through the Akt/mTOR signalling pathway in rat cerebellar granule cells

    PubMed Central

    Wang, Chang-Ying; Huang, An-Qi; Zhou, Meng-Hua; Mei, Yan-Ai

    2014-01-01

    GDF15 (growth/differentiation factor 15), a novel member of the TGFβ (transforming growth factor β) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK (extracellular-signal-regulated kinase), Akt and mTOR (mammalian target of rapamycin), but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFβR2 (TGFβ receptor 2), not TGFβR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFβRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFβRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling. PMID:24597762

  16. Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons.

    PubMed

    Bobba, Antonella; Canu, Nadia; Atlante, Anna; Petragallo, Vito; Calissano, Pietro; Marra, Ersilia

    2002-03-27

    In order to find out whether and how proteasomes participate in the processes leading cerebellar granule cells to death either in necrosis, due to glutamate neurotoxicity, or in apoptosis, due to K(+) shift, we measured the three proteasome activities by using specific fluorescent probes and investigated the effect of several proteasome inhibitors, including MG132, on the cytochrome c release taking place in the early phase of both apoptosis and necrosis. We show that differently from apoptosis, the early phase of necrosis does not require proteasome activation. Inhibition of proteasome activity can prevent cytochrome c release in cerebellar granule cells undergoing apoptosis, thus improving cell survival, but not necrosis. These findings show that proteasomes play an important role in the early phase of apoptosis but not that of necrosis, and that these two types of cell death differ from each other in their mechanism of cytochrome c release. PMID:11943185

  17. 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

  18. The Immp2l mutation causes age-dependent degeneration of cerebellar granule neurons prevented by antioxidant treatment.

    PubMed

    Liu, Chunlian; Li, Xue; Lu, Baisong

    2016-02-01

    Reactive oxygen species are implicated in age-associated neurodegeneration, although direct in vivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2-like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age-associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (> 16 months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria-targeted antioxidant SkQ1 from 6 weeks to 21 months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age-matched normal control mice or SkQ1-treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4-hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age-associated neurodegeneration. PMID:26616244

  19. Gene expression as a sensitive endpoint to evaluate cell differentiation and maturation of the developing central nervous system in primary cultures of rat cerebellar granule cells (CGCs) exposed to pesticides

    SciTech Connect

    Hogberg, Helena T.; Kinsner-Ovaskainen, Agnieszka; Hartung, Thomas; Coecke, Sandra; Bal-Price, Anna K.

    2009-03-15

    The major advantage of primary neuronal cultures for developmental neurotoxicity (DNT) testing is their ability to replicate the crucial stages of neurodevelopment. In our studies using primary culture of cerebellar granule cells (CGCs) we have evaluated whether the gene expression relevant to the most critical developmental processes such as neuronal differentiation (NF-68 and NF-200) and functional maturation (NMDA and GABA{sub A} receptors), proliferation and differentiation of astrocytes (GFAP and S100{beta}) as well as the presence of neural precursor cells (nestin and Sox10) could be used as an endpoint for in vitro DNT. The expression of these genes was assessed after exposure to various pesticides (paraquat parathion, dichlorvos, pentachlorophenol and cycloheximide) that could induce developmental neurotoxicity through different mechanisms. All studied pesticides significantly modified the expression of selected genes, related to the different stages of neuronal and/or glial cell development and maturation. The most significant changes were observed after exposure to paraquat and parathion (i.e. down-regulation of mRNA expression of NF-68 and NF-200, NMDA and GABA{sub A} receptors). Similarly, dichlorvos affected mainly neurons (decreased mRNA expression of NF-68 and GABA{sub A} receptors) whereas cycloheximide had an effect on neurons and astrocytes, as significant decreases in the mRNA expression of both neurofilaments (NF-68 and NF-200) and the astrocyte marker (S100{beta}) were observed. Our results suggest that toxicity induced by pesticides that target multiple pathways of neurodevelopment can be identified by studying expression of genes that are involved in different stages of cell development and maturation, and that gene expression could be used as a sensitive endpoint for initial screening to identify the compounds with the potential to cause developmental neurotoxicity.

  20. Profilin1 activity in cerebellar granule neurons is required for radial migration in vivo

    PubMed Central

    Kullmann, Jan A; Wickertsheim, Ines; Minnerup, Lara; Costell, Mercedes; Friauf, Eckhard; Rust, Marco B

    2015-01-01

    Neuron migration defects are an important aspect of human neuropathies. The underlying molecular mechanisms of such migration defects are largely unknown. Actin dynamics has been recognized as an important determinant of neuronal migration, and we recently found that the actin-binding protein profilin1 is relevant for radial migration of cerebellar granule neurons (CGN). As the exploited brain-specific mutants lacked profilin1 in both neurons and glial cells, it remained unknown whether profilin1 activity in CGN is relevant for CGN migration in vivo. To test this, we capitalized on a transgenic mouse line that expresses a tamoxifen-inducible Cre variant in CGN, but no other cerebellar cell type. In these profilin1 mutants, the cell density was elevated in the molecular layer, and ectopic CGN occurred. Moreover, 5-bromo-2′-deoxyuridine tracing experiments revealed impaired CGN radial migration. Hence, our data demonstrate the cell autonomous role of profilin1 activity in CGN for radial migration. PMID:25495756

  1. RNA Granules in Germ Cells

    PubMed Central

    Voronina, Ekaterina; Seydoux, Geraldine; Sassone-Corsi, Paolo; Nagamori, Ippei

    2011-01-01

    “Germ granules” are cytoplasmic, nonmembrane-bound organelles unique to germline. Germ granules share components with the P bodies and stress granules of somatic cells, but also contain proteins and RNAs uniquely required for germ cell development. In this review, we focus on recent advances in our understanding of germ granule assembly, dynamics, and function. One hypothesis is that germ granules operate as hubs for the posttranscriptional control of gene expression, a function at the core of the germ cell differentiation program. PMID:21768607

  2. Isoforms of alpha1E voltage-gated calcium channels in rat cerebellar granule cells--detection of major calcium channel alpha1-transcripts by reverse transcription-polymerase chain reaction.

    PubMed

    Schramm, M; Vajna, R; Pereverzev, A; Tottene, A; Klöckner, U; Pietrobon, D; Hescheler, J; Schneider, T

    1999-01-01

    In primary cultures of rat cerebellar granule cells, transcripts of voltage-gated Ca2+ channels have been amplified by reverse transcription-polymerase chain reaction and identified by sequencing of subcloned polymerase chain reaction products. In these neurons cultured for six to eight days in vitro, fragments of the three major transcripts alpha1C, alpha1A, and alpha1E are detected using degenerated oligonucleotide primer pairs under highly stringent conditions. Whole-cell Ca2+ current recordings from six to eight days in vitro granule cells show that most of the current is due to L-type (25%), P-type (33%) and R-type (30%) Ca2+ channels. These data support the correlation between alpha1A and P-type Ca2+ channels (G1) and between alpha1E and R-type channels (G2 and G3). By including specific primer pairs for alpha1E the complimentary DNA fragments of indicative regions of alpha1E isoforms are amplified corresponding to the three most variable regions of alpha1E, the 5'-end, the II/III-loop, and the central part of the 3'-end. Although the complementary DNA fragments of the 5'-end of rat alpha1E yield a uniform reverse transcription-polymerase chain reaction product, its structure is unusual in the sense that it is longer than in the cloned rat alpha1E complementary DNA. It corresponds to the alpha1E isoform reported for mouse and human brain and is also expressed in cerebellum and cerebrum of rat brain as the major or maybe even the only variant of alpha1E. While fragments of a new rat alpha1E isoform are amplified from the 5'-end, three known fragments of the II/III-loop and two known isoforms homologue to the 3'-coding region are detected, which in the last case are discriminated by a 129 base pair insertion. The shift of the alpha1E expression from a pattern seen in cerebellum (alpha1Ee) to a pattern identified in other regions of the brain (alpha1E-3) is discussed. These data show that: (i) alpha1E is expressed in rat brain as a structural homologue to the

  3. Assessment of GaN chips for culturing cerebellar granule neurons.

    PubMed

    Young, Tai-Horng; Chen, Chi-Ruei

    2006-06-01

    In this work, the behaviors of cerebellar granule neurons prepared from 7-day-old Wistar rats on gallium nitride (GaN) were investigated. We believe that this is the first time that the GaN has been used as a substrate for neuron cultures to examine its effect on cell response in vitro. The GaN surface structure and its relationship with cells were examined by atomic force microscopy (AFM), metallography microscopy, scanning electron microscopy (SEM), lactate dehydrogenase (LDH) release and Western blot analysis. GaN is a so-called III-V compound semiconductor material with a wide bandgap and a relatively high bandgap voltage. Compared with silicon used for most neural chips, neurons seeded on GaN were able to form an extensive neuritic network and expressed very high levels of GAP-43 coincident with the neurite outgrowth. Therefore, the GaN structure may spatially mediate cellular response that can promote neuronal cell attachment, differentiation and neuritic growth. The favorable biocompatibility characteristics of GaN can be used to measure electric signals from networks of neuronal cells in culture to make it a possible candidate for use in a microelectrode array. PMID:16516287

  4. Transcriptional Analysis of Apoptotic Cerebellar Granule Neurons Following Rescue by Gastric Inhibitory Polypeptide

    PubMed Central

    Maino, Barbara; Ciotti, Maria Teresa; Calissano, Pietro; Cavallaro, Sebastiano

    2014-01-01

    Apoptosis triggered by exogenous or endogenous stimuli is a crucial phenomenon to determine the fate of neurons, both in physiological and in pathological conditions. Our previous study established that gastric inhibitory polypeptide (Gip) is a neurotrophic factor capable of preventing apoptosis of cerebellar granule neurons (CGNs), during its pre-commitment phase. In the present study, we conducted whole-genome expression profiling to obtain a comprehensive view of the transcriptional program underlying the rescue effect of Gip in CGNs. By using DNA microarray technology, we identified 65 genes, we named survival related genes, whose expression is significantly de-regulated following Gip treatment. The expression levels of six transcripts were confirmed by real-time quantitative polymerase chain reaction. The proteins encoded by the survival related genes are functionally grouped in the following categories: signal transduction, transcription, cell cycle, chromatin remodeling, cell death, antioxidant activity, ubiquitination, metabolism and cytoskeletal organization. Our data outline that Gip supports CGNs rescue via a molecular framework, orchestrated by a wide spectrum of gene actors, which propagate survival signals and support neuronal viability. PMID:24694544

  5. PSD-95 regulates NMDA receptors in developing cerebellar granule neurons of the rat

    PubMed Central

    Losi, Gabriele; Prybylowski, Kate; Fu, Zhanyan; Luo, Jianhong; Wenthold, Robert J; Vicini, Stefano

    2003-01-01

    We transfected a green fluorescent protein-tagged PSD-95 (PSD-95gfp) into cultured rat cerebellar granule cells (CGCs) to investigate the role of PSD-95 in excitatory synapse maturation. Cells were grown in low potassium to favour functional synapse formation in vitro. Transfected cells displayed clear clusters of PSD-95gfp, often at the extremities of the short dendritic trees. We recorded NMDA and AMPA miniature excitatory postsynaptic currents (NMDA- and AMPA-mESPCs) in the presence of TTX and bicuculline. At days in vitro (DIV) 7–8 PSD-95gfp-transfected cells had NMDA-mEPSCs with faster decay and smaller amplitudes than matching controls. In contrast, AMPA-mEPSC frequencies and amplitudes were increased. Whole-cell current density and ifenprodil sensitivity were reduced in PSD-95gfp cells, indicating a reduction of NR2B subunits containing NMDA receptors. No changes were observed compared to control when cells were transfected with cDNA for PSD-95gfp with palmitoylation site mutations that prevent targeting to the synapse. Overexpression of the NMDA receptor NR2A subunit, but not the NR2B subunit, prevented NMDA-mEPSC amplitude reduction when cotransfected with PSD-95gfp. PSD-95gfp overexpression produced faster NMDA-mEPSC decay when transfected alone or with either NR2 subunit. Surface staining of the epitope-tagged NR2 subunits revealed that colocalization with PSD-95gfp was higher for flag-tagged NR2A subunit clusters than for flag-tagged NR2B subunit clusters. These data suggest that PSD-95 overexpression in CGCs favours synaptic maturation by allowing synaptic insertion of NR2A and depressing expression of NR2B subunits. PMID:12576494

  6. Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling.

    PubMed

    Wigdal, Susan S; Kirkland, Rebecca A; Franklin, James L; Haak-Frendscho, Mary

    2002-09-01

    It has been suggested that release of cytochrome c (Cyt c) from mitochondria during apoptotic death is through opening of the mitochondrial permeability transition pore followed by swelling-induced rupture of the mitochondrial outer membrane. However, this remains controversial and may vary with cell type and model system. We determined that in mouse cerebellar granule neurons, Cyt c redistribution preceded the loss of mitochondrial membrane potential during the apoptotic process, suggesting that the pore did not open prior to release. Furthermore, when mitochondria were morphologically assessed by electron microscopy, they were not obviously swollen during the period of Cyt c release. This indicates that the pore mechanism of action, if any, is not through mitochondrial outer membrane rupture. While bongkrekic acid, an inhibitor of pore opening, modestly delayed apoptotic death, it also caused a significant (p < 0.05) suppression of protein synthesis. An equivalent suppression of protein synthesis by cycloheximide had a similar delaying effect, suggesting that bongkrekic acid was acting non-specifically. These findings suggest that mitochondrial permeability transition pore is not involved in Cyt c release from mitochondria during the apoptotic death of cerebellar granule neurons. PMID:12358750

  7. NAAG fails to antagonize synaptic and extrasynaptic NMDA receptors in cerebellar granule neurons.

    PubMed

    Losi, G; Vicini, S; Neale, J

    2004-03-01

    The peptide transmitter N-acetylaspartylglutamate (NAAG) selectively activates the group II metabotropic glutamate receptors. Several reports also suggest that this peptide acts as a partial agonist at N-methyl-D-aspartate (NMDA) receptors but its putative antagonist effects have not been directly tested. To do this, we used whole cell recordings from cerebellar granule cells (CGC) in culture that allow the highest possible resolution of NMDA channel activation. When CGC were activated with equimolar concentrations of NMDA and NAAG, the peptide failed to alter the peak current elicited by NMDA. Very high concentrations of NAAG (100-200 microM) did not significantly reduce the current elicited by 10 microM NMDA or 0.1 microM glutamate, while 400 microM NAAG produced only a very small (less than 15%) reduction in these whole cell currents. Similarly, NAAG (400 microM) failed to significantly alter the average decay time constant or the peak amplitude of NMDA receptor-mediated miniature excitatory post-synaptic currents (mEPSCs). We conclude that high concentrations of the peptide do not exert physiologically relevant antagonist actions on synaptic NMDA receptor activation following vesicular release of glutamate. As an agonist, purified NAAG was found to be at least 10,000-fold less potent than glutamate in increasing "background" current via NMDA receptors on CGC. Inasmuch as it is difficult to confirm that NAAG preparations are completely free from contamination with glutamate at the 0.01% level, the peptide itself appears unlikely to have a direct agonist activity at the NMDA receptor subtypes found in CGC. Recent reports indicate that enhancing the activity of endogenous NAAG may be an important therapeutic approach to excitotoxicity and chronic pain perception. These effects are likely mediated by group II mGluRs, not NMDA receptors. PMID:14975672

  8. Afferent-target cell interactions in the cerebellum: negative effect of granule cells on Purkinje cell development in lurcher mice.

    PubMed

    Doughty, M L; Lohof, A; Selimi, F; Delhaye-Bouchaud, N; Mariani, J

    1999-05-01

    Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor gene that results in a large, constitutive inward current in the cerebellar Purkinje cells of +/Lc mice. +/Lc Purkinje cells fail to differentiate fully and die during postnatal development. In normal mice, interactions with granule cells promote Purkinje cell dendritic differentiation. Partial destruction of the granule cell population in young +/Lc mice by x irradiation resulted in a significant increase in Purkinje cell dendritic growth and improved cytoplasmic structure but did not prevent Purkinje cell death. These results indicate two components to Purkinje cell abnormalities in +/Lc mice: a retardation/blockade of dendritic development that is mediated by interactions with granule cells and the death of the cell. Thus, the normal trophic effects of granule cell interaction on Purkinje cell development are absent in the +/Lc cerebellum, suggesting that granule cells are powerful regulators of Purkinje cell differentiation. PMID:10212305

  9. A Mathematical Model of Granule Cell Generation During Mouse Cerebellum Development.

    PubMed

    Leffler, Shoshana R; Legué, Emilie; Aristizábal, Orlando; Joyner, Alexandra L; Peskin, Charles S; Turnbull, Daniel H

    2016-05-01

    Determining the cellular basis of brain growth is an important problem in developmental neurobiology. In the mammalian brain, the cerebellum is particularly amenable to studies of growth because it contains only a few cell types, including the granule cells, which are the most numerous neuronal subtype. Furthermore, in the mouse cerebellum granule cells are generated from granule cell precursors (gcps) in the external granule layer (EGL), from 1 day before birth until about 2 weeks of age. The complexity of the underlying cellular processes (multiple cell behaviors, three spatial dimensions, time-dependent changes) requires a quantitative framework to be fully understood. In this paper, a differential equation-based model is presented, which can be used to estimate temporal changes in granule cell numbers in the EGL. The model includes the proliferation of gcps and their differentiation into granule cells, as well as the process by which granule cells leave the EGL. Parameters describing these biological processes were derived from fitting the model to histological data. This mathematical model should be useful for understanding altered gcp and granule cell behaviors in mouse mutants with abnormal cerebellar development and cerebellar cancers. PMID:27125657

  10. Effects on K+ currents in rat cerebellar granule neurones of a membrane-permeable analogue of the calcium chelator BAPTA.

    PubMed Central

    Watkins, C. S.; Mathie, A.

    1996-01-01

    1. Whole cell recordings of voltage-activated K+ currents were made with the amphotericin B perforated patch technique from cerebellar granule (CG) neurones of 6-8 days rats that had been in culture for 1 to 16 days. By use of appropriate voltage protocols, the effects of the membrane-permeant form of BAPTA, 1,2-bis-(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), on the transient A current (IKA), the delayed rectifier current (IKV) and a standing outward current (IKSO) were investigated. 2. Bath application of 25 microM BAPTA-AM inhibited both IKV and IKSO in cultured neurones, but did not seem to affect IKA. Neither 25 microM BAPTA (free acid) nor 25 microM ethylenediaminetetraacetic acid acetoxymethyl ester (EDTA-AM) had any significant effect on the magnitude of IKSO. Similarly in short-term (1-2 days) cultured CG neurones IKV, but not IKA, was inhibited by 25 microM BAPTA-AM. 3. BAPTA-AM (2.5 microM) reduced IKV in short-term culture CG neurones, with further inhibition being seen when the perfusate was changed to one containing 25 microM BAPTA-AM. 4. Tetraethylammonium ions (TEA) (10 mM) reversibly inhibited IKV in these cells with a similar rate of block of IKV to that induced by 25 microM BAPTA-AM. 5. The degree of inhibition of IKV by 25 microM BAPTA-AM was both time- and voltage-dependent, in contrast to the inhibition of this current by TEA. 6. These data indicate that BAPTA-AM reduces K+ currents in cerebellar granule neurones and that this inhibition cannot be explained in terms of intracellular Ca2+ chelation, but is a direct effect on the underlying channels. PMID:8842443

  11. Iron granules in plasma cells.

    PubMed Central

    Cook, M K; Madden, M

    1982-01-01

    The curious and unusual finding of coarse iron granules in marrow plasma cells is reported in 13 patients, in whom the finding was incidental. In 10 of these patients there was known alcohol abuse and serious medical complications of that abuse. Previous reports of the finding are reviewed. Haematological data of the 13 patients are presented. A hypothesis is outlined which may account for the finding. Images PMID:7068907

  12. A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity

    PubMed Central

    Ankri, Lea; Husson, Zoé; Pietrajtis, Katarzyna; Proville, Rémi; Léna, Clément; Yarom, Yosef; Dieudonné, Stéphane; Uusisaari, Marylka Yoe

    2015-01-01

    The cerebellum, a crucial center for motor coordination, is composed of a cortex and several nuclei. The main mode of interaction between these two parts is considered to be formed by the inhibitory control of the nuclei by cortical Purkinje neurons. We now amend this view by showing that inhibitory GABA-glycinergic neurons of the cerebellar nuclei (CN) project profusely into the cerebellar cortex, where they make synaptic contacts on a GABAergic subpopulation of cerebellar Golgi cells. These spontaneously firing Golgi cells are inhibited by optogenetic activation of the inhibitory nucleo-cortical fibers both in vitro and in vivo. Our data suggest that the CN may contribute to the functional recruitment of the cerebellar cortex by decreasing Golgi cell inhibition onto granule cells. DOI: http://dx.doi.org/10.7554/eLife.06262.001 PMID:25965178

  13. NF1 regulation of RAS/ERK signaling is required for appropriate granule neuron progenitor expansion and migration in cerebellar development.

    PubMed

    Sanchez-Ortiz, Efrain; Cho, Woosung; Nazarenko, Inga; Mo, Wei; Chen, Jian; Parada, Luis F

    2014-11-01

    Cerebellar development is regulated by a coordinated spatiotemporal interplay between granule neuron progenitors (GNPs), Purkinje neurons, and glia. Abnormal development can trigger motor deficits, and more recent data indicate important roles in aspects of memory, behavior, and autism spectrum disorders (ASDs). Germline mutation in the NF1 tumor suppressor gene underlies Neurofibromatosis type 1, a complex disease that enhances susceptibility to certain cancers and neurological disorders, including intellectual deficits and ASD. The NF1 gene encodes for neurofibromin, a RAS GTPase-activating protein, and thus negatively regulates the RAS signaling pathway. Here, using mouse models to direct conditional NF1 ablation in either embryonic cerebellar progenitors or neonatal GNPs, we show that neurofibromin is required for appropriate development of cerebellar folia layering and structure. Remarkably, neonatal administration of inhibitors of the ERK pathway reversed the morphological defects. Thus, our findings establish a critical cell-autonomous role for the NF1-RAS-ERK pathway in the appropriate regulation of cerebellar development and provide a basis for using neonatal ERK inhibitor-based therapies to treat NF1-induced cerebellar disorders. PMID:25367036

  14. Igf1 and Pacap rescue cerebellar granule neurons from apoptosis via a common transcriptional program

    PubMed Central

    Maino, Barbara; D'Agata, Velia; Severini, Cinzia; Ciotti, Maria T.; Calissano, Pietro; Copani, Agata; Chang, Yi-Chien; DeLisi, Charles; Cavallaro, Sebastiano

    2016-01-01

    A shift of the delicate balance between apoptosis and survival-inducing signals determines the fate of neurons during the development of the central nervous system and its homeostasis throughout adulthood. Both pathways, promoting or protecting from apoptosis, trigger a transcriptional program. We conducted whole-genome expression profiling to decipher the transcriptional regulatory elements controlling the apoptotic/survival switch in cerebellar granule neurons following the induction of apoptosis by serum and potassium deprivation or their rescue by either insulin-like growth factor-1 (Igf1) or pituitary adenylyl cyclase-activating polypeptide (Pacap). Although depending on different upstream signaling pathways, the survival effects of Igf1 and Pacap converged into common transcriptional cascades, thus suggesting the existence of a general transcriptional program underlying neuronal survival. PMID:26941962

  15. Interleukin-6 protects cerebellar granule neurons from NMDA-induced neurotoxicity.

    PubMed

    Wang, Xiao-Chun; Qiu, Yi-Hua; Peng, Yu-Ping

    2007-04-25

    Interleukin-6 (IL-6) is an important cytokine that participates in inflammation reaction and cell growth and differentiation in the immune and nervous systems. However, the neuroprotection of IL-6 against N-methyl-D-aspartate (NMDA)-induced neurotoxicity and the related underlying mechanisms are still not identified. In the present study, the cultured cerebellar granule neurons (CGNs) from postnatal (8-day) infant rats were chronically exposed to IL-6 for 8 d, and then NMDA (100 micromol/L) was applied to the cultured CGNs for 30 min. Methyl-thiazole-tetrazolium (MTT) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and confocal laser scanning microscope (CLSM) were used to detect neuronal vitality, apoptosis and dynamic changes of intracellular Ca(2+) levels in the neurons, respectively. Anti-gp130 monoclonal antibody (75 ng/mL) was employed to the cultured CGNs with IL-6 to inhibit IL-6 activity so as to evaluate the role of gp130 (a 130 kDa glucoprotein transducing IL-6 signal) in mediating IL-6 neuroprotection. Western blot was used to measure the expressions of phospho-signal transducer and activator of transcription 3 (STAT3) and phospho-extracellular signal regulated kinase 1/2 (ERK1/2) in the cultured CGNs. The NMDA stimulation of the cultured CGNs without IL-6 pretreatment resulted in a significant reduction of the neuronal vitality, notable enhancement of the neuronal apoptosis and intracellular Ca(2+) overload in the neurons. The NMDA stimulation of the CGNs chronically pretreated with IL-6 caused a remarkable increase in the neuronal vitality, marked suppression of neuronal apoptosis and intracellular Ca(2+) overload in the neurons, compared with that in the control neurons without IL-6 pretreatment. Furthermore, anti-gp130 antibody blocked the inhibitory effect of IL-6 on NMDA-induced intracellular Ca(2+) overload in the neurons. The levels of phospho-STAT3 and phospho-ERK1/2 were significantly higher in IL-6

  16. Intracellular acidification by inhibition of the Na+/H+-exchanger leads to caspase-independent death of cerebellar granule neurons resembling paraptosis.

    PubMed

    Schneider, D; Gerhardt, E; Bock, J; Müller, M M; Wolburg, H; Lang, F; Schulz, J B

    2004-07-01

    Potassium withdrawal is commonly used to induce caspase-mediated apoptosis in cerebellar granule neurons in vitro. However, the underlying and cell death-initiating mechanisms are unknown. We firstly investigated potassium efflux through the outward delayed rectifier K+ current (Ik) as a potential mediator. However, tetraethylammoniumchloride, an inhibitor of Ik, was ineffective to block apoptosis after potassium withdrawal. Since potassium withdrawal reduced intracellular pH (pHi) from 7.4 to 7.2, we secondly investigated the effects of intracellular acidosis. To study intracellular acidosis in cerebellar granule neurons, we inhibited the Na+/H+ exchanger (NHE) with 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE 642) and 5-(N-ethyl-N-isopropyl)-amiloride. Both inhibitors concentration-dependently induced cell death and potentiated cell death after potassium withdrawal. Although inhibition of the NHE induced cell death with morphological criteria of apoptosis in light and electron microscopy including chromatin condensation, positive TUNEL staining and cell shrinkage, no internucleosomal DNA cleavage or activation of caspases was detected. In contrast to potassium withdrawal-induced apoptosis, cell death induced by intracellular acidification was not prevented by insulin-like growth factor-1, cyclo-adenosine-monophosphate, caspase inhibitors and transfection with an adenovirus expressing Bcl-XL. However, cycloheximide protected cerebellar granule neurons from death induced by potassium withdrawal as well as from death after treatment with HOE 642. Therefore, the molecular mechanisms leading to cell death after acidification appear to be different from the mechanisms after potassium withdrawal and resemble the biochemical but not the morphological characteristics of paraptosis. PMID:15017383

  17. Dynamics of fast and slow inhibition from cerebellar Golgi cells allow flexible control of synaptic integration

    PubMed Central

    Crowley, John J.; Fioravante, Diasynou; Regehr, Wade G.

    2011-01-01

    Throughout the brain, multiple interneuron types influence distinct aspects of synaptic processing. Interneuron diversity can thereby promote differential firing from neurons receiving common excitation. In contrast, Golgi cells are the sole interneurons regulating granule cell spiking evoked by mossy fibers, thereby gating inputs to the cerebellar cortex. Here, we examine how this single interneuron type modifies activity in its targets. We find that GABAA-mediated transmission at unitary Golgi cellgranule cell synapses consists of varying contributions of fast synaptic currents and sustained inhibition. Fast IPSCs depress and slow IPSCs gradually build during high frequency Golgi cell activity. Consequently, fast and slow inhibition differentially influence granule cell spike timing during persistent mossy fiber input. Furthermore, slow inhibition reduces the gain of the mossy fiber → granule cell input-output curve, while fast inhibition increases the threshold. Thus, a lack of interneuron diversity need not prevent flexible inhibitory control of synaptic processing. PMID:19778512

  18. Pharmacological properties and H+ sensitivity of excitatory amino acid receptor channels in rat cerebellar granule neurones.

    PubMed Central

    Traynelis, S F; Cull-Candy, S G

    1991-01-01

    1. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate receptor channels have been examined in rat cerebellar granule neurones with whole-cell and single-channel patch-clamp methods. The whole-cell peak and steady-state aspartate and NMDA currents were reversibly inhibited by extracellular protons; the IC50 (concentration producing half-maximal inhibition) for the full H+ inhibition curve for NMDA receptors corresponded to pH 7.3, near to physiological pH. (S)-AMPA and kainate whole-cell currents were inhibited by protons with IC50 values that corresponded to pH 6.3 and 5.7, respectively; these receptors were, however, insensitive to H+ concentrations that inhibited NMDA receptor responses. 2. Proton inhibition of the NMDA, AMPA and kainate receptor-mediated responses was voltage insensitive, and did not involve a shift in reversal potential. 3. The EC50 (concentration producing half-maximal effect) for aspartate calculated from the whole-cell dose-response curve was similar at pH 6.8 and 7.6 (mean 11.2 microM). Although the EC50 for glycine potentiation of the aspartate response was marginally increased from 273 nM at pH 7.6 to 373 nM at pH 6.8, H+ inhibition was not overcome by up to 1 mM-external glycine. Inhibiting concentrations of H+ appropriate for AMPA and kainate receptors did not markedly alter the EC50 values determined for (S)-AMPA (3.4 microM) and kainate (114 microM) at pH 7.2. 4. Treatment of neurones with N-ethylmaleimide, iodoacetic acid, dithiothretiol or diethyl pyrocarbonate did not influence proton inhibition of NMDA receptor responses. However, treatment with diethyl pyrocarbonate, which potentiated aspartate responses, appeared to reduce the effectiveness of Zn2+ inhibition of NMDA receptors. 5. Desensitization of whole-cell NMDA and (S)-AMPA currents was studied with ionophoretic application of agonist to the cell soma. Whole-cell aspartate currents desensitized rapidly, irrespective of the

  19. The ALIAmide palmitoylethanolamide and cannabinoids, but not anandamide, are protective in a delayed postglutamate paradigm of excitotoxic death in cerebellar granule neurons.

    PubMed Central

    Skaper, S D; Buriani, A; Dal Toso, R; Petrelli, L; Romanello, S; Facci, L; Leon, A

    1996-01-01

    The amino acid L-glutamate is a neurotransmitter that mediates fast neuronal excitation in a majority of synapses in the central nervous system. Glutamate stimulates both N-methyl-D-aspartate (NMDA) and non-NMDA receptors. While activation of NMDA receptors has been implicated in a variety of neurophysiologic processes, excessive NMDA receptor stimulation (excitotoxicity) is thought to be primarily responsible for neuronal injury in a wide variety of acute neurological disorders including hypoxia-ischemia, seizures, and trauma. Very little is known about endogenous molecules and mechanisms capable of modulating excitotoxic neuronal death. Saturated N-acylethanolamides like palmitoylethanolamide accumulate in ischemic tissues and are synthesized by neurons upon excitatory amino acid receptor activation. Here we report that palmitoylethanolamide, but not the cognate N-acylamide anandamide (the ethanolamide of arachidonic acid), protects cultured mouse cerebellar granule cells against glutamate toxicity in a delayed postagonist paradigm. Palmitoylethanolamide reduced this injury in a concentration-dependent manner and was maximally effective when added 15-min postglutamate. Cannabinoids, which like palmitoylethanolamide are functionally active at the peripheral cannabinoid receptor CB2 on mast cells, also prevented neuron loss in this delayed postglutamate model. Furthermore, the neuroprotective effects of palmitoylethanolamide, as well as that of the active cannabinoids, were efficiently antagonized by the candidate central cannabinoid receptor (CB1) agonist anandamide. Analogous pharmacological behaviors have been observed for palmitoylethanolamide (ALI-Amides) in downmodulating mast cell activation. Cerebellar granule cells expressed mRNA for CB1 and CB2 by in situ hybridization, while two cannabinoid binding sites were detected in cerebellar membranes. The results suggest that (i) non-CB1 cannabinoid receptors control, upon agonist binding, the downstream

  20. Inverse Stochastic Resonance in Cerebellar Purkinje Cells

    PubMed Central

    Häusser, Michael; Gutkin, Boris S.; Roth, Arnd

    2016-01-01

    Purkinje neurons play an important role in cerebellar computation since their axons are the only projection from the cerebellar cortex to deeper cerebellar structures. They have complex internal dynamics, which allow them to fire spontaneously, display bistability, and also to be involved in network phenomena such as high frequency oscillations and travelling waves. Purkinje cells exhibit type II excitability, which can be revealed by a discontinuity in their f-I curves. We show that this excitability mechanism allows Purkinje cells to be efficiently inhibited by noise of a particular variance, a phenomenon known as inverse stochastic resonance (ISR). While ISR has been described in theoretical models of single neurons, here we provide the first experimental evidence for this effect. We find that an adaptive exponential integrate-and-fire model fitted to the basic Purkinje cell characteristics using a modified dynamic IV method displays ISR and bistability between the resting state and a repetitive activity limit cycle. ISR allows the Purkinje cell to operate in different functional regimes: the all-or-none toggle or the linear filter mode, depending on the variance of the synaptic input. We propose that synaptic noise allows Purkinje cells to quickly switch between these functional regimes. Using mutual information analysis, we demonstrate that ISR can lead to a locally optimal information transfer between the input and output spike train of the Purkinje cell. These results provide the first experimental evidence for ISR and suggest a functional role for ISR in cerebellar information processing. PMID:27541958

  1. Inverse Stochastic Resonance in Cerebellar Purkinje Cells.

    PubMed

    Buchin, Anatoly; Rieubland, Sarah; Häusser, Michael; Gutkin, Boris S; Roth, Arnd

    2016-08-01

    Purkinje neurons play an important role in cerebellar computation since their axons are the only projection from the cerebellar cortex to deeper cerebellar structures. They have complex internal dynamics, which allow them to fire spontaneously, display bistability, and also to be involved in network phenomena such as high frequency oscillations and travelling waves. Purkinje cells exhibit type II excitability, which can be revealed by a discontinuity in their f-I curves. We show that this excitability mechanism allows Purkinje cells to be efficiently inhibited by noise of a particular variance, a phenomenon known as inverse stochastic resonance (ISR). While ISR has been described in theoretical models of single neurons, here we provide the first experimental evidence for this effect. We find that an adaptive exponential integrate-and-fire model fitted to the basic Purkinje cell characteristics using a modified dynamic IV method displays ISR and bistability between the resting state and a repetitive activity limit cycle. ISR allows the Purkinje cell to operate in different functional regimes: the all-or-none toggle or the linear filter mode, depending on the variance of the synaptic input. We propose that synaptic noise allows Purkinje cells to quickly switch between these functional regimes. Using mutual information analysis, we demonstrate that ISR can lead to a locally optimal information transfer between the input and output spike train of the Purkinje cell. These results provide the first experimental evidence for ISR and suggest a functional role for ISR in cerebellar information processing. PMID:27541958

  2. Mast cell secretory granules: armed for battle.

    PubMed

    Wernersson, Sara; Pejler, Gunnar

    2014-07-01

    Mast cells are important effector cells of the immune system and recent studies show that they have immunomodulatory roles in diverse processes in both health and disease. Mast cells are distinguished by their high content of electron-dense secretory granules, which are filled with large amounts of preformed and pre-activated immunomodulatory compounds. When appropriately activated, mast cells undergo degranulation, a process by which these preformed granule compounds are rapidly released into the surroundings. In many cases, the effects that mast cells have on an immune response are closely associated with the biological actions of the granule compounds that they release, as exemplified by the recent studies showing that mast cell granule proteases account for many of the protective and detrimental effects of mast cells in various inflammatory settings. In this Review, we discuss the current knowledge of mast cell secretory granules. PMID:24903914

  3. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    SciTech Connect

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia are motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.

  4. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    DOE PAGESBeta

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; Howell, Danielle; Kerekes, Ryan A.; Solecki, David J.

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

  5. Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

    PubMed Central

    Holubowska, Anna; Mukherjee, Chaitali; Vadhvani, Mayur; Stegmüller, Judith

    2014-01-01

    Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration. PMID:24686379

  6. Glucose deprivation stimulates Cu(2+) toxicity in cultured cerebellar granule neurons and Cu(2+)-dependent zinc release.

    PubMed

    Isaev, Nickolay K; Genrikhs, Elisaveta E; Aleksandrova, Olga P; Zelenova, Elena A; Stelmashook, Elena V

    2016-05-27

    Copper chloride (0.01mM, 2h) did not have significant influence on the survival of cerebellar granule neurons (CGNs) incubated in balanced salt solution. However, CuCl2 caused severe neuronal damage by glucose deprivation (GD). The glutamate NMDA-receptors blocker MK-801 partially and antioxidant N-acetyl-l-cysteine (NAC) or Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) almost entirely protected CGNs from this toxic effect. Measurements of intracellular calcium ions using Fluo-4 AM, or zinc ions with FluoZin-3 AM demonstrated that 1 h-exposure to GD induced intensive increase of Fluo-4 but not FluoZin-3 fluorescence in neurons. The supplementation of solution with CuCl2 caused an increase of FluoZin-3, Fluo-4 and CellROX Green (reactive oxygen species probe) fluorescence by GD. The stimulation of Fluo-4 but not FluoZin-3 fluorescence by copper could be prevented partially by MK-801 and as well as CellROX Green fluorescence by NAC at GD. This data imply that during GD copper ions induce intense displacement zinc ions from intracellular stores, in addition free radical production, glutamate release and Ca(2+) overload of CGNs, that causes death of neurons as a result. PMID:27063646

  7. Paired-pulse facilitation of multivesicular release and intersynaptic spillover of glutamate at rat cerebellar granule cell–interneurone synapses

    PubMed Central

    Satake, Shin’Ichiro; Inoue, Tsuyoshi; Imoto, Keiji

    2012-01-01

    A simple form of presynaptic plasticity, paired-pulse facilitation (PPF), has been explained as a transient increase in the probability of vesicular release. Using the whole-cell patch-clamp technique to record synaptic activity in rat cerebellar slices, we found different forms of presynaptically originated short-term plasticity during glutamatergic excitatory neurotransmission from granule cells (GCs) to molecular-layer interneurones (INs). Paired-pulse activation of GC axons at short intervals (30–100 ms) elicited not only a facilitation in the peak amplitude (PPFamp), but also a prolongation in the decay-time constant (PPPdecay) of the EPSCs recorded from INs. The results of pharmacological tests and kinetics analyses suggest that the mechanisms underlying the respective types of short-term plasticity were different. PPFamp was elicited by a transient increase in the number of released vesicles. On the other hand, PPPdecay was caused not only by delayed release as has been reported but also by extrasynaptic spillover of the GC transmitter and the subsequent intersynaptic pooling. Both PPFamp and PPPdecay closely rely on repetitive-activation-induced multivesicular release. Using a dynamic clamp technique, we further examined the physiological significance of different presynaptic plasticity, and found that PPFamp and PPPdecay can differentially encode and process neuronal information by influencing the total synaptic charge transferred to postsynaptic INs to reflect activation frequency of the presynaptic GCs. PMID:22930264

  8. Sigma-1 Receptor Enhances Neurite Elongation of Cerebellar Granule Neurons via TrkB Signaling

    PubMed Central

    Kimura, Yuriko; Fujita, Yuki; Shibata, Kumi; Mori, Megumi; Yamashita, Toshihide

    2013-01-01

    Sigma-1 receptor (Sig-1R) is an integral membrane protein predominantly expressed in the endoplasmic reticulum. Sig-1R demonstrates a high affinity to various synthetic compounds including well-known psychotherapeutic drugs in the central nervous system (CNS). For that, it is considered as an alternative target for psychotherapeutic drugs. On the cellular level, when Sig-1R is activated, it is known to play a role in neuroprotection and neurite elongation. These effects are suggested to be mediated by its ligand-operated molecular chaperone activity, and/or upregulation of various Ca2+ signaling. In addition, recent studies show that Sig-1R activation induces neurite outgrowth via neurotrophin signaling. Here, we tested the hypothesis that Sig-1R activation promotes neurite elongation through activation of tropomyosin receptor kinase (Trk), a family of neurotrophin receptors. We found that 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate (PRE-084), a selective Sig-1R agonist, significantly promoted neurite outgrowth, and K252a, a Trk inhibitor, attenuated Sig-1R-mediated neurite elongation in cerebellar granule neurons (CGNs). Moreover, we revealed that Sig-1R interacts with TrkB, and PRE-084 treatment enhances phosphorylation of Y515, but not Y706. Thus, our results indicate that Sig-1R activation promotes neurite outgrowth in CGNs through Y515 phosphorylation of TrkB. PMID:24116072

  9. Peroxynitrite is Involved in the Apoptotic Death of Cultured Cerebellar Granule Neurons Induced by Staurosporine, but not by Potassium Deprivation.

    PubMed

    Olguín-Albuerne, Mauricio; Ramos-Pittol, José Miguel; Coyoy, Angélica; Martínez-Briseño, Carlos Patricio; Domínguez, Guadalupe; Morán, Julio

    2016-02-01

    Nitric oxide (NO) regulates numerous physiological process and is the main source of reactive nitrogen species (RNS). NO promotes cell survival, but it also induces apoptotic death having been involved in the pathogenesis of several neurodegenerative diseases. NO and superoxide anion react to form peroxynitrite, which accounts for most of the deleterious effects of NO. The mechanisms by which these molecules regulate the apoptotic process are not well understood. In this study, we evaluated the role of NO and peroxynitrite in the apoptotic death of cultured cerebellar granule neurons (CGN), which are known to experience apoptosis by staurosporine (St) or potassium deprivation (K5). We found that CGN treated with the peroxynitrite catalyst, FeTTPs were completely rescued from St-induced death, but not from K5-induced death. On the other hand, the inhibition of the inducible nitric oxide synthase partially protected cell viability in CGN treated with K5, but not with St, while the inhibitor L-NAME further reduced the cell viability in St, but it did not affect K5. Finally, an inhibitor of the soluble guanylate cyclase (sGC) diminished the cell viability in K5, but not in St. Altogether, these results shows that NO promotes cell survival in K5 through sGC-cGMP and promotes cell death by other mechanisms, while in St NO promotes cell survival independently of cGMP and peroxynitrite results critical for St-induced death. Our results suggest that RNS are differentially handled by CGN during cell death depending on the death-inducing conditions. PMID:26700430

  10. The cerebellar Golgi cell and spatiotemporal organization of granular layer activity

    PubMed Central

    D'Angelo, Egidio; Solinas, Sergio; Mapelli, Jonathan; Gandolfi, Daniela; Mapelli, Lisa; Prestori, Francesca

    2013-01-01

    The cerebellar granular layer has been suggested to perform a complex spatiotemporal reconfiguration of incoming mossy fiber signals. Central to this role is the inhibitory action exerted by Golgi cells over granule cells: Golgi cells inhibit granule cells through both feedforward and feedback inhibitory loops and generate a broad lateral inhibition that extends beyond the afferent synaptic field. This characteristic connectivity has recently been investigated in great detail and been correlated with specific functional properties of these neurons. These include theta-frequency pacemaking, network entrainment into coherent oscillations and phase resetting. Important advances have also been made in terms of determining the membrane and synaptic properties of the neuron, and clarifying the mechanisms of activation by input bursts. Moreover, voltage sensitive dye imaging and multi-electrode array (MEA) recordings, combined with mathematical simulations based on realistic computational models, have improved our understanding of the impact of Golgi cell activity on granular layer circuit computations. These investigations have highlighted the critical role of Golgi cells in: generating dense clusters of granule cell activity organized in center-surround structures, implementing combinatorial operations on multiple mossy fiber inputs, regulating transmission gain, and cut-off frequency, controlling spike timing and burst transmission, and determining the sign, intensity and duration of long-term synaptic plasticity at the mossy fiber-granule cell relay. This review considers recent advances in the field, highlighting the functional implications of Golgi cells for granular layer network computation and indicating new challenges for cerebellar research. PMID:23730271

  11. Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons.

    PubMed

    Stella, Roberto; Cifani, Paolo; Peggion, Caterina; Hansson, Karin; Lazzari, Cristian; Bendz, Maria; Levander, Fredrik; Sorgato, Maria Catia; Bertoli, Alessandro; James, Peter

    2012-02-01

    Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes. PMID:22023170

  12. The Small GTPases RhoA and Rac1 Regulate Cerebellar Development by Controlling Cell Morphogenesis, Migration and Foliation

    PubMed Central

    Mulherkar, Shalaka; Uddin, Mohammad Danish; Couvillon, Anthony D.; Sillitoe, Roy V.; Tolias, Kimberley F.

    2014-01-01

    The small GTPases RhoA and Rac1 are key cytoskeletal regulators that function in a mutually antagonistic manner to control the migration and morphogenesis of a broad range of cell types. However, their role in shaping the cerebellum, a unique brain structure composed of an elaborate set of folia separated by fissures of different lengths, remains largely unexplored. Here we show that dysregulation of both RhoA and Rac1 signaling results in abnormal cerebellar ontogenesis. Ablation of RhoA from neuroprogenitor cells drastically alters the timing and placement of fissure formation, the migration and positioning of granule and Purkinje cells, the alignment of Bergmann glia, and the integrity of the basement membrane, primarily in the anterior lobules. Furthermore, in the absence of RhoA, granule cell precursors located at the base of fissures fail to undergo cell shape changes required for fissure initiation. Many of these abnormalities can be recapitulated by deleting RhoA specifically from granule cell precursors but not postnatal glia, indicating that RhoA functions in granule cell precursors to control cerebellar morphogenesis. Notably, mice with elevated Rac1 activity due to loss of the Rac1 inhibitors Bcr and Abr show similar anterior cerebellar deficits, including ectopic neurons and defects in fissure formation, Bergmann glia organization and basement membrane integrity. Together, our results suggest that RhoA and Rac1 play indispensable roles in patterning cerebellar morphology. PMID:25128586

  13. Murine cerebellar neurons express a novel gene encoding a protein related to cell cycle control and cell fate determination proteins.

    PubMed

    Taoka, M; Isobe, T; Okuyama, T; Watanabe, M; Kondo, H; Yamakawa, Y; Ozawa, F; Hishinuma, F; Kubota, M; Minegishi, A

    1994-04-01

    We cloned cDNAs of a novel protein (designated V-1) that has been identified from among the developmentally regulated proteins in the rat cerebellum. Protein sequencing analysis (Taoka, M., Yamakuni, T., Song, S.-Y., Yamakawa, Y., Seta, K., Okuyama, T., and Isobe, T. (1992) Eur. J. Biochem. 207, 615-620) and cDNA sequence analysis revealed that the V-1 protein consists of 117 amino acids and contains 2.5 contiguous repeats of the cdc10/SWI6 motif, which was originally found in the products of the cell cycle control genes of yeasts and the cell fate determination genes in Drosophila and Caenorhabditis elegans. In situ hybridization histochemistry revealed that the expression of the V-1 gene is transiently increased in postmigratory granule cells during postnatal rat cerebellar development and thereafter is markedly suppressed, whereas Purkinje cells constitutively express V-1 mRNA. In contrast, cerebellar granule cells of the staggerer mutant mouse continue to express the V-1 gene even when the granule cells of the normal mouse have ceased to express the V-1 gene, suggesting that the expression of the V-1 gene in granule cells is regulated through the interaction with Purkinje cells. On the basis of these results, we postulate that the V-1 protein has a potential role in the differentiation of granule cells. PMID:8144589

  14. Calpain plays a central role in 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in cerebellar granule neurons.

    PubMed

    Harbison, Richard A; Ryan, Kristen R; Wilkins, Heather M; Schroeder, Emily K; Loucks, F Alexandra; Bouchard, Ron J; Linseman, Daniel A

    2011-04-01

    1-Methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity has previously been attributed to either caspase-dependent apoptosis or caspase-independent cell death. In the current study, we found that MPP(+) induces a unique, non-apoptotic nuclear morphology coupled with a caspase-independent but calpain-dependent mechanism of cell death in primary cultures of rat cerebellar granule neurons (CGNs). Using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay in CGNs exposed to MPP(+), we observed that these neurons are essentially devoid of caspase-dependent DNA fragments indicative of apoptosis. Moreover, proteolysis of a well recognized caspase-3 substrate, poly (ADP ribose) polymerase (PARP), was not observed in CGNs exposed to MPP(+). In contrast, calpain-dependent proteolysis of fodrin and pro-caspases-9 and -3 occurred in this model coupled with inhibition of caspase-3/-7 activities. Notably, several key members of the Bcl-2 protein family appear to be prominent calpain targets in MPP(+)-treated CGNs. Bid and Bax were proteolyzed to truncated forms thought to have greater pro-death activity at mitochondria. Moreover, the pro-survival Bcl-2 protein was degraded to a form predicted to be inactive at mitochondria. Cyclin E was also cleaved by calpain to an active low MW fragment capable of facilitating cell cycle re-entry. Finally, MPP(+)-induced neurotoxicity in CGNs was significantly attenuated by a cocktail of calpain and caspase inhibitors in combination with the antioxidant glutathione. Collectively, these results demonstrate that caspases do not play a central role in CGN toxicity induced by exposure to MPP(+), whereas calpain cleavage of key protein targets, coupled with oxidative stress, plays a critical role in MPP(+)-induced neurotoxicity. Our findings underscore the complexity of MPP(+)-induced neurotoxicity and suggest that calpain may play a fundamental role in causing neuronal death downstream of mitochondrial oxidative stress

  15. DS-03SONIC HEDGEHOG ANTAGONISTS POTENTLY INDUCE APOPTOSIS IN THE CEREBELLAR EXTERNAL GRANULE LAYER: IMPLICATIONS FOR MEDULLOBLASTOMA TREATMENT

    PubMed Central

    Noguchi, Kevin; Cabrera, Omar; Swiney, Brant; Smith, Julie; Farber, Nuri

    2014-01-01

    There is a great interest in Hedgehog signaling both for its role in cerebellar development and medulloblastoma (MB) treatment. The cerebellum maintains its own proliferative layer called the external granule layer (EGL) that produces over 90% of its neurons. During development, the established dogma views Hedgehog signaling as a robust mitogenic stimulator of EGL proliferation. However, in other regions of the body, Hedgehog stimulation acts as a survival signal by potently inducing NPC apoptosis when signaling is lost. In this manner, the sonic hedgehog ligand's concentration gradient determines NPC survival or death thereby morphologically sculpting the developing nervous system. Therefore, we tested whether Hedgehog signaling also acts as a survival signal in the EGL by administering several Hedgehog antagonists (vismodegib, cyclopamine, and jervine). Remarkably, we found all Hedgehog antagonists (HAs) potently induced EGL apoptosis within a few hours of administration. This suggests a large portion of the HAs' anti-proliferative effects are due to the apoptotic loss of a large number of EGL NPCs. This research may also have important implications for MB formation and treatment. There is convincing evidence that EGL neural progenitor cells (NPCs) can be the tumor initiating cells for MBs (the most common malignant brain tumor in children). Therefore, we examined if HAs can also produce apoptosis in Patched mice which exhibit constitutive Hedgehog stimulation and are prone to MB formation. We found HA administration also potently increased apoptosis in both EGL NPCs and preneoplasms. This may have important implications for the treatment of MBs with HAs. For example, apoptosis involves signaling mechanisms distinct from proliferation that may need to be disabled for malignant transformation. In addition, the requirement for Hedgehog signaling may prevent metastasis by killing tumor cells as they spread to regions where such signaling is absent.

  16. Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

    PubMed Central

    Giordano, G.; Klintworth, H.M.; Kavanagh, T.J.; Costa, L.G.

    2008-01-01

    In mouse cerebellar granule neurons (CGN) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (−/−) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase, phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs. PMID:18164102

  17. GDF-15 enhances intracellular Ca2+ by increasing Cav1.3 expression in rat cerebellar granule neurons

    PubMed Central

    Lu, Jun-Mei; Wang, Chang-Ying; Hu, Changlong; Fang, Yan-Jia; Mei, Yan-Ai

    2016-01-01

    GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-β superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K+ currents and Kv2.1 α subunit expression through TβRII (TGF-β receptor II) to activate Src kinase and Akt/mTOR (mammalian target of rapamycin) signalling in rat CGNs (cerebellar granule neurons). In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca2+ concentration ([Ca2+]i) in response to membrane depolarization, as determined by Ca2+ imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca2+ current (ICa) without altering steady-state activation of Ca2+ channels. Treatment with nifedipine, an inhibitor of L-type Ca2+ channels, abrogated GDF-15-induced increases in [Ca2+]i and ICa. The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK (extracellular-signal-regulated kinase) pathways and by pharmacological inhibition of Src-mediated TβRII phosphorylation. Given that Cav1.3 is not only a channel for Ca2+ influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TβRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons. PMID:27114559

  18. Role of glutamate receptors in tetrabrominated diphenyl ether (BDE-47) neurotoxicity in mouse cerebellar granule neurons.

    PubMed

    Costa, Lucio G; Tagliaferri, Sara; Roqué, Pamela J; Pellacani, Claudia

    2016-01-22

    The polybrominated diphenyl ether (PBDE) flame retardants are developmental neurotoxicants, as evidenced by numerous in vitro, animal and human studies. PBDEs can alter the homeostasis of thyroid hormone and directly interact with brain cells. Induction of oxidative stress, leading to DNA damage and apoptotic cell death is a prominent mechanism of PBDE neurotoxicity, though other mechanisms have also been suggested. In the present study we investigated the potential role played by glutamate receptors in the in vitro neurotoxicity of the tetrabromodiphenyl ether BDE-47, one of the most abundant PBDE congeners. Toxicity of BDE-47 in mouse cerebellar neurons was diminished by antagonists of glutamate ionotropic receptors, but not by antagonists of glutamate metabotropic receptors. Antagonists of NMDA and AMPA/Kainate receptors also inhibited BDE-47-induced oxidative stress and increases in intracellular calcium. The calcium chelator BAPTA-AM also inhibited BDE-47 cytotoxicity and oxidative stress. BDE-47 caused a rapid increase of extracellular glutamate levels, which was not antagonized by any of the compounds tested. The results suggest that BDE-47, by still unknown mechanisms, increases extracellular glutamate which in turn activates ionotropic glutamate receptors leading to increased calcium levels, oxidative stress, and ultimately cell death. PMID:26640238

  19. Vaccine adjuvants: Tailor-made mast-cell granules

    NASA Astrophysics Data System (ADS)

    Gunzer, Matthias

    2012-03-01

    Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.

  20. Efficient differentiation of human embryonic stem cells into functional cerebellar-like cells.

    PubMed

    Erceg, Slaven; Ronaghi, Mohammad; Zipancic, Ivan; Lainez, Sergio; Roselló, Mireia Gárcia; Xiong, Chen; Moreno-Manzano, Victoria; Rodríguez-Jiménez, Fernando Javier; Planells, Rosa; Alvarez-Dolado, Manuel; Bhattacharya, Shom Shanker; Stojkovic, Miodrag

    2010-11-01

    The cerebellum has critical roles in motor and sensory learning and motor coordination. Many cerebellum-related disorders indicate cell therapy as a possible treatment of neural loss. Here we show that application of inductive signals involved in early patterning of the cerebellar region followed by application of different factors directs human embryonic stem cell differentiation into cerebellar-like cells such as granule neurons, Purkinje cells, interneuron, and glial cells. Neurons derived using our protocol showed a T-shaped polarity phenotype and express similar markers to the developed human cerebellum. Electrophysiological measurements confirmed functional electrical properties compatible with these cells. In vivo implantation of differentiated human embryonic stem cells transfected with MATH1-GFP construct into neonatal mice resulted in cell migration across the molecular and the Purkinje cell layers and settlement in the internal molecular layers. Our findings demonstrate that the universal mechanisms involved in the development of cerebellum can be efficiently recapitulated in vitro, which enables the design of new strategies for cell replacement therapy, to study early human development and pathogenesis of neurodegenerative diseases. PMID:20521974

  1. Repeated intermittent alcohol exposure during the third trimester-equivalent increases expression of the GABAA receptor δ subunit in cerebellar granule neurons and delays motor development in rats

    PubMed Central

    Diaz, Marvin R.; Vollmer, Cyndel C.; Zamudio-Bulcock, Paula A.; Vollmer, William; Blomquist, Samantha; Morton, Russell A.; Everett, Julie C.; Zurek, Agnieszka A.; Yu, Jieying; Orser, Beverley A.; Valenzuela, C. Fernando

    2014-01-01

    Exposure to ethanol (EtOH) during fetal development can lead to long-lasting alterations, including deficits in fine motor skills and motor learning. Studies suggest that these are, in part, a consequence of cerebellar damage. Cerebellar granule neurons (CGNs) are the gateway of information into the cerebellar cortex. Functionally, CGNs are heavily regulated by phasic and tonic GABAergic inhibition from Golgi cell interneurons; however, the effect of EtOH exposure on the development of GABAergic transmission in immature CGNs has not been investigated. To model EtOH exposure during the 3rd trimester-equivalent of human pregnancy, neonatal pups were exposed intermittently to high levels of vaporized EtOH from postnatal day (P) 2 to P12. This exposure gradually increased pup serum EtOH concentrations (SECs) to ~60 mM (~0.28 g/dl) during the 4 hours of exposure. EtOH levels gradually decreased to baseline 8 hrs after the end of exposure. Surprisingly, basal tonic and phasic GABAergic currents in CGNs were not significantly affected by postnatal alcohol exposure (PAE). However, PAE increased the expression of δ subunit expression at P28 as detected by immunohistochemical and western blot analyses. Also, electrophysiological studies with an agonist that is highly selective for δ-containing GABAA receptors, 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol (THIP), showed an increase in THIP-induced tonic current. Behavioral studies of PAE rats did not reveal any deficits in motor coordination, except for a delay in the acquisition of the mid-air righting reflex that was apparent at P15 to P18. These findings demonstrate that repeated intermittent exposure to high levels of EtOH during the equivalent of the last trimester of human pregnancy has significant but relatively subtle effects on motor coordination and GABAergic transmission in CGNs in rats. PMID:24316160

  2. The compartmental restriction of cerebellar interneurons

    PubMed Central

    Consalez, G. Giacomo; Hawkes, Richard

    2013-01-01

    The Purkinje cells (PC's) of the cerebellar cortex are subdivided into multiple different molecular phenotypes that form an elaborate array of parasagittal stripes. This array serves as a scaffold around which afferent topography is organized. The ways in which cerebellar interneurons may be restricted by this scaffolding are less well-understood. This review begins with a brief survey of cerebellar topography. Next, it reviews the development of stripes in the cerebellum with a particular emphasis on the embryological origins of cerebellar interneurons. These data serve as a foundation to discuss the hypothesis that cerebellar compartment boundaries also restrict cerebellar interneurons, both excitatory [granule cells, unipolar brush cells (UBCs)] and inhibitory (e.g., Golgi cells, basket cells). Finally, it is proposed that the same PC scaffold that restricts afferent terminal fields to stripes may also act to organize cerebellar interneurons. PMID:23346049

  3. Changes in mitogen-activated protein kinase in cerebellar granule neurons by polybrominated diphenyl ethers and polychlorinated biphenyls

    SciTech Connect

    Fan Chunyang; Besas, Jonathan

    2010-05-15

    Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants and have been detected in human blood, adipose tissue, and breast milk. Both in vitro and in vivo studies have shown that the effects of PBDEs are similar to the known human developmental neurotoxicants such as polychlorinated biphenyls (PCBs) on a molar basis. Previously, we reported that PBDE mixtures and congeners, perturbed calcium homeostasis which is critical for the development and function of the nervous system. In the present study, we tested whether environmentally relevant PBDE/PCB mixtures and congeners affected mitogen-activated protein kinase (MAPK) pathways, which are down-stream events of calcium signaling in cerebellar granule neuronal cultures. In this study, phosphorylated extracellular signal-regulated kinase (pERK)1/2, a widely studied MAPK cascade and known to be involved in learning and memory, levels were quantitated using western blot technique with phospho-specific antibodies. Glutamate (a positive control) increased pERK1/2 in a time- and concentration-dependent manner reaching maximum activation at 5-30 min of exposure and at doses >= 10 muM. Both Aroclor 1254 (a commercial penta PCB mixture) and DE-71 (a commercial penta PBDE mixture) elevated phospho-ERK1/2, producing maximum stimulation at 30 min and at concentrations >= 3 mug/ml; Aroclor 1254 was more efficacious than DE-71. DE-79 (an octabrominated diphenyl ether mixture) also elevated phospho-ERK1/2, but to a lesser extent than that of DE-71. PBDE congeners 47, 77, 99, and 153 also increased phospo-ERK1/2 in a concentration-dependent manner. The data indicated that PBDE congeners are more potent than the commercial mixtures. PCB 47 also increased phospho-ERK1/2 like its structural analog PBDE 47, but to a lesser extent, suggesting that these chemicals affect similar pathways. Cytotoxicity, measured as %LDH release, data showed that higher concentrations (> 30 muM) and longer exposures (> 30 min) are

  4. Alcohol Excites Cerebellar Golgi Cells by Inhibiting the Na+/K+ ATPase

    PubMed Central

    Botta, Paolo; de Souza, Fabio M Simões; Sangrey, Thomas; De Schutter, Erik; Valenzuela, C Fernando

    2010-01-01

    Alcohol-induced alterations of cerebellar function cause motor coordination impairments that are responsible for millions of injuries and deaths worldwide. Cognitive deficits associated with alcoholism are also a consequence of cerebellar dysfunction. The mechanisms responsible for these effects of ethanol are poorly understood. Recent studies have identified neurons in the input layer of the cerebellar cortex as important ethanol targets. In this layer, granule cells (GrCs) receive the majority of sensory inputs to the cerebellum through the mossy fibers. Information flow at these neurons is gated by a specialized pacemaker interneuron known as the Golgi cell, which provides divergent GABAergic input to thousands of GrCs. In vivo electrophysiological experiments have previously shown that acute ethanol exposure abolishes GrC responsiveness to sensory inputs carried by mossy fibers. Slice electrophysiological studies suggest that ethanol causes this effect by potentiating GABAergic transmission at Golgi cell-to-GrC synapses through an increase in Golgi cell excitability. Using patch-clamp electrophysiological techniques in cerebellar slices and computer modeling, we show here that ethanol excites Golgi cells by inhibiting the Na+/K+ ATPase. Voltage-clamp recordings of Na+/K+ ATPase currents indicated that ethanol partially inhibits this pump and this effect could be mimicked by low concentrations of ouabain. Partial inhibition of Na+/K+ ATPase function in a computer model of the Golgi cell reproduced these experimental findings. These results establish a novel mechanism of action of ethanol on neuronal excitability, which likely has a role in ethanol-induced cerebellar dysfunction and may also contribute to neuronal functional alterations in other brain regions. PMID:20520600

  5. PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure

    PubMed Central

    LaSarge, Candi L.; Santos, Victor R; Danzer, Steve C.

    2015-01-01

    Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offset the reduction in boutons per axon length. These morphological changes predicts a net increase in granule cell >> CA3 innervation. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell >> CA3 communication. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. PMID:25600212

  6. Role of glycogen in processes of cerebellar glial cells under conditions of its damage with sodium nitrite.

    PubMed

    Samosudova, N V; Reutov, V P; Larionova, N P

    2010-12-01

    Ultrastructure of processes of glial cell, astrocytes of the molecular layer of cerebellar cortex in Rana temporaria frog, under conditions of damage to the cerebellum caused by NO-generating compound sodium nitrite was studied under an electron microscope. It was found that astrocytes have at least two types of processes: the first (fibrillar) primarily contained numerous fibrils and few glycogen granules and the second (granular) primarily containing glycogen granules. In the presence of NO-generating compound in toxic doses, fibrillar processes are damaged or completely degrade more rapidly than granular ones. The processes containing glycogen can protect both damaged synapses and individual synaptic buttons by forming a compact structure, wrapping, around them. We analyzed the possible role of glycogen of cerebellar glial cell processes in neuroglial interactions in the presence of sodium nitrite. PMID:21240384

  7. Single granule cells excite Golgi cells and evoke feedback inhibition in the cochlear nucleus.

    PubMed

    Yaeger, Daniel B; Trussell, Laurence O

    2015-03-18

    In cerebellum-like circuits, synapses from thousands of granule cells converge onto principal cells. This fact, combined with theoretical considerations, has led to the concept that granule cells encode afferent input as a population and that spiking in individual granule cells is relatively unimportant. However, granule cells also provide excitatory input to Golgi cells, each of which provide inhibition to hundreds of granule cells. We investigated whether spiking in individual granule cells could recruit Golgi cells and thereby trigger widespread inhibition in slices of mouse cochlear nucleus. Using paired whole-cell patch-clamp recordings, trains of action potentials at 100 Hz in single granule cells was sufficient to evoke spikes in Golgi cells in ∼40% of paired granule-to-Golgi cell recordings. High-frequency spiking in single granule cells evoked IPSCs in ∼5% of neighboring granule cells, indicating that bursts of activity in single granule cells can recruit feedback inhibition from Golgi cells. Moreover, IPSPs mediated by single Golgi cell action potentials paused granule cell firing, suggesting that inhibitory events recruited by activity in single granule cells were able to control granule cell firing. These results suggest a previously unappreciated relationship between population coding and bursting in single granule cells by which spiking in a small number of granule cells may have an impact on the activity of a much larger number of granule cells. PMID:25788690

  8. Transplantation and Stem Cell Therapy for Cerebellar Degenerations.

    PubMed

    Cendelin, Jan

    2016-02-01

    Stem cell-based and regenerative therapy may become a hopeful treatment for neurodegenerative diseases including hereditary cerebellar degenerations. Neurotransplantation therapy mainly aims to substitute lost cells, but potential effects might include various mechanisms including nonspecific trophic effects and stimulation of endogenous regenerative processes and neural plasticity. Nevertheless, currently, there remain serious limitations. There is a wide spectrum of human hereditary cerebellar degenerations as well as numerous cerebellar mutant mouse strains that serve as models for the development of effective therapy. By now, transplantation has been shown to ameliorate cerebellar function, e.g. in Purkinje cell degeneration mice, Lurcher mutant mice and mouse models of spinocerebellar ataxia type 1 and type 2 and Niemann-Pick disease type C. Despite the lack of direct comparative studies, it appears that there might be differences in graft development and functioning between various types of cerebellar degeneration. Investigation of the relation of graft development to specific morphological, microvascular or biochemical features of the diseased host tissue in various cerebellar degenerations may help to identify factors determining the fate of grafted cells and potential of their functional integration. PMID:26155762

  9. Cell death and neurodegeneration in the postnatal development of cerebellar vermis in normal and Reeler mice.

    PubMed

    Castagna, Claudia; Merighi, Adalberto; Lossi, Laura

    2016-09-01

    Programmed cell death (PCD) was demonstrated in neurons and glia in normal brain development, plasticity, and aging, but also in neurodegeneration. (Macro)autophagy, characterized by cytoplasmic vacuolization and activation of lysosomal hydrolases, and apoptosis, typically entailing cell shrinkage, chromatin and nuclear condensation, are the two more common forms of PCD. Their underlying intracellular pathways are partly shared and neurons can die following both modalities, according to the type of death-triggering stimulus. Reelin is an extracellular protein necessary for proper neuronal migration and brain lamination. In the mutant Reeler mouse, its absence causes neuronal mispositioning, with a notable degree of cerebellar hypoplasia that was tentatively related to an increase in PCD. We have carried out an ultrastructural analysis on the occurrence and type of postnatal PCD affecting the cerebellar neurons in normal and Reeler mice. In the forming cerebellar cortex, PCD took the form of apoptosis or autophagy and mainly affected the cerebellar granule cells (CGCs). Densities of apoptotic CGCs were comparable in both mouse strains at P0-P10, while, in mutants, they increased to become significantly higher at P15. In WT mice the density of autophagic neurons did not display statistically significant differences in the time interval examined in this study, whereas it was reduced in Reeler in the P0-P10 interval, but increased at P15. Besides CGCs, the Purkinje neurons also displayed autophagic features in both WT and Reeler mice. Therefore, cerebellar neurons undergo different types of PCD and a Reelin deficiency affects the type and degree of neuronal death during postnatal development of the cerebellum. PMID:26931496

  10. Ultra-rapid axon-axon ephaptic inhibition of cerebellar Purkinje cells by the pinceau.

    PubMed

    Blot, Antonin; Barbour, Boris

    2014-02-01

    Excitatory synaptic activity in the brain is shaped and balanced by inhibition. Because inhibition cannot propagate, it is often recruited with a synaptic delay by incoming excitation. Cerebellar Purkinje cells are driven by long-range excitatory parallel fiber inputs, which also recruit local inhibitory basket cells. The axon initial segment of each Purkinje cell is ensheathed by basket cell axons in a structure called the pinceau, which is largely devoid of chemical synapses. In mice, we found at the single-cell level that the pinceau mediates ephaptic inhibition of Purkinje cell firing at the site of spike initiation. The reduction of firing rate was synchronous with the presynaptic action potential, eliminating a synaptic delay and allowing granule cells to inhibit Purkinje cells without a preceding phase of excitation. Axon-axon ephaptic intercellular signaling can therefore mediate near-instantaneous feedforward and lateral inhibition. PMID:24413696

  11. Role of granule-cell transmission in memory trace of cerebellum-dependent optokinetic motor learning.

    PubMed

    Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

    2014-04-01

    Adaptation of the optokinetic response (OKR) is an eye movement enhanced by repeated motion of a surrounding visual field and represents a prototype of cerebellum-dependent motor learning. Purkinje cells and vestibular nuclei (VN) receive optokinetic and retinal slip signals via the mossy fiber-granule cell pathway and climbing-fiber projections, respectively. To explore the neural circuits and mechanisms responsible for OKR adaptation, we adopted the reversible neurotransmission-blocking (RNB) technique, in which granule-cell transmission to Purkinje cells was selectively and reversibly blocked by doxycycline-dependent expression of transmission-blocking tetanus toxin in granule cells. Blockade of granule-cell inputs abolished both short-term and long-term OKR adaptation induced by repeated OKR training, but normal levels of both responses were immediately evoked in the pretrained RNB mice by OKR retraining once granule-cell transmission had recovered. Importantly, eye movement elicited by electrical stimulation of the cerebellar focculus was elevated by long-term but not by short-term OKR training in adaptive OKR-negative RNB mice. Furthermore, when the flocculus of adaptive OKR-negative RNB mice was electrically excited in-phase with OKR stimulation, these mice exhibited long-term adaptive OKR. These results indicate that convergent information to the VN was critical for acquisition and storage of long-term OKR adaptation with conjunctive action of Purkinje cells for OKR expression. Interestingly, in contrast to conditioned eyeblink memory, the expression of once acquired adaptive long-term OKR was not abrogated by blockade of granule-cell transmission, suggesting that distinct forms of neural plasticity would operate in different forms of cerebellum-dependent motor learning. PMID:24706878

  12. Rapid Signaling Actions of Environmental Estrogens in Developing Granule Cell Neurons Are Mediated by Estrogen Receptor β

    PubMed Central

    Le, Hoa H.; Belcher, Scott M.

    2010-01-01

    Estrogenic endocrine disrupting chemicals (EDCs) constitute a diverse group of man-made chemicals and natural compounds derived from plants and microbial metabolism. Estrogen-like actions are mediated via the nuclear hormone receptor activity of estrogen receptor (ER)α and ERβ and rapid regulation of intracellular signaling cascades. Previous study defined cerebellar granule cell neurons as estrogen responsive and that granule cell precursor viability was developmentally sensitive to estrogens. In this study experiments using Western blot analysis and pharmacological approaches have characterized the receptor and signaling modes of action of selective and nonselective estrogen ligands in developing cerebellar granule cells. Estrogen treatments were found to briefly increase ERK1/2-phosphorylation and then cause prolonged depression of ERK1/2 activity. The sensitivity of granule cell precursors to estrogen-induced cell death was found to require the integrated activation of membrane and intracellular ER signaling pathways. The sensitivity of granule cells to selective and nonselective ER agonists and a variety of estrogenic and nonestrogenic EDCs was also examined. The ERβ selective agonist DPN, but not the ERα selective agonist 4,4′,4′-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol or other ERα-specific ligands, stimulated cell death. Only EDCs with selective or nonselective ERβ activities like daidzein, equol, diethylstilbestrol, and bisphenol A were observed to induce E2-like neurotoxicity supporting the conclusion that estrogen sensitivity in granule cells is mediated via ERβ. The presented results also demonstrate the utility of estrogen sensitive developing granule cells as an in vitro assay for elucidating rapid estrogen-signaling mechanisms and to detect EDCs that act at ERβ to rapidly regulate intracellular signaling. PMID:20926581

  13. Robustness effect of gap junctions between Golgi cells on cerebellar cortex oscillations

    PubMed Central

    2011-01-01

    Background Previous one-dimensional network modeling of the cerebellar granular layer has been successfully linked with a range of cerebellar cortex oscillations observed in vivo. However, the recent discovery of gap junctions between Golgi cells (GoCs), which may cause oscillations by themselves, has raised the question of how gap-junction coupling affects GoC and granular-layer oscillations. To investigate this question, we developed a novel two-dimensional computational model of the GoC-granule cell (GC) circuit with and without gap junctions between GoCs. Results Isolated GoCs coupled by gap junctions had a strong tendency to generate spontaneous oscillations without affecting their mean firing frequencies in response to distributed mossy fiber input. Conversely, when GoCs were synaptically connected in the granular layer, gap junctions increased the power of the oscillations, but the oscillations were primarily driven by the synaptic feedback loop between GoCs and GCs, and the gap junctions did not change oscillation frequency or the mean firing rate of either GoCs or GCs. Conclusion Our modeling results suggest that gap junctions between GoCs increase the robustness of cerebellar cortex oscillations that are primarily driven by the feedback loop between GoCs and GCs. The robustness effect of gap junctions on synaptically driven oscillations observed in our model may be a general mechanism, also present in other regions of the brain. PMID:22330240

  14. Cerebellar Zonal Patterning Relies on Purkinje Cell Neurotransmission

    PubMed Central

    White, Joshua J.; Arancillo, Marife; Stay, Trace L.; George-Jones, Nicholas A.; Levy, Sabrina L.; Heck, Detlef H.

    2014-01-01

    Cerebellar circuits are patterned into an array of topographic parasagittal domains called zones. The proper connectivity of zones is critical for motor coordination and motor learning, and in several neurological diseases cerebellar circuits degenerate in zonal patterns. Despite recent advances in understanding zone function, we still have a limited understanding of how zones are formed. Here, we focused our attention on Purkinje cells to gain a better understanding of their specific role in establishing zonal circuits. We used conditional mouse genetics to test the hypothesis that Purkinje cell neurotransmission is essential for refining prefunctional developmental zones into sharp functional zones. Our results show that inhibitory synaptic transmission in Purkinje cells is necessary for the precise patterning of Purkinje cell zones and the topographic targeting of mossy fiber afferents. As expected, blocking Purkinje cell neurotransmission caused ataxia. Using in vivo electrophysiology, we demonstrate that loss of Purkinje cell communication altered the firing rate and pattern of their target cerebellar nuclear neurons. Analysis of Purkinje cell complex spike firing revealed that feedback in the cerebellar nuclei to inferior olive to Purkinje cell loop is obstructed. Loss of Purkinje neurotransmission also caused ectopic zonal expression of tyrosine hydroxylase, which is only expressed in adult Purkinje cells when calcium is dysregulated and if excitability is altered. Our results suggest that Purkinje cell inhibitory neurotransmission establishes the functional circuitry of the cerebellum by patterning the molecular zones, fine-tuning afferent circuitry, and shaping neuronal activity. PMID:24920627

  15. Motor learning of mice lacking cerebellar Purkinje cells.

    PubMed

    Porras-García, M Elena; Ruiz, Rocío; Pérez-Villegas, Eva M; Armengol, José Á

    2013-01-01

    The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input-output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum. PMID:23630472

  16. Motor learning of mice lacking cerebellar Purkinje cells

    PubMed Central

    Porras-García, M. Elena; Ruiz, Rocío; Pérez-Villegas, Eva M.; Armengol, José Á.

    2013-01-01

    The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question (cerebellar lesions, pharmacological agonist and/or antagonist of cerebellar neurotransmitters, virus tract tracings, etc.). One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer (i.e., the Purkinje cell). In this review, we discuss the results obtained in our laboratory in motor learning of both Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum. PMID:23630472

  17. Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

    PubMed Central

    Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

    2008-01-01

    Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547

  18. Developmental features of rat cerebellar neural cells cultured in a chemically defined medium

    SciTech Connect

    Gallo, V.; Ciotti, M.T.; Aloisi, F.; Levi, G.

    1986-01-01

    We studied some aspects of the differentiation of rat cerebellar neural cells obtained from 8-day postnatal animals and cultured in a serum-free, chemically defined medium (CDM). The ability of the cells to take up radioactive transmitter amino acids was analyzed autoradiographically. The L-glutamate analogue /sup 3/H-D-aspartate was taken up by astroglial cells, but not by granule neurons, even in late cultures (20 days in vitro). This is in agreement with the lack of depolarization-induced release of /sup 3/H-D-aspartate previously observed in this type of culture. In contrast, /sup 3/H-(GABA) was scarcely accumulated by glial-fibrillary-acidic-protein (GFAP)-positive astrocytes, but taken up by glutamate-decarboxylase-positive inhibitory interneurons and was released in a Ca2+-dependent way upon depolarization: /sup 3/H-GABA evoked release progressively increased with time in culture. Interestingly, the expression of the vesicle-associated protein synapsin I was much reduced in granule cells cultured in CDM as compared to those maintained in the presence of serum. These data would indicate that in CDM the differentiation of granule neurons is not complete, while that of GABAergic neurons is not greatly affected. Whether the diminished differentiation of granule cells must be attributed only to serum deprivation or also to other differences in the composition of the culture medium remains to be established. /sup 3/H-GABA was avidly taken up also by a population of cells which were not recognized by antibodies raised against GFAP, glutamate decarboxylase, and microtubule-associated protein 2. These cells have been characterized as bipotential precursors of oligodendrocytes and of a subpopulation of astrocytes bearing a stellate shape and capable of high-affinity /sup 3/H-GABA uptake.

  19. Ectopic cerebellar cell migration causes maldevelopment of Purkinje cells and abnormal motor behaviour in Cxcr4 null mice.

    PubMed

    Huang, Guo-Jen; Edwards, Andrew; Tsai, Cheng-Yu; Lee, Yi-Shin; Peng, Lei; Era, Takumi; Hirabayashi, Yoshio; Tsai, Ching-Yen; Nishikawa, Shin-Ichi; Iwakura, Yoichiro; Chen, Shu-Jen; Flint, Jonathan

    2014-01-01

    SDF-1/CXCR4 signalling plays an important role in neuronal cell migration and brain development. However, the impact of CXCR4 deficiency in the postnatal mouse brain is still poorly understood. Here, we demonstrate the importance of CXCR4 on cerebellar development and motor behaviour by conditional inactivation of Cxcr4 in the central nervous system. We found CXCR4 plays a key role in cerebellar development. Its loss leads to defects in Purkinje cell dentritogenesis and axonal projection in vivo but not in cell culture. Transcriptome analysis revealed the most significantly affected pathways in the Cxcr4 deficient developing cerebellum are involved in extra cellular matrix receptor interactions and focal adhesion. Consistent with functional impairment of the cerebellum, Cxcr4 knockout mice have poor coordination and balance performance in skilled motor tests. Together, these results suggest ectopic the migration of granule cells impairs development of Purkinje cells, causes gross cerebellar anatomical disruption and leads to behavioural motor defects in Cxcr4 null mice. PMID:24516532

  20. Ultrastructural similarity between bat and human mast cell secretory granules.

    PubMed

    Oliani, S M; Vugman, I; Jamur, M C

    1993-01-01

    Mast cells in the tongue of the bat (Artibeus lituratus) show a well-developed Golgi area and abundant mitochondria in the granule-free perinuclear cytoplasm. Rough endoplasmic reticulum profiles, free ribosomes, mitochondria, bundles of filaments and a great number of secretory granules are found throughout the remaining cytoplasm. The granules, of various shapes and sizes, are simple containing an electron-dense, homogeneous matrix, coarse particles or cylindrical scrolls, or combinations (cylindrical scrolls with either electron-dense, homogeneous matrix or coarse particle contents). Up to now, scroll-containing granules have been considered to be a unique feature of human mast cells. PMID:8453310

  1. Chlorpyrifos Toxicity in Mouse Cultured Cerebellar Granule Neurons at Different Stages of Development: Additive Effect on Glutamate-Induced Excitotoxicity

    PubMed Central

    Amani, Nahid; Soodi, Maliheh; Daraei, Bahram; Dashti, Abolfazl

    2016-01-01

    Objective Chlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide. Its mechanism of action includes oxidative stress, excitotoxicity, and inhibition of the acetylcholinesterase enzyme (AChE). The aim of the present study is to investigate CPF toxicity in mature and immature cerebellar granule neurons (CGNs), as well as its effect on glutamate induced excitotoxicity. Materials and Methods This study was an in vitro experimental study performed on mice cultured CGNs. Immature and mature neurons were exposed to different concentrations of CPF (1-1000 µM) and glutamate (10-600 µM) for 48 hours after which we used the MTT assay to measure cytotoxicity. Immature neurons had exposure to CPF for 5 days in order to evaluate the cytotoxic effect on developing neurons. Mature neurons received sub-lethal concentrations of CPF (10, 100 µM) combined with different concentrations of glutamate. AChE activity and reactive oxygen species (ROS) generation were assessed after treatments. Results Immature CGNs had increased sensitivity to CPF toxicity compared to mature neurons. We observed significantly greater ROS production in immature compared to mature neurons, however AChE activity was more inhibited in mature neurons. Although CPF toxicity was not well correlated with AChE inhibition, it correlated well with ROS production. Glutamate toxicity was potentiated by sub-lethal concentration of CPF, however glutamate induced ROS production was not affected. The results suggested that CPF potentiated glutamate toxicity by mechanisms other than oxidative stress. Conclusion CPF toxicity differed in mature and immature neurons. Potentiated glutamate toxicity by CPF implied that CPF exposure might be a risk factor for neurodegenerative disease. PMID:27602329

  2. N-terminal cleavage of the mitochondrial fusion GTPase OPA1 occurs via a caspase-independent mechanism in cerebellar granule neurons exposed to oxidative or nitrosative stress

    PubMed Central

    Gray, Josie J.; Zommer, Amelia E.; Bouchard, Ron J.; Duval, Nathan; Blackstone, Craig; Linseman, Daniel A.

    2013-01-01

    Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. Here, we demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from the N-terminal cleavage of OPA1 observed in CGNs undergoing caspase-mediated apoptosis (Loucks et al., 2009) and results in removal of a key lysine residue (K301) within the GTPase domain. OPA1 cleavage in CGNs occurs coincident with extensive mitochondrial fragmentation, disruption of the microtubule network, and cell death. In contrast to OPA1 cleavage induced in CGNs by removing depolarizing extracellular potassium (5K apoptotic conditions), oxidative or nitrosative stress-induced OPA1 cleavage caused by complex I inhibition or nitric oxide, respectively, is caspase-independent. N-terminal cleavage of OPA1 is also observed in vivo in aged rat and mouse midbrain and hippocampal tissues. We conclude that N-terminal cleavage and subsequent inactivation of OPA1 may be a contributing factor in the neuronal cell death processes underlying neurodegenerative diseases, particularly those associated with aging. Furthermore, these data suggest that OPA1 cleavage is a likely convergence point for mitochondrial dysfunction and imbalances in mitochondrial fission and fusion induced by oxidative or nitrosative stress. PMID:23220553

  3. N-terminal cleavage of the mitochondrial fusion GTPase OPA1 occurs via a caspase-independent mechanism in cerebellar granule neurons exposed to oxidative or nitrosative stress.

    PubMed

    Gray, Josie J; Zommer, Amelia E; Bouchard, Ron J; Duval, Nathan; Blackstone, Craig; Linseman, Daniel A

    2013-02-01

    Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. Here, we demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from the N-terminal cleavage of OPA1 observed in CGNs undergoing caspase-mediated apoptosis (Loucks et al., 2009) and results in removal of a key lysine residue (K301) within the GTPase domain. OPA1 cleavage in CGNs occurs coincident with extensive mitochondrial fragmentation, disruption of the microtubule network, and cell death. In contrast to OPA1 cleavage induced in CGNs by removing depolarizing extracellular potassium (5K apoptotic conditions), oxidative or nitrosative stress-induced OPA1 cleavage caused by complex I inhibition or nitric oxide, respectively, is caspase-independent. N-terminal cleavage of OPA1 is also observed in vivo in aged rat and mouse midbrain and hippocampal tissues. We conclude that N-terminal cleavage and subsequent inactivation of OPA1 may be a contributing factor in the neuronal cell death processes underlying neurodegenerative diseases, particularly those associated with aging. Furthermore, these data suggest that OPA1 cleavage is a likely convergence point for mitochondrial dysfunction and imbalances in mitochondrial fission and fusion induced by oxidative or nitrosative stress. PMID:23220553

  4. Proteoglycans support proper granule formation in pancreatic acinar cells.

    PubMed

    Aroso, Miguel; Agricola, Brigitte; Hacker, Christian; Schrader, Michael

    2015-10-01

    Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas. PMID:26105026

  5. Mapping the development of cerebellar Purkinje cells in zebrafish.

    PubMed

    Hamling, Kyla R; Tobias, Zachary J C; Weissman, Tamily A

    2015-11-01

    The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. PMID:25655100

  6. Mitigation of cerebellar neuropathy in globoid cell leukodystrophy mice by AAV-mediated gene therapy.

    PubMed

    Lin, Dar-Shong; Hsiao, Chung-Der; Lee, Allan Yueh-Luen; Ho, Che-Sheng; Liu, Hsuan-Liang; Wang, Tuen-Jen; Jian, Yuan-Ren; Hsu, Jui-Cheng; Huang, Zon-Darr; Lee, Tsung-Han; Chiang, Ming-Fu

    2015-10-15

    Globoid cell leukodystrophy (GLD) is an autosomal recessive, lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). The absence of GALC activity leads to the accumulation of the toxic substance psychosine and the preferential loss of myelinating cells in the central and peripheral nervous systems. Profound demyelination, astrogliosis and axonopathy are the hallmarks of the pathogenesis of GLD, and cerebellar ataxia is one of the dominant manifestations in adolescents and adults affected with GLD. To date, studies regarding cerebellar degeneration in GLD are limited. In this study, the efficacy of cerebellum-targeted gene therapy on the cerebellar neuropathology in twitcher mice (a murine model of GLD) has been validated. We observed degeneration of Purkinje cells, Bergmann glia, and granule cells in addition to astrocytosis and demyelination in the cerebellum of the twitcher mice. Ultrastructural analysis revealed dark cell degeneration and disintegration of the cellular composition of Purkinje cells in untreated twitcher mice. In addition, the expressions of neurotrophic factors CNTF, GDNF and IGF-I were up-regulated and the expression of BDNF was down-regulated. Intracerebellar-mediated gene therapy efficiently corrected enzymatic deficiency by direct transduction to Purkinje cells and cross-correction in other cell types in the cerebellum, leading to the amelioration of both neuroinflammation and demyelination. The population, dendritic territory, and axonal processes of Purkinje cells remained normal in the cerebellum of treated twitcher mice, where radial fibers of Bergmann glia spanned the molecular layer and collateral branches ensheathed the dendritic processes of Purkinje cells. Moreover, the aberrant expressions of neurotrophic factors were mitigated in the cerebellum of treated twitcher mice, indicating the preservation of cellular function in addition to maintaining the neuronal architecture. The life span of the

  7. Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons

    PubMed Central

    Reddy, C E; Albanito, L; De Marco, P; Aiello, D; Maggiolini, M; Napoli, A; Musti, A M

    2013-01-01

    Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a

  8. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control.

    PubMed

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections). PMID:25983678

  9. A realistic bi-hemispheric model of the cerebellum uncovers the purpose of the abundant granule cells during motor control

    PubMed Central

    Pinzon-Morales, Ruben-Dario; Hirata, Yutaka

    2015-01-01

    The cerebellar granule cells (GCs) have been proposed to perform lossless, adaptive spatio-temporal coding of incoming sensory/motor information required by downstream cerebellar circuits to support motor learning, motor coordination, and cognition. Here we use a physio-anatomically inspired bi-hemispheric cerebellar neuronal network (biCNN) to selectively enable/disable the output of GCs and evaluate the behavioral and neural consequences during three different control scenarios. The control scenarios are a simple direct current motor (1 degree of freedom: DOF), an unstable two-wheel balancing robot (2 DOFs), and a simulation model of a quadcopter (6 DOFs). Results showed that adequate control was maintained with a relatively small number of GCs (< 200) in all the control scenarios. However, the minimum number of GCs required to successfully govern each control plant increased with their complexity (i.e., DOFs). It was also shown that increasing the number of GCs resulted in higher robustness against changes in the initialization parameters of the biCNN model (i.e., synaptic connections and synaptic weights). Therefore, we suggest that the abundant GCs in the cerebellar cortex provide the computational power during the large repertoire of motor activities and motor plants the cerebellum is involved with, and bring robustness against changes in the cerebellar microcircuit (e.g., neuronal connections). PMID:25983678

  10. Sonic hedgehog patterning during cerebellar development.

    PubMed

    De Luca, Annarita; Cerrato, Valentina; Fucà, Elisa; Parmigiani, Elena; Buffo, Annalisa; Leto, Ketty

    2016-01-01

    The morphogenic factor sonic hedgehog (Shh) actively orchestrates many aspects of cerebellar development and maturation. During embryogenesis, Shh signaling is active in the ventricular germinal zone (VZ) and represents an essential signal for proliferation of VZ-derived progenitors. Later, Shh secreted by Purkinje cells sustains the amplification of postnatal neurogenic niches: the external granular layer and the prospective white matter, where excitatory granule cells and inhibitory interneurons are produced, respectively. Moreover, Shh signaling affects Bergmann glial differentiation and promotes cerebellar foliation during development. Here we review the most relevant functions of Shh during cerebellar ontogenesis, underlying its role in physiological and pathological conditions. PMID:26499980

  11. Curcumin Pretreatment Induces Nrf2 and an Antioxidant Response and Prevents Hemin-Induced Toxicity in Primary Cultures of Cerebellar Granule Neurons of Rats

    PubMed Central

    González-Reyes, Susana; Guzmán-Beltrán, Silvia; Medina-Campos, Omar Noel; Pedraza-Chaverri, José

    2013-01-01

    Curcumin is a bifunctional antioxidant derived from Curcuma longa. This study identifies curcumin as a neuroprotectant against hemin-induced damage in primary cultures of cerebellar granule neurons (CGNs) of rats. Hemin, the oxidized form of heme, is a highly reactive compound that induces cellular injury. Pretreatment of CGNs with 5–30 μM curcumin effectively increased by 2.3–4.9 fold heme oxygenase-1 (HO-1) expression and by 5.6–14.3-fold glutathione (GSH) levels. Moreover, 15 μM curcumin attenuated by 55% the increase in reactive oxygen species (ROS) production, by 94% the reduction of GSH/glutathione disulfide (GSSG) ratio, and by 49% the cell death induced by hemin. The inhibition of heme oxygenase system or GSH synthesis with tin mesoporphyrin and buthionine sulfoximine, respectively, suppressed the protective effect of curcumin against hemin-induced toxicity. These data strongly suggest that HO-1 and GSH play a major role in the protective effect of curcumin. Furthermore, it was found that 24 h of incubation with curcumin increases by 1.4-, 2.3-, and 5.2-fold the activity of glutathione reductase, glutathione S-transferase and superoxide dismutase, respectively. Additionally, it was found that curcumin was capable of inducing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) translocation into the nucleus. These data suggest that the pretreatment with curcumin induces Nrf2 and an antioxidant response that may play an important role in the protective effect of this antioxidant against hemin-induced neuronal death. PMID:24454990

  12. UNUSUAL EOSINOPHILIC GRANULE CELL PROLIFERATION IN COHO SALMON (ONCHORHYNCHUS KISUTCH)

    EPA Science Inventory

    Proliferative lesions comprised of eosinophilic granule cells (EGCs) extended throughout the gastrointestinal tract of several mature, spawning coho salmon Oncorhynchus kisutch (Walbaum). istological examination of the tumour showed extensive proliferation and infiltration of EGC...

  13. Human NK cell lytic granules and regulation of their exocytosis

    PubMed Central

    Krzewski, Konrad; Coligan, John E.

    2012-01-01

    Natural killer (NK) cells form a subset of lymphocytes that play a key role in immuno-surveillance and host defense against cancer and viral infections. They recognize stressed cells through a variety of germline-encoded activating cell surface receptors and utilize their cytotoxic ability to eliminate abnormal cells. Killing of target cells is a complex, multi-stage process that concludes in the directed secretion of lytic granules, containing perforin and granzymes, at the immunological synapse. Upon delivery to a target cell, perforin mediates generation of pores in membranes of target cells, allowing granzymes to access target cell cytoplasm and induce apoptosis. Therefore, lytic granules of NK cells are indispensable for normal NK cell cytolytic function. Indeed, defects in lytic granule secretion lead or are related to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis (FHL) type 2–5 or Griscelli syndrome type 2. A number of reports highlight the role of several proteins involved in lytic granule release and NK cell-mediated killing of tumor cells. This review focuses on lytic granules of human NK cells and the advancements in understanding the mechanisms controlling their exocytosis. PMID:23162553

  14. Regulation of Tlx3 by Pax6 is required for the restricted expression of Chrnα3 in Cerebellar Granule Neuron progenitors during development

    PubMed Central

    Divya, Thulasi Sheela; Lalitha, Soundararajan; Parvathy, Surendran; Subashini, Chandramohan; Sanalkumar, Rajendran; Dhanesh, Sivadasan Bindu; Rasheed, Vazhanthodi Abdul; Divya, Mundackal Sivaraman; Tole, Shubha; James, Jackson

    2016-01-01

    Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6−/−Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD). PMID:27452274

  15. Purkinje cell apoptosis in arabian horses with cerebellar abiotrophy.

    PubMed

    Blanco, A; Moyano, R; Vivo, J; Flores-Acuña, R; Molina, A; Blanco, C; Monterde, J G

    2006-08-01

    Purkinje cerebellar cells were studied in three Arabian horses aged between 6 and 8 months with clinical disorders in their movements, tremors and ataxia; the occurrence of apoptosis in this cell population was investigated by the (terminal deoxynucleotidyl transferase biotin-dUTP nick-end labelling (TUNEL) method. Both optical and electron microscopical images showed a scant number of Purkinje cells, most of them with morphological features of apoptosis such as condensation of the nucleus and cytoplasm as well as segregation and fragmentation of the nucleus into apoptotic bodies. The TUNEL technique revealed a substantial number (65%) of positive immunoreactive Purkinje cells. PMID:16901270

  16. Young Dentate Granule Cells Mediate Pattern Separation whereas Old Granule Cells Contribute to Pattern Completion

    PubMed Central

    Nakashiba, Toshiaki; Cushman, Jesse D.; Pelkey, Kenneth A.; Renaudineau, Sophie; Buhl, Derek L.; McHugh, Thomas J.; Barrera, Vanessa Rodriguez; Chittajallu, Ramesh; Iwamoto, Keisuke S.; McBain, Chris J.; Fanselow, Michael S.; Tonegawa, Susumu

    2012-01-01

    Summary Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks following functional integration into the neuronal network (young GCs), distinguishing them from less active older adult-born GCs and the major population of dentate GCs generated developmentally (together, old GCs). We created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts that was reduced following removal of young GCs by X-ray irradiation. Furthermore, mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation of similar contexts requires adult-born young GCs while old GCs are unnecessary, whereas older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion. PMID:22365813

  17. 2', 3'-cyclic nucleotide 3'-phosphodiesterase is expressed in dissociated rat cerebellar cells and included in the postsynaptic density fraction.

    PubMed

    Cho, Sun-Jung; Jung, Jae Seob; Jin, IngNyol; Moon, Il Soo

    2003-08-31

    We have shown by protein sequencing that the phosphotyrosine-containing 48 kDa protein band of the rat cerebellar postsynaptic density fraction (CBL-PSD) is 2', 3'-cyclic nucleotide 3'-phosphodiesterase 2 (CNP2). Immunoblot analysis indicated that both CNP1 and CNP2 isoforms are present in the CBL-PSD fraction, whereas there is little CNP2 in the forebrain (FB)-PSD fraction. Both isoforms in the CBL-PSD fraction were tyrosine-phosphorylated to a basal extent. They were efficiently dissociated from the complexes in the PSD fraction by salt, but not by non-ionic detergents such as n-octyl glucoside (OG) and Triton X-100. Immunocytochemistry of dissociated cerebellar cultures revealed patchy CNP staining in oligodendrocytes (OLs), Purkinje cells (PCs), and unidentified PSD95-positive cells, but no staining in granule cells (GCs). Our results indicate that both CNP1 and CNP2 are expressed in cerian populations of cerebellar cells in addition to OL, and that they are associated with complexes that are co-isolated with the PSD. PMID:14503857

  18. Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons

    PubMed Central

    Amarillo, Yimy; De Santiago-Castillo, Jose A; Dougherty, Kevin; Maffie, Jonathon; Kwon, Elaine; Covarrubias, Manuel; Rudy, Bernardo

    2008-01-01

    Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (ISA) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the ISA in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K+ currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native ISA-mediating channels. PMID:18276729

  19. Characterization of Mast Cell Secretory Granules and Their Cell Biology

    PubMed Central

    Azouz, Nurit Pereg; Hammel, Ilan

    2014-01-01

    Exocytosis and secretion of secretory granule (SG) contained inflammatory mediators is the primary mechanism by which mast cells exert their protective immune responses in host defense, as well as their pathological functions in allergic reactions and anaphylaxis. Despite their central role in mast cell function, the molecular mechanisms underlying the biogenesis and secretion of mast cell SGs remain largely unresolved. Early studies have established the lysosomal nature of the mast cell SGs and implicated SG homotypic fusion as an important step occurring during both their biogenesis and compound secretion. However, the molecular mechanisms that account for key features of this process largely remain to be defined. A novel high-resolution imaging based methodology allowed us to screen Rab GTPases for their phenotypic and functional impact and identify Rab networks that regulate mast cell secretion. This screen has identified Rab5 as a novel regulator of homotypic fusion of the mast cell SGs that thereby regulates their size and cargo composition. PMID:24988214

  20. Primary cerebellar extramedullary myeloid cell tumor mimicking oligodendroglioma.

    PubMed

    Ho, D M; Wong, T T; Guo, W Y; Chang, K P; Yen, S H

    1997-10-01

    Extramedullary myeloid cell tumors (EMCTs) are tumors consisting of immature cells of the myeloid series that occur outside the bone marrow. Most of them are associated with acute myelogenous leukemia or other myeloproliferative disorders, and a small number occur as primary lesions, i.e., are not associated with hematological disorders. Occurrence inside the cranium is rare, and there has been only one case of primary EMCT involving the cerebellum reported in the literature. The case we report here is a blastic EMCT occurring in the cerebellum of a 3-year-old boy who had no signs of leukemia or any hematological disorder throughout the entire course. The cerebellar tumor was at first misdiagnosed as an "oligodendroglioma" because of the uniformity and "fried egg" artifact of the tumor cells. The tumor disappeared during chemotherapy consisting of 12 treatments. However, it recurred and metastasized to the cerebrospinal fluid (CSF) shortly after the therapy was completed. A diagnosis of EMCT was suspected because of the presence of immature myeloid cells in the CSF, and was confirmed by anti-myeloperoxidase and anti-lysozyme immunoreactivity of the cerebellar tumor. The patient succumbed 1 year and 3 months after the first presentation of the disease. PMID:9341943

  1. Differential effects of polybrominated diphenyl ethers and polychlorinated biphenyls on [3H]arachidonic acid release in rat cerebellar granule neurons.

    PubMed

    Kodavanti, Prasada Rao S; Derr-Yellin, Ethel C

    2002-08-01

    Polybrominated diphenyl ethers (PBDEs), which are widely used as flame-retardants, have been increasing in environmental and human tissue samples during the past 20-30 years, while other structurally related, persistent organic pollutants such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (on a TEQ basis), have decreased. PBDEs have been detected in human blood, adipose tissue, and breast milk, and developmental and long-term exposure to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that PCBs, which cause neurotoxic effects, including changes in learning and memory, stimulated the release of [(3)H]arachidonic acid ([(3)H]AA) by a cPLA(2)/iPLA(2)-dependent mechanism. PLA(2)(phospholipase A(2)) activity has been associated with learning and memory, and AA has been identified as a second messenger involved in synaptic plasticity. The objective of the present study was to test whether PBDE mixtures (DE-71 and DE-79), like other organohalogen mixtures, have a similar action on [(3)H]AA release in an in vitro neuronal culture model. Cerebellar granule cells at 7 days in culture were labeled with [(3)H]AA for 16-20 h and then exposed in vitro to PBDEs. DE-71, a mostly pentabromodiphenyl ether mixture, significantly stimulated [(3)H]AA release at concentrations as low as 10 microg/ml, while DE-79, a mostly octabromodiphenyl ether mixture, did not stimulate [(3)H]AA release, even at 50 microg/ml. The release of [(3)H]AA by DE-71 is time-dependent, and a significant increase was seen after only 5-10 min of exposure. The removal and chelation of calcium from the exposure buffer, using 0.3 mM EGTA, significantly attenuated the DE-71-stimulated [(3)H]AA release; however, only an 18% inhibition of the release was demonstrated for the calcium replete conditions at 30 microg/ml DE-71. Methyl arachidonylfluorophosphonate (5 microM), an inhibitor of cPLA(2)/iPLA(2), completely attenuated the DE-71

  2. Purkinje Cell Activity in the Cerebellar Anterior Lobe after Rabbit Eyeblink Conditioning

    ERIC Educational Resources Information Center

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

    The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus.…

  3. Memory trace and timing mechanism localized to cerebellar Purkinje cells.

    PubMed

    Johansson, Fredrik; Jirenhed, Dan-Anders; Rasmussen, Anders; Zucca, Riccardo; Hesslow, Germund

    2014-10-14

    The standard view of the mechanisms underlying learning is that they involve strengthening or weakening synaptic connections. Learned response timing is thought to combine such plasticity with temporally patterned inputs to the neuron. We show here that a cerebellar Purkinje cell in a ferret can learn to respond to a specific input with a temporal pattern of activity consisting of temporally specific increases and decreases in firing over hundreds of milliseconds without a temporally patterned input. Training Purkinje cells with direct stimulation of immediate afferents, the parallel fibers, and pharmacological blocking of interneurons shows that the timing mechanism is intrinsic to the cell itself. Purkinje cells can learn to respond not only with increased or decreased firing but also with an adaptively timed activity pattern. PMID:25267641

  4. Development and specification of cerebellar stem and progenitor cells in zebrafish: from embryo to adult

    PubMed Central

    2013-01-01

    Background Teleost fish display widespread post-embryonic neurogenesis originating from many different proliferative niches that are distributed along the brain axis. During the development of the central nervous system (CNS) different cell types are produced in a strict temporal order from increasingly committed progenitors. However, it is not known whether diverse neural stem and progenitor cell types with restricted potential or stem cells with broad potential are maintained in the teleost fish brain. Results To study the diversity and output of neural stem and progenitor cell populations in the zebrafish brain the cerebellum was used as a model brain region, because of its well-known architecture and development. Transgenic zebrafish lines, in vivo imaging and molecular markers were used to follow and quantify how the proliferative activity and output of cerebellar progenitor populations progress. This analysis revealed that the proliferative activity and progenitor marker expression declines in juvenile zebrafish before they reach sexual maturity. Furthermore, this correlated with the diminished repertoire of cell types produced in the adult. The stem and progenitor cells derived from the upper rhombic lip were maintained into adulthood and they actively produced granule cells. Ventricular zone derived progenitor cells were largely quiescent in the adult cerebellum and produced a very limited number of glia and inhibitory inter-neurons. No Purkinje or Eurydendroid cells were produced in fish older than 3 months. This suggests that cerebellar cell types are produced in a strict temporal order from distinct pools of increasingly committed stem and progenitor cells. Conclusions Our results in the zebrafish cerebellum show that neural stem and progenitor cell types are specified and they produce distinct cell lineages and sub-types of brain cells. We propose that only specific subtypes of brain cells are continuously produced throughout life in the teleost fish

  5. Optogenetic Manipulation of Cerebellar Purkinje Cell Activity In Vivo

    PubMed Central

    Tsubota, Tadashi; Ohashi, Yohei; Tamura, Keita; Sato, Ayana; Miyashita, Yasushi

    2011-01-01

    Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex. Although their anatomical connections and physiological response properties have been extensively studied, the causal role of their activity in behavioral, cognitive and autonomic functions is still unclear because PC activity cannot be selectively controlled. Here we developed a novel technique using optogenetics for selective and rapidly reversible manipulation of PC activity in vivo. We injected into rat cerebellar cortex lentiviruses expressing either the light-activated cationic channel channelrhodopsin-2 (ChR2) or light-driven chloride pump halorhodopsin (eNpHR) under the control of the PC-specific L7 promoter. Transgene expression was observed in most PCs (ChR2, 92.6%; eNpHR, 95.3%), as determined by immunohistochemical analysis. In vivo electrophysiological recordings showed that all light-responsive PCs in ChR2-transduced rats increased frequency of simple spike in response to blue laser illumination. Similarly, most light-responsive PCs (93.8%) in eNpHR-transduced rats decreased frequency of simple spike in response to orange laser illumination. We then applied these techniques to characterize the roles of rat cerebellar uvula, one of the cardiovascular regulatory regions in the cerebellum, in resting blood pressure (BP) regulation in anesthetized rats. ChR2-mediated photostimulation and eNpHR-mediated photoinhibition of the uvula had opposite effects on resting BP, inducing depressor and pressor responses, respectively. In contrast, manipulation of PC activity within the neighboring lobule VIII had no effect on BP. Blue and orange laser illumination onto PBS-injected lobule IX didn't affect BP, indicating the observed effects on BP were actually due to PC activation and inhibition. These results clearly demonstrate that the optogenetic method we developed here will provide a powerful way to elucidate a causal relationship between local PC activity and functions of the cerebellum

  6. Distribution and phenotypes of unipolar brush cells in relation to the granule cell system of the rat cochlear nuclear nucleus

    PubMed Central

    Diño, Maria. R.; Mugnaini, Enrico

    2009-01-01

    In most mammals the cochlear nuclear complex (CN) contains a distributed system of granule cells (GCS), whose parallel fiber axons innervate the dorsal cochlear nucleus (DCN). Like their counterpart in cerebellum, CN granules are innervated by mossy fibers of various origins. The GCS is complemented by unipolar brush (UBCs) and Golgi cells, and by stellate and cartwheel cells of the DCN. This cerebellum-like microcircuit modulates the activity of the DCN’s main projection neurons, the pyramidal, giant and tuberculoventral neurons, and is thought to improve auditory performance by integrating acoustic and proprioceptive information. In this paper, we focus on the UBCs, a chemically heterogeneous neuronal population, using antibodies to calretinin, mGluR1α epidermal growth factor substrate 8 (Eps8) and the transcription factor Tbr2. Eps8 and Tbr2 labeled most of the CN’s UBCs, if not the entire population, while calretinin and mGluR1α distinguished two largely separate subsets with overlapping distributions. By double labeling with antibodies to Tbr2 and the α6 GABAA-receptor subunit, we found that UBCs populate all regions of the GCS and occur at remarkably high densities in the DCN and subpeduncular corner, but rarely in the lamina. Although GCS subregions likely share the same microcircuitry, their dissimilar UBC densities suggest they may be functionally distinct. UBCs and granules are also present in regions previously not included in the GCS, namely the rostrodorsal magnocellular portions of VCN, vestibular nerve root, trapezoid body, spinal tract and sensory and principal nuclei of the trigeminal nerve, and cerebellar peduncles. The UBC’s dendritic brush receives AMPA- and NMDA-mediated input from an individual mossy fiber, favoring singularity of input, and its axon most likely forms several mossy fiber-like endings that target numerous granule cells and other UBCs, as in the cerebellum. The UBCs therefore, may amplify afferent signals temporally and

  7. Implications on cerebellar function from information coding.

    PubMed

    Huang, Chiming

    2008-01-01

    One function of the cerebellar cortex is to process information. There are at least two types of information. Temporal information is encoded in the timing pattern of action and synaptic potentials, whereas structural information is encoded in the spatial pattern of the cerebellar synaptic circuitry. Intuitively, analysis of highly complex information in the time domain would require a cerebellar cortex with structural complexity to match. Information theory offers a way to estimate quantitatively both types of information and thereby helps to test hypotheses or advance theories of cerebellar neurobiology. These estimates suggest: (i) That the mossy-fiber-granule-cell system carries far more (temporal) information than the climbing fiber system, (ii) that Purkinje cells extract only a fraction of the (temporal) information from their afferents, and (iii) that the cerebellar cortex has a large (spatial) information coding capacity. Concerning information, one can argue that the cerebellar cortex analyzes temporal information in its afferents as a search engine, in search of coincidental mossy fiber events based on timing cues provided by climbing fiber events. Results of successive searches are continuously being converted into structural information encoded in the spatial distribution pattern of granule-cell-Purkinje-cell synapses along granule cell axons, thereby providing an adaptive and indeed self-correcting dimension to the structural information code. The search engine operation involves cellular mechanisms acting on temporal events and is part of an associative learning process. The conversion and generation of structural information involves neuroplasticity mechanisms acting at the synaptic level, with electrophysiological as well as structural consequences, and may be part of the short- and long-term memory process. These and other attributes qualify the cerebellar cortex as a dynamic information processing center, contributing to memory and learning while

  8. Unusual morphological damage of Purkinje cells following postnatal BrdU administration in the cerebellar cortex of mouse.

    PubMed

    Takács, T

    2012-01-01

    Postnatal development of the cerebellum lasts for weeks in rodents and can be disturbed by systemic 5-bromo-2'-deoxyuridine (BrdU) administration. This thymidine analogue incorporates into the DNA of proliferating cells, and result in more or less serious damage or death granule cells, the most actively dividing neuronal population in the developing cerebellar cortex. Further consequences of postnatal BrdU administration are the interrupted postnatal migration and integrations as well as partial loss of cerebellar Purkinje cells. In the present study, C57B16 mice were administered with 50 μg/g body weight BrdU, one sc. injection daily, between P0 and P11 postnatal days, respectively.Large "cavities" appeared in the cytoplasm of a subpopulation of Purkinje cells by P7 in about one-third of administered animals, their number are size of the cavities (and PCs exhibiting unusual morphology) decreased. EM studies revealed that the unusual Purkinje cells received numerous axonal inputs of unknown origin, first of all on their somatic and dendritic spines. The transitory appearance of a subpopulation of Purkinje cells possessing unusual morphology refers to the influence of other (neuronal, glial, or both) cells on their regular differentiation. PMID:22514871

  9. Encoding of whisker input by cerebellar Purkinje cells

    PubMed Central

    Bosman, Laurens W J; Koekkoek, Sebastiaan K E; Shapiro, Joël; Rijken, Bianca F M; Zandstra, Froukje; van der Ende, Barry; Owens, Cullen B; Potters, Jan-Willem; de Gruijl, Jornt R; Ruigrok, Tom J H; De Zeeuw, Chris I

    2010-01-01

    The cerebellar cortex is crucial for sensorimotor integration. Sensorimotor inputs converge on cerebellar Purkinje cells via two afferent pathways: the climbing fibre pathway triggering complex spikes, and the mossy fibre–parallel fibre pathway, modulating the simple spike activities of Purkinje cells. We used, for the first time, the mouse whisker system as a model system to study the encoding of somatosensory input by Purkinje cells. We show that most Purkinje cells in ipsilateral crus 1 and crus 2 of awake mice respond to whisker stimulation with complex spike and/or simple spike responses. Single-whisker stimulation in anaesthetised mice revealed that the receptive fields of complex spike and simple spike responses were strikingly different. Complex spike responses, which proved to be sensitive to the amplitude, speed and direction of whisker movement, were evoked by only one or a few whiskers. Simple spike responses, which were not affected by the direction of movement, could be evoked by many individual whiskers. The receptive fields of Purkinje cells were largely intermingled, and we suggest that this facilitates the rapid integration of sensory inputs from different sources. Furthermore, we describe that individual Purkinje cells, at least under anaesthesia, may be bound in two functional ensembles based on the receptive fields and the synchrony of the complex spike and simple spike responses. The ‘complex spike ensembles’ were oriented in the sagittal plane, following the anatomical organization of the climbing fibres, while the ‘simple spike ensembles’ were oriented in the transversal plane, as are the beams of parallel fibres. PMID:20724365

  10. Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

    SciTech Connect

    Baconnais, S.; Delavoie, F. |; Zahm, J.M.; Milliot, M.; Castillon, N.; Terryn, C.; Banchet, V.; Michel, J.; Danos, O.; Merten, M.; Chinet, T.; Zierold, K.; Bonnet, N.; Puchelle, E. , E-Mail: edith.puchelle@univ-reims.fr; Balossier, G.

    2005-10-01

    The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na{sup +} absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na{sup +}, Mg{sup 2+}, P, S and Cl{sup -}) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR{sub inh}-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.

  11. Asymmetric cell division of granule neuron progenitors in the external granule layer of the mouse cerebellum

    PubMed Central

    Haldipur, Parthiv; Sivaprakasam, Iswariya; Periasamy, Vinod; Govindan, Subashika; Mani, Shyamala

    2015-01-01

    ABSTRACT The plane of division of granule neuron progenitors (GNPs) was analysed with respect to the pial surface in P0 to P14 cerebellum and the results showed that there was a significant bias towards the plane of cell division being parallel to pial surface across this developmental window. In addition, the distribution of β-Catenin in anaphase cells was analysed, which showed that there was a significant asymmetry in the distribution of β-Catenin in dividing GNPs. Further, inhibition of Sonic Hedgehog (Shh) signalling had an effect on plane of cell division. Asymmetric distribution of β-Catenin was shown to occur towards the source of a localized extracellular cue. PMID:25979710

  12. Signal transduction pathways in mast cell granule-mediated endothelial cell activation.

    PubMed Central

    Chi, Luqi; Stehno-Bittel, Lisa; Smirnova, Irina; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2003-01-01

    BACKGROUND: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8. AIMS: The objective of the present study was to identify candidate molecules and signal transduction pathways involved in the synergy between mast cell granules and lipopolysaccharide on endothelial cell activation. METHODS: Human umbilical vein endothelial cells were incubated with rat mast cell granules in the presence and absence of lipopolysaccharide, and IL-6 production was quantified. The status of c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2 activation, nuclear factor-kappaB translocation and intracellular calcium levels were determined to identify the mechanism of synergy between mast cell granules and lipopolysaccaride. RESULTS: Mast cell granules induced low levels of interleukin-6 production by endothelial cells, and this effect was markedly enhanced by lipopolysaccharide. The results revealed that both serine proteases and histamine present in mast cell granules were involved in this activation process. Mast cell granules increased intracellular calcium, and activated c-Jun amino-terminal kinase and extracellular signal-regulated kinase 1/2. The combination of lipopolysaccharide and mast cell granules prolonged c-Jun amino-terminal kinase activity beyond the duration of induction by either stimulant alone and was entirely due to active proteases. However, both proteases and histamine contributed to calcium mobilization and extracellular signal-regulated kinase 1/2 activation. The nuclear translocation of nuclear factor-kappaB proteins was of greater magnitude in endothelial cells treated with the combination of mast cell granules and lipopolysaccharide. CONCLUSIONS:Mast cell granule serine proteases and histamine can amplify lipopolysaccharide-induced endothelial cell activation, which involves calcium mobilization, mitogen

  13. Cerebellar Globular Cells Receive Monoaminergic Excitation and Monosynaptic Inhibition from Purkinje Cells

    PubMed Central

    Hirono, Moritoshi; Saitow, Fumihito; Kudo, Moeko; Suzuki, Hidenori; Yanagawa, Yuchio; Yamada, Masahisa; Nagao, Soichi; Konishi, Shiro; Obata, Kunihiko

    2012-01-01

    Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control. PMID:22235322

  14. Rapid Feedforward Inhibition and Asynchronous Excitation Regulate Granule Cell Activity in the Mammalian Main Olfactory Bulb

    PubMed Central

    Burton, Shawn D.

    2015-01-01

    Granule cell-mediated inhibition is critical to patterning principal neuron activity in the olfactory bulb, and perturbation of synaptic input to granule cells significantly alters olfactory-guided behavior. Despite the critical role of granule cells in olfaction, little is known about how sensory input recruits granule cells. Here, we combined whole-cell patch-clamp electrophysiology in acute mouse olfactory bulb slices with biophysical multicompartmental modeling to investigate the synaptic basis of granule cell recruitment. Physiological activation of sensory afferents within single glomeruli evoked diverse modes of granule cell activity, including subthreshold depolarization, spikelets, and suprathreshold responses with widely distributed spike latencies. The generation of these diverse activity modes depended, in part, on the asynchronous time course of synaptic excitation onto granule cells, which lasted several hundred milliseconds. In addition to asynchronous excitation, each granule cell also received synchronous feedforward inhibition. This inhibition targeted both proximal somatodendritic and distal apical dendritic domains of granule cells, was reliably recruited across sniff rhythms, and scaled in strength with excitation as more glomeruli were activated. Feedforward inhibition onto granule cells originated from deep short-axon cells, which responded to glomerular activation with highly reliable, short-latency firing consistent with tufted cell-mediated excitation. Simulations showed that feedforward inhibition interacts with asynchronous excitation to broaden granule cell spike latency distributions and significantly attenuates granule cell depolarization within local subcellular compartments. Collectively, our results thus identify feedforward inhibition onto granule cells as a core feature of olfactory bulb circuitry and establish asynchronous excitation and feedforward inhibition as critical regulators of granule cell activity. SIGNIFICANCE

  15. Calcicludine, a venom peptide of the Kunitz-type protease inhibitor family, is a potent blocker of high-threshold Ca2+ channels with a high affinity for L-type channels in cerebellar granule neurons.

    PubMed Central

    Schweitz, H; Heurteaux, C; Bois, P; Moinier, D; Romey, G; Lazdunski, M

    1994-01-01

    Calcicludine (CaC) is a 60-amino acid polypeptide from the venom of Dendroaspis angusticeps. It is structurally homologous to the Kunitz-type protease inhibitor, to dendrotoxins, which block K+ channels, and to the protease inhibitor domain of the amyloid beta protein that accumulates in Alzheimer disease. Voltage-clamp experiments on a variety of excitable cells have shown that CaC specifically blocks most of the high-threshold Ca2+ channels (L-, N-, or P-type) in the 10-100 nM range. Particularly high densities of specific 125I-labeled CaC binding sites were found in the olfactory bulb, in the molecular layer of the dentate gyrus and the stratum oriens of CA3 field in the hippocampal formation, and in the granular layer of the cerebellum. 125I-labeled CaC binds with a high affinity (Kd = 15 pM) to a single class of noninteracting sites in rat olfactory bulb microsomes. The distribution of CaC binding sites in cerebella of three mutant mice (Weaver, Reeler, and Purkinje cell degeneration) clearly shows that the specific high-affinity labeling is associated with granule cells. Electrophysiological experiments on rat cerebellar granule neurons in primary culture have shown that CaC potently blocks the L-type component of the Ca2+ current (K0.5 = 0.2 nM). Then CaC, in the nanomolar range, appears to be a highly potent blocker of an L-subtype of neuronal Ca2+ channels. Images PMID:8302860

  16. Behavioral experience induces zif268 expression in mature granule cells but suppresses its expression in immature granule cells

    PubMed Central

    Huckleberry, Kylie A.; Kane, Gary A.; Mathis, Rita J.; Cook, Sarah G.; Clutton, Jonathan E.; Drew, Michael R.

    2015-01-01

    Thousands of neurons are born each day in the dentate gyrus (DG), but many of these cells die before reaching maturity. Both death and survival of adult-born neurons are regulated by neuronal activity in the DG. The immediate-early gene (IEG) zif268 appears to be an important mediator of these effects, as its expression can be induced by neural activity and knockout of zif268 impairs survival of adult-born neurons (Richardson et al., 1992; Veyrac et al., 2013). Despite the apparent importance of zif268 for adult neurogenesis, its behavior-induced expression has not been fully characterized in adult-born neurons. Here we characterize behavior-evoked expression of zif268 in mature and newborn dentate granule cells (DGCs). We first quantified zif268 expression in doublecortin-positive (DCX+) immature neurons and in the general granule cell population after brief exposure to a novel environment (NE). In the general granule cell population, zif268 expression peaked 1 h after NE exposure and returned to baseline by 8 h post-exposure. However, in the DCX+ cells, zif268 expression was suppressed relative to home cage for at least 8 h post-exposure. We next asked whether suppression of zif268 in DCX+ immature cells occurs in other behavioral paradigms that recruit the hippocampus. Exposure to Morris water maze (MWM) training, an enriched environment, or a NE caused approximately equal suppression of zif268 expression in DCX+ cells and approximately equal activation of zif268 expression among the general granule cell population. The same behavioral procedures activated zif268 expression in 6-week-old BrdU-labeled adult-born neurons, indicating that zif268 suppression is specific to immature neurons. Finally, we asked whether zif268 suppression varied as a function of age within the DCX+ population, which ranges in age from 0 to approximately 4 weeks. NE exposure had no significant effect on zif268 expression in 2- or 4-week-old BrdU-labeled neurons, but it significantly

  17. Survival of interneurons and parallel fiber synapses in a cerebellar cortex deprived of Purkinje cells: studies in the double mutant mouse Grid2Lc/+;Bax(-/-).

    PubMed

    Zanjani, S Hadi; Selimi, Fekrije; Vogel, Michael W; Haeberlé, Anne-Marie; Boeuf, Julien; Mariani, Jean; Bailly, Yannick J

    2006-08-01

    The Lurcher mutation in the Grid2 gene causes the cell autonomous death of virtually all cerebellar Purkinje cells and the target-related death of 90% of the granule cells and 60-75% of the olivary neurons. Inactivation of Bax, a pro-apoptotic gene of the Bcl-2 family, in heterozygous Lurcher mutants (Grid2Lc/+) rescues approximately 60% of the granule cells, but does not rescue Purkinje or olivary neurons. Given the larger size of the cerebellar molecular layer in Grid2Lc/+;Bax(-/-) double mutants compared to Grid2Lc/+ mutants, we analyzed the survival of the stellate and basket interneurons as well as the synaptic connectivity of parallel fibers originating from the surviving granule cells in the absence of their Purkinje cell targets in the Grid2Lc/+;Bax(-/-) cerebellum. Quantification showed a significantly higher density of interneurons ( approximately 60%) in the molecular layer of the Grid2Lc/+;Bax(-/-) mice compared to Grid2Lc/+, suggesting that interneurons are subject to a BAX-dependent target-related death in the Lurcher mutants. Furthermore, electron microscopy showed the normal ultrastructural aspect of a number of parallel fibers in the molecular layer of the Grid2Lc/+; Bax(-/-) double mutant mice and preserved their numerous synaptic contacts on interneurons, suggesting that interneurons could play a trophic role for axon terminals of surviving granule cells. Finally, parallel fibers varicosities in the double mutant established "pseudo-synapses" on glia as well as displayed autophagic profiles, suggesting that the connections established by the parallel fibers in the absence of their Purkinje cell targets were subject to a high turnover involving autophagy. PMID:16739195

  18. Clonal analysis reveals granule cell behaviors and compartmentalization that determine the folded morphology of the cerebellum

    PubMed Central

    Legué, Emilie; Riedel, Elyn; Joyner, Alexandra L.

    2015-01-01

    The mammalian cerebellum consists of folds of different sizes and shapes that house distinct neural circuits. A crucial factor underlying foliation is the generation of granule cells (gcs), the most numerous neuron type in the brain. We used clonal analysis to uncover global as well as folium size-specific cellular behaviors that underlie cerebellar morphogenesis. Unlike most neural precursors, gc precursors divide symmetrically, accounting for their massive expansion. We found that oriented cell divisions underlie an overall anteroposteriorly polarized growth of the cerebellum and gc clone geometry. Clone geometry is further refined by mediolateral oriented migration and passive dispersion of differentiating gcs. Most strikingly, the base of each fissure acts as a boundary for gc precursor dispersion, which we propose allows each folium to be regulated as a developmental unit. Indeed, the geometry and size of clones in long and short folia are distinct. Moreover, in engrailed 1/2 mutants with shorter folia, clone cell number and geometry are most similar to clones in short folia of wild-type mice. Thus, the cerebellum has a modular mode of development that allows the plane of cell division and number of divisions to be differentially regulated to ensure that the appropriate number of cells are partitioned into each folium. PMID:25834018

  19. FIB/SEM cell sectioning for intracellular metal granules characterization

    NASA Astrophysics Data System (ADS)

    Milani, Marziale; Brundu, Claudia; Santisi, Grazia; Savoia, Claudio; Tatti, Francesco

    2009-05-01

    Focused Ion Beams (FIBs) provide a cross-sectioning tool for submicron dissection of cells and subcellular structures. In combination with Scanning Electron Microscope (SEM), FIB provides complementary morphological information, that can be further completed by EDX (Energy Dispersive X-ray Spectroscopy). This study focus onto intracellular microstructures, particularly onto metal granules (typically Zn, Cu and Fe) and on the possibility of sectioning digestive gland cells of the terrestrial isopod P. scaber making the granules available for a compositional analysis with EDX. Qualitative and quantitative analysis of metal granules size, amount and distribution are performed. Information is made available of the cellular storing pattern and, indirectly, metal metabolism. The extension to human level is of utmost interest since some pathologies of relevance are metal related. Apart from the common metal-overload-diseases (hereditary hemochromatosis, Wilson's and Menkes disease) it has been demonstrated that metal in excess can influence carcinogenesis in liver, kidney and breast. Therefore protocols will be established for the observation of mammal cells to improve our knowledge about the intracellular metal amount and distribution both in healthy cells and in those affected by primary or secondary metal overload or depletion.

  20. Localization of human intestinal defensin 5 in Paneth cell granules.

    PubMed Central

    Porter, E M; Liu, L; Oren, A; Anton, P A; Ganz, T

    1997-01-01

    Antibiotic peptides of higher animals include the defensins, first discovered in phagocytic cells but recently also found to be produced by epithelial cells. We biosynthesized recombinant human intestinal defensin 5 (rHD-5) using the baculovirus-insect cell expression system. Since insect cells process defensin incompletely and secrete the precursor proHD-5, we substituted a methionine for an alanine at a likely processing site to allow selective chemical cleavage with cyanogen bromide, and rHD-5 was used to elicit polyclonal antibodies. By the immunoperoxidase-staining technique, the antibodies selectively stained Paneth cells of the normal adult small intestine. Immunogold electron microscopy further localized HD-5 to the Paneth cell secretory granules. Since some defensins exert activity cytotoxic to mammalian cells, we assayed the effect of rHD-5 on the human intestinal cell lines Caco2 and Int407. proHD-5 did not exert cytotoxic activity, and rHD-5 showed only minimal activity against Int407 and was inert against Caco2. Since Paneth cells release their granules adjacent to the mitotic cells of the intestinal crypts, HD could protect this cell population against invasion and parasitization by microbes. PMID:9169779

  1. Menin immunoreactivity in secretory granules of human pancreatic islet cells.

    PubMed

    Debelenko, Larisa V; Agarwal, Sunita; Du, Qiang; Yan, Wusheng; Erickson, Heidi S; Abu-Asab, Mones; Raffeld, Mark A; Libutti, Steven K; Marx, Stephen J; Emmert-Buck, Michael R

    2014-01-01

    The protein product of the Multiple Endocrine Neoplasia Type I (MEN1) gene is thought to be involved in predominantly nuclear functions; however, immunohistochemical (IHC) analysis data on cellular localization are conflicting. To further investigate menin expression, we analyzed human pancreas (an MEN1 target organ) using IHC analyses and 6 antibodies raised against full-length menin or its peptides. In 10 normal pancreas specimens, 2 independently raised antibodies showed unexpected cytoplasmic immunoreactivity in peripheral cells in each islet examined (over 100 total across all 10 patients). The staining exhibited a distinct punctate pattern and subsequent immunoelectron microscopy indicated the target antigen was in secretory granules. Exocrine pancreas and pancreatic stroma were not immunoreactive. In MEN1 patients, unaffected islets stained similar to those in normal samples but with a more peripheral location of positive cells, whereas hyperplastic islets and tumorlets showed increased and diffuse cytoplasmic staining, respectively. Endocrine tumors from MEN1 patients were negative for menin, consistent with a 2-hit loss of a tumor suppressor gene. Secretory granule localization of menin in a subset of islet cells suggests a function of the protein unique to a target organ of familial endocrine neoplasia, although the IHC data must be interpreted with some caution because of the possibility of antibody cross-reaction. The identity, cellular trafficking, and role of this putative secretory granule-form of menin warrant additional investigation. PMID:25153502

  2. Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge

    PubMed Central

    Chaumont, Joseph; Guyon, Nicolas; Valera, Antoine M.; Dugué, Guillaume P.; Popa, Daniela; Marcaggi, Paikan; Gautheron, Vanessa; Reibel-Foisset, Sophie; Dieudonné, Stéphane; Stephan, Aline; Barrot, Michel; Cassel, Jean-Christophe; Dupont, Jean-Luc; Doussau, Frédéric; Poulain, Bernard; Selimi, Fekrije; Léna, Clément; Isope, Philippe

    2013-01-01

    Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning. PMID:24046366

  3. Clusters of cerebellar Purkinje cells control their afferent climbing fiber discharge.

    PubMed

    Chaumont, Joseph; Guyon, Nicolas; Valera, Antoine M; Dugué, Guillaume P; Popa, Daniela; Marcaggi, Paikan; Gautheron, Vanessa; Reibel-Foisset, Sophie; Dieudonné, Stéphane; Stephan, Aline; Barrot, Michel; Cassel, Jean-Christophe; Dupont, Jean-Luc; Doussau, Frédéric; Poulain, Bernard; Selimi, Fekrije; Léna, Clément; Isope, Philippe

    2013-10-01

    Climbing fibers, the projections from the inferior olive to the cerebellar cortex, carry sensorimotor error and clock signals that trigger motor learning by controlling cerebellar Purkinje cell synaptic plasticity and discharge. Purkinje cells target the deep cerebellar nuclei, which are the output of the cerebellum and include an inhibitory GABAergic projection to the inferior olive. This pathway identifies a potential closed loop in the olivo-cortico-nuclear network. Therefore, sets of Purkinje cells may phasically control their own climbing fiber afferents. Here, using in vitro and in vivo recordings, we describe a genetically modified mouse model that allows the specific optogenetic control of Purkinje cell discharge. Tetrode recordings in the cerebellar nuclei demonstrate that focal stimulations of Purkinje cells strongly inhibit spatially restricted sets of cerebellar nuclear neurons. Strikingly, such stimulations trigger delayed climbing-fiber input signals in the stimulated Purkinje cells. Therefore, our results demonstrate that Purkinje cells phasically control the discharge of their own olivary afferents and thus might participate in the regulation of cerebellar motor learning. PMID:24046366

  4. Cr (VI) induced oxidative stress and toxicity in cultured cerebellar granule neurons at different stages of development and protective effect of Rosmarinic acid.

    PubMed

    Dashti, Abolfazl; Soodi, Maliheh; Amani, Nahid

    2016-03-01

    Chromium (Cr) is a widespread metal ion in the workplace, industrial effluent, and water. The toxicity of chromium (VI) on various organs including the liver, kidneys, and lung were studied, but little is known about neurotoxicity. In this study, neurotoxic effects of Cr (VI) have been investigated by cultured cerebellar granule neurons (CGNs). Immature and mature neurons were exposed to different concentrations of potassium dichromate for 24 h and cytotoxicity was measured by MTT assay. In addition, immature neurons were exposed for 5 days as regards cytotoxic effect in development stages. The reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and the protective effect of Rosmarinic acid on mature and immature neurons exposed to potassium dichromate, were measured. Furthermore, lipid peroxidation, glutathione peroxidase (GPx), and acetylcholinesterase activity in mature neurons were assessed following exposure to potassium dichromate. The results indicate that toxicity of Cr (VI) dependent on maturation steps. Cr (VI) was less toxic for immature neurons. Also, Cr (VI) induced MMP reduction and ROS production in both immature and mature neurons. In Cr (VI) treated neurons, increased lipid peroxidation and GPx activity but not acetylcholinesterase activity was observed. Interestingly, Rosmarinic acid, as a natural antioxidant, could protect mature but not immature neurons against Cr (VI) induced toxicity. Our findings revealed vulnerability of mature neurons to Cr (VI) induced toxicity and oxidative stress. PMID:25213303

  5. Reciprocal autoregulation by NFI occupancy and ETV1 promotes the developmental expression of dendrite-synapse genes in cerebellar granule neurons

    PubMed Central

    Ding, Baojin; Cave, John W.; Dobner, Paul R.; Mullikin-Kilpatrick, Debra; Bartzokis, Marina; Zhu, Hong; Chow, Chi-Wing; Gronostajski, Richard M.; Kilpatrick, Daniel L.

    2016-01-01

    Nuclear Factor One (NFI) transcription factors regulate temporal gene expression required for dendritogenesis and synaptogenesis via delayed occupancy of target promoters in developing cerebellar granule neurons (CGNs). Mechanisms that promote NFI temporal occupancy have not been previously defined. We show here that the transcription factor ETV1 directly binds to and is required for expression and NFI occupancy of a cohort of NFI-dependent genes in CGNs maturing in vivo. Expression of ETV1 is low in early postnatal cerebellum and increases with maturation, mirroring NFI temporal occupancy of coregulated target genes. Precocious expression of ETV1 in mouse CGNs accelerated onset of expression and NFI temporal occupancy of late target genes and enhanced Map2(+) neurite outgrowth. ETV1 also activated expression and NFI occupancy of the Etv1 gene itself, and this autoregulatory loop preceded ETV1 binding and activation of other coregulated target genes in vivo. These findings suggest a potential model in which ETV1 activates NFI temporal binding to a subset of late-expressed genes in a stepwise manner by initial positive feedback regulation of the Etv1 gene itself followed by activation of downstream coregulated targets as ETV1 expression increases. Sequential transcription factor autoregulation and subsequent binding to downstream promoters may provide an intrinsic developmental timer for dendrite/synapse gene expression. PMID:26941328

  6. Reciprocal autoregulation by NFI occupancy and ETV1 promotes the developmental expression of dendrite-synapse genes in cerebellar granule neurons.

    PubMed

    Ding, Baojin; Cave, John W; Dobner, Paul R; Mullikin-Kilpatrick, Debra; Bartzokis, Marina; Zhu, Hong; Chow, Chi-Wing; Gronostajski, Richard M; Kilpatrick, Daniel L

    2016-05-01

    Nuclear Factor One (NFI) transcription factors regulate temporal gene expression required for dendritogenesis and synaptogenesis via delayed occupancy of target promoters in developing cerebellar granule neurons (CGNs). Mechanisms that promote NFI temporal occupancy have not been previously defined. We show here that the transcription factor ETV1 directly binds to and is required for expression and NFI occupancy of a cohort of NFI-dependent genes in CGNs maturing in vivo. Expression of ETV1 is low in early postnatal cerebellum and increases with maturation, mirroring NFI temporal occupancy of coregulated target genes. Precocious expression of ETV1 in mouse CGNs accelerated onset of expression and NFI temporal occupancy of late target genes and enhanced Map2(+) neurite outgrowth. ETV1 also activated expression and NFI occupancy of the Etv1 gene itself, and this autoregulatory loop preceded ETV1 binding and activation of other coregulated target genes in vivo. These findings suggest a potential model in which ETV1 activates NFI temporal binding to a subset of late-expressed genes in a stepwise manner by initial positive feedback regulation of the Etv1 gene itself followed by activation of downstream coregulated targets as ETV1 expression increases. Sequential transcription factor autoregulation and subsequent binding to downstream promoters may provide an intrinsic developmental timer for dendrite/synapse gene expression. PMID:26941328

  7. Transplantation of human induced cerebellar granular-like cells improves motor functions in a novel mouse model of cerebellar ataxia

    PubMed Central

    Zhu, Tongming; Tang, Hailiang; Shen, Yiwen; Tang, Qisheng; Chen, Luping; Wang, Zhifu; Zhou, Ping; Xu, Feng; Zhu, Jianhong

    2016-01-01

    Stem cell-based reparative approaches have been applied to cerebellum-related disorders during the last two decades. Direct lineage reprogramming of human fibroblasts into functional granular neurons holds great promise for biomedical applications such as cerebellum regeneration and cellbased disease modeling. In the present study, we showed that a combination of Ascl1, Sox2 and OCT4, in a culture subsequently treated with secreted factors (BMP4, Wnt3a and FGF8b), was capable of converting human fibroblasts from the scalp tissue of patients with traumatic brain injury (TBI) into functional human induced cerebellar granular-like cells (hiCGCs). Morphological analysis, immunocytochemistry, gene expression and electrophysiological analysis were performed to identify the similarity of induced neuronal cells to human cerebellum granular cells. Our strategy improved the efficiency for hiCGCs induction, which gave the highest conversion efficiency 12.30±0.88%, and Ath1+/Tuj1+ double positive cells to 5.56±0.80%. We transplanted hiCGCs into the cerebellum of NmycTRE/TRE: tTS mice, a novel mouse model of cerebellar ataxia, and demonstrated that the hiCGCs were able to survive, migrate, proliferate and promote mild functional recovery after been grafted into cerebellum. PMID:27158363

  8. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice

    PubMed Central

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

  9. Disinhibition of olfactory bulb granule cells accelerates odour discrimination in mice.

    PubMed

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

    Granule cells are the dominant cell type of the olfactory bulb inhibiting mitral and tufted cells via dendrodendritic synapses; yet the factors regulating the strength of their inhibitory output, and, therefore, their impact on odour discrimination, remain unknown. Here we show that GABAAR β3-subunits are distributed in a somatodendritic pattern, mostly sparing the large granule cell spines also known as gemmules. Granule cell-selective deletion of β3-subunits nearly abolishes spontaneous and muscimol-induced currents mediated by GABAA receptors in granule cells, yet recurrent inhibition of mitral cells is strongly enhanced. Mice with disinhibited granule cells require less time to discriminate both dissimilar as well as highly similar odourants, while discrimination learning remains unaffected. Hence, granule cells are controlled by an inhibitory drive that in turn tunes mitral cell inhibition. As a consequence, the olfactory bulb inhibitory network adjusts the speed of early sensory processing. PMID:26592770

  10. Functional Properties of Dendritic Gap Junctions in Cerebellar Golgi Cells.

    PubMed

    Szoboszlay, Miklos; Lőrincz, Andrea; Lanore, Frederic; Vervaeke, Koen; Silver, R Angus; Nusser, Zoltan

    2016-06-01

    The strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells. PMID:27133465

  11. Different Degrees of Iodine Deficiency Inhibit Differentiation of Cerebellar Granular Cells in Rat Offspring, via BMP-Smad1/5/8 Signaling.

    PubMed

    Dong, Jing; Lei, Xibing; Wang, Yi; Wang, Yuan; Song, Heling; Li, Min; Min, Hui; Yu, Ye; Xi, Qi; Teng, Weiping; Chen, Jie

    2016-09-01

    Iodine deficiency (ID) during development results in dysfunction of the central nervous system (CNS) and affects psychomotor and motor function. It is worth noting that maternal mild and marginal ID tends to be the most common reason of preventable neurodevelopmental impairment, via a mechanism that has not been elucidated. Therefore, our aim was to study the effects of developmental mild and marginal ID on the differentiation of cerebellar granule cells (GCs) and investigate the activation of BMP-Smad1/5/8 signaling, which is crucial for the development and differentiation of cerebellum. Three developmental rat models were created by feeding dam rats with a diet deficient in iodine and deionized water supplemented with potassium iodide. Our results showed that different degrees of ID inhibited and delayed the differentiation of cerebellar GCs on postnatal day (PN) 7, PN14, and PN21. Moreover, mild and severe ID reduced the expression of BMP2 and p-Smad1/5/8, and increased the levels of Id2 on PN7, PN14, and PN21. However, marginal ID rarely altered expression of these proteins in the offspring. Our study supports the hypothesis that mild and severe ID during development inhibits the differentiation of cerebellar GCs, which may be ascribed to the down-regulation of BMP-Smad1/5/8 signaling and the overexpression of Id2. Furthermore, it was speculated that maternal marginal ID rarely affected the differentiation of cerebellar GCs in the offspring. PMID:26307610

  12. Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

    PubMed Central

    Strasser, Markus J; Mackenzie, Natalia C; Dumstrei, Karin; Nakkrasae, La-Iad; Stebler, Jürg; Raz, Erez

    2008-01-01

    Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7) is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network. PMID:18507824

  13. Defects in the CAPN1 Gene Result in Alterations in Cerebellar Development and Cerebellar Ataxia in Mice and Humans.

    PubMed

    Wang, Yubin; Hersheson, Joshua; Lopez, Dulce; Hammer, Monia; Liu, Yan; Lee, Ka-Hung; Pinto, Vanessa; Seinfeld, Jeff; Wiethoff, Sarah; Sun, Jiandong; Amouri, Rim; Hentati, Faycal; Baudry, Neema; Tran, Jennifer; Singleton, Andrew B; Coutelier, Marie; Brice, Alexis; Stevanin, Giovanni; Durr, Alexandra; Bi, Xiaoning; Houlden, Henry; Baudry, Michel

    2016-06-28

    A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous or heterozygous CAPN1-null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knockout (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1-mediated cleavage of PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans. PMID:27320912

  14. Defects in the CAPN1 gene result in alterations in cerebellar development and in cerebellar ataxia in mice and humans

    PubMed Central

    Wang, Yubin; Hersheson, Joshua; Lopez, Dulce; Hamad, Monia Ben; Liu, Yan; Lee, Ka-Hung; Pinto, Vanessa; Seinfeld, Jeff; Wiethoff, Sarah; Sun, Jiandong; Amouri, Rim; Hentati, Faycal; Baudry, Neema; Tran, Jennifer; Singleton, Andrew B; Coutelier, Marie; Brice, Alexis; Stevanin, Giovanni; Durr, Alexandra; Bi, Xiaoning; Houlden, Henry; Baudry, Michel

    2016-01-01

    SUMMARY A CAPN1 missense mutation in Parson Russell Terrier dogs is associated with spinocerebellar ataxia. We now report that homozygous CAPN1 null mutations in humans result in cerebellar ataxia and limb spasticity in four independent pedigrees. Calpain-1 knock-out (KO) mice also exhibit a mild form of ataxia due to abnormal cerebellar development, including enhanced neuronal apoptosis, decreased number of cerebellar granule cells, and altered synaptic transmission. Enhanced apoptosis is due to absence of calpain-1 mediated cleavage of PH domain and Leucine rich repeat Protein Phosphatase 1 (PHLPP1), which results in inhibition of the Akt pro-survival pathway in developing granule cells. Injection of neonatal mice with the indirect Akt activator, bisperoxovanadium, or crossing calpain-1 KO mice with PHLPP1 KO mice prevented increased postnatal cerebellar granule cell apoptosis, and restored granule cell density and motor coordination in adult mice. Thus, mutations in CAPN1 are an additional cause of ataxia in mammals, including humans. PMID:27320912

  15. Impaired motor coordination and disrupted cerebellar architecture in Fgfr1 and Fgfr2 double knockout mice

    PubMed Central

    Smith, Karen Müller; Williamson, Theresa L.; Schwartz, Michael L.

    2012-01-01

    Fibroblast growth factor receptor (FGFR) signaling determines the size of the cerebral cortex by regulating the amplification of radial glial stem cells, and participates in the formation of midline glial structures. We show that Fgfr1 and Fgfr2 double knockouts (FGFR DKO) generated by Cre mediated recombination driven by the human GFAP promoter (hGFAP) have reduced cerebellar size due to reduced proliferation of radial glia and other glial precursors in late embryonic and neonatal FGFR DKO mice. The proliferation of granule cell progenitors (GCPs) in the EGL was also reduced, leading to reduced granule cell numbers. Furthermore, both inward migration of granule cells into the inner granule cell layer (IGL) and outward migration of GABA interneurons into the molecular layer (ML) were arrested, disrupting layer and lobular morphology. Purkinje neurons and their dendrites, which were not targeted by Cre mediated recombination of Fgf receptors, were also misplaced in FGFR DKO mice, possibly as a consequence of altered Bergmann glia orientation or reduced granule cell number. Our findings indicate a dual role for FGFR signaling in cerebellar morphogenesis. The first role is to amplify the number of granule neuron precursors in the external granular layer and glial precursor cells throughout the cerebellum. The second is to establish the correct Bergmann glia morphology, which is crucial for granule cell migration. The disrupted cerebellar size and laminar architecture resulting from loss of FGFR signaling impairs motor learning and coordination in FGFR DKO mice. PMID:22578469

  16. Ethanol-Induced Cerebellar Ataxia: Cellular and Molecular Mechanisms.

    PubMed

    Dar, M Saeed

    2015-08-01

    The cerebellum is an important target of ethanol toxicity given that cerebellar ataxia is the most consistent physical manifestation of acute ethanol consumption. Despite the significance of the cerebellum in ethanol-induced cerebellar ataxia (EICA), the cellular and molecular mechanisms underlying EICA are incompletely understood. However, two important findings have shed greater light on this phenomenon. First, ethanol-induced blockade of cerebellar adenosine uptake in rodent models points to a role for adenosinergic A1 modulation of EICA. Second, the consistent observation that intracerebellar administration of nicotine in mice leads to antagonism of EICA provides evidence for a critical role of cerebellar nitric oxide (NO) in EICA reversal. Based on these two important findings, this review discusses the potential molecular events at two key synaptic sites (mossy fiber-granule cell-Golgi cell (MGG synaptic site) and granule cell parallel fiber-Purkinje cell (GPP synaptic site) that lead to EICA. Specifically, ethanol-induced neuronal NOS inhibition at the MGG synaptic site acts as a critical trigger for Golgi cell activation which leads to granule cell deafferentation. Concurrently, ethanol-induced inhibition of adenosine uptake at the GPP synaptic site produces adenosine accumulation which decreases glutamate release and leads to the profound activation of Purkinje cells (PCs). These molecular events at the MGG and GPP synaptic sites are mutually reinforcing and lead to cerebellar dysfunction, decreased excitatory output of deep cerebellar nuclei, and EICA. The critical importance of PCs as the sole output of the cerebellar cortex suggests normalization of PC function could have important therapeutic implications. PMID:25578036

  17. Imaging of zymogen granules in fully wet cells: evidence for restricted mechanism of granule growth.

    PubMed

    Hammel, Ilan; Anaby, Debbie

    2007-09-01

    The introduction of wet SEM imaging technology permits electron microscopy of wet samples. Samples are placed in sealed specimen capsules and are insulated from the vacuum in the SEM chamber by an impermeable, electron-transparent membrane. The complete insulation of the sample from the vacuum allows direct imaging of fully hydrated, whole-mount tissue. In the current work, we demonstrate direct inspection of thick pancreatic tissue slices (above 400 mum). In the case of scanning of the pancreatic surface, the boundaries of intracellular features are seen directly. Thus no unfolding is required to ascertain the actual particle size distribution based on the sizes of the sections. This method enabled us to investigate the true granule size distribution and confirm early studies of improved conformity to a Poisson-like distribution, suggesting that the homotypic granule growth results from a mechanism, which favors the addition of a single unit granule to mature granules. PMID:17557275

  18. In vivo imaging of dendritic pruning in dentate granule cells.

    PubMed

    Gonçalves, J Tiago; Bloyd, Cooper W; Shtrahman, Matthew; Johnston, Stephen T; Schafer, Simon T; Parylak, Sarah L; Tran, Thanh; Chang, Tina; Gage, Fred H

    2016-06-01

    We longitudinally imaged the developing dendrites of adult-born mouse dentate granule cells (DGCs) in vivo and found that they underwent over-branching and pruning. Exposure to an enriched environment and constraint of dendritic growth by disrupting Wnt signaling led to increased branch addition and accelerated growth, which were, however, counteracted by earlier and more extensive pruning. Our results indicate that pruning is regulated in a homeostatic fashion to oppose excessive branching and promote a similar dendrite structure in DGCs. PMID:27135217

  19. Exposure to 50 Hz magnetic field modulates GABAA currents in cerebellar granule neurons through an EP receptor-mediated PKC pathway.

    PubMed

    Yang, Guang; Ren, Zhen; Mei, Yan-Ai

    2015-10-01

    Previous work from both our lab and others have indicated that exposure to 50 Hz magnetic fields (ELF-MF) was able to modify ion channel functions. However, very few studies have investigated the effects of MF on γ-aminobutyric acid (GABA) type A receptors (GABA(A) Rs) channel functioning, which are fundamental to overall neuronal excitability. Here, our major goal is to reveal the potential effects of ELF-MF on GABA(A) Rs activity in rat cerebellar granule neurons (CGNs). Our results indicated that exposing CGNs to 1 mT ELF-MF for 60 min. significantly increased GABA(A) R currents without modifying sensitivity to GABA. However, activation of PKA by db-cAMP failed to do so, but led to a slight decrease instead. On the other hand, PKC activation or inhibition by PMA or Bis and Docosahexaenoic acid (DHA) mimicked or eliminated the field-induced-increase of GABA(A) R currents. Western blot analysis indicated that the intracellular levels of phosphorylated PKC (pPKC) were significantly elevated after 60 min. of ELF-MF exposure, which was subsequently blocked by application of DHA or EP1 receptor-specific (prostaglandin E receptor 1) antagonist (SC19220), but not by EP2-EP4 receptor-specific antagonists. SC19220 also significantly inhibited the ELF-MF-induced elevation on GABA(A) R currents. Together, these data obviously demonstrated for the first time that neuronal GABA(A) currents are significantly increased by ELF-MF exposure, and also suggest that these effects are mediated via an EP1 receptor-mediated PKC pathway. Future work will focus on a more comprehensive analysis of the physiological and/or pathological consequences of these effects. PMID:26176998

  20. Exposure to 50 Hz magnetic field modulates GABAA currents in cerebellar granule neurons through an EP receptor-mediated PKC pathway

    PubMed Central

    Yang, Guang; Ren, Zhen; Mei, Yan-Ai

    2015-01-01

    Previous work from both our lab and others have indicated that exposure to 50 Hz magnetic fields (ELF-MF) was able to modify ion channel functions. However, very few studies have investigated the effects of MF on γ-aminobutyric acid (GABA) type A receptors (GABAARs) channel functioning, which are fundamental to overall neuronal excitability. Here, our major goal is to reveal the potential effects of ELF-MF on GABAARs activity in rat cerebellar granule neurons (CGNs). Our results indicated that exposing CGNs to 1 mT ELF-MF for 60 min. significantly increased GABAAR currents without modifying sensitivity to GABA. However, activation of PKA by db-cAMP failed to do so, but led to a slight decrease instead. On the other hand, PKC activation or inhibition by PMA or Bis and Docosahexaenoic acid (DHA) mimicked or eliminated the field-induced-increase of GABAAR currents. Western blot analysis indicated that the intracellular levels of phosphorylated PKC (pPKC) were significantly elevated after 60 min. of ELF-MF exposure, which was subsequently blocked by application of DHA or EP1 receptor-specific (prostaglandin E receptor 1) antagonist (SC19220), but not by EP2-EP4 receptor-specific antagonists. SC19220 also significantly inhibited the ELF-MF-induced elevation on GABAAR currents. Together, these data obviously demonstrated for the first time that neuronal GABAA currents are significantly increased by ELF-MF exposure, and also suggest that these effects are mediated via an EP1 receptor-mediated PKC pathway. Future work will focus on a more comprehensive analysis of the physiological and/or pathological consequences of these effects. PMID:26176998

  1. Sustained Arc expression in adult-generated granule cells.

    PubMed

    Meconi, Alicia; Lui, Erika; Marrone, Diano F

    2015-08-31

    The dentate gyrus (DG) plays a critical role in memory formation and maintenance. Fitting this specialized role, the DG has many unique characteristics. In addition to being one of the few places in which new neurons are continually added in adulthood, the region also shows a unique long-term sustained transcriptional response of the immediate-early gene Arc to sensory input. Although we know that adult-generated granule cells are reliably recruited into behaviorally-driven neuronal network, it remains unknown whether they display robust late-phase sustained transcription in response to activity like their developmentally-generated counterparts. Since this late-phase of transcription is required for enduring plasticity, knowing if sustained transcription appears as soon as these cells are incorporated provides information on their potential for plasticity. To address this question, adult F344 rats were injected with BrdU (50mg/kg/day for 5 days) and 4 weeks later explored a novel environment. Arc expression in both BrdU- and BrdU+ neurons was determined 0.5h, 1h, 2h, 6h, 8h, 12h, or 24h following this behavior. Recently-generated granule cells showed a robust sustained Arc expression following a discrete behavioral experience. These data provide information on a potential mechanism to sculpt the representations of events occurring within hours of each other to create uncorrelated representations of episodes despite a highly excitable population of neurons. PMID:26219984

  2. Effect of Cell Adhesion Molecule 1 Expression on Intracellular Granule Movement in Pancreatic α Cells.

    PubMed

    Yokawa, Satoru; Furuno, Tadahide; Suzuki, Takahiro; Inoh, Yoshikazu; Suzuki, Ryo; Hirashima, Naohide

    2016-09-01

    Although glucagon secreted from pancreatic α cells plays a role in increasing glucose concentrations in serum, the mechanism regulating glucagon secretion from α cells remains unclear. Cell adhesion molecule 1 (CADM1), identified as an adhesion molecule in α cells, has been reported not only to communicate among α cells and between nerve fibers, but also to prevent excessive glucagon secretion from α cells. Here, we investigated the effect of CADM1 expression on the movement of intracellular secretory granules in α cells because the granule transport is an important step in secretion. Spinning disk microscopic analysis showed that granules moved at a mean velocity of 0.236 ± 0.010 μm/s in the mouse α cell line αTC6 that expressed CADM1 endogenously. The mean velocity was significantly decreased in CADM1-knockdown (KD) cells (mean velocity: 0.190 ± 0.016 μm/s). The velocity of granule movement decreased greatly in αTC6 cells treated with the microtubule-depolymerizing reagent nocodazole, but not in αTC6 cells treated with the actin-depolymerizing reagent cytochalasin D. No difference in the mean velocity was observed between αTC6 and CADM1-KD cells treated with nocodazole. These results suggest that intracellular granules in pancreatic α cells move along the microtubule network, and that CADM1 influences their velocity. PMID:27262873

  3. An active membrane model of the cerebellar Purkinje cell II. Simulation of synaptic responses.

    PubMed

    De Schutter, E; Bower, J M

    1994-01-01

    1. Both excitatory and inhibitory postsynaptic channels were added to a previously described complex compartmental model of a cerebellar Purkinje cell to examine model responses to synaptic inputs. All model parameters remained as described previously, leaving maximum synaptic conductance as the only parameter that was tuned in the studies described in this paper. Under these conditions the model was capable of reproducing physiological recorded responses to each of the major types of synaptic input. 2. When excitatory synapses were activated on the smooth dendrites of the model, the model generated a complex dendritic Ca2+ spike similar to that generated by climbing fiber inputs. Examination of the model showed that activation of P-type Ca2+ channels in both the smooth and spiny dendrites augmented the depolarization during the complex spike and that Ca(2+)-activated K+ channels in the same dendritic regions determined the duration of the spike. When these synapses were activated under simulated current-clamp conditions the model also generated the characteristic dual reversal potential of the complex spike. The shape of the dendritic complex spike could be altered by changing the maximum conductance of the climbing fiber synapse and thus the amount of Ca2+ entering the cell. 3. To explore the background simple spike firing properties of Purkinje cells in vivo we added excitatory "parallel fiber" synapses to the spiny dendritic branches of the model. Continuous asynchronous activation of these granule cell synapses resulted in the generation of spontaneous sodium spikes. However, very low asynchronous input frequencies produced a highly regular, very fast rhythm (80-120 Hz), whereas slightly higher input frequencies resulted in Purkinje cell bursting. Both types of activity are uncharacteristic of in vivo Purkinje cell recordings. 4. Inhibitory synapses of the sort presumably generated by stellate cells were also added to the dendritic tree. When asynchronous

  4. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    SciTech Connect

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU{sup +} cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU{sup +} cells, very few are mash1{sup +} or nestin{sup +} stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1{sup +} microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition.

  5. Using human induced pluripotent stem cells to model cerebellar disease: Hope and hype

    PubMed Central

    Wiethoff, Sarah; Arber, Charles; Li, Abi; Wray, Selina; Houlden, Henry; Patani, Rickie

    2015-01-01

    The cerebellum forms a highly ordered and indispensible component of motor function within the adult neuraxis, consisting of several distinct cellular subtypes. Cerebellar disease, through a variety of genetic and acquired causes, results in the loss of function of defined subclasses of neurons, and remains a significant and untreatable health care burden. The scarcity of therapies in this arena can partially be explained by unresolved disease mechanisms due to inaccessibility of human cerebellar neurons in a relevant experimental context where initiating disease mechanisms could be functionally elucidated, or drug screens conducted. In this review we discuss the potential promise of human induced pluripotent stem cells (hiPSCs) for regenerative neurology, with a particular emphasis on in vitro modelling of cerebellar degeneration. We discuss progress made thus far using hiPSC-based models of neurodegeneration, noting the relatively slower pace of discovery made in modelling cerebellar dysfunction. We conclude by speculating how strategies attempting cerebellar differentiation from hiPSCs can be refined to allow the generation of accurate disease models. This in turn will permit a greater understanding of cerebellar pathophysiology to inform mechanistically rationalised therapies, which are desperately needed in this field. PMID:25985846

  6. Oligodendrocyte ablation affects the coordinated interaction between granule and Purkinje neurons during cerebellum development

    SciTech Connect

    Collin, Ludovic; Doretto, Sandrine; Malerba, Monica; Ruat, Martial; Borrelli, Emiliana . E-mail: borrelli@uci.edu

    2007-08-01

    Oligodendrocytes (OLs) are the glial cells of the central nervous system (CNS) classically known to be devoted to the formation of myelin sheaths around most axons of the vertebrate brain. We have addressed the role of these cells during cerebellar development, by ablating OLs in vivo. Previous analyses had indicated that OL ablation during the first six postnatal days results into a striking cerebellar phenotype, whose major features are a strong reduction of granule neurons and aberrant Purkinje cells development. These two cell types are highly interconnected during cerebellar development through the production of molecules that help their proliferation, differentiation and maintenance. In this article, we present data showing that OL ablation has major effects on the physiology of Purkinje (PC) and granule cells (GC). In particular, OL ablation results into a reduction of sonic hedgehog (Shh), Brain Derived Neurotrophic Factor (BDNF), and Reelin (Rln) expression. These results indicate that absence of OLs profoundly alters the normal cerebellar developmental program.

  7. Only a Minority of the Inhibitory Inputs to Cerebellar Golgi Cells Originates from Local GABAergic Cells123

    PubMed Central

    2016-01-01

    Abstract Cerebellar Golgi cells (GoCs) efficiently control the spiking activity of granule cells through GABAA receptor-mediated tonic and phasic inhibition. Recent experiments provided compelling evidence for the extensive interconnection of GoCs through electrical synapses, but their chemical inhibitory synaptic inputs are debated. Here, we investigated the GABAergic synaptic inputs of GoCs using in vitro electrophysiology and quantitative light microscopy (LM) and electron microscopy (EM). We characterized GABAA receptor-mediated IPSCs in GoCs and Lugaro cells (LuCs), and found that IPSCs in GoCs have lower frequencies, smaller amplitudes, and much slower decay kinetics. Pharmacological and LM immunolocalization experiments revealed that GoCs express α3, whereas LuCs express α1 subunit-containing GABAA receptors. The selective expression and clustered distribution of the α3 subunit in GoCs allowed the quantitative analysis of GABAergic synapses on their dendrites in the molecular layer (ML). EM and LM experiments in rats, and wild-type and GlyT2-GFP transgenic mice revealed that only one third of axon terminals establishing GABAergic synapses on GoC dendrites contain GlyT2, ruling out LuCs, globular cells, and any noncortical glycinergic inputs as major inhibitory sources. We also show that axon terminals of stellate/basket cells very rarely innervate GlyT2-GFP-expressing GoCs, indicating that only a minority of the inhibitory inputs to GoCs in the ML originates from local interneurons, and the majority of their inhibitory inputs exclusively releases GABA. PMID:27257627

  8. Neurotrophic effects of PACAP in the cerebellar cortex.

    PubMed

    Botia, Béatrice; Basille, Magali; Allais, Aurélie; Raoult, Emilie; Falluel-Morel, Anthony; Galas, Ludovic; Jolivel, Valérie; Wurtz, Olivier; Komuro, Hitoshi; Fournier, Alain; Vaudry, Hubert; Burel, Delphine; Gonzalez, Bruno J; Vaudry, David

    2007-09-01

    In the rodent cerebellum, PACAP is expressed by Purkinje neurons and PAC1 receptors are present on granule cells during both the development period and in adulthood. Treatment of granule neurons with PACAP inhibits proliferation, slows migration, promotes survival and induces differentiation. PACAP also protects cerebellar granule cells against the deleterious effects of neurotoxic agents. Most of the neurotrophic effects of PACAP are mediated through the cAMP/PKA signaling pathway and often involve the ERK MAPkinase. Caspase-3 is one of the key enzymes implicated in the neuroprotective action of PACAP but PACAP also inhibits caspase-9 activity and increases Bcl-2 expression. PACAP and functional PAC1 receptors are expressed in the monkey and human cerebellar cortex with a pattern of expression very similar to that described in rodents, suggesting that PACAP could also exert neurodevelopmental and neuroprotective functions in the cerebellum of primates including human. PMID:17544170

  9. Biochemical and microscopic evidence for the internalization and degradation of heparin-containing mast cell granules by bovine endothelial cells

    SciTech Connect

    Atkins, F.M.; Friedman, M.M.; Metcalfe, D.D.

    1985-03-01

    Incubation of (/sup 35/S)heparin-containing mast cell granules with cultured bovine endothelial cells was followed by the appearance of /sup 35/S-granule-associated radioactivity within the endothelial cells and a decrease in radioactivity in the extracellular fluid. These changes occurred during the first 24 hours of incubation and suggested ingestion of the mast cell granules by the endothelial cells. Periodic electron microscopic examination of the monolayers confirmed this hypothesis by demonstrating apposition of the granules to the plasmalemma of endothelial cells, which was followed by the engulfment of the granules by cytoplasmic projections. Under light microscopic examination, mast cell granules within endothelial cells then appeared to undergo degradation. The degradation of (/sup 35/S)heparin in mast cell granules was demonstrated by a decrease in the amount of intracellular (/sup 35/S)heparin proteoglycan after 24 hours and the appearance of free (/sup 35/S)sulfate in the extracellular compartment. Intact endothelial cells were more efficient at degrading (/sup 35/S)heparin than were cell lysates or cell supernatants. These data provide evidence of the ability of endothelial cells to ingest mast cell granules and degrade native heparin that is presented as a part of the mast cell granule.

  10. Enhanced CREB phosphorylation in immature dentate gyrus granule cells precedes neurotrophin expression and indicates a specific role of CREB in granule cell differentiation

    PubMed Central

    Bender, R. A.; Lauterborn, J. C.; Gall, C. M.; Cariaga, W.; Baram, T. Z.

    2011-01-01

    Differentiation and maturation of dentate gyrus granule cells requires coordinated interactions of numerous processes. These must be regulated by protein factors capable of integrating signals mediated through diverse signalling pathways. Such integrators of inter and intracellular physiological stimuli include the cAMP-response element binding protein (CREB), a leucine-zipper class transcription factor that is activated through phosphorylation. Neuronal activity and neurotrophic factors, known to be involved in granule cell differentiation, are major physiologic regulators of CREB function. To examine whether CREB may play a role in governing coordinated gene transcription during granule cell differentiation, we determined the spatial and temporal profiles of phosphorylated (activated) CREB throughout postnatal development in immature rat hippocampus. We demonstrate that CREB activation is confined to discrete, early stages of granule cell differentiation. In addition, CREB phosphorylation occurs prior to expression of the neurotrophins BDNF and NT-3. These data indicate that in a signal transduction cascade connecting CREB and neurotrophins in the process of granule cell maturation, CREB is located upstream of neurotrophins. Importantly, CREB may be a critical component of the machinery regulating the coordinated transcription of genes contributing to the differentiation of granule cells and their integration into the dentate gyrus network. PMID:11207803

  11. Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

    PubMed Central

    Lavut, Anna; Raveh, Dina

    2012-01-01

    Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance

  12. Nav2 hypomorphic mutant mice are ataxic and exhibit abnormalities in cerebellar development

    PubMed Central

    McNeill, Elizabeth M.; Klöckner-Bormann, Mariana; Roesler, Elizabeth C.; Talton, Lynn E.; Moechars, Dieder; Clagett-Dame, Margaret

    2011-01-01

    Development of the cerebellum involves a coordinated program of neuronal process outgrowth and migration resulting in a foliated structure that plays a key role in motor function. Neuron navigator 2 (Nav2) is a cytoskeletal-interacting protein that functions in neurite outgrowth and axonal elongation. Herein we show that hypomorphic mutant mice lacking the full-length Nav2 transcript exhibit ataxia and defects in cerebellar development. At embryonic day (E)17.5, the mutant cerebellum is reduced in size and exhibits defects in vermal foliation. Reduction in cell proliferation at early times (E12.5 and E14.5) may contribute to this size reduction. The full-length Nav2 transcript is expressed in the premigratory zone of the external granule layer (EGL). Granule cells in the germinal zone of the EGL appear to proliferate normally, however, due to the reduction in cerebellar circumference there are fewer total BrdU-labeled granule cells in the mutants, and these fail to migrate normally toward the interior of the cerebellum. In Nav2 hypomorphs, fewer granule cells migrate out of cerebellar EGL explants and neurite outgrowth from both explants and isolated external granule cell cultures is reduced. This suggests the formation of parallel axon fibers and neuronal migration is disrupted in Nav2 mutants. This work supports an essential role for full-length Nav2 in cerebellar development, including axonal elongation and migration of the EGL neurons. PMID:21419114

  13. Nav2 hypomorphic mutant mice are ataxic and exhibit abnormalities in cerebellar development.

    PubMed

    McNeill, Elizabeth M; Klöckner-Bormann, Mariana; Roesler, Elizabeth C; Talton, Lynn E; Moechars, Dieder; Clagett-Dame, Margaret

    2011-05-15

    Development of the cerebellum involves a coordinated program of neuronal process outgrowth and migration resulting in a foliated structure that plays a key role in motor function. Neuron navigator 2 (Nav2) is a cytoskeletal-interacting protein that functions in neurite outgrowth and axonal elongation. Herein we show that hypomorphic mutant mice lacking the full-length Nav2 transcript exhibit ataxia and defects in cerebellar development. At embryonic day (E)17.5, the mutant cerebellum is reduced in size and exhibits defects in vermal foliation. Reduction in cell proliferation at early times (E12.5 and E14.5) may contribute to this size reduction. The full-length Nav2 transcript is expressed in the premigratory zone of the external granule layer (EGL). Granule cells in the germinal zone of the EGL appear to proliferate normally, however, due to the reduction in cerebellar circumference there are fewer total BrdU-labeled granule cells in the mutants, and these fail to migrate normally toward the interior of the cerebellum. In Nav2 hypomorphs, fewer granule cells migrate out of cerebellar EGL explants and neurite outgrowth from both explants and isolated external granule cell cultures is reduced. This suggests that the formation of parallel axon fibers and neuronal migration is disrupted in Nav2 mutants. This work supports an essential role for full-length Nav2 in cerebellar development, including axonal elongation and migration of the EGL neurons. PMID:21419114

  14. Innovative macroporous granules of nanostructured-hydroxyapatite agglomerates: bioactivity and osteoblast-like cell behaviour.

    PubMed

    Laranjeira, M S; Fernandes, M H; Monteiro, F J

    2010-12-01

    To modulate the biological response of implantable granules, two types of bioactive porous granules composed of nanostructured-hydroxyapatite (HA) agglomerates and microstructured-HA, respectively, were prepared using a polyurethane sponge impregnation and burnout method. The resulting granules presented a highly porous structure with interconnected porosity. Both types of granules were characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and mercury intrusion porosimetry. Results showed that nanostructed-HA granules presented higher surface area and porosity than microstructured-HA granules. In vitro testing using MG63 human osteoblast-like cells showed that on both types of surfaces cells were able to adhere, proliferate, and migrate through the macropores, and a higher growth rate was achieved on nanostructured-HA granules than on microstructured-HA granules (76 and 40%, respectively). In addition, these cells maintained similar expression levels of osteoblastic-associated markers namely collagen type I, alkaline phosphatase, bone morphogenetic protein-2, macrophage colony-stimulating factor, and osteoprotegerin. These innovative nanostructured-HA granules may be considered as promising bioceramic alternative matrixes for bone regeneration and drug release application. PMID:20845490

  15. The stealthy nano-machine behind mast cell granule size distribution.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2015-01-01

    The classical model of mast cell secretory granule formation suggests that newly synthesized secretory mediators, transported from the rough endoplasmic reticulum to the Golgi complex, undergo post-transitional modification and are packaged for secretion by condensation within membrane-bound granules of unit size. These unit granules may fuse with other granules to form larger granules that reside in the cytoplasm until secreted. A novel stochastic model for mast cell granule growth and elimination (G&E) as well as inventory management is presented. Resorting to a statistical mechanics approach in which SNAP (Soluble NSF Attachment Protein) REceptor (SNARE) components are viewed as interacting particles, the G&E model provides a simple 'nano-machine' of SNARE self-aggregation that can perform granule growth and secretion. Granule stock is maintained as a buffer to meet uncertainty in demand by the extracellular environment and to serve as source of supply during the lead time to produce granules of adaptive content. Experimental work, mathematical calculations, statistical modeling and a rationale for the emergence of nearly last-in, first out inventory management, are discussed. PMID:24629227

  16. Characterization of the T-cell subpopulations in the granulation tissues of chronic suppurative otitis media

    PubMed Central

    WANG, BING; CHENG, YING; XU, MIN

    2016-01-01

    The present study aimed to investigate the potential involvement of specific T-cell subpopulations in granulation tissue formation in chronic suppurative otitis media (CSOM). Fifteen patients with CSOM were enrolled in this study. Granulation tissues were obtained from the middle ear cavity. Hematoxylin and eosin staining was performed for histopathological observation, and different T-cell subpopulations were characterized by immunohistochemistry. No evident association was identified between granulation tissue formation and disease course. The number of cluster of differentiation 8+ (CD8+) T cells, forkhead box P3+ (FOXP3+) regulatory T (Treg) cells and OX40+ T cells were significantly higher in granulation tissues from patients with ear discharge within the last 6 months compared to those without (P<0.05). Fresh granulation tissues had more CD8+ T cells and FOXP3+ Treg cells compared to the mature granulation tissues (P<0.05). There was a differential abundance of specific T-cell subpopulations in the granulation tissues in CSOM with different disease courses or with ear discharge, suggesting that T cell-mediated cellular immunity is involved in lesion formation of CSOM. PMID:27313854

  17. Clonal Analysis of Newborn Hippocampal Dentate Granule Cell Proliferation and Development in Temporal Lobe Epilepsy123

    PubMed Central

    LaSarge, Candi L.; McAuliffe, John J.

    2015-01-01

    Abstract Hippocampal dentate granule cells are among the few neuronal cell types generated throughout adult life in mammals. In the normal brain, new granule cells are generated from progenitors in the subgranular zone and integrate in a typical fashion. During the development of epilepsy, granule cell integration is profoundly altered. The new cells migrate to ectopic locations and develop misoriented “basal” dendrites. Although it has been established that these abnormal cells are newly generated, it is not known whether they arise ubiquitously throughout the progenitor cell pool or are derived from a smaller number of “bad actor” progenitors. To explore this question, we conducted a clonal analysis study in mice expressing the Brainbow fluorescent protein reporter construct in dentate granule cell progenitors. Mice were examined 2 months after pilocarpine-induced status epilepticus, a treatment that leads to the development of epilepsy. Brain sections were rendered translucent so that entire hippocampi could be reconstructed and all fluorescently labeled cells identified. Our findings reveal that a small number of progenitors produce the majority of ectopic cells following status epilepticus, indicating that either the affected progenitors or their local microenvironments have become pathological. By contrast, granule cells with “basal” dendrites were equally distributed among clonal groups. This indicates that these progenitors can produce normal cells and suggests that global factors sporadically disrupt the dendritic development of some new cells. Together, these findings strongly predict that distinct mechanisms regulate different aspects of granule cell pathology in epilepsy. PMID:26756038

  18. Studies on the pH gradient and histamine uptake of isolated mast cell granules

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1986-05-01

    A purified preparation of mast cell granules with intact perigranular membranes was obtained using a method involving probe sonication of rat serosal mast cells followed by differential centrifugation and Percoll gradient separation of the granules. Purification was assessed with histamine and mast cell granule protease assays. Granule integrity was demonstrated by light and electron microscopy and quantitated with a ruthenium red binding assay. The low yield of granules (20 ..mu..g protein/4 rats) necessitated the development of two microanalytical techniques to demonstrate the existence of a pH gradient across the membrane: 9-aminoacridine fluorescence studies in a cuvet with 50 ..mu..l capacity and /sup 14/C-methylamine distribution studies on microgram quantities of granule protein. Quantitation of results from isotope studies were confounded by the presence of oil used for separating granules from the aqueous phase. Nonetheless, an extrapolation procedure calibrated by external pH yielded an internal pH value of 5.46 +/- .03 (n = 4), consistent with values observed in granules obtained from other secretory cells. Collapse of the pH gradient by NH/sub 4//sup +/ or nigericin/KCl was demonstrated using either technique. Addition of histamine depressed intragranular pH, suggesting that histamine transport may utilize the ..delta..pH as a driving force.

  19. Internalized Tau sensitizes cells to stress by promoting formation and stability of stress granules

    PubMed Central

    Brunello, Cecilia A.; Yan, Xu; Huttunen, Henri J.

    2016-01-01

    Stress granules are membrane-less RNA- and RNA-binding protein-containing complexes that are transiently assembled in stressful conditions to promote cell survival. Several stress granule-associated RNA-binding proteins have been associated with neurodegenerative diseases. In addition, a close link was recently identified between the stress granule core-nucleating protein TIA-1 and Tau. Tau is a central pathological protein in Alzheimer’s disease and other tauopathies, and misfolded, aggregated Tau is capable of propagating pathology via cell-to-cell transmission. Here we show that following internalization hyperphosphorylated extracellular Tau associates with stress granules in a TIA-1 dependent manner. Cytosolic Tau normally only weakly interacts with TIA-1 but mutations mimicking abnormal phosphorylation promote this interaction. We show that internalized Tau significantly delays normal clearance of stress granules in the recipient cells sensitizing them to secondary stress. These results suggest that secreted Tau species may have properties, likely related to its hyperphosphorylation and oligomerization, which promote pathological association of internalized Tau with stress granules altering their dynamics and reducing cell viability. We suggest that stress granules and TIA-1 play a central role in the cell-to-cell transmission of Tau pathology. PMID:27460788

  20. Odontogenic Differentiation of Human Dental Pulp Stem Cells Stimulated by the Calcium Phosphate Porous Granules

    PubMed Central

    Nam, Sunyoung; Won, Jong-Eun; Kim, Cheol-Hwan; Kim, Hae-Won

    2011-01-01

    Effects of three-dimensional (3D) calcium phosphate (CaP) porous granules on the growth and odontogenic differentiation of human dental pulp stem cells (hDPSCs) were examined for dental tissue engineering. hDPSCs isolated from adult human dental pulps were cultured for 3-4 passages, and populated on porous granules. Cell growth on the culture dish showed an ongoing increase for up to 21 days, whereas the growth on the 3D granules decreased after 14 days. This reduction in proliferative potential on the 3D granules was more conspicuous under the osteogenic medium conditions, indicating that the 3D granules may induce the odontogenic differentiation of hDPSCs. Differentiation behavior on the 3D granules was confirmed by the increased alkaline phosphatase activity, up-regulation of odontoblast-specific genes, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1) by quantitative polymerase chain reaction, and greater level of dentin sialoprotein synthesis by western blot. Moreover, the cellular mineralization, as assessed by Alizarin red S and calcium quantification, was significantly higher in the 3D CaP granules than in the culture dish. Taken all, the 3D CaP porous granules should be useful for dental tissue engineering in combination with hDPSCs by providing favorable 3D substrate conditions for cell growth and odontogenic development. PMID:21772958

  1. Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

    SciTech Connect

    Gianotti, A.J.; Clark, D.T.; Dash, J. )

    1991-04-01

    A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent 'sphaerocrystals' located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.

  2. Granulated peripolar epithelial cells in the renal corpuscle of marine elasmobranch fish.

    PubMed

    Lacy, E R; Reale, E

    1989-07-01

    Granulated epithelial cells at the vascular pole of the renal corpuscle, peripolar cells, have been found in the kidneys of five species of elasmobranchs, the little skate (Raja erinacea), the smooth dogfish shark (Mustelus canis), the Atlantic sharpnose shark (Rhizoprionodon terraenovae), the scalloped hammerhead shark (Sphyrna lewini), and the cow-nosed ray (Rhinoptera bonasus). In a sixth elasmobranch, the spiny dogfish shark (Squalus acanthias), the peripolar cells could not be identified among numerous other granulated epithelial cells. The peripolar cells are located at the transition between the parietal epithelium of Bowman's capsule and the visceral epithelium (podocytes) of the glomerulus, thus forming a cuff-like arrangement surrounding the hilar vessels of the renal corpuscle. These cells may have granules and/or vacuoles. Electron microscopy shows that the granules are membrane-bounded, and contain either a homogeneous material or a paracrystalline structure with a repeating period of about 18 nm. The vacuoles are electron lucent or may contain remnants of a granule. These epithelial cells lie close to the granulated cells of the glomerular afferent arteriole. They correspond to the granular peripolar cells of the mammalian, avian and amphibian kidney. The present study is the first reported occurrence of peripolar cells in a marine organism or in either bony or cartilagenous fish. PMID:2519933

  3. Plasticity of intrinsic excitability in mature granule cells of the dentate gyrus

    PubMed Central

    Lopez-Rojas, Jeffrey; Heine, Martin; Kreutz, Michael R.

    2016-01-01

    The dentate gyrus is the main entry gate for cortical input to the hippocampus and one of the few brain areas where adult neurogenesis occurs. Several studies have shown that it is relatively difficult to induce synaptic plasticity in mature but not in newborn dentate granule cells. In the present work we have systematically addressed how classical protocols to induce synaptic plasticity affect action potential firing and intrinsic excitability in mature granule cells. We found that stimulation paradigms considered to be relevant for learning processes consistently modified the probability to generate action potentials in response to a given synaptic input in mature cells, in some paradigms even without any modification of synaptic strength. Collectively the results suggest that plasticity of intrinsic dendritic excitability has a lower induction-threshold than synaptic plasticity in mature granule cells and that this form of plasticity might be an important mechanism by which mature granule cells contribute to hippocampal function. PMID:26857841

  4. Formation of tRNA granules in the nucleus of heat-induced human cells

    SciTech Connect

    Miyagawa, Ryu; Mizuno, Rie; Watanabe, Kazunori; Ijiri, Kenichi

    2012-02-03

    Highlights: Black-Right-Pointing-Pointer tRNAs are tranlocated into the nucleus in heat-induced HeLa cells. Black-Right-Pointing-Pointer tRNAs form the unique granules in the nucleus. Black-Right-Pointing-Pointer tRNA ganules overlap with nuclear stress granules. -- Abstract: The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA{sup Met} (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA{sup Met} was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

  5. BK Channels Control Cerebellar Purkinje and Golgi Cell Rhythmicity In Vivo

    PubMed Central

    Cheron, Guy; Sausbier, Matthias; Sausbier, Ulrike; Neuhuber, Winfried; Ruth, Peter; Dan, Bernard; Servais, Laurent

    2009-01-01

    Calcium signaling plays a central role in normal CNS functioning and dysfunction. As cerebellar Purkinje cells express the major regulatory elements of calcium control and represent the sole integrative output of the cerebellar cortex, changes in neural activity- and calcium-mediated membrane properties of these cells are expected to provide important insights into both intrinsic and network physiology of the cerebellum. We studied the electrophysiological behavior of Purkinje cells in genetically engineered alert mice that do not express BK calcium-activated potassium channels and in wild-type mice with pharmacological BK inactivation. We confirmed BK expression in Purkinje cells and also demonstrated it in Golgi cells. We demonstrated that either genetic or pharmacological BK inactivation leads to ataxia and to the emergence of a beta oscillatory field potential in the cerebellar cortex. This oscillation is correlated with enhanced rhythmicity and synchronicity of both Purkinje and Golgi cells. We hypothesize that the temporal coding modification of the spike firing of both Purkinje and Golgi cells leads to the pharmacologically or genetically induced ataxia. PMID:19956720

  6. Consensus Paper: Neuroimmune Mechanisms of Cerebellar Ataxias.

    PubMed

    Mitoma, Hiroshi; Adhikari, Keya; Aeschlimann, Daniel; Chattopadhyay, Partha; Hadjivassiliou, Marios; Hampe, Christiane S; Honnorat, Jérôme; Joubert, Bastien; Kakei, Shinji; Lee, Jongho; Manto, Mario; Matsunaga, Akiko; Mizusawa, Hidehiro; Nanri, Kazunori; Shanmugarajah, Priya; Yoneda, Makoto; Yuki, Nobuhiro

    2016-04-01

    In the last few years, a lot of publications suggested that disabling cerebellar ataxias may develop through immune-mediated mechanisms. In this consensus paper, we discuss the clinical features of the main described immune-mediated cerebellar ataxias and address their presumed pathogenesis. Immune-mediated cerebellar ataxias include cerebellar ataxia associated with anti-GAD antibodies, the cerebellar type of Hashimoto's encephalopathy, primary autoimmune cerebellar ataxia, gluten ataxia, Miller Fisher syndrome, ataxia associated with systemic lupus erythematosus, and paraneoplastic cerebellar degeneration. Humoral mechanisms, cell-mediated immunity, inflammation, and vascular injuries contribute to the cerebellar deficits in immune-mediated cerebellar ataxias. PMID:25823827

  7. Dysfunctional cerebellar Purkinje cells contribute to autism-like behaviour in Shank2-deficient mice.

    PubMed

    Peter, Saša; Ten Brinke, Michiel M; Stedehouder, Jeffrey; Reinelt, Claudia M; Wu, Bin; Zhou, Haibo; Zhou, Kuikui; Boele, Henk-Jan; Kushner, Steven A; Lee, Min Goo; Schmeisser, Michael J; Boeckers, Tobias M; Schonewille, Martijn; Hoebeek, Freek E; De Zeeuw, Chris I

    2016-01-01

    Loss-of-function mutations in the gene encoding the postsynaptic scaffolding protein SHANK2 are a highly penetrant cause of autism spectrum disorders (ASD) involving cerebellum-related motor problems. Recent studies have implicated cerebellar pathology in the aetiology of ASD. Here we evaluate the possibility that cerebellar Purkinje cells (PCs) represent a critical locus of ASD-like pathophysiology in mice lacking Shank2. Absence of Shank2 impairs both PC intrinsic plasticity and induction of long-term potentiation at the parallel fibre to PC synapse. Moreover, inhibitory input onto PCs is significantly enhanced, most prominently in the posterior lobe where simple spike (SS) regularity is most affected. Using PC-specific Shank2 knockouts, we replicate alterations of SS regularity in vivo and establish cerebellar dependence of ASD-like behavioural phenotypes in motor learning and social interaction. These data highlight the importance of Shank2 for PC function, and support a model by which cerebellar pathology is prominent in certain forms of ASD. PMID:27581745

  8. Ethanol modulates facial stimulation-evoked outward currents in cerebellar Purkinje cells in vivo in mice.

    PubMed

    Wu, Mao-Cheng; Bing, Yan-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2016-01-01

    Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs. PMID:27489024

  9. Ethanol modulates facial stimulation-evoked outward currents in cerebellar Purkinje cells in vivo in mice

    PubMed Central

    Wu, Mao-Cheng; Bing, Yan-Hua; Chu, Chun-Ping; Qiu, De-Lai

    2016-01-01

    Acute ethanol overdose can induce dysfunction of cerebellar motor regulation and cerebellar ataxia. In this study, we investigated the effect of ethanol on facial stimulation-evoked inhibitory synaptic responses in cerebellar Purkinje cells (PCs) in urethane-anesthetized mice, using in vivo patch-clamp recordings. Under voltage-clamp conditions, ethanol (300 mM) decreased the amplitude, half-width, rise time and decay time of facial stimulation-evoked outward currents in PCs. The ethanol-induced inhibition of facial stimulation-evoked outward currents was dose-dependent, with an IC50 of 148.5 mM. Notably, the ethanol-induced inhibition of facial stimulation-evoked outward currents were significantly abrogated by cannabinoid receptor 1 (CB1) antagonists, AM251 and O-2050, as well as by the CB1 agonist WIN55212-2. Moreover, the ethanol-induced inhibition of facial stimulation-evoked outward currents was prevented by cerebellar surface perfusion of the PKA inhibitors H-89 and Rp-cAMP, but not by intracellular administration of the PKA inhibitor PKI. Our present results indicate that ethanol inhibits the facial stimulation-evoked outward currents by activating presynaptic CB1 receptors via the PKA signaling pathway. These findings suggest that ethanol overdose impairs sensory information processing, at least in part, by inhibiting GABA release from molecular layer interneurons onto PCs. PMID:27489024

  10. Cerebellar cortical degeneration in three English bulldogs: clinical and neuropathological findings.

    PubMed

    Gandini, G; Botteron, C; Brini, E; Fatzer, R; Diana, A; Jaggy, A

    2005-06-01

    This case report describes the clinical and neuropathological findings in three young English bulldogs affected by cerebellar cortical degeneration. The dogs, born from the same parents, were presented with clinical signs indicating progressive cerebellar dysfunction: a wide-based stance, severe cerebellar ataxia characterised by marked hypermetria, spasticity, and intention tremors of the head and trunk with loss of balance. On histopathological examination, lesions were confined to the cerebellum and consisted of diffuse degenerative cortical lesions, and there was a loss of Purkinje and granule cells. The history, clinical signs and neuropathological findings confirmed the diagnosis of cerebellar cortical degeneration. To the authors' knowledge, this is the first report of cerebellar cortical degeneration in the English bulldog. PMID:15971900

  11. Minocycline fails to protect cerebellar granular cell cultures against malonate-induced cell death.

    PubMed

    Fernandez-Gomez, F J; Gomez-Lazaro, M; Pastor, D; Calvo, S; Aguirre, N; Galindo, M F; Jordán, J

    2005-11-01

    Experimental and clinical studies support the view that the semisynthetic tetracycline minocycline exhibits neuroprotective roles in several models of neurodegenerative diseases, including ischemia, Huntington, Parkinson diseases, and amyotrophic lateral sclerosis. However, recent evidence indicates that minocycline does not always present beneficial actions. For instance, in an in vivo model of Huntington's disease, it fails to afford protection after malonate intrastriatal injection. Moreover, it reverses the neuroprotective effect of creatine in nigrostriatal dopaminergic neurons. This apparent contradiction prompted us to analyze the effect of this antibiotic on malonate-induced cell death. We show that, in rat cerebellar granular cells, the succinate dehydrogenase inhibitor malonate induces cell death in a concentration-dependent manner. By using DFCA, monochlorobimane and 10-N-nonyl-Acridin Orange to measure, respectively, H2O2-derived oxidant species and reduced forms of GSH and cardiolipin, we observed that malonate induced reactive oxygen species (ROS) production to an extent that surpasses the antioxidant defense capacity of the cells, resulting in GSH depletion and cardiolipin oxidation. The pre-treatment for 4 h with minocycline (10-100 microM) did not present cytoprotective actions. Moreover, minocycline failed to block ROS production and to abrogate malonate-induced oxidation of GSH and cardiolipin. Additional experiments revealed that minocycline was also unsuccessful to prevent the mitochondrial swelling induced by malonate. Furthermore, malonate did not induce the expression of the iNOS, caspase-3, -8, and -9 genes which have been shown to be up-regulated in several models where minocycline resulted cytoprotective. In addition, malonate-induced down-regulation of the antiapoptotic gene Bcl-2 was not prevented by minocycline, controversially the mechanism previously proposed to explain minocycline protective action. These results suggest that the

  12. Control of a simulated arm using a novel combination of Cerebellar learning mechanisms

    NASA Technical Reports Server (NTRS)

    Assad, C.; Hartmann, M.; Paulin, M. G.

    2001-01-01

    We present a model of cerebellar cortex that combines two types of learning: feedforward predicitve association based on local Hebbian-type learning between granule cell ascending branch and parallel fiber inputs, and reinforcement learning with feedback error correction based on climbing fiber activity.

  13. Activity-dependent plasticity of spike pauses in cerebellar Purkinje cells

    PubMed Central

    Grasselli, Giorgio; He, Qionger; Wan, Vivian; Adelman, John P.; Ohtsuki, Gen; Hansel, Christian

    2016-01-01

    Summary Plasticity of intrinsic excitability has been described in several types of neurons, but the significance of non-synaptic mechanisms in brain plasticity and learning remains elusive. Cerebellar Purkinje cells are inhibitory neurons that spontaneously fire action potentials at high frequencies and regulate activity in their target cells in the cerebellar nuclei by generating a characteristic spike burst–pause sequence upon synaptic activation. Using patch-clamp recordings from mouse Purkinje cells, we find that depolarization-triggered intrinsic plasticity enhances spike firing and shortens the duration of spike pauses. Pause plasticity is absent from mice lacking SK2-type potassium channels (SK2−/− mice) and in occlusion experiments using the SK channel blocker apamin, while apamin wash-in mimics pause reduction. Our findings demonstrate that spike pauses can be regulated through an activity-dependent, exclusively non-synaptic, SK2 channel-dependent mechanism and suggest that pause plasticity—by altering the Purkinje cell output—may be crucial to cerebellar information storage and learning. PMID:26972012

  14. Competition from newborn granule cells does not drive axonal retraction of silenced old granule cells in the adult hippocampus

    PubMed Central

    Lopez, Carla M.; Pelkey, Kenneth A.; Chittajallu, Ramesh; Nakashiba, Toshiaki; Tóth, Katalin; Tonegawa, Susumu; McBain, Chris J.

    2012-01-01

    In the developing nervous system synaptic refinement, typified by the neuromuscular junction where supernumerary connections are eliminated by axon retraction leaving the postsynaptic target innervated by a single dominant input, critically regulates neuronal circuit formation. Whether such competition-based pruning continues in established circuits of mature animals remains unknown. This question is particularly relevant in the context of adult neurogenesis where newborn cells must integrate into preexisting circuits, and thus, potentially compete with functionally mature synapses to gain access to their postsynaptic targets. The hippocampus plays an important role in memory formation/retrieval and the dentate gyrus (DG) subfield exhibits continued neurogenesis into adulthood. Therefore, this region contains both mature granule cells (old GCs) and immature recently born GCs that are generated throughout adult life (young GCs), providing a neurogenic niche model to examine the role of competition in synaptic refinement. Recent work from an independent group in developing animals indicated that embryonically/early postnatal generated GCs placed at a competitive disadvantage by selective expression of tetanus toxin (TeTX) to prevent synaptic release rapidly retracted their axons, and that this retraction was driven by competition from newborn GCs lacking TeTX. In contrast, following 3–6 months of selective TeTX expression in old GCs of adult mice we did not observe any evidence of axon retraction. Indeed ultrastructural analyses indicated that the terminals of silenced GCs even maintained synaptic contact with their postsynaptic targets. Furthermore, we did not detect any significant differences in the electrophysiological properties between old GCs in control and TeTX conditions. Thus, our data demonstrate a remarkable stability in the face of a relatively prolonged period of altered synaptic competition between two populations of neurons within the adult brain

  15. EFFECTS OF ORGANOTINS ON RACTIVE OXYGEN SPECIES AND INTRACELLULAR CALCIUM IN CEREBELLAR GRANULE CELLS IN CULTURE.

    EPA Science Inventory

    Use of organotins has increased drastically in the past decade, including their use as stabilizers in polyvinylchloride pipes. Monomethyl- (MMT), dimethyl- (DMT), monobutyl- (MBT), and dibutyltin (DBT) have been found in home water samples and in human blood at concentrations up...

  16. Metabolic pathways for the degradation of phosphatidic acid in isolated nuclei from cerebellar cells.

    PubMed

    Gaveglio, Virginia L; Pasquaré, Susana J; Giusto, Norma M

    2011-03-15

    The aim of the present research was to analyse the pathways for phosphatidic acid metabolism in purified nuclei from cerebellar cells. Lipid phosphate phosphatase and diacylglyceride lipase activities were detected in nuclei from cerebellar cells. It was observed that DAGL activity makes up 50% of LPP activity and that PtdOH can also be metabolised to lysophosphatidic acid. With a nuclear protein content of approximately 40 μg, the production of diacylglycerol and monoacylglycerol was linear for 30 min and 5 min, respectively, whereas it increased with PtdOH concentrations of up to 250 μM. LysoPtdOH, sphingosine 1-phosphate and ceramide 1-phosphate, which are alternative substrates for LPP, significantly reduced DAG production from PA. DAG and MAG production increased in the presence of Triton X-100 (1 mM) whereas no modifications were observed in the presence of ionic detergent sodium deoxycholate. Ca²+ and Mg²+ stimulated MAG production without affecting DAG formation whereas fluoride and vanadate inhibited the generation of both products. Specific PtdOH-phospholipase A1 and PtdOH-phospholipase A2 were also detected in nuclei. Our findings constitute the first reported evidence of active PtdOH metabolism involving LPP, DAGL and PtdOH-selective PLA activities in purified nuclei prepared from cerebellar cells. PMID:21216221

  17. Mesenchymal cell activation is the rate-limiting step of granulation tissue induction.

    PubMed Central

    McClain, S. A.; Simon, M.; Jones, E.; Nandi, A.; Gailit, J. O.; Tonnesen, M. G.; Newman, D.; Clark, R. A.

    1996-01-01

    During wound repair a 3-day lag occurs between injury and granulation tissue development. When full-thickness, 8-mm-round, excisional wounds were made in the paravertebral skin of outbred Yorkshire pigs and harvested at various times, no granulation tissue was observed before day 4. Day 4 wounds were 3% filled with granulation tissue, day 5 wounds 48% filled, and day 7 wounds 88% filled. The prerequisites for granulation tissue induction are not known but hypothetically include fibrin matrix maturation or cell activation. To examine whether matrix maturation was necessary, wounds were allowed to heal for 5 or 7 days and then aggressively curetted, resulting in the formation of fresh fibrin clots in the newly formed wound spaces. In contrast to original wounds, no lag phase was observed; wounds curetted on day 5 were 23% filled with granulation tissue 1 day later and 99% filled 3 days later, whereas wounds curetted on day 7 were 47% filled 1 day later and completely filled within 2 days. Thus, granulation tissue formation resumed promptly and independently of fibrin clot matrix maturation. This observation suggested that mesenchymal cell activation might be the rate-limiting step in granulation tissue formation. To address this hypothesis more directly, cultured porcine or human fibroblasts, grown to 80% confluence in Dulbecco's minimal essential medium plus 10% fetal calf serum, were added to new wounds. These wounds were sealed with a freshly made exogenous fibrin clot. In some wounds, platelet releasate was added to the fibrin clot. Granulation tissue did not form in day 3 wounds, which had received either fibrin alone, fibrin and platelet releasate, or fibrin and fibroblasts. In contrast, granulation tissue was observed in wounds receiving fibrin, human fibroblasts, and platelet releasate. By day 4, wounds receiving cultured human fibroblasts, fibrin, and platelet releasate were 14% filled with granulation tissue compared with less than 4% granulation tissue in

  18. Excitatory Cerebellar Nucleocortical Circuit Provides Internal Amplification during Associative Conditioning

    PubMed Central

    Gao, Zhenyu; Proietti-Onori, Martina; Lin, Zhanmin; ten Brinke, Michiel M.; Boele, Henk-Jan; Potters, Jan-Willem; Ruigrok, Tom J.H.; Hoebeek, Freek E.; De Zeeuw, Chris I.

    2016-01-01

    Summary Closed-loop circuitries between cortical and subcortical regions can facilitate precision of output patterns, but the role of such networks in the cerebellum remains to be elucidated. Here, we characterize the role of internal feedback from the cerebellar nuclei to the cerebellar cortex in classical eyeblink conditioning. We find that excitatory output neurons in the interposed nucleus provide efference-copy signals via mossy fibers to the cerebellar cortical zones that belong to the same module, triggering monosynaptic responses in granule and Golgi cells and indirectly inhibiting Purkinje cells. Upon conditioning, the local density of nucleocortical mossy fiber terminals significantly increases. Optogenetic activation and inhibition of nucleocortical fibers in conditioned animals increases and decreases the amplitude of learned eyeblink responses, respectively. Our data show that the excitatory nucleocortical closed-loop circuitry of the cerebellum relays a corollary discharge of premotor signals and suggests an amplifying role of this circuitry in controlling associative motor learning. PMID:26844836

  19. Measurement of the internal pH of mast cell granules using microvolumetric fluorescence and isotopic techniques

    SciTech Connect

    De Young, M.B.; Nemeth, E.F.; Scarpa, A.

    1987-04-01

    The intragranular pH of isolated mast cell granules was measured. Because of the minute amounts of isolated granules available, two techniques were developed by modifying aminoacridine fluorescence and (/sup 14/C)methylamine accumulation techniques to permit measurements with microliter sample volumes. Granule purity was demonstrated by electron microscopy, ruthenium red exclusion, and biochemical (histamine, mast cell granule protease) analysis. The internal pH was determined to be 5.55 +/- 0.06, indicating that the pH environment within mast cell granules is not significantly different from that of previously studied granule types (i.e., chromaffin, platelet, pancreatic islet, and pituitary granules). Collapse of the pH gradient by NH+4 was demonstrated with both techniques. No evidence of Cl-/OH- or specific cation/H+ transport was found, and major chloride permeability could not be unequivocably demonstrated. Ca/sup 2 +/ and Cl- at concentrations normally present extracellularly destabilized granules in the presence of NH+4, but this phenomenon does not necessarily indicate a role for these ions in the exocytotic release of granule contents from intact cells. The pH measurement techniques developed for investigating the properties of granules in mast cells may be useful for studying other granules that can be obtained only in limited quantities.

  20. Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

    PubMed Central

    Sánchez, J; Buendía, A J; Salinas, J; Bernabé, A; Rodolakis, A; Stewart, I J

    1996-01-01

    Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells. PMID:8751945

  1. Changes in Spontaneous Firing Patterns of Cerebellar Purkinje Cells in p75 Knockout Mice

    PubMed Central

    Tian, Jinbin; Tep, Chhavy; Zhu, Michael X.; Yoon, Sung Ok

    2013-01-01

    The p75 neurotrophin receptor is highly expressed in the developing nervous system and is required for neuronal survival, growth and synaptic transmission. In young mice, p75 is present in both granular cells and Purkinje cells of the cerebellum. Although p75 has been implicated in modulation of neuronal excitability in several neuronal types, whether and how it affects the excitability of cerebellar Purkinje neurons remained unclear. Using extracellular recordings of spontaneous firing of Purkinje neurons in cerebellar slices prepared from wild type and p75 knockout mice, we measured intrinsic firing properties in the presence of fast synaptic blockers of more than 200 Purkinje cells, each for a period of 5 min, for each genotype. We detected a significant increase in the mean firing frequency in p75−/− neurons comparing to the wild type littermates. Upon separating tonically firing from phasically firing cells, i.e. cells with firing pauses of longer than 300 msec, we observed that the change mainly arose from phasic firing cells and can be explained by an increase in the firing/silence ratio and a decrease in the number of long pauses during the 5-min recording period. We conclude that p75 plays an important role in regulating the firing-to-silence transition during the phasic firing period of the spontaneous firing of Purkinje cells. Thus, p75 exerts a modulatory function on Purkinje cell firing patterns, through which it may act as a key player in motor coordination and other cerebellum-regulated activities since Purkinje cells represent the sole neuronal output of the cerebellar cortex. PMID:23307658

  2. Quantitative and morphological analysis of dentate granule cells with recurrent basal dendrites from normal and epileptic rats.

    PubMed

    Dashtipour, Khashayar; Yan, Xiao-Xin; Dinh, Trinh T; Okazaki, Maxine M; Nadler, J Victor; Ribak, Charles E

    2002-01-01

    Granule cells with recurrent basal dendrites (RBDs) were previously reported in both control and epileptic rats. RBDs are dendrites that arise from the basal half of granule cell bodies and curve toward and extend into the molecular layer. They are increased in frequency in the pilocarpine model of epilepsy. The present study was undertaken to analyze the distribution and morphology of granule cells with RBDs and the synaptic connections of RBDs. Granule cells were labeled by retrograde transport of biocytin. Those with an RBD were found throughout the granule cell layer, but were most numerous at the hilar border. The morphology of these cells varied in the different depths of the granule cell layer; the angle of their cell body's long axis was mainly vertical at the hilar margin, and changed to virtually horizontal close to the molecular layer border. Quantitative data on the distribution of granule cells with RBDs and the angle of the cell body's long axis confirmed these descriptions. At the electron microscopic level, RBDs showed the typical features of dendrites and formed numerous axodendritic and axospinous synapses with labeled and unlabeled axon terminals. These results showed that RBDs of granule cells from epileptic rats are postsynaptic to axon terminals, including mossy fibers, and thus are involved in a similar synaptic circuitry as apical dendrites of granule cells from these animals. PMID:12000120

  3. Ultrastructural pathology of human peritumoural oedematous cerebellar cortex.

    PubMed

    Castejón, O J

    2016-01-01

    Cerebellar cortical biopsies of the peritumoural region of seven patients with cerebellar haemangioma, mesencephalic meningioma, cerebellopontine astrocytoma, cerebellopontine meningioma, and medulloblastoma of cerebellar vermis were examined by means of conventional transmission electron microscopy. Granule cells showed oedematous cytoplasm and mitochondria. Swollen Golgi cells exhibited lipofuscin granules and intranuclear inclusions. Both neuron cell types displayed swollen dendritic digits synapsing with afferent mossy fibre endings. Degenerated myelinated axons corresponding to afferent mossy and climbing fibres and efferent Purkinje cell axons were observed at the granular layer. Dense and clear ischaemic Purkinje cells established degenerated synapses with swollen parallel fibre synaptic varicosities. Degenerated Purkinje cell recurrent axonal collaterals were found at the molecular layer. Swollen and clear Bergmann glial cell cytoplasm was observed closely applied to the oedematous clear and dark Purkinje cell body, dendritic trunk, secondary and tertiary dendritic branches. Swollen climbing fibre endings featured by numerous microtubules and neurofilaments, and a decreased number of synaptic vesicles were observed making degenerated axo-spinodendritic synapses with clear and swollen dendritic spines from Purkinje, Golgi, basket and stellate cell dendrites. Swollen stellate neurons showed oedematous mitochondria. Lipofuscin-rich astrocytes and reactive phagocytic astrocytes were observed. The latter appeared engulfing haematogenous proteinaceous oedema fluid. All cerebellar neurons showed stress endoplasmic reticulum dysfunction featured by focal dilated cisterns and detachment of associated ribosomes. Myelin sheath degeneration was related with oligodendrocyte degenerating hydropic changes. The peritumoural ischaemic cerebellar nerve and glial cell abnormalities were related with neurobehavioral changes, tremor, nystagmus, dismetry and gait disturbance

  4. Single cell ganglioside catabolism in primary cerebellar neurons and glia

    PubMed Central

    Essaka, David C.; Prendergast, Jillian; Keithley, Richard B.; Hindsgaul, Ole; Palcic, Monica M.

    2013-01-01

    Cell-to-cell heterogeneity in ganglioside catabolism was determined by profiling fluorescent tetramethylrhodamine-labeled GM1 (TMR-GM1) breakdown in individual primary neurons and glia from the rat cerebellum. Cells isolated from 5–6 day old rat cerebella were cultured for 7 days, and then incubated for 14 h with TMR-GM1. Intact cells were recovered from cultures by mild proteolysis, paraformaldehyde fixed, and subjected to single cell analysis. Individual cells were captured in a capillary, lysed, and the released single-cell contents subjected to capillary electrophoresis with quantitative laser-induced fluorescent detection of the catabolic products. Non-neuronal cells on average took up much more exogenous TMR-GM1 than neuronal cells, and catabolized it more extensively. After 14 h of incubation, non-neuronal cells retained only 14% of the TMR products as GM1 and GM2, compared to >50% for neurons. On average, non-neuronal cells contained 74% of TMR-labeled product as TMR-ceramide, compared to only 42% for neurons. Non-neuronal cells retained seven times as much TMR-GM3 (7%) compared to neuronal cells (1%). To confirm the observed single cell metabolomics, we lysed and compared TMR-GM1 catabolic profiles from mixed neuron/glial cell cultures and from cultures depleted of non-neuronal cells by treatment with the antimitotic agent cytosine arabinoside. The whole culture catabolic profiles were consistent with the average profiles of single neurons and glia. We conclude that the ultrasensitive analytic methods described accurately reflect single cell ganglioside catabolism in different cell populations from the brain. PMID:22407243

  5. FGF-2 signal promotes proliferation of cerebellar progenitor cells and their oligodendrocytic differentiation at early postnatal stage

    SciTech Connect

    Naruse, Masae; Shibasaki, Koji; Ishizaki, Yasuki

    2015-08-07

    The origins and developmental regulation of cerebellar oligodendrocytes are largely unknown, although some hypotheses of embryonic origins have been suggested. Neural stem cells exist in the white matter of postnatal cerebellum, but it is unclear whether these neural stem cells generate oligodendrocytes at postnatal stages. We previously showed that cerebellar progenitor cells, including neural stem cells, widely express CD44 at around postnatal day 3. In the present study, we showed that CD44-positive cells prepared from the postnatal day 3 cerebellum gave rise to neurospheres, while CD44-negative cells prepared from the same cerebellum did not. These neurospheres differentiated mainly into oligodendrocytes and astrocytes, suggesting that CD44-positive neural stem/progenitor cells might generate oligodendrocytes in postnatal cerebellum. We cultured CD44-positive cells from the postnatal day 3 cerebellum in the presence of signaling molecules known as mitogens or inductive differentiation factors for oligodendrocyte progenitor cells. Of these, only FGF-2 promoted survival and proliferation of CD44-positive cells, and these cells differentiated into O4+ oligodendrocytes. Furthermore, we examined the effect of FGF-2 on cerebellar oligodendrocyte development ex vivo. FGF-2 enhanced proliferation of oligodendrocyte progenitor cells and increased the number of O4+ and CC1+ oligodendrocytes in slice cultures. These results suggest that CD44-positive cells might be a source of cerebellar oligodendrocytes and that FGF-2 plays important roles in their development at an early postnatal stage. - Highlights: • CD44 is expressed in cerebellar neural stem/progenitor cells at postnatal day 3 (P3). • FGF-2 promoted proliferation of CD44-positive progenitor cells from P3 cerebellum. • FGF-2 promoted oligodendrocytic differentiation of CD44-positive progenitor cells. • FGF-2 increased the number of oligodendrocytes in P3 cerebellar slice culture.

  6. Serglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity.

    PubMed

    Sutton, Vivien R; Brennan, Amelia J; Ellis, Sarah; Danne, Jill; Thia, Kevin; Jenkins, Misty R; Voskoboinik, Ilia; Pejler, Gunnar; Johnstone, Ricky W; Andrews, Daniel M; Trapani, Joseph A

    2016-03-01

    The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means. PMID:26756195

  7. Effects of norepinephrine on spontaneous firing activity of cerebellar Purkinje cells in vivo in mice.

    PubMed

    Guo, Ao; Feng, Jun-Yang; Li, Jia; Ding, Nan; Li, Ying-Jun; Qiu, De-Lai; Piao, Ri-Long; Chu, Chun-Ping

    2016-08-26

    Norepinephrine (NE), from the locus coeruleus (LC), has been supported to affect GABAergic system and parallel fiber (PF)-Purkinje cell (PC) synaptic transmission via adrenoceptor in cerebellum cortex. However, the effects of NE on the spontaneous spike activity of cerebellar PCs in living mouse have not yet been fully understood. We here examined the effects of NE on the spontaneous activity of PC in urethane-anesthetized mice by electrophysiological and pharmacological methods. Cerebellar surface application of NE (2.5-25μM) reduced the PC simple spike (SS) firing rate in a dose-dependent manner. The half-inhibitory concentration (IC50) was 5.97μM. In contrast, NE significantly increased the spontaneous firing rate of molecular layer interneuron (MLI). Application of GABAA receptor antagonist, gabazine (SR95531, 20μM) not only blocked the NE-induced inhibition of PC SS firing but also revealed NE-induced excitation of cerebellar PC. Blocking AMPA receptors activity enhanced NE-induced inhibition of PC spontaneous activity. Moreover, the effects of NE on PC spontaneous activity were abolished by simultaneously blocking GABAA and AMPA receptors activity. These results indicated that NE bidirectional modulated the spontaneous activity of PCs via enhancing both inhibitory inputs from MLIs and excitatory inputs of parallel fibers, but NE-induced enhance of inhibitory inputs overwhelmed the excitatory inputs under in vivo conditions. PMID:27369323

  8. Linalool suppresses voltage-gated currents in sensory neurons and cerebellar Purkinje cells.

    PubMed

    Narusuye, K; Kawai, F; Matsuzaki, K; Miyachi, E

    2005-02-01

    Linalool is a major component of essential oils and possesses various biological effects in sensory or central nervous systems. To investigate the pharmacological and biophysical effects of linalool on voltage-gated currents in sensory neurons, we used the whole-cell patch clamp and the Ca(2+) imaging techniques. Under the voltage clamp, membrane depolarization generated time- and voltage-dependent current responses in newt olfactory receptor cells (ORCs). Linalool significantly and reversibly suppressed the voltage-gated currents in ORCs. The dose-suppression relation of linalool for the voltage-gated Na(+) current could be fitted by the Hill equation with a half-blocking concentration of 0.56 mM and a Hill coefficient of 1.2. To test whether linalool suppresses voltage-gated currents in ORCs specifically or suppresses currents in other neurons generally, we next examined the effects of linalool on voltage-gated currents in newt retinal neurons and rat cerebellar Purkinje cells. Linalool suppressed the voltage-gated currents not only in retinal horizontal cells and ganglion cells but also in Purkinje cells. Furthermore, bath application of linalool inhibited the KCl-induced [Ca(2+)](i) response of ORCs, suggesting that linalool suppresses Ca(2+) currents in ORCs. These results suggest that linalool non-selectively suppresses the voltage-gated currents in newt sensory neurons and rat cerebellar Purkinje cells. PMID:15365786

  9. Protein Kinases Are Associated with Multiple, Distinct Cytoplasmic Granules in Quiescent Yeast Cells

    PubMed Central

    Shah, Khyati H.; Nostramo, Regina; Zhang, Bo; Varia, Sapna N.; Klett, Bethany M.; Herman, Paul K.

    2014-01-01

    The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival. PMID:25342717

  10. Nos2 Inactivation Promotes the Development of Medulloblastoma in Ptch1+/− Mice by Deregulation of Gap43–Dependent Granule Cell Precursor Migration

    PubMed Central

    Westrich, Viola; Karra, Daniela; Pfleger, Karin; Toedt, Grischa; Blond, Frederik; Delhomme, Nicolas; Hahn, Meinhard; Reifenberger, Julia; Reifenberger, Guido; Lichter, Peter

    2012-01-01

    Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1+/− mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1+/− mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1+/− Nos2−/− mice compared to Ptch1+/− Nos2+/+ mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1+/+ Nos2−/− mice but not from Ptch1+/− Nos2−/− mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1+/+ Nos2−/− mice but increased in Ptch1+/− Nos2−/− mice relative to Ptch1+/− Nos2+/+ mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1+/− mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during

  11. Dissociation of locomotor and cerebellar deficits in a murine Angelman syndrome model.

    PubMed

    Bruinsma, Caroline F; Schonewille, Martijn; Gao, Zhenyu; Aronica, Eleonora M A; Judson, Matthew C; Philpot, Benjamin D; Hoebeek, Freek E; van Woerden, Geeske M; De Zeeuw, Chris I; Elgersma, Ype

    2015-11-01

    Angelman syndrome (AS) is a severe neurological disorder that is associated with prominent movement and balance impairments that are widely considered to be due to defects of cerebellar origin. Here, using the cerebellar-specific vestibulo-ocular reflex (VOR) paradigm, we determined that cerebellar function is only mildly impaired in the Ube3am-/p+ mouse model of AS. VOR phase-reversal learning was singularly impaired in these animals and correlated with reduced tonic inhibition between Golgi cells and granule cells. Purkinje cell physiology, in contrast, was normal in AS mice as shown by synaptic plasticity and spontaneous firing properties that resembled those of controls. Accordingly, neither VOR phase-reversal learning nor locomotion was impaired following selective deletion of Ube3a in Purkinje cells. However, genetic normalization of αCaMKII inhibitory phosphorylation fully rescued locomotor deficits despite failing to improve cerebellar learning in AS mice, suggesting extracerebellar circuit involvement in locomotor learning. We confirmed this hypothesis through cerebellum-specific reinstatement of Ube3a, which ameliorated cerebellar learning deficits but did not rescue locomotor deficits. This double dissociation of locomotion and cerebellar phenotypes strongly suggests that the locomotor deficits of AS mice do not arise from impaired cerebellar cortex function. Our results provide important insights into the etiology of the motor deficits associated with AS. PMID:26485287

  12. Sun1 deficiency leads to cerebellar ataxia in mice

    PubMed Central

    Wang, Jing-Ya; Yu, I.-Shing; Huang, Chien-Chi; Chen, Chia-Yen; Wang, Wan-Ping; Lin, Shu-Wha; Jeang, Kuan-Teh; Chi, Ya-Hui

    2015-01-01

    ABSTRACT Migration and organization of the nucleus are essential for the proliferation and differentiation of cells, including neurons. However, the relationship between the positioning of the nucleus and cellular morphogenesis remains poorly understood. Inherited recessive cerebellar ataxia has been attributed to mutations in SYNE1, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. Regardless, Syne1-mutant mice present with normal cerebellar development. The Sad1-Unc-84 homology (SUN)-domain proteins are located at the inner nuclear membrane and recruit Syne proteins through the KASH domain to the outer nuclear membrane. Here, we report an unrecognized contribution of Sun1 and Sun2 to the postnatal development of murine cerebellum. Mice depleted of Sun1 showed a marked reduction in the cerebellar volume, and this phenotype is exacerbated with additional loss of a Sun2 allele. Consistent with these histological changes, Sun1−/− and Sun1−/−Sun2+/− mice exhibited defective motor coordination. Results of immunohistochemical analyses suggested that Sun1 is highly expressed in Purkinje cells and recruits Syne2 to the periphery of the nucleus. Approximately 33% of Purkinje cells in Sun1−/− mice and 66% of Purkinje cells in Sun1−/−Sun2+/− mice were absent from the surface of the internal granule layer (IGL), whereas the proliferation and migration of granule neurons were unaffected. Furthermore, the Sun1−/−Sun2+/− Purkinje cells exhibited retarded primary dendrite specification, reduced dendritic complexity and aberrant patterning of synapses. Our findings reveal a cell-type-specific role for Sun1 and Sun2 in nucleokinesis during cerebellar development, and we propose the use of Sun-deficient mice as a model for studying cerebellar ataxia that is associated with mutation of human SYNE genes or loss of Purkinje cells. PMID:26035387

  13. Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment

    PubMed Central

    Orcinha, Catarina; Münzner, Gert; Gerlach, Johannes; Kilias, Antje; Follo, Marie; Egert, Ulrich; Haas, Carola A.

    2016-01-01

    Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3–6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons. PMID:27516734

  14. Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment.

    PubMed

    Orcinha, Catarina; Münzner, Gert; Gerlach, Johannes; Kilias, Antje; Follo, Marie; Egert, Ulrich; Haas, Carola A

    2016-01-01

    Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons. PMID:27516734

  15. Cerebellar control of gait and interlimb coordination.

    PubMed

    Vinueza Veloz, María Fernanda; Zhou, Kuikui; Bosman, Laurens W J; Potters, Jan-Willem; Negrello, Mario; Seepers, Robert M; Strydis, Christos; Koekkoek, Sebastiaan K E; De Zeeuw, Chris I

    2015-11-01

    Synaptic and intrinsic processing in Purkinje cells, interneurons and granule cells of the cerebellar cortex have been shown to underlie various relatively simple, single-joint, reflex types of motor learning, including eyeblink conditioning and adaptation of the vestibulo-ocular reflex. However, to what extent these processes contribute to more complex, multi-joint motor behaviors, such as locomotion performance and adaptation during obstacle crossing, is not well understood. Here, we investigated these functions using the Erasmus Ladder in cell-specific mouse mutant lines that suffer from impaired Purkinje cell output (Pcd), Purkinje cell potentiation (L7-Pp2b), molecular layer interneuron output (L7-Δγ2), and granule cell output (α6-Cacna1a). We found that locomotion performance was severely impaired with small steps and long step times in Pcd and L7-Pp2b mice, whereas it was mildly altered in L7-Δγ2 and not significantly affected in α6-Cacna1a mice. Locomotion adaptation triggered by pairing obstacle appearances with preceding tones at fixed time intervals was impaired in all four mouse lines, in that they all showed inaccurate and inconsistent adaptive walking patterns. Furthermore, all mutants exhibited altered front-hind and left-right interlimb coordination during both performance and adaptation, and inconsistent walking stepping patterns while crossing obstacles. Instead, motivation and avoidance behavior were not compromised in any of the mutants during the Erasmus Ladder task. Our findings indicate that cell type-specific abnormalities in cerebellar microcircuitry can translate into pronounced impairments in locomotion performance and adaptation as well as interlimb coordination, highlighting the general role of the cerebellar cortex in spatiotemporal control of complex multi-joint movements. PMID:25139623

  16. Doppel Induces Degeneration of Cerebellar Purkinje Cells Independently of Bax

    PubMed Central

    Dong, Jiaxin; Li, Aimin; Yamaguchi, Naohiro; Sakaguchi, Suehiro; Harris, David A.

    2007-01-01

    Doppel (Dpl) is a prion protein paralog that causes neurodegeneration when expressed ectopically in the brain. To investigate the cellular mechanism underlying this effect, we analyzed Dpl-expressing transgenic mice in which the gene for the proapoptotic protein Bax had been deleted. We found that Bax deletion does not alter either clinical symptoms or Purkinje cell degeneration in Dpl transgenic mice. In addition, we observed that degenerating Purkinje cells in these animals do not display DNA fragmentation or caspase-3 activation. Our results suggest that non-Bax-dependent pathways mediate the toxic effects of Dpl in Purkinje cells, highlighting a possible role for nonapoptotic mechanisms in the death of these neurons. PMID:17569776

  17. The econobiology of pancreatic acinar cells granule inventory and the stealthy nano-machine behind it.

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2016-03-01

    The pancreatic gland secretes most of the enzymes and many other macromolecules needed for food digestion in the gastrointestinal tract. These molecules play an important role in digestion, host defense and lubrication. The secretion of pancreatic proteins ensures the availability of the correct mix of proteins when needed. This review describes model systems available for the study of the econobiology of secretory granule content. The secretory pancreatic molecules are stored in large dense-core secretory granules that may undergo either constitutive or evoked secretion, and constitute the granule inventory of the cell. It is proposed that the Golgi complex functions as a distribution center for secretory proteins in pancreatic acinar cells, packing the newly formed secretory molecules into maturing secretory granules, also known functionally as condensing vacuoles. Mathematical modelling brings forward a process underlying granule inventory maintenance at various physiological states of condensation and aggregation by homotypic fusion. These models suggest unique but simple mechanisms accountable for inventory buildup and size, as well as for the distribution of secretory molecules into different secretory pathways in pancreatic acinar cells. PMID:26702787

  18. Dependence of structure stability and integrity of aerobic granules on ATP and cell communication.

    PubMed

    Jiang, Bo; Liu, Yu

    2013-06-01

    Aerobic granules are dense and compact microbial aggregates with various bacterial species. Recently, aerobic granulation technology has been extensively explored for treatment of municipal and industrial wastewaters. However, little information is currently available with regard to their structure stability and integrity at levels of energy metabolism and cell communication. In the present study, a typical chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide with the power to dissipate proton motive force and subsequently inhibit adenosine triphosphate (ATP) generation, was used to investigate possible roles of ATP and cell communication in maintaining the structure stability and integrity of aerobic granules. It was found that inhibited ATP synthesis resulted in the reduced production of autoinducer-2 and N-acylhomoserine lactones essential for cell communication, while lowered extracellular polymeric substance (EPS) production was also observed. As a consequence, aerobic granules appeared to break up. This study showed that ATP-dependent quorum sensing and EPS were essential for sustaining the structure stability and integrity of aerobic granules. PMID:23011346

  19. Stimulation of mast cells leads to cholesterol accumulation in macrophages in vitro by a mast cell granule-mediated uptake of low density lipoprotein

    SciTech Connect

    Kokkonen, J.O.; Kovanen, P.T.

    1987-04-01

    The uptake of low density lipoprotein (LDL) by cultured mouse macrophages was markedly promoted by isolated rat mast cell granules present in the culture medium. The granule-mediated uptake of /sup 125/I-LDL enhanced the rate of cholesteryl ester synthesis in the macrophages, the result being accumulation of cholesteryl esters in these cells. Binding of LDL to the granules was essential for the granule-mediated uptake of LDL by macrophages, for the uptake process was prevented by treating the granules with avidin or protamine chloride or by treating LDL with 1,2-cyclohexanedione, all of which inhibit the binding of LDL to the granules. Inhibition of granule phagocytosis by the macrophages with cytochalasin B also abolished the granule-mediated uptake of LDL. Finally, mouse macrophage monolayers and LDL were incubated in the presence of isolated rat serosal mast cells. Stimulation of the mast cells with compound 48/80, a degranulating agent, resulted in dose-dependent release of secretory granules from the mast cells and a parallel increase in /sup 14/C cholesteryl ester synthesis in the macrophages. The results show that, in this in vitro model, the sequence of events leading to accumulation of cholesteryl esters in macrophages involves initial stimulation of mast cells, subsequent release of their secretory granules, binding of LDL to the exocytosed granules, and, finally, phagocytosis of the LDL-containing granules by macrophages.

  20. Interneuron- and GABAA receptor-specific inhibitory synaptic plasticity in cerebellar Purkinje cells

    NASA Astrophysics Data System (ADS)

    He, Qionger; Duguid, Ian; Clark, Beverley; Panzanelli, Patrizia; Patel, Bijal; Thomas, Philip; Fritschy, Jean-Marc; Smart, Trevor G.

    2015-07-01

    Inhibitory synaptic plasticity is important for shaping both neuronal excitability and network activity. Here we investigate the input and GABAA receptor subunit specificity of inhibitory synaptic plasticity by studying cerebellar interneuron-Purkinje cell (PC) synapses. Depolarizing PCs initiated a long-lasting increase in GABA-mediated synaptic currents. By stimulating individual interneurons, this plasticity was observed at somatodendritic basket cell synapses, but not at distal dendritic stellate cell synapses. Basket cell synapses predominantly express β2-subunit-containing GABAA receptors; deletion of the β2-subunit ablates this plasticity, demonstrating its reliance on GABAA receptor subunit composition. The increase in synaptic currents is dependent upon an increase in newly synthesized cell surface synaptic GABAA receptors and is abolished by preventing CaMKII phosphorylation of GABAA receptors. Our results reveal a novel GABAA receptor subunit- and input-specific form of inhibitory synaptic plasticity that regulates the temporal firing pattern of the principal output cells of the cerebellum.

  1. The glycosylation pattern of secretory granules in binucleate trophoblast cells is highly conserved in ruminants.

    PubMed

    Klisch, K; Wooding, F B P; Jones, C J P

    2010-01-01

    The binucleate trophoblast cells (BNCs) in the ruminant placenta are a unique feature of this taxon. These cells produce several secretory proteins and transfer these across the fetomaternal barrier into the dam. We used lectin histochemistry with a panel of 24 lectins to characterise the glycosylation pattern of BNC secretory granules in a variety of ruminants. Seven species out of three ruminant families were thus investigated: greater malayan chevrotain (Tragulidae); fallow deer, red deer, chinese water deer (Cervidae); and domestic goat, springbok, impala (Bovidae). BNC granules in all species studied strongly expressed tri-/tetraantennary complex N-glycans and bisecting N-acetylglucosamine [GlcNAc] as shown by binding of leuco- and erythroagglutins of Phaseolus vulgaris respectively. The presence of terminal N-acetylgalactosamine [GalNAc]) in BNC granules is shown by intense staining with lectins from Dolichos biflorus, Vicia villosa and Wisteria floribunda. Terminal galactose or GalNAc was also present, bound by Glycine max agglutinin. Treatment of slides with neuraminidase strongly intensified staining of Erythrina cristagalli lectin (ECA) to terminal lactosamine in all species studied; this was otherwise absent except in goat. Sambucus nigra-1 lectin bound to BNC granules in all species except in Impala, indicating the presence of abundant alpha2,6 linked sialic acid. These results indicate that these unusual highly branched glycans, with bisecting GlcNAc and terminal GalNAc are a general feature of BNC granules in Ruminants, including the most basal Tragulid branch. It therefore appears that the specific glycosylation pattern of BNC granules evolved early in ruminant phylogenesis, together with the appearance of BNC. The conserved glycan structure in BNC secretory granules indicates that this pattern of glycosylation is likely to be of considerable functional importance for the secretory glycoproteins of ruminant BNC. PMID:19959226

  2. Cytocompatibility of porous biphasic calcium phosphate granules with human mesenchymal cells by a multiparametric assay.

    PubMed

    Mitri, Fabio; Alves, Gutemberg; Fernandes, Gustavo; König, Bruno; Rossi, Alexandre J R; Granjeiro, Jose

    2012-06-01

    This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (β-TCP) (60:40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100°C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/β-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications. PMID:22372877

  3. High frequency burst firing of granule cells ensures transmission at the parallel fiber to purkinje cell synapse at the cost of temporal coding.

    PubMed

    van Beugen, Boeke J; Gao, Zhenyu; Boele, Henk-Jan; Hoebeek, Freek; De Zeeuw, Chris I

    2013-01-01

    Cerebellar granule cells (GrCs) convey information from mossy fibers (MFs) to Purkinje cells (PCs) via their parallel fibers (PFs). MF to GrC signaling allows transmission of frequencies up to 1 kHz and GrCs themselves can also fire bursts of action potentials with instantaneous frequencies up to 1 kHz. So far, in the scientific literature no evidence has been shown that these high-frequency bursts also exist in awake, behaving animals. More so, it remains to be shown whether such high-frequency bursts can transmit temporally coded information from MFs to PCs and/or whether these patterns of activity contribute to the spatiotemporal filtering properties of the GrC layer. Here, we show that, upon sensory stimulation in both un-anesthetized rabbits and mice, GrCs can show bursts that consist of tens of spikes at instantaneous frequencies over 800 Hz. In vitro recordings from individual GrC-PC pairs following high-frequency stimulation revealed an overall low initial release probability of ~0.17. Nevertheless, high-frequency burst activity induced a short-lived facilitation to ensure signaling within the first few spikes, which was rapidly followed by a reduction in transmitter release. The facilitation rate among individual GrC-PC pairs was heterogeneously distributed and could be classified as either "reluctant" or "responsive" according to their release characteristics. Despite the variety of efficacy at individual connections, grouped activity in GrCs resulted in a linear relationship between PC response and PF burst duration at frequencies up to 300 Hz allowing rate coding to persist at the network level. Together, these findings support the hypothesis that the cerebellar granular layer acts as a spatiotemporal filter between MF input and PC output (D'Angelo and De Zeeuw, 2009). PMID:23734102

  4. Neuroprotective Effect of Carnosine on Primary Culture of Rat Cerebellar Cells under Oxidative Stress.

    PubMed

    Lopachev, A V; Lopacheva, O M; Abaimov, D A; Koroleva, O V; Vladychenskaya, E A; Erukhimovich, A A; Fedorova, T N

    2016-05-01

    Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells. PMID:27297901

  5. Role of Tet1/3 Genes and Chromatin Remodeling Genes in Cerebellar Circuit Formation.

    PubMed

    Zhu, Xiaodong; Girardo, David; Govek, Eve-Ellen; John, Keisha; Mellén, Marian; Tamayo, Pablo; Mesirov, Jill P; Hatten, Mary E

    2016-01-01

    Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are upregulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of upregulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNAi in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry. PMID:26711116

  6. Cerebellar cortex development in the weaver condition presents regional and age-dependent abnormalities without differences in Purkinje cells neurogenesis.

    PubMed

    Martí, Joaquín; Santa-Cruz, María C; Hervás, José P; Bayer, Shirley A; Villegas, Sandra

    2016-01-01

    Ataxias are neurological disorders associated with the degeneration of Purkinje cells (PCs). Homozygous weaver mice (wv/wv) have been proposed as a model for hereditary cerebellar ataxia because they present motor abnormalities and PC loss. To ascertain the physiopathology of the weaver condition, the development of the cerebellar cortex lobes was examined at postnatal day (P): P8, P20 and P90. Three approaches were used: 1) quantitative determination of several cerebellar features; 2) qualitative evaluation of the developmental changes occurring in the cortical lobes; and 3) autoradiographic analyses of PC generation and placement. Our results revealed a reduction in the size of the wv/wv cerebellum as a whole, confirming previous results. However, as distinguished from these reports, we observed that quantified parameters contribute differently to the abnormal growth of the wv/wv cerebellar lobes. Qualitative analysis showed anomalies in wv/wv cerebellar cytoarchitecture, depending on the age and lobe analyzed. Such abnormalities included the presence of the external granular layer after P20 and, at P90, ectopic cells located in the molecular layer following several placement patterns. Finally, we obtained autoradiographic evidence that wild-type and wv/wv PCs presented similar neurogenetic timetables, as reported. However, the innovative character of this current work lies in the fact that the neurogenetic gradients of wv/wv PCs were not modified from P8 to P90. A tendency for the accumulation of late-formed PCs in the anterior and posterior lobes was found, whereas early-generated PCs were concentrated in the central and inferior lobes. These data suggested that wv/wv PCs may migrate properly to their final destinations. The extrapolation of our results to patients affected with cerebellar ataxias suggests that all cerebellar cortex lobes are affected with several age-dependent alterations in cytoarchitectonics. We also propose that PC loss may be regionally

  7. The Changeable Nervous System: Studies On Neuroplasticity In Cerebellar Cultures

    PubMed Central

    Seil, Fredrick J.

    2014-01-01

    Circuit reorganization after injury was studied in a cerebellar culture model. When cerebellar cultures derived from newborn mice were exposed at explantation to a preparation of cytosine arabinoside that destroyed granule cells and oligodendrocytes and compromised astrocytes, Purkinje cells surviving in greater than usual numbers were unensheathed by astrocytic processes and received twice the control number of inhibitory axosomatic synapses. Purkinje cell axon collaterals sprouted and many of their terminals formed heterotypical synapses with other Purkinje cell dendritic spines. The resulting circuit reorganization preserved inhibition in the cerebellar cortex. Following this reorganization, replacement of the missing granule cells and glia was followed by a restitution of the normal circuitry. Most of these developmental and reconstructive changes were not dependent on neuronal activity, the major exception being inhibitory synaptogenesis. The full complement of inhibitory synapses did not develop in the absence of neuronal activity, which could be mitigated by application of exogenous TrkB receptor ligands. Inhibitory synaptogenesis could also be promoted by activity-induced release of endogenous TrkB receptor ligands or by antibody activation of the TrkB receptor. PMID:24933693

  8. Requirement of TrkB for synapse elimination in developing cerebellar Purkinje cells

    PubMed Central

    Bosman, Laurens W. J.; Hartmann, Jana; Barski, Jaroslaw J.; Lepier, Alexandra; Noll-Hussong, Michael; Reichardt, Louis F.; Konnerth, Arthur

    2009-01-01

    The receptor tyrosine kinase TrkB and its ligands, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), are critically important for growth, survival and activity-dependent synaptic strengthening in the central nervous system. These TrkB-mediated actions occur in a highly cell-type specific manner. Here we report that cerebellar Purkinje cells, which are richly endowed with TrkB receptors, develop a normal morphology in trkB-deficient mice. Thus, in contrast to other types of neurons, Purkinje cells do not need TrkB for dendritic growth and spine formation. Instead, we find a moderate delay in the maturation of GABAergic synapses and, more importantly, an abnormal multiple climbing fiber innervation in Purkinje cells in trkB-deficient mice. Thus, our results demonstrate an involvement of TrkB receptors in synapse elimination and reveal a new role for receptor tyrosine kinases in the brain. PMID:17940915

  9. Stereotyped spatial patterns of functional synaptic connectivity in the cerebellar cortex.

    PubMed

    Valera, Antoine M; Binda, Francesca; Pawlowski, Sophie A; Dupont, Jean-Luc; Casella, Jean-François; Rothstein, Jeffrey D; Poulain, Bernard; Isope, Philippe

    2016-01-01

    Motor coordination is supported by an array of highly organized heterogeneous modules in the cerebellum. How incoming sensorimotor information is channeled and communicated between these anatomical modules is still poorly understood. In this study, we used transgenic mice expressing GFP in specific subsets of Purkinje cells that allowed us to target a given set of cerebellar modules. Combining in vitro recordings and photostimulation, we identified stereotyped patterns of functional synaptic organization between the granule cell layer and its main targets, the Purkinje cells, Golgi cells and molecular layer interneurons. Each type of connection displayed position-specific patterns of granule cell synaptic inputs that do not strictly match with anatomical boundaries but connect distant cortical modules. Although these patterns can be adjusted by activity-dependent processes, they were found to be consistent and predictable between animals. Our results highlight the operational rules underlying communication between modules in the cerebellar cortex. PMID:26982219

  10. Radioresistance of granulation tissue-derived cells from skin wounds combined with total body irradiation.

    PubMed

    Dai, Tingyu; Chen, Zelin; Tan, Li; Shi, Chunmeng

    2016-04-01

    Combined radiation and wound injury (CRWI) occurs following nuclear explosions and accidents, radiological or nuclear terrorism, and radiation therapy combined with surgery. CRWI is complicated and more difficult to heal than single injuries. Stem cell‑based therapy is a promising treatment strategy for CRWI, however, sourcing stem cells remains a challenge. In the present study, the granulation tissue-derived cells (GTCs) from the skin wounds (SWs) of CRWI mice (C‑GTCs) demonstrated a higher radioresistance to the damage caused by combined injury, and were easier to isolate and harvest when compared with bone marrow‑derived mesenchymal stromal cells (BMSCs). Furthermore, the C-GTCs exhibited similar stem cell-associated properties, such as self-renewal and multilineage differentiation capacity, when compared with neonatal dermal stromal cells (DSCs) and GTCs from unirradiated SWs. Granulation tissue, which is easy to access, may present as an optimal autologous source of stem/progenitor cells for therapeutic applications in CRWI. PMID:26936439

  11. Plasticity of cerebellar Purkinje cells in behavioral training of body balance control

    PubMed Central

    Lee, Ray X.; Huang, Jian-Jia; Huang, Chiming; Tsai, Meng-Li; Yen, Chen-Tung

    2015-01-01

    Neural responses to sensory inputs caused by self-generated movements (reafference) and external passive stimulation (exafference) differ in various brain regions. The ability to differentiate such sensory information can lead to movement execution with better accuracy. However, how sensory responses are adjusted in regard to this distinguishability during motor learning is still poorly understood. The cerebellum has been hypothesized to analyze the functional significance of sensory information during motor learning, and is thought to be a key region of reafference computation in the vestibular system. In this study, we investigated Purkinje cell (PC) spike trains as cerebellar cortical output when rats learned to balance on a suspended dowel. Rats progressively reduced the amplitude of body swing and made fewer foot slips during a 5-min balancing task. Both PC simple (SSs; 17 of 26) and complex spikes (CSs; 7 of 12) were found to code initially on the angle of the heads with respect to a fixed reference. Using periods with comparable degrees of movement, we found that such SS coding of information in most PCs (10 of 17) decreased rapidly during balance learning. In response to unexpected perturbations and under anesthesia, SS coding capability of these PCs recovered. By plotting SS and CS firing frequencies over 15-s time windows in double-logarithmic plots, a negative correlation between SS and CS was found in awake, but not anesthetized, rats. PCs with prominent SS coding attenuation during motor learning showed weaker SS-CS correlation. Hence, we demonstrate that neural plasticity for filtering out sensory reafference from active motion occurs in the cerebellar cortex in rats during balance learning. SS-CS interaction may contribute to this rapid plasticity as a form of receptive field plasticity in the cerebellar cortex between two receptive maps of sensory inputs from the external world and of efference copies from the will center for volitional movements

  12. Structural, mass and elemental analyses of storage granules in methanogenic archaeal cells

    PubMed Central

    Toso, Daniel B.; Henstra, Anne M.; Gunsalus, Robert P.; Zhou, Z. Hong

    2013-01-01

    Summary Storage granules are an important component of metabolism in many organisms spanning the bacterial, eukaryal and archaeal domains, but systematic analysis of their organization inside cells is lacking. In this study, we identify and characterize granulelike inclusion bodies in a methanogenic archaeon, Methanospirillum hungatei, an anaerobic microorganism that plays an important role in nutrient recycling in the ecosystem. Using cryo electron microscopy, we show that granules in mature M. hungatei are amorphous in structure with a uniform size. Energy dispersive X-ray spectroscopy analysis establishes that each granule is a polyphosphate body (PPB) that consists of high concentrations of phosphorous and oxygen, and increased levels of iron and magnesium. By scanning transmission electron tomography, we further estimate that the mass density within a PPB is a little less than metal titanium at room temperature and is about four times higher than that of the surrounding cytoplasm. Finally, three-dimensional cryo electron tomography reveals that PPBs are positioned off-centre in their radial locations relative to the cylindrical axis of the cell, and almost uniformly placed near cell ends. This positioning ability points to a genetic program that spatially and temporally directs the accumulation of polyphosphate into a storage granule, perhaps for energy-consuming activities, such as cell maintenance, division or motility. PMID:21854518

  13. Induced pluripotent stem cell technology for modelling and therapy of cerebellar ataxia

    PubMed Central

    Watson, Lauren M.; Wong, Maggie M. K.; Becker, Esther B. E.

    2015-01-01

    Induced pluripotent stem cell (iPSC) technology has emerged as an important tool in understanding, and potentially reversing, disease pathology. This is particularly true in the case of neurodegenerative diseases, in which the affected cell types are not readily accessible for study. Since the first descriptions of iPSC-based disease modelling, considerable advances have been made in understanding the aetiology and progression of a diverse array of neurodegenerative conditions, including Parkinson's disease and Alzheimer's disease. To date, however, relatively few studies have succeeded in using iPSCs to model the neurodegeneration observed in cerebellar ataxia. Given the distinct neurodevelopmental phenotypes associated with certain types of ataxia, iPSC-based models are likely to provide significant insights, not only into disease progression, but also to the development of early-intervention therapies. In this review, we describe the existing iPSC-based disease models of this heterogeneous group of conditions and explore the challenges associated with generating cerebellar neurons from iPSCs, which have thus far hindered the expansion of this research. PMID:26136256

  14. Numb deficiency in cerebellar Purkinje cells impairs synaptic expression of metabotropic glutamate receptor and motor coordination.

    PubMed

    Zhou, Liang; Yang, Dong; Wang, De-Juan; Xie, Ya-Jun; Zhou, Jia-Huan; Zhou, Lin; Huang, Hao; Han, Shuo; Shao, Chong-Yu; Li, Hua-Shun; Zhu, J Julius; Qiu, Meng-Sheng; De Zeeuw, Chris I; Shen, Ying

    2015-12-15

    Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated. Here we show that cerebellar Purkinje cells (PCs) express high levels of Numb during adulthood and that conditional deletion of Numb in PCs is sufficient to impair motor coordination despite maintenance of a normal cerebellar cyto-architecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlu1) in PCs. A significant decrease of mGlu1 and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlu1 induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlu1-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlu1 plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlu1 trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlu1. PMID:26621723

  15. Pyridoxine may protect the cerebellar granular cells against glutamate-induced toxicity.

    PubMed

    Büyükokuroglu, Mehmet Emin; Gepdiremen, Akcahan; Taştekin, Ayhan; Ors, Rahmi

    2007-09-01

    In the present study, the possible protective effect of the pyridoxine against glutamate-induced neurotoxicity in cerebellar granular cell culture of rat pups is investigated for its therapeutic potential. Glutamate (10(-7) M) was administered to cerebellar granular cell cultures that were prepared from one-day-old Sprague-Dawley rats. The neuroprotective effect of pyridoxine was examined. Pyridoxine at the doses of 10(-8), 10(-7), 10(-6), and 10(-5) M was introduced into the culture flasks before inclusion of glutamate. Pyridoxine at the doses of 10(-8) M and 10(-7) M significantly reduced glutamate cytotoxicity. A 10(-7) M dose of pyridoxine proved to be more effective than a 10(-8) M dose. The present study demonstrates that pyridoxine may protect glutamate-induced neurotoxicity. Neuroprotective effect of pyridoxine, at least in part, may result from its anti-glutamatergic activity. Pyridoxine merits further investigation as a therapeutic option in hypoxic-ischemic brain injury. PMID:18453318

  16. Migration behavior of rodent granule neurons in the presence of antibody to the 4C5 antigen.

    PubMed

    Yfanti, E; Nagata, I; Patsavoudi, E

    1998-10-01

    We have reported the production of monoclonal antibody 4C5, which recognizes a cell surface antigen, the 4C5 antigen, involved in granule cell migration processes. In the present study, we investigated in a more precise manner the role of the 4C5 antigen in the different types of granule cell migrations that take place during cerebellar development. When cerebellar explant cultures derived from 10-day-old rats were performed for 2 days in the presence of monoclonal antibody 4C5, vertical granule cell migration, occurring in the presence of glia, was not significantly inhibited. In contrast, when monoclonal antibody 4C5 was included in the medium of microexplant cultures derived from 4-day-old mice and maintained for 4 days in vitro, granule cell migrations that occurred both parallel and perpendicular to the neurite bundles that were free of glia were inhibited. Moreover, a stronger inhibitory effect of the antibody was observed on migration perpendicular to the neurite bundles compared with the parallel type of migration. Our results indicate that the 4C5 antigen differentially affects the different developmental stages and types of granule cell migration during rodent cerebellar development. PMID:9751168

  17. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    NASA Technical Reports Server (NTRS)

    Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.; Rhodes, Christopher J.

    2002-01-01

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  18. Cerebellar cortex and cerebellar nuclei are concomitantly activated during eyeblink conditioning: a 7T fMRI study in humans.

    PubMed

    Thürling, Markus; Kahl, Fabian; Maderwald, Stefan; Stefanescu, Roxana M; Schlamann, Marc; Boele, Henk-Jan; De Zeeuw, Chris I; Diedrichsen, Jörn; Ladd, Mark E; Koekkoek, Sebastiaan K E; Timmann, Dagmar

    2015-01-21

    There are controversies whether learning of conditioned eyeblink responses primarily takes place within the cerebellar cortex, the interposed nuclei, or both. It has also been suggested that the cerebellar cortex may be important during early stages of learning, and that there is a shift to the cerebellar nuclei during later stages. As yet, human studies have provided little to resolve this question. In the present study, we established a setup that allows ultra-high-field 7T functional magnetic resonance imaging (fMRI) of the cerebellar cortex and interposed cerebellar nuclei simultaneously during delay eyeblink conditioning in humans. Event-related fMRI signals increased concomitantly in the cerebellar cortex and nuclei during early acquisition of conditioned eyeblink responses in 20 healthy human subjects. ANOVAs with repeated-measures showed significant effects of time across five blocks of 20 conditioning trials in the cortex and nuclei (p < 0.05, permutation corrected). Activations were most pronounced in, but not limited to, lobules VI and interposed nuclei. Increased activations were most prominent at the first time the maximum number of conditioned responses was achieved. Our data are consistent with a simultaneous and synergistic two-site model of learning during acquisition of classically conditioned eyeblinks. Because increased MRI signal reflects synaptic activity, concomitantly increased signals in the cerebellar nuclei and cortex are consistent with findings of learning related potentiation at the mossy fiber to nuclear cell synapse and mossy fiber to granule cell synapse. Activity related to the expression of conditioned responses, however, cannot be excluded. PMID:25609637

  19. Immunohistological comparison of granulated cell proteins in induced immediate urticarial dermographism and delayed pressure urticaria lesions.

    PubMed

    McEvoy, M T; Peterson, E A; Kobza-Black, A; English, J S; Dover, J S; Murphy, G M; Bhogal, B; Greaves, M W; Winkelmann, R K; Leiferman, K M

    1995-12-01

    Urticarial dermographism and delayed pressure urticaria are two forms of physical urticaria which are well defined clinically and histologically. Previous studies have shown eosinophil granule protein deposition in urticarial reactions, including chronic urticaria, solar urticaria and delayed pressure urticaria. To evaluate and compare the involvement of granulated inflammatory cells in urticarial dermographism and delayed pressure urticaria, we studied sequential biopsies of induced lesions of urticarial dermographism and delayed pressure urticaria by indirect immunofluorescence, to detect eosinophil granule major basic protein (MBP) and neutrophil granule elastase. Biopsies from dermographic lesions at time 0, 5 min, 15 min, 2 h and 24 h, showed few infiltrating eosinophils, with minimal extracellular MBP deposition, and a few infiltrating neutrophils, with minimal neutrophil elastase deposition, throughout the evolution of the lesions. Sequential biopsies of delayed pressure urticaria at time 0, 20 min, 6, 12 and 24 h, showed eosinophil infiltration with extensive MBP deposition beginning at 20 min, and neutrophil infiltration with variable elastase deposition beginning at 20 min. Control tissue specimens from normal volunteers showed neutrophil infiltration and slight degranulation, but no eosinophil infiltration or degranulation. Comparison of urticarial dermographism with delayed pressure urticaria showed marked differences in the patterns of infiltration. Delayed pressure urticaria, with eosinophil and neutrophil degranulation, was strikingly similar to the IgE-mediated late phase reaction. In contrast, eosinophil and neutrophil involvement in urticarial dermographism was minimal. Considering the extent of eosinophil granule protein deposition and the biological activities of the eosinophil granule proteins, the findings in delayed pressure urticaria point to an important pathophysiological role of eosinophils in the disease. PMID:8547035

  20. Stereological study of the effects of maternal diabetes on cerebellar cortex development in rat.

    PubMed

    Hami, Javad; Vafaei-Nezhad, Saeed; Ghaemi, Kazem; Sadeghi, Akram; Ivar, Ghasem; Shojae, Fatemeh; Hosseini, Mehran

    2016-06-01

    Diabetes during pregnancy is associated with the deficits in balance and motor coordination and altered social behaviors in offspring. In the present study, we have investigated the effect of maternal diabetes and insulin treatment on the cerebellar volume and morphogenesis of the cerebellar cortex of rat neonates during the first two postnatal weeks. Sprague Dawley female rats were maintained diabetic from a week before pregnancy through parturition. At the end of pregnancy, the male offspring euthanized on postnatal days (P) 0, 7, and 14. Cavalieri's principle and fractionator methods were used to estimate the cerebellar volume, the thickness and the number of cells in the different layers of the cerebellar cortex. In spite of P0, there was a significant reduction in the cerebellar volume and the thickness of the external granule, molecular, and internal granule layers between the diabetic and the control animals. In diabetic group, the granular and purkinje cell densities were increased at P0. Moreover, the number of granular and purkinje cells in the cerebellum of diabetic neonates was reduced in comparison with the control group at P7 and P14. There were no significant differences in either the volume and thickness or the number of cells in the different layers of the cerebellar cortex between the insulin-treated diabetic group and controls. Our data indicate that diabetes in pregnancy disrupts the morphogenesis of cerebellar cortex. This dysmorphogenesis may be part of the cascade of events through which diabetes during pregnancy affects motor coordination and social behaviors in offspring. PMID:26842601

  1. Local postsynaptic voltage-gated sodium channel activation in dendritic spines of olfactory bulb granule cells.

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Pálfi, Dénes; Rózsa, Balázs; Egger, Veronica

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function. PMID:25619656

  2. Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

    PubMed Central

    Hammel, Ilan; Shoichetman, Tanya; Amihai, Dina; Galli, Stephen J.; Skutelsky, Ehud

    2013-01-01

    We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower adivin gold binding density (by approximately 50%, P<0.001). The latter result provides additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild type mice. In both wild type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild type cells (P < 0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in the bm/bm cells. PMID:20127366

  3. Optogenetic Control of Motor Coordination by Gi/o Protein-coupled Vertebrate Rhodopsin in Cerebellar Purkinje Cells*

    PubMed Central

    Gutierrez, Davina V.; Mark, Melanie D.; Masseck, Olivia; Maejima, Takashi; Kuckelsberg, Denise; Hyde, Robert A.; Krause, Martin; Kruse, Wolfgang; Herlitze, Stefan

    2011-01-01

    G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific Gi/o protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the Gi/o protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of Gi/o modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar Gi/o-coupled GABAB receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via Gi/o-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner. PMID:21628464

  4. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

    PubMed Central

    Gao, Fei; Song, Xueying; Zhu, Dexiao; Wang, Xiaochen; Hao, Aijun; Nadler, J. Victor; Zhan, Ren-Zhi

    2015-01-01

    To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells. PMID:26500490

  5. Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

    PubMed

    Safina, Dina R; Surin, Alexander M; Pinelis, Vsevolod G; Kostrov, Sergey V

    2015-12-01

    Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²⁺ homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ±  0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period. PMID:26346533

  6. Comparative sensitivity of rat cerebellar neurons to dysregulation of divalent cation homeostasis and cytotoxicity caused by methylmercury

    SciTech Connect

    Edwards, Joshua R.; Marty, M. Sue; Atchison, William D. . E-mail: atchiso1@msu.edu

    2005-11-01

    The objective of the present study was to determine the relative effectiveness of methylmercury (MeHg) to alter divalent cation homeostasis and cause cell death in MeHg-resistant cerebellar Purkinje and MeHg-sensitive granule neurons. Application of 0.5-5 {mu}M MeHg to Purkinje and granule cells grown in culture caused a concentration- and time-dependent biphasic increase in fura-2 fluorescence. At 0.5 and 1 {mu}M MeHg, the elevations of fura-2 fluorescence induced by MeHg were biphasic in both cell types, but significantly delayed in Purkinje as compared to granule cells. Application of the heavy-metal chelator, TPEN, to Purkinje cells caused a precipitous decline in a proportion of the fura-2 fluorescence signal, indicating that MeHg causes release of Ca{sup 2+} and non-Ca{sup 2+} divalent cations. Purkinje cells were also more resistant than granule cells to the neurotoxic effects of MeHg. At 24.5 h after-application of 5 {mu}M MeHg, 97.7% of Purkinje cells were viable. At 3 {mu}M MeHg there was no detectable loss of Purkinje cell viability. In contrast, only 40.6% of cerebellar granule cells were alive 24.5 h after application of 3 {mu}M MeHg. In conclusion, Purkinje neurons in primary cultures appear to be more resistant to MeHg-induced dysregulation of divalent cation homeostasis and subsequent cell death when compared to cerebellar granule cells. There is a significant component of non-Ca{sup 2+} divalent cation released by MeHg in Purkinje neurons.

  7. ERBB3-mediated regulation of Bergmann glia proliferation in cerebellar lamination

    PubMed Central

    Sathyamurthy, Anupama; Yin, Dong-Min; Barik, Arnab; Shen, Chengyong; Bean, Jonathan C.; Figueiredo, Dwight; She, Jin-Xiong; Xiong, Wen-Cheng; Mei, Lin

    2015-01-01

    Cortical lamination is crucial for the assembly of cerebellar circuitry. In this process, granule neurons (GNs) migrate along Bergmann glia (BG), which are specialized astroglial cells, from the external granule layer to the internal granule layer. However, the molecular mechanisms underlying BG development are not well understood. Here, we show that GFAP::Cre;Erbb3F/F mice, which lack Erbb3 in both radial glia and neurons, exhibit impairments in balance and motor coordination. Cerebellar lamination is aberrant, with misplaced Purkinje neurons and GN clusters. These phenotypes were not observed in Math1::CreERT2;Erbb3F/F mice, where the Erbb3 gene was deleted in GNs, suggesting involvement of non-neuronal Erbb3 in cerebellar lamination. Mechanistic studies indicate that ERBB3 is crucial for the proliferation of BG, which are required for GN migration. These observations identify a crucial role for ERBB3 in cerebellar lamination and reveal a novel mechanism that regulates BG development. PMID:25564653

  8. Mature granule cells of the dentate gyrus-Passive bystanders or principal performers in hippocampal function?

    PubMed

    Lopez-Rojas, Jeffrey; Kreutz, Michael R

    2016-05-01

    The dentate gyrus is the main entrance of highly processed information to the hippocampus which derives from associative cortices and it is one of the few privileged areas in the brain where adult neurogenesis occurs. This creates the unique situation that neurons of diverse maturation stages are part of one neuronal network at any given point in life. While recently adult-born cells have a low induction threshold for long-term potentiation several studies suggest that following maturation granule cells are poorly excitable and they exhibit reduced Hebbian synaptic plasticity to an extent that it was even suggested that they functionally retire. Here, we review the functional properties of mature granule cells and discuss how plasticity of intrinsic excitability and alterations in excitation-inhibition balance might impact on their role in hippocampal information processing. PMID:26949226

  9. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells.

    PubMed

    Madroñal, Noelia; Delgado-García, José M; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  10. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells

    PubMed Central

    Madroñal, Noelia; Delgado-García, José M.; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T.; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  11. Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells

    PubMed Central

    Wang, Lei; Arcasoy, Murat O.; Watowich, Stephanie S.; Forget, Bernard G.

    2008-01-01

    Objective In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells. Materials and Methods Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis. Results We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBPε. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBPε genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction. Conclusion These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to

  12. Re-defining the cerebellar cortex as an assembly of non-uniform Purkinje cell microcircuits

    PubMed Central

    Cerminara, Nadia L; Lang, Eric J; Sillitoe, Roy V; Apps, Richard

    2015-01-01

    The adult mammalian cerebellar cortex is generally assumed to have a uniform cytoarchitecture. Differences in cerebellar function are thought to arise, in the main, through distinct patterns of input and output connectivity, rather than as a result of variations in cortical microcircuitry. However, evidence from anatomical, physiological and genetic studies is increasingly challenging this orthodoxy and there are now various lines of evidence that the cerebellar cortex is non uniform. Here we develop the hypothesis that regional differences in cerebellar cortical microcircuit properties lead to important differences in information processing. PMID:25601779

  13. Role of Pax6 in development of the cerebellar system.

    PubMed

    Engelkamp, D; Rashbass, P; Seawright, A; van Heyningen, V

    1999-08-01

    Post-mitotic neurons generated at the rhombic lip undertake long distance migration to widely dispersed destinations, giving rise to cerebellar granule cells and the precerebellar nuclei. Here we show that Pax6, a key regulator in CNS and eye development, is strongly expressed in rhombic lip and in cells migrating away from it. Development of some structures derived from these cells is severely affected in Pax6-null Small eye (Pax6(Sey)/Pax6(Sey)) embryos. Cell proliferation and initial differentiation seem unaffected, but cell migration and neurite extension are disrupted in mutant embryos. Three of the five precerebellar nuclei fail to form correctly. In the cerebellum the pre-migratory granule cell sub-layer and fissures are absent. Some granule cells are found in ectopic positions in the inferior colliculus which may result from the complete absence of Unc5h3 expression in Pax6(Sey)/Pax6(Sey) granule cells. Our results suggest that Pax6 plays a strong role during hindbrain migration processes and at least part of its activity is mediated through regulation of the netrin receptor Unc5h3. PMID:10409504

  14. Enterovirus 71 induces anti-viral stress granule-like structures in RD cells.

    PubMed

    Zhu, Yuanmei; Wang, Bei; Huang, He; Zhao, Zhendong

    2016-08-01

    Stress granules (SGs) are dynamic cytoplasmic granules formed in response to a variety of stresses, including viral infection. Several viruses can modulate the formation of SG with different effects, but the relationship between SG formation and EV71 infection is poorly understood. In this study, we report that EV71 inhibits canonical SGs formation in infected cells and induces the formation of novel RNA granules that were distinguished from canonical SGs in composition and morphology, which we termed 'SG like structures'. Our results also demonstrated that EV71 triggered formation of SG-like structures is dependent on PKR and eIF2α phosphorylation and requires ongoing cellular mRNA synthesis. Finally, we found that SG-like structures are antiviral RNA granules that promote cellular apoptosis and suppress EV71 propagation. Taken together, our findings explain the formation mechanism of SG-like structures induced by EV71 and shed light on virus-host interaction and molecular mechanism underlying EV71 pathogenesis. PMID:27216457

  15. GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury.

    PubMed

    Zonouzi, Marzieh; Scafidi, Joseph; Li, Peijun; McEllin, Brian; Edwards, Jorge; Dupree, Jeffrey L; Harvey, Lloyd; Sun, Dandan; Hübner, Christian A; Cull-Candy, Stuart G; Farrant, Mark; Gallo, Vittorio

    2015-05-01

    Diffuse white matter injury (DWMI), a leading cause of neurodevelopmental disabilities in preterm infants, is characterized by reduced oligodendrocyte formation. NG2-expressing oligodendrocyte precursor cells (NG2 cells) are exposed to various extrinsic regulatory signals, including the neurotransmitter GABA. We investigated GABAergic signaling to cerebellar white matter NG2 cells in a mouse model of DWMI (chronic neonatal hypoxia). We found that hypoxia caused a loss of GABAA receptor-mediated synaptic input to NG2 cells, extensive proliferation of these cells and delayed oligodendrocyte maturation, leading to dysmyelination. Treatment of control mice with a GABAA receptor antagonist or deletion of the chloride-accumulating transporter NKCC1 mimicked the effects of hypoxia. Conversely, blockade of GABA catabolism or GABA uptake reduced NG2 cell numbers and increased the formation of mature oligodendrocytes both in control and hypoxic mice. Our results indicate that GABAergic signaling regulates NG2 cell differentiation and proliferation in vivo, and suggest that its perturbation is a key factor in DWMI. PMID:25821912

  16. Reappraisal of Bergmann glial cells as modulators of cerebellar circuit function

    PubMed Central

    De Zeeuw, Chris I.; Hoogland, Tycho M.

    2015-01-01

    Just as there is a huge morphological and functional diversity of neuron types specialized for specific aspects of information processing in the brain, astrocytes have equally distinct morphologies and functions that aid optimal functioning of the circuits in which they are embedded. One type of astrocyte, the Bergmann glial cell (BG) of the cerebellum, is a prime example of a highly diversified astrocyte type, the architecture of which is adapted to the cerebellar circuit and facilitates an impressive range of functions that optimize information processing in the adult brain. In this review we expand on the function of the BG in the cerebellum to highlight the importance of astrocytes not only in housekeeping functions, but also in contributing to plasticity and information processing in the cerebellum. PMID:26190972

  17. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

    PubMed Central

    Ueki, Shigeharu; Melo, Rossana C. N.; Ghiran, Ionita; Spencer, Lisa A.; Dvorak, Ann M.; Weller, Peter F.

    2013-01-01

    Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets. PMID:23303825

  18. Electrophysiological, morphological, and topological properties of two histochemically distinct subpopulations of cerebellar unipolar brush cells.

    PubMed

    Kim, Jin-Ah; Sekerková, Gabriella; Mugnaini, Enrico; Martina, Marco

    2012-12-01

    Unipolar brush cells (UBCs) are excitatory cerebellar granular layer interneurons whose brush-like dendrites receive one-to-one mossy fiber inputs. Subclasses of UBCs differ primarily by expressing metabotropic glutamate receptor (mGluR) 1α or calretinin. We used GENSAT Tg(Grp-EGFP) BAC transgenic mice, which selectively express enhanced green fluorescent protein (EGFP) in mGluR1α-positive UBCs to compare the functional properties of the two subclasses. Compared to EGFP-negative UBCs, which include the calretinin-positive cells, EGFP-positive UBCs had smaller somata (area 48 vs 63 μm(2)), lower specific membrane resistance (6.4 vs. 13.7 KΩ cm(2)), were less prone to intrinsic firing, and showed more irregular firing (in cell-attached ~49 % were firing vs. ~88 %, and the CV was 0.53 vs. 0.32 for EGFP-negative cells). Some of these differences are attributable to higher density of background K(+) currents in EGFP-positive cells (at -120 mV, the barium-sensitive current was 94 vs. 37 pA in EGFP-negative cells); Ih, on the contrary, was more abundantly expressed in EGFP-negative cells (at -140 mV, it was -122 vs. -54 pA in EGFP-positive neurons); furthermore, while group II mGluR modulation of the background potassium current in EGFP-negative UBCs was maintained after intracellular dialysis, mGluR modulation in EGFP-positive UBCs was lost in whole-cell recordings. Finally, cell-attached firing was reversibly abolished by the GABA(B) activation in EGFP-positive, but not in EGFP-negative UBCs. Immunohistochemistry showed that EGFP-negative UBCs express GIRK2 at high density, while mGluR1α UBCs are GIRK2 negative, suggesting that GIRK2 mediates the mGluR-sensitive current in EGFP-negative UBCs. These data suggest that the two subclasses perform different functions in the cerebellar microcircuits. PMID:22528965

  19. Elemental levels in mast cell granules differ in sections from normal and diabetic rats: an X-ray microanalysis study

    SciTech Connect

    Kendall, M.D.

    1988-03-01

    Mast cells around the thymus of rats stain red with alcian blue and safranin indicating that the mast cells are probably of the peritoneal (connective tissue) type. After the onset of streptozotocin induced diabetes some cells contain both red and blue granules and blue staining cells may appear. X-ray microanalysis of frozen freeze-dried sections from diabetic male CSE Wistar rats showed electron dense granules to have similar amounts of S to normal rat mast cell granules but reduced levels of Na, Mg, P, Cl and K. Two cells also had electron lucent granules with very high levels of Na, Cl, K and Ca and reduced concentrations of S. The differences in elemental composition suggest that the mast cells from diabetic rats are not immature, but are related to the condition of induced diabetes, and that granules of very different composition can occur within a single cell. X-ray microanalysis has given an insight into mast cell granule elemental content which was not possible by conventional biochemical methods.

  20. Downregulation of Glutamate Transporter EAAT4 by Conditional Knockout of Rheb1 in Cerebellar Purkinje Cells.

    PubMed

    Jiang, Nan-Wei; Wang, De-Juan; Xie, Ya-Jun; Zhou, Liang; Su, Li-Da; Li, Huashun; Wang, Qin-Wen; Shen, Ying

    2016-06-01

    Excitatory amino acid transporter 4 (EAAT4) is believed to be critical to the synaptic activity of cerebellar Purkinje cells by limiting extracellular glutamate concentrations and facilitating the induction of long-term depression. However, the modulation of EAAT4 expression has not been elucidated. It has been shown that Ras homolog enriched in brain (Rheb)/mammalian target of rapamycin (mTOR) signaling plays essential roles in the regulation of protein translation, cell size, and cell growth. In addition, we previously found that a cascade including mTOR suppression and Akt activation induces increased expression of EAAT2 in astrocytes. In the present work, we explored whether Rheb/mTOR signaling is involved in the regulation of EAAT4 expression using conditional Rheb1 knockout mice. Our results demonstrated that Rheb1 deficiency resulted in the downregulation of EAAT4 expression, as well as decreased activity of mTOR and increased activity of Akt. The downregulation of EAAT4 was also confirmed by reduced EAAT4 currents and slowed kinetics of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor-mediated currents. On the other hand, conditional knockout of Rheb1 did not alter the morphology of Purkinje cell layer and the number of Purkinje cells. Overall, our findings suggest that small GTPase Rheb1 is a modulator in the expression of EAAT4 in Purkinje cells. PMID:26194056

  1. Purkinje-cell plasticity and cerebellar motor learning are graded by complex-spike duration.

    PubMed

    Yang, Yan; Lisberger, Stephen G

    2014-06-26

    Behavioural learning is mediated by cellular plasticity, such as changes in the strength of synapses at specific sites in neural circuits. The theory of cerebellar motor learning relies on movement errors signalled by climbing-fibre inputs to cause long-term depression of synapses from parallel fibres to Purkinje cells. However, a recent review has called into question the widely held view that the climbing-fibre input is an 'all-or-none' event. In anaesthetized animals, there is wide variation in the duration of the complex spike (CS) caused in Purkinje cells by a climbing-fibre input. Furthermore, the amount of plasticity in Purkinje cells is graded according to the duration of electrically controlled bursts in climbing fibres. The duration of bursts depends on the 'state' of the inferior olive and therefore may be correlated across climbing fibres. Here we provide a potential functional context for these mechanisms during motor learning in behaving monkeys. The magnitudes of both plasticity and motor learning depend on the duration of the CS responses. Furthermore, the duration of CS responses seems to be a meaningful signal that is correlated across the Purkinje-cell population during motor learning. We suggest that during learning, longer bursts in climbing fibres lead to longer-duration CS responses in Purkinje cells, more calcium entry into Purkinje cells, larger synaptic depression, and stronger learning. The same graded impact of instructive signals for plasticity and learning might occur throughout the nervous system. PMID:24814344

  2. Early-onset Purkinje cell dysfunction underlies cerebellar ataxia in peroxisomal multifunctional protein-2 deficiency.

    PubMed

    De Munter, Stephanie; Verheijden, Simon; Vanderstuyft, Esther; Malheiro, Ana Rita; Brites, Pedro; Gall, David; Schiffmann, Serge N; Baes, Myriam

    2016-10-01

    The cerebellar pathologies in peroxisomal diseases underscore that these organelles are required for the normal development and maintenance of the cerebellum, but the mechanisms have not been resolved. Here we investigated the origins of the early-onset coordination impairment in a mouse model with neural selective deficiency of multifunctional protein-2, the central enzyme of peroxisomal β-oxidation. At the age of 4weeks, Nestin-Mfp2(-/-) mice showed impaired motor learning on the accelerating rotarod and underperformed on the balance beam test. The gross morphology of the cerebellum and Purkinje cell arborization were normal. However, electrophysiology revealed a reduced Purkinje cell firing rate, a decreased excitability and an increased membrane capacitance. The distribution of climbing and parallel fiber synapses on Purkinje cells was immature and was accompanied by an increased spine length. Despite normal myelination, Purkinje cell axon degeneration was evident from the occurrence of axonal swellings containing accumulated organelles. In conclusion, the electrical activity, axonal integrity and wiring of Purkinje cells are exquisitely dependent on intact peroxisomal β-oxidation in neural cells. PMID:27353294

  3. Parallel Computational Subunits in Dentate Granule Cells Generate Multiple Place Fields

    PubMed Central

    Ujfalussy, Balázs; Kiss, Tamás; Érdi, Péter

    2009-01-01

    A fundamental question in understanding neuronal computations is how dendritic events influence the output of the neuron. Different forms of integration of neighbouring and distributed synaptic inputs, isolated dendritic spikes and local regulation of synaptic efficacy suggest that individual dendritic branches may function as independent computational subunits. In the present paper, we study how these local computations influence the output of the neuron. Using a simple cascade model, we demonstrate that triggering somatic firing by a relatively small dendritic branch requires the amplification of local events by dendritic spiking and synaptic plasticity. The moderately branching dendritic tree of granule cells seems optimal for this computation since larger dendritic trees favor local plasticity by isolating dendritic compartments, while reliable detection of individual dendritic spikes in the soma requires a low branch number. Finally, we demonstrate that these parallel dendritic computations could contribute to the generation of multiple independent place fields of hippocampal granule cells. PMID:19750211

  4. Effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules

    SciTech Connect

    Cervera, J.; Baguena-Cervellera, R.; Martinez, A.

    1985-12-01

    The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.

  5. Adult-born hippocampal dentate granule cells undergoing maturation modulate learning and memory in the brain

    PubMed Central

    Deng, Wei; Saxe, Michael D.; Gallina, Iryna S.; Gage, Fred H.

    2009-01-01

    Adult-born dentate granule cells (DGCs) contribute to learning and memory, yet it remains unknown when adult-born DGCs become involved in the cognitive processes. During neurogenesis, immature dentate granule cells (DGCs) display distinctive physiological characteristics while undergoing morphological maturation before final integration into the neural circuits. The survival and activity of the adult-born DGCs can be influenced by the experience of the animal during a critical period when newborn DGCs are still immature. To assess the temporal importance of adult neurogenesis, we developed a transgenic mouse model that allowed us to transiently reduce the numbers of adult-born DGCs in a temporally regulatable manner. We found that mice with a reduced population of adult-born DGCs at the immature stage were deficient in forming robust, long-term spatial memory and displayed impaired performance in extinction tasks. These results suggest that immature DGCs that undergo maturation make important contributions to learning and memory. PMID:19864566

  6. Cerebellar neurones: differentiation and modulation of sensitivity to excitotoxic treatment.

    PubMed

    Mercanti, D; Angelini, A; Ciotti, M T; Eboli, M L; Galli, C; Battistini, L; Merlo, D; Calissano, P

    1993-01-01

    The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C18 column. SDS-PAGE analysis of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate. PMID:7763737

  7. Cerebellar neurones: Differentiation and modulation of sensitivity to excitotoxic treatment.

    PubMed

    Mercanti, D; Angelini, A; Ciotti, M; Eboli, M; Galli, C; Battistini, L; Merlo, D; Calissano, P

    1993-01-01

    The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C(18) column. SDS-PAGE analisys of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate. PMID:22358672

  8. Tethering of ICAM on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization

    PubMed Central

    Gross, Catharina C.; Brzostowski, Joseph A.; Liu, Dongfang; Long, Eric O.

    2013-01-01

    αLβ2 integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion, but also polarization of cytolytic granules in NK cells. Here we tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adapter molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, while NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes. PMID:20675589

  9. Fine Tuning of Synaptic Plasticity and Filtering by GABA Released from Hippocampal Autaptic Granule Cells.

    PubMed

    Valente, Pierluigi; Orlando, Marta; Raimondi, Andrea; Benfenati, Fabio; Baldelli, Pietro

    2016-03-01

    The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ∼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits. PMID:25576534

  10. Final steps in exocytosis observed in a cell with giant secretory granules.

    PubMed Central

    Breckenridge, L J; Almers, W

    1987-01-01

    Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis. Images PMID:3470768

  11. Neuroligins Sculpt Cerebellar Purkinje-Cell Circuits by Differential Control of Distinct Classes of Synapses.

    PubMed

    Zhang, Bo; Chen, Lulu Y; Liu, Xinran; Maxeiner, Stephan; Lee, Sung-Jin; Gokce, Ozgun; Südhof, Thomas C

    2015-08-19

    Neuroligins are postsynaptic cell-adhesion molecules that bind presynaptic neurexins and are genetically linked to autism. Neuroligins are proposed to organize synaptogenesis and/or synaptic transmission, but no systematic analysis of neuroligins in a defined circuit is available. Here, we show that conditional deletion of all neuroligins in cerebellar Purkinje cells caused loss of distal climbing-fiber synapses and weakened climbing-fiber but not parallel-fiber synapses, consistent with alternative use of neuroligins and cerebellins as neurexin ligands for the excitatory climbing-fiber versus parallel-fiber synapses. Moreover, deletion of neuroligins increased the size of inhibitory basket/stellate-cell synapses but simultaneously severely impaired their function. Multiple neuroligin isoforms differentially contributed to climbing-fiber and basket/stellate-cell synapse functions, such that inhibitory synapse-specific neuroligin-2 was unexpectedly essential for maintaining normal climbing-fiber synapse numbers. Using systematic analyses of all neuroligins in a defined neural circuit, our data thus show that neuroligins differentially contribute to various Purkinje-cell synapses in the cerebellum in vivo. PMID:26291161

  12. Epithelial Cell Transforming 2 and Aurora Kinase B Modulate Formation of Stress Granule-Containing Transcripts from Diverse Cellular Pathways in Astrocytoma Cells.

    PubMed

    Weeks, Adrienne; Agnihotri, Sameer; Lymer, Jennifer; Chalil, Alan; Diaz, Roberto; Isik, Semra; Smith, Christian; Rutka, James T

    2016-06-01

    Stress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins epithelial cell transforming 2 and Aurora kinase B (AurkB) are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed using high-throughput fluorescent-based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells, and subsequent analysis revealed that astrocytoma stress granules harbor unique mRNAs for various cellular pathways, including cellular migration, metabolism, translation, and transcriptional regulation. Human astrocytoma cell stress granules contain mRNAs that are known to be involved in glioma signaling and the mammalian target of rapamycin pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptibility to conventional therapy, such as radiation and chemotherapy. PMID:27106762

  13. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells.

    PubMed

    Rudolf, Rüdiger; Kögel, Tanja; Kuznetsov, Sergei A; Salm, Thorsten; Schlicker, Oliver; Hellwig, Andrea; Hammer, John A; Gerdes, Hans-Hermann

    2003-04-01

    Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells. PMID:12615975

  14. Rearing conditions differently affect the motor performance and cerebellar morphology of prenatally stressed juvenile rats.

    PubMed

    Ulupinar, Emel; Erol, Kevser; Ay, Hakan; Yucel, Ferruh

    2015-02-01

    The cerebellum is one of the most vulnerable parts of the brain to environmental changes. In this study, the effect of diverse environmental rearing conditions on the motor performances of prenatally stressed juvenile rats and its reflection to the cerebellar morphology were investigated. Prenatally stressed Wistar rats were grouped according to different rearing conditions (Enriched=EC, Standard=SC and Isolated=IC) after weaning. Six weeks later, male and female offspring from different litters were tested behaviorally. In rotarod and string suspension tests, females gained better scores than males. Significant gender and housing effects were observed especially on the motor functions requiring fine skills with the best performance by enriched females, but the worst by enriched males. The susceptibility of cerebellar macro- and micro-neurons to environmental conditions was compared using stereological methods. In female groups, no differences were observed in the volume proportions of cerebellar layers, soma sizes and the numerical densities of granule or Purkinje cells. However, a significant interaction between housing and gender was observed in the granule to Purkinje cell ratio of males, due to the increased numerical densities of the granule cells in enriched males. These data imply that proper functioning of the cerebellum relies on its well organized and evolutionarily conserved structure and circuitry. Although early life stress leads to long term behavioral and neurobiological consequences in the offspring, diverse rearing conditions can alter the motor skills of animals and synaptic connectivity between Purkinje and granular cells in a gender dependent manner. PMID:25315128

  15. Sprouty genes prevent excessive FGF signalling in multiple cell types throughout development of the cerebellum

    PubMed Central

    Yu, Tian; Yaguchi, Yuichiro; Echevarria, Diego; Martinez, Salvador; Basson, M. Albert

    2011-01-01

    Fibroblast growth factors (FGFs) and regulators of the FGF signalling pathway are expressed in several cell types within the cerebellum throughout its development. Although much is known about the function of this pathway during the establishment of the cerebellar territory during early embryogenesis, the role of this pathway during later developmental stages is still poorly understood. Here, we investigated the function of sprouty genes (Spry1, Spry2 and Spry4), which encode feedback antagonists of FGF signalling, during cerebellar development in the mouse. Simultaneous deletion of more than one of these genes resulted in a number of defects, including mediolateral expansion of the cerebellar vermis, reduced thickness of the granule cell layer and abnormal foliation. Analysis of cerebellar development revealed that the anterior cerebellar neuroepithelium in the early embryonic cerebellum was expanded and that granule cell proliferation during late embryogenesis and early postnatal development was reduced. We show that the granule cell proliferation deficit correlated with reduced sonic hedgehog (SHH) expression and signalling. A reduction in Fgfr1 dosage during development rescued these defects, confirming that the abnormalities are due to excess FGF signalling. Our data indicate that sprouty acts both cell autonomously in granule cell precursors and non-cell autonomously to regulate granule cell number. Taken together, our data demonstrate that FGF signalling levels have to be tightly controlled throughout cerebellar development in order to maintain the normal development of multiple cell types. PMID:21693512

  16. Mitochondrial fission protein Drp1 regulates mitochondrial transport and dendritic arborization in cerebellar Purkinje cells.

    PubMed

    Fukumitsu, Kansai; Hatsukano, Tetsu; Yoshimura, Azumi; Heuser, John; Fujishima, Kazuto; Kengaku, Mineko

    2016-03-01

    Mitochondria dynamically change their shape by repeated fission and fusion in response to physiological and pathological conditions. Recent studies have uncovered significant roles of mitochondrial fission and fusion in neuronal functions, such as neurotransmission and spine formation. However, the contribution of mitochondrial fission to the development of dendrites remains controversial. We analyzed the function of the mitochondrial fission GTPase Drp1 in dendritic arborization in cerebellar Purkinje cells. Overexpression of a dominant-negative mutant of Drp1 in postmitotic Purkinje cells enlarged and clustered mitochondria, which failed to exit from the soma into the dendrites. The emerging dendrites lacking mitochondrial transport remained short and unstable in culture and in vivo. The dominant-negative Drp1 affected neither the basal respiratory function of mitochondria nor the survival of Purkinje cells. Enhanced ATP supply by creatine treatment, but not reduced ROS production by antioxidant treatment, restored the hypomorphic dendrites caused by inhibition of Drp1 function. Collectively, our results suggest that Drp1 is required for dendritic distribution of mitochondria and thereby regulates energy supply in growing dendritic branches in developing Purkinje cells. PMID:26689905

  17. Ischemia deteriorates the spike encoding of rat cerebellar Purkinje cells by raising intracellular Ca{sup 2+}

    SciTech Connect

    Zhao Shidi; Chen Na; Yang Zhilai; Huang Li; Zhu Yan; Guan Sudong; Chen Qianfen; Wang Jinhui

    2008-02-08

    Ischemia-induced excitotoxicity at cerebellar Purkinje cells is presumably due to a persistent glutamate action. To the fact that they are more vulnerable to ischemia than other glutamate-innervated neurons, we studied whether additional mechanisms are present and whether cytoplasm Ca{sup 2+} plays a key role in their ischemic excitotoxicity. Ischemic changes in the excitability of Purkinje cells were measured by whole-cell recording in cerebellar slices of rats with less glutamate action. The role of cytoplasm Ca{sup 2+} was examined by two-photon cellular imaging and BAPTA infusion in Purkinje cells. Lowering perfusion rate to cerebellar slices deteriorated spike timing and raised spike capacity of Purkinje cells. These changes were associated with the reduction of spike refractory periods and threshold potentials, as well as the loss of their control to spike encoding. Ischemia-induced functional deterioration at Purkinje neurons was accompanied by cytoplasm Ca{sup 2+} rise and prevented by BAPTA infusion. Therefore, the ischemia destabilizes the spike encoding of Purkinje cells via raising cytoplasm Ca{sup 2+} without a need for glutamate, which subsequently causes their excitotoxic death.

  18. Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

    PubMed

    Yoo, Yeong-Yung; Jung, Goo-Eun; Kwon, Hee-Min; Bae, Kyoung-Hee; Cho, Sang-Joon; Soh, Kwang-Sup

    2015-12-01

    Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy. PMID:26742911

  19. Synthetic mast-cell granules as adjuvants to promote and polarize immunity in lymph nodes

    NASA Astrophysics Data System (ADS)

    St. John, Ashley L.; Chan, Cheryl Y.; Staats, Herman F.; Leong, Kam W.; Abraham, Soman N.

    2012-03-01

    Granules of mast cells (MCs) enhance adaptive immunity when, on activation, they are released as stable particles. Here we show that submicrometre particles modelled after MC granules augment immunity when used as adjuvants in vaccines. The synthetic particles, which consist of a carbohydrate backbone with encapsulated inflammatory mediators such as tumour necrosis factor, replicate attributes of MCs in vivo including the targeting of draining lymph nodes and the timed release of the encapsulated mediators. When used as an adjuvant during vaccination of mice with haemagglutinin from the influenza virus, the particles enhanced adaptive immune responses and increased survival of mice on lethal challenge. Furthermore, differential loading of the particles with the cytokine IL-12 directed the character of the response towards Th1 lymphocytes. The synthetic MC adjuvants replicate and enhance the functions of MCs during vaccination, and can be extended to polarize the resulting immunity.

  20. Selective autophagic degradation of maternally-loaded germline P granule components in somatic cells during C. elegans embryogenesis.

    PubMed

    Zhao, Yu; Tian, E; Zhang, Hong

    2009-07-01

    Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development. PMID:19372764

  1. The Cerebellum and SIDS: Disordered Breathing in a Mouse Model of Developmental Cerebellar Purkinje Cell Loss during Recovery from Hypercarbia

    PubMed Central

    Calton, Michele A.; Howard, Jeremy R.; Harper, Ronald M.; Goldowitz, Dan; Mittleman, Guy

    2016-01-01

    The cerebellum assists coordination of somatomotor, respiratory, and autonomic actions. Purkinje cell alterations or loss appear in sudden infant death and sudden death in epilepsy victims, possibly contributing to the fatal event. We evaluated breathing patterns in 12 wild-type (WT) and Lurcher mutant mice with 100% developmental cerebellar Purkinje cell loss under baseline (room air), and recovery from hypercapnia, a concern in sudden death events. Six mutant and six WT mice were exposed to 4-min blocks of increasing CO2 (2, 4, 6, and 8%), separated by 4-min recovery intervals in room air. Breath-by-breath patterns, including depth of breathing and end-expiratory pause (EEP) durations during recovery, were recorded. No baseline genotypic differences emerged. However, during recovery, EEP durations significantly lengthened in mutants, compared to WT mice, following the relatively low levels of CO2 exposure. Additionally, mutant mice exhibited signs of post-sigh disordered breathing during recovery following each exposure. Developmental cerebellar Purkinje cell loss significantly affects compensatory breathing patterns following mild CO2 exposure, possibly by inhibiting recovery from elevated CO2. These data implicate cerebellar Purkinje cells in the ability to recover from hypercarbia, suggesting that neuropathologic changes or loss of these cells contribute to inadequate ventilatory recovery to increased environmental CO2. Multiple disorders, including sudden infant death syndrome (SIDS) and sudden unexpected death in epilepsy (SUDEP), appear to involve both cardiorespiratory failure and loss or injury to cerebellar Purkinje cells; the findings support the concept that such neuropathology may precede and exert a prominent role in these fatal events. PMID:27242661

  2. Simvastatin induces cell death in a mouse cerebellar slice culture (CSC) model of developmental myelination

    PubMed Central

    Xiang, Zhongmin; Reeves, Steven A.

    2009-01-01

    Statins (inhibitors of HMG-CoA reductase) have shown promise in treating multiple sclerosis (MS). However, their effect on oligodendrocyte remyelination of demyelinated axons has not been clarified. Since developmental myelination shares many features with the remyelination process, we investigated the effect of lipophilic simvastatin on developmental myelination in organotypic cerebellar slice cultures (CSC). In this study, we first characterized developmental myelination in CSC from postnatal day (P)5 and P10 mice that express enhanced green fluorescence protein (eGFP) in oligodendrocyte-lineage cells. We then examined the effect of simvastain on three developmental myelination stages: early myelination (P5 CSC, 2DIV), late myelination (P10 CSC, 2DIV) and full myelination (P10 CSC, 10DIV). We found that treatment with simvastatin (0.1 μM) for 6 days decreased the survival of Purkinje cells and oligodendrocytes drastically during the early myelination stage, while moderately during the late and full myelination stages. Oligodendrocytes are more resistant than Purkinje cells. The toxic effect of simvastatin could be rescued by the product of HMG-CoA reductase mevalonate but not low-density lipoprotein (LDL). Additionally, this toxic effect is independent of isoprenylation since farnesyl pyrophosphate (Fpp) but not geranylgeranyl pyrophosphate (GGpp) provided partial rescue. Our findings therefore suggest that inhibition of cholesterol synthesis is detrimental to neuronal tissue. PMID:18929563

  3. Differential dendritic Ca2+ signalling in young and mature hippocampal granule cells

    PubMed Central

    Stocca, Gabriella; Schmidt-Hieber, Christoph; Bischofberger, Josef

    2008-01-01

    Neuronal activity is critically important for development and plasticity of dendrites, axons and synaptic connections. Although Ca2+ is an important signal molecule for these processes, not much is known about the regulation of the dendritic Ca2+ concentration in developing neurons. Here we used confocal Ca2+ imaging to investigate dendritic Ca2+ signalling in young and mature hippocampal granule cells, identified by the expression of the immature neuronal marker polysialated neural cell adhesion molecule (PSA-NCAM). Using the Ca2+-sensitive fluorescent dye OGB-5N, we found that both young and mature granule cells showed large action-potential evoked dendritic Ca2+ transients with similar amplitude of ∼200 nm, indicating active backpropagation of action potentials. However, the decay of the dendritic Ca2+ concentration back to baseline values was substantially different with a decay time constant of 550 ms in young versus 130 ms in mature cells, leading to a more efficient temporal summation of Ca2+ signals during theta-frequency stimulation in the young neurons. Comparison of the peak Ca2+ concentration and the decay measured with different Ca2+ indicators (OGB-5N, OGB-1) in the two populations of neurons revealed that the young cells had an ∼3 times smaller endogenous Ca2+-binding ratio (∼75 versus∼220) and an ∼10 times slower Ca2+ extrusion rate (∼170 s−1versus∼1800 s−1). These data suggest that the large dendritic Ca2+ signals due to low buffer capacity and slow extrusion rates in young granule cells may contribute to the activity-dependent growth and plasticity of dendrites and new synaptic connections. This will finally support differentiation and integration of young neurons into the hippocampal network. PMID:18591186

  4. The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa).

    PubMed Central

    Howard, B; Mitchell, P C; Ritchie, A; Simkiss, K; Taylor, M

    1981-01-01

    Certain cells in the hepatopancreas of the common garden snail (Helix aspersa) contain intracellular granules that are sites of metal-ion accumulation. These granules have been extracted and investigated by u.v. and i.r. spectroscopy, atomic-absorption spectroscopy, X-ray microanalysis, thermogravimetric analysis, enzymic assay and microanalysis. The deposits contain about 18% (w/w) water, 5% (w/w) organic matter and 76% (w/w) inorganic material of which the main components are Ca2+, Mg2+ and P2O7(4)-. The possible origin of these granules is discussed, as is their role in detoxifying heavy-metal ions. PMID:6272732

  5. Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-04

    To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

  6. Cerebellar Hypoplasia

    MedlinePlus

    ... disorders that begin in early childhood, such as ataxia telangiectasia. In an infant or young child, symptoms of a disorder that features cerebellar hypoplasia might include floppy muscle tone, developmental or ...

  7. Cerebellar Degeneration

    MedlinePlus

    ... Degeneration? Cerebellar degeneration is a process in which neurons in the cerebellum - the area of the brain ... proteins that are necessary for the survival of neurons. Associated diseases: Diseases that are specific to the ...

  8. Cerebellar liponeurocytoma.

    PubMed

    Owler, Brian K; Makeham, John M; Shingde, Meena; Besser, Michael

    2005-04-01

    A case of cerebellar liponeurocytoma in a 34-year-old man is reported. There are only 19 other cases reporting this entity in the medical literature. The diagnostic, radiological and clinical features associated with this tumour are reviewed and discussed in relation to our case. The differences in behaviour and prognosis between medulloblastoma and cerebellar liponeurocytoma are presented with the corresponding implications for management. PMID:15851097

  9. Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

    PubMed

    Lopachev, Alexander V; Lopacheva, Olga M; Osipova, Ekaterina A; Vladychenskaya, Elizaveta A; Smolyaninova, Larisa V; Fedorova, Tatiana N; Koroleva, Olga V; Akkuratov, Evgeny E

    2016-07-01

    Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 μM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-μM and 10-μM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27338714

  10. Autistic-Like Traits and Cerebellar Dysfunction in Purkinje Cell PTEN Knock-Out Mice.

    PubMed

    Cupolillo, Dario; Hoxha, Eriola; Faralli, Alessio; De Luca, Annarita; Rossi, Ferdinando; Tempia, Filippo; Carulli, Daniela

    2016-05-01

    Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits. PMID:26538449

  11. Lurcher GRID2-induced death and depolarization can be dissociated in cerebellar Purkinje cells.

    PubMed

    Selimi, Fekrije; Lohof, Ann M; Heitz, Stéphane; Lalouette, Alexis; Jarvis, Christopher I; Bailly, Yannick; Mariani, Jean

    2003-03-01

    The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1. PMID:12628171

  12. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans

    PubMed Central

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S.; Greenstein, David; Navarro, Rosa E.

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline—the immortal cell lineage required for sexual reproduction—protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  13. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    PubMed

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  14. Staufen1 impairs stress granule formation in skeletal muscle cells from myotonic dystrophy type 1 patients

    PubMed Central

    Ravel-Chapuis, Aymeric; Klein Gunnewiek, Amanda; Bélanger, Guy; Crawford Parks, Tara E.; Côté, Jocelyn; Jasmin, Bernard J.

    2016-01-01

    Myotonic dystrophy (DM1) is caused by an expansion of CUG repeats (CUGexp) in the DMPK mRNA 3′UTR. CUGexp-containing mRNAs become toxic to cells by misregulating RNA-binding proteins. Here we investigated the consequence of this RNA toxicity on the cellular stress response. We report that cell stress efficiently triggers formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells, as shown by the appearance of distinct cytoplasmic TIA-1– and DDX3-containing foci. We show that Staufen1 is also dynamically recruited into these granules. Moreover, we discovered that DM1 myoblasts fail to properly form SGs in response to arsenite. This blockage was not observed in DM1 fibroblasts, demonstrating a cell type–specific defect. DM1 myoblasts display increased expression and sequestration of toxic CUGexp mRNAs compared with fibroblasts. Of importance, down-regulation of Staufen1 in DM1 myoblasts rescues SG formation. Together our data show that Staufen1 participates in the inhibition of SG formation in DM1 myoblasts. These results reveal that DM1 muscle cells fail to properly respond to stress, thereby likely contributing to the complex pathogenesis of DM1. PMID:27030674

  15. Acute cerebellar ataxia

    MedlinePlus

    Cerebellar ataxia; Ataxia - acute cerebellar; Cerebellitis; Post-varicella acute cerebellar ataxia; PVACA ... Acute cerebellar ataxia in children, especially younger than age 3, may occur several weeks after an illness caused by a virus. ...

  16. BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

    PubMed

    Finzi, Giovanna; Franzi, Francesca; Placidi, Claudia; Acquati, Francesco; Palumbo, Elisa; Russo, Antonella; Taramelli, Roberto; Sessa, Fausto; La Rosa, Stefano

    2008-01-01

    BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis. PMID:19117266

  17. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. PMID:26845357

  18. Entorhinal Denervation Induces Homeostatic Synaptic Scaling of Excitatory Postsynapses of Dentate Granule Cells in Mouse Organotypic Slice Cultures

    PubMed Central

    Vlachos, Andreas; Becker, Denise; Jedlicka, Peter; Winkels, Raphael; Roeper, Jochen; Deller, Thomas

    2012-01-01

    Denervation-induced changes in excitatory synaptic strength were studied following entorhinal deafferentation of hippocampal granule cells in mature (≥3 weeks old) mouse organotypic entorhino-hippocampal slice cultures. Whole-cell patch-clamp recordings revealed an increase in excitatory synaptic strength in response to denervation during the first week after denervation. By the end of the second week synaptic strength had returned to baseline. Because these adaptations occurred in response to the loss of excitatory afferents, they appeared to be in line with a homeostatic adjustment of excitatory synaptic strength. To test whether denervation-induced changes in synaptic strength exploit similar mechanisms as homeostatic synaptic scaling following pharmacological activity blockade, we treated denervated cultures at 2 days post lesion for 2 days with tetrodotoxin. In these cultures, the effects of denervation and activity blockade were not additive, suggesting that similar mechanisms are involved. Finally, we investigated whether entorhinal denervation, which removes afferents from the distal dendrites of granule cells while leaving the associational afferents to the proximal dendrites of granule cells intact, results in a global or a local up-scaling of granule cell synapses. By using computational modeling and local electrical stimulations in Strontium (Sr2+)-containing bath solution, we found evidence for a lamina-specific increase in excitatory synaptic strength in the denervated outer molecular layer at 3–4 days post lesion. Taken together, our data show that entorhinal denervation results in homeostatic functional changes of excitatory postsynapses of denervated dentate granule cells in vitro. PMID:22403720

  19. Enhanced acoustic startle responding in rats with radiation-induced hippocampal granule cell hypoplasia

    SciTech Connect

    Mickley, G.A.; Ferguson, J.L.

    1989-01-01

    Irradiation of the neonatal rat hippocampus reduces the proliferation of granule cells in the dentate gyrus and results in locomotor hyperactivity, behavioral preservation, and deficits on some learned tasks. In order to address the role of changes in stimulus salience and behavioral inhibition in animals with this type of brain damage, irradiated and normal rats were compared in their startle reactions to an acoustic stimulus. Irradiated rats startled with a consistently higher amplitude than control and were more likely to exhibit startle responses. These animals with hippocampal damage also failed to habituate to the startle stimulus and, under certain circumstances, showed potentiated startle responses after many tone presentations.

  20. [Microspectrofluorometric analysis of the effect of centrophenoxine on lipofuscin granules of hybridoma (Retrovirus-transformed) cells].

    PubMed

    Tatariunas, A B

    1990-04-01

    The increase of the own luminescence lipofuscin granules (LG) in the course of single and repeated ultraviolet (UV) excitations (365 nm) in hybridoma (retrovirus transformed) cells cultured with or without 5 x 10(-4) M centrophenoxine (CP) was studied by microspectrofluorometric method. It was shown that CP influences only the rate of photochemical changes of chromophores in LG. Kinetic patterns of the own luminescence intensity of LG at the wavelength of 540 nm during excitation by UV permit one to suggest the occurrence of the cyclic chromophore changes. PMID:2117472

  1. TIA1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis

    PubMed Central

    Arimoto-Matsuzaki, Kyoko; Saito, Haruo; Takekawa, Mutsuhiro

    2016-01-01

    Cytoplasmic stress granules (SGs) are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of misfolded proteins, and that are formed in response to certain types of stress including ER stress. SG formation contributes to cell survival not only by suppressing translation but also by sequestering some apoptosis regulatory factors. Because cells can be exposed to various stresses simultaneously in vivo, the regulation of SG assembly under multiple stress conditions is important but unknown. Here we report that reactive oxygen species (ROS) such as H2O2 oxidize the SG-nucleating protein TIA1, thereby inhibiting SG assembly. Thus, when cells are confronted with a SG-inducing stress such as ER stress caused by protein misfolding, together with ROS-induced oxidative stress, they cannot form SGs, resulting in the promotion of apoptosis. We demonstrate that the suppression of SG formation by oxidative stress may underlie the neuronal cell death seen in neurodegenerative diseases. PMID:26738979

  2. Myeloperoxidase and Crystalline Bodies in the Granules of DMBA-Induced Rat Chloroma Cells

    PubMed Central

    Ioachim, Harry L.; Keller, Steven; Sabbath, Marlene; Andersson, Barbro; Dorsett, Brent; Essner, Edward

    1972-01-01

    Chloroma (chloroleukemia) was induced in a splenectomized rat by repeatedly administering dimethylbenz(a)anthracene (DMBA) and was serially transplanted thereafter. Composed of immature myeloid cells, the tumor imparted a green discoloration to the tissues that it infiltrated extensively. Chloroma cells fluoresced red in ultraviolet light, produced a characteristic curve in spectrophotometry, and contained large amounts of myeloperoxidase. They included numerous intracytoplasmic granules of both types A and B, which contained occasional crystalline bars. Permanent lines of chloroma cells were established in tissue culture. These cells, while maintaining their initial morphology, ceased producing myeloperoxidase and subsequently induced white tumors when they were isotransplanted. ImagesFig 12Fig 13Fig 4Fig 5Fig 6Fig 7Fig 8Fig 9Fig 10Fig 11Fig 1Fig 2Fig 3 PMID:4333120

  3. Different subsets of newborn granule cells: a possible role in epileptogenesis?

    PubMed

    Bielefeld, Pascal; van Vliet, Erwin A; Gorter, Jan A; Lucassen, Paul J; Fitzsimons, Carlos P

    2014-01-01

    Several factors, including epileptic seizures, can strongly stimulate ongoing neurogenesis in the adult hippocampus. Although adult-born granule cells generated after seizure activity have different physiological properties from their normal counterparts, they integrate into the existing, mature network of the adult hippocampal dentate gyrus. However, the exact role of the neurogenic response during epilepsy and its possible involvement in epileptogenesis have remained elusive. Here, we discuss recent studies shedding new light on the interplay between epilepsy and neurogenesis, and try to explain discrepancies in this literature by proposing seizure severity-dependent induction of two subsets of newborn cells with different properties. We hypothesise that a low seizure intensity would stimulate neurogenesis to a 'physiological plasticity' level and have few pathological consequences. In contrast, a high initial seizure intensity may induce a specific subset of altered and/or ectopically located new granule cells with different electrophysiological properties that could initiate hyperexcitatory recurrent networks that could, in turn, contribute to chronic epilepsy. This hypothesis may clarify previously contradictory data in the literature, and could thereby aid in our understanding of the role of neurogenesis in epileptogenesis, and open up promising avenues for therapeutic intervention. PMID:24387591

  4. Calcium buffering in rodent olfactory bulb granule cells and mitral cells.

    PubMed

    Egger, Veronica; Stroh, Olga

    2009-09-15

    In the mammalian olfactory bulb, axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between mitral cells (MCs) via reciprocal dendrodendritic synapses. Calcium signals in the GC dendrites and reciprocal spines appear to decay unusually slowly, hence GC calcium handling might contribute to the known asynchronous release at this synapse. By recording fluorescence transients of different Ca(2+)-sensitive dyes at variable concentrations evoked by backpropagating action potentials (APs) and saturating AP trains we extrapolated Ca(2+) dynamics to conditions of zero added buffer for juvenile rat GC apical dendrites and spines and MC lateral dendrites. Resting [Ca(2+)] was at approximately 50 nM in both GC dendrites and spines. The average endogenous GC buffer capacities (kappa(E)) were within a range of 80-90 in the dendrites and 110-140 in the spines. The extrusion rate (gamma) was estimated as 570 s(-1) for dendrites and 870 s(-1) for spines and the decay time constant as approximately 200 ms for both. Single-current-evoked APs resulted in a [Ca(2+)] elevation of approximately 250 nM. Calcium handling in juvenile and adult mouse GCs appeared mostly similar. In MC lateral dendrites, we found AP-mediated [Ca(2+)] elevations of approximately 130 nM with a similar decay to that in GC dendrites, while kappa(E) and gamma were roughly 4-fold higher. In conclusion, the slow GC Ca(2+) dynamics are due mostly to sluggish Ca(2+) extrusion. Under physiological conditions this slow removal may well contribute to delayed release and also feed into other Ca(2+)-dependent mechanisms that foster asynchronous output from the reciprocal spine. PMID:19635818

  5. Cerebellar transcriptional alterations with Purkinje cell dysfunction and loss in mice lacking PGC-1α.

    PubMed

    Lucas, Elizabeth K; Reid, Courtney S; McMeekin, Laura J; Dougherty, Sarah E; Floyd, Candace L; Cowell, Rita M

    2014-01-01

    Alterations in the expression and activity of the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (ppargc1a or PGC-1α) have been reported in multiple movement disorders, yet it is unclear how a lack of PGC-1α impacts transcription and function of the cerebellum, a region with high PGC-1α expression. We show here that mice lacking PGC-1α exhibit ataxia in addition to the previously described deficits in motor coordination. Using q-RT-PCR in cerebellar homogenates from PGC-1α(-/-) mice, we measured expression of 37 microarray-identified transcripts upregulated by PGC-1α in SH-SY5Y neuroblastoma cells with neuroanatomical overlap with PGC-1α or parvalbumin (PV), a calcium buffer highly expressed by Purkinje cells. We found significant reductions in transcripts with synaptic (complexin1, Cplx1; Pacsin2), structural (neurofilament heavy chain, Nefh), and metabolic (isocitrate dehydrogenase 3a, Idh3a; neutral cholesterol ester hydrolase 1, Nceh1; pyruvate dehydrogenase alpha 1, Pdha1; phytanoyl-CoA hydroxylase, Phyh; ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1, Uqcrfs1) functions. Using conditional deletion of PGC-1α in PV-positive neurons, we determined that 50% of PGC-1α expression and a reduction in a subset of these transcripts could be explained by its concentration in PV-positive neuronal populations in the cerbellum. To determine whether there were functional consequences associated with these changes, we conducted stereological counts and spike rate analysis in Purkinje cells, a cell type rich in PV, from PGC-1α(-/-) mice. We observed a significant loss of Purkinje cells by 6 weeks of age, and the remaining Purkinje cells exhibited a 50% reduction in spike rate. Together, these data highlight the complexity of PGC-1α's actions in the central nervous system and suggest that dysfunction in multiple cell types contribute to motor deficits in the context of PGC-1α deficiency. PMID:25610371

  6. [Cerebellar stroke].

    PubMed

    Paradowski, Michał; Zimny, Anna; Paradowski, Bogusław

    2015-01-01

    Cerebellar stroke belongs to a group of rare diseases of vascular origin. Cerebellum, supplied by three pairs of arteries (AICA, PICA, SCA) with many anastomoses between them is less susceptible for a stroke, especially ischemic one. Diagnosis of the stroke in this region is harder due to lower sensibility of commonly used CT of the head in case of stroke suspicion. The authors highlight clinical symptoms distinguishing between vascular territories or topographical locations of the stroke, diagnostic procedures, classical and surgical treatment, the most common misdiagnoses are also mentioned. The authors suggest a diagnostic and therapeutic algorithm development, including rtPA treatment criteria for ischemic cerebellar stroke. PMID:26181157

  7. BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells

    PubMed Central

    Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R.; Trimmer, James S.

    2015-01-01

    In myelinated axons, K+ channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na+ channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K+ channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K+ channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni2+ elicited a similar effect on APs, indicating the involvement of Ni2+-sensitive Ca2+ channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex. PMID:25948259

  8. Cholinergic afferent stimulation induces axonal function plasticity in adult hippocampal granule cells.

    PubMed

    Martinello, Katiuscia; Huang, Zhuo; Lujan, Rafael; Tran, Baouyen; Watanabe, Masahiko; Cooper, Edward C; Brown, David A; Shah, Mala M

    2015-01-21

    Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca(2+) via T-type Ca(2+) channels, as indicated by two-photon imaging. The enhanced Ca(2+) levels inhibited an axonal KV7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, CaV3.2, and KV7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing. PMID:25578363

  9. The perinuclear factor, a rheumatoid arthritis-specific autoantigen, is not present in keratohyalin granules of cultured buccal mucosa cells.

    PubMed Central

    Hoet, R M; Voorsmit, R A; Van Venrooij, W J

    1991-01-01

    Rheumatoid arthritis patients have antibodies in their serum directed against the perinuclear factor, a protein component present in keratohyalin granules in the cytoplasm of human buccal mucosa cells. The anti-perinuclear factor (APF) can only be detected by an indirect immunofluorescence test performed on fresh buccal mucosa cells from 'selected donors'. To obtain a more reliable antigen source and to gain more insight into the origin and nature of the perinuclear factor we attempted to culture perinuclear factor-containing buccal mucosa cells. Here we describe the successful culturing of such cells, which, however, did not contain keratohyalin granules nor the perinuclear factor. By adding the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA) we were able to induce keratohyalin granules in both cultured primary buccal mucosa cells and a squamous carcinoma cell line of the cheek (SqCC/Y1). These induced keratohyalin granules do contain the protein profilaggrin, which in vivo, in fresh buccal mucosa cells, co-localizes with the perinuclear factor. However, we were not able to demonstrate the presence of the perinuclear factor, not even after induction of terminal differentiation of the cultured cells nor after Epstein-Barr virus infection. Our results suggest that the perinuclear factor, in contrast to profilaggrin, is not an integral component of buccal mucosa cells. Images Fig. 1 PMID:1849807

  10. In vitro atrazine-exposure inhibits human natural killer cell lytic granule release

    SciTech Connect

    Rowe, Alexander M.; Brundage, Kathleen M.; Barnett, John B. . E-mail: jbarnett@hsc.wvu.edu

    2007-06-01

    The herbicide atrazine is a known immunotoxicant and an inhibitor of human natural killer (NK) cell lytic function. The precise changes in NK cell lytic function following atrazine exposure have not been fully elucidated. The current study identifies the point at which atrazine exerts its affect on the stepwise process of human NK cell-mediated lyses of the K562 target cell line. Using intracellular staining of human peripheral blood lymphocytes, it was determined that a 24-h in vitro exposure to atrazine did not decrease the level of NK cell lytic proteins granzyme A, granzyme B or perforin. Thus, it was hypothesized that atrazine exposure was inhibiting the ability of the NK cells to bind to the target cell and subsequently inhibit the release of lytic protein from the NK cell. To test this hypothesis, flow cytometry and fluorescent microscopy were employed to analyze NK cell-target cell co-cultures following atrazine exposure. These assays demonstrated no significant decrease in the level of target cell binding. However, the levels of NK intracellular lytic protein retained and the amount of lytic protein released were assessed following a 4-h incubation with K562 target cells. The relative level of intracellular lytic protein was 25-50% higher, and the amount of lytic protein released was 55-65% less in atrazine-treated cells than vehicle-treated cells following incubation with the target cells. These results indicate that ATR exposure inhibits the ability of NK cells to lyse target cells by blocking lytic granule release without affecting the ability of the NK cell to form stable conjugates with target cells.

  11. Telocytes as a Source of Progenitor Cells in Regeneration and Repair Through Granulation Tissue.

    PubMed

    Díaz-Flores, Lucio; Gutiérrez, Ricardo; Pino García, Maria; González, Miriam; Díaz-Flores, Lucio; Francisco Madrid, Juan

    2016-01-01

    This review outlines the role of CD34+ stromal cells/telocytes (CD34+ SC/TCs) in repair and considers the following issues. Firstly, the conceptual aspects of repair, including regeneration and repair through granulation tissue (RTGT) as two types of repair, RTGT stages (inflammatory, proliferative, and remodeling), and tissue in repair as a substrate to assess the in vivo behavior of activated CD34+ SC/TCs. Subsequently, current knowledge of CD34+ SC/TCs, such as identification, characteristics, and functions, as well as possible stages (quiescent and activated) are taken into account. We then consider the role in regeneration of quiescent CD34+ SC/TCs (in unperturbed physiological conditions) as a nurse of stem cells (e.g., in the heart, skin, respiratory tree, gastrointestinal tract, liver, eye, and choroid plexus). Special attention is paid to the characteristics of activated CD34+ SC/TCs and the overlapping steps of activation with and without loss of CD34 expression and with and without gain of αSMA expression. With this contribution, we establish the role of CD34+ SC/TCs as progenitor cells and as a source of fibroblasts and myofibroblasts in repair through granulation tissue, fibrosis, and tumor stroma. Activated CD34+ SC/TCs in encapsulation and other processes (e.g., Reinke's edema, cutaneous myxoid cyst, mixomatous mitral valve degeneration, and fibrous papula of the face) are also outlined. Finally, similarities between modifications of CD34+ SC/TCs during in vivo activation and of multipotent mesenchymal stromal/stem cells in culture are examined in order to correlate the growing literature on CD34+ SC/TCs and the exponential research in cultured mesenchymal stromal/stem cells. PMID:26423297

  12. Cerebellar abiotrophy.

    PubMed

    DeBowes, R M; Leipold, H W; Turner-Beatty, M

    1987-08-01

    Cerebellar abiotrophy is a degenerative condition of Arabian horses that produces signs of head tremors and ataxia. Affected foals demonstrate clinical signs between the time of birth and 6 months of age. The condition is untreatable, although some animals have reportedly improved to varying degrees. The disease is believed to be inherited; however, definitive evidence is lacking at this time. PMID:3497695

  13. Role of Synchronous Activation of Cerebellar Purkinje Cell Ensembles in Multi-joint Movement Control

    PubMed Central

    Hoogland, Tycho M.; De Gruijl, Jornt R.; Witter, Laurens; Canto, Cathrin B.; De Zeeuw, Chris I.

    2015-01-01

    Summary It is a longstanding question in neuroscience how elaborate multi-joint movements are coordinated coherently. Microzones of cerebellar Purkinje cells (PCs) are thought to mediate this coordination by controlling the timing of particular motor domains. However, it remains to be elucidated to what extent motor coordination deficits can be correlated with abnormalities in coherent activity within these microzones and to what extent artificially evoked synchronous activity within PC ensembles can elicit multi-joint motor behavior. To study PC ensemble correlates of limb, trunk, and tail movements, we developed a transparent disk treadmill that allows quantitative readout of locomotion and posture parameters in head-fixed mice and simultaneous cellular-resolution imaging and/or optogenetic manipulation. We show that PC ensembles in the ataxic and dystonic mouse mutant tottering have a reduced level of complex spike co-activation, which is delayed relative to movement onset and co-occurs with prolonged swing duration and reduced phase coupling of limb movements as well as with enlarged deflections of body-axis and tail movements. Using optogenetics to increase simple spike rate in PC ensembles, we find that preferred locomotion and posture patterns can be elicited or perturbed depending on the behavioral state. At rest, preferred sequences of limb movements can be elicited, whereas during locomotion, preferred gait-inhibition patterns are evoked. Our findings indicate that synchronous activation of PC ensembles can facilitate initiation and coordination of limb and trunk movements, presumably by tuning downstream systems involved in the execution of behavioral patterns. PMID:25843032

  14. Long-term climbing fibre activity induces transcription of microRNAs in cerebellar Purkinje cells.

    PubMed

    Barmack, Neal H; Qian, Zuyuan; Yakhnitsa, Vadim

    2014-09-26

    Synaptic activation of central neurons is often evoked by electrical stimulation leading to post-tetanic potentiation, long-term potentiation or long-term depression. Even a brief electrical tetanus can induce changes in as many as 100 proteins. Since climbing fibre activity is often associated with cerebellar behavioural plasticity, we used horizontal optokinetic stimulation (HOKS) to naturally increase synaptic input to floccular Purkinje cells in mice for hours, not minutes, and investigated how this activity influenced the transcription of microRNAs, small non-coding nucleotides that reduce transcripts of multiple, complementary mRNAs. A single microRNA can reduce the translation of as many as 30 proteins. HOKS evoked increases in 12 microRNA transcripts in floccular Purkinje cells. One of these microRNAs, miR335, increased 18-fold after 24 h of HOKS. After HOKS stopped, miR335 transcripts decayed with a time constant of approximately 2.5 h. HOKS evoked a 28-fold increase in pri-miR335 transcripts compared with an 18-fold increase in mature miR335 transcripts, confirming that climbing fibre-evoked increases in miR335 could be attributed to increases in transcription. We used three screens to identify potential mRNA targets for miR335 transcripts: (i) nucleotide complementarity, (ii) detection of increased mRNAs following microinjection of miR335 inhibitors into the cerebellum, and (iii) detection of decreased mRNAs following HOKS. Two genes, calbindin and 14-3-3-θ, passed these screens. Transfection of N2a cells with miR335 inhibitors or precursors inversely regulated 14-3-3-θ transcripts. Immunoprecipitation of 14-3-3-θ co-immunoprecipitated PKC-γ and GABAAγ2. Knockdown of either 14-3-3-θ or PKC-γ decreased the serine phosphorylation of GABAAγ2, suggesting that 14-3-3-θ and PKC-γ under the control of miR335 homeostatically regulate the phosphorylation and insertion of GABAAγ2 into the Purkinje cell post-synaptic membrane. PMID:25135969

  15. Calcium-binding proteins in the cerebellar cortex of the bottlenose dolphin and harbour porpoise.

    PubMed

    Kalinichenko, Sergei G; Pushchin, Igor I

    2008-07-01

    Studying the distribution of Ca2+-binding proteins allows one to discover specific neuron chemotypes involved in the regulation of the activity of various neural elements. While extensive data exist on Ca2+-binding proteins in the nervous system, in particular, in the cerebellar cortex of terrestrial mammals, the localization of these proteins in the cerebellar cortex of marine mammals has not been studied. We studied the localization of calretinin, calbindin, and parvalbumin immunoreactivity in the cerebellar cortex of the bottlenose dolphin Tursiops truncates and harbour porpoise Phocoena phocoena. In both species, most Purkinje cells were calbindin-immunoreactive, while calretinin and parvalbumin were expressed in a small portion of Purkinje cells. In addition, calretinin-immunoreactive unipolar brush and granule cells and calbindin- and parvalbumin-immunoreactive basket, stellate, and Golgi cells were observed. Calretinin-immunoreactive corticopetal (mossy and climbing) fibers were found. Based on the length of the primary dendrite, short-, middle-, and long-dendrite unipolar brush cells could be distinguished. The validity of this classification was supported using cluster analysis suggesting the presence of several natural types of these cells. The distribution of Ca2+-binding proteins in the cerebellar cortex of the cetaceans studied was generally similar to that reported for terrestrial mammals, suggesting that this trait is evolutionarily conservative in mammals. PMID:18455363

  16. Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously.

    PubMed

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H J; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals. PMID:24205250

  17. Revisiting the Single Cell Protein Application of Cupriavidus necator H16 and Recovering Bioplastic Granules Simultaneously

    PubMed Central

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H. J.; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals. PMID:24205250

  18. EFFECTS OF SELECTED NEUROACTIVE CHEMICALS ON CALCIUM TRANSPORTING SYSTEMS IN RAT CEREBELLUM AND ON SURVIVAL OF CEREBELLAR GRANULE CELLS

    EPA Science Inventory

    This investigation examined the effects of several neuroactive chemicals on Ca2+ -transporting systems and cytotoxicity in vitro. 5Ca2+ -uptake as a measure of Ca2+s -sequestration, was determined n mitochondria and microsomes, isolated from cerebella of adult male Long-Evans hoo...

  19. DIFFERENTIAL EFFECTS OF POLYCHLORINATED BIPHENYL CONGENERS ON PHOSPHOINOSITIDE HYDROLYSIS AND PROTEIN KINASE C TRANSLOCATION IN RAT CEREBELLAR GRANULE CELLS

    EPA Science Inventory

    Previous reports from our laboratory have suggested that the neuroactivity of some polychlorinated biphenyl (PCB) congeners is associated with perturbations in cellular Ca2+-homeostasis. o characterize the neurochemical effects of PCBs further, the present experiments found that ...

  20. Cerebellar Cortical Lamination and Foliation Require Cyclin A2

    PubMed Central

    Otero, José Javier; Kalaszczynska, Ilona; Michowski, Wojciech; Wong, Michael; Gygli, Patrick Edwin; Gokozan, Hamza Numan; Griveau, Amélie; Odajima, Junko; Czeisler, Catherine; Catacutan, Fay Patsy; Murnen, Alice; Schüler, Ulrich; Sicinski, Piotr; Rowitch, David

    2014-01-01

    The mammalian genome encodes two A-type cyclins, which are considered potentially redundant yet essential regulators of the cell cycle. Here, we tested requirements for cyclin A1 and cyclin A2 function in cerebellar development. Compound conditional loss of cyclin A1/A2 in neural progenitors resulted in severe cerebellar hypoplasia, decreased proliferation of cerebellar granule neuron progenitors (CGNP), and Purkinje (PC) neuron dyslamination. Deletion of cyclin A2 alone showed an identical phenotype, demonstrating that cyclin A1 does not compensate for cyclin A2 loss in neural progenitors. Cyclin A2 loss lead to increased apoptosis at early embryonic time points but not at post-natal time points. In contrast, neural progenitors of the VZ/SVZ did not undergo increased apoptosis, indicating that VZ/SVZ-derived and rhombic lip-derived progenitor cells show differential requirements to cyclin A2. Conditional knockout of cyclin A2 or the SHH proliferative target Nmyc in CGNP also resulted in PC neuron dyslamination. Although cyclin E1 has been reported to compensate for cyclin A2 function in fibroblasts and is upregulated in cyclin A2 null cerebella, cyclin E1 expression was unable to compensate for loss-of cyclin A2 function. PMID:24184637

  1. Processing of Multi-dimensional Sensorimotor Information in the Spinal and Cerebellar Neuronal Circuitry: A New Hypothesis

    PubMed Central

    Spanne, Anton; Jörntell, Henrik

    2013-01-01

    Why are sensory signals and motor command signals combined in the neurons of origin of the spinocerebellar pathways and why are the granule cells that receive this input thresholded with respect to their spike output? In this paper, we synthesize a number of findings into a new hypothesis for how the spinocerebellar systems and the cerebellar cortex can interact to support coordination of our multi-segmented limbs and bodies. A central idea is that recombination of the signals available to the spinocerebellar neurons can be used to approximate a wide array of functions including the spatial and temporal dependencies between limb segments, i.e. information that is necessary in order to achieve coordination. We find that random recombination of sensory and motor signals is not a good strategy since, surprisingly, the number of granule cells severely limits the number of recombinations that can be represented within the cerebellum. Instead, we propose that the spinal circuitry provides useful recombinations, which can be described as linear projections through aspects of the multi-dimensional sensorimotor input space. Granule cells, potentially with the aid of differentiated thresholding from Golgi cells, enhance the utility of these projections by allowing the Purkinje cell to establish piecewise-linear approximations of non-linear functions. Our hypothesis provides a novel view on the function of the spinal circuitry and cerebellar granule layer, illustrating how the coordinating functions of the cerebellum can be crucially supported by the recombinations performed by the neurons of the spinocerebellar systems. PMID:23516353

  2. Processing of multi-dimensional sensorimotor information in the spinal and cerebellar neuronal circuitry: a new hypothesis.

    PubMed

    Spanne, Anton; Jörntell, Henrik

    2013-01-01

    Why are sensory signals and motor command signals combined in the neurons of origin of the spinocerebellar pathways and why are the granule cells that receive this input thresholded with respect to their spike output? In this paper, we synthesize a number of findings into a new hypothesis for how the spinocerebellar systems and the cerebellar cortex can interact to support coordination of our multi-segmented limbs and bodies. A central idea is that recombination of the signals available to the spinocerebellar neurons can be used to approximate a wide array of functions including the spatial and temporal dependencies between limb segments, i.e. information that is necessary in order to achieve coordination. We find that random recombination of sensory and motor signals is not a good strategy since, surprisingly, the number of granule cells severely limits the number of recombinations that can be represented within the cerebellum. Instead, we propose that the spinal circuitry provides useful recombinations, which can be described as linear projections through aspects of the multi-dimensional sensorimotor input space. Granule cells, potentially with the aid of differentiated thresholding from Golgi cells, enhance the utility of these projections by allowing the Purkinje cell to establish piecewise-linear approximations of non-linear functions. Our hypothesis provides a novel view on the function of the spinal circuitry and cerebellar granule layer, illustrating how the coordinating functions of the cerebellum can be crucially supported by the recombinations performed by the neurons of the spinocerebellar systems. PMID:23516353

  3. Stress Granules Modulate SYK to Cause Microglial Cell Dysfunction in Alzheimer's Disease

    PubMed Central

    Ghosh, Soumitra; Geahlen, Robert L.

    2015-01-01

    Microglial cells in the brains of Alzheimer's patients are known to be recruited to amyloid-beta (Aβ) plaques where they exhibit an activated phenotype, but are defective for plaque removal by phagocytosis. In this study, we show that microglia stressed by exposure to sodium arsenite or Aβ(1–42) peptides or fibrils form extensive stress granules (SGs) to which the tyrosine kinase, SYK, is recruited. SYK enhances the formation of SGs, is active within the resulting SGs and stimulates the production of reactive oxygen and nitrogen species that are toxic to neuronal cells. This sequestration of SYK inhibits the ability of microglial cells to phagocytose Escherichia coli or Aβ fibrils. We find that aged microglial cells are more susceptible to the formation of SGs; and SGs containing SYK and phosphotyrosine are prevalent in the brains of patients with severe Alzheimer's disease. Phagocytic activity can be restored to stressed microglial cells by treatment with IgG, suggesting a mechanism to explain the therapeutic efficacy of intravenous IgG. These studies describe a mechanism by which stress, including exposure to Aβ, compromises the function of microglial cells in Alzheimer's disease and suggest approaches to restore activity to dysfunctional microglial cells. PMID:26870803

  4. Anti-Yo Associated Paraneoplastic Cerebellar Degeneration in a Man with Large Cell Cancer of the Lung

    PubMed Central

    Lee, James; Wang, Bonnie H.; Yekkirala, Lalitha

    2013-01-01

    Purkinje cell cytoplasmic antibody type 1 (PCA-1), or anti-Yo, is the most frequently detected autoantibody in paraneoplastic cerebellar degeneration (PCD). The vast majority of cases of anti-Yo PCD, however, occur in females over 60 years old and are associated with gynecologic tumors. Only 10 cases have been reported in males, and only 2 were associated with cancer of the lung. Here we describe the youngest known case of PCA-1 positive PCD in a male, whose lung tumor was undetectable even on FDG-PET. PMID:24167748

  5. Sorafenib, a multikinase inhibitor, induces formation of stress granules in hepatocarcinoma cells

    PubMed Central

    Adjibade, Pauline; St-Sauveur, Valérie Grenier; Huberdeau, Miguel Quevillon; Fournier, Marie-Josée; Savard, Andreanne; Coudert, Laetitia; Khandjian, Edouard W.; Mazroui, Rachid

    2015-01-01

    Stress granules (SGs) are cytoplasmic RNA multimeric bodies that form under stress conditions known to inhibit translation initiation. In most reported stress cases, the formation of SGs was associated with the cell recovery from stress and survival. In cells derived from cancer, SGs formation was shown to promote resistance to either proteasome inhibitors or 5-Fluorouracil used as chemotherapeutic agents. Despite these studies, the induction of SGs by chemotherapeutic drugs contributing to cancer cells resistance is still understudied. Here we identified sorafenib, a tyrosine kinase inhibitor used to treat hepatocarcinoma, as a potent chemotherapeutic inducer of SGs. The formation of SGs in sorafenib-treated hepatocarcionoma cells correlates with inhibition of translation initiation; both events requiring the phosphorylation of the translation initiation factor eIF2α. Further characterisation of the mechanism of sorafenib-induced SGs revealed PERK as the main eIF2α kinase responsible for SGs formation. Depletion experiments support the implication of PERK-eIF2α-SGs pathway in hepatocarcinoma cells resistance to sorafenib. This study also suggests the existence of an unexpected complex regulatory balance between SGs and phospho-eIF2α where SGs dampen the activation of the phospho-eIF2α-downstream ATF4 cell death pathway. PMID:26556863

  6. The Critical Role of Golgi Cells in Regulating Spatio-Temporal Integration and Plasticity at the Cerebellum Input Stage

    PubMed Central

    D'Angelo, Egidio

    2008-01-01

    The discovery of the Golgi cell is bound to the foundation of the Neuron Doctrine. Recently, the excitable mechanisms of this inhibitory interneuron have been investigated with modern experimental and computational techniques raising renewed interest for the implications it might have for cerebellar circuit functions. Golgi cells are pacemakers with preferential response frequency and phase-reset in the theta-frequency band and can therefore impose specific temporal dynamics to granule cell responses. Moreover, through their connectivity, Golgi cells determine the spatio-temporal organization of cerebellar activity. Finally, Golgi cells, by controlling granule cell depolarization and NMDA channel unblock, regulate the induction of long-term synaptic plasticity at the mossy fiber – granule cell synapse. Thus, the Golgi cells can exert an extensive control on spatio-temporal signal organization and information storage in the granular layer playing a critical role for cerebellar computation. PMID:18982105

  7. Cerebellar Structure and Function in Male Wistar-Kyoto Hyperactive Rats

    PubMed Central

    Thanellou, Alexandra; Green, John T.

    2014-01-01

    Previous research has suggested that the Wistar-Kyoto Hyperactive (WKHA) rat strain may model some of the behavioral features associated with attention-deficit/hyperactivity disorder (ADHD). We have shown that, in cerebellar-dependent eyeblink conditioning, WKHA emit eyeblink CRs with shortened onset latencies. To further characterize the shortened CR onset latencies seen in WKHA rats, we examined 750-ms delay conditioning with either a tone CS or a light CS, we extended acquisition training, and we included Wistar rats as an additional, outbred control strain. Our results indicated that WKHAs learned more quickly and showed a shortened CR onset latency to a tone CS compared to both Wistar-Kyoto Hypertensive (WKHT) and Wistars. WKHAs and Wistars show a lengthening of CR onset latency over conditioning with a tone CS and an increasing confinement of CRs to the later part of the tone CS (inhibition of delay). WKHAs learned more quickly to a light CS only in comparison to WKHTs and showed a shortened CR onset latency only in comparison to Wistars. Wistars showed an increasing confinement of CRs to the late part of the light CS over conditioning. We used unbiased stereology to estimate the number of Purkinje and granule cells in the cerebellar cortex of the three strains. Our results indicated that WKHAs have more granule cells than Wistars and WKHTs and more Purkinje cells than Wistars. Results are discussed in terms of CS processing and cerebellar cortical contributions to EBC. PMID:23398437

  8. Lesion-induced and activity-dependent structural plasticity of Purkinje cell dendritic spines in cerebellar vermis and hemisphere.

    PubMed

    Gelfo, Francesca; Florenzano, Fulvio; Foti, Francesca; Burello, Lorena; Petrosini, Laura; De Bartolo, Paola

    2016-09-01

    Neuroplasticity allows the brain to encode experience and learn behaviors, and also to re-acquire lost functions after damage. The cerebellum is a suitable structure to address this topic because of its strong involvement in learning processes and compensation of lesion-induced deficits. This study was aimed to characterize the effects of a hemicerebellectomy (HCb) combined or not with the exposition to environmental enrichment (EE) on dendritic spine density and size in Purkinje cell proximal and distal compartments of cerebellar vermian and hemispherical regions. Male Wistar rats were housed in enriched or standard environments from the 21st post-natal day (pnd) onwards. At the 75th pnd, rats were submitted to HCb or sham lesion. Neurological symptoms and spatial performance in the Morris water maze were evaluated. At the end of testing, morphological analyses assessed dendritic spine density, area, length, and head diameter on vermian and hemispherical Purkinje cells. All hemicerebellectomized (HCbed) rats showed motor compensation, but standard-reared HCbed animals exhibited cognitive impairment that was almost completely compensated in enriched HCbed rats. The standard-reared HCbed rats showed decreased density with augmented size of Purkinje cell spines in the vermis, and augmented both density and size in the hemisphere. Enriched HCbed rats almost completely maintained the spine density and size induced by EE. Both lesion-induced and activity-dependent cerebellar plastic changes may be interpreted as "beneficial" brain reactions, aimed to support behavioral performance rescuing. PMID:26420278

  9. Mast cell procarboxypeptidase A. Molecular modeling and biochemical characterization of its processing within secretory granules.

    PubMed

    Springman, E B; Dikov, M M; Serafin, W E

    1995-01-20

    Previously, we characterized murine mast cell procarboxypeptidase A (MC-proCPA) as an inactive zymogen. To investigate the mechanisms for this lack of enzymatic activity and the processing of the zymogen to the active form, we now have performed molecular modeling of the tertiary structure of murine MC-proCPA based on the x-ray crystallographic structures of porcine pancreatic procarboxypeptidases A and B. Our model predicts that MC-proCPA retains a high degree of structural similarity to its pancreatic homologues. The globular propeptide physically blocks access to the fully formed active site of the catalytic domain and contains a salt bridge to the substrate-binding region that precludes docking of even small substrates. Based on consideration of the predicted tertiary structure and charge field characteristics of the model, the activation site (between GluA94 and Ile1) appears to be highly exposed even after MC-proCPA binds to secretory granule proteoglycans. Based on the steady-state levels of MC-proCPA versus MC-CPA, cycloheximide inhibition of protein synthesis, and brefeldin A blockage of protein sorting, we show that MC-proCPA is processed rapidly in murine mast cell line KiSV-MC14 with a half-life of 26 +/- 5 min (mean +/- S.D., n = 3), and the processing occurs within the secretory granules. The enzyme responsible for this processing may be a thiol protease since treatment of the KiSV-MC14 with 200 microM E-64d, a selective thiol-protease inhibitor, increases MC-proCPA by 2.7 +/- 0.2-fold (mean +/- S.D., n = 3) within 6 h of application. PMID:7836395

  10. Object/Context-Specific Memory Deficits Associated with Loss of Hippocampal Granule Cells after Adrenalectomy in Rats

    ERIC Educational Resources Information Center

    Spanswick, Simon C.; Sutherland, Robert J.

    2010-01-01

    Chronic adrenalectomy (ADX) causes a gradual and selective loss of granule cells in the dentate gyrus (DG) of the rat. Here, we administered replacement corticosterone to rats beginning 10 wk after ADX. We then tested them in three discrimination tasks based on object novelty, location, or object/context association. Only during testing of the…

  11. Transient degradation of NF-kappaB proteins in macrophages after interaction with mast cell granules.

    PubMed Central

    Ito, N; Li, Y; Suzuki, T; Stechschulte, D J; Dileepan, K N

    1998-01-01

    The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the formation of altered nuclear transcription factor proteins (NF-kappaBx), which had faster electrophoretic mobility than the p50 homodimer of NF-KB, but retained comparable DNA binding capacity. Antibodies to N-terminal peptides of p50, p52, p65 or c-Rel supershifted only a fraction of NF-kappaBx. Western blot analyses revealed that nuclear p65 and c-Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c-Rel as well as IkBalpha remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of mouse peritoneal macrophages with MCG resulted in more extensive alterations to NF-KB proteins. The alterations in NF-KB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF-alpha mRNA inJ774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c-Rel, which retain intact N-terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF-KB proteins with associated inhibition of iNOS or TNF-alpha mRNA expression. PMID:9927232

  12. PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

    PubMed

    Ho, Yeung; Li, Xiting; Jamison, Stephanie; Harding, Heather P; McKinnon, Peter J; Ron, David; Lin, Wensheng

    2016-07-01

    Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation. PMID:27181404

  13. Regulation of synaptic transmission in the mossy fibre-granule cell pathway of rat cerebellum by metabotropic glutamate receptors.

    PubMed

    Vetter, P; Garthwaite, J; Batchelor, A M

    1999-06-01

    The role of metabotropic glutamate receptors (mGluRs) in the mossy fibre-granule cell pathway in rat cerebellum was studied using slice preparations and electrophysiological techniques. Application of the group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked, in a concentration-dependent manner (EC50 = 33 microM), a depolarising/hyperpolarising complex response from granule cells which was preferentially inhibited by the group I selective antagonist (S)-4-carboxyphenylglycine (4CPG). The group III selective agonist L-amino-4-phosphonobutyrate (AP4) evoked a hyperpolarising response (EC50 = 10 microM) which was inhibited by the group II/III selective antagonist (S)-alpha-methyl-4-phosphonophenylglycine (MPPG). The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV) elicited no measurable voltage change. The amplitude of the synaptically-mediated mossy fibre response in granule cells was unaffected during application of AP4, was reduced by DHPG and was enhanced by DCG-IV (EC50 = 80 nM). These effects were inhibited by the group selective antagonists 4CPG and (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-4), respectively. Further investigation using patch-clamp recording revealed that DCG-IV potently inhibited spontaneous GABAergic currents. We conclude that group I and III (but not group II) mGluRs are functionally expressed by granule cells, whereas unexpectedly group II or III mGluRs do not appear to be present presynaptically on mossy fibre terminals. Group II mGluRs are located on Golgi cell terminals; when activated these receptors cause disinhibition, a function which may be important for gating information transfer from the mossy fibres to the granule cells. PMID:10465684

  14. Widespread State-Dependent Shifts in Cerebellar Activity in Locomoting Mice

    PubMed Central

    Ozden, Ilker; Dombeck, Daniel A.; Hoogland, Tycho M.; Tank, David W.; Wang, Samuel S.-H.

    2012-01-01

    Excitatory drive enters the cerebellum via mossy fibers, which activate granule cells, and climbing fibers, which activate Purkinje cell dendrites. Until now, the coordinated regulation of these pathways has gone unmonitored in spatially resolved neuronal ensembles, especially in awake animals. We imaged cerebellar activity using functional two-photon microscopy and extracellular recording in awake mice locomoting on an air-cushioned spherical treadmill. We recorded from putative granule cells, molecular layer interneurons, and Purkinje cell dendrites in zone A of lobule IV/V, representing sensation and movement from trunk and limbs. Locomotion was associated with widespread increased activity in granule cells and interneurons, consistent with an increase in mossy fiber drive. At the same time, dendrites of different Purkinje cells showed increased co-activation, reflecting increased synchrony of climbing fiber activity. In resting animals, aversive stimuli triggered increased activity in granule cells and interneurons, as well as increased Purkinje cell co-activation that was strongest for neighboring dendrites and decreased smoothly as a function of mediolateral distance. In contrast with anesthetized recordings, no 1–10 Hz oscillations in climbing fiber activity were evident. Once locomotion began, responses to external stimuli in all three cell types were strongly suppressed. Thus climbing and mossy fiber representations can shift together within a fraction of a second, reflecting in turn either movement-associated activity or external stimuli. PMID:22880068

  15. mGlu1 receptor mediates homeostatic control of intrinsic excitability through Ih in cerebellar Purkinje cells.

    PubMed

    Shim, Hyun Geun; Jang, Sung-Soo; Jang, Dong Cheol; Jin, Yunju; Chang, Wonseok; Park, Joo Min; Kim, Sang Jeong

    2016-06-01

    Homeostatic intrinsic plasticity is a cellular mechanism for maintaining a stable neuronal activity level in response to developmental or activity-dependent changes. Type 1 metabotropic glutamate receptor (mGlu1 receptor) has been widely known to monitor neuronal activity, which plays a role as a modulator of intrinsic and synaptic plasticity of neurons. Whether mGlu1 receptor contributes to the compensatory adjustment of Purkinje cells (PCs), the sole output of the cerebellar cortex, in response to chronic changes in excitability remains unclear. Here, we demonstrate that the mGlu1 receptor is involved in homeostatic intrinsic plasticity through the upregulation of the hyperpolarization-activated current (Ih) in cerebellar PCs. This plasticity was prevented by inhibiting the mGlu1 receptor with Bay 36-7620, an mGlu1 receptor inverse agonist, but not with CPCCOEt, a neutral antagonist. Chronic inactivation with tetrodotoxin (TTX) increased the components of Ih in the PCs, and ZD 7288, a hyperpolarization-activated cyclic nucleotide-gated channel selective inhibitor, fully restored reduction of firing rates in the deprived neurons. The homeostatic elevation of Ih was also prevented by BAY 36-7620, but not CPCCOEt. Furthermore, KT 5720, a blocker of protein kinase A (PKA), prevented the effect of TTX reducing the evoked firing rates, indicating the reduction in excitability of PCs due to PKA activation. Our study shows that both the mGlu1 receptor and the PKA pathway are involved in the homeostatic intrinsic plasticity of PCs after chronic blockade of the network activity, which provides a novel understanding on how cerebellar PCs can preserve the homeostatic state under activity-deprived conditions. PMID:26912592

  16. Morphometric studies of secretory granule formation in mouse pancreatic acinar cells. Dissecting the early structural changes following pilocarpine injection

    PubMed Central

    HAMMEL, ILAN; SHOR-HAZAN, OSNAT; ELDAR, TORA; AMIHAI, DINA; LEW, SYLVIA

    1999-01-01

    Secretory granule formation in pancreatic acinar cells is known to involve massive membrane flow. In previous studies we have undertaken morphometry of the regranulation mechanism in these cells and in mast cells as a model for cellular membrane movement. In our current work, electron micrographs of pancreatic acinar cells from ICR mice were taken at several time points after extensive degranulation induced by pilocarpine injection in order to investigate the volume changes of rough endoplasmic reticulum (RER), nucleus, mitochondria and autophagosomes. At 2–4 h after stimulation, when the pancreatic cells demonstrated a complete loss of granules, this was accompanied by an increased proportion of autophagosomal activity. This change primarily reflected a greatly increased proportion of profiles retaining autophagic vacuoles containing recognisable cytoplasmic structures such as mitochondria, granule profiles and fragments of RER. The mitochondrial structures reached a significant maximal size 4 h following injection (before degranulation 0.178±0.028 μm3; at 4 h peak value, 0.535±0.109 μm3). Nucleus size showed an early volume increase approaching a maximum value 2 h following degranulation. The regranulation span was thus divided into 3 stages. The first was the membrane remodelling stage (0–2 h). During this period the volume of the RER and secretory granules was greatly decreased. At the intermediate stage (2–4 h) a significant increase of the synthesis zone was observed within the nucleus. The volume of the mitochondria was increasing. At the last step, the major finding was a significant granule accumulation in parallel with an active Golgi zone. PMID:10227666

  17. Late onset cerebellar cortical degeneration in a koala.

    PubMed

    Kuwamura, M; Murai, F; Nishioka, S; Aoki, M; Ohashi, F; Yamate, J; Kotani, T; Summers, B A

    2009-08-01

    A 10-year-old male koala started to fall from the tree while sleeping. Subsequently, the koala often fell down while walking and showed a gait abnormality, abnormal nystagmus and hypersalivation. At 12 years of age, the koala became ataxic and seemed blind. At 13 years of age, the koala exhibited signs of dysstasia and was euthanased. Necropsy revealed marked symmetrical atrophy of the cerebellum. Histopathologically, a severe loss of Purkinje and granule cells was evident in the cerebellum, while the molecular layer was more cellular than normal with cells resembling small neurons, which were positively stained with parvalbumin immunohistochemistry. Reactive Bergmann glial cells (astrocytes) were present adjacent to the depleted Purkinje cell zone. The very late onset and slow progression of the cerebellar cortical degeneration in this case is particularly interesting and appears to be the first report in the koala. PMID:19673852

  18. Translation suppression promotes stress granule formation and cell survival in response to cold shock

    PubMed Central

    Hofmann, Sarah; Cherkasova, Valeria; Bankhead, Peter; Bukau, Bernd; Stoecklin, Georg

    2012-01-01

    Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia. PMID:22875991

  19. Tonic inhibition sets the state of excitability in olfactory bulb granule cells

    PubMed Central

    Labarrera, Christina; London, Michael; Angelo, Kamilla

    2013-01-01

    GABAergic granule cells (GCs) regulate, via mitral cells, the final output from the olfactory bulb to piriform cortex and are central for the speed and accuracy of odour discrimination. However, little is known about the local circuits in which GCs are embedded and how GCs respond during functional network activity. We recorded inhibitory and excitatory currents evoked during a single sniff-like odour presentation in GCs in vivo. We found that synaptic excitation was extensively activated across cells, whereas phasic inhibition was rare. Furthermore, our analysis indicates that GCs are innervated by a persistent firing of deep short axon cells that mediated the inhibitory evoked responses. Blockade of GABAergic synaptic input onto GCs revealed a tonic inhibitory current mediated by furosemide-sensitive GABAA receptors. The average current associated with this tonic GABAergic conductance was 3-fold larger than that of phasic inhibitory postsynaptic currents. We show that the pharmacological blockage of tonic inhibition markedly increased the occurrence of supra-threshold responses during an odour-stimulated sniff. Our findings suggest that GCs mediate recurrent or lateral inhibition, depending on the ambient level of extracellular GABA. PMID:23318869

  20. Synaptotagmin-7 Functions to Replenish Insulin Granules for Exocytosis in Human Islet β-Cells.

    PubMed

    Dolai, Subhankar; Xie, Li; Zhu, Dan; Liang, Tao; Qin, Tairan; Xie, Huanli; Kang, Youhou; Chapman, Edwin R; Gaisano, Herbert Y

    2016-07-01

    Synaptotagmin (Syt)-7, a major component of the exocytotic machinery in neurons, is also the major Syt in rodent pancreatic β-cells shown to mediate glucose-stimulated insulin secretion (GSIS). However, Syt-7's precise exocytotic actions in β-cells remain unknown. We show that Syt-7 is abundant in human β-cells. Adenovirus-short hairpin RNA knockdown (KD) of Syt-7 in human islets reduced first- and second-phase GSIS attributed to the reduction of exocytosis of predocked and newcomer insulin secretory granules (SGs). Glucose stimulation expectedly induced Syt-7 association in a Ca(2+)-dependent manner with syntaxin-3 and syntaxin-1A soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes known to mediate exocytosis of newcomer and predocked SGs, respectively. However, Syt-7-KD did not disrupt SNARE complex assembly. Instead, electron microscopy analysis showed that Syt-7-KD reduced the recruitment of SGs to the plasma membrane after glucose-stimulated depletion, which could not be rescued by glucagon-like peptide 1 pretreatment. To assess the possibility that this new action of Syt-7 on SG recruitment may involve calmodulin (CaM), pretreatment of islets with CaM blocker calmidazolium showed effects very similar to those of Syt-7-KD. Syt-7 therefore plays a novel more dominant function in the replenishment of releasable SG pools in human β-cells than its previously purported role in exocytotic fusion per se. PMID:27207520

  1. Potential implications of a monosynaptic pathway from mossy cells to adult-born granule cells of the dentate gyrus

    PubMed Central

    Scharfman, Helen E.; Bernstein, Hannah L.

    2015-01-01

    The dentate gyrus (DG) is important to many aspects of hippocampal function, but there are many aspects of the DG that are incompletely understood. One example is the role of mossy cells (MCs), a major DG cell type that is glutamatergic and innervates the primary output cells of the DG, the granule cells (GCs). MCs innervate the GCs as well as local circuit neurons that make GABAergic synapses on GCs, so the net effect of MCs on GCs – and therefore the output of the DG – is unclear. Here we first review fundamental information about MCs and the current hypotheses for their role in the normal DG and in diseases that involve the DG. Then we review previously published data which suggest that MCs are a source of input to a subset of GCs that are born in adulthood (adult-born GCs). In addition, we discuss the evidence that adult-born GCs may support the normal inhibitory ‘gate’ functions of the DG, where the GCs are a filter or gate for information from the entorhinal cortical input to area CA3. The implications are then discussed in the context of seizures and temporal lobe epilepsy (TLE). In TLE, it has been suggested that the DG inhibitory gate is weak or broken and MC loss leads to insufficient activation of inhibitory neurons, causing hyperexcitability. That idea was called the “dormant basket cell hypothesis.” Recent data suggest that loss of normal adult-born GCs may also cause disinhibition, and seizure susceptibility. Therefore, we propose a reconsideration of the dormant basket cell hypothesis with an intervening adult-born GC between the MC and basket cell and call this hypothesis the “dormant immature granule cell hypothesis.” PMID:26347618

  2. Recovery of amorphous polyhydroxybutyrate granules from Cupriavidus necator cells grown on used cooking oil.

    PubMed

    Martino, Lucrezia; Cruz, Madalena V; Scoma, Alberto; Freitas, Filomena; Bertin, Lorenzo; Scandola, Mariastella; Reis, Maria A M

    2014-11-01

    Used cooking oil (UCO) was employed as the sole carbon source for the production of polyhydroxybutyrate (PHB) by cultivation in batch mode of Cupriavidus necator DSM 428. The produced biomass was used for extraction of the PHB granules with a solvent-free approach using sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), and the enzyme Alcalase in an aqueous medium. The recovered PHB granules showed a degree of purity higher than 90% and no crystallization (i.e., granules were recovered in their 'native' amorphous state) as demonstrated by wide angle X-ray diffraction (WAXS). Granules were characterized according to their thermal properties and stability by differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Results show that UCO can be used as a renewable resource to produce amorphous PHB granules with excellent properties in a biocompatible manner. PMID:24751509

  3. Model of very fast (>75 Hz) network oscillations generated by electrical coupling between the proximal axons of cerebellar Purkinje cells

    PubMed Central

    Traub, Roger D; Middleton, Steven J; Knöpfel, Thomas; Whittington, Miles A

    2009-01-01

    Very fast oscillations (VFO, >75 Hz) occur transiently in vivo, in the cerebellum of mice genetically modified to model Angelman syndrome, and in a mouse model of fetal alcohol syndrome. We recently reported VFO in slices of mouse cerebellar cortex (Crus I and II of ansiform and paramedian lobules), either in association with gamma oscillations (~40 Hz, evoked by nicotine), or in isolation (evoked by nicotine in combination with GABAA receptor blockade). The experimental data suggest a role for electrical coupling between Purkinje cells (blockade of VFO by drugs reducing gap junction conductance, and spikelets in some Purkinje cells); and the data suggest the specific involvement of Purkinje cell axons (because of field oscillation maxima in the granular layer). We show here that a detailed network model (1,000 multicompartment Purkinje cells) replicates the experimental data, when gap junctions are located on the proximal axons of Purkinje cells, provided sufficient spontaneous firing is present. Unlike other VFO models, most somatic spikelets do not correspond to axonal spikes in the parent axon, but reflect spikes in electrically coupled axons. The model predicts gating of VFO frequency by gNa inactivation, and experiments prolonging this inactivation time constant, with β-pompilidotoxin, are consistent with this prediction. The model also predicts that cerebellar VFO can be explained as an electrically coupled system of axons which are not intrinsic oscillators: the electrically uncoupled cells do not individually oscillate (in the model), and axonal firing rates are much lower in the uncoupled state than in the coupled state. PMID:18973579

  4. Granule membrane protein 140 (GMP140) binds to carcinomas and carcinoma-derived cell lines.

    PubMed Central

    Aruffo, A; Dietsch, M T; Wan, H; Hellström, K E; Hellström, I

    1992-01-01

    The glycoproteins granule membrane protein 140 (GMP140), endothelial-leukocyte adhesion molecule 1 (ELAM-1), and Leu-8 are members of a family of glycoprotein receptors (selectins or LEC-CAMs) that play an important role in adhesive interactions between circulating leukocytes and vascular endothelium. Recently it has been reported that ELAM-1 is able to mediate the binding of the colon carcinoma cell line HT-29 to cytokine-activated vascular endothelium, suggesting that tumor cell adhesion to vascular endothelium, a prerequisite for tumor extravasation and metastasis, is in part the result of adhesive interactions between blood-borne tumor cells and cell surface proteins expressed by vascular endothelium. Here, using an approach in which soluble immunoglobulin chimeras of the GMP140 and ELAM-1 receptors were prepared and used to carry out immunohistological studies, we establish that GMP140 binds to tumor cells in a variety of human carcinoma tissue sections (colon, lung, and breast), whereas ELAM-1 binds exclusively to tumor cells in colon carcinoma tissue sections. In addition, GMP140 was found to bind to the cell surface of a number of cell lines derived from various carcinomas but not from melanomas, whereas ELAM-1 bound only colon carcinoma cell lines. We further investigated the nature of the ligands of GMP140 and ELAM-1 on the surface of the carcinoma cells and found that the GMP140 ligand on the surface of tumor cells appears to be distinct from that expressed on the myeloid cell line HL-60. Neuraminidase treatment of a breast carcinoma cell line does not affect, or in some instances increases, GMP140 binding, whereas it completely abolishes GMP140 binding to HL-60 cells. On the other hand, the ligand of ELAM-1 on both the colon carcinoma and HL-60 cells is neuraminidase sensitive in accord with its identification as sialyl-CD15. Parallel results were obtained with neuraminidase-treated frozen carcinoma tissue sections. The present findings form the basis

  5. Adult-born granule cells mature through two functionally distinct states

    PubMed Central

    Brunner, János; Neubrandt, Máté; Van-Weert, Susan; Andrási, Tibor; Kleine Borgmann, Felix B; Jessberger, Sebastian; Szabadics, János

    2014-01-01

    Adult-born granule cells (ABGCs) are involved in certain forms of hippocampus-dependent learning and memory. It has been proposed that young but functionally integrated ABGCs (4-weeks-old) specifically contribute to pattern separation functions of the dentate gyrus due to their heightened excitability, whereas old ABGCs (>8 weeks old) lose these capabilities. Measuring multiple cellular and integrative characteristics of 3- 10-week-old individual ABGCs, we show that ABGCs consist of two functionally distinguishable populations showing highly distinct input integration properties (one group being highly sensitive to narrow input intensity ranges while the other group linearly reports input strength) that are largely independent of the cellular age and maturation stage, suggesting that ‘classmate’ cells (born during the same period) can contribute to the network with fundamentally different functions. Thus, ABGCs provide two temporally overlapping but functionally distinct neuronal cell populations, adding a novel level of complexity to our understanding of how life-long neurogenesis contributes to adult brain function. DOI: http://dx.doi.org/10.7554/eLife.03104.001 PMID:25061223

  6. Adult-born granule cells mature through two functionally distinct states.

    PubMed

    Brunner, János; Neubrandt, Máté; Van-Weert, Susan; Andrási, Tibor; Kleine Borgmann, Felix B; Jessberger, Sebastian; Szabadics, János

    2014-01-01

    Adult-born granule cells (ABGCs) are involved in certain forms of hippocampus-dependent learning and memory. It has been proposed that young but functionally integrated ABGCs (4-weeks-old) specifically contribute to pattern separation functions of the dentate gyrus due to their heightened excitability, whereas old ABGCs (>8 weeks old) lose these capabilities. Measuring multiple cellular and integrative characteristics of 3- 10-week-old individual ABGCs, we show that ABGCs consist of two functionally distinguishable populations showing highly distinct input integration properties (one group being highly sensitive to narrow input intensity ranges while the other group linearly reports input strength) that are largely independent of the cellular age and maturation stage, suggesting that 'classmate' cells (born during the same period) can contribute to the network with fundamentally different functions. Thus, ABGCs provide two temporally overlapping but functionally distinct neuronal cell populations, adding a novel level of complexity to our understanding of how life-long neurogenesis contributes to adult brain function. PMID:25061223

  7. Cytochrome c is released from mitochondria in a reactive oxygen species (ROS)-dependent fashion and can operate as a ROS scavenger and as a respiratory substrate in cerebellar neurons undergoing excitotoxic death.

    PubMed

    Atlante, A; Calissano, P; Bobba, A; Azzariti, A; Marra, E; Passarella, S

    2000-11-24

    In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome c take place during glutamate toxicity. This investigation was aimed (i) to ascertain whether and how these two processes are related and (ii) to gain insight into the role played by the released cytochrome c in the onset of neurotoxicity. Cytochrome c release takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome c release was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome c was able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochrome c release can be part of a cellular and mitochondrial defense mechanism against oxidative stress. PMID:10980192

  8. Restricted distribution of mrg-1 mRNA in C. elegans primordial germ cells through germ granule-independent regulation.

    PubMed

    Miwa, Takashi; Takasaki, Teruaki; Inoue, Kunio; Sakamoto, Hiroshi

    2015-11-01

    The chromodomain protein MRG-1 is an essential maternal factor for proper germline development that protects germ cells from cell death in C. elegans. Unlike germ granules, which are exclusively segregated to the germline blastomeres at each cell division from the first cleavage of the embryo, MRG-1 is abundant in all cells in early embryos and is then gradually restricted to the primordial germ cells (PGCs) by the morphogenesis stage. Here, we show that this characteristic spatiotemporal expression pattern is dictated by the mrg-1 3'UTR and is differentially regulated at the RNA level between germline and somatic cells. Asymmetric segregation of germ granules is not necessary to localize MRG-1 to the PGCs. We found that MES-4, an essential chromatin regulator in germ cells, also accumulates in the PGCs in a germ granule-independent manner. We propose that C.elegans PGCs have a novel mechanism to accumulate at least some chromatin-associated proteins that are essential for germline immortality. PMID:26537333

  9. Lgr4 protein deficiency induces ataxia-like phenotype in mice and impairs long term depression at cerebellar parallel fiber-Purkinje cell synapses.

    PubMed

    Guan, Xin; Duan, Yanhong; Zeng, Qingwen; Pan, Hongjie; Qian, Yu; Li, Dali; Cao, Xiaohua; Liu, Mingyao

    2014-09-19

    Cerebellar dysfunction causes ataxia characterized by loss of balance and coordination. Until now, the molecular and neuronal mechanisms of several types of inherited cerebellar ataxia have not been completely clarified. Here, we report that leucine-rich G protein-coupled receptor 4 (Lgr4/Gpr48) is highly expressed in Purkinje cells (PCs) in the cerebellum. Deficiency of Lgr4 leads to an ataxia-like phenotype in mice. Histologically, no obvious morphological changes were observed in the cerebellum of Lgr4 mutant mice. However, the number of PCs was slightly but significantly reduced in Lgr4(-/-) mice. In addition, in vitro electrophysiological analysis showed an impaired long term depression (LTD) at parallel fiber-PC (PF-PC) synapses in Lgr4(-/-) mice. Consistently, immunostaining experiments showed that the level of phosphorylated cAMP-responsive element-binding protein (Creb) was significantly decreased in Lgr4(-/-) PCs. Furthermore, treatment with forskolin, an adenylyl cyclase agonist, rescued phospho-Creb in PCs and reversed the impairment in PF-PC LTD in Lgr4(-/-) cerebellar slices, indicating that Lgr4 is an upstream regulator of Creb signaling, which is underlying PF-PC LTD. Together, our findings demonstrate for first time an important role for Lgr4 in motor coordination and cerebellar synaptic plasticity and provide a potential therapeutic target for certain types of inherited cerebellar ataxia. PMID:25063812

  10. Intracisternal Cyclodextrin Prevents Cerebellar Dysfunction and Purkinje Cell Death in Feline Niemann-Pick type C1 disease

    PubMed Central

    Vite, C. H.; Bagel, J. H.; Swain, G. P.; Prociuk, M.; Sikora, T. U.; Stein, V. M.; O’Donnell, P.; Ruane, T.; Ward, S.; Crooks, A.; Li, S.; Mauldin, E.; Stellar, S.; De Meulder, M.; Kao, M. L.; Ory, D. S.; Davidson, C.; Vanier, M. T.; Walkley, S. U.

    2015-01-01

    Niemann-Pick type C1 (NPC) disease is a lysosomal storage disease caused by mutations in the NPC1 gene, leading to an increase in unesterified cholesterol and several sphingolipids, and resulting in hepatic disease and progressive neurological disease. Whereas subcutaneous administration of the pharmaceutical excipient 2-hydroxypropyl-beta-cyclodextrin (HPβCD) ameliorated hepatic disease, doses sufficient to reduce neurological disease resulted in pulmonary toxicity. In contrast, direct administration of HPβCD into the cisterna magna of presymptomatic cats with NPC disease prevented the onset of cerebellar dysfunction for greater than a year and resulted in a reduction in Purkinje cell loss and near normal concentrations of cholesterol and sphingolipids. Moreover, administration of intracisternal HPβCD to NPC cats with ongoing cerebellar dysfunction slowed disease progression, increased survival time, and decreased the accumulation of brain gangliosides. An increase in hearing threshold was identified as a potential adverse effect. Together, these studies in the feline animal model have provided critical data on efficacy and safety of drug administration directly into the CNS that will be important for advancing HPβCD into clinical trials. PMID:25717099

  11. Primary Signet Ring Cell Carcinoma of the Lung with Cerebellar Metastasis Showing Full Response to Cisplatin and Docetaxel Therapy

    PubMed Central

    Selcukbiricik, Fatih; Bilici, Ahmet; Kanıtez, Metin; Yildiz, Serdar; Avci, Suna; Tanik, Canan

    2014-01-01

    Introduction. Primary signet ring cell carcinoma (SRCC) of the lung is a very rare disease. We describe a new case of primary SRCC of the lung with cerebellar metastasis, which responded well to the therapeutic approach with cisplatin and docetaxel. Case Report. A 41-year-old female patient (nonsmoker) was consulted to our oncology outpatient clinic after cerebellar metastasectomy. The histopathological diagnosis was SRCC metastasis. The primary tumor was unknown. The PET-CT imaging showed a hypermetabolic mass in the right middle lobe of the lung and hypermetabolic mediastinal lymph node stations. Oesophagogastroduodenoscopy and colonoscopy showed no evidence of gastrointestinal system tumor. The clinical diagnosis of primary SRCC of the lung was made and the administration of six rounds of cisplatin and docetaxel treatment was planned. After the chemotherapy the PET-CT scan to evaluate the therapy response showed full metabolic regression of the primary tumor and the mediastinal lymph nodes. There was no evidence of new metastasis. Conclusion. Primary SRCC of the lung is a very rare disease with poor prognosis. There are not many cases in literature and no standardized chemotherapy protocols. Cisplatin and docetaxel may be a good treatment option. PMID:24716057

  12. Accumulation of abnormal adult-generated hippocampal granule cells predicts seizure frequency and severity

    PubMed Central

    Hester, Michael S.; Danzer, Steve C.

    2013-01-01

    Accumulation of abnormally integrated, adult-born, hippocampal dentate granule cells (DGC) is hypothesized to contribute to the development of temporal lobe epilepsy (TLE). DGCs have long been implicated in TLE, as they regulate excitatory signaling through the hippocampus and exhibit neuroplastic changes during epileptogenesis. Furthermore, DGCs are unusual in that they are continually generated throughout life, with aberrant integration of new cells underlying the majority of restructuring in the dentate during epileptogenesis. While it is known that these abnormal networks promote abnormal neuronal firing and hyperexcitability, it has yet to be established whether they directly contribute to seizure generation. If abnormal DGCs do contribute, a reasonable prediction would be that the severity of epilepsy will be correlated with the number or load of abnormal DGCs. To test this prediction, we utilized a conditional, inducible transgenic mouse model to fate-map adult-generated DGCs. Mossy cell loss, also implicated in epileptogenesis, was assessed as well. Transgenic mice rendered epileptic using the pilocarpine-status epilepticus model of epilepsy were monitored 24/7 by video/EEG for four weeks to determine seizure frequency and severity. Positive correlations were found between seizure frequency and: 1) the percentage of hilar ectopic DGCs, 2) the amount of mossy fiber sprouting and 3) the extent of mossy cell death. In addition, mossy fiber sprouting and mossy cell death were correlated with seizure severity. These studies provide correlative evidence in support of the hypothesis that abnormal DGCs contribute to the development of TLE, and also support a role for mossy cell loss. PMID:23699504

  13. A method for detailed analysis of the structure of mast cell secretory granules by negative contrast imaging

    PubMed Central

    Tanaka, Shotaro; Takakuwa, Yuichi

    2016-01-01

    Secretory granules (SGs) in mast cells contain various molecules that elicit allergy symptoms and are generally considered therapeutic targets. However, the biogenesis, maintenance, regulation, and recycling of these granules remain controversial, mainly due to the lack of suitable live-cell imaging methods. In this study, we applied negative contrast imaging with soluble green fluorescent protein (GFP) expressed in the cytoplasm as a method to validate structural information of mast cell SGs. We evaluated the accuracy of the method in detail, and we demonstrated that it can be used for quantitative analysis. Using this technique, secretory granules, the nucleus, mitochondria, and the cell body were visualized in individual RBL-2H3 mast cells without any influence. When combined with conventional multicolor fluorescence imaging, visualization of SG-associated proteins and SG–SG fusion was achieved. Moreover, 3D images were constructed based on this method, and detailed information on the number, size, and shape of individual SGs was obtained. We found that cell volume was correlated with SG number. In summary, the technique provides valuable and unique data, and will therefore advance SG research. PMID:26997316

  14. Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

    PubMed Central

    Greenlee, John E.; Clawson, Susan A.; Hill, Kenneth E.; Wood, Blair; Clardy, Stacey L.; Tsunoda, Ikuo; Carlson, Noel G.

    2015-01-01

    Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not

  15. SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells

    PubMed Central

    Kebede, Melkam A.; Oler, Angie T.; Gregg, Trillian; Balloon, Allison J.; Johnson, Adam; Mitok, Kelly; Rabaglia, Mary; Schueler, Kathryn; Stapleton, Donald; Thorstenson, Candice; Wrighton, Lindsay; Floyd, Brendan J.; Richards, Oliver; Raines, Summer; Eliceiri, Kevin; Seidah, Nabil G.; Rhodes, Christopher; Keller, Mark P.; Coon, Joshua L.; Audhya, Anjon; Attie, Alan D.

    2014-01-01

    We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptinob mutation (ob/ob), developed diabetes. β Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis. PMID:25157818

  16. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo.

    PubMed

    McDole, B; Isgor, C; Pare, C; Guthrie, K

    2015-09-24

    Olfactory bulb granule cells (GCs) are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on GC spines. These contacts are established in the distal apical dendritic compartment, while GC basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong GC neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb GC spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF in transgenic mice produces a marked increase in GC spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on GCs, suggesting a role for this factor in modulating GC functional connectivity within adult olfactory circuitry. PMID:26211445

  17. Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826).

    PubMed

    dos Santos, Daiane Cristina Marques; Cupertino, Marli do Carmo; Fialho, Maria do Carmo Queiroz; Barbosa, Alfredo Jose Afonso; Fonseca, Cláudio Cesar; Sartori, Sirlene Souza Rodrigues; da Matta, Sérgio Luis Pinto

    2014-02-01

    This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats. PMID:24359801

  18. Cerebellar EBV-associated diffuse large B cell lymphoma following angioimmunoblastic T cell lymphoma

    PubMed Central

    Zhou, Yi; Rosenblum, Marc K.; Dogan, Ahmet; Jungbluth, Achim A.; Chiu, April

    2016-01-01

    Epstein-Barr virus (EBV)-associated B cell lymphoproliferative disorders may be seen in patients with angioimmunoblastic T cell lymphoma (AITL). Although both nodal and extranodal sites of involvement have been described, central nervous system involvement by B cell lymphoma following AITL has not previously been documented. We report a first example of such unusual presentation, in which an 80-year-old man developed diffuse large B cell lymphoma (DLBCL) in the cerebellum 4 months after the initial diagnosis of AITL. EBV-encoded RNAs were detected in the DLBCL, suggesting that EBV played a pivotal role in the pathogenesis of high-grade histologic progression of AITL. The patient survived less than 9 months after his initial diagnosis of AITL. We believe that this case expands the spectrum of extranodal manifestation of EBV-positive B cell lymphoma associated with AITL and illustrates the importance of recognition of this association when encountering unusual central nervous system lesions in patients with known AITL.

  19. Modeling possible effects of atypical cerebellar processing on eyeblink conditioning in autism.

    PubMed

    Radell, Milen L; Mercado, Eduardo

    2014-09-01

    Autism is unique among other disorders in that acquisition of conditioned eyeblink responses is enhanced in children, occurring in a fraction of the trials required for control participants. The timing of learned responses is, however, atypical. Two animal models of autism display a similar phenotype. Researchers have hypothesized that these differences in conditioning reflect cerebellar abnormalities. The present study used computer simulations of the cerebellar cortex, including inhibition by the molecular layer interneurons, to more closely examine whether atypical cerebellar processing can account for faster conditioning in individuals with autism. In particular, the effects of inhibitory levels on delay eyeblink conditioning were simulated, as were the effects of learning-related synaptic changes at either parallel fibers or ascending branch synapses from granule cells to Purkinje cells. Results from these simulations predict that whether molecular layer inhibition results in an enhancement or an impairment of acquisition, or changes in timing, may depend on (1) the sources of inhibition, (2) the levels of inhibition, and (3) the locations of learning-related changes (parallel vs. ascending branch synapses). Overall, the simulations predict that a disruption in the balance or an overall increase of inhibition within the cerebellar cortex may contribute to atypical eyeblink conditioning in children with autism and in animal models of autism. PMID:24590391

  20. AMP Kinase Activation Alters Oxidant-Induced Stress Granule Assembly by Modulating Cell Signaling and Microtubule Organization.

    PubMed

    Mahboubi, Hicham; Koromilas, Antonis E; Stochaj, Ursula

    2016-10-01

    Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules. PMID:27430620

  1. Cognitive Enhancing Treatment with a PPARγ Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

    PubMed Central

    Nenov, Miroslav N.; Laezza, Fernanda; Haidacher, Sigmund J.; Zhao, Yingxin; Sadygov, Rovshan G.; Starkey, Jonathan M.; Spratt, Heidi; Luxon, Bruce A.; Dineley, Kelly T.

    2014-01-01

    Hippocampal network hyperexcitability is considered an early indicator of Alzheimer's disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I–O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I–O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity. PMID:24431460

  2. Responses of Purkinje-cells of the cerebellar anterior vermis to stimulation of vestibular and somatosensory receptors.

    PubMed

    Bruschini, L; Andre, P; Pompeiano, O; Manzoni, D

    2006-09-29

    In decerebrate cats, sinusoidal rotation of the forepaw around the wrist modifies the activity of the ipsilateral forelimb extensor triceps brachii (TB) and leads to plastic changes of adaptive nature in the gain of vestibulospinal (VS) reflexes (VSRs). Both effects are depressed by functional inactivation of the cerebellar anterior vermis, which also decreases the gain of VSRs. In order to better understand the mechanisms of these phenomena, the simple spike activity of Purkinje (P-) cells was recorded from the vermal cortex of the cerebellar anterior lobe during individual and/or combined stimulation of somatosensory wrist, neck and vestibular receptors. About one third of the recorded units were affected by sinusoidal rotation of the ipsilateral forepaw around the wrist axis (0.16 Hz, +/-10 degrees ). Most of these neurons ( approximately 60%) increased their activity during ventral flexion of the wrist and decreased it during the oppositely directed movement, with an average phase lag of -141 degrees with respect to the position of maximal dorsiflexion. The remaining cells ( approximately 40%) were excited during dorsiflexion of the wrist, with an average phase lead of 59 degrees with respect to the extreme dorsal flexion. Both populations showed comparable response gains, with an average value of 0.42+/-0.52, S.D., imp/s/deg. About half of the recorded units were also tested during sinusoidal roll tilt of the animal around the longitudinal axis (0.16 Hz, +/-10 degrees ), leading to stimulation of labyrinthine receptors. When both stimuli were applied simultaneously, the responses to combined stimulation usually corresponded to the sum of individual responses. While the phase distribution of somatosensory responses was clearly bimodal, vestibular responses showed phase angle values uniformly scattered between +/-180 degrees and 0 degrees , so that, during combined stimulation, each neuron could be maximally activated by coupling the two stimuli with a

  3. Inhibition of Protease-Activated Receptor 1 Does not Affect Dendritic Homeostasis of Cultured Mouse Dentate Granule Cells

    PubMed Central

    Schuldt, Gerlind; Galanis, Christos; Strehl, Andreas; Hick, Meike; Schiener, Sabine; Lenz, Maximilian; Deller, Thomas; Maggio, Nicola; Vlachos, Andreas

    2016-01-01

    Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS). While a firm link between PAR1-activation and functional synaptic and intrinsic neuronal properties exists, studies on the role of PAR1 in neural structural plasticity are scarce. The physiological function of PAR1 in the brain remains not well understood. We here sought to determine whether prolonged pharmacologic PAR1-inhibition affects dendritic morphologies of hippocampal neurons. To address this question we employed live-cell microscopy of mouse dentate granule cell dendrites in 3-week old entorhino-hippocampal slice cultures prepared from Thy1-GFP mice. A subset of cultures were treated with the PAR1-inhibitor SCH79797 (1 μM; up to 3 weeks). No major effects of PAR1-inhibition on static and dynamic parameters of dentate granule cell dendrites were detected under control conditions. Granule cells of PAR1-deficient slice cultures showed unaltered dendritic morphologies, dendritic spine densities and excitatory synaptic strength. Furthermore, we report that PAR1-inhibition does not prevent dendritic retraction following partial deafferentation in vitro. Consistent with this finding, no major changes in PAR1-mRNA levels were detected in the denervated dentate gyrus (DG). We conclude that neural PAR1 is not involved in regulating the steady-state dynamics or deafferentation-induced adaptive changes of cultured dentate granule cell dendrites. These results indicate that drugs targeting neural PAR1-signals may not affect the stability and structural integrity of neuronal networks in healthy brain regions. PMID:27378862

  4. Inhibition of Protease-Activated Receptor 1 Does not Affect Dendritic Homeostasis of Cultured Mouse Dentate Granule Cells.

    PubMed

    Schuldt, Gerlind; Galanis, Christos; Strehl, Andreas; Hick, Meike; Schiener, Sabine; Lenz, Maximilian; Deller, Thomas; Maggio, Nicola; Vlachos, Andreas

    2016-01-01

    Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS). While a firm link between PAR1-activation and functional synaptic and intrinsic neuronal properties exists, studies on the role of PAR1 in neural structural plasticity are scarce. The physiological function of PAR1 in the brain remains not well understood. We here sought to determine whether prolonged pharmacologic PAR1-inhibition affects dendritic morphologies of hippocampal neurons. To address this question we employed live-cell microscopy of mouse dentate granule cell dendrites in 3-week old entorhino-hippocampal slice cultures prepared from Thy1-GFP mice. A subset of cultures were treated with the PAR1-inhibitor SCH79797 (1 μM; up to 3 weeks). No major effects of PAR1-inhibition on static and dynamic parameters of dentate granule cell dendrites were detected under control conditions. Granule cells of PAR1-deficient slice cultures showed unaltered dendritic morphologies, dendritic spine densities and excitatory synaptic strength. Furthermore, we report that PAR1-inhibition does not prevent dendritic retraction following partial deafferentation in vitro. Consistent with this finding, no major changes in PAR1-mRNA levels were detected in the denervated dentate gyrus (DG). We conclude that neural PAR1 is not involved in regulating the steady-state dynamics or deafferentation-induced adaptive changes of cultured dentate granule cell dendrites. These results indicate that drugs targeting neural PAR1-signals may not affect the stability and structural integrity of neuronal networks in healthy brain regions. PMID:27378862

  5. Improved performance of microbial fuel cell using combination biocathode of graphite fiber brush and graphite granules

    NASA Astrophysics Data System (ADS)

    Zhang, Guo-dong; Zhao, Qing-liang; Jiao, Yan; Zhang, Jin-na; Jiang, Jun-qiu; Ren, Nanqi; Kim, Byung Hong

    2011-08-01

    The efficiency and sustainability of microbial fuel cell (MFC) are heavily dependent on the cathode performance. We show here that the use of graphite fiber brush (GBF) together with graphite granules (GGs) as a basal material for biocathode (MFC reactor type R1) significantly improve the performance of a MFC compared with MFCs using GGs (MFC reactor type R2) or GFB (MFC reactor type R3) individually. Compared with R3, the use of the combination biocathode (R1) can shorten the start-up time by 53.75%, improve coulombic efficiencies (CEs) by 21.0 ± 2.7% at external resistance (REX) of 500 Ω, and increase maximum power densities by 38.2 ± 12.6%. Though the start-up time and open circuit voltage (OCV) of the reactor R2 are similar to R1, the CE (REX = 500 Ω) and maximum power density of R2 are 21.4 ± 1.7% and 38.2 ± 15.6% lower than that of R1. Fluorescence in situ hybridization (FISH) analyses indicate the bacteria on cathodes of R1 and R2 are richer than that of R3. Molecular taxonomic analyses reveal that the biofilm formed on the biocathode surface is dominated by strains belonging to Nitrobacter, Achromobacter, Acinetobacter, and Bacteroidetes. Combination of GFB and GGs as biocathode material in MFC is more efficient and can achieve sustainable electricity recovery from organic substances, which substantially increases the viability and sustainability of MFCs.

  6. Glyceraldehyde-3-phosphate dehydrogenase antisense oligodeoxynucleotides protect against cytosine arabinonucleoside-induced apoptosis in cultured cerebellar neurons.

    PubMed Central

    Ishitani, R; Chuang, D M

    1996-01-01

    Cytosine arabinonucleoside (AraC) is a pyrimidine antimetabolite that kills proliferating cells by inhibiting DNA synthesis and, importantly, is also an inducer of apoptosis. We recently reported that age-induced apoptotic cell death of cultured cerebellar neurons is directly associated with an over-expression of a particulate 38-kDa protein, identified by us as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). We now show that the AraC-induced neuronal death of immature cerebellar granule cells in culture is effectively delayed by actinomycin-D, cycloheximide, or aurintricarboxylic acid (a DNase inhibitor). Furthermore, two GAPDH antisense, but not their corresponding sense, oligodeoxyribonucleotides markedly arrested AraC-induced apoptosis. This protection was more effective than that induced by the above-mentioned classical inhibitors of apoptosis. Prior to AraC-induced neuronal death, GAPDH mRNA levels increased by approximately 2.5-fold, and this mRNA accumulation was blocked by actinomycin-D and the GAPDH antisense (but not sense) oligonucleotide. Like actinomycin-D, a GAPDH antisense oligonucleotide also suppressed the AraC-induced over-expression of the 38-kDa particulate protein (i.e., GAPDH), while the corresponding sense oligonucleotide was totally ineffective. Thus, the present results show that GAPDH over-expression is involved in AraC-induced apoptosis of cultured cerebellar granule cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:8790435

  7. PACAP modulation of calcium ion activity in developing granule cells of the neonatal mouse olfactory bulb

    PubMed Central

    Irwin, Mavis; Greig, Ann; Tvrdik, Petr

    2014-01-01

    Ca2+ activity in the CNS is critical for the establishment of developing neuronal circuitry prior to and during early sensory input. In developing olfactory bulb (OB), the neuromodulators that enhance network activity are largely unknown. Here we provide evidence that pituitary adenylate cyclase-activating peptide (PACAP)-specific PAC1 receptors (PAC1Rs) expressed in postnatal day (P)2–P5 mouse OB are functional and enhance network activity as measured by increases in calcium in genetically identified granule cells (GCs). We used confocal Ca2+ imaging of OB slices from Dlx2-tdTomato mice to visualize GABAergic GCs. To address whether the PACAP-induced Ca2+ oscillations were direct or indirect effects of PAC1R activation, we used antagonists for the GABA receptors (GABARs) and/or glutamate receptors (GluRs) in the presence and absence of PACAP. Combined block of GABARs and GluRs yielded a 66% decrease in the numbers of PACAP-responsive cells, suggesting that 34% of OB neurons are directly activated by PACAP. Similarly, immunocytochemistry using anti-PAC1 antibody showed that 34% of OB neurons express PAC1R. Blocking either GluRs or GABARs alone indirectly showed that PACAP stimulates release of both glutamate and GABA, which activate GCs. The appearance of PACAP-induced Ca2+ activity in immature GCs suggests a role for PACAP in GC maturation. To conclude, we find that PACAP has both direct and indirect effects on neonatal OB GABAergic cells and may enhance network activity by promoting glutamate and GABA release. Furthermore, the numbers of PACAP-responsive GCs significantly increased between P2 and P5, suggesting that PACAP-induced Ca2+ activity contributes to neonatal OB development. PMID:25475351

  8. Smad2 protein disruption in the central nervous system leads to aberrant cerebellar development and early postnatal ataxia in mice.

    PubMed

    Wang, Lixiang; Nomura, Masatoshi; Goto, Yutaka; Tanaka, Kimitaka; Sakamoto, Ryuichi; Abe, Ichiro; Sakamoto, Shohei; Shibata, Atsushi; Enciso, Patricio L M; Adachi, Masahiro; Ohnaka, Keizo; Kawate, Hisaya; Takayanagi, Ryoichi

    2011-05-27

    Smad2 is a critical mediator of TGF-β signals that are known to play an important role in a wide range of biological processes in various cell types. Its role in the development of the CNS, however, is largely unknown. Mice lacking Smad2 in the CNS (Smad2-CNS-KO) were generated by a Cre-loxP approach. These mice exhibited behavioral abnormalities in motor coordination from an early postnatal stage and mortality at approximately 3 weeks of age, suggestive of severe cerebellar dysfunction. Gross observation of Smad2-CNS-KO cerebella demonstrated aberrant foliations in lobule IX and X. Further analyses revealed increased apoptotic cell death, delayed migration and maturation of granule cells, and retardation of dendritic arborization of Purkinje cells. These findings indicate that Smad2 plays a key role in cerebellar development and motor function control. PMID:21464123

  9. Impaired Eye-Blink Conditioning in waggler, a Mutant Mouse With Cerebellar BDNF Deficiency

    PubMed Central

    Bao, Shaowen; Chen, Lu; Qiao, Xiaoxi; Knusel, Beat; Thompson, Richard F.

    1998-01-01

    In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expression of brain-derived neurotrophic factor (BDNF) was selectively absent in the cerebellar granule cells. The cytoarchitecture of the waggler cerebellum appeared to be normal at the light microscope level. The mutant mice exhibited no sensory deficits to auditory stimuli or heat-induced pain. However, they were massively impaired in classic eye-blink conditioning. These results suggest that BDNF may have a role in normal cerebellar neuronal function, which, in turn, is essential for classic eye-blink conditioning. PMID:10454360

  10. Network Structure within the Cerebellar Input Layer Enables Lossless Sparse Encoding

    PubMed Central

    Billings, Guy; Piasini, Eugenio; Lőrincz, Andrea; Nusser, Zoltan; Silver, R. Angus

    2014-01-01

    Summary The synaptic connectivity within neuronal networks is thought to determine the information processing they perform, yet network structure-function relationships remain poorly understood. By combining quantitative anatomy of the cerebellar input layer and information theoretic analysis of network models, we investigated how synaptic connectivity affects information transmission and processing. Simplified binary models revealed that the synaptic connectivity within feedforward networks determines the trade-off between information transmission and sparse encoding. Networks with few synaptic connections per neuron and network-activity-dependent threshold were optimal for lossless sparse encoding over the widest range of input activities. Biologically detailed spiking network models with experimentally constrained synaptic conductances and inhibition confirmed our analytical predictions. Our results establish that the synaptic connectivity within the cerebellar input layer enables efficient lossless sparse encoding. Moreover, they provide a functional explanation for why granule cells have approximately four dendrites, a feature that has been evolutionarily conserved since the appearance of fish. PMID:25123311

  11. Synaptic Multivesicular Release in the Cerebellar Cortex: Its Mechanism and Role in Neural Encoding and Processing.

    PubMed

    Satake, Shin'Ichiro; Inoue, Tsuyoshi; Imoto, Keiji

    2016-04-01

    The number of synaptic vesicles released during fast release plays a major role in determining the strength of postsynaptic response. However, it remains unresolved how the number of vesicles released in response to action potentials is controlled at a single synapse. Recent findings suggest that the Cav2.1 subtype (P/Q-type) of voltage-gated calcium channels is responsible for inducing presynaptic multivesicular release (MVR) at rat cerebellar glutamatergic synapses from granule cells to molecular layer interneurons. The topographical distance from Cav2.1 channels to exocytotic Ca(2+) sensors is a critical determinant of MVR. In physiological trains of presynaptic neurons, MVR significantly impacts the excitability of postsynaptic neurons, not only by increasing peak amplitude but also by prolonging decay time of the postsynaptic currents. Therefore, MVR contributes additional complexity to neural encoding and processing in the cerebellar cortex. PMID:25971904

  12. A nuclear factor containing the leucine-rich repeats expressed in murine cerebellar neurons.

    PubMed Central

    Matsuoka, K; Taoka, M; Satozawa, N; Nakayama, H; Ichimura, T; Takahashi, N; Yamakuni, T; Song, S Y; Isobe, T

    1994-01-01

    A nuclear protein, termed leucine-rich acidic nuclear protein (LANP), has been isolated from among rat cerebellar proteins whose expression was transiently increased during an early stage of postnatal development. The amino acid sequence, deduced from its cDNA, showed that LANP contains 247 amino acids consisting of two distinct structural domains: the N-terminal domain characterized by "leucine-rich repeat," which is found in many eukaryotic proteins and which potentially functions in mediating protein-protein interactions, and the C-terminal domain characterized by a cluster of acidic amino acids with a putative nuclear localization signal. Immunohistochemical study using an antibody against LANP revealed that the protein is localized mainly in nuclei of Purkinje cells. In the rat cerebellum on postnatal day 7, LANP mRNA was expressed moderately in the external granule and Purkinje cells and weakly in the internal granule cells. The expression in these cells, especially in Purkinje cells, increased in the second postnatal week and thereafter decreased to an adult level. The structural characteristics, localization, and the stage- and cell type-specific expression suggest a potential role of LANP in a signal transduction pathway that directs differentiation of cerebellar neurons. Images PMID:7937870

  13. Characterisation of the biosynthesis and processing of the neutrophil granule membrane protein CD63 in myeloid cells.

    PubMed

    Ageberg, M; Lindmark, A

    2003-10-01

    The biosynthesis and processing of the neutrophil granule membrane protein CD63, present in azurophil granules, was investigated in four myeloid cell lines. The amount of CD63 synthesised differed, so did the amount of protein processed to high molecular weight forms, with the demonstration of a more prominent synthesis of CD63 in K562 cells. Newly synthesised CD63 was initially detected as two precursor forms with molecular weight of 32 and 35 kDa, respectively. These two initial forms were processed further to yield high molecular weight forms of CD63 with a mean molecular weight of 50 kDa. Treatment with endoglycosidase H or N-glycosidase F revealed a protein core, free from asparagine-linked carbohydrates, with a molecular weight of 23 kDa. Newly synthesised CD63 was susceptible to digestion with endoglycosidase H, and the protein was not completely resistant to endoglycosidase H until after 4 h of chase, indicating that transport through the medial and trans-Golgi complex with conversion of high-mannose carbohydrates to complex oligosaccharide side chains had occurred. This finding indicates a relatively long processing time for CD63 compared to that of soluble azurophil granule proteins. By digestion with O-glycanase, the existence of O-linked oligosaccharides on CD63 could not be demonstrated. Biosynthetic labelling of cells in the presence of brefeldin A showed the importance of a functional Golgi apparatus for processing of the protein to its high molecular weight forms. PMID:12974720

  14. Interaction and colocalization of HERMES/RBPMS with NonO, PSF, and G3BP1 in neuronal cytoplasmic RNP granules in mouse retinal line cells.

    PubMed

    Furukawa, Mari T; Sakamoto, Hiroshi; Inoue, Kunio

    2015-04-01

    HERMES, also called RBPMS, is a conserved RNA binding protein with a single RNA recognition motif (RRM) that is abundantly expressed in retinal ganglion cells (RGCs) and in the heart in vertebrates. Here, we identified NonO and PSF as the interacting proteins of HERMES only when the neuronal differentiation of the retinal cell line RGC-5 was induced. Although NonO and PSF are nuclear paraspeckle components, these proteins formed cytoplasmic granules with HERMES in the neurites. G3BP1, a component of stress granules, was also colocalized to the granules, interacting with NonO and HERMES even in the absence of cellular stress. Consistent with a previous report that KIF5 interacts with neuronal granules, the localization of KIF5A overlapped with the cytoplasmic granules in differentiated RGC-5 cells. Thus, our study strongly suggests that the cytoplasmic granule containing HERMES, NonO, PSF, and G3BP1 is a neuronal RNA-protein granule that is transported in neurites during retinal differentiation. PMID:25651939

  15. Effects of formaldehyde exposure on granule cell number and volume of dentate gyrus: a histopathological and stereological study.

    PubMed

    Aslan, Huseyin; Songur, Ahmet; Tunc, Ayten Turkkani; Ozen, Oguz Aslan; Bas, Orhan; Yagmurca, Murat; Turgut, Mehmet; Sarsilmaz, Mustafa; Kaplan, Suleyman

    2006-11-29

    The hippocampal formation is a complex region of the brain related to memory and learning. The purpose of the present study was to determine whether exposure of neonatal rats to formaldehyde (FA) had either early or delayed effects on the numbers of granule cells in the dentate gyrus (DG). After birth, the neonatal male Wistar rats were exposed throughout a 30-day period to various concentrations of FA: 0 (control group), 6 ppm (low concentration group) and 12 ppm (high concentration group). This was done by placing them for 6 h/day and 5 days per week in a glass chamber containing FA vapor. Then, five animals from each group were anesthetized and decapitated on postnatal day (PND) 30, and the remaining five animals were sacrificed on PND 90 by intracardiac perfusion using 10% neutral buffered FA solution. The Cavalieri principle of stereological approaches was used to determine the volume of the DG in these sections. The optical fractionator counting method was used to estimate the total number of granule cells in the DG. The appearance of granule cells was normal under light microscopy in all PND 30 and PND 90 groups. There were significant age-related reductions in the volume of the DG at PND 90 irrespective of which group was examined. Significant age-related neuron loss was also determined at PND 90 compared to that at PND 30. Rats treated with a high concentration FA were found to have fewer granule cells than either the animals treated with a low concentration FA or the control group (p<0.01 and p<0.01, respectively) at PND 90 but not at PND 30. These findings clearly indicate that granule cells in the DG may be vulnerable to stress and the concentration of FA to which they are exposed during early postnatal life, and also that a neurotoxic effect of high dose FA on cell number is only seen after a long time period. These results may explain why some disorders do not appear until later life. PMID:17011527

  16. Genetic ablation of homeodomain-interacting protein kinase 2 selectively induces apoptosis of cerebellar Purkinje cells during adulthood and generates an ataxic-like phenotype

    PubMed Central

    Anzilotti, S; Tornincasa, M; Gerlini, R; Conte, A; Brancaccio, P; Cuomo, O; Bianco, G; Fusco, A; Annunziato, L; Pignataro, G; Pierantoni, G M

    2015-01-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2−/− mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2−/− mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2−/− mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype. PMID:26633710

  17. Stereotyped spatial patterns of functional synaptic connectivity in the cerebellar cortex

    PubMed Central

    Valera, Antoine M; Binda, Francesca; Pawlowski, Sophie A; Dupont, Jean-Luc; Casella, Jean-François; Rothstein, Jeffrey D; Poulain, Bernard; Isope, Philippe

    2016-01-01

    Motor coordination is supported by an array of highly organized heterogeneous modules in the cerebellum. How incoming sensorimotor information is channeled and communicated between these anatomical modules is still poorly understood. In this study, we used transgenic mice expressing GFP in specific subsets of Purkinje cells that allowed us to target a given set of cerebellar modules. Combining in vitro recordings and photostimulation, we identified stereotyped patterns of functional synaptic organization between the granule cell layer and its main targets, the Purkinje cells, Golgi cells and molecular layer interneurons. Each type of connection displayed position-specific patterns of granule cell synaptic inputs that do not strictly match with anatomical boundaries but connect distant cortical modules. Although these patterns can be adjusted by activity-dependent processes, they were found to be consistent and predictable between animals. Our results highlight the operational rules underlying communication between modules in the cerebellar cortex. DOI: http://dx.doi.org/10.7554/eLife.09862.001 PMID:26982219

  18. Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients

    PubMed Central

    Tree, T I M; O'Byrne, D; Tremble, J M; Macfarlane, W M; Haskins, K; James, R F L; Docherty, K; Hutton, J C; Banga, J P

    2000-01-01

    Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet β-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet β-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet β-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in β-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet β-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis. PMID:10886245

  19. High dosage of monosodium glutamate causes deficits of the motor coordination and the number of cerebellar Purkinje cells of rats.

    PubMed

    Prastiwi, D; Djunaidi, A; Partadiredja, G

    2015-11-01

    Monosodium glutamate (MSG) has been widely used throughout the world as a flavoring agent of food. However, MSG at certain dosages is also thought to cause damage to many organs, including cerebellum. This study aimed at investigating the effects of different doses of MSG on the motor coordination and the number of Purkinje cells of the cerebellum of Wistar rats. A total of 24 male rats aged 4 to 5 weeks were divided into four groups, namely, control (C), T2.5, T3, and T3.5 groups, which received intraperitoneal injection of 0.9% sodium chloride solution, 2.5 mg/g body weight (bw) of MSG, 3.0 mg/g bw of MSG, and 3.5 mg/g bw of MSG, respectively, for 10 consecutive days. The motor coordination of the rats was examined prior and subsequent to the treatment. The number of cerebellar Purkinje cells was estimated using physical fractionator method. It has been found that the administration of MSG at a dosage of 3.5 mg/g bw, but not at lower dosages, caused a significant decrease of motor coordination and the estimated total number of Purkinje cells of rats. There was also a significant correlation between motor coordination and the total number of Purkinje cells. PMID:25697849

  20. Cerebellar associative sensory learning defects in five mouse autism models

    PubMed Central

    Kloth, Alexander D; Badura, Aleksandra; Li, Amy; Cherskov, Adriana; Connolly, Sara G; Giovannucci, Andrea; Bangash, M Ali; Grasselli, Giorgio; Peñagarikano, Olga; Piochon, Claire; Tsai, Peter T; Geschwind, Daniel H; Hansel, Christian; Sahin, Mustafa; Takumi, Toru; Worley, Paul F; Wang, Samuel S-H

    2015-01-01

    Sensory integration difficulties have been reported in autism, but their underlying brain-circuit mechanisms are underexplored. Using five autism-related mouse models, Shank3+/ΔC, Mecp2R308/Y, Cntnap2−/−, L7-Tsc1 (L7/Pcp2Cre::Tsc1flox/+), and patDp(15q11-13)/+, we report specific perturbations in delay eyeblink conditioning, a form of associative sensory learning requiring cerebellar plasticity. By distinguishing perturbations in the probability and characteristics of learned responses, we found that probability was reduced in Cntnap2−/−, patDp(15q11-13)/+, and L7/Pcp2Cre::Tsc1flox/+, which are associated with Purkinje-cell/deep-nuclear gene expression, along with Shank3+/ΔC. Amplitudes were smaller in L7/Pcp2Cre::Tsc1flox/+ as well as Shank3+/ΔC and Mecp2R308/Y, which are associated with granule cell pathway expression. Shank3+/ΔC and Mecp2R308/Y also showed aberrant response timing and reduced Purkinje-cell dendritic spine density. Overall, our observations are potentially accounted for by defects in instructed learning in the olivocerebellar loop and response representation in the granule cell pathway. Our findings indicate that defects in associative temporal binding of sensory events are widespread in autism mouse models. DOI: http://dx.doi.org/10.7554/eLife.06085.001 PMID:26158416

  1. Influence of thyroid hormone and thyroid hormone receptors in the generation of cerebellar gamma-aminobutyric acid-ergic interneurons from precursor cells.

    PubMed

    Manzano, Jimena; Cuadrado, Maria; Morte, Beatriz; Bernal, Juan

    2007-12-01

    Thyroid hormones have important actions in the developing central nervous system. We describe here a novel action of thyroid hormone and its nuclear receptors on maturation of cerebellar gamma-aminobutyric acid (GABA)-ergic interneurons from their precursor cells. In rats, the density of GABAergic terminals in the cerebellum was decreased by hypothyroidism,