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Sample records for adult-generated granule cells

  1. 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

  2. High-frequency stimulation induces gradual immediate early gene expression in maturing adult-generated hippocampal granule cells.

    PubMed

    Jungenitz, Tassilo; Radic, Tijana; Jedlicka, Peter; Schwarzacher, Stephan W

    2014-07-01

    Increasing evidence shows that adult neurogenesis of hippocampal granule cells is advantageous for learning and memory. We examined at which stage of structural maturation and age new granule cells can be activated by strong synaptic stimulation. High-frequency stimulation of the perforant pathway in urethane-anesthetized rats elicited expression of the immediate early genes c-fos, Arc, zif268 and pCREB133 in almost 100% of mature, calbindin-positive granule cells. In contrast, it failed to induce immediate early gene expression in immature doublecortin-positive granule cells. Furthermore, doublecortin-positive neurons did not react with c-fos or Arc expression to mild theta-burst stimulation or novel environment exposure. Endogenous expression of pCREB133 was increasingly present in young cells with more elaborated dendrites, revealing a close correlation to structural maturation. Labeling with bromodeoxyuridine revealed cell age dependence of stimulation-induced c-fos, Arc and zif268 expression, with only a few cells reacting at 21 days, but with up to 75% of cells activated at 35-77 days of cell age. Our results indicate an increasing synaptic integration of maturing granule cells, starting at 21 days of cell age, but suggest a lack of ability to respond to activation with synaptic potentiation on the transcriptional level as long as immature cells express doublecortin.

  3. Hippocampal granule cells opt for early retirement.

    PubMed

    Alme, C B; Buzzetti, R A; Marrone, D F; Leutgeb, J K; Chawla, M K; Schaner, M J; Bohanick, J D; Khoboko, T; Leutgeb, S; Moser, E I; Moser, M-B; McNaughton, B L; Barnes, C A

    2010-10-01

    Increased excitability and plasticity of adult-generated hippocampal granule cells during a critical period suggests that they may "orthogonalize" memories according to time. One version of this "temporal tag" hypothesis suggests that young granule cells are particularly responsive during a specific time period after their genesis, allowing them to play a significant role in sculpting CA3 representations, after which they become much less responsive to any input. An alternative possibility is that the granule cells active during their window of increased plasticity, and excitability become selectively tuned to events that occurred during that time and participate in later reinstatement of those experiences, to the exclusion of other cells. To discriminate between these possibilities, rats were exposed to different environments at different times over many weeks, and cell activation was subsequently assessed during a single session in which all environments were revisited. Dispersing the initial experiences in time did not lead to the increase in total recruitment at reinstatement time predicted by the selective tuning hypothesis. The data indicate that, during a given time frame, only a very small number of granule cells participate in many experiences, with most not participating significantly in any. Based on these and previous data, the small excitable population of granule cells probably correspond to the most recently generated cells. It appears that, rather than contributing to the recollection of long past events, most granule cells, possibly 90-95%, are effectively "retired." If granule cells indeed sculpt CA3 representations (which remains to be shown), then a possible consequence of having a new set of granule cells participate when old memories are reinstated is that new representations of these experiences might be generated in CA3. Whatever the case, the present data may be interpreted to undermine the standard "orthogonalizer" theory of the role of

  4. Ectopic Granule Cells of the Rat Dentate Gyrus

    PubMed Central

    Scharfman, Helen; Goodman, Jeffrey; McCloskey, Daniel

    2007-01-01

    Granule cells of the mammalian dentate gyrus normally form a discrete layer, and virtually all granule cells migrate to this location. Exceptional granule cells that are positioned incorrectly, in ‘ectopic’ locations, are rare. Although the characteristics of such ectopic granule cells appear similar in many respects to granule cells located in the granule cell layer, their rare occurrence has limited a full evaluation of their structure and function. More information about ectopic granule cells has been obtained by studying those that develop after experimental manipulations that increase their number. For example, after severe seizures, the number of ectopic granule cells located in the hilus increases dramatically. These experimentally induced ectopic granule cells may not be equivalent to normal ectopic granule cells necessarily, but the vastly increased numbers have allowed much more information to be obtained. Remarkably, the granule cells that are positioned ectopically develop intrinsic properties and an axonal projection that are similar to granule cells that are located normally, i.e., in the granule cell layer. However, dendritic structure and synaptic structure/function appear to differ. These studies have provided new insight into a rare type of granule cell in the dentate gyrus, and the plastic characteristics of dentate granule cells that appear to depend on the location of the cell body. PMID:17148946

  5. 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.

  6. Single Granule Cells Excite Golgi Cells and Evoke Feedback Inhibition in the Cochlear Nucleus

    PubMed Central

    Yaeger, Daniel B.

    2015-01-01

    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

  7. Integration of quanta in cerebellar granule cells during sensory processing.

    PubMed

    Chadderton, Paul; Margrie, Troy W; Häusser, Michael

    2004-04-22

    To understand the computations performed by the input layers of cortical structures, it is essential to determine the relationship between sensory-evoked synaptic input and the resulting pattern of output spikes. In the cerebellum, granule cells constitute the input layer, translating mossy fibre signals into parallel fibre input to Purkinje cells. Until now, their small size and dense packing have precluded recordings from individual granule cells in vivo. Here we use whole-cell patch-clamp recordings to show the relationship between mossy fibre synaptic currents evoked by somatosensory stimulation and the resulting granule cell output patterns. Granule cells exhibited a low ongoing firing rate, due in part to dampening of excitability by a tonic inhibitory conductance mediated by GABA(A) (gamma-aminobutyric acid type A) receptors. Sensory stimulation produced bursts of mossy fibre excitatory postsynaptic currents (EPSCs) that summate to trigger bursts of spikes. Notably, these spike bursts were evoked by only a few quantal EPSCs, and yet spontaneous mossy fibre inputs triggered spikes only when inhibition was reduced. Our results reveal that the input layer of the cerebellum balances exquisite sensitivity with a high signal-to-noise ratio. Granule cell bursts are optimally suited to trigger glutamate receptor activation and plasticity at parallel fibre synapses, providing a link between input representation and memory storage in the cerebellum.

  8. Event-driven simulation of cerebellar granule cells.

    PubMed

    Carrillo, Richard R; Ros, Eduardo; Tolu, Silvia; Nieus, Thierry; D'Angelo, Egidio

    2008-01-01

    Around half of the neurons of a human brain are granule cells (approximately 10(11)granule neurons) [Kandel, E.R., Schwartz, J.H., Jessell, T.M., 2000. Principles of Neural Science. McGraw-Hill Professional Publishing, New York]. In order to study in detail the functional role of the intrinsic features of this cell we have developed a pre-compiled behavioural model based on the simplified granule-cell model of Bezzi et al. [Bezzi, M., Nieus, T., Arleo, A., D'Angelo, E., Coenen, O.J.-M.D., 2004. Information transfer at the mossy fiber-granule cell synapse of the cerebellum. 34th Annual Meeting. Society for Neuroscience, San Diego, CA, USA]. We can use an efficient event-driven simulation scheme based on lookup tables (EDLUT) [Ros, E., Carrillo, R.R., Ortigosa, E.M., Barbour, B., Ags, R., 2006. Event-driven simulation scheme for spiking neural networks using lookup tables to characterize neuronal dynamics. Neural Computation 18 (12), 2959-2993]. For this purpose it is necessary to compile into tables the data obtained through a massive numerical calculation of the simplified cell model. This allows network simulations requiring minimal numerical calculation. There are three major features that are considered functionally relevant in the simplified granule cell model: bursting, subthreshold oscillations and resonance. In this work we describe how the cell model is compiled into tables keeping these key properties of the neuron model.

  9. Platelet Granule Exocytosis: A Comparison with Chromaffin Cells

    PubMed Central

    Fitch-Tewfik, Jennifer L.; Flaumenhaft, Robert

    2013-01-01

    The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli. PMID:23805129

  10. Characterization of mast cell secretory granules and their cell biology.

    PubMed

    Azouz, Nurit Pereg; Hammel, Ilan; Sagi-Eisenberg, Ronit

    2014-10-01

    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.

  11. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development.

    PubMed

    Dengler, Christopher G; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A

    2017-02-20

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses.

  12. Massively augmented hippocampal dentate granule cell activation accompanies epilepsy development

    PubMed Central

    Dengler, Christopher G.; Yue, Cuiyong; Takano, Hajime; Coulter, Douglas A.

    2017-01-01

    In a mouse model of temporal lobe epilepsy, multicellular calcium imaging revealed that disease emergence was accompanied by massive amplification in the normally sparse, afferent stimulation-induced activation of hippocampal dentate granule cells. Patch recordings demonstrated reductions in local inhibitory function within the dentate gyrus at time points where sparse activation was compromised. Mimicking changes in inhibitory synaptic function and transmembrane chloride regulation was sufficient to elicit the dentate gyrus circuit collapse evident during epilepsy development. Pharmacological blockade of outward chloride transport had no effect during epilepsy development, and significantly increased granule cell activation in both control and chronically epileptic animals. This apparent occlusion effect implicates reduction in chloride extrusion as a mechanism contributing to granule cell hyperactivation specifically during early epilepsy development. Glutamine plays a significant role in local synthesis of GABA in synapses. In epileptic mice, sparse granule cell activation could be restored by glutamine application, implicating compromised GABA synthesis. Glutamine had no effect on granule cell activation earlier, during epilepsy development. We conclude that compromised feedforward inhibition within the local circuit generates the massive dentate gyrus circuit hyperactivation evident in animals during and following epilepsy development. However, the mechanisms underlying this disinhibition diverge significantly as epilepsy progresses. PMID:28218241

  13. Isolating stromal stem cells from periodontal granulation tissues.

    PubMed

    Hung, Tzu-Yuan; Lin, Hsiang-Chun; Chan, Ying-Jen; Yuan, Kuo

    2012-08-01

    Stem cell therapy is a promising area in regenerative medicine. Periodontal granulation tissues are often discarded during conventional surgery. If stromal stem cells can be isolated from these tissues, they can be used for subsequent surgery on the same patient. Fifteen human periodontal granulation tissue samples were obtained from intrabony defects during surgery. Immunohistochemistry (IHC) was carried out on five of the samples to identify STRO-1, a marker of mesenchymal stem cells. Five samples underwent flow cytometry analysis for the same marker. The remaining five samples were characterized by "colony formation unit-fibroblast" (CFU-f) assay and selected for proliferation assay, flow cytometry of stem cell markers, immunocytochemistry (ICC), multipotent differentiation assays, and repairing critical-size defects in mice. The ratio of STRO-1(+) cells detected by IHC was 5.91 ± 1.50%. The analysis of flow cytometry for STRO-1 was 6.70 ± 0.81%. Approximately two thirds of the CFU-f colonies had a strong reaction to STRO-1 in ICC staining. The cells were multipotent both in vitro and in vivo. Mice given bone grafts and stem cells showed significantly better bone healing than those without stem cells. Multipotent stromal stem cells can be isolated from human periodontal granulation tissues. These cells improve new bone formation when transplanted in mouse calvarial defects. Isolating stem cells from relatively accessible sites without extra procedures is clinically advantageous. This study demonstrated that human periodontal granulation tissues contain isolatable multipotent stem cells. The cells may be a good source for autotransplantation in subsequent treatment.

  14. Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens

    PubMed Central

    Ogbomo, Henry; Mody, Christopher H.

    2017-01-01

    Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed. PMID:28123389

  15. Transcriptional Profiling of Newly Generated Dentate Granule Cells Using TU Tagging Reveals Pattern Shifts in Gene Expression during Circuit Integration1,2

    PubMed Central

    Chatzi, Christina; Shen, Rongkun; Goodman, Richard H.

    2016-01-01

    Abstract Despite representing only a small fraction of hippocampal granule cells, adult-generated newborn granule cells have been implicated in learning and memory (Aimone et al., 2011). Newborn granule cells undergo functional maturation and circuit integration over a period of weeks. However, it is difficult to assess the accompanying gene expression profiles in vivo with high spatial and temporal resolution using traditional methods. Here we used a novel method [“thiouracil (TU) tagging”] to map the profiles of nascent mRNAs in mouse immature newborn granule cells compared with mature granule cells. We targeted a nonmammalian uracil salvage enzyme, uracil phosphoribosyltransferase, to newborn neurons and mature granule cells using retroviral and lentiviral constructs, respectively. Subsequent injection of 4-TU tagged nascent RNAs for analysis by RNA sequencing. Several hundred genes were significantly enhanced in the retroviral dataset compared with the lentiviral dataset. We compared a selection of the enriched genes with steady-state levels of mRNAs using quantitative PCR. Ontology analysis revealed distinct patterns of nascent mRNA expression, with newly generated immature neurons showing enhanced expression for genes involved in synaptic function, and neural differentiation and development, as well as genes not previously associated with granule cell maturation. Surprisingly, the nascent mRNAs enriched in mature cells were related to energy homeostasis and metabolism, presumably indicative of the increased energy demands of synaptic transmission and their complex dendritic architecture. The high spatial and temporal resolution of our modified TU-tagging method provides a foundation for comparison with steady-state RNA analyses by traditional transcriptomic approaches in defining the functional roles of newborn neurons. PMID:27011954

  16. 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.

  17. Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells

    PubMed Central

    Bosch, Carles; Masachs, Nuria; Exposito-Alonso, David; Martínez, Albert; Teixeira, Cátia M.; Fernaud, Isabel; Pujadas, Lluís; Ulloa, Fausto; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2016-01-01

    The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pathway controls astroglial ensheathment of synapses. Thus, the Reelin pathway is a key regulator of adult-generated GC integration, by controlling dendritic spine types and shapes, their synaptic innervation patterns, and glial ensheathment. These findings may help to better understanding of hippocampal circuit alterations in neurological disorders in which the Reelin pathway is implicated. Significance Statement The extracellular protein Reelin has an important role in neurological diseases, including epilepsy, Alzheimer's disease and psychiatric diseases, targeting hippocampal circuits. Here we address the role of Reelin in the development of synaptic contacts in adult-generated granule cells (GCs), a neuronal population that is crucial for learning and memory and implicated in neurological and psychiatric diseases. We found that the Reelin pathway controls the shapes, sizes, and types of dendritic

  18. Newborn granule cells in the ageing dentate gyrus

    PubMed Central

    Morgenstern, Nicolás A; Lombardi, Gabriela; Schinder, Alejandro F

    2008-01-01

    The dentate gyrus of the hippocampus generates neurons throughout life, but adult neurogenesis exhibits a marked age-dependent decline. Although the decrease in the rate of neurogenesis has been extensively documented in the ageing hippocampus, the specific characteristics of dentate granule cells born in such a continuously changing environment have received little attention. We have used retroviral labelling of neural progenitor cells of the adult mouse dentate gyrus to study morphological properties of neurons born at different ages. Dendritic spine density was measured to estimate glutamatergic afferent connectivity. Fully mature neurons born at the age of 2 months display ∼2.3 spines μm−1 and maintain their overall morphology and spine density in 1-year-old mice. Surprisingly, granule cells born in 10-month-old mice, at which time the rate of neurogenesis has decreased by ∼40-fold, reach a density of dendritic spines similar to that of neurons born in young adulthood. Therefore, in spite of the sharp decline in cell proliferation, differentiation and overall neuronal number, the ageing hippocampus presents a suitable environment for new surviving neurons to reach a high level of complexity, comparable to that of all other dentate granule cells. PMID:18565998

  19. Neuroligin-1 Overexpression in Newborn Granule Cells In Vivo

    PubMed Central

    Schnell, Eric; Bensen, AeSoon L.; Washburn, Eric K.; Westbrook, Gary L.

    2012-01-01

    Adult-born dentate granule cells integrate into the hippocampal network, extend neurites and form synapses in otherwise mature tissue. Excitatory and inhibitory inputs innervate these new granule cells in a stereotyped, temporally segregated manner, which presents a unique opportunity to study synapse development in the adult brain. To examine the role of neuroligins as synapse-inducing molecules in vivo, we infected dividing neural precursors in adult mice with a retroviral construct that increased neuroligin-1 levels during granule cell differentiation. By 21 days post-mitosis, exogenous neuroligin-1 was expressed at the tips of dendritic spines and increased the number of dendritic spines. Neuroligin-1-overexpressing cells showed a selective increase in functional excitatory synapses and connection multiplicity by single afferent fibers, as well as an increase in the synaptic AMPA/NMDA receptor ratio. In contrast to its synapse-inducing ability in vitro, neuroligin-1 overexpression did not induce precocious synapse formation in adult-born neurons. However, the dendrites of neuroligin-1-overexpressing cells did have more thin protrusions during an early period of dendritic outgrowth, suggesting enhanced filopodium formation or stabilization. Our results indicate that neuroligin-1 expression selectively increases the degree, but not the onset, of excitatory synapse formation in adult-born neurons. PMID:23110172

  20. Spatial Representations of Granule Cells and Mossy Cells of the Dentate Gyrus.

    PubMed

    GoodSmith, Douglas; Chen, Xiaojing; Wang, Cheng; Kim, Sang Hoon; Song, Hongjun; Burgalossi, Andrea; Christian, Kimberly M; Knierim, James J

    2017-02-08

    Granule cells in the dentate gyrus of the hippocampus are thought to be essential to memory function by decorrelating overlapping input patterns (pattern separation). A second excitatory cell type in the dentate gyrus, the mossy cell, forms an intricate circuit with granule cells, CA3c pyramidal cells, and local interneurons, but the influence of mossy cells on dentate function is often overlooked. Multiple tetrode recordings, supported by juxtacellular recording techniques, showed that granule cells fired very sparsely, whereas mossy cells in the hilus fired promiscuously in multiple locations and in multiple environments. The activity patterns of these cell types thus represent different environments through distinct computational mechanisms: sparse coding in granule cells and changes in firing field locations in mossy cells.

  1. 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.

  2. Calcium dynamics in bovine adrenal medulla chromaffin cell secretory granules.

    PubMed

    Santodomingo, Jaime; Vay, Laura; Camacho, Marcial; Hernández-Sanmiguel, Esther; Fonteriz, Rosalba I; Lobatón, Carmen D; Montero, Mayte; Moreno, Alfredo; Alvarez, Javier

    2008-10-01

    The secretory granules constitute one of the less well-known compartments in terms of Ca2+ dynamics. They contain large amounts of total Ca2+, but the free intragranular [Ca2+] ([Ca2+]SG), the mechanisms for Ca2+ uptake and release from the granules and their physiological significance regarding exocytosis are still matters of debate. We used in the present work an aequorin chimera targeted to the granules to investigate [Ca2+]SG homeostasis in bovine adrenal chromaffin cells. We found that most of the intracellular aequorin chimera is present in a compartment with 50-100 microM Ca2+. Ca2+ accumulation into this compartment takes place mainly through an ATP-dependent mechanism, namely, a thapsigargin-sensitive Ca2+-ATPase. In addition, fast Ca2+ release was observed in permeabilized cells after addition of inositol 1,4,5-trisphosphate (InsP3) or caffeine, suggesting the presence of InsP3 and ryanodine receptors in the vesicular membrane. Stimulation of intact cells with the InsP3-producing agonist histamine or with caffeine also induced Ca2+ release from the vesicles, whereas acetylcholine or high-[K+] depolarization induced biphasic changes in vesicular[Ca2+], suggesting heterogeneous responses of different vesicle populations, some of them releasing and some taking up Ca2+during stimulation. In conclusion, our data show that chromaffin cell secretory granules have the machinery required for rapid uptake and release of Ca2+, and this strongly supports the hypothesis that granular Ca2+ may contribute to its own secretion.

  3. Single granule cells reliably discharge targets in the hippocampal CA3 network in vivo.

    PubMed

    Henze, Darrell A; Wittner, Lucia; Buzsáki, György

    2002-08-01

    Processing of neuronal information depends on interactions between the anatomical connectivity and cellular properties of single cells. We examined how these computational building blocks work together in the intact rat hippocampus. Single spikes in dentate granule cells, controlled intracellularly, generally failed to discharge either interneurons or CA3 pyramidal cells. In contrast, trains of spikes effectively discharged both CA3 cell types. Increasing the discharge rate of the granule cell increased the discharge probability of its target neuron and decreased the delay between the onset of a granule cell train and evoked firing in postsynaptic targets. Thus, we conclude that the granule cell to CA3 synapses are 'conditional detonators,' dependent on granule cell firing pattern. In addition, we suggest that information in single granule cells is converted into a temporal delay code in target CA3 pyramidal cells and interneurons. These data demonstrate how a neural circuit of the CNS may process information.

  4. Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells.

    PubMed

    Bosch, Carles; Masachs, Nuria; Exposito-Alonso, David; Martínez, Albert; Teixeira, Cátia M; Fernaud, Isabel; Pujadas, Lluís; Ulloa, Fausto; Comella, Joan X; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2016-09-13

    The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pathway controls astroglial ensheathment of synapses. Thus, the Reelin pathway is a key regulator of adult-generated GC integration, by controlling dendritic spine types and shapes, their synaptic innervation patterns, and glial ensheathment. These findings may help to better understanding of hippocampal circuit alterations in neurological disorders in which the Reelin pathway is implicated.

  5. Cerebellar granule cells acquire a widespread predictive feedback signal during motor learning.

    PubMed

    Giovannucci, Andrea; Badura, Aleksandra; Deverett, Ben; Najafi, Farzaneh; Pereira, Talmo D; Gao, Zhenyu; Ozden, Ilker; Kloth, Alexander D; Pnevmatikakis, Eftychios; Paninski, Liam; De Zeeuw, Chris I; Medina, Javier F; Wang, Samuel S-H

    2017-03-20

    Cerebellar granule cells, which constitute half the brain's neurons, supply Purkinje cells with contextual information necessary for motor learning, but how they encode this information is unknown. Here we show, using two-photon microscopy to track neural activity over multiple days of cerebellum-dependent eyeblink conditioning in mice, that granule cell populations acquire a dense representation of the anticipatory eyelid movement. Initially, granule cells responded to neutral visual and somatosensory stimuli as well as periorbital airpuffs used for training. As learning progressed, two-thirds of monitored granule cells acquired a conditional response whose timing matched or preceded the learned eyelid movements. Granule cell activity covaried trial by trial to form a redundant code. Many granule cells were also active during movements of nearby body structures. Thus, a predictive signal about the upcoming movement is widely available at the input stage of the cerebellar cortex, as required by forward models of cerebellar control.

  6. 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

  7. LKB1 Regulates Cerebellar Development by Controlling Sonic Hedgehog-mediated Granule Cell Precursor Proliferation and Granule Cell Migration

    PubMed Central

    Men, Yuqin; Zhang, Aizhen; Li, Haixiang; Jin, Yecheng; Sun, Xiaoyang; Li, Huashun; Gao, Jiangang

    2015-01-01

    The Liver Kinase B1 (LKB1) gene plays crucial roles in cell differentiation, proliferation and the establishment of cell polarity. We created LKB1 conditional knockout mice (LKB1Atoh1 CKO) to investigate the function of LKB1 in cerebellar development. The LKB1Atoh1 CKO mice displayed motor dysfunction. In the LKB1Atoh1 CKO cerebellum, the overall structure had a larger volume and morelobules. LKB1 inactivationled to an increased proliferation of granule cell precursors (GCPs), aberrant granule cell migration and overproduction of unipolar brush cells. To investigate the mechanism underlying the abnormal foliation, we examined sonic hedgehog signalling (Shh) by testing its transcriptional mediators, the Gli proteins, which regulate the GCPs proliferation and cerebellar foliation during cerebellar development. The expression levels of Gli genes were significantly increased in the mutant cerebellum. In vitro assays showed that the proliferation of cultured GCPs from mutant cerebellum significantly increased, whereas the proliferation of mutant GCPs significantly decreased in the presence of a Shh inhibitor GDC-0049. Thus, LKB1 deficiency in the LKB1Atoh1 CKO mice enhanced Shh signalling, leading to the excessive GCP proliferation and the formation of extra lobules. We proposed that LKB1 regulates cerebellar development by controlling GCPs proliferation through Shh signalling during cerebellar development. PMID:26549569

  8. 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.

  9. Automated insulin granule segmentation from electron photomicrographs of rat pancreatic β-cells

    NASA Astrophysics Data System (ADS)

    McClanahan, Timothy P.; Straub, Susanne G.; Sharp, Geoffrey W. G.; Loew, Murray

    2005-04-01

    Increased blood glucose stimulates pancreatic β-cells and induces an exocytotic release of insulin. The β-cell, which contains ~10^4 insulin-containing granules, releases only a few percent of the granules during a given stimulus such as a meal. The temporal response function to a square wave increase in the concentration of glucose is characteristically biphasic. It is not known whether the granules exhibit random or directed migration patterns as a function of phase. Directed migration would suggest the development of an intracellular gradient directing the path and velocity of insulin granule movement. Our ongoing research investigates this process using manual morphometric analysis of electron micrographs of rat pancreatic β-cells. This is a tedious and time-consuming stereological process. Consequently, we have developed an automated algorithm for accurately segmenting and deriving granule counts, areas, and measuring distance to the plasma membrane. The method is a data-driven image processing approach that implements Mahalanobis classifiers to hierarchically classify pixel candidates and subsequently pixel aggregates as insulin granules. Granule cores and halos are classified independently and fused by intersecting the convex difference of granule halos with core candidates. Once fused, total and individual granule areas and distance metrics to the β-cell plasma membrane are obtained. This algorithm provides a rapid and accurate method for the determination of granule numbers, location, and potential gradients in the pancreatic β-cell under different experimental conditions.

  10. Differentiation of apical and basal dendrites in pyramidal cells and granule cells in dissociated hippocampal cultures.

    PubMed

    Wu, You Kure; Fujishima, Kazuto; Kengaku, Mineko

    2015-01-01

    Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.

  11. Thrombopoietin-induced Dami cells as a model for alpha-granule biogenesis.

    PubMed

    Briquet-Laugier, Véronique; El Golli, Nargès; Nurden, Paquita; Lavenu-Bombled, Cécile; Dubart-Kupperschmitt, Anne; Nurden, Alan; Rosa, Jean-Philippe

    2004-09-01

    Megakaryocytic alpha-granules contain secretory proteins relevant to megakaryocyte and platelet functions. Understanding alpha-granule biogenesis is hampered because human primary megakaryocytes are difficult to manipulate. Existing promegakaryocytic cell lines do not spontaneously exhibit mature alpha-granules. Dami cells, transfected with the megakaryocytic platelet factor 4, fused to GFP (PF4-GFP), were induced in the presence of thrombopoietin (TPO), a megakaryocyte cytokine and PMA. Using confocal microscopy, PF4-GFP colocalized with von Willebrand Factor (vWF) in newly formed storage granules. Immunoelectron microscopy demonstrated alpha-granule-like features, including a dense core or parallel tubules and colocalization of PF4-GFP and vWF. Hence, TPO-treated Dami cells are a suitable model to study alpha-granules and their biogenesis.

  12. BDNF over-expression increases olfactory bulb granule cell dendritic spine density in vivo

    PubMed Central

    McDole, Brittnee; Isgor, Ceylan; Pare, Christopher; Guthrie, Kathleen

    2015-01-01

    Olfactory bulb granule cells 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 granule cell spines. These contacts are established in the distal apical dendritic compartment, while granule cell 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 granule cell 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 granule cell spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF produces a marked increase in granule cell 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 granule cells, suggesting a role for this factor in modulating granule cell functional connectivity within adult olfactory circuitry. PMID:26211445

  13. 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.

  14. Three-Dimensional Tracking of Single Granules in Living PC-12 Cells Employing TIRFM and WFFM.

    PubMed

    Xiong, Jun; Li, Dongdong; Zhu, Dan; Qu, Anlian

    2005-01-01

    A comparative study was carried out on evaluating the performance of total internal reflection fluorescence microscopy (TIRFM) and deconvolution wide-field fluorescence microscopy (WFFM) in tracking single secretory granules. Both techniques have been applied to follow the three-dimensional mobility of single secretory granules in living neuroendocrine PC-12 cells. Both techniques return the similar result that most acridine orange-labeled granules were found to travel in random and caged diffusion, and only a small fraction of granules traveled in directed diffusion. Furthermore, the size and 3-D diffusion coefficient of secretory granules, obtained by these two imaging techniques, yield the same value. Together, our results demonstrate the potential of the combination TIRFM and WFFM in tracking long-termed motion of granules throughout live whole cells.

  15. 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

  16. Synthesis of Prostaglandins and Eicosanoids by the Mast Cell Secretory Granule

    DTIC Science & Technology

    1988-01-01

    various lipid-derived mediators during exocytosis. MATERIALS AND METHODS The procedure for granule preparation is similar to that which has been described...Press, Inc. Printed in U.S.A. SYNTHESIS OF PROSTACLANDINS AND KICOSANOIDS BY THE M&ST CELL SECRETORY GRANULE Stephen P. Chock and Elsa A. Schmauder-Chock...SCISNTIFIC XeiOT Received September 30, 1988 SR88-32 The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of

  17. Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

    PubMed Central

    Chen, Huan-Yuan; Chiang, Dapi Meng-Lin; Lin, Zi-Jing; Hsieh, Chia-Chun; Yin, Gung-Chian; Weng, I.-Chun; Guttermann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Lai, Lee-Jene; Liu, Fu-Tong

    2016-01-01

    Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level. PMID:27748356

  18. Sparse activity of identified dentate granule cells during spatial exploration

    PubMed Central

    Diamantaki, Maria; Frey, Markus; Berens, Philipp; Preston-Ferrer, Patricia; Burgalossi, Andrea

    2016-01-01

    In the dentate gyrus – a key component of spatial memory circuits – granule cells (GCs) are known to be morphologically diverse and to display heterogeneous activity profiles during behavior. To resolve structure–function relationships, we juxtacellularly recorded and labeled single GCs in freely moving rats. We found that the vast majority of neurons were silent during exploration. Most active GCs displayed a characteristic spike waveform, fired at low rates and showed spatial activity. Primary dendritic parameters were sufficient for classifying neurons as active or silent with high accuracy. Our data thus support a sparse coding scheme in the dentate gyrus and provide a possible link between structural and functional heterogeneity among the GC population. DOI: http://dx.doi.org/10.7554/eLife.20252.001 PMID:27692065

  19. Ultrastructure and cytochemistry of lipid granules in the many-celled magnetotactic prokaryote, 'Candidatus Magnetoglobus multicellularis'.

    PubMed

    Silva, Karen Tavares; Abreu, Fernanda; Keim, Carolina N; Farina, Marcos; Lins, Ulysses

    2008-12-01

    Conspicuous cytoplasmic granules are reported in a magnetotactic multicellular prokaryote named 'Candidatus Magnetoglobus multicellularis'. Unfortunately, this microorganism, which consists of an assembly of gram-negative bacterial cells, cannot yet be cultivated, limiting the biochemical analysis of the granules and preventing in vitro studies with starvation/excess of nutrients. In this scenario, light and electron microscopy techniques were used to partially address the nature of the granules. Besides magnetosomes, three types of inclusions were observed: small (mean diameter=124 nm) polyhydroxyalkanoate-like (PHA) granules, large (diameters ranging from 0.11 to 2.5 microm) non-PHA lipid granules, and rare phosphorus-rich granules, which probably correspond to polyphosphate bodies. The PHA granules were rounded in projection, non-reactive with OsO(4), and suffered the typical plastic deformation of PHAs after freeze fracturing. The nature of the large granules, consisting of round globular structures (mean diameter=0.76 microm), was classified as non-PHA based on the following data: (a) multilayered structure in freeze-fracture electron microscopy, typical of non-PHA lipids; (b) Nile blue fluorescence imaging detected non-PHA lipids; (c) imidazole buffered osmium tetroxide and ruthenium red cytochemistry stained the globules, which appeared as electron-dense granules instead of electron lucent as PHAs do. Most likely, 'Candidatus Magnetoglobus multicellularis' stores carbon mainly as unusual lipid granules, together with smaller amounts of PHAs.

  20. Delayed release of neurotransmitter from cerebellar granule cells.

    PubMed

    Atluri, P P; Regehr, W G

    1998-10-15

    At fast chemical synapses the rapid release of neurotransmitter that occurs within a few milliseconds of an action potential is followed by a more sustained elevation of release probability, known as delayed release. Here we characterize the role of calcium in delayed release and test the hypothesis that facilitation and delayed release share a common mechanism. Synapses between cerebellar granule cells and their postsynaptic targets, stellate cells and Purkinje cells, were studied in rat brain slices. Presynaptic calcium transients were measured with calcium-sensitive fluorophores, and delayed release was detected with whole-cell recordings. Calcium influx, presynaptic calcium dynamics, and the number of stimulus pulses were altered to assess their effect on delayed release and facilitation. Following single stimuli, delayed release can be separated into two components: one lasting for tens of milliseconds that is steeply calcium-dependent, the other lasting for hundreds of milliseconds that is driven by low levels of calcium with a nearly linear calcium dependence. The amplitude, calcium dependence, and magnitude of delayed release do not correspond to those of facilitation, indicating that these processes are not simple reflections of a shared mechanism. The steep calcium dependence of delayed release, combined with the large calcium transients observed in these presynaptic terminals, suggests that for physiological conditions delayed release provides a way for cells to influence their postsynaptic targets long after their own action potential activity has subsided.

  1. Giant cytoplasmic granules in Langerhans cells of Chediak-Higashi syndrome.

    PubMed

    Carrillo-Farga, J; Gutiérrez-Palomera, G; Ruiz-Maldonado, R; Rondán, A; Antuna, S

    1990-02-01

    Giant membrane-bound cytoplasmic granules were found in the epidermal Langerhans cells of a patient with the Chediak-Higashi syndrome. These cells also contained normal-appearing Birbeck granules. The giant granules had a granular or sometimes globular internal structure; they are believed to derive from fusion of lysosomes or some portion of Birbeck granules. It is unclear whether this morphologic change in Langerhans cell interferes with their antigen-presenting function; it may be, in part, responsible for the frequent infections seen in patients with Chediak-Higashi syndrome that are otherwise more clearly related to the abnormalities in neutrophils and lymphocytes. The Langerhans cell is another cellular type in Chediak-Higashi syndrome in which giant cytoplasmic granules are found.

  2. Differentiated cytoplasmic granule formation in quiescent and non-quiescent cells upon chronological aging

    PubMed Central

    Lee, Hsin-Yi; Cheng, Kuo-Yu; Chao, Jung-Chi; Leu, Jun-Yi

    2016-01-01

    Stationary phase cultures represent a complicated cell population comprising at least two different cell types, quiescent (Q) and non-quiescent (NQ) cells. Q and NQ cells have different lifespans and cell physiologies. However, less is known about the organization of cytosolic protein structures in these two cell types. In this study, we examined Q and NQ cells for the formation of several stationary phase-prevalent granule structures including actin bodies, proteasome storage granules, stress granules, P-bodies, the compartment for unconventional protein secretion (CUPS), and Hsp42-associated stationary phase granules (Hsp42-SPGs). Most of these structures preferentially form in NQ cells, except for Hsp42-SPGs, which are enriched in Q cells. When nutrients are provided, NQ cells enter mitosis less efficiently than Q cells, likely due to the time requirement for reorganizing some granule structures. We observed that heat shock-induced misfolded proteins often colocalize to Hsp42-SPGs, and Q cells clear these protein aggregates more efficiently, suggesting that Hsp42-SPGs may play an important role in the stress resistance of Q cells. Finally, we show that the cell fate of NQ cells is largely irreversible even if they are allowed to reenter mitosis. Our results reveal that the formation of different granule structures may represent the early stage of cell type differentiation in yeast stationary phase cultures. PMID:28357341

  3. 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.

  4. Analysis of Spine Motility of Newborn Granule Cells in Acute Brain Slices.

    PubMed

    Tashiro, Ayumu; Zhao, Chunmei; Suh, Hoonkyo; Gage, Fred H

    2015-10-01

    In this protocol, acute brain slices are prepared from mice in which newborn granule cells have been labeled using retroviral vector technology. Using a live-cell imaging stage and confocal microscopy coupled to imaging software, dendritic spines are analyzed.

  5. 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.

  6. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus.

    PubMed

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-03-19

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7*nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7*nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2-3 week-old Wistar rats, and 2-9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7*nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7*nicotinic receptor modulator, which were blocked by a specific α7*nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7*nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7*nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain.

  7. Functional alpha7 nicotinic receptors are expressed on immature granule cells of the postnatal dentate gyrus

    PubMed Central

    John, Danielle; Shelukhina, Irina; Yanagawa, Yuchio; Deuchars, Jim; Henderson, Zaineb

    2015-01-01

    Neurogenesis occurs throughout life in the subgranular zone of the dentate gyrus, and postnatal-born granule cells migrate into the granule cell layer and extend axons to their target areas. The α7⁎nicotinic receptor has been implicated in neuronal maturation during development of the brain and is abundant in interneurons of the hippocampal formation of the adult brain. Signalling through these same receptors is believed also to promote maturation and integration of adult-born granule cells in the hippocampal formation. We therefore aimed to determine whether functional α7⁎nicotinic receptors are expressed in developing granule cells of the postnatal dentate gyrus. For these experiments we used 2–3 week-old Wistar rats, and 2–9 week old transgenic mice in which GABAergic interneurons were marked by expression of green fluorescent protein. Immunohistochemistry indicated the presence of α7⁎nicotinic receptor subunits around granule cells close around the subgranular zone which correlated with the distribution of developmental markers for immature granule cells. Whole-cell patch clamp recording showed that a proportion of granule cells responded to puffed ACh in the presence of atropine, and that these cells possessed electrophysiological properties found in immature granule cells. The nicotinic responses were potentiated by an allosteric α7⁎nicotinic receptor modulator, which were blocked by a specific α7⁎nicotinic receptor antagonist and were not affected by ionotropic glutamate or GABA receptor antagonists. These results suggest the presence of functional somato-dendritic α7⁎nicotinic receptors on immature granule cells of the postnatal dentate gyrus, consistent with studies implicating α7⁎nicotinic receptors in dendritic maturation of dentate gyrus neurons in adult brain. PMID:25553616

  8. 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

  9. Three-dimensional tracking of single secretory granules in live PC12 cells.

    PubMed

    Li, Dongdong; Xiong, Jun; Qu, Anlian; Xu, Tao

    2004-09-01

    Deconvolution wide-field fluorescence microscopy and single-particle tracking were used to study the three-dimensional mobility of single secretory granules in live PC12 cells. Acridine orange-labeled granules were found to travel primarily in random and caged diffusion, whereas only a small fraction of granules traveled in directed fashion. High K(+) stimulation increased significantly the percentage of granules traveling in directed fashion. By dividing granules into the near-membrane group (within 1 microm from the plasma membrane) and cytosolic group, we have revealed significant differences between these two groups of granules in their mobility. The mobility of these two groups of granules is also differentially affected by disruption of F-actin, suggesting different mechanisms are involved in the motion of the two groups of granules. Our results demonstrate that combined deconvolution and single-particle tracking may find its application in three-dimensional tracking of long-term motion of granules and elucidating the underlying mechanisms.

  10. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells.

    PubMed

    Barg, S; Knowles, M K; Chen, X; Midorikawa, M; Almers, Wolfhard

    2010-11-30

    Syntaxin resides in the plasma membrane, where it helps to catalyze membrane fusion during exocytosis. The protein also forms clusters in cell-free and granule-free plasma-membrane sheets. We imaged the interaction between syntaxin and single secretory granules by two-color total internal reflection microscopy in PC12 cells. Syntaxin-GFP assembled in clusters at sites where single granules had docked at the plasma membrane. Clusters were intermittently present at granule sites, as syntaxin molecules assembled and disassembled in a coordinated fashion. Recruitment to granules required the N-terminal domain of syntaxin, but not the entry of syntaxin into SNARE complexes. Clusters facilitated exocytosis and disassembled once exocytosis was complete. Syntaxin cluster formation defines an intermediate step in exocytosis.

  11. Somatic translocation: a novel mechanism of granule cell dendritic dysmorphogenesis and dispersion

    PubMed Central

    Murphy, Brian L.; Danzer, Steve C.

    2011-01-01

    Pronounced neuronal remodeling is a hallmark of temporal lobe epilepsy. Here, we use real-time confocal imaging of tissue from mouse brain to demonstrate that remodeling can involve fully-differentiated granule cells following translocation of the soma into an existing apical dendrite. Somatic translocation converts dendritic branches into primary dendrites and shifts adjacent apical dendrites to the basal pole of the cell. Moreover, somatic translocation contributes to the dispersion of the granule cell body layer in vitro, and when granule cell dispersion is induced in vivo, the dispersed cells exhibit virtually identical derangements of their dendritic structures. Together, these findings identify novel forms of neuronal plasticity which contribute to granule cell dysmorphogenesis in the epileptic brain. PMID:21414917

  12. 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.

  13. Potassium currents in acutely isolated human hippocampal dentate granule cells.

    PubMed Central

    Beck, H; Clusmann, H; Kral, T; Schramm, J; Heinemann, U; Elger, C E

    1997-01-01

    1. Properties of voltage- and Ca(2+)-dependent K+ currents were investigated in thirty-four dentate granule cells acutely isolated from the resected hippocampus of eleven patients with therapy-refractory temporal lobe epilepsy (TLE). 2. When intracellular Ca2+ was strongly buffered with 11.5 mM EGTA-1 mM Ca2+ in the recording pipette, K+ currents (IK) with a slow activation and biexponential time-dependent decay could be elicited, which showed a threshold for activation around -30 mV. 3. A contribution of Ca(2+)-dependent K+ currents became apparent with intracellular solution containing 1 mM BAPTA-0.1 mM Ca2+. Superfusion of low-Ca2+ extracellular solution blocked 43% of outward currents in this recording configuration. Outward current components could also be blocked by substituting 5 mM Ba2+ for extracellular Ca2+ (78%), or by application of 100 microM Cd2+ (25%). 4. The Ca(2+)-dependent K+ currents could be pharmacologically subdivided into two components. One component was sensitive to 500 microM tetraethylammmonium (TEA; 41%) and 10 nM charybdotoxin (CTX; 47.2%). The blocking effects of 10 nM CTX and 500 microM TEA were not additive, suggesting that both agents block the same conductance. A second, smaller outward current component was blocked by 50 nM apamin (13%). 5. A transient A-type K+ current could be observed in six neurones and showed a fast monoexponential time-dependent inactivation with a steady-state voltage dependence that was distinct from that of IK. The A-type current was blocked by 4-aminopyridine (4-AP) but not by TEA or low-Ca2+ solution. 6. We conclude that outward currents in human hippocampal dentate granule cells can be separated into at least four types by their kinetic and pharmacological properties. These include at least one voltage-dependent current similar to those observed in mammalian hippocampal neurones, and two Ca(2+)-dependent K+ currents that most probably correspond to SK- and BK-type currents. A classical A-type current

  14. Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells

    PubMed Central

    Linster, Christiane

    2015-01-01

    Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and afterpotentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of afterdepolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function. PMID:26334007

  15. Functional differentiation of cholinergic and noradrenergic modulation in a biophysical model of olfactory bulb granule cells.

    PubMed

    Li, Guoshi; Linster, Christiane; Cleland, Thomas A

    2015-12-01

    Olfactory bulb granule cells are modulated by both acetylcholine (ACh) and norepinephrine (NE), but the effects of these neuromodulators have not been clearly distinguished. We used detailed biophysical simulations of granule cells, both alone and embedded in a microcircuit with mitral cells, to measure and distinguish the effects of ACh and NE on cellular and microcircuit function. Cholinergic and noradrenergic modulatory effects on granule cells were based on data obtained from slice experiments; specifically, ACh reduced the conductance densities of the potassium M current and the calcium-dependent potassium current, whereas NE nonmonotonically regulated the conductance density of an ohmic potassium current. We report that the effects of ACh and NE on granule cell physiology are distinct and functionally complementary to one another. ACh strongly regulates granule cell firing rates and afterpotentials, whereas NE bidirectionally regulates subthreshold membrane potentials. When combined, NE can regulate the ACh-induced expression of afterdepolarizing potentials and persistent firing. In a microcircuit simulation developed to investigate the effects of granule cell neuromodulation on mitral cell firing properties, ACh increased spike synchronization among mitral cells, whereas NE modulated the signal-to-noise ratio. Coapplication of ACh and NE both functionally improved the signal-to-noise ratio and enhanced spike synchronization among mitral cells. In summary, our computational results support distinct and complementary roles for ACh and NE in modulating olfactory bulb circuitry and suggest that NE may play a role in the regulation of cholinergic function.

  16. 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.

  17. 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.

  18. Prox1 postmitotically defines dentate gyrus cells by specifying granule cell identity over CA3 pyramidal cell fate in the hippocampus.

    PubMed

    Iwano, Tomohiko; Masuda, Aki; Kiyonari, Hiroshi; Enomoto, Hideki; Matsuzaki, Fumio

    2012-08-01

    The brain is composed of diverse types of neurons that fulfill distinct roles in neuronal circuits, as manifested by the hippocampus, where pyramidal neurons and granule cells constitute functionally distinct domains: cornu ammonis (CA) and dentate gyrus (DG), respectively. Little is known about how these two types of neuron differentiate during hippocampal development, although a set of transcription factors that is expressed in progenitor cells is known to be required for the survival of granule cells. Here, we demonstrate in mice that Prox1, a transcription factor constitutively expressed in the granule cell lineage, postmitotically functions to specify DG granule cell identity. Postmitotic elimination of Prox1 caused immature DG neurons to lose the granule cell identity and in turn terminally differentiate into the pyramidal cell type manifesting CA3 neuronal identity. By contrast, Prox1 overexpression caused opposing effects on presumptive hippocampal pyramidal cells. These results indicate that the immature DG cell has the potential to become a granule cell or a pyramidal cell, and Prox1 defines the granule cell identity. This bi-potency is lost in mature DG cells, although Prox1 is still required for correct gene expression in DG granule cells. Thus, our data indicate that Prox1 acts as a postmitotic cell fate determinant for DG granule cells over the CA3 pyramidal cell fate and is crucial for maintenance of the granule cell identity throughout the life.

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

    PubMed Central

    Fu, Zhanyan; Vicini, Stefano

    2009-01-01

    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(NLG2) in cultured mouse cerebellar granule cells (CGCs), and recorded GABAA (γ-aminobutyric acid) receptor mediated miniature postsynaptic currents (mISPCs). NLG2 transfected cells had mIPSCs with faster decay than matching GFP expressing controls at young culture ages (days in vitro, DIV 7-8). Down-regulation of NLG2 by the isoform specific shRNA-NLG2 resulted in an opposite effect. We and others have shown that the switch of α subunits of GABAA Rs from α2/3 to α1 underlies developmental speeding of the IPSC decay in various CNS regions, including the cerebellum. To assess whether the reduced decay time of mIPSCs by NLG2 is due to the recruitment of more α1 containing GABAARs at the synapses, we examined the prolongation of current decay by the zolpidem, which has been shown to preferentially enhance the activity of α1 subunit containing GABA channel. The application of zolpidem resulted in a significantly greater prolongation kinetics of synaptic currents in NLG2 over-expressing cells than control cells, suggesting that NLG2 over-expression accelerates synapse maturation by promoting incorporation of the α1 subunit-containing GABAARs at postsynaptic sites in immature cells. In addition, the effect of NLG2 on the speeding of decay time course of synaptic currents was abolished when we used CGC cultures from α1-/- mice. Lastly, to exclude the possibility that the fast decay of mIPSCs induced by NLG2 could be also due to the impacts of NLG2 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 NLG2 over-expressing CGCs both at young age (DIV8) and old age (DIV14) of cultures. However, we confirm our previous

  20. Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy.

    PubMed

    Kobayashi, Masayuki; Buckmaster, Paul S

    2003-03-15

    Patients and models of temporal lobe epilepsy have fewer inhibitory interneurons in the dentate gyrus than controls, but it is unclear whether granule cell inhibition is reduced. We report the loss of GABAergic inhibition of granule cells in the temporal dentate gyrus of pilocarpine-induced epileptic rats. In situ hybridization for GAD65 mRNA and immunocytochemistry for parvalbumin and somatostatin confirmed the loss of inhibitory interneurons. In epileptic rats, granule cells had prolonged EPSPs, and they discharged more action potentials than controls. Although the conductances of evoked IPSPs recorded in normal ACSF were not significantly reduced and paired-pulse responses showed enhanced inhibition of granule cells from epileptic rats, more direct measures of granule cell inhibition revealed significant deficiencies. In granule cells from epileptic rats, evoked monosynaptic IPSP conductances were <40% of controls, and the frequency of GABA(A) receptor-mediated spontaneous and miniature IPSCs (mIPSCs) was <50% of controls. Within 3-7 d after pilocarpine-induced status epilepticus, miniature IPSC frequency had decreased, and it remained low, without functional evidence of compensatory synaptogenesis by GABAergic axons in chronically epileptic rats. Both parvalbumin- and somatostatin-immunoreactive interneuron numbers and the frequency of both fast- and slow-rising GABA(A) receptor-mediated mIPSCs were reduced, suggesting that loss of inhibitory synaptic input to granule cells occurred at both proximal/somatic and distal/dendritic sites. Reduced granule cell inhibition in the temporal dentate gyrus preceded the onset of spontaneous recurrent seizures by days to weeks, so it may contribute, but is insufficient, to cause epilepsy.

  1. Two modes of lytic granule fusion during degranulation by natural killer cells.

    PubMed

    Liu, Dongfang; Martina, Jose A; Wu, Xufeng S; Hammer, John A; Long, Eric O

    2011-08-01

    Lytic granules in cytotoxic lymphocytes, which include T cells and natural killer (NK) cells, are secretory lysosomes that release their content upon fusion with the plasma membrane (PM), a process known as degranulation. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Here, we used total internal reflection fluorescence microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand (FasL) in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptor-ligand interactions. Two fusion modes were observed: complete fusion, characterized by loss of granule content and rapid diffusion of FasL at the PM; and incomplete fusion, characterized by transient fusion pore opening and retention of FasL at the fusion site. The pH-sensitive green fluorescence protein (pHluorin) fused to the lumenal domain of FasL was used to visualize fusion pore opening with a time resolution of 30 ms. Upon incomplete fusion, pHluorin emission lasted several seconds in the absence of noticeable diffusion. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the PM, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.

  2. Secretory granules of mast cells accumulate mature and immature MHC class II molecules.

    PubMed

    Vincent-Schneider, H; Théry, C; Mazzeo, D; Tenza, D; Raposo, G; Bonnerot, C

    2001-01-01

    Bone marrow-derived mast cells as well as dendritic cells, macrophages and B lymphocytes express major histocompatibility complex (MHC) class II molecules. In mast cells, the majority of MHC class II molecules reside in intracellular cell type-specific compartments, secretory granules. To understand the molecular basis for the localisation of MHC class II molecules in secretory granules, MHC class II molecules were expressed, together with the invariant chain, in the mast cell line, RBL-2H3. Using electron and confocal microscopy, we observed that in RBL-2H3 cells, mature and immature class II molecules accumulate in secretory granules. Two particular features of class II transport accounted for this intracellular localization: first, a large fraction of newly synthesized MHC class II molecules remained associated with invariant chain fragments. This defect, resulting in a slower rate of MHC class II maturation, was ascribed to a low cathepsin S activity. Second, although a small fraction of class II dimers matured (i.e. became free of invariant chain), allowing their association with antigenic peptides, they were retained in secretory granules. As a consequence of this intracellular localization, cell surface expression of class II molecules was strongly increased by cell activation stimuli which induced the release of the contents of secretory granules. Our results suggest that antigen presentation, and thereby antigen specific T cell stimulation, are regulated in mast cells by stimuli which induce mast cell activation.

  3. A hypothesis for temporal coding of young and mature granule cells

    PubMed Central

    Rangel, Lara M.; Quinn, Laleh K.; Chiba, Andrea A.; Gage, Fred H.; Aimone, James B.

    2013-01-01

    While it has been hypothesized that adult neurogenesis (NG) plays a role in the encoding of temporal information at long time-scales, the temporal relationship of immature cells to the highly rhythmic network activity of the hippocampus has been largely unexplored. Here, we present a theory for how the activity of immature adult-born granule cells relates to hippocampal oscillations. Our hypothesis is that theta rhythmic (5–10 Hz) excitatory and inhibitory inputs into the hippocampus could differentially affect young and mature granule cells due to differences in intrinsic physiology and synaptic inhibition between the two cell populations. Consequently, immature cell activity may occur at broader ranges of theta phase than the activity of their mature counterparts. We describe how this differential influence on young and mature granule cells could separate the activity of differently aged neurons in a temporal coding regime. Notably, this process could have considerable implications on how the downstream CA3 region interprets the information conveyed by young and mature granule cells. To begin to investigate the phasic behavior of granule cells, we analyzed in vivo recordings of the rat dentate gyrus (DG), observing that the temporal behavior of granule cells with respect to the theta rhythm is different between rats with normal and impaired levels of NG. Specifically, in control animals, granule cells exhibit both strong and weak coupling to the phase of the theta rhythm. In contrast, the distribution of phase relationships in NG-impaired rats is shifted such that they are significantly stronger. These preliminary data support our hypothesis that immature neurons could distinctly affect the temporal dynamics of hippocampal encoding. PMID:23717259

  4. The transcription factor Cux1 in cerebellar granule cell development and medulloblastoma pathogenesis.

    PubMed

    Topka, Sabine; Glassmann, Alexander; Weisheit, Gunnar; Schüller, Ulrich; Schilling, Karl

    2014-12-01

    Cux1, also known as Cutl1, CDP or Cut is a homeodomain transcription factor implicated in the regulation of normal and oncogenic development in diverse peripheral tissues and organs. We studied the expression and functional role of Cux1 in cerebellar granule cells and medulloblastoma. Cux1 is robustly expressed in proliferating granule cell precursors and in postmitotic, migrating granule cells. Expression is lost as postmigratory granule cells mature. Moreover, Cux1 is also strongly expressed in a well-established mouse model of medulloblastoma. In contrast, expression of CUX1 in human medulloblastoma tissue samples is lower than in normal fetal cerebellum. In these tumors, CUX1 expression tightly correlates with a set of genes which, when mapped on a global protein-protein interaction dataset, yields a tight network that constitutes a cell cycle control signature and may be related to p53 and the DNA damage response pathway. Antisense-mediated reduction of CUX1 levels in two human medulloblastoma cell lines led to a decrease in proliferation and altered motility. The developmental expression of Cux1 in the cerebellum and its action in cell lines support a role in granule cell and medulloblastoma proliferation. Its expression in human medulloblastoma shifts that perspective, suggesting that CUX1 is part of a network involved in cell cycle control and maintenance of DNA integrity. The constituents of this network may be rational targets to therapeutically approach medulloblastomas.

  5. Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

    PubMed

    Rindler, Michael J; Xu, Chong-Feng; Gumper, Iwona; Cen, Chuan; Sonderegger, Peter; Neubert, Thomas A

    2008-04-01

    Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.

  6. 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.

  7. Intracisternal granules in the adipokinetic cells of locusts are not degraded and apparently function as supplementary stores of secretory material.

    PubMed

    Harthoorn, L F; Diederen, J H; Oudejans, R C; Verstegen, M M; Vullings, H G; Van der Horst, D J

    2000-01-01

    The intracisternal granules in locust adipokinetic cells appear to represent accumulations of secretory material within cisternae of the rough endoplasmic reticulum. An important question is whether these granules are destined for degradation or represent stores of (pro)hormones. Two strategies were used to answer this question. First, cytochemistry was applied to elucidate the properties of intracisternal granules. The endocytic tracers horseradish peroxidase and wheat-germ agglutinin-conjugated horseradish peroxidase were used to facilitate the identification of endocytic, autophagic, and lysosomal organelles, which may be involved in the degradation of intracisternal granules. No intracisternal granules could be found within autophagosomes, and granules fused with endocytic and lysosomal organelles were not observed, nor could tracer be found within the granules. The lysosomal enzyme acid phosphatase was absent from the granules. Second, biochemical analysis of the content of intracisternal granules revealed that these granules contain prohormones as well as hormones. Prohormones were present in relatively higher amounts compared with ordinary secretory granules. Since the intracisternal granules in locust adipokinetic cells are not degraded and contain intact (pro)hormones it is concluded that they function as supplementary stores of secretory material.

  8. Disrupted Dentate Granule Cell Chloride Regulation Enhances Synaptic Excitability during Development of Temporal Lobe Epilepsy

    PubMed Central

    Pathak, Hemal R.; Weissinger, Florian; Terunuma, Miho; Carlson, Gregory C.; Hsu, Fu-Chun; Moss, Stephen J.; Coulter, Douglas A.

    2008-01-01

    GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl− concentration ([Cl−]in ) at low levels. In neurons in the developing CNS, [Cl−]in is elevated, EGABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1–60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that EGABA was positively shifted in granule cells. This shift in EGABA altered synaptic integration, increased granule cell excitability, and resulted in compromised “gate” function of the dentate gyrus. EGABA recovered to control values at longer latencies post-STEP (2–8 weeks), when animals had developed epilepsy. During this period of shifted EGABA, expression of the Cl− extruding K+/Cl− cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked EGABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on EGABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy. PMID:18094240

  9. Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells

    PubMed Central

    Smith, T Caitlin; Wang, Lu-Yang; Howe, James R

    1999-01-01

    Although glutamate receptors have been shown to be involved in neuronal maturation, a developmental role for kainate-type receptors has not been described. In addition, the single-channel properties of native kainate receptors have not been studied in situ. We have characterized the electrophysiological properties of native kainate receptors of granule cell neurons at two distinct stages in postnatal development, using whole-cell and outside-out patch-clamp recordings in acute cerebellar slices. Kainate-type currents were detected in both immature and mature granule cells. However, noise analysis showed that the apparent unitary conductance of kainate-type channels is significantly higher in proliferating than post-migratory granule cells. The conductance and rectification behaviour of the channels in immature granule cells indicate that they contain unedited GluR5 and GluR6 subunits and are likely to be calcium permeable. Single-channel kainate-type currents were observed in outside-out patches from proliferating granule cells in the external germinal layer. The kinetic behaviour of kainate receptors in immature cells was complex. Openings to multiple conductance levels were observed, although our analysis indicates that the channels spend most of their open time in a 4 pS state. PMID:10226148

  10. Depletion of primary cilia from mature dentate granule cells impairs hippocampus-dependent contextual memory

    PubMed Central

    Rhee, Soyoung; Kirschen, Gregory W.; Gu, Yan; Ge, Shaoyu

    2016-01-01

    The primary cilium, a sensory organelle, regulates cell proliferation and neuronal development of dentate granule cells in the hippocampus. However, its role in the function of mature dentate granule cells remains unknown. Here we specifically depleted and disrupted ciliary proteins IFT20 and Kif3A (respectively) in mature dentate granule cells and investigated hippocampus-dependent contextual memory and long-term plasticity at mossy fiber synapses. We found that depletion of IFT20 in these cells significantly impaired context-dependent fear-related memory. Furthermore, we tested synaptic plasticity of mossy fiber synapses in area CA3 and found increased long-term potentiation upon depletion of IFT20 or disruption of Kif3A. Our findings suggest a role of primary cilia in the memory function of mature dentate granule cells, which may result from abnormal mossy fiber synaptic plasticity. A direct link between the primary cilia of mature dentate granule cells and behavior will require further investigation using independent approaches to manipulate primary cilia. PMID:27678193

  11. Altered morphology of hippocampal dentate granule cell presynaptic and postsynaptic terminals following conditional deletion of TrkB.

    PubMed

    Danzer, Steve C; Kotloski, Robert J; Walter, Cynthia; Hughes, Maya; McNamara, James O

    2008-01-01

    Dentate granule cells play a critical role in the function of the entorhinal-hippocampal circuitry in health and disease. Dentate granule cells are situated to regulate the flow of information into the hippocampus, a structure required for normal learning and memory. Correspondingly, impaired granule cell function leads to memory deficits, and, interestingly, altered granule cell connectivity may contribute to the hyperexcitability of limbic epilepsy. It is important, therefore, to understand the molecular determinants of synaptic connectivity of these neurons. Brain-derived neurotrophic factor and its receptor TrkB are expressed at high levels in the dentate gyrus (DG) of the hippocampus, and are implicated in regulating neuronal development, neuronal plasticity, learning, and the development of epilepsy. Whether and how TrkB regulates granule cell structure, however, is incompletely understood. To begin to elucidate the role of TrkB in regulating granule cell morphology, here we examine conditional TrkB knockout mice crossed to mice expressing green fluorescent protein in subsets of dentate granule cells. In stratum lucidum, where granule cell mossy fiber axons project, the density of giant mossy fiber boutons was unchanged, suggesting similar output to CA3 pyramidal cell targets. However, filopodial extensions of giant boutons, which contact inhibitory interneurons, were increased in number in TrkB knockout mice relative to wildtype controls, predicting enhanced feedforward inhibition of CA3 pyramidal cells. In knockout animals, dentate granule cells possessed fewer primary dendrites and enlarged dendritic spines, indicative of disrupted excitatory synaptic input to the granule cells. Together, these findings demonstrate that TrkB is required for development and/or maintenance of normal synaptic connectivity of the granule cells, thereby implying an important role for TrkB in the function of the granule cells and hippocampal circuitry.

  12. An LRRTM4-HSPG complex mediates excitatory synapse development on dentate gyrus granule cells.

    PubMed

    Siddiqui, Tabrez J; Tari, Parisa Karimi; Connor, Steven A; Zhang, Peng; Dobie, Frederick A; She, Kevin; Kawabe, Hiroshi; Wang, Yu Tian; Brose, Nils; Craig, Ann Marie

    2013-08-21

    Selective synapse development determines how complex neuronal networks in the brain are formed. Complexes of postsynaptic neuroligins and LRRTMs with presynaptic neurexins contribute widely to excitatory synapse development, and mutations in these gene families increase the risk of developing psychiatric disorders. We find that LRRTM4 has distinct presynaptic binding partners, heparan sulfate proteoglycans (HSPGs). HSPGs are required to mediate the synaptogenic activity of LRRTM4. LRRTM4 shows highly selective expression in the brain. Within the hippocampus, we detected LRRTM4 specifically at excitatory postsynaptic sites on dentate gyrus granule cells. LRRTM4(-/-) dentate gyrus granule cells, but not CA1 pyramidal cells, exhibit reductions in excitatory synapse density and function. Furthermore, LRRTM4(-/-) dentate gyrus granule cells show impaired activity-regulated AMPA receptor trafficking. These results identifying cell-type-specific functions and multiple presynaptic binding partners for different LRRTM family members reveal an unexpected complexity in the design and function of synapse-organizing proteins.

  13. Olfactory experiences dynamically regulate plasticity of dendritic spines in granule cells of Xenopus tadpoles in vivo

    PubMed Central

    Zhang, Li; Huang, Yubin; Hu, Bing

    2016-01-01

    Granule cells, rich in dendrites with densely punctated dendritic spines, are the most abundant inhibitory interneurons in the olfactory bulb. The dendritic spines of granule cells undergo remodeling during the development of the nervous system. The morphological plasticity of the spines’ response to different olfactory experiences in vivo is not fully known. In initial studies, a single granule cell in Xenopus tadpoles was labeled with GFP plasmids via cell electroporation; then, morphologic changes of the granule cell spines were visualized by in vivo confocal time-lapse imaging. With the help of long-term imaging, the total spine density, dynamics, and stability of four types of dendritic spines (mushroom, stubby, thin and filopodia) were obtained. Morphological analysis demonstrated that odor enrichment produced a remarkable increase in the spine density and stability of large mushroom spine. Then, with the help of short-term imaging, we analyzed the morphological transitions among different spines. We found that transitions between small spines (thin and filopodia) were more easily influenced by odor stimulation or olfactory deprivation. These results indicate that different olfactory experiences can regulate the morphological plasticity of different dendritic spines in the granule cell. PMID:27713557

  14. Cortical granule exocytosis in C. elegans is regulated by cell cycle components including separase.

    PubMed

    Bembenek, Joshua N; Richie, Christopher T; Squirrell, Jayne M; Campbell, Jay M; Eliceiri, Kevin W; Poteryaev, Dmitry; Spang, Anne; Golden, Andy; White, John G

    2007-11-01

    In many organisms, cortical granules undergo exocytosis following fertilization, releasing cargo proteins that modify the extracellular covering of the zygote. We identified cortical granules in Caenorhabditis elegans and have found that degranulation occurs in a wave that initiates in the vicinity of the meiotic spindle during anaphase I. Previous studies identified genes that confer an embryonic osmotic sensitivity phenotype, thought to result from abnormal eggshell formation. Many of these genes are components of the cell cycle machinery. When we suppressed expression of several of these genes by RNAi, we observed that cortical granule trafficking was disrupted and the eggshell did not form properly. We conclude that osmotic sensitivity phenotypes occur because of defects in trafficking of cortical granules and the subsequent formation of an impermeable eggshell. We identified separase as a key cell cycle component that is required for degranulation. Separase localized to cortically located filamentous structures in prometaphase I upon oocyte maturation. After fertilization, separase disappeared from these structures and appeared on cortical granules by anaphase I. RNAi of sep-1 inhibited degranulation in addition to causing extensive chromosomal segregation failures. Although the temperature-sensitive sep-1(e2406) allele exhibited similar inhibition of degranulation, it had minimal effects on chromosome segregation. These observations lead us to speculate that SEP-1 has two separable yet coordinated functions: to regulate cortical granule exocytosis and to mediate chromosome separation.

  15. MicroRNAs Promote Granule Cell Expansion in the Cerebellum Through Gli2.

    PubMed

    Constantin, Lena; Wainwright, Brandon J

    2015-12-01

    MicroRNAs (miRNAs) are important regulators of cerebellar function and homeostasis. Their deregulation results in cerebellar neuronal degeneration and spinocerebellar ataxia type 1 and contributes to medulloblastoma. Canonical miRNA processing involves Dicer, which cleaves precursor miRNAs into mature double-stranded RNA duplexes. In order to address the role of miRNAs in cerebellar granule cell precursor development, loxP-flanked exons of Dicer1 were conditionally inactivated using the granule cell precursor-specific Atoh1-Cre recombinase. A reduction of 87% in Dicer1 transcript was achieved in this conditional Dicer knockdown model. Although knockdown resulted in normal survival, mice had disruptions to the cortical layering of the anterior cerebellum, which resulted from the premature differentiation of granule cell precursors in this region during neonatal development. This defect manifested as a thinner external granular layer with ectopic mature granule cells, and a depleted internal granular layer. We found that expression of the activator components of the Hedgehog-Patched pathway, the Gli family of transcription factors, was perturbed in conditional Dicer knockdown mice. We propose that loss of Gli2 mRNA mediated the anterior-restricted defect in conditional Dicer knockdown mice and, as proof of principle, were able to show that miR-106b positively regulated Gli2 mRNA expression. These findings confirm the importance of miRNAs as positive mediators of Hedgehog-Patched signalling during granule cell precursor development.

  16. Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells.

    PubMed

    Ohashi, Ryo; Sakata, Shin-ichi; Naito, Asami; Hirashima, Naohide; Tanaka, Masahiko

    2014-04-01

    Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.

  17. Culturing of cerebellar granule cells to study neuronal migration: gradient and local perfusion assays.

    PubMed

    Guijarro, Patricia; Jiang, Jian; Yuan, Xiao-bing

    2012-07-01

    Cultures of cerebellar granule cells are a suitable model to analyze the mechanisms governing neuronal migration. In this unit, we describe a protocol to obtain cultures of dissociated granule cells at a low density, where individual cells can be easily observed. In addition, we include a protocol for studying neuronal migration in these cultures, using single, actively migrating cerebellar granule cells. Following this protocol, a factor of interest can be applied either in a gradient concentration by means of a micropipet located near the neuron, or in a homogeneous concentration by locally perfusing a certain region of the neuron. Time-lapse images are taken to analyze changes in the speed and/or directionality of the observed neuron. Overall, the two protocols take more or less a day and a half to perform, and are a useful way to evaluate a certain factor/drug for its chemotactic activity or its capacity to alter migration speed.

  18. Identification of miRNAs differentially expressed in human epilepsy with or without granule cell pathology.

    PubMed

    Zucchini, Silvia; Marucci, Gianluca; Paradiso, Beatrice; Lanza, Giovanni; Roncon, Paolo; Cifelli, Pierangelo; Ferracin, Manuela; Giulioni, Marco; Michelucci, Roberto; Rubboli, Guido; Simonato, Michele

    2014-01-01

    The microRNAs (miRNAs) are small size non-coding RNAs that regulate expression of target mRNAs at post-transcriptional level. miRNAs differentially expressed under pathological conditions may help identifying mechanisms underlying the disease and may represent biomarkers with prognostic value. However, this kind of studies are difficult in the brain because of the cellular heterogeneity of the tissue and of the limited access to fresh tissue. Here, we focused on a pathology affecting specific cells in a subpopulation of epileptic brains (hippocampal granule cells), an approach that bypasses the above problems. All patients underwent surgery for intractable temporal lobe epilepsy and had hippocampal sclerosis associated with no granule cell pathology in half of the cases and with type-2 granule cell pathology (granule cell layer dispersion or bilamination) in the other half. The expression of more than 1000 miRNAs was examined in the laser-microdissected dentate granule cell layer. Twelve miRNAs were differentially expressed in the two groups. One of these, miR487a, was confirmed to be expressed at highly differential levels in an extended cohort of patients, using RT-qPCR. Bioinformatics searches and RT-qPCR verification identified ANTXR1 as a possible target of miR487a. ANTXR1 may be directly implicated in granule cell dispersion because it is an adhesion molecule that favors cell spreading. Thus, miR487a could be the first identified element of a miRNA signature that may be useful for prognostic evaluation of post-surgical epilepsy and may drive mechanistic studies leading to the identification of therapeutic targets.

  19. Neural injury alters proliferation and integration of adult-generated neurons in the dentate gyrus

    PubMed Central

    Perederiy, Julia V.; Luikart, Bryan W.; Washburn, Eric K.; Schnell, Eric; Westbrook, Gary L.

    2013-01-01

    Neural plasticity following brain injury illustrates the potential for regeneration in the central nervous system. Lesioning of the perforant path, which innervates the outer 2/3rds of the molecular layer of the dentate gyrus, was one of the first models to demonstrate structural plasticity of mature granule cells (Parnavelas, 1974; Caceres and Steward, 1983; Diekmann et al., 1996). The dentate gyrus also harbors a continuously proliferating population of neuronal precursors that can integrate into functional circuits and show enhanced short-term plasticity (Schmidt-Hieber et al., 2004; Abrous et al., 2005). To examine the response of adult-generated granule cells to unilateral complete transection of the perforant path in vivo, we tracked these cells using transgenic POMC-EGFP mice or by retroviral expression of GFP. Lesioning triggered a marked proliferation of newborn neurons. Subsequently, the dendrites of newborn neurons showed reduced complexity within the denervated zone, but dendritic spines still formed in the absence of glutamatergic nerve terminals. Electron micrographs confirmed the lack of intact presynaptic terminals apposing spines on mature cells and on newborn neurons. Newborn neurons, but not mature granule cells, had a higher density of dendritic spines in the inner molecular layer post-lesion, accompanied by an increase in miniature EPSC amplitudes and rise times. Our results indicate that injury causes an increase in newborn neurons and lamina-specific synaptic reorganization, indicative of enhanced plasticity. The presence of de novo dendritic spines in the denervated zone suggests that the post-lesion environment provides the necessary signals for spine formation. PMID:23486947

  20. 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

  1. 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.

  2. 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.

  3. Avian minor salivary glands: an ultrastructural study of the secretory granules in mucous and seromucous cells.

    PubMed

    Olmedo, L A; Samar, M E; Avila, R E; de Crosa, M G; Dettin, L

    2000-01-01

    Ultrastructural descriptions in birds are scarce thus, in this study we have characterized the secretory granules of mucous and seromucous cells from the palatine and lingual salivary glands of birds with different diets. The samples were taken from the tongue and palatine mucosa of chicken (Gallus gallus), quail (Coturnix coturnix), chimango (Milvago chimango) and white heron (Egretta thula). The samples were processed for observation by transmission electron microscopy (TEM) employing 4% Karnovsky solution for fixation. The most noteworthy finding was the heterogeneous ultrastructural appearance of the secretory granules. Differences in substructure were found between the four species, between the palatine and lingual glands in the same species and even within the same acinus and the same cell. At variance with other authors, these differences cannot be attributed to the type of fixative solution used taking into account that all the samples were processed in the same way. Previous histochemical studies have shown the presence of sulfated and non sulfated glycoconjugates in these glands which can be associated to the maturation of the granules. These granules are probably representative of peculiar storage of the secretory products that would give rise to a heterogeneous and complex ultrastructural pattern of granules in the mucosa and seromucosa cells of these avian species.

  4. 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.

  5. Mast cell differentiation depends on T cells and granule synthesis on fibroblasts.

    PubMed Central

    Davidson, S; Mansour, A; Gallily, R; Smolarski, M; Rofolovitch, M; Ginsburg, H

    1983-01-01

    Mast cell differentiation was generated in the following three experimental situations: (i) infection of mice with Schistosoma Mansoni or with Nippostrongylus brasiliensis and growth of the lymph node cells in the presence of the corresponding helminth antigen; (ii) immunization with horse serum and growth of blood and lymph node cells in the presence of the horse serum; (iii) exposure of T-cell-depleted suspensions of lymph node cells from unimmunized mice to T-cell factor (TCF) released into medium of the young cultures of (i) and (ii). This differentiation was also obtained when lymph node cells from athymic nude mice were exposed to TCF. The cell suspensions were plated on X-irradiated fibroblast monolayers prepared from embryonic mouse skin. Screening of the suspensions before plating on the fibroblasts in culture revealed no young forms of mast cells, and none were present in culture of nude mice lymph node cells maintained without TCF. Primordial appearance of metachromatic granules generally in the golgi zone was first seen in many 'large lymphoid cells' as early as 18 hr after plating. This was followed by increase in the cytoplasm volume, increase in granule number and mitosis, ending at 10-18 days with homogeneous populations of mature mast cells. When the mesenteric lymph node cells from mice infected with the helminths were grown in the absence of fibroblasts but in the presence of the antigen, homogeneous populations of cells with extended cytoplasm, filled with unstained vacuoles developed during days 7-13. These cells did not contain histamine (or at most 0.2 microgram per 10(6) vacuolated cells). When these cells were plated on fibroblast monolayers clear granule formation in all the vacuoles was seen 2 days later. It increased progressively in size and staining intensity, until the vacuoles transformed into typical mast cell granules. By the fourth day the vacuolated cells attained the typical mast cell morphology and the histamine content greatly

  6. [Basic proteins in the granules of mast cells. Demonstration of masked proteins, acidophilic staining of the granules].

    PubMed

    Anikó, K; Lajos, K

    1976-07-01

    Basic proteins of the granules of mast cells in nativ, formalin-, alcohol- and aceton fixed preparations without any preliminary treatment, when stained with acidic dye at the pH 9 cytochemically seem to be masked. After various preliminary treatment (treatment with acid, with cetylpiridinumchlorid, CPC) mast-cell granula stained with acidic-dye at pH 9 appear intensively acidophile. This phenomenon can be explained by the presence of basic proteins in the mast cell granula. Preliminary treatment with CPC inhibits acid radicals of the heparin. This may lead to the disintegration of the linkage between proteins of the heparin, thus amino-group of the basic proteins become reactivated and can be identified by acidic dyes. It can not be excluded as well, that CPC linked to the heparin with free positive radicals reveals acidic-dye-binding capacity. In cases of preliminary treatment with various acids this mechanism does not seem possible to lay on the base of changing of the dye binding capacity.

  7. 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.

  8. Role of actin cortex in the subplasmalemmal transport of secretory granules in PC-12 cells.

    PubMed Central

    Lang, T; Wacker, I; Wunderlich, I; Rohrbach, A; Giese, G; Soldati, T; Almers, W

    2000-01-01

    In neuroendocrine PC-12 cells, evanescent-field fluorescence microscopy was used to track motions of green fluorescent protein (GFP)-labeled actin or GFP-labeled secretory granules in a thin layer of cytoplasm where cells adhered to glass. The layer contained abundant filamentous actin (F-actin) locally condensed into stress fibers. More than 90% of the granules imaged lay within the F-actin layer. One-third of the granules did not move detectably, while two-thirds moved randomly; the average diffusion coefficient was 23 x 10(-4) microm(2)/s. A small minority (<3%) moved rapidly and in a directed fashion over distances more than a micron. Staining of F-actin suggests that such movement occurred along actin bundles. The seemingly random movement of most other granules was not due to diffusion since it was diminished by the myosin inhibitor butanedione monoxime, and blocked by chelating intracellular Mg(2+) and replacing ATP with AMP-PNP. Mobility was blocked also when F-actin was stabilized with phalloidin, and was diminished when the actin cortex was degraded with latrunculin B. We conclude that the movement of granules requires metabolic energy, and that it is mediated as well as limited by the actin cortex. Opposing actions of the actin cortex on mobility may explain why its degradation has variable effects on secretion. PMID:10827968

  9. Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells

    PubMed Central

    Huang, Cheng-Chiu; Sugino, Ken; Shima, Yasuyuki; Guo, Caiying; Bai, Suxia; Mensh, Brett D; Nelson, Sacha B; Hantman, Adam W

    2013-01-01

    Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control. DOI: http://dx.doi.org/10.7554/eLife.00400.001 PMID:23467508

  10. Mechanisms of granule membrane recapture following exocytosis in intact mast cells.

    PubMed

    Cabeza, Jose M; Acosta, Jorge; Alés, Eva

    2013-07-12

    In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified "large-capacitance flickers" that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent "compound cavicapture," most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event.

  11. Mechanisms of Granule Membrane Recapture following Exocytosis in Intact Mast Cells*

    PubMed Central

    Cabeza, Jose M.; Acosta, Jorge; Alés, Eva

    2013-01-01

    In secretory cells, several exocytosis-coupled forms of endocytosis have been proposed including clathrin-mediated endocytosis, kiss-and-run endocytosis, cavicapture, and bulk endocytosis. These forms of endocytosis can be induced under different conditions, but their detailed molecular mechanisms and functions are largely unknown. We studied exocytosis and endocytosis in mast cells with both perforated-patch and whole-cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric serotonin detection. We found that intact mast cells exhibit an early endocytosis that follows exocytosis induced by compound 48/80. Direct observation of individual exocytic and endocytic events showed a higher percentage of capacitance flickers (27.3%) and off-steps (11.4%) in intact mast cells than in dialyzed cells (5.4% and 2.9%, respectively). Moreover, we observed a type of endocytosis of large pieces of membrane that were likely formed by cumulative fusion of several secretory granules with the cell membrane. We also identified “large-capacitance flickers” that occur after large endocytosis events. Pore conductance analysis indicated that these transient events may represent “compound cavicapture,” most likely due to the flickering of a dilated fusion pore. Using fluorescence imaging of individual exocytic and endocytic events we observed that granules can fuse to granules already fused with the plasma membrane, and then the membranes and dense cores of fused granules are internalized. Altogether, our results suggest that stimulated exocytosis in intact mast cells is followed by several forms of compensatory endocytosis, including kiss-and-run endocytosis and a mechanism for efficient retrieval of the compound membrane of several secretory granules through a single membrane fission event. PMID:23709219

  12. Mouse granulated metrial gland cells require contact with stromal cells to maintain viability

    PubMed Central

    STEWART, I. J.

    2000-01-01

    Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown. In vivo, GMG cells share an intimate association with fibroblast-like stromal cells. The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro. When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture. The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d. When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later. Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells. The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function. PMID:11117633

  13. Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype.

    PubMed

    Malosio, Maria Luisa; Giordano, Tiziana; Laslop, Andrea; Meldolesi, Jacopo

    2004-02-15

    Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B

  14. Axonal plasticity of age-defined dentate granule cells in a rat model of mesial temporal lobe epilepsy.

    PubMed

    Althaus, A L; Zhang, H; Parent, J M

    2016-02-01

    Dentate granule cell (DGC) mossy fiber sprouting (MFS) in mesial temporal lobe epilepsy (mTLE) is thought to underlie the creation of aberrant circuitry which promotes the generation or spread of spontaneous seizure activity. Understanding the extent to which populations of DGCs participate in this circuitry could help determine how it develops and potentially identify therapeutic targets for regulating aberrant network activity. In this study, we investigated how DGC birthdate influences participation in MFS and other aspects of axonal plasticity using the rat pilocarpine-induced status epilepticus (SE) model of mTLE. We injected a retrovirus (RV) carrying a synaptophysin-yellow fluorescent protein (syp-YFP) fusion construct to birthdate DGCs and brightly label their axon terminals, and compared DGCs born during the neonatal period with those generated in adulthood. We found that both neonatal and adult-born DGC populations participate, to a similar extent, in SE-induced MFS within the dentate gyrus inner molecular layer (IML). SE did not alter hilar MF bouton density compared to sham-treated controls, but adult-born DGC bouton density was greater in the IML than in the hilus after SE. Interestingly, we also observed MF axonal reorganization in area CA2 in epileptic rats, and these changes arose from DGCs generated both neonatally and in adulthood. These data indicate that both neonatal and adult-generated DGCs contribute to axonal reorganization in the rat pilocarpine mTLE model, and indicate a more complex relationship between DGC age and participation in seizure-related plasticity than was previously thought.

  15. AXONAL PLASTICITY OF AGE-DEFINED DENTATE GRANULE CELLS IN A RAT MODEL OF MESIAL TEMPORAL LOBE EPILEPSY

    PubMed Central

    Althaus, AL; Zhang, H; Parent, JM

    2016-01-01

    Dentate granule cell (DGC) mossy fiber sprouting (MFS) in mesial temporal lobe epilepsy (mTLE) is thought to underlie the creation of aberrant circuitry which promotes the generation or spread of spontaneous seizure activity. Understanding the extent to which populations of DGCs participate in this circuitry could help determine how it develops and potentially identify therapeutic targets for regulating aberrant network activity. In this study, we investigated how DGC birthdate influences participation in MFS and other aspects of axonal plasticity using the rat pilocarpine-induced status epilepticus (SE) model of mTLE. We injected a retrovirus (RV) carrying a synaptophysin-yellow fluorescent protein (syp-YFP) fusion construct to birthdate DGCs and brightly label their axon terminals, and compared DGCs born during the neonatal period with those generated in adulthood. We found that both neonatal and adult-born DGC populations participate, to a similar extent, in SE-induced MFS within the dentate gyrus inner molecular layer (IML). SE did not alter hilar MF bouton density compared to sham-treated controls, but adult-born DGC bouton density was greater in the IML than in the hilus after SE. Interestingly, we also observed MF axonal reorganization in area CA2 in epileptic rats, and these changes arose from DGCs generated both neonatally and in adulthood. These data indicate that both neonatal and adult-generated DGCs contribute to axonal reorganization in the rat pilocarpine mTLE model, and indicate a more complex relationship between DGC age and participation in seizure-related plasticity than was previously thought. PMID:26644085

  16. Linking local circuit inhibition to olfactory behavior: a critical role for granule cells in olfactory discrimination.

    PubMed

    Strowbridge, Ben W

    2010-02-11

    In this issue of Neuron, Abraham et al. report a direct connection between inhibitory function and olfactory behavior. Using molecular methods to alter glutamate receptor subunit composition in olfactory bulb granule cells, the authors found a selective modulation in the time required for difficult, but not simple, olfactory discrimination tasks.

  17. Morphological changes among hippocampal dentate granule cells exposed to early kindling-epileptogenesis

    PubMed Central

    Singh, Shatrunjai P.; He, Xiaoping; McNamara, James O.; Danzer, Steve C.

    2013-01-01

    Temporal lobe epilepsy is associated with changes in the morphology of hippocampal dentate granule cells. These changes are evident in numerous models that are associated with substantial neuron loss and spontaneous recurrent seizures. By contrast, previous studies have shown that in the kindling model, it is possible to administer a limited number of stimulations sufficient to produce a lifelong enhanced sensitivity to stimulus evoked seizures without associated spontaneous seizures and minimal neuronal loss. Here we examined whether stimulation of the amygdala sufficient to evoke five convulsive seizures (class IV or greater on Racine’s scale) produce morphological changes similar to those observed in models of epilepsy associated with substantial cell loss. The morphology of GFP-expressing granule cells from Thy-1 GFP mice was examined either one day or one month after the last evoked seizure. Interestingly, significant reductions in dendritic spine density were evident one day after the last seizure, the magnitude of which had diminished by one month. Further, there was an increase in the thickness of the granule cell layer one day after the last evoked seizure, which was absent a month later. We also observed an increase in the area of the proximal axon, which again returned to control levels a month later. No differences in the number of basal dendrites were detected at either time point. These findings demonstrate that the early stages of kindling epileptogenesis produce transient changes in the granule cell body layer thickness, molecular layer spine density and axon proximal area, but do not produce striking rearrangements of granule cell structure. PMID:23893783

  18. Recruitment of an inhibitory hippocampal network after bursting in a single granule cell.

    PubMed

    Mori, Masahiro; Gähwiler, Beat H; Gerber, Urs

    2007-05-01

    The hippocampal CA3 area, an associational network implicated in memory function, receives monosynaptic excitatory as well as disynaptic inhibitory input through the mossy-fiber axons of the dentate granule cells. Synapses made by mossy fibers exhibit low release probability, resulting in high failure rates at resting discharge frequencies of 0.1 Hz. In recordings from functionally connected pairs of neurons, burst firing of a granule cell increased the probability of glutamate release onto both CA3 pyramidal cells and inhibitory interneurons, such that subsequent low-frequency stimulation evoked biphasic excitatory/inhibitory responses in a CA3 pyramidal cell, an effect lasting for minutes. Analysis of the unitary connections in the circuit revealed that granule cell bursting caused powerful activation of an inhibitory network, thereby transiently suppressing excitatory input to CA3 pyramidal cells. This phenomenon reflects the high incidence of spike-to-spike transmission at granule cell to interneuron synapses, the numerically much greater targeting by mossy fibers of inhibitory interneurons versus principal cells, and the extensively divergent output of interneurons targeting CA3 pyramidal cells. Thus, mossy-fiber input to CA3 pyramidal cells appears to function in three distinct modes: a resting mode, in which synaptic transmission is ineffectual because of high failure rates; a bursting mode, in which excitation predominates; and a postbursting mode, in which inhibitory input to the CA3 pyramidal cells is greatly enhanced. A mechanism allowing the transient recruitment of inhibitory input may be important for controlling network activity in the highly interconnected CA3 pyramidal cell region.

  19. Hedgehog antagonist REN(KCTD11) regulates proliferation and apoptosis of developing granule cell progenitors.

    PubMed

    Argenti, Beatrice; Gallo, Rita; Di Marcotullio, Lucia; Ferretti, Elisabetta; Napolitano, Maddalena; Canterini, Sonia; De Smaele, Enrico; Greco, Azzura; Fiorenza, Maria Teresa; Maroder, Marella; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2005-09-07

    During the early development of the cerebellum, a burst of granule cell progenitor (GCP) proliferation occurs in the outer external granule layer (EGL), which is sustained mainly by Purkinje cell-derived Sonic Hedgehog (Shh). Shh response is interrupted once GCPs move into the inner EGL, where granule progenitors withdraw proliferation and start differentiating and migrating toward the internal granule layer (IGL). Failure to interrupt Shh signals results in uncoordinated proliferation and differentiation of GCPs and eventually leads to malignancy (i.e., medulloblastoma). The Shh inhibitory mechanisms that are responsible for GCP growth arrest and differentiation remain unclear. Here we report that REN, a putative tumor suppressor frequently deleted in human medulloblastoma, is expressed to a higher extent in nonproliferating inner EGL and IGL granule cells than in highly proliferating outer EGL cells. Accordingly, upregulated REN expression occurs along GCP differentiation in vitro, and, in turn, REN overexpression promotes growth arrest and increases the proportion of p27/Kip1+ GCPs. REN also impairs both Gli2-dependent gene transcription and Shh-enhanced expression of the target Gli1 mRNA, thus antagonizing the Shh-induced effects on the proliferation and differentiation of cultured GCPs. Conversely, REN functional knock-down impairs Hedgehog antagonism and differentiation and sustains the proliferation of GCPs. Finally, REN enhances caspase-3 activation and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling apoptotic GCP numbers; therefore, the pattern of REN expression, its activity, and its antagonism on the Hedgehog pathway suggest that this gene may represent a restraint of Shh signaling at the outer to inner EGL GCP transitions. Medulloblastoma-associated REN loss of function might withdraw such a limiting signal for immature cell expansion, thus favoring tumorigenesis.

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

    PubMed

    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

    2002-07-05

    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.

  1. 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.

  2. Preservation of perisomatic inhibitory input of granule cells in the epileptic human dentate gyrus.

    PubMed

    Wittner, L; Maglóczky, Z; Borhegyi, Z; Halász, P; Tóth, S; Eross, L; Szabó, Z; Freund, T F

    2001-01-01

    Temporal lobe epilepsy is known to be associated with hyperactivity that is likely to be generated or amplified in the hippocampal formation. The majority of granule cells, the principal cells of the dentate gyrus, are found to be resistant to damage in epilepsy, and may serve as generators of seizures if their inhibition is impaired. Therefore, the parvalbumin-containing subset of interneurons, known to provide the most powerful inhibitory input to granule cell somata and axon initial segments, were examined in human control and epileptic dentate gyrus. A strong reduction in the number of parvalbumin-containing cells was found in the epileptic samples especially in the hilar region, although in some patches of the granule cell layer parvalbumin-positive terminals that form vertical clusters characteristic of axo-axonic cells were more numerous than in controls. Analysis of the postsynaptic target elements of parvalbumin-positive axon terminals showed that they form symmetric synapses with somata, dendrites, axon initial segments and spines as in the control, but the ratio of axon initial segment synapses was increased in the epileptic tissue (control: 15.9%, epileptic: 31.3%). Furthermore, the synaptic coverage of granule cell axon initial segments increased more than three times (control: 0.52, epileptic: 2.10 microm synaptic length/100 microm axon initial segment membrane) in the epileptic samples, whereas the amount of somatic symmetric synapses did not change significantly. Although the number of parvalbumin-positive interneurons is decreased, the perisomatic inhibitory input of dentate granule cells is preserved in temporal lobe epilepsy. Basket and axo-axonic cell terminals - whether positive or negative for parvalbumin - are present, moreover, the axon collaterals targeting axon initial segments sprout in the epileptic dentate gyrus. We suggest that perisomatic inhibitory interneurons survive in epilepsy, but their somadendritic compartment and partly the

  3. Active dentate granule cells encode experience to promote the addition of adult-born hippocampal neurons.

    PubMed

    Kirschen, Gregory W; Shen, Jia; Tian, Mu; Schroeder, Bryce; Wang, Jia; Man, Guoming; Wu, Song; Ge, Shaoyu

    2017-04-03

    The continuous addition of new dentate granule cells, exquisitely regulated by brain activity, renders the hippocampus plastic. However, how neural circuits encode experiences to impact the addition of adult-born neurons remains unknown. Here, we used endoscopic Ca(2+) imaging to track the real-time activity of individual dentate granule cells in freely-behaving mice. For the first time, we found that active dentate granule cells responded to a novel experience by preferentially increasing their Ca(2+) event frequency. This elevated activity, which we found to be associated with object exploration, returned to baseline by one hour in the same environment, but could be dishabituated via introduction to a novel environment. To seamlessly transition between environments, we next established a freely-controllable virtual reality system for unrestrained mice. We again observed increased firing of active neurons in a virtual enriched environment. Interestingly, multiple novel virtual experiences accumulatively increased the number of newborn neurons when compared to a single experience. Finally, optogenetic silencing of existing dentate granule cells during novel environmental exploration perturbed experience-induced neuronal addition. Together, our study shows that the adult brain conveys novel, enriched experiences to increase the addition of adult-born hippocampal neurons by increasing the firing of active dentate granule cells.SIGNIFICANCE STATEMENTAdult brains are constantly reshaping themselves from synapses to circuits as we encounter novel experiences from moment to moment. Importantly, this reshaping includes the addition of newborn hippocampal neurons. However, it remains largely unknown how our circuits encode experience-induced brain activity to govern the addition of new hippocampal neurons. By coupling in vivo Ca(2+) imaging of dentate granule neurons with a novel unrestrained virtual reality system for rodents, we discovered that a new experience rapidly

  4. Mechanisms and benefits of granule cell latency coding in the mouse olfactory bulb

    PubMed Central

    Giridhar, Sonya; Urban, Nathaniel N.

    2012-01-01

    Inhibitory circuits are critical for shaping odor representations in the olfactory bulb. There, individual granule cells can respond to brief stimulation with extremely long (up to 1000 ms), input-specific latencies that are highly reliable. However, the mechanism and function of this long timescale activity remain unknown. We sought to elucidate the mechanism responsible for long-latency activity, and to understand the impact of widely distributed interneuron latencies on olfactory coding. We used a combination of electrophysiological, optical, and pharmacological techniques to show that long-latency inhibition is driven by late onset synaptic excitation to granule cells. This late excitation originates from tufted cells, which have intrinsic properties that favor longer latency spiking than mitral cells. Using computational modeling, we show that widely distributed interneuron latency increases the discriminability of similar stimuli. Thus, long-latency inhibition in the olfactory bulb requires a combination of circuit- and cellular-level mechanisms that function to improve stimulus representations. PMID:22754503

  5. Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

    PubMed Central

    Urbé, Sylvie; Page, Lesley J.; Tooze, Sharon A.

    1998-01-01

    The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [35S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process. PMID:9864358

  6. Recurrent mossy fibers preferentially innervate parvalbumin-immunoreactive interneurons in the granule cell layer of the rat dentate gyrus.

    PubMed

    Blasco-Ibáñez, J M; Martínez-Guijarro, F J; Freund, T F

    2000-09-28

    Detection of vesicular zinc and immunohistochemistry against markers for different interneuron subsets were combined to study the postsynaptic target selection of zinc-containing recurrent mossy fiber collaterals in the dentate gyrus. Mossy fiber collaterals in the granule cell layer selectively innervated parvalbumin-containing cells, with numerous contacts per cell, whereas the granule cells were avoided. Under the electron microscope, those boutons made asymmetrical contacts on dendrites and somata. These findings suggest that, in addition to the hilar perforant path-associated (HIPP) interneurons, the basket and chandelier cells also receive a powerful feed-back drive from the granule cells, and thereby are able to control population synchrony in the dentate gyrus. On the other hand, the amount of monosynaptic excitatory feed-back among granule cells is shown to be negligible.

  7. On the origins of the universal dynamics of endogenous granules in mammalian cells.

    PubMed

    Vanapalli, Siva A; Li, Yixuan; Mugele, Frieder; Duits, Michel H G

    2009-12-01

    Endogenous granules (EGs) that consist of lipid droplets and mitochondria have been commonly used to assess intracellular mechanical properties via multiple particle tracking microrheology (MPTM). Despite their widespread use, the nature of interaction of EGs with the cytoskeletal network and the type of forces driving their dynamics--both of which are crucial for the interpretation of the results from MPTM technique--are yet to be resolved. In this report, we study the dynamics of endogenous granules in mammalian cells using particle tracking methods. We find that the ensemble dynamics of EGs is diffusive in three types of mammalian cells (endothelial cells, smooth muscle cells and fibroblasts), thereby suggesting an apparent universality in their dynamical behavior. Moreover, in a given cell, the amplitude of the mean-squared displacement for EGs is an order of magnitude larger than that of injected particles. This observation along with results from ATP depletion and temperature intervention studies suggests that cytoskeletal active forces drive the dynamics of EGs. To elucidate the dynamical origin of the diffusive-like nonthermal motion, we consider three active force generation mechanisms--molecular motor transport, actomyosin contractility and microtubule polymerization forces. We test these mechanisms using pharmacological interventions. Experimental evidence and model calculations suggest that EGs are intimately linked to microtubules and that microtubule polymerization forces drive their dynamics. Thus, endogenous granules could serve as non-invasive probes for microtubule network dynamics in mammalian cells.

  8. Pasteurella multocida toxin: Targeting mast cell secretory granules during kiss-and-run secretion.

    PubMed

    Danielsen, Elisabeth M; Christiansen, Nina; Danielsen, E Michael

    2016-02-01

    Pasteurella multocida toxin (PMT), a virulence factor of the pathogenic Gram-negative bacterium P. multocida, is a 146 kDa protein belonging to the A-B class of toxins. Once inside a target cell, the A domain deamidates the α-subunit of heterotrimeric G-proteins, thereby activating downstream signaling cascades. However, little is known about how PMT selects and enters its cellular targets. We therefore studied PMT binding and uptake in porcine cultured intestinal mucosal explants to identify susceptible cells in the epithelium and underlying lamina propria. In comparison with Vibrio cholera B-subunit, a well-known enterotoxin taken up by receptor-mediated endocytosis, PMT binding to the epithelial brush border was scarce, and no uptake into enterocytes was detected by 2h, implying that none of the glycolipids in the brush border are a functional receptor for PMT. However, in the lamina propria, PMT distinctly accumulated in the secretory granules of mast cells. This also occurred at 4 °C, ruling out endocytosis, but suggestive of uptake via pores that connect the granules to the cell surface. Mast cell granules are known to secrete their contents by a "kiss-and-run" mechanism, and we propose that PMT may exploit this secretory mechanism to gain entry into this particular cell type.

  9. Conditional induction of Math1 specifies embryonic stem cells to cerebellar granule neuron lineage and promotes differentiation into mature granule neurons.

    PubMed

    Srivastava, Rupali; Kumar, Manoj; Peineau, Stéphane; Csaba, Zsolt; Mani, Shyamala; Gressens, Pierre; El Ghouzzi, Vincent

    2013-04-01

    Directing differentiation of embryonic stem cells (ESCs) to specific neuronal subtype is critical for modeling disease pathology in vitro. An attractive means of action would be to combine regulatory differentiation factors and extrinsic inductive signals added to the culture medium. In this study, we have generated mature cerebellar granule neurons by combining a temporally controlled transient expression of Math1, a master gene in granule neuron differentiation, with inductive extrinsic factors involved in cerebellar development. Using a Tetracyclin-On transactivation system, we overexpressed Math1 at various stages of ESCs differentiation and found that the yield of progenitors was considerably increased when Math1 was induced during embryonic body stage. Math1 triggered expression of Mbh1 and Mbh2, two target genes directly involved in granule neuron precursor formation and strong expression of early cerebellar territory markers En1 and NeuroD1. Three weeks after induction, we observed a decrease in the number of glial cells and an increase in that of neurons albeit still immature. Combining Math1 induction with extrinsic factors specifically increased the number of neurons that expressed Pde1c, Zic1, and GABAα6R characteristic of mature granule neurons, formed "T-shaped" axons typical of granule neurons, and generated synaptic contacts and action potentials in vitro. Finally, in vivo implantation of Math1-induced progenitors into young adult mice resulted in cell migration and settling of newly generated neurons in the cerebellum. These results show that conditional induction of Math1 drives ESCs toward the cerebellar fate and indicate that acting on both intrinsic and extrinsic factors is a powerful means to modulate ESCs differentiation and maturation into a specific neuronal lineage.

  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. Cerebellar cortical degeneration with selective granule cell loss in Bavarian mountain dogs.

    PubMed

    Flegel, T; Matiasek, K; Henke, D; Grevel, V

    2007-08-01

    Three Bavarian mountain dogs aged between 18 and 20 months, not related to each other, were presented with chronic signs of cerebellar dysfunction. On sagittal T2-weighted magnetic resonance imaging brain images, the tentative diagnosis of cerebellar hypoplasia was established based on an enlarged cerebrospinal fluid space around the cerebellum and an increased cerebrospinal fluid signal between the folia. Post-mortem examination was performed in one dog and did show an overall reduction of cerebellar size. On histopathologic examination, a selective loss of cerebellar granule cells with sparing of Purkinje cells was evident. Therefore, the Bavarian mountain dog is a breed where cerebellar cortical degeneration caused by the rather exceptional selective granule cell loss can be seen as cause of chronic, slowly progressive cerebellar dysfunction starting at an age of several months.

  12. Granule cargo release from bone marrow-derived cells sustains cardiac hypertrophy.

    PubMed

    Yang, Fanmuyi; Dong, Anping; Ahamed, Jasimuddin; Sunkara, Manjula; Smyth, Susan S

    2014-11-15

    Bone marrow-derived inflammatory cells, including platelets, may contribute to the progression of pressure overload-induced left ventricular hypertrophy (LVH). However, the underlying mechanisms for this are still unclear. One potential mechanism is through release of granule cargo. Unc13-d(Jinx) (Jinx) mice, which lack Munc13-4, a limiting factor in vesicular priming and fusion, have granule secretion defects in a variety of hematopoietic cells, including platelets. In the current study, we investigated the role of granule secretion in the development of LVH and cardiac remodeling using chimeric mice specifically lacking Munc13-4 in marrow-derived cells. Pressure overload was elicited by transverse aortic constriction (TAC). Chimeric mice were created by bone marrow transplantation. Echocardiography, histology staining, immunohistochemistry, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and mass spectrometry were used to study LVH progression and inflammatory responses. Wild-type (WT) mice that were transplanted with WT bone marrow (WT→WT) and WT mice that received Jinx bone marrow (Jinx→WT) developed LVH and a classic fetal reprogramming response early (7 days) after TAC. However, at late times (5 wk), mice lacking Munc13-4 in bone marrow-derived cells (Jinx→WT) failed to sustain the cardiac hypertrophy observed in WT chimeric mice. No difference in cardiac fibrosis was observed at early or late time points. Reinjection of WT platelets or platelet releasate partially restored cardiac hypertrophy in Jinx chimeric mice. These results suggest that sustained LVH in the setting of pressure overload depends on one or more factors secreted from bone marrow-derived cells, possibly from platelets. Inhibiting granule cargo release may represent a novel target for preventing sustained LVH.

  13. 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.

  14. Secretory granule biogenesis and neuropeptide sorting to the regulated secretory pathway in neuroendocrine cells.

    PubMed

    Loh, Y Peng; Kim, Taeyoon; Rodriguez, Yazmin M; Cawley, Niamh X

    2004-01-01

    Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the granules of the regulated secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of secretory granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a

  15. Structural Plasticity of Dentate Granule Cell Mossy Fibers During the Development of Limbic Epilepsy

    PubMed Central

    Danzer, Steve C.; He, Xiaoping; Loepke, Andreas W.; McNamara, James O.

    2009-01-01

    Altered granule cell≫CA3 pyramidal cell synaptic connectivity may contribute to the development of limbic epilepsy. To explore this possibility, granule cell giant mossy fiber bouton plasticity was examined in the kindling and pilocarpine models of epilepsy using green fluorescent protein-expressing transgenic mice. These studies revealed significant increases in the frequency of giant boutons with satellite boutons 2 days and 1 month after pilocarpine status epilepticus, and increases in giant bouton area at 1 month. Similar increases in giant bouton area were observed shortly after kindling. Finally, both models exhibited plasticity of mossy fiber giant bouton filopodia, which contact GABAergic interneurons mediating feedforward inhibition of CA3 pyramids. In the kindling model, however, all changes were fleeting, having resolved by 1 month after the last evoked seizure. Together, these findings demonstrate striking structural plasticity of granule cell mossy fiber synaptic terminal structure in two distinct models of adult limbic epileptogenesis. We suggest that these plasticities modify local connectivities between individual mossy fiber terminals and their targets, inhibitory interneurons, and CA3 pyramidal cells potentially altering the balance of excitation and inhibition during the development of epilepsy. PMID:19294647

  16. Induction of stress granule-like structures in vesicular stomatitis virus-infected cells.

    PubMed

    Dinh, Phat X; Beura, Lalit K; Das, Phani B; Panda, Debasis; Das, Anshuman; Pattnaik, Asit K

    2013-01-01

    Previous studies from our laboratory revealed that cellular poly(C) binding protein 2 (PCBP2) downregulates vesicular stomatitis virus (VSV) gene expression. We show here that VSV infection induces the formation of granular structures in the cytoplasm containing cellular RNA-binding proteins, including PCBP2, T-cell-restricted intracellular antigen 1 (TIA1), and TIA1-related protein (TIAR). Depletion of TIA1 via small interfering RNAs (siRNAs), but not depletion of TIAR, results in enhanced VSV growth and gene expression. The VSV-induced granules appear to be similar to the stress granules (SGs) generated in cells triggered by heat shock or oxidative stress but do not contain some of the bona fide SG markers, such as eukaryotic initiation factor 3 (eIF3) or eIF4A, or the processing body (PB) markers, such as mRNA-decapping enzyme 1A (DCP1a), and thus may not represent canonical SGs or PBs. Our results revealed that the VSV-induced granules, called SG-like structures here, contain the viral replicative proteins and RNAs. The formation and maintenance of the SG-like structures required viral replication and ongoing protein synthesis, but an intact cytoskeletal network was not necessary. These results suggest that cells respond to VSV infection by aggregating the antiviral proteins, such as PCBP2 and TIA1, to form SG-like structures. The functional significance of these SG-like structures in VSV-infected cells is currently under investigation.

  17. Selective nucleotide-release from dense-core granules in insulin-secreting cells.

    PubMed

    Obermüller, Stefanie; Lindqvist, Anders; Karanauskaite, Jovita; Galvanovskis, Juris; Rorsman, Patrik; Barg, Sebastian

    2005-09-15

    Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by approximately 0.1 and approximately 2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.

  18. Activation of basophil and mast cell histamine release by eosinophil granule major basic protein

    PubMed Central

    1983-01-01

    Major basic protein (MBP) is a primary constituent of eosinophil granules. In this report, we demonstrate that MBP from human eosinophil granules initiates a nonlytic histamine release from human leukocytes. A direct effect of MBP on basophils was confirmed using purified human basophils. The kinetics of release were similar to those reported for poly-L-arginine, although MBP was less potent than poly-L-arginine of similar molecular weight. Reduction and alkylation of MBP diminished both the potency and efficacy of the molecule. Native MBP also stimulated histamine secretion from purified rat peritoneal mast cells in a manner characteristic of other polycations. These results emphasize the bidirectional nature of the basophil/mast cell-eosinophil regulatory axis. PMID:6854212

  19. Dentate granule cell modulation in freely moving rats: vigilance state effects.

    PubMed

    Bronzino, J D; Blaise, J H; Mokler, D J; Morgane, P J

    1999-04-12

    Dentate granule cell population responses to paired-pulse stimulation applied to the perforant pathway across a range of interpulse intervals (IPIs) were examined during different vigilance states-quiet waking (QW), slow-wave sleep (SWS), and rapid-eye movement (REM) sleep-in freely moving rats at 15, 30 and 90 days of age. Using these evoked field potentials, the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability, was computed and shown to be altered as a function of age. Animals, 15 days old, showed significantly lower levels of early inhibition (20-40 ms IPIs), i.e., greater PPI values, during all three vigilance states when compared to both the 30- and 90-day old animals. Adult, i.e, 90-day old animals, on the other hand, showed significantly greater levels of late inhibition (300-1000 ms IPIs), i.e., lower PPI values, than the younger animals (15- and 30-day old) during QW and SWS. These results indicate that as the dentate field of the hippocampal formation matures there are significant alterations in the modulation of dentate granule cell activity.

  20. 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.

  1. Studies of dentate granule cell modulation: paired-pulse responses in freely moving rats at three ages.

    PubMed

    Bronzino, J D; Blaise, J H; Austin-LaFrance, R J; Morgane, P J

    1996-10-23

    Dentate granule cell population responses to paired-pulse stimulations applied to the perforant pathway across a range of interpulse intervals (IPI) were examined in freely moving rats at 15, 30, and 90 days of age. The profile of the paired-pulse index (PPI), a measure of the type and degree of modulation of dentate granule cell excitability, was shown to change significantly as a function of age.

  2. Effects of prenatal protein malnutrition on kindling-induced alterations in dentate granule cell excitability. II. Paired-pulse measures.

    PubMed

    Bronzino, J D; Austin-LaFrance, R J; Morgane, P J; Galler, J R

    1991-05-01

    The effects of prenatal protein malnutrition on kindling-induced changes in inhibitory modulation of dentate granule cell activity were examined by analysis of extracellular field potentials recorded from the granule cell layer of the dentate gyrus in response to paired-pulse stimulation of the perforant pathway in freely-moving rats. Since we have shown that kindling results in enhanced synaptic transmission at the level of the perforant path/granule cell synapse (see preceding paper), we sought to determine if the kindling process might induce changes in inhibitory modulation of granule cell excitability which could be involved in the slower acquisition of the kindled state we have previously reported in malnourished animals. Beginning at 120-150 days of age, the response of dentate granule cells to paired-pulse stimulation of the perforant path was examined at interpulse intervals (IPIs) ranging from 20-1000 ms. A paired-pulse index (PPI) was constructed based on the mean percent change in population spike amplitudes of the two responses resulting from application of the pulse pair. PPI measures obtained during the kindling process were compared with individual prekindling measures to determine the mean percent change in excitatory/inhibitory modulation of granule cell activity. Significant inhibition of the second population response was apparent at all IPIs tested for both diet groups following the first kindled afterdischarge.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. 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.

  4. Use of granzyme B-based fluorescent protein reporters to monitor granzyme distribution and granule integrity in live cells.

    PubMed

    Bird, Catherina H; Rizzitelli, Alexandra; Harper, Ian; Prescott, Mark; Bird, Phillip I

    2010-08-01

    Reporter proteins comprising granzyme B (GrB) fused to eGFP, ecliptic pHluorin or mCherry, were generated and used to study granule (lysosome) distribution and properties in COS-1 cells and natural killer cells. The reporters resembled native GrB in biosynthesis and localization, and accumulated in granules. In live cells both the eGFP and pHluorin reporters were dark in lysosomes, but fluoresced when the granule integrity or pH was perturbed by Leu-Leu methyl ester, hydrogen peroxide, naphthazarin, or sphingosine treatment. By contrast, fluorescence of the mCherry reporter was not pH-dependent. The quenching of eGFP within granules indicates that this commonly-used fluorescent protein is not appropriate as a vital intra-lysosomal marker.

  5. 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.

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

    PubMed

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

    2016-07-19

    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 (p75(NTR)) 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 p75(NTR) 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 p75(NTR), GCPs continue to proliferate beyond their normal period, resulting in a larger cerebellum that persists into adulthood, with consequent motor deficits.

  7. 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.

  8. Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the 'irritable mossy cell' hypothesis.

    PubMed

    Santhakumar, V; Bender, R; Frotscher, M; Ross, S T; Hollrigel, G S; Toth, Z; Soltesz, I

    2000-04-01

    1. Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. 2. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. 3. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. 4. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. 5. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. 6. In addition, the late EPSCs were not present in low (0.5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. 7. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. 8. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus.

  9. 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

  10. Excitability changes within transverse lamellae of dentate granule cells and their longitudinal spread following orthodromic or antidromic activation.

    PubMed

    Lømo, Terje

    2009-07-01

    The functional organization of the perforant path input to the dentate gyrus of the exposed hippocampus was studied in adult rabbits anesthetized with urethane and chloralose. Electrical stimulation of perforant path fibers caused excitation of granule cells along narrow, nearly transverse strips (lamellae) of tissue. Stimulation of granule cell axons (mossy fibers) in CA3 caused antidromic activation of granule cells along similar strips. Paired-pulse stimulation revealed marked changes in granule cell excitability both within a lamella (on-line) and for several mm off-line along the septo-temporal axis of the dentate gyrus. After the first pulse, granule cells were inhibited for up to about 100 ms and then facilitated for up to hundreds of ms. Feedback activity along mossy fiber collaterals exciting local inhibitory and excitatory neurons appeared to dominate in producing on- and off-line inhibition and facilitation. Neurons mediating these effects could be inhibitory basket cells and other inhibitory interneurons targeting granule cells on- and off-line. In addition, excitatory mossy cells with far reaching, longitudinally running axons could affect off-line granule cells by exciting them directly or inhibit them indirectly by exciting local inhibitory interneurons. A scheme for dentate gyrus function is proposed whereby information to the dentate gyrus becomes split into interacting transverse strips of neuronal assemblies along which temporal processing occurs. A matrix of neuronal assemblies thus arises within which fragments of events and experiences is stored through the plasticity of synapses within and between the assemblies. Similar fragments may then be recognized at later times allowing memories of the whole to be created by pattern completion at subsequent computational stages in the hippocampus.

  11. Transient fusion ensures granule replenishment to enable repeated release after IgE-mediated mast cell degranulation.

    PubMed

    Balseiro-Gomez, Santiago; Flores, Juan A; Acosta, Jorge; Ramirez-Ponce, M Pilar; Ales, Eva

    2016-11-01

    To ensure normal immune function, mast cells employ different pathways to release mediators. Here, we report a thus far unknown capacity of mast cells to recycle and reuse secretory granules after an antigen-evoked degranulation process under physiological conditions; this phenomenon involves the existence of a recycling secretory granule pool that is available for release in a short time scale. Rapid endocytic modes contributed to the recycling of ∼60% of the total secretory granule population, which involved kiss-and-run and cavicapture mechanisms, causing retention of the intragranular matrix. We found the presence of normal-size granules and giant actomyosin- and dynamin-dependent granules, which were characterized by large quantal content. These large structures allowed the recovered mast cells to release a large amount of 5-HT, compensating for the decrease in the number of exocytosed secretory granules. This work uncovers a new physiological role of the exo-endocytosis cycle in the immunological plasticity of mast cells and reveals a new property of their biological secretion.

  12. Mefloquine, an anti-malaria agent, causes reactive oxygen species-dependent cell death in mast cells via a secretory granule-mediated pathway

    PubMed Central

    Paivandy, Aida; Calounova, Gabriela; Zarnegar, Behdad; Öhrvik, Helena; Melo, Fabio R; Pejler, Gunnar

    2014-01-01

    Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells. PMID:25505612

  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

    2016-04-07

    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.

  14. 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

  15. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

    NASA Astrophysics Data System (ADS)

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-02-01

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

  16. 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

  17. Serine proteinases of mast cell and leukocyte granules. A league of their own.

    PubMed

    Caughey, G H

    1994-12-01

    Serine proteinases are hydrolases that use serine's side chain hydroxyl group to attack and cleave internal peptide bonds in peptides and proteins. They reside in all mammalian tissues, including the lung and airway. As a group, they vary tremendously in form and target specificity and have a vast repertoire of functions, many of which are critical for life. A subset of these proteinases is expressed primarily in the cytosolic granules of leukocytes from bone marrow, including mast cells. Examples are elastase-related proteinases and cathepsin G of monocytes and neutrophils, the many "granzymes" of cytotoxic T lymphocytes and natural killer (NK) cells, and the tryptases and chymases of mast cells. The pace of discovery and characterization of these granule-associated serine proteinases, fueled by technical advances in molecular biology, has accelerated rapidly in the past few years. Progress has been made in assigning possible functions to individual proteinases. However, the burgeoning numbers of these enzymes; their cell, tissue and species-dependent differences in expression; and their variety of action in vitro (despite, in many cases, shared modes of activation and recent divergence in protein evolution) have vexed and challenged those of us who are anxious to establish their roles in mammalian biology. Certainly, much remains to be discovered and clarified. The purpose of this overview is to capture the state of the art in this field, stressing the similarities as well as the differences among individual granule-associated proteinases and focusing particularly on those enzymes likely to be important in the human lung and airways.

  18. 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.

  19. Glucose Toxic Effects on Granulation Tissue Productive Cells: The Diabetics' Impaired Healing

    PubMed Central

    Berlanga-Acosta, Jorge; Schultz, Gregory S.; López-Mola, Ernesto; Guillen-Nieto, Gerardo; García-Siverio, Marianela; Herrera-Martínez, Luis

    2013-01-01

    Type 2 diabetes mellitus is a metabolic noncommunicable disease with an expanding pandemic magnitude. Diabetes predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Diabetes also dismantles innate immunity favoring wound infection. Amputation is therefore acknowledged as one of the disease's complications. Hyperglycemia is the proximal detonator of systemic and local toxic effectors including proinflammation, acute-phase proteins elevation, and spillover of reactive oxygen and nitrogen species. Insulin axis deficiency weakens wounds' anabolism and predisposes to inflammation. The systemic accumulation of advanced glycation end-products irreversibly impairs the entire physiology from cells-to-organs. These factors in concert hamper fibroblasts and endothelial cells proliferation, migration, homing, secretion, and organization of a productive granulation tissue. Diabetic wound bed may turn chronically inflammed, procatabolic, and an additional source of circulating pro-inflammatory cytokines, establishing a self-perpetuating loop. Diabetic fibroblasts and endothelial cells may bear mitochondrial damages becoming prone to apoptosis, which impairs granulation tissue cellularity and perfusion. Endothelial progenitor cells recruitment and tubulogenesis are also impaired. Failure of wound reepithelialization remains a clinical challenge while it appears to be biologically multifactorial. Ulcer prevention by primary care surveillance, education, and attention programs is of outmost importance to reduce worldwide amputation figures. PMID:23484099

  20. Increased expression of BDNF and proliferation of dentate granule cells after bacterial meningitis.

    PubMed

    Tauber, Simone C; Stadelmann, Christine; Spreer, Annette; Brück, Wolfgang; Nau, Roland; Gerber, Joachim

    2005-09-01

    Proliferation and differentiation of neural progenitor cells is increased after bacterial meningitis. To identify endogenous factors involved in neurogenesis, expression of brain-derived neurotrophic factor (BDNF), TrkB, nerve growth factor (NGF), and glial cell line-derived neurotrophic factor (GDNF) was investigated. C57BL/6 mice were infected by intracerebral injection of Streptococcus pneumoniae. Mice were killed 30 hours later or treated with ceftriaxone and killed 4 days after infection. Hippocampal BDNF mRNA levels were increased 2.4-fold 4 days after infection (p = 0.026). Similarly, BDNF protein levels in the hippocampal formation were higher in infected mice than in control animals (p = 0.0003). This was accompanied by an elevated proliferation of dentate granule cells (p = 0.0002). BDNF protein was located predominantly in the hippocampal CA3/4 area and the hilus of the dentate gyrus. The density of dentate granule cells expressing the BDNF receptor TrkB as well as mRNA levels of TrkB in the hippocampal formation were increased 4 days after infection (p = 0.027 and 0.0048, respectively). Conversely, NGF mRNA levels at 30 hours after infection were reduced by approximately 50% (p = 0.004). No significant changes in GDNF expression were observed. In conclusion, increased synthesis of BDNF and TrkB suggests a contribution of this neurotrophic factor to neurogenesis after bacterial meningitis.

  1. 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.

  2. Organization of spinocerebellar projection map in three types of agranular cerebellum: Purkinje cells vs. granule cells as organizer element

    SciTech Connect

    Arsenio Nunes, M.L.; Sotelo, C.; Wehrle, R.

    1988-07-01

    The organization of the spinocerebellar projection was analysed by the anterograde axonal WGA-HRP (horseradish peroxidase-wheat germ agglutinin conjugate) tracing method in three different types of agranular cerebellar cortex either induced experimentally by X-irradiation or occurring spontaneously in weaver (wv/wv) and staggerer (sg/sg) mutant mice. The results of this study show that in the X-irradiated rat and weaver mouse, in both of which the granule cells are directly affected and die early in development, the spinal axons reproduce, with few differences, the normal spinocerebellar pattern. Conversely, in staggerer mouse, in which the Purkinje cells are intrinsically affected and granule neurons do not seem to be primarily perturbed by the staggerer gene action, the spinocerebellar organization is severely modified. These findings appear somewhat paradoxical because if granule cells, the synaptic targets of mossy spinocerebellar fibers, were necessary for the organization of spinocerebellar projection, the staggerer cerebellum would exhibit a much more normal projectional map than the weaver and the X-irradiated cerebella. It is, therefore, obvious that granule cells, and even specific synaptogenesis, are not essential for the establishment of the normal spinocerebellar topography. On the other hand, the fact that the Purkinje cells are primarily affected in the unique agranular cortex in which the spinocerebellar organization is severely modified suggests that these neurons could be the main element in the organization of the spinocerebellar projection map. This hypothesis is discussed in correlation with already-reported findings on the zonation of the cerebellar cortex by biochemically different clusters of Purkinje cells.

  3. Altered patterning of dentate granule cell mossy fiber inputs onto CA3 pyramidal cells in limbic epilepsy

    PubMed Central

    McAuliffe, John J.; Bronson, Stefanie L.; Hester, Michael S.; Murphy, Brian L.; Dahlquist-Topalá, Renée; Richards, David A.; Danzer, Steve C.

    2009-01-01

    Impaired gating by hippocampal dentate granule cells may promote the development of limbic epilepsy by facilitating seizure spread through the hippocampal trisynaptic circuit. The second synapse in this circuit, the dentate granule cell≫CA3 pyramidal cell connection, may be of particular importance because pathological changes occurring within the dentate likely exert their principal effect on downstream CA3 pyramids. Here, we utilized GFP-expressing mice and immunolabeling for the zinc transporter ZnT-3 to reveal the pre- and postsynaptic components of granule cell≫CA3 pyramidal cell synapses following pilocarpine-epileptogenesis. Confocal analyses of these terminals revealed that while granule cell presynaptic giant boutons increased in size and complexity one month after status epilepticus, individual thorns making up the postsynaptic thorny excrescences of the CA3 pyramidal cells were reduced in number. This reduction, however, was transient, and three months after status, thorn density recovered. This recovery was accompanied by a significant change in the distribution of thorns along pyramidal cells dendrites. While thorns in control animals tended to be tightly clustered, thorns in epileptic animals were more evenly distributed. Computational modeling of thorn distributions predicted an increase in the number of boutons required to cover equivalent numbers of thorns in epileptic vs. control mice. Confirming this prediction, ZnT-3 labeling of presynaptic giant boutons apposed to GFP-expressing thorns revealed a near doubling in bouton density, while the number of individual thorns per bouton was reduced by half. Together, these data provide clear evidence of novel plastic changes occurring within the epileptic hippocampus. PMID:20014385

  4. High-pressure potato starch granule gelatinization: synchrotron radiation micro-SAXS/WAXS using a diamond anvil cell.

    PubMed

    Gebhardt, R; Hanfland, M; Mezouar, M; Riekel, C

    2007-07-01

    Potato starch granules have been examined by synchrotron radiation small- and wide-angle scattering in a diamond anvil cell (DAC) up to 750 MPa. Use of a 1 microm synchrotron radiation beam allowed the mapping of individual granules at several pressure levels. The data collected at 183 MPa show an increase in the a axis and lamellar period from the edge to the center of the granule, probably due to a gradient in water content of the crystalline and amorphous lamellae. The average granules radius increases up to the onset of gelatinization at about 500 MPa, but the a axis and the lamellar periodicity remain constant or even show a decrease, suggesting an initial hydration of amorphous growth rings. The onset of gelatinization is accompanied by (i) an increase in the average a axis and lamellar periodicity, (ii) the appearance of an equatorial SAXS streak, and (iii) additional short-range order peaks.

  5. The paired-pulse index: a measure of hippocampal dentate granule cell modulation.

    PubMed

    Bronzino, J D; Blaise, J H; Morgane, P J

    1997-01-01

    This study was undertaken to assess whether the paired-pulse index (PPI) is an effective measure of the modulation of dentate granule cell excitability during normal development. Paired-pulse stimulations of the perforant path were, therefore, used to construct a PPI for 15-, 30-, and 90-day old, freely moving male rats. Significant age-dependent differences in the PPI were obtained. Fifteen-day old rats showed significantly less inhibition at short interpulse intervals [interpulse interval (IPI): 20 to 30 msec), a lack of facilitation at intermediate IPIs (50 to 150 msec), and significantly less inhibition at longer IPIs (300 to 1,000 msec) than adults.

  6. 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.

  7. MMP-13 Regulates Growth of Wound Granulation Tissue and Modulates Gene Expression Signatures Involved in Inflammation, Proteolysis, and Cell Viability

    PubMed Central

    Toriseva, Mervi; Laato, Matti; Carpén, Olli; Ruohonen, Suvi T.; Savontaus, Eriika; Inada, Masaki; Krane, Stephen M.; Kähäri, Veli-Matti

    2012-01-01

    Proteinases play a pivotal role in wound healing by regulating cell-matrix interactions and availability of bioactive molecules. The role of matrix metalloproteinase-13 (MMP-13) in granulation tissue growth was studied in subcutaneously implanted viscose cellulose sponge in MMP-13 knockout (Mmp13−/−) and wild type (WT) mice. The tissue samples were harvested at time points day 7, 14 and 21 and subjected to histological analysis and gene expression profiling. Granulation tissue growth was significantly reduced (42%) at day 21 in Mmp13−/− mice. Granulation tissue in Mmp13−/− mice showed delayed organization of myofibroblasts, increased microvascular density at day 14, and virtual absence of large vessels at day 21. Gene expression profiling identified differentially expressed genes in Mmp13−/− mouse granulation tissue involved in biological functions including inflammatory response, angiogenesis, cellular movement, cellular growth and proliferation and proteolysis. Among genes linked to angiogenesis, Adamts4 and Npy were significantly upregulated in early granulation tissue in Mmp13−/− mice, and a set of genes involved in leukocyte motility including Il6 were systematically downregulated at day 14. The expression of Pdgfd was downregulated in Mmp13−/− granulation tissue in all time points. The expression of matrix metalloproteinases Mmp2, Mmp3, Mmp9 was also significantly downregulated in granulation tissue of Mmp13−/− mice compared to WT mice. Mmp13−/− mouse skin fibroblasts displayed altered cell morphology and impaired ability to contract collagen gel and decreased production of MMP-2. These results provide evidence for an important role for MMP-13 in wound healing by coordinating cellular activities important in the growth and maturation of granulation tissue, including myofibroblast function, inflammation, angiogenesis, and proteolysis. PMID:22880047

  8. Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

    PubMed

    Lavado, Alfonso; Lagutin, Oleg V; Chow, Lionel M L; Baker, Suzanne J; Oliver, Guillermo

    2010-08-17

    The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.

  9. Dense-core granules in neuroendocrine cells and neurons release their secretory constituents by piecemeal degranulation (review).

    PubMed

    Crivellato, Enrico; Nico, Beatrice; Bertelli, Eugenio; Nussdorfer, Gastone G; Ribatti, Domenico

    2006-12-01

    The term piecemeal degranulation (PMD) refers to a slow releasing process mediated by vesicular transport of stored secretory granule contents. This form of cell secretion was first proposed for basophils, mast cells and eosinophils, but evidence has begun to accumulate that PMD also occurs in dense-core granules of neuroendocrine cells and neurons. This review summarizes the electron-microscopic evidence that has been gathered in support of this view and also discusses the possible physiological significance of PMD in this class of secretory organelles in comparison with 'full fusion' and 'kiss-and-run' exocytosis.

  10. Increased excitatory synaptic input to granule cells from hilar and CA3 regions in a rat model of temporal lobe epilepsy

    PubMed Central

    Zhang, Wei; Huguenard, John R.; Buckmaster, Paul S.

    2012-01-01

    One potential mechanism of temporal lobe epilepsy is recurrent excitation of dentate granule cells through aberrant sprouting of their axons (mossy fibers), which is found in many patients and animal models. However, correlations between the extent of mossy fiber sprouting and seizure frequency are weak. Additional potential sources of granule cell recurrent excitation that would not have been detected by markers of mossy fiber sprouting in previous studies include surviving mossy cells and proximal CA3 pyramidal cells. To test those possibilities in hippocampal slices from epileptic pilocarpine-treated rats, laser scanning glutamate uncaging was used to randomly and focally activate neurons in the granule cell layer, hilus, and proximal CA3 pyramidal cell layer while measuring evoked excitatory postsynaptic currents (EPSCs) in normotopic granule cells. Consistent with mossy fiber sprouting, a higher proportion of glutamate-uncaging spots in the granule cell layer evoked EPSCs in epileptic rats compared to controls. In addition, stimulation spots in the hilus and proximal CA3 pyramidal cell layer were more likely to evoke EPSCs in epileptic rats, despite significant neuron loss in those regions. Furthermore, synaptic strength of recurrent excitatory inputs to granule cells from CA3 pyramidal cells and other granule cells was increased in epileptic rats. These findings reveal substantial levels of excessive, recurrent, excitatory synaptic input to granule cells from neurons in the hilus and proximal CA3 field. The aberrant development of these additional positive-feedback circuits might contribute to epileptogenesis in temporal lobe epilepsy. PMID:22279204

  11. Ontogeny of calbindin immunoreactivity in the human hippocampal formation with a special emphasis on granule cells of the dentate gyrus.

    PubMed

    Abrahám, Hajnalka; Veszprémi, Béla; Kravják, András; Kovács, Krisztina; Gömöri, Eva; Seress, László

    2009-04-01

    Calbindin (CB) is a calcium-binding protein that is present in principal cells as well as in interneurons of the hippocampal formation of various species including humans. Studies with transgenic mice revealed that CB is essential for long-term potentiation and synaptic plasticity which are the cellular basis of learning and memory. In a previous study we have shown that CB expression in granule cells of the dentate gyrus correlates with the functional maturation of the hippocampal formation in the rat. In the present study we examined the ontogeny of CB using immunohistochemistry in the human hippocampal formation paying special attention to the granule cells of the dentate gyrus. As early as the 14(th) week of gestation (GW), CB was being expressed by pyramidal cells of CA1-3 regions in the deepest cell rows of the pyramidal layer towards the ventricular zone. Later, CB sequentially appears in more superficial cell rows. After midgestation, CB disappears from CA3 pyramidal neurons. Expression of CB by granule cells starts at the 22(nd)-23(rd) GW, first by the most superficial neurons of the ectal end of the dorsal blade. At the 24(th) GW, CB is expressed by granule cells of the crest and medial portion of the ventral blade whereas later the entire ventral blade revealed CB immunoreactivity. At term, and in the first few postnatal months, CB-immunoreaction is detected in granule cells of both blades except for those neurons in the deepest cell rows at the hilar border. At around 2-3 years of age, all granule cells of the entire cell layer are CB-immunoreactive. Axons of granule cells, the mossy fibers, start to express CB around the 30(th) GW in stratum lucidum of CA3a. With further development, CB is expressed in CA3b and c, as well as in the hilus. An adult-like pattern of CB-immunoreactivity could be observed at 11 years of age. Our results indicate that (i) CB is expressed by hippocampal pyramidal cells a few weeks before midgestation; (ii) similarly to

  12. Accumulation of Major Histocompatibility Complex Class II Molecules in Mast Cell Secretory Granules and Their Release upon Degranulation

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Mecheri, Salahedine; Peronet, Roger; Bonnerot, Christian; Desaymard, Catherine

    1997-01-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60–80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles. PMID:9398681

  13. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation.

    PubMed

    Raposo, G; Tenza, D; Mecheri, S; Peronet, R; Bonnerot, C; Desaymard, C

    1997-12-01

    To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

  14. Targeted deletion of AKAP7 in dentate granule cells impairs spatial discrimination

    PubMed Central

    Weisenhaus, Michael; Sanford, Christina A; Slack, Margaret C; Chin, Jenesa; Nachmanson, Daniela; McKennon, Alex; Castillo, Pablo E; McKnight, G Stanley

    2016-01-01

    Protein Kinase A (PKA) mediates synaptic plasticity and is widely implicated in learning and memory. The hippocampal dentate gyrus (DG) is thought to be responsible for processing and encoding distinct contextual associations in response to highly similar inputs. The mossy fiber (MF) axons of the dentate granule cells convey strong excitatory drive to CA3 pyramidal neurons and express presynaptic, PKA-dependent forms of plasticity. Here, we demonstrate an essential role for the PKA anchoring protein, AKAP7, in mouse MF axons and terminals. Genetic ablation of AKAP7 specifically from dentate granule cells results in disruption of MF-CA3 LTP directly initiated by cAMP, and the AKAP7 mutant mice are selectively deficient in pattern separation behaviors. Our results suggest that the AKAP7/PKA complex in the MF projections plays an essential role in synaptic plasticity and contextual memory formation. DOI: http://dx.doi.org/10.7554/eLife.20695.001 PMID:27911261

  15. Interleukin-1 mediates long-term hippocampal dentate granule cell loss following postnatal viral infection.

    PubMed

    Orr, Anna G; Sharma, Anup; Binder, Nikolaus B; Miller, Andrew H; Pearce, Bradley D

    2010-05-01

    Viral infections of the developing CNS can cause long-term neuropathological sequela through undefined mechanisms. Proinflammatory cytokines such as IL-1beta have gained attention in mediating neurodegeneration in corticohippocampal structures due to a variety of insults in adults, though there is less information on the developing brain. Little is known concerning the spatial-temporal pattern of IL-1beta induction in the developing hippocampus following live virus infection, and there are few studies addressing the long-term consequences of this cytokine induction. We report that infection of rats with lymphocytic choriomeningitis virus on postnatal day 4 induces IL-1beta protein in select regions of the hippocampus on 6, 15, 21, and 45 days after infection. This infection resulted in a 71% reduction of dentate granule cell neurons by the time the rats reached mid-adulthood. We further investigated the causative role of IL-1 in this dentate granule cell loss by blocking IL-1 activity using an IL-1ra-expressing adenoviral vector administered at the time of infection. Blockade of IL-1 abrogated the infection-associated neuron loss in this vivo model. Considering that IL-1 can be triggered by multiple perinatal insults, our findings suggest that early therapy with anti-inflammatory agents that block IL-1 may be effective for reducing adulthood neuropathology.

  16. Antimicrobial peptide gramicidin S is accumulated in granules of producer cells for storage of bacterial phosphagens

    PubMed Central

    Berditsch, Marina; Trapp, Mareike; Afonin, Sergii; Weber, Christian; Misiewicz, Julia; Turkson, Joana; Ulrich, Anne S.

    2017-01-01

    Many antimicrobial peptides are synthesized non-ribosomally in bacteria, but little is known about their subcellular route of biosynthesis, their mode of intracellular accumulation, or their role in the physiology of the producer cells. Here, we present a comprehensive view on the biosynthesis of gramicidin S (GS) in Aneurinibacillus migulanus, having observed a peripheral membrane localization of its synthetases. The peptide gets accumulated in nano-globules, which mature by fusion into larger granules and end up within vacuolar structures. These granules serve as energy storage devices, as they contain GS molecules that are non-covalently attached to alkyl phosphates and protect them from dephosphorylation and premature release of energy. This finding of a fundamentally new type of high-energy phosphate storage mechanism can explain the curious role of GS biosynthesis in the physiology of the bacterial producer cells. The unknown role of the GrsT protein, which is part of the non-ribosomal GS synthetase operon, can thus be assumed to be responsible for the biosynthesis of alkyl phosphates. GS binding to alkyl phosphates may suggest its general affinity to phosphagens such as ATP and GTP, which can represent the important intracellular targets in pathogenic bacteria. PMID:28295017

  17. 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.

  18. The immunomodulatory properties of periodontal ligament stem cells isolated from inflamed periodontal granulation.

    PubMed

    Li, Chenghua; Wang, Xinwen; Tan, Jun; Wang, Tao; Wang, Qintao

    2014-01-01

    Periodontitis is currently the main cause of tooth loss and as yet there is no appropriate method for establishing a functional and predictable periodontal regeneration. Tissue engineering involving seed cells provides a new prospect for periodontal regeneration. While periodontal ligament stem cells (PDLSCs) are a good choice for seed cells, it is not always possible to obtain the patients' own PDLSCs. We and others have found a type of stromal cells from inflamed periodontal granulation. These cells displayed similar differentiation properties to PDLSCs. Inflammation has a profound influence on the immunomodulatory properties of mesenchymal stem cells, which may affect therapeutic outcome. In this study, we assessed the immunomodulatory characteristics of these inflamed human (ih)PDLSCs. Along with the similarity in cell surface marker expressions, they also displayed immunomodulatory properties comparable to those in healthy human (hh)PDLSCs. Both hhPDLSCs and ihPDLSCs can suppress the proliferation and secretion of IFN-γ in peripheral blood mononuclear cells by indirect soluble mediators and direct cell-cell contact. Albeit with some quantitative variances, the gene expressions of inducible nitric oxide synthases, indoleamine 2,3 dioxygenase, cyclooxygenase-2, TNF-α-induced protein 6 and IL-10 in ihPDLSCs displayed similar patterns as those in hhPDLSCs. Taken together, our results suggest that ihPDLSCs can provide a promising alternative to hhPDLSCs in terms of evident similarities in immunomodulatory properties as well as their easier accessibility and availability.

  19. The chemokine growth-related gene product β protects rat cerebellar granule cells from apoptotic cell death through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors

    PubMed Central

    Limatola, Cristina; Ciotti, Maria Teresa; Mercanti, Delio; Vacca, Fabrizio; Ragozzino, Davide; Giovannelli, Aldo; Santoni, Angela; Eusebi, Fabrizio; Miledi, Ricardo

    2000-01-01

    Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K+. We have found that the growth-related gene product β (GROβ) partially prevents the K+-depletion-induced cell death, and that the neuroprotective action of GROβ on granule cells is mediated through the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GROβ-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-d-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 μM) treatment. Moreover, GROβ-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GROβ is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors. PMID:10811878

  20. Remodelling of cortical actin where lytic granules dock at natural killer cell immune synapses revealed by super-resolution microscopy.

    PubMed

    Brown, Alice C N; Oddos, Stephane; Dobbie, Ian M; Alakoskela, Juha-Matti; Parton, Richard M; Eissmann, Philipp; Neil, Mark A A; Dunsby, Christopher; French, Paul M W; Davis, Ilan; Davis, Daniel M

    2011-09-01

    Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ~100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology.

  1. Developmental changes in membrane properties and postsynaptic currents of granule cells in rat dentate gyrus.

    PubMed

    Liu, Y B; Lio, P A; Pasternak, J F; Trommer, B L

    1996-08-01

    1. Whole cell patch-clamp recordings were used to study dentate gyrus granule cells in hippocampal slices from juvenile rats (postnatal days 8-32). Membrane properties were measured with the use of current-clamp recordings and were correlated with the morphology of a subgroup of neurons filled with biocytin. The components of the postsynaptic currents (PSCs) induced by medial perforant path stimulation were characterized with the use of specific receptor antagonists in voltage-clamp recordings. 2. Granule cells located in the middle third of the superior blade of stratum granulosum from the rostral third of hippocampus were divided into three groups according to their input resistance (IR). Neurons with low IR (206 +/- 182 M omega, mean +/- SD) had hyperpolarized resting membrane potentials (-82 +/- 7 mV) and high-amplitude action potentials (108 +/- 23 mV). Neurons were high IR (1,259 +/- 204 M omega) had more depolarized resting membrane potentials (-54 +/- 6 mV) and lower-amplitude action potentials (71 +/- 10 mV). Neurons with intermediate IR (619 +/- 166 M omega) also had intermediate resting membrane potentials (-63 +/- 7 mV) and action potential amplitudes (86 +/- 14 mV). Low-IR neurons became increasingly prevalent with advancing postnatal age, but neurons from each group could be found throughout the entire period under study. 3. Morphological studies of low-IR neurons revealed an extensive dendritic arborization that traversed the entire molecular layer and was characteristic of mature granule cells. High-IR cells had smaller somata and short, simple dendritic arborization that incompletely penetrated the molecular layer and were classified as immature. Intermediate-IR cells had morphological features of intermediate maturity. 4. The initial phase of the PSC evoked at -80 mV was a fast inward current that was comparable with respect to latency to peak, latency to onset, and 10-90% rise time in neurons of all maturities held at -80 mV. This current was 6

  2. The role of calcium and cyclic nucleotide signaling in cerebellar granule cell migration under normal and pathological conditions.

    PubMed

    Komuro, Yutaro; Galas, Ludovic; Lebon, Alexis; Raoult, Emilie; Fahrion, Jennifer K; Tilot, Amanda; Kumada, Tasturo; Ohno, Nobuhiko; Vaudry, David; Komuro, Hitoshi

    2015-04-01

    In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca(2+) and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca(2+) and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca(2+) and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease.

  3. Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

    PubMed

    Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

    2016-09-15

    CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process.

  4. 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.

  5. Dissecting the molecular mechanisms that impair stress granule formation in aging cells.

    PubMed

    Moujaber, Ossama; Mahboubi, Hicham; Kodiha, Mohamed; Bouttier, Manuella; Bednarz, Klaudia; Bakshi, Ragini; White, John; Larose, Louise; Colmegna, Inés; Stochaj, Ursula

    2017-03-01

    Aging affects numerous aspects of cell biology, but the senescence-associated changes in the stress response are only beginning to emerge. To obtain mechanistic insights into these events, we examined the formation of canonical and non-canonical stress granules (SGs) in the cytoplasm. SG generation is a key event after exposure to physiological or environmental stressors. It requires the SG-nucleating proteins G3BP1 and TIA-1/TIAR and stress-related signaling events. To analyze SG formation, we used two independent models of somatic cell aging. In both model systems, cellular senescence impaired the assembly of two SG classes: (i) it compromised the formation of canonical SGs, and (ii) skewed the production of non-canonical SGs. We dissected the mechanisms underlying these senescence-dependent changes in granule biogenesis and identified several specific targets that were modulated by aging. Thus, we demonstrate a depletion of G3BP1 and TIA-1/TIAR in senescent cells and show that the loss of G3BP1 contributed to impaired SG formation. We further reveal that aging reduced Sp1 levels; this transcription factor regulated G3BP1 and TIA-1/TIAR abundance. The assembly of canonical SGs relies on the phosphorylation of translation initiation factor eIF2α. We show that senescence can cause eIF2α hyperphosphorylation. CReP is a subunit of protein phosphatase 1 and critical to reverse the stress-dependent phosphorylation of eIF2α. We demonstrate that the loss of CReP correlated with the aging-related hyperphosphorylation of eIF2α. Together, we have identified significant changes in the stress response of aging cells and provide mechanistic insights. Based on our work, we propose that the decline in SG formation can provide a new biomarker to evaluate cellular aging.

  6. Demonstration of Birbeck (Langerhans cells) granules in the normal chicken epidermis

    PubMed Central

    PÉREZ-TORRES, ARMANDO; USTARROZ-CANO, MARTHA

    2001-01-01

    Mammalian Langerhans cells (LC) are epidermal dendritic cells which originate in bone marrow and migrate toward the T cell area of lymph nodes, where they act as professional antigen-presenting cells. A variety of cell surface markers, such as the ectoenzyme adenosine triphosphatase (ATPase), Ia and CD1a antigens, have been used extensively to identify LC. Ultrastructural identification of this cell type in the mammalian epidermis is made by the demonstration of a typical and unique cytoplasmic organelle, the Birbeck granule (BG). Although we had earlier demonstrated the coexpression of ATPase and Ia antigens on epidermal dendritic cells of the chicken epidermis, the presence of the BG has not previously been documented. The aim of the present study was to investigate whether chicken epidermal LC-like cells possess an organelle similar to the BG, and thus to complete their identification. Our findings are the first demonstration of characteristic rod-shaped, racket-shaped and disc-shaped intracytoplasmic organelles, morphologically similar to the mammalian BG, in avian LC. PMID:11693310

  7. Regional development of Langerhans cells and formation of Birbeck granules in human embryonic and fetal skin.

    PubMed

    Fujita, M; Furukawa, F; Horiguchi, Y; Ueda, M; Kashihara-Sawami, M; Imamura, S

    1991-07-01

    The regional development of Langerhans cells (LC) and the formation of Birbeck granules (BG) were examined in human embryonic and fetal skin. Samples were obtained from multiple anatomic sites and stained with anti-CD36, anti-CD1a, and anti-HLA-DR antibody as well as Lag antibody specifically reactive to BG and some vacuoles of human LC. In the first trimester, CD36+ dendritic epidermal cells were identified before the appearance of CD1a+ cells and Lag+ cells. Some of the former co-expressed HLA-DR antigens but not CD1a antigens. In the second trimester, regional variations in LC development were observed. Epidermal LC of palms and soles reached a peak in number in the first trimester but were rarely detected after 18 weeks estimated gestation age (EGA), whereas, in other regions, their number increased with age. In the second trimester, CD1a+ cells and Lag+ cells were also identified in the epidermis, although Lag+ cells appeared later than CD1a+ cells. The Lag+ cells until 17 weeks EGA showed a variety of staining intensities and immunoelectron microscopy revealed that they contained various amounts of Lag-reactive BG. Flow cytometric analysis showed that relative amounts of Lag antigens in LC increased during the second trimester and that fetal LC of 18 weeks EGA expressed the same amounts of HLA-DR, CD1a, and Lag antigens as did adult human LC. In the dermis, in the second trimester, numerous CD36+ cells and HLA-DR+ cells were found, whereas CD1a+ cells and Lag+ cells were rarely detected. Taken together, it is suggested that HLA-DR+ dendritic cells acquire CD1a+ antigens first and then form BG after migration to the epidermis and that fetal LC are phenotypically mature in the second trimester.

  8. 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

  9. Expression of the AMPA Receptor Subunits GluR1 and GluR2 is Associated with Granule Cell Maturation in the Dentate Gyrus

    PubMed Central

    Hagihara, Hideo; Ohira, Koji; Toyama, Keiko; Miyakawa, Tsuyoshi

    2011-01-01

    The dentate gyrus produces new granule neurons throughout adulthood in mammals from rodents to humans. During granule cell maturation, defined markers are expressed in a highly regulated sequential process, which is necessary for directed neuronal differentiation. In the present study, we show that α-amino-3-hydroxy-5-methy-4-isoxazole propionate (AMPA) receptor subunits GluR1 and GluR2 are expressed in differentiated granule cells, but not in stem cells, in neonatal, and adult dentate gyrus. Using markers for neural progenitors, immature and mature granule cells, we found that GluR1 and GluR2 were expressed mainly in mature cells and in some immature cells. A time-course analysis of 5-bromo-2′-deoxyuridine staining revealed that granule cells express GluR1 around 3 weeks after being generated. In mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II, a putative animal model of schizophrenia and bipolar disorder in which dentate gyrus granule cells fail to mature normally, GluR1 and GluR2 immunoreactivities were substantially downregulated in the dentate gyrus granule cells. In the granule cells of mutant mice, the expression of both presynaptic and postsynaptic markers was decreased, suggesting that GluR1 and GluR2 are also associated with synaptic maturation. Moreover, GluR1 and GluR2 were also expressed in mature granule cells of the neonatal dentate gyrus. Taken together, these findings indicate that GluR1 and GluR2 expression closely correlates with the neuronal maturation state, and that GluR1 and GluR2 are useful markers for mature granule cells in the dentate gyrus. PMID:21927594

  10. Granulator Selection

    SciTech Connect

    Gould, T H; Armantrout, G

    1999-08-02

    Following our detailed review of the granulation reports and additional conversations with process and development personnel, we have reached a consensus position regarding granulator selection. At this time, we recommend going forward with implementation of the tumbling granulator approach (GEMCO) based on our assessment of the tested granulation techniques using the established criteria. The basis for this selection is summarized in the following sections, followed by our recommendations for proceeding with implementation of the tumbling granulation approach. All five granulation technologies produced granulated products that can be made into acceptable sintered pucks. A possible exception is the product from the fluidized bed granulator. This material has been more difficult to press into uniform pucks without subsequent cracking of the puck during the sintering cycle for the pucks in this series of tests. This problem may be an artifact of the conditions of the particular granulation demonstration run involved, but earlier results have also been mixed. All granulators made acceptable granulated feed from the standpoint of transfer and press feeding, though the roller compactor and fluidized bed products were dustier than the rest. There was also differentiation among the granulators in the operational areas of (1) potential for process upset, (2) plant implementation and operational complexity, and (3) maintenance concerns. These considerations will be discussed further in the next section. Note that concerns also exist regarding the extension of the granulation processes to powders containing actinides. Only the method that involves tumbling and moisture addition has been tested with uranium, and in that instance, significant differences were found in the granulation behavior of the powders.

  11. Increased number of cerebellar granule cells and astrocytes in the internal granule layer in sheep following prenatal intra-amniotic injection of lipopolysaccharide.

    PubMed

    Strackx, Eveline; Gantert, Markus; Moers, Veronique; van Kooten, Imke A J; Rieke, Rebecca; Hürter, Hanna; Lemmens, Marijke A M; Steinbusch, Harry W M; Zimmermann, L J I; Vles, Johannes S H; Garnier, Yves; Gavilanes, Antonio W D; Kramer, Boris W

    2012-03-01

    Chorioamnionitis is an important problem in perinatology today, leading to brain injury and neurological handicaps. However, there are almost no data available regarding chorioamnionitis and a specific damage of the cerebellum. Therefore, this study aimed at determining if chorioamnionitis causes cerebellar morphological alterations. Chorioamnionitis was induced in sheep by the intra-amniotic injection of lipopolysaccharide (LPS) at a gestational age (GA) of 110 days. At a GA of 140 days, we assessed the mean total and layer-specific volume and the mean total granule cell (GCs) and Purkinje cell (PC) number in the cerebelli of LPS-exposed and control animals using high-precision design-based stereology. Astrogliosis was assessed in the gray and white matter (WM) using a glial fibrillary acidic protein staining combined with gray value image analysis. The present study showed an unchanged volume of the total cerebellum as well as the molecular layer, outer and inner granular cell layers (OGL and IGL, respectively), and WM. Interestingly, compared with controls, the LPS-exposed brains showed a statistically significant increase (+20.4%) in the mean total number of GCs, whereas the number of PCs did not show any difference between the two groups. In addition, LPS-exposed animals showed signs of astrogliosis specifically affecting the IGL. Intra-amniotic injection of LPS causes morphological changes in the cerebellum of fetal sheep still detectable at full-term birth. In this study, changes were restricted to the inner granule layer. These cerebellar changes might correspond to some of the motor or non-motor deficits seen in neonates from compromised pregnancies.

  12. Stress Granules contribute to α-globin homeostasis in differentiating erythroid cells

    PubMed Central

    Ghisolfi, Laura; Dutt, Shilpee; McConkey, Marie E.; Ebert, Benjamin L.; Anderson, Paul

    2012-01-01

    Hemoglobin is the major biosynthetic product of developing erythroid cells. Assembly of hemoglobin requires the balanced production of globin protein and the oxygen-carrying heme moiety. The heme-regulated inhibitor kinase (HRI) participates in this process by phosphorylating eIF2α and inhibiting the translation of globin protein when levels of free heme are limiting. HRI is also activated in erythroid cells subjected to oxidative stress. Phospho-eIF2α-mediated translational repression induces the assembly of stress granules (SG), cytoplasmic foci that harbor untranslated mRNAs and promote the survival of cells subjected to adverse environmental conditions. We have found that differentiating erythroid, but not myelomonocytic or megakaryocytic, murine and human progenitor cells assemble SGs, in vitro and in vivo. Targeted knockdown of HRI or G3BP, a protein required for SG assembly, inhibits spontaneous and arsenite-induced assembly of SGs in erythroid progenitor cells. This is accompanied by reduced globin production and increased apoptosis suggesting that G3BP+ SGs facilitate the survival of developing erythroid cells. PMID:22452989

  13. Dendrites of dentate gyrus granule cells contribute to pattern separation by controlling sparsity

    PubMed Central

    Chavlis, Spyridon; Petrantonakis, Panagiotis C.

    2016-01-01

    ABSTRACT The hippocampus plays a key role in pattern separation, the process of transforming similar incoming information to highly dissimilar, nonverlapping representations. Sparse firing granule cells (GCs) in the dentate gyrus (DG) have been proposed to undertake this computation, but little is known about which of their properties influence pattern separation. Dendritic atrophy has been reported in diseases associated with pattern separation deficits, suggesting a possible role for dendrites in this phenomenon. To investigate whether and how the dendrites of GCs contribute to pattern separation, we build a simplified, biologically relevant, computational model of the DG. Our model suggests that the presence of GC dendrites is associated with high pattern separation efficiency while their atrophy leads to increased excitability and performance impairments. These impairments can be rescued by restoring GC sparsity to control levels through various manipulations. We predict that dendrites contribute to pattern separation as a mechanism for controlling sparsity. © 2016 The Authors Hippocampus Published by Wiley Periodicals, Inc. PMID:27784124

  14. FHF-independent conduction of action potentials along the leak-resistant cerebellar granule cell axon

    PubMed Central

    Dover, Katarzyna; Marra, Christopher; Solinas, Sergio; Popovic, Marko; Subramaniyam, Sathyaa; Zecevic, Dejan; D'Angelo, Egidio; Goldfarb, Mitchell

    2016-01-01

    Neurons in vertebrate central nervous systems initiate and conduct sodium action potentials in distinct subcellular compartments that differ architecturally and electrically. Here, we report several unanticipated passive and active properties of the cerebellar granule cell's unmyelinated axon. Whereas spike initiation at the axon initial segment relies on sodium channel (Nav)-associated fibroblast growth factor homologous factor (FHF) proteins to delay Nav inactivation, distal axonal Navs show little FHF association or FHF requirement for high-frequency transmission, velocity and waveforms of conducting action potentials. In addition, leak conductance density along the distal axon is estimated as <1% that of somatodendritic membrane. The faster inactivation rate of FHF-free Navs together with very low axonal leak conductance serves to minimize ionic fluxes and energetic demand during repetitive spike conduction and at rest. The absence of FHFs from Navs at nodes of Ranvier in the central nervous system suggests a similar mechanism of current flux minimization along myelinated axons. PMID:27666389

  15. 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

  16. 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…

  17. Palmitoylethanolamide protects dentate gyrus granule cells via peroxisome proliferator-activated receptor-α.

    PubMed

    Koch, Marco; Kreutz, Susanne; Böttger, Charlotte; Benz, Alexander; Maronde, Erik; Ghadban, Chalid; Korf, Horst-Werner; Dehghani, Faramarz

    2011-02-01

    Endocannabinoids like 2-arachidonoylglycerol strongly modulate the complex machinery of secondary neuronal damage and are shown to improve neuronal survival after excitotoxic lesion. Palmitoylethanolamide (PEA), the naturally occurring fatty acid amide of ethanolamine and palmitic acid, is an endogenous lipid known to mimic several effects of endocannabinoids even without binding to cannabinoid receptors. Here we show that PEA (0.001-1 μM) and the synthetic peroxisome proliferator-activated receptor (PPAR)-alpha agonist 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio acetic acid (Wy-14,643; 0.1-1 μM) reduced the number of microglial cells and protected dentate gyrus granule cells in excitotoxically lesioned organotypic hippocampal slice cultures (OHSCs). Treatment with the PPAR-alpha antagonist N-((2S)-2-(((1Z)-1-Methyl-3-oxo-3-(4-(trifluoromethyl)phenyl)prop-1-enyl)amino)-3-(4-(2-(5-methyl-2-phenyl-1,3-oxazol-4-yl)ethoxy)phenyl)propyl)propanamide (GW6471; 0.05-5 μM) blocked PEA-mediated neuroprotection and reduction of microglial cell numbers whereas the PPAR-gamma antagonist 2-chloro-5-nitro-N-phenyl-benzamide (GW9662; 0.01-1 μM) showed no effects. Immunocytochemistry and Western blot analyses revealed a strong PPAR-alpha immunoreaction in BV-2 microglial cells and in HT22 hippocampal cells. Intensity and location of PPAR-alpha immunoreaction remained constant during stimulation with PEA (0.01 μM; 1-36 h). In conclusion our data provide evidence that (1) PEA counteracted excitotoxically induced secondary neuronal damage of dentate gyrus granule cells, (2) PPAR-alpha but not PPAR-gamma is the endogenous binding site for PEA-mediated neuroprotection, and (3) PEA may activate PPAR-alpha in microglial cells and hippocampal neurons to exert its neuroprotective effects. In addition to classical endocannabinoids, PEA-mediated PPAR-alpha activation represents a possible target for therapeutic interventions to mitigate symptoms of secondary neuronal damage.

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

    PubMed

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

    2015-05-01

    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.

  19. 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.

  20. Identification of a Chromogranin A Domain That Mediates Binding to Secretogranin III and Targeting to Secretory Granules in Pituitary Cells and Pancreatic β-Cells

    PubMed Central

    Hosaka, Masahiro; Watanabe, Tsuyoshi; Sakai, Yuko; Uchiyama, Yasuo; Takeuchi, Toshiyuki

    2002-01-01

    Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca2+-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48–111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214–373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells. PMID:12388744

  1. Formation of secretory granules by chromogranins.

    PubMed

    Inomoto, Chie; Osamura, Robert Yoshiyuki

    2009-12-01

    This review article covers the molecular mechanisms of secretory granule formation by chromogranin transfection. Recently, a few investigators have reported that the transfection of chromogranin A and B produces the structures of secretory granules. We used the GFP-chromogranin A transfection method to nonendocrine cells, COS-7 cells, which are not equipped with secretory granules. Despite the absence of endogenous secretory granules in nontransfected COS-7 cells, COS-7 cells transfected with chromogranin A contained granule-like structures in electron micrographs. The granules were composed of an outer limiting membrane with core structures that were interpreted as secretory granules. Human chromogranin A (CgA) labeled with 5-nm gold particles was present in several dense-core granules in our previous electron microscopy study. This review depicts the role of chromogranin A in the formation of secretory granules. It emphasizes the application of recently developed new technologies and the genesis of secretory granules.

  2. Cytoskeletal Dependence of Insulin Granule Movement Dynamics in INS-1 Beta-Cells in Response to Glucose

    PubMed Central

    Heaslip, Aoife T.; Nelson, Shane R.; Lombardo, Andrew T.; Beck Previs, Samantha; Armstrong, Jessica; Warshaw, David M.

    2014-01-01

    For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited. PMID:25310693

  3. FYCO1 and autophagy control the integrity of the haploid male germ cell-specific RNP granules

    PubMed Central

    Da Ros, Matteo; Lehtiniemi, Tiina; Olotu, Opeyemi; Fischer, Daniel; Zhang, Fu-Ping; Vihinen, Helena; Jokitalo, Eija; Sironen, Anu; Toppari, Jorma; Kotaja, Noora

    2017-01-01

    ABSTRACT Ribonucleoprotein (RNP) granules play a major role in compartmentalizing cytoplasmic RNA regulation. Haploid round spermatids that have exceptionally diverse transcriptomes are characterized by a unique germ cell-specific RNP granule, the chromatoid body (CB). The CB shares many characteristics with somatic RNP granules but also has germline-specific features. The CB appears to be a central structure in PIWI-interacting RNA (piRNA)-targeted RNA regulation. Here, we identified a novel CB component, FYCO1, which is involved in the intracellular transport of autophagic vesicles in somatic cells. We demonstrated that the CB is associated with autophagic activity. Induction of autophagy leads to the recruitment of lysosomal vesicles onto the CB in a FYCO1-dependent manner as demonstrated by the analysis of a germ cell-specific Fyco1 conditional knockout mouse model. Furthermore, in the absence of FYCO1, the integrity of the CB was affected and the CB was fragmented. Our results suggest that RNP granule homeostasis is regulated by FYCO1-mediated autophagy. PMID:27929729

  4. 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

  5. Adult-generated hippocampal neurons allow the flexible use of spatially precise learning strategies.

    PubMed

    Garthe, Alexander; Behr, Joachim; Kempermann, Gerd

    2009-01-01

    Despite enormous progress in the past few years the specific contribution of newly born granule cells to the function of the adult hippocampus is still not clear. We hypothesized that in order to solve this question particular attention has to be paid to the specific design, the analysis, and the interpretation of the learning test to be used. We thus designed a behavioral experiment along hypotheses derived from a computational model predicting that new neurons might be particularly relevant for learning conditions, in which novel aspects arise in familiar situations, thus putting high demands on the qualitative aspects of (re-)learning.In the reference memory version of the water maze task suppression of adult neurogenesis with temozolomide (TMZ) caused a highly specific learning deficit. Mice were tested in the hidden platform version of the Morris water maze (6 trials per day for 5 days with a reversal of the platform location on day 4). Testing was done at 4 weeks after the end of four cycles of treatment to minimize the number of potentially recruitable new neurons at the time of testing. The reduction of neurogenesis did not alter longterm potentiation in CA3 and the dentate gyrus but abolished the part of dentate gyrus LTP that is attributed to the new neurons. TMZ did not have any overt side effects at the time of testing, and both treated mice and controls learned to find the hidden platform. Qualitative analysis of search strategies, however, revealed that treated mice did not advance to spatially precise search strategies, in particular when learning a changed goal position (reversal). New neurons in the dentate gyrus thus seem to be necessary for adding flexibility to some hippocampus-dependent qualitative parameters of learning.Our finding that a lack of adult-generated granule cells specifically results in the animal's inability to precisely locate a hidden goal is also in accordance with a specialized role of the dentate gyrus in generating a metric

  6. P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells

    PubMed Central

    1992-01-01

    P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P- selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P- selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules. PMID:1370497

  7. Granule cell hyperexcitability in the early post-traumatic rat dentate gyrus: the ‘irritable mossy cell’ hypothesis

    PubMed Central

    Santhakumar, Vijayalakshmi; Bender, Roland; Frotscher, Michael; Ross, Stephen T; Hollrigel, Greg S; Toth, Zsolt; Soltesz, Ivan

    2000-01-01

    Cytochemical and in vitro whole-cell patch clamp techniques were used to investigate granule cell hyperexcitability in the dentate gyrus 1 week after fluid percussion head trauma. The percentage decrease in the number of hilar interneurones labelled with either GAD67 or parvalbumin mRNA probes following trauma was not different from the decrease in the total population of hilar cells, indicating no preferential survival of interneurones with respect to the non-GABAergic hilar cells, i.e. the mossy cells. Dentate granule cells following trauma showed enhanced action potential discharges, and longer-lasting depolarizations, in response to perforant path stimulation, in the presence of the GABAA receptor antagonist bicuculline. There was no post-traumatic alteration in the perforant path-evoked monosynaptic excitatory postsynaptic currents (EPSCs), or in the intrinsic properties of granule cells. However, after trauma, the monosynaptic EPSC was followed by late, polysynaptic EPSCs, which were not present in controls. The late EPSCs in granule cells from fluid percussion-injured rats were not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV), but were eliminated by both the non-NMDA glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the AMPA receptor antagonist GYKI 53655. In addition, the late EPSCs were not present in low (0·5 mM) extracellular calcium, and they were also eliminated by the removal of the dentate hilus from the slice. Mossy hilar cells in the traumatic dentate gyrus responded with significantly enhanced, prolonged trains of action potential discharges to perforant path stimulation. These data indicate that surviving mossy cells play a crucial role in the hyperexcitable responses of the post-traumatic dentate gyrus. PMID:10747187

  8. 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

  9. Spontaneous proliferative lesions of the adrenal medulla in aging Long-Evans rats. Comparison to PC12 cells, small granule-containing cells, and human adrenal medullary hyperplasia.

    PubMed

    Tischler, A S; DeLellis, R A; Perlman, R L; Allen, J M; Costopoulos, D; Lee, Y C; Nunnemacher, G; Wolfe, H J; Bloom, S R

    1985-10-01

    Aging rats of the Long-Evans strain spontaneously develop diffuse and nodular hyperplasia of the adrenal medulla in association with other abnormalities commonly encountered in human multiple endocrine neoplasia syndromes. The cells which comprise the adrenal nodules resemble those in the parent tumor of the rat PC12 pheochromocytoma cell line in that they show varying degrees of spontaneous or nerve growth factor-induced neurite outgrowth in culture and they contain little or no epinephrine. In addition, cells from at least some of the nodules contain immunoreactive neurotensin and neuropeptide-Y, which are also found in PC12 cells. There are a number of striking resemblances between the cells in adrenal nodules and the small granule-containing cells in the normal rodent adrenal. The findings suggest that spontaneous rat adrenal medullary nodules and PC12 cells might be derived from small granule-containing cells, or that cells within the nodules might regain properties of immature chromaffin cells and acquire characteristics of small granule-containing cells and of PC12 cells in the course of neoplastic progression. They further suggest a possible relationship between proliferative capacity and neurotransmitter phenotype in the adult rat adrenal medulla. By virtue of their sparse epinephrine content and their small granules, the cells in adrenal medullary nodules of Long-Evans rats differ from those in adrenal medullary nodules of humans with multiple endocrine neoplasia syndromes.

  10. 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.

  11. Differential inhibition onto developing and mature granule cells generates high-frequency filters with variable gain

    PubMed Central

    Pardi, María Belén; Ogando, Mora Belén; Schinder, Alejandro F; Marin-Burgin, Antonia

    2015-01-01

    Adult hippocampal neurogenesis provides the dentate gyrus with heterogeneous populations of granule cells (GC) originated at different times. The contribution of these cells to information encoding is under current investigation. Here, we show that incoming spike trains activate different populations of GC determined by the stimulation frequency and GC age. Immature GC respond to a wider range of stimulus frequencies, whereas mature GC are less responsive at high frequencies. This difference is dictated by feedforward inhibition, which restricts mature GC activation. Yet, the stronger inhibition of mature GC results in a higher temporal fidelity compared to that of immature GC. Thus, hippocampal inputs activate two populations of neurons with variable frequency filters: immature cells, with wide‐range responses, that are reliable transmitters of the incoming frequency, and mature neurons, with narrow frequency response, that are precise at informing the beginning of the stimulus, but with a sparse activity. DOI: http://dx.doi.org/10.7554/eLife.08764.001 PMID:26163657

  12. Cell type-dependent trafficking of neuropeptide Y-containing dense core granules in CNS neurons.

    PubMed

    Ramamoorthy, Prabhu; Wang, Qian; Whim, Matthew D

    2011-10-12

    Neuropeptide transmitters are synthesized throughout the CNS and play important modulatory roles. After synthesis in the neuronal cell body, it is generally assumed that peptides are transported to nonspecialized sites of release. However, apart from a few cases, this scenario has not been thoroughly examined. Using wild-type and NPY(GFP) transgenic mice, we have studied the subcellular distribution of neuropeptide Y (NPY), a prototypical and broadly expressed neuropeptide. NPY puncta were found in the dendrites and axons of hippocampal GABAergic interneurons in situ. In contrast in hypothalamic GABAergic interneurons, NPY was restricted to the axon. Surprisingly this differential trafficking was preserved when the neurons were maintained in vitro. When hippocampal and hypothalamic neurons were transfected with NPY-Venus, the distribution of the fluorescent puncta replicated the cell type-specific distribution of endogenous neuropeptide Y. The NPY puncta in the axons of hippocampal and hypothalamic neurons colocalized with the sites of classical transmitter release (identified by staining for synapsin and the vesicular GABAergic transporter, VGAT). In hippocampal neurons, most of the postsynaptic NPY puncta were clustered opposite synapsin-containing varicosities. When neurons were stained for a second neuropeptide, agouti-related protein, immunoreactivity was found in the axon and dendrites of hippocampal neurons but only in the axons of hypothalamic neurons, thus mimicking the polarized distribution of NPY. These results indicate that the trafficking of neuropeptide-containing dense core granules is markedly cell type specific and is not determined entirely by the characteristics of the particular peptide per se.

  13. Brefeldin A sensitive mechanisms contribute to endocytotic membrane retrieval and vesicle recycling in cerebellar granule cells.

    PubMed

    Rampérez, Alberto; Sánchez-Prieto, José; Torres, Magdalena

    2017-03-11

    The recycling of synaptic vesicle (SV) proteins and transmitter release both occur at multiple sites along the axon. These processes are sensitive to inhibition of the small GTP binding protein ARF1, which regulates the AP-1/AP-3 complex. As the axon matures, SV recycling becomes restricted to the presynaptic bouton, and its machinery undergoes a complex process of maturation. We used the styryl dye FM1-43 to highlight differences in the efficiency of membrane recycling at different sites in cerebellar granule cells cultured for 7 days in vitro. We used Brefeldin A (BFA) to inhibit AP-1/AP-3-mediated recycling and to test the contribution of this pathway to the heterogeneity of the responses when these cells are strongly stimulated. Combining imaging techniques and ultrastructural analyses, we found a significant decrease in the density of functional boutons and an increase in the presence of endosome-like structures within the boutons of cells incubated with BFA prior to FM1-43 loading. Such effects were not observed when BFA was added 5 minutes after the end of the loading step, when endocytosis was almost fully completed. In this situation, vesicles were found closer to the active zone (AZ) in boutons exposed to BFA. Together, these data suggest that the AP-1/AP-3 pathway contributes to SV recycling, affecting different steps in all boutons but not equally, and thus being partly responsible for the heterogeneity of the different recycling efficiencies. This article is protected by copyright. All rights reserved.

  14. Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells

    PubMed Central

    Redhai, Siamak; Hellberg, Josephine E. E. U.; Wainwright, Mark; Perera, Sumeth W.; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C. I.

    2016-01-01

    Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion. PMID:27727275

  15. Regulation of Dense-Core Granule Replenishment by Autocrine BMP Signalling in Drosophila Secondary Cells.

    PubMed

    Redhai, Siamak; Hellberg, Josephine E E U; Wainwright, Mark; Perera, Sumeth W; Castellanos, Felix; Kroeger, Benjamin; Gandy, Carina; Leiblich, Aaron; Corrigan, Laura; Hilton, Thomas; Patel, Benjamin; Fan, Shih-Jung; Hamdy, Freddie; Goberdhan, Deborah C I; Wilson, Clive

    2016-10-01

    Regulated secretion by glands and neurons involves release of signalling molecules and enzymes selectively concentrated in dense-core granules (DCGs). Although we understand how many secretagogues stimulate DCG release, how DCG biogenesis is then accelerated to replenish the DCG pool remains poorly characterised. Here we demonstrate that each prostate-like secondary cell (SC) in the paired adult Drosophila melanogaster male accessory glands contains approximately ten large DCGs, which are loaded with the Bone Morphogenetic Protein (BMP) ligand Decapentaplegic (Dpp). These DCGs can be marked in living tissue by a glycophosphatidylinositol (GPI) lipid-anchored form of GFP. In virgin males, BMP signalling is sporadically activated by constitutive DCG secretion. Upon mating, approximately four DCGs are typically released immediately, increasing BMP signalling, primarily via an autocrine mechanism. Using inducible knockdown specifically in adult SCs, we show that secretion requires the Soluble NSF Attachment Protein, SNAP24. Furthermore, mating-dependent BMP signalling not only promotes cell growth, but is also necessary to accelerate biogenesis of new DCGs, restoring DCG number within 24 h. Our analysis therefore reveals an autocrine BMP-mediated feedback mechanism for matching DCG release to replenishment as secretion rates fluctuate, and might explain why in other disease-relevant systems, like pancreatic β-cells, BMP signalling is also implicated in the control of secretion.

  16. Changes in the Paneth cell population of human small intestine assessed by image analysis of the secretory granule area.

    PubMed Central

    Elmes, M E; Jones, J G; Stanton, M R

    1983-01-01

    Estimates of the Paneth cell population in human jejunum and ileum were made using measurement of the granule area in micron2 by image analysis in a defined number of crypts. This figure was preferable to granule area per mm as there was a significant difference in crypts per mm between biopsies and surgical samples. In the jejunum no significant difference was found between normal children and adults with and without peptic ulcer. In adults with subtotal or partial villous atrophy the decrease in area was not statistically significant and there was no decrease in area in children with partial villous atrophy and coeliac disease. There was a marked increase in granule area in the jejunum of patients who had had a previous partial gastrectomy which was statistically significant. In the ileum patients with carcinoma of the caecum had higher values than patients with non-inflammatory non-malignant conditions but this was not statistically significant and two patients with Crohn's disease had an increased granule area. Paneth cell populations are affected by alterations in the intestinal luminal environment due to previous surgery or neoplastic or inflammatory disease. Images PMID:6875016

  17. Incoming synapses and size of small granule-containing cells in a rat sympathetic ganglion after post-ganglionic axotomy.

    PubMed Central

    Case, C P; Matthews, M R

    1986-01-01

    A quantitative ultrastructural study has been made of the reaction of the incoming synapses of small granule-containing cells after axotomy of the major post-ganglionic branches of the superior cervical ganglion of the young adult rat. These cells are intrinsic and interneurone-like in this ganglion, receiving a preganglionic input and giving outgoing synapses to principal post-ganglionic neurones. Unlike their outgoing synapses, which are lost after post-ganglionic axotomy (Case & Matthews, 1986), the incoming synapses of the small granule-containing cells in axotomized ganglia increased in incidence post-operatively. The increase first became clearly evident 5-7 days post-operatively and was greater, being both more sustained and progressive, after bilateral than after unilateral axotomy. After bilateral axotomy the incidence of incoming synapses rose to more than four times that of normal ganglia and was still elevated at 128 days post-operatively, but was within normal limits at 390 days. After a unilateral lesion, increases of similar extent and time course to those in the axotomized ganglia were seen in the incoming synapses of small granule-containing cells in the uninjured contralateral ganglia. The incoming synapses of the small granule-containing cells are multifocal, i.e. show several points or active foci of synaptic specialization. The increase in synapses expressed itself both through an increased incidence of these synaptic active foci per nerve terminal and through an increase in the number of presynaptic nerve terminal profiles associated with the cells. Control observations indicated that the increase in synapses was not due to surgical stress, nor was it attributable solely to post-operative ageing. The nerve terminals which were presynaptic to the small granule-containing cells post-operatively were all of preganglionic origin: no incoming synapses or presynaptic nerve terminals remained at 2 days after a preganglionic denervation of axotomized

  18. Fibroblasts from the inner granulation tissue of the pseudocapsule in hips at revision arthroplasty induce osteoclast differentiation, as do stromal cells

    PubMed Central

    Sakai, H; Jingushi, S; Shuto, T; Urabe, K; Ikenoue, T; Okazaki, K; Kukita, T; Kukita, A; Iwamoto, Y

    2002-01-01

    Background: It has previously been shown that many osteoclast precursors are included in the granulation tissue within the pseudocapsule obtained at revision arthroplasty from hips with osteolysis. In vitro culture of only cells isolated from the granulation tissue has been previously shown to generate many mature osteoclasts. Objective: To investigate the presence or otherwise of supporting cells, similar to stromal cells, which differentiate osteoclasts within the granulation tissue. Methods: Cells isolated from the granulation tissue were cultured alone, and after four weeks fibroblast-like cells (granulation fibroblasts) remained. Rat non-adherent bone marrow cells (NA-BMCs) were co-cultured with the granulation fibroblasts with or without 1α,25(OH)2D3 (10-8 M) or heat treated ROS 17/2.8 cell conditioned medium (ht ROSCM), or both. Multinucleated cells (MNCs), which formed, were assessed by biochemical and functional characterisation of osteoclasts. Receptor activator of NFκB ligand (RANKL) was investigated by immunohistochemistry. Results: Co-culture of NA-BMCs and granulation fibroblasts caused the formation of tartrate resistant acid phosphatase (TRAP) positive MNCs, which had the calcitonin receptor (CTR), the Kat-1 antigen, which is specific to the surface of rat osteoclasts, and the ability to form pits in the presence of both 1α,25(OH)2D3 and ht ROSCM or in the presence of just ht ROSCM. RANKL was detected in fibroblast-like cells in the granulation tissue. Conclusion: These data suggest that granulation fibroblasts support osteoclast differentiation, as do osteoblasts/stromal cells, and may play a part in aseptic loosening. PMID:11796394

  19. 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

  20. Simulated Responses of Cerebellar Purkinje Cells are Independent of the Dendritic Location of Granule Cell Synaptic Inputs

    NASA Astrophysics Data System (ADS)

    de Schutter, Erik; Bower, James M.

    1994-05-01

    Cerebellar Purkinje cell responses to granule cell synaptic inputs were examined with a computer model including active dendritic conductances. Dendritic P-type Ca2+ channels amplified postsynaptic responses when the model was firing at a physiological rate. Small synchronous excitatory inputs applied distally on the large dendritic tree resulted in somatic responses of similar size to those generated by more proximal inputs. In contrast, in a passive model the somatic postsynaptic potentials to distal inputs were 76% smaller. The model predicts that the somatic firing response of Purkinje cells is relatively insensitive to the exact dendritic location of synaptic inputs. We describe a mechanism of Ca2+-mediated synaptic amplification, based on the subspiking threshold recruitment of P-type Ca2+ channels in the dendritic branches surrounding the input site.

  1. Urethral Reconstruction Using Mesothelial Cell-Seeded Autogenous Granulation Tissue Tube: An Experimental Study in Male Rabbits

    PubMed Central

    Jiang, Shiwei; Xu, Zhonghua; Zhao, Yuanyuan; Yan, Lei; Zhou, Zunlin

    2017-01-01

    Objective. This study was to evaluate the utility of the compound graft for tubularized urethroplasty by seeding mesothelial cells onto autogenous granulation tissue. Methods. Silastic tubes were implanted subcutaneously in 18 male rabbits, of which nine underwent omentum biopsies simultaneously for in vitro expansion of mesothelial cells. The granulation tissue covering the tubes was harvested 2 weeks after operation. Mesothelial cells were seeded onto and cocultured with the tissue for 7 days. A pendulous urethral segment of 1.5 cm was totally excised. Urethroplasty was performed with mesothelial cell-seeded tissue tubes in an end-to-end fashion in nine rabbits and with unseeded grafts in others as controls. Serial urethrograms were performed at 1, 2, and 6 months postoperatively. Meanwhile, the neourethra was harvested and analyzed grossly and histologically. Results. Urethrograms showed cell-seeded grafts maintained wide at each time point, while strictures formation was found in unseeded grafts. Histologically, layers of urothelium surrounded by increasingly organized smooth muscles were observed in seeded grafts. In contrast, myofibroblasts accumulation and extensive scarring occurred in unseeded grafts. Conclusions. Mesothelial cell-seeded granulation tissue tube can be successfully used for tubularized urethroplasty in male rabbits. PMID:28337443

  2. 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.

  3. Cancer metastasis-suppressing peptide metastin upregulates excitatory synaptic transmission in hippocampal dentate granule cells.

    PubMed

    Arai, Amy C; Xia, Yan-Fang; Suzuki, Erika; Kessler, Markus; Civelli, Olivier; Nothacker, Hans-Peter

    2005-11-01

    Metastin is an antimetastatic peptide encoded by the KiSS-1 gene in cancer cells. Recent studies found that metastin is a ligand for the orphan G-protein-coupled receptor GPR54, which is highly expressed in specific brain regions such as the hypothalamus and parts of the hippocampus. This study shows that activation of GPR54 by submicromolar concentrations of metastin reversibly enhances excitatory synaptic transmission in hippocampal dentate granule cells in a mitogen-activated protein (MAP) kinase-dependent manner. Synaptic enhancement by metastin was suppressed by intracellular application of the G-protein inhibitor GDP-beta-S and the calcium chelator BAPTA. Analysis of miniature excitatory postsynaptic currents (mEPSCs) revealed an increase in the mean amplitude but no change in event frequency. This indicates that GPR54 and the mechanism responsible for the increase in EPSCs are postsynaptic. Metastin-induced synaptic potentiation was abolished by 50 microM PD98059 and 20 microM U0126, two inhibitors of the MAP kinases ERK1 and ERK2. The effect was also blocked by inhibitors of calcium/calmodulin-dependent kinases and tyrosine kinases. RT-PCR experiments showed that both KiSS-1 and GPR54 are expressed in the hippocampal dentate gyrus. Metastin is thus a novel endogenous factor that modulates synaptic excitability in the dentate gyrus through mechanisms involving MAP kinases, which in turn may be controlled upstream by calcium-activated kinases and tyrosine kinases.

  4. Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells

    PubMed Central

    Duan, Yuntao; Wang, Shih-Hsiu; Song, Juan; Mironova, Yevgeniya; Ming, Guo-li; Kolodkin, Alex L; Giger, Roman J

    2014-01-01

    Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A−/− GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A−/− mutants, PlexinA2−/− mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A−/− mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders. DOI: http://dx.doi.org/10.7554/eLife.04390.001 PMID:25313870

  5. 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.

  6. Electrophysiological characterization of granule cells in the dentate gyrus immediately after birth

    PubMed Central

    Pedroni, Andrea; Minh, Do Duc; Mallamaci, Antonello; Cherubini, Enrico

    2014-01-01

    Granule cells (GCs) in the dentate gyrus are generated mainly postnatally. Between embryonic day 10 and 14, neural precursors migrate from the primary dentate matrix to the dentate gyrus where they differentiate into neurons. Neurogenesis reaches a peak at the end of the first postnatal week and it is completed at the end of the first postnatal month. This process continues at a reduced rate throughout life. Interestingly, immediately after birth, GCs exhibit a clear GABAergic phenotype. Only later they integrate the classical glutamatergic trisynaptic hippocampal circuit. Here, whole cell patch clamp recordings, in current clamp mode, were performed from immature GCs, intracellularly loaded with biocytin (in hippocampal slices from P0 to P3 old rats) in order to compare their morphological characteristics with their electrophysiological properties. The vast majority of GCs were very immature with small somata, few dendritic branches terminating with small varicosities and growth cones. In spite of their immaturity their axons reached often the cornu ammonis 3 area. Immature GCs generated, upon membrane depolarization, either rudimentary sodium spikes or more clear overshooting action potentials that fired repetitively. They exhibited also low threshold calcium spikes. In addition, most spiking neurons showed spontaneous synchronized network activity, reminiscent of giant depolarizing potentials (GDPs) generated in the hippocampus by the synergistic action of glutamate and GABA, both depolarizing and excitatory. This early synchronized activity, absent during adult neurogenesis, may play a crucial role in the refinement of local neuronal circuits within the developing dentate gyrus. PMID:24592213

  7. 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.

  8. Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells.

    PubMed

    Masoli, Stefano; Rizza, Martina F; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

    2017-01-01

    In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models.

  9. Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells

    PubMed Central

    Masoli, Stefano; Rizza, Martina F.; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

    2017-01-01

    In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (Gi-max) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of Gi-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of Gi-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental Gi-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models. PMID:28360841

  10. Neurotoxicity of amphetamine derivatives is mediated by caspase pathway activation in rat cerebellar granule cells.

    PubMed

    Jiménez, Andrés; Jordà, Elvira G; Verdaguer, Ester; Pubill, David; Sureda, Francesc X; Canudas, Anna M; Escubedo, Elena; Camarasa, Jordi; Camins, Antoni; Pallàs, Mercè

    2004-04-15

    The neurotoxic action of the abuse drugs methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) on cerebellar granule neurones (CGNs) culture was examined. Treatment for 48 h with METH or MDMA (1-5 mM) induced a higher decrease in viability than 24 h treatment. z.VAD.fmk (100 microM) but not MK-801 nor NBQX recovered control viability values. In both cases, cell death was characterised as apoptotic rather than necrotic by morphology cell observation. Apoptosis measured by flow cytometry indicated an increase in the hypodiploid population after 48 h treatment with METH and MDMA. Apoptosis was reverted by the presence of z.VAD.fmk (100 microM) but not by 10 microM MK-801 or NBQX. Similar results were obtained by analysing nuclear chromatine condensation. These results ruled out excitotoxic participation in amphetamine derivative-induced neurotoxicity in CGNs. Participation of radical oxygen species (ROS) was evaluated using alpha-tocopherol (1-15 microM) and cytometric studies. The co-treatment with 4 mM METH or MDMA for 48 h partially reverted neurotoxic action and apoptotic features, indicating ROS implication in CGNs death by amphetamine derivatives. Alteration of mitochondrial function induced cytochrome C (Cyt C) release after 48-h treatment with METH and MDMA (4 mM). There was also indication of caspase-3-like activation, measured by immunoanalysis and biochemically. Finally, neurodegenerative action caused by amphetamine derivatives may be prevented by using caspase inhibitors.

  11. Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

    PubMed

    Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

    2012-07-01

    Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

  12. Nanocrystalline spherical hydroxyapatite granules for bone repair: in vitro evaluation with osteoblast-like cells and osteoclasts.

    PubMed

    Bernhardt, A; Dittrich, R; Lode, A; Despang, F; Gelinsky, M

    2013-07-01

    Conventionally sintered hydroxyapatite-based materials for bone repair show poor resorbability due to the loss of nanocrystallinity. The present study describes a method to establish nanocrystalline hydroxyapatite granules. The material was prepared by ionotropic gelation of an alginate sol containing hydroxyapatite (HA) powder. Subsequent thermal elimination of alginate at 650 °C yielded non-sintered, but unexpectedly stable hydroxyapatite granules. By adding stearic acid as an organic filler to the alginate/HA suspension, the granules exhibited macropores after thermal treatment. A third type of material was achieved by additional coating of the granules with silica particles. Microstructure and specific surface area of the different materials were characterized in comparison to the already established granular calcium phosphate material Cerasorb M(®). Cytocompatibility and potential for bone regeneration of the materials was evaluated by in vitro examinations with osteosarcoma cells and osteoclasts. Osteoblast-like SaOS-2 cells proliferated on all examined materials and showed the typical increase of alkaline phosphatase (ALP) activity during cultivation. Expression of bone-related genes coding for ALP, osteonectin, osteopontin, osteocalcin and bone sialoprotein II on the materials was proven by RT-PCR. Human monocytes were seeded onto the different granules and osteoclastogenesis was examined by activity measurement of tartrate-specific acid phosphatase (TRAP). Gene expression analysis after 23 days of cultivation revealed an increased expression of osteoclast-related genes TRAP, vitronectin receptor and cathepsin K, which was on the same level for all examined materials. These results indicate, that the nanocrystalline granular materials are of clinical interest, especially for bone regeneration.

  13. Tracing CD34+ Stromal Fibroblasts in Palatal Mucosa and Periodontal Granulation Tissue as a Possible Cell Reservoir for Periodontal Regeneration.

    PubMed

    Roman, Alexandra; Páll, Emőke; Mihu, Carmen M; Petruţiu, Adrian S; Barbu-Tudoran, Lucian; Câmpian, Radu S; Florea, Adrian; Georgiu, Carmen

    2015-08-01

    The aim of the present research was to trace CD34+ stromal fibroblastic cells (CD34+ SFCs) in the palatal connective tissue harvested for muco-gingival surgical procedures and in granulation tissues from periodontal pockets using immunohistochemical and transmission electron microscopy. Immunohistochemical analysis targeted the presence of three antigens: CD31, α-smooth muscle actin (α-SMA), and CD34. In the palate, CD31 staining revealed a colored inner ring of the vessels representing the endothelium, α-SMA+ was located in the medial layer of the vasculature, and CD34 was intensely expressed by endothelial cells and artery adventitial cells (considered to be CD34+ SFCs). Granulation tissue showed the same pattern for CD31+ and α-SMA, but a different staining pattern for CD34. Ultrastructural examination of the palatal tissue highlighted perivascular cells with fibroblast-like characteristics and pericytes in close spatial relationship to endothelial cells. The ultrastructural evaluation of granulation tissue sections confirmed the presence of neovasculature and the inflammatory nature of this tissue. The present study traced the presence of CD34+ SFCs and of pericytes in the palatal connective tissue thus highlighting once more its intrinsic regenerative capabilities. The clinical and systemic factors triggering mobilization and influencing the fate of local CD34+SCFs and other progenitors are issues to be further investigated.

  14. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

    PubMed

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-Ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-Ichiro

    2016-10-31

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

  15. Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution

    PubMed Central

    Yoshitomi, Munetake; Ohta, Keisuke; Kanazawa, Tomonoshin; Togo, Akinobu; Hirashima, Shingo; Uemura, Kei-ichiro; Okayama, Satoko; Morioka, Motohiro; Nakamura, Kei-ichiro

    2016-01-01

    Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules. PMID:27796315

  16. Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata).

    PubMed

    Cavalcante, Moisés C M; de Andrade, Leonardo R; Du Bocage Santos-Pinto, Claudia; Straus, Anita H; Takahashi, Hélio K; Allodi, Silvana; Pavão, Mauro S G

    2002-03-01

    In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.

  17. Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice.

    PubMed

    Ghosh, Ajoy Kumar; Hirasawa, Noriyasu; Ohtsu, Hiroshi; Watanabe, Takehiko; Ohuchi, Kazuo

    2002-04-15

    We have analyzed the role of histamine in the angiogenesis of the granulation tissue in histidine decarboxylase-deficient (HDC(-/-)) mice, mast cell-deficient mice (WBB6F1-W/W(V)), and their corresponding wild-type mice (HDC(+/+) and WBB6F(1)(+/+)). In HDC(+/+) mice, subcutaneous implantation of a cotton thread in the dorsum induced granulation tissue formation with angiogenesis, while the topical injection of anti-vascular endothelial growth factor (VEGF) IgG strongly suppressed them. In HDC(-/-) mice which showed lower VEGF levels in the granulation tissue, there was notably less angiogenesis and granulation tissue formation than in HDC(+/+) mice. The topical injection of histamine or the H(2) agonist dimaprit rescued the defective angiogenesis and granulation tissue formation in HDC(-/-) mice. There was no significant difference in the granulation tissue formation and angiogenesis between WBB6F1-W/W(V) and WBB6F1(+/+) mice. In addition, macrophages in the granulation tissue were found to express HDC. Our findings indicate that histamine derived from non-mast cells plays a significant role in the angiogenesis of the inflammatory granulation tissue.

  18. Elements of the nitric oxide/cGMP pathway expressed in cerebellar granule cells: biochemical and functional characterisation.

    PubMed

    Jurado, Sandra; Sánchez-Prieto, José; Torres, Magdalena

    2004-11-01

    It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.

  19. Posttranslational regulation of BCL2 levels in cerebellar granule cells: A mechanism of neuronal survival.

    PubMed

    Lossi, Laura; Gambino, Graziana; Ferrini, Francesco; Alasia, Silvia; Merighi, Adalberto

    2009-11-01

    Apoptosis can be modulated by K(+) and Ca(2+) inside the cell and/or in the extracellular milieu. In murine organotypic cultures, membrane potential-regulated Ca(2+) signaling through calcineurin phosphatase has a pivotal role in development and maturation of cerebellar granule cells (CGCs). P8 cultures were used to analyze the levels of expression of B cell lymphoma 2 (BCL2) protein, and, after particle-mediated gene transfer in CGCs, to study the posttranslational modifications of BCL2 fused to a fluorescent tag in response to a perturbation of K(+)/Ca(2+) homeostasis. There are no changes in Bcl2 mRNA after real time PCR, whereas the levels of the fusion protein (monitored by calculating the density of transfected CGCs under the fluorescence microscope) and of BCL2 (inWestern blotting) are increased. After using a series of agonists/antagonists for ion channels at the cell membrane or the endoplasmic reticulum (ER), and drugs affecting protein synthesis/degradation, accumulation of BCL2 was related to a reduction in posttranslational cleavage by macroautophagy. The ER functionally links the [K(+)](e) and [Ca(2+)](i) to the BCL2 content in CGCs along two different pathways. The first, triggered by elevated [K(+)](e) under conditions of immaturity, is independent of extracellular Ca(2+) and operates via IP3 channels. The second leads to influx of extracellular Ca(2+) following activation of ryanodine channels in the presence of physiological [K(+)](e), when CGCs are maintained in mature status. This study identifies novel mechanisms of neuroprotection in immature and mature CGCs involving the posttranslational regulation of BCL2.

  20. 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

  1. Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus.

    PubMed

    Katoh-Semba, Ritsuko; Asano, Tomiko; Ueda, Hiroshi; Morishita, Rika; Takeuchi, Ikuo K; Inaguma, Yutaka; Kato, Kanefusa

    2002-08-01

    The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain-derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF-specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

  2. Influence of brain-derived neurotrophic factor on pathfinding of dentate granule cell axons, the hippocampal mossy fibers.

    PubMed

    Tamura, Makoto; Tamura, Naohiro; Ikeda, Takamitsu; Koyama, Ryuta; Ikegaya, Yuji; Matsuki, Norio; Yamada, Maki K

    2009-01-31

    Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced aberrant extension of mossy fibers into ectopic regions. BDNF overexpression in granule cells ameliorated the mossy fiber pathway abnormalities caused by a submaximal dose of K252a. A similar rescue was observed when BDNF was expressed in CA3 pyramidal cells, most notably in mossy fibers distal to the expression site. These findings are the first to clarify the role of BDNF in mossy fiber pathfinding, not as an attractant cue but as a regulator, possibly acting in a paracrine manner. This effect of BDNF may be as a signal for new fibers to fasciculate and extend further to form synapses with neurons that are far from active BDNF-expressing synapses. This mechanism would ensure the emergence of new independent dentate gyrus-CA3 circuits by the axons of new-born granule cells.

  3. Expression of NR2B in cerebellar granule cells specifically facilitates effect of motor training on motor learning.

    PubMed

    Jiao, Jianwei; Nakajima, Akira; Janssen, William G M; Bindokas, Vytautas P; Xiong, Xiaoli; Morrison, John H; Brorson, James R; Tang, Ya-Ping

    2008-02-27

    It is believed that gene/environment interaction (GEI) plays a pivotal role in the development of motor skills, which are acquired via practicing or motor training. However, the underlying molecular/neuronal mechanisms are still unclear. Here, we reported that the expression of NR2B, a subunit of NMDA receptors, in cerebellar granule cells specifically enhanced the effect of voluntary motor training on motor learning in the mouse. Moreover, this effect was characterized as motor learning-specific and developmental stage-dependent, because neither emotional/spatial memory was affected nor was the enhanced motor learning observed when the motor training was conducted starting at the age of 3 months old in these transgenic mice. These results indicate that changes in the expression of gene(s) that are involved in regulating synaptic plasticity in cerebellar granule cells may constitute a molecular basis for the cerebellum to be involved in the GEI by facilitating motor skill learning.

  4. 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.

  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. Hyperpolarization-Activated Currents and Subthreshold Resonance in Granule Cells of the Olfactory Bulb

    PubMed Central

    Hu, Ruilong; Ferguson, Katie A.; Meijer, Dimphna H.

    2016-01-01

    Abstract An important contribution to neural circuit oscillatory dynamics is the ongoing activation and inactivation of hyperpolarization-activated currents (Ih). Network synchrony dynamics play an important role in the initial processing of odor signals by the main olfactory bulb (MOB) and accessory olfactory bulb (AOB). In the mouse olfactory bulb, we show that Ih is present in granule cells (GCs), the most prominent inhibitory neuron in the olfactory bulb, and that Ih underlies subthreshold resonance in GCs. In accord with the properties of Ih, the currents exhibited sensitivity to changes in extracellular K+ concentration and ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidin chloride), a blocker of Ih. ZD7288 also caused GCs to hyperpolarize and increase their input resistance, suggesting that Ih is active at rest in GCs. The inclusion of cAMP in the intracellular solution shifted the activation of Ih to less negative potentials in the MOB, but not in the AOB, suggesting that channels with different subunit composition mediate Ih in these regions. Furthermore, we show that mature GCs exhibit Ih-dependent subthreshold resonance in the theta frequency range (4–12 Hz). Another inhibitory subtype in the MOB, the periglomerular cells, exhibited Ih-dependent subthreshold resonance in the delta range (1–4 Hz), while principal neurons, the mitral cells, do not exhibit Ih-dependent subthreshold resonance. Importantly, Ih size, as well as the strength and frequency of resonance in GCs, exhibited a postnatal developmental progression, suggesting that this development of Ih in GCs may differentially contribute to their integration of sensory input and contribution to oscillatory circuit dynamics. PMID:27844056

  7. Exocytosis from pancreatic β-cells: mathematical modelling of the exit of low-molecular-weight granule content.

    PubMed

    Galvanovskis, Juris; Braun, Matthias; Rorsman, Patrik

    2011-02-06

    Pancreatic β-cells use Ca(2+)-dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in β-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the β-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents.

  8. 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.

  9. BDNF-Mediated Cerebellar Granule Cell Development is Impaired in Mice Null for CaMKK2 or CaMKIV

    PubMed Central

    Kokubo, Manabu; Nishio, Masahiro; Ribar, Thomas J.; Anderson, Kristin A.; West, Anne E.; Means, Anthony R.

    2009-01-01

    The Ca2+/calmodulin-activated kinases CamKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca2+. To address the biological functions of Ca2+ signaling via these kinases during brain development we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of CREB correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Further, loss of CaMKK2 or CaMKIV results in decreased pCREB, Bdnf exon I and IV-containing mRNAs and BDNF protein in cerebellar granule cell neurons. Re-expression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca2+-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process. PMID:19605628

  10. Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

    PubMed Central

    Tabilio, A; Rosa, J P; Testa, U; Kieffer, N; Nurden, A T; Del Canizo, M C; Breton-Gorius, J; Vainchenker, W

    1984-01-01

    We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12-O-tetradecanoylphorbol-13-acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha-granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha-granule proteins (von Willebrand factor, platelet factor-4 and beta-thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS-polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet-specific proteins and glycoproteins. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:6201359

  11. Blockade of excitatory synaptogenesis with proximal dendrites of dentate granule cells following rapamycin treatment in a mouse model of temporal lobe epilepsy

    PubMed Central

    Yamawaki, Ruth; Thind, Khushdev; Buckmaster, Paul S.

    2014-01-01

    Inhibiting the mTOR signaling pathway with rapamycin blocks granule cell axon (mossy fiber) sprouting after epileptogenic injuries, including pilocarpine-induced status epilepticus. However, it remains unclear whether axons from other types of neurons sprout into the inner molecular layer and synapse with granule cell dendrites despite rapamycin treatment. If so, other aberrant positive-feedback networks might develop. To test this possibility stereological electron microscopy was used to estimate numbers of excitatory synapses in the inner molecular layer per hippocampus in pilocarpine-treated control mice, in mice 5 d after pilocarpine-induced status epilepticus, and after status epilepticus and daily treatment beginning 24 h later with rapamycin or vehicle for 2 months. The optical fractionator method was used to estimate numbers of granule cells in Nissl-stained sections so that numbers of excitatory synapses in the inner molecular layer per granule cell could be calculated. Control mice had an average of 2280 asymmetric synapses in the inner molecular layer per granule cell, which was reduced to 63% of controls 5 d after status epilepticus, recovered to 93% of controls in vehicle-treated mice 2 months after status epilepticus, but remained at only 63% of controls in rapamycin-treated mice. These findings reveal that rapamycin prevented excitatory axons from synapsing with proximal dendrites of granule cells and raise questions about the recurrent excitation hypothesis of temporal lobe epilepsy. PMID:25234294

  12. Brain-derived neurotrophic factor (BDNF) ameliorates the suppression of thyroid hormone-induced granule cell neurite extension by hexabromocyclododecane (HBCD).

    PubMed

    Ibhazehiebo, Kingsley; Iwasaki, Toshiharu; Xu, Ming; Shimokawa, Noriaki; Koibuchi, Noriyuki

    2011-04-08

    Thyroid hormone (TH) plays an essential role in growth and differentiation of the central nervous system. Deficiency of TH during perinatal period results in abnormal brain development known as cretinism in human. We recently reported that an environmental chemical 1,2,5,6,9,10-α-hexabromocyclododecane (HBCD) suppressed TH receptor (TR)-mediated transcription. To examine the effect of HBCD on cerebellar granule cells, we used purified rat cerebellar granule cells in reaggregate culture. Low dose HBCD (10(-10)M) significantly suppressed TH-induced neurite extension of granule cell aggregate. To clarify further the mechanisms of such suppression, we added brain-derived neurotrophic factor (BDNF) into culture medium, since BDNF plays a critical role in promoting granule cell development and is regulated by TH. BDNF completely rescued HBCD-induced suppression of granule cell neurite extension in the presence of T3. These results indicate that HBCD may disrupt TH-mediated brain development at least in part due to a disruption of the T3 stimulated increase in BDNF and BDNF may possess ability to ameliorate the effect of HBCD in granule cells.

  13. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    PubMed

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function.

  14. Platelets can neutralize hydrogen peroxide in an acute toxicity model with cells involved in granulation tissue formation.

    PubMed

    Kandler, Barbara; Maitz, Philipp; Fischer, Michael B; Watzek, Georg; Gruber, Reinhard

    2005-04-01

    Platelets play a key role in the replacement of the blood clot with granulation tissue during the early steps of bone regeneration. We hypothesized that activated platelets can neutralize locally produced reactive oxygen species, thereby protecting cells involved in granulation tissue formation. The potential of platelet-released supernatant (PRS) to neutralize hydrogen peroxide (H(2)O(2)) was tested in an acute toxicity model with osteogenic, inflammatory, and endothelial cells. In the human fetal osteoblastic cell line 1.19 (hFOB), considerable morphological changes, cell shedding, and dysfunction of the respiratory chain were observed when cells were exposed to 3 mM H(2)O(2). Caspase-3 and poly-(ADP-ribose)-polymerase were not activated, suggesting that cell death occurred by necrosis. Preincubation of osteogenic cells, leukocytes, or endothelial cells with PRS decreased the acute toxicity of H(2)O(2). The capacity of platelets to release H(2)O(2)-detoxifying activity was retained for up to 72 h. Aminotriazole, an inhibitor of catalase, decreased the cytoprotective activity of PRS, whereas blocking of glutathione peroxidase by mercaptosuccinate had no effect. These results suggest that platelet-released catalase can rapidly neutralize cytotoxic amounts of H(2)O(2), a process that may play a role during the early stages of bone regeneration.

  15. Synaptosomal-associated protein 25 mutation induces immaturity of the dentate granule cells of adult mice

    PubMed Central

    2013-01-01

    Background Synaptosomal-associated protein, 25 kDa (SNAP-25) regulates the exocytosis of neurotransmitters. Growing evidence suggests that SNAP-25 is involved in neuropsychiatric disorders, such as schizophrenia, attention-deficit/hyperactivity disorder, and epilepsy. Recently, increases in anxiety-related behaviors and epilepsy have been observed in SNAP-25 knock-in (KI) mice, which have a single amino acid substitution of Ala for Ser187. However, the molecular and cellular mechanisms underlying the abnormalities in this mutant remain unknown. Results In this study, we found that a significant number of dentate gyrus (DG) granule cells was histologically and electrophysiologically similar to immature DG neurons in the dentate gyrus of the adult mutants, a phenomenon termed the “immature DG” (iDG). SNAP-25 KI mice and other mice possessing the iDG phenotype, i.e., alpha-calcium/calmodulin-dependent protein kinase II heterozygous mice, Schnurri-2 knockout mice, and mice treated with the antidepressant fluoxetine, showed similar molecular expression patterns, with over 100 genes similarly altered. A working memory deficit was also identified in mutant mice during a spontaneous forced alternation task using a modified T-maze, a behavioral task known to be dependent on hippocampal function. Chronic treatments with the antiepileptic drug valproate abolished the iDG phenotype and the working memory deficit in mutants. Conclusions These findings suggest that the substitution of Ala for Ser187 in SNAP-25 induces the iDG phenotype, which can also be caused by epilepsy, and led to a severe working memory deficit. In addition, the iDG phenotype in adulthood is likely an endophenotype for at least a part of some common psychiatric disorders. PMID:23497716

  16. In vivo 7 Tesla imaging of the dentate granule cell layer in Schizophrenia

    PubMed Central

    Kirov, Ivan I.; Hardy, Caitlin J.; Matsuda, Kant; Messinger, Julie; Cankurtaran, Ceylan Z.; Warren, Melina; Wiggins, Graham C.; Perry, Nissa N.; Babb, James S.; Goetz, Raymond R.; George, Ajax; Malaspina, Dolores; Gonen, Oded

    2013-01-01

    PURPOSE The hippocampus is central to the pathophysiology of schizophrenia. Histology shows abnormalities in the dentate granule cell layer (DGCL), but its small size (~100 micron thickness) has precluded in vivo human studies. We used ultra high field magnetic resonance imaging (MRI) to compare DGCL morphology of schizophrenic patients to matched controls’. METHOD Bilateral hippocampi of 16 schizophrenia patients (10 male) 40.7±10.6 years old (mean ±standard deviation) were imaged at 7 Tesla MRI with heavily T2*-weighted gradient-echo sequence at 232 micron in-plane resolution (0.08 μL image voxels). Fifteen matched controls (8 male, 35.6±9.4 years old) and one ex vivo post mortem hippocampus (that also underwent histopathology) were scanned with same protocol. Three blinded neuroradiologists rated each DGCL on a qualitative scale of 1 to 6 (from “not discernible” to “easily visible, appearing dark gray or black”) and mean left and right DGCL scores were compared using a non-parametric Mann-Whitney test. RESULTS MRI identification of the DGCL was validated with histopathology. Mean right and left DGCL ratings in patients (3.2±1.0 and 3.5±1.2) were not statistically different from controls’ (3.9±1.1 and 3.8±0.8), but patients’ had a trend for lower right DGCL score (p=0.07), which was significantly associated with patient diagnosis (p=0.05). The optimal 48% sensitivity and 80% specificity for schizophrenia was achieved with a DGCL rating of ≤2. CONCLUSION Decreased contrast in the right DGCL in schizophrenia was predictive of schizophrenia diagnosis. Better utility of this metric as a schizophrenia biomarker may be achieved in future studies of patients with homogeneous disease subtypes and progression rates. PMID:23664589

  17. Membrane properties of the granule cells of the islands of Calleja of the rat studied in vitro.

    PubMed Central

    Halliwell, J V; Horne, A L

    1995-01-01

    1. Using patch-clamp techniques, we have studied granule neurones from the islands of Calleja in vitro: as isolated cells or as groups of varying numbers following enzymic digestion, or within untreated slices of approximately 100 microns thickness. 2. Recordings were made with patch pipettes in conventional or nystatin-perforated whole-cell mode. Current-clamp recordings indicated that these granule cells are excitable and at resting potential produce irregular spontaneous activity. In voltage clamp the transient inward current underlying these action potentials could be evoked. This current had a threshold for activation of about -50 mV and was sensitive to TTX. In some cells a TTX-resistant transient inward current was observed with a threshold for activation of about -70 mV. 3. Island of Calleja granule cells also exhibited outward currents. A rapidly activating transient current was observed that was resistant to TEA and sensitive to 4-AP, and therefore resembled IA. The current was half-maximally activated at -6 mV and steady-state inactivation was half-complete at -65 mV. 4. More sustained outward currents were also observed. Although some cells appeared to express a Ca(2+)-activated K+ current, the most common finding was a rapidly activating, slowly inactivating, voltage-dependent K+ current that was sensitive to TEA and Ba2+. This current resembled M-current more than delayed rectifier but displayed a number of idiosyncratic kinetic properties. Chief amongst these was the accumulation of an inactivating process when the current was repeatedly evoked from potentials near the cells' resting value by voltage steps that by themselves produced no observable inactivation during the voltage command; this behaviour was similar to the 'C-terminal' inactivation exhibited by lymphocytes and certain expressed K+ channel clones (Kv1.3). 5. These results indicate that the granule cells of the islands of Calleja are excitable and contain a number of additional

  18. Complementary Postsynaptic Activity Patterns Elicited in Olfactory Bulb by Stimulation of Mitral/Tufted and Centrifugal Fiber Inputs to Granule Cells

    PubMed Central

    Laaris, Nora; Puche, Adam; Ennis, Matthew

    2009-01-01

    Main olfactory bulb (MOB) granule cells receive spatially segregated glutamatergic synaptic inputs from the dendrites of mitral/tufted cells as well as from the axons of centrifugal fibers (CFFs) originating in olfactory cortical areas. Dendrodendritic synapses from mitral/tufted cells occur on granule cell distal dendrites in the external plexiform layer (EPL), whereas CFFs preferentially target the somata/proximal dendrites of granule cells in the granule cell layer (GCL). In the present study, tract tracing, and recordings of field potentials and voltage-sensitive dye optical signals were used to map activity patterns elicited by activation of these two inputs to granule cells in mouse olfactory bulb slices. Stimulation of the lateral olfactory tract (LOT) produced a negative field potential in the EPL and a positivity in the GCL. CFF stimulation produced field potentials of opposite polarity in the EPL and GCL to those elicited by LOT. LOT-evoked optical signals appeared in the EPL and spread subsequently to deeper layers, whereas CFF-evoked responses appeared in the GCL and then spread superficially. Evoked responses were reduced by N-methyl-d-aspartate (NMDA) receptor antagonists and completely suppressed by AMPA receptor antagonists. Reduction of extracellular Mg2+ enhanced the strength and spatiotemporal extent of the evoked responses. These and additional findings indicate that LOT- and CFF-evoked field potentials and optical signals reflect postsynaptic activity in granule cells, with moderate NMDA and dominant AMPA receptor components. Taken together, these results demonstrate that LOT and CFF stimulation in MOB slices selectively activate glutamatergic inputs to the distal dendrites versus somata/proximal dendrites of granule cells. PMID:17035366

  19. Excitatory tonus is required for the survival of granule cell precursors during postnatal development within the cerebellum.

    PubMed

    Kanungo, A K; Liadis, N; Robertson, J; Woo, M; Henderson, J T

    2009-02-18

    In addition to protective effects within the adult central nervous system (CNS), in vivo application of N-methyl-d-aspartate inhibitors such as (+) MK-801 have been shown to induce neurodegeneration in neonatal rats over a specific developmental period. We have systematically mapped the nature and extent of MK-801-induced neurodegeneration throughout the neonatal murine brain in order to genetically dissect the mechanism of these effects. Highest levels of MK-801-induced neurodegeneration are seen in the cerebellar external germinal layer; while mature neurons of the internal granule layer are unaffected by MK-801 treatment. Examination of external germinal layer neurons by electron microscopy, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and bromodeoxyuridine (BrdU) labeling, and caspase-3 activation demonstrate that these neurons die through the process of programmed cell death soon after they exit from the cell cycle. Significantly, ablation of caspase-3 activity completely inhibited the MK-801-induced (and developmental) programmed cell death of external germinal layer neurons. Similar to caspase-3, inactivation of muscarinic acetylcholine receptors in vivo using scopolamine inhibited MK-801-induced programmed cell death. By contrast, the GABAergic agonist diazepam, either alone or in combination with MK-801, enhanced programmed cell death within external germinal layer neurons. These data demonstrate that, in vivo, cerebellar granule neurons undergo a dramatic change in intracellular signaling in response to molecules present in the local cellular milieu during their first 24 h following exit from the cell cycle.

  20. Identification of a C1q family member associated with cortical granules and follicular cell apoptosis in Carassius auratus gibelio.

    PubMed

    Mei, Jie; Chen, Bo; Yue, Huamei; Gui, Jian-Fang

    2008-07-16

    C1q family proteins with C1q domain have been reported in vertebrates, but their biological roles are currently unknown. In this study, a C1q-like factor, designated Carassius auratus gibelio ovary-specific C1q-like factor (CagOC1q-like), was identified as a cortical granules component. Immunofluorescence localization revealed that the C1q family member was specifically expressed in follicular epithelial cells, and associated with cortical granules in fully grown oocytes. Moreover, it was discharged to the perivitelline space and egg envelope upon fertilization. As it is the first identified C1q family member that is expressed in follicular cells that surround oocyte, CagOC1q-like was applied to detection of follicular cell apoptosis and deletion. The entire cytological process of follicular cell apoptosis and deletion was clearly seen from double visualizations of follicular cells with CagOC1q-like immunofluorescence and apoptotic follicular cells labeled by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) during oocyte maturation and ovulation.

  1. Anion exchange polymer coated graphite granule electrodes for improving the performance of anodes in unbuffered microbial fuel cells

    NASA Astrophysics Data System (ADS)

    Wang, Xu; Li, Dengfeng; Mao, Xuhui; Yu, Eileen Hao; Scott, Keith; Zhang, Enren; Wang, Dihua

    2016-10-01

    In this paper, graphite granule composite electrodes are prepared for microbial fuel cells (MFCs) by coating commercial graphite granules with the mixture of quaternary DABCO polysulfone or Nafion ion exchange polymer and carbon black. The results of electrochemical impedance spectroscopy (EIS) suggest that the addition of carbon black could significantly improve the electrical conductivity of graphite granule anodes. When phosphate buffer solution (PBS) is replaced by NaCl solution, the current densities of the pristine anode, 0.08 g Nafion coated anode and 0.16 g QDPSU coated anode decrease by 52.6%, 20.6% and 10.3% at -0.2 V (vs. Ag/AgCl), respectively. The solution resistance of ion exchange polymer coated anodes is more stable in comparison with that of pristine anode. After 40 operational days, the performance drop of 0.16 g QDPSU coated anode when switching the solution from PBS to NaCl is still smaller than that of pristine anode. However, 0.08 g Nafion coated anode shows the similar performance in NaCl solution to the pristine anode after long term operation. This study reveals that QDPSU anion exchange polymer is more suitable for the anode modification. The QDPSU coated anode promises a great potential for three-dimensional anode based MFCs to treat domestic wastewater.

  2. Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis

    PubMed Central

    Griffin, Nicole G.; Wang, Yu; Hulette, Christine M.; Halvorsen, Matt; Cronin, Kenneth D.; Walley, Nicole M.; Haglund, Michael M.; Radtke, Rodney A.; Pate Skene, J. H.; Sinha, Saurabh R.; Heinzen, Erin L.

    2015-01-01

    Summary Objective Hippocampal sclerosis is the most common neuropathological finding in medically intractable cases of mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of mesial temporal lobe epilepsy patients with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations and to shed light on the transcriptional changes associated with hippocampal sclerosis. Methods RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from mesial temporal lobe epilepsy patients with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. Results Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes upregulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. Significance By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from mesial temporal lobe epilepsy patients with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology

  3. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats

    PubMed Central

    1992-01-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats. PMID:1371773

  4. Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats.

    PubMed

    Husmann, K; Faissner, A; Schachner, M

    1992-03-01

    The extracellular matrix molecule tenascin has been implicated in neuron-glia recognition in the developing central and peripheral nervous system and in regeneration. In this study, its role in Bergmann glial process-mediated neuronal migration was assayed in vitro using tissue explants of the early postnatal mouse cerebellar cortex. Of the five mAbs reacting with nonoverlapping epitopes on tenascin, mAbs J1/tn1, J1/tn4, and J1/tn5, but not mAbs J1/tn2 and J1/tn3 inhibited granule cell migration. Localization of the immunoreactive domains by EM of rotary shadowed tenascin molecules revealed that the mAbs J1/tn4 and J1/tn5, like the previously described J1/tn1 antibody, bound between the third and fifth fibronectin type III homologous repeats and mAb J1/tn3 bound between the third and fifth EGF-like repeats. mAb J1/tn2 had previously been found to react between fibronectin type III homologous repeats 10 and 11 of the mouse molecule (Lochter, A., L. Vaughan, A. Kaplony, A. Prochiantz, M. Schachner, and A. Faissner. 1991. J. Cell Biol. 113:1159-1171). When postnatal granule cell neurons were cultured on tenascin adsorbed to polyornithine, both the percentage of neurite-bearing cells and the length of outgrowing neurites were increased when compared to neurons growing on polyornithine alone. This neurite outgrowth promoting effect of tenascin was abolished only by mAb J1/tn2 or tenascin added to the culture medium in soluble form. The other antibodies did not modify the stimulatory or inhibitory effects of the molecule. These observations indicate that tenascin influences neurite outgrowth and migration of cerebellar granule cells by different domains in the fibronectin type III homologous repeats.

  5. Effects of spaced learning in the water maze on development of dentate granule cells generated in adult mice.

    PubMed

    Trinchero, Mariela F; Koehl, Muriel; Bechakra, Malik; Delage, Pauline; Charrier, Vanessa; Grosjean, Noelle; Ladeveze, Elodie; Schinder, Alejandro F; Abrous, D Nora

    2015-11-01

    New dentate granule cells (GCs) are generated in the hippocampus throughout life. These adult-born neurons are required for spatial learning in the Morris water maze (MWM). In rats, spatial learning shapes the network by regulating their number and dendritic development. Here, we explored whether such modulatory effects exist in mice. New GCs were tagged using thymidine analogs or a GFP-expressing retrovirus. Animals were exposed to a reference memory protocol for 10-14 days (spaced training) at different times after newborn cells labeling. Cell proliferation, cell survival, cell death, neuronal phenotype, and dendritic and spine development were examined using immunohistochemistry. Surprisingly, spatial learning did not modify any of the parameters under scrutiny including cell number and dendritic morphology. These results suggest that although new GCs are required in mice for spatial learning in the MWM, they are, at least for the developmental intervals analyzed here, refractory to behavioral stimuli generated in the course of learning in the MWM.

  6. Functional and molecular analysis of transient voltage-dependent K+ currents in rat hippocampal granule cells

    PubMed Central

    Riazanski, Vladimir; Becker, Albert; Chen, Jian; Sochivko, Dmitry; Lie, Ailing; Wiestler, Otmar D; Elger, Christian E; Beck, Heinz

    2001-01-01

    We have investigated voltage-dependent outward K+ currents of dentate granule cells (DGCs) in acute brain slices from young and adult rats using nucleated and outside-out patch recordings. In adult DGCs, the outward current pattern was dominated by a transient K+ current component. One portion of this current (∼60 %) was blocked by micromolar concentrations of tetraethylammonium (TEA; IC50 42 μm) and BDS-I, a specific blocker of Kv3.4 subunits (2.5 μm). A second component was insensitive to tetraethylammonium (10 mm) and BDS-I. The transient outward current could be completely blocked by 4-aminopyridine (IC50 296 μm). The TEA- and BDS-I-sensitive and the TEA-resistant current components were isolated pharmacologically. The current component that was blocked by BDS-I and TEA showed a depolarized threshold of activation (∼-30 mV) reminiscent of Kv3.4 subunits, while the current component resistant to TEA activated at more hyperpolarized potentials (∼-60 mV). In nucleated patches obtained by placing the patch pipette adjacent to the apical dendrite, only small Na+ currents and small BDS-I-sensitive transient currents were detected. Nucleated patches obtained from either the cell soma (see above) or the axon hillock showed significantly larger amplitude Na+ currents as well as larger BDS-I-sensitive currents, indicating that this current was predominantly localized within the axosomatic compartment. This result was in good agreement with the distribution of Kv3.4 protein as determined by immunohistochemistry. Current-clamp as well as mock action potential-clamp experiments revealed that the BDS-sensitive current component contributes to action potential repolarization. A comparison of the two age groups (4-10 days and 60-100 days) revealed a marked developmental up-regulation of the BDS-I-sensitive component. These functional changes are paralleled by a developmental increase in Kv3.4 mRNA expression determined by quantitative real-time RT-PCR, as well as a

  7. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    PubMed

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  8. Environmental Pb2+ exposure during early life alters granule cell neurogenesis and morphology in the hippocampus of young adult rats

    PubMed Central

    Verina, Tatyana; Rohde, Charles A.; Guilarte, Tomás R.

    2007-01-01

    Exposure to environmentally relevant levels of lead (Pb2+) during early life produces deficits in hippocampal synaptic plasticity in the form of long-term potentiation (LTP) and spatial learning in young adult rats (Nihei et al., 2000; Guilarte et al., 2003). Other evidence suggests that the performance of rats in the Morris water maze spatial learning tasks is associated with the level of granule cell neurogenesis in the dentate gyrus (DG) (Drapeau et al., 2003). In this study, we examined whether continuous exposure to environmentally relevant levels of Pb2+ during early life altered granule cell neurogenesis and morphology in the rat hippocampus. Control and Pb2+-exposed rats received BrdU injections (100 mg/kg; i.p.) for five consecutive days starting at postnatal day 45 and were sacrificed either one day or four weeks after the last injection. The total number of newborn cells in the DG of Pb2+-exposed rats was significantly decreased (13%; p<0.001) one day after BrdU injections relative to controls. Further, the survival of newborn cells in Pb2+-exposed rats was significantly decreased by 22.7% (p<0.001) relative to control animals. Co-localization of BrdU with neuronal or astrocytic markers did not reveal a significant effect of Pb2+ exposure on cellular fate. In Pb2+-exposed rats, immature granule cells immunolabeled with doublecortin (DCX) displayed aberrant dendritic morphology. That is, the overall length-density of the DCX-positive apical dendrites in the outer portion of the DG molecular layer was significantly reduced up to 36% in the suprapyramidal blade only. We also found that the area of Timm’s-positive staining representative of the mossy fibers terminal fields in the CA3 stratum oriens (SO) was reduced by 26% in Pb2+-exposed rats. These findings demonstrate that exposure to environmentally relevant levels of Pb2+ during early life alter granule cell neurogenesis and morphology in the rat hippocampus. They provide a cellular and morphological

  9. The granule cell density of the dentate gyrus following administration of Urtica dioica extract to young diabetic rats.

    PubMed

    Fazeli, S A; Gharravi, A M; Ghafari, S; Jahanshahi, M; Golalipour, M J

    2008-08-01

    Urtica dioica L. Stinging nettle has long been known worldwide as a medicinal plant. To study the benefits of the nettle in diabetic encephalopathy, the granule cell density of the dentate gyrus of diabetic rats was studied following administration of Urtica dioica extract. A total of 24 male albino Wistar rats were allocated equally to normal, diabetic, preventive and treatment groups. Hyperglycaemia was induced by streptozotocin (80 mg/kg) in the animals of the diabetic and treatment groups. One week after injection of the streptozotocin the animals in the treatment group received a hydroalcoholic extract of Urtica dioica (100 mg/kg/day) for 4 weeks intraperitoneally. The rats of the preventive group received hydroalcoholic extract of U. dioica (100 mg/kg/day) IP for the first 5 days and an injection of streptozotocin (80 mg/kg) on the 6th day. After 5 weeks of study all the rats were sacrificed and coronal sections were taken from the dorsal hippocampal formation of the right cerebral hemispheres and stained with cresyl violet. The area densities of the granule cells were measured and compared in the four groups. The density was lower in the diabetic rats compared with the controls (p > 0.05). The preventive group showed lower cell density than the controls (p > 0.05). The densities in the treated rats were higher than in the diabetic rats (p > 0.05). Furthermore, the control and treated rats showed similar densities (p > 0.05). It seems that U. dioica extract can help compensate for granule cell loss in the diabetic rat dentate gyrus, which can ameliorate cognitive impairment in diabetes. However, preventive use of the extract showed no significant benefit.

  10. Activation of κ opioid receptors increases intrinsic excitability of dentate gyrus granule cells

    PubMed Central

    McDermott, Carmel M; Schrader, Laura A

    2011-01-01

    Abstract The dentate gyrus of the hippocampus is thought to control information flow into the rest of the hippocampus. Under pathological conditions, such as epilepsy, this protective feature is circumvented and uninhibited activity flows throughout the hippocampus. Many factors can modulate excitability of the dentate gyrus and ultimately, the hippocampus. It is therefore of critical importance to understand the mechanisms involved in regulating excitability in the dentate gyrus. Dynorphin, the endogenous ligand for the kappa (κ) opioid receptor (KOR), is thought to be involved in neuromodulation in the dentate gyrus. Both dynorphin and its receptor are widely expressed in the dentate gyrus and have been implicated in epilepsy and other complex behaviours such as stress-induced deficits in learning and stress-induced depression-like behaviours. Administration of KOR agonists can prevent both the behavioural and electroencephalographic measures of seizures in several different models of epilepsy. Antagonism of the KORs also prevents stress-induced behaviours. This evidence suggests the KORs as possible therapeutic targets for various pathological conditions. In addition, KOR agonists prevent the induction of LTP. Although there are several mechanisms through which dynorphin could mediate these effects, no studies to date investigated the effects of KOR activation on intrinsic membrane properties and cell excitability. We used whole-cell, patch-clamp recordings from acute mouse hippocampus slices to investigate the effect of KOR activation on dentate gyrus granule cell excitability. The agonist U69,593 (U6, 1 μm) resulted in a lower spike threshold, a decreased latency to first spike, an increased spike half-width, and an overall increase in spike number with current injections ranging from 15 to 45 pA. There was also a reduction in the interspike interval (ISI) both early and late in the spike train, with no change in membrane potential or input resistance

  11. Hydrogen sulfide induces hyperpolarization and decreases the exocytosis of secretory granules of rat GH3 pituitary tumor cells.

    PubMed

    Mustafina, Alsu N; Yakovlev, Aleksey V; Gaifullina, Aisylu Sh; Weiger, Thomas M; Hermann, Anton; Sitdikova, Guzel F

    2015-10-02

    The aim of the present study was to evaluate the effects of hydrogen sulfide (H2S) on the membrane potential, action potential discharge and exocytosis of secretory granules in neurosecretory pituitary tumor cells (GH3). The H2S donor - sodium hydrosulfide (NaHS) induced membrane hyperpolarization, followed by truncation of spontaneous electrical activity and decrease of the membrane resistance. The NaHS effect was dose-dependent with an EC50 of 152 μM (equals effective H2S of 16-19 μM). NaHS effects were not altered after inhibition of maxi conductance calcium-activated potassium (BK) channels by tetraethylammonium or paxilline, but were significantly reduced after inhibition or activation of ATP-dependent potassium channels (KATP) by glibenclamide or by diazoxide, respectively. In whole-cell recordings NaHS increased the amplitude of KATP currents, induced by hyperpolarizing pulses and subsequent application of glibenclamide decreased currents to control levels. Using the fluorescent dye FM 1-43 exocytosis of secretory granules was analyzed in basal and stimulated conditions (high K(+) external solution). Prior application of NaHS decreased the fluorescence of the cell membrane in both conditions which links with activation of KATP currents (basal secretion) and activation of KATP currents and BK-currents (stimulated exocytosis). We suggest that H2S induces hyperpolarization of GH3 cells by activation of KATP channels which results in a truncation of spontaneous action potentials and a decrease of hormone release.

  12. Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells

    PubMed Central

    Li, Xiang-Zhou

    2016-01-01

    Eucommia ulmoides Oliv. (E. ulmoides Oliv.) and moso bamboo (Phyllostachys pubescens) leaves are used as folk medicines in central-western China to treat diabetes. To investigate the hypoglycemic activity of the effervescent granules prepared using E. ulmoides Oliv. and moso bamboo leaves (EBEG) in HepG2 cells, EBEG were prepared with 5% of each of polysaccharides and chlorogenic acids from moso bamboo and E. ulmoides Oliv. leaves, respectively. HepG2 cells cultured in a high-glucose medium were classified into different groups. The results displayed EBEG-treated cells showed better glucose utilization than the negative controls; thus, the hypoglycemic effect of EBEG was much greater than that of granules prepared using either component alone, thereby indicating that this effect was due to a synergistic action of the components. Further, glucose consumption levels in the cells treated with EBEG (156.35% at 200 μg/mL) and the positive controls (metformin, 162.29%; insulin, 161.52%) were similar. Thus, EBEG exhibited good potential for use as a natural antidiabetic agent. The hypoglycemic effect of EBEG could be due to the synergistic action of polysaccharides from the moso bamboo leaves and chlorogenic acids from E. ulmoides Oliv. leaves via the inhibition of alpha-glucosidase and glucose-6-phosphate displacement enzyme. PMID:27656239

  13. Effervescent Granules Prepared Using Eucommia ulmoides Oliv. and Moso Bamboo Leaves: Hypoglycemic Activity in HepG2 Cells.

    PubMed

    Li, Xiang-Zhou; Zhang, Sheng

    2016-01-01

    Eucommia ulmoides Oliv. (E. ulmoides Oliv.) and moso bamboo (Phyllostachys pubescens) leaves are used as folk medicines in central-western China to treat diabetes. To investigate the hypoglycemic activity of the effervescent granules prepared using E. ulmoides Oliv. and moso bamboo leaves (EBEG) in HepG2 cells, EBEG were prepared with 5% of each of polysaccharides and chlorogenic acids from moso bamboo and E. ulmoides Oliv. leaves, respectively. HepG2 cells cultured in a high-glucose medium were classified into different groups. The results displayed EBEG-treated cells showed better glucose utilization than the negative controls; thus, the hypoglycemic effect of EBEG was much greater than that of granules prepared using either component alone, thereby indicating that this effect was due to a synergistic action of the components. Further, glucose consumption levels in the cells treated with EBEG (156.35% at 200 μg/mL) and the positive controls (metformin, 162.29%; insulin, 161.52%) were similar. Thus, EBEG exhibited good potential for use as a natural antidiabetic agent. The hypoglycemic effect of EBEG could be due to the synergistic action of polysaccharides from the moso bamboo leaves and chlorogenic acids from E. ulmoides Oliv. leaves via the inhibition of alpha-glucosidase and glucose-6-phosphate displacement enzyme.

  14. 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.

  15. Interactions between Inhibitory Interneurons and Excitatory Associational Circuitry in Determining Spatio-Temporal Dynamics of Hippocampal Dentate Granule Cells: A Large-Scale Computational Study

    PubMed Central

    Hendrickson, Phillip J.; Yu, Gene J.; Song, Dong; Berger, Theodore W.

    2015-01-01

    This paper reports on findings from a million-cell granule cell model of the rat dentate gyrus that was used to explore the contributions of local interneuronal and associational circuits to network-level activity. The model contains experimentally derived morphological parameters for granule cells, which each contain approximately 200 compartments, and biophysical parameters for granule cells, basket cells, and mossy cells that were based both on electrophysiological data and previously published models. Synaptic input to cells in the model consisted of glutamatergic AMPA-like EPSPs and GABAergic-like IPSPs from excitatory and inhibitory neurons, respectively. The main source of input to the model was from layer II entorhinal cortical neurons. Network connectivity was constrained by the topography of the system, and was derived from axonal transport studies, which provided details about the spatial spread of axonal terminal fields, as well as how subregions of the medial and lateral entorhinal cortices project to subregions of the dentate gyrus. Results of this study show that strong feedback inhibition from the basket cell population can cause high-frequency rhythmicity in granule cells, while the strength of feedforward inhibition serves to scale the total amount of granule cell activity. Results furthermore show that the topography of local interneuronal circuits can have just as strong an impact on the development of spatio-temporal clusters in the granule cell population as the perforant path topography does, both sharpening existing clusters and introducing new ones with a greater spatial extent. Finally, results show that the interactions between the inhibitory and associational loops can cause high frequency oscillations that are modulated by a low-frequency oscillatory signal. These results serve to further illustrate the importance of topographical constraints on a global signal processing feature of a neural network, while also illustrating how rich

  16. Cysteine string protein (CSP) is an insulin secretory granule-associated protein regulating beta-cell exocytosis.

    PubMed Central

    Brown, H; Larsson, O; Bränström, R; Yang, S N; Leibiger, B; Leibiger, I; Fried, G; Moede, T; Deeney, J T; Brown, G R; Jacobsson, G; Rhodes, C J; Braun, J E; Scheller, R H; Corkey, B E; Berggren, P O; Meister, B

    1998-01-01

    Cysteine string proteins (CSPs) are novel synaptic vesicle-associated protein components characterized by an N-terminal J-domain and a central palmitoylated string of cysteine residues. The cellular localization and functional role of CSP was studied in pancreatic endocrine cells. In situ hybridization and RT-PCR analysis demonstrated CSP mRNA expression in insulin-producing cells. CSP1 mRNA was present in pancreatic islets; both CSP1 and CSP2 mRNAs were seen in insulin-secreting cell lines. Punctate CSP-like immunoreactivity (CSP-LI) was demonstrated in most islets of Langerhans cells, acinar cells and nerve fibers of the rat pancreas. Ultrastructural analysis showed CSP-LI in close association with membranes of secretory granules of cells in the endo- and exocrine pancreas. Subcellular fractionation of insulinoma cells showed CSP1 (34/36 kDa) in granular fractions; the membrane and cytosol fractions contained predominantly CSP2 (27 kDa). The fractions also contained proteins of 72 and 70 kDa, presumably CSP dimers. CSP1 overexpression in INS-1 cells or intracellular administration of CSP antibodies into mouse ob/ob beta-cells did not affect voltage-dependent Ca2+-channel activity. Amperometric measurements showed a significant decrease in insulin exocytosis in individual INS-1 cells after CSP1 overexpression. We conclude that CSP is associated with insulin secretory granules and that CSP participates in the molecular regulation of insulin exocytosis by mechanisms not involving changes in the activity of voltage-gated Ca2+-channels. PMID:9724640

  17. Pattern of rise in subplasma membrane Ca{sup 2+} concentration determines type of fusing insulin granules in pancreatic {beta} cells

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Nakamichi, Yoko; Nishiwaki, Chiyono; Sakurai, Takashi; Nagamatsu, Shinya

    2009-07-31

    We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca{sup 2+} concentration ([Ca{sup 2+}]{sub PM}) with the Ca{sup 2+} indicator Fura Red in rat {beta} cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca{sup 2+}]{sub PM} caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca{sup 2+}]{sub PM} induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca{sup 2+}]{sub PM} rise directly determines the types of fusing granules.

  18. Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest.

    PubMed

    Diederen, J H; Vullings, H G

    1995-03-01

    The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the trans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horse-radish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.

  19. Tonic Inhibitory Control of Dentate Gyrus Granule Cells by α5-Containing GABAA Receptors Reduces Memory Interference

    PubMed Central

    Zarnowska, Ewa D.; Benke, Dietmar; Tsvetkov, Evgeny; Sigal, Maksim; Keist, Ruth; Bolshakov, Vadim Y.; Pearce, Robert A.; Rudolph, Uwe

    2015-01-01

    Interference between similar or overlapping memories formed at different times poses an important challenge on the hippocampal declarative memory system. Difficulties in managing interference are at the core of disabling cognitive deficits in neuropsychiatric disorders. Computational models have suggested that, in the normal brain, the sparse activation of the dentate gyrus granule cells maintained by tonic inhibitory control enables pattern separation, an orthogonalization process that allows distinct representations of memories despite interference. To test this mechanistic hypothesis, we generated mice with significantly reduced expression of the α5-containing GABAA (α5-GABAARs) receptors selectively in the granule cells of the dentate gyrus (α5DGKO mice). α5DGKO mice had reduced tonic inhibition of the granule cells without any change in fast phasic inhibition and showed increased activation in the dentate gyrus when presented with novel stimuli. α5DGKO mice showed impairments in cognitive tasks characterized by high interference, without any deficiencies in low-interference tasks, suggesting specific impairment of pattern separation. Reduction of fast phasic inhibition in the dentate gyrus through granule cell-selective knock-out of α2-GABAARs or the knock-out of the α5-GABAARs in the downstream CA3 area did not detract from pattern separation abilities, which confirms the anatomical and molecular specificity of the findings. In addition to lending empirical support to computational hypotheses, our findings have implications for the treatment of interference-related cognitive symptoms in neuropsychiatric disorders, particularly considering the availability of pharmacological agents selectively targeting α5-GABAARs. SIGNIFICANCE STATEMENT Interference between similar memories poses a significant limitation on the hippocampal declarative memory system, and impaired interference management is a cognitive symptom in many disorders. Thus, understanding

  20. 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.

  1. BOULE, a Deleted in Azoospermia Homolog, Is Recruited to Stress Granules in the Mouse Male Germ Cells

    PubMed Central

    Kim, Byunghyuk; Rhee, Kunsoo

    2016-01-01

    High temperature adversely affects normal development of male germ cells in mammals. Acute heat stress induces the formation of stress granules (SGs) in a set of male germ cells, and the SGs have been proposed to protect those cells from heat-induced apoptosis. DAZL, one of DAZ (Deleted in Azoospermia) family proteins, was shown to be an essential component of SGs, which is required for SG formation in the mouse testis. In the present study, we asked whether BOULE, the founding member of DAZ family proteins, is a component of the SGs. We show that BOULE is recruited to the SGs upon heat stress, and that these SGs are developmental stage-specific. These results suggest that DAZ family proteins may have conserved roles in the SGs of male germ cells. PMID:27632217

  2. Somatically expressed germ-granule components, PGL-1 and PGL-3, repress programmed cell death in C. elegans

    PubMed Central

    Al-Amin, Mohammad; Min, Hyemin; Shim, Yhong-Hee; Kawasaki, Ichiro

    2016-01-01

    We previously reported that germline apoptosis in C. elegans increased by loss of PGL-1 and PGL-3, members of a family of constitutive germ-granule components, from germ cells in adult hermaphrodite gonads. In this study, we found that somatic apoptosis was reduced in synthetic multivulva class B (synMuv B) mutants due to ectopic expression of PGL-1 and PGL-3 in the soma. In synMuv B-mutant somatic cells, CED-4 expression level was reduced due to ectopic expression of PGL-1. Furthermore, in contrast to wild type, somatic apoptosis in synMuv B mutants increased following DNA damage in a SIR-2.1-dependent manner. Intriguingly, somatic apoptosis was repressed not only in synMuv B mutants but also by ectopically expressing pgl-1 and/or pgl-3 transgenes in wild-type somatic cells. Our study demonstrates that germ-granule components, PGL-1 and PGL-3, can serve as negative regulators of apoptosis not only in the germline but also in the soma in C. elegans. PMID:27650246

  3. Chronic corticosterone administration reduces dendritic complexity in mature, but not young granule cells in the rat dentate gyrus

    PubMed Central

    Yau, Suk-Yu; Li, Ang; Tong, Jian-Bin; Bostrom, Crystal; Christie, Brian R.; Lee, Tatia M.C.; So, Kwok-Fai

    2016-01-01

    Background: Our previous work has shown that exposure to the stress hormone corticosterone (40 mg/kg CORT) for two weeks induces dendritic atrophy of pyramidal neurons in the hippocampal CA3 region and behavioral deficits. However, it is unclear whether this treatment also affects the dentate gyrus (DG), a subregion of the hippocampus comprising a heterogeneous population of young and mature neurons. Objective: We examined the effect of CORT treatment on the dendritic complexity of mature and young granule cells in the DG. Methods: We utilized a Golgi staining method to investigate the dendritic morphology and spine density of young neurons in the inner granular cell layer (GCL) and mature neurons in the outer GCL in response to CORT application. The expressions of glucocorticoid receptors during neuronal maturation were examined using Western blot analysis in a primary hippocampal neuronal culture. Results: Sholl analysis revealed that CORT treatment decreased the number of intersections and shortened the dendritic length in mature, but not young, granule cells. However, the spine density of mature and young neurons was not affected. Western blot analysis showed a progressive increase in the protein levels of glucocorticoid receptors (GRs) in the cultured primary hippocampal neurons during neuronal maturation. Conclusion: These data suggest that mature neurons are likely more vulnerable to chronic exposure to CORT; this may be due to their higher expression of GRs when compared to younger DG neurons. PMID:27567758

  4. Preventing Effect of L-Type Calcium Channel Blockade on Electrophysiological Alterations in Dentate Gyrus Granule Cells Induced by Entorhinal Amyloid Pathology

    PubMed Central

    Pourbadie, Hamid Gholami; Naderi, Nima; Mehranfard, Nasrin; Janahmadi, Mahyar; Khodagholi, Fariba; Motamedi, Fereshteh

    2015-01-01

    The entorhinal cortex (EC) is one of the earliest affected brain regions in Alzheimer’s disease (AD). EC-amyloid pathology induces synaptic failure in the dentate gyrus (DG) with resultant behavioral impairment, but there is little known about its impact on neuronal properties in the DG. It is believed that calcium dyshomeostasis plays a pivotal role in the etiology of AD. Here, the effect of the EC amyloid pathogenesis on cellular properties of DG granule cells and also possible neuroprotective role of L-type calcium channel blockers (CCBs), nimodipine and isradipine, were investigated. The amyloid beta (Aβ) 1–42 was injected bilaterally into the EC of male rats and one week later, electrophysiological properties of DG granule cells were assessed. Voltage clamp recording revealed appearance of giant sIPSC in combination with a decrease in sEPSC frequency which was partially reversed by CCBs in granule cells from Aβ treated rats. EC amyloid pathogenesis induced a significant reduction of input resistance (Rin) accompanied by a profound decreased excitability in the DG granule cells. However, daily administration of CCBs, isradipine or nimodipine (i.c.v. for 6 days), almost preserved the normal excitability against Aβ. In conclusion, lower tendency to fire AP along with reduced Rin suggest that DG granule cells might undergo an alteration in the membrane ion channel activities which finally lead to the behavioral deficits observed in animal models and patients with early-stage Alzheimer’s disease. PMID:25689857

  5. Differential expression of cytoskeletal proteins in the dendrites of parvalbumin-positive interneurons versus granule cells in the adult rat dentate gyrus.

    PubMed

    de Haas Ratzliff, A; Soltesz, I

    2000-01-01

    Parvalbumin-positive interneurons and granule cells of the dentate gyrus exhibit characteristic differences in morphological, cytochemical, physiological, and pathophysiological properties. Several of these defining features, including dendritic morphology, spine density, and sensitivity to insults, are likely to be influenced by the neuronal cytoskeleton. The data in this paper demonstrate striking differences in the expression levels of all three neurofilament triplet proteins, as well as alpha-internexin and beta-tubulin III, between the parvalbumin-positive interneurons and dentate granule cells. Therefore, the molecular composition of intermediate filaments and microtubules in the dendritic domain of parvalbumin-positive dentate interneurons is distinct from the cytoskeleton of neighboring granule cells, indicating the existence of highly cell type-specific cytoskeletal architecture within the dentate gyrus.

  6. Delayed dendritic development in newly generated dentate granule cells by cell-autonomous expression of the amyloid precursor protein.

    PubMed

    Morgenstern, Nicolás A; Giacomini, Damiana; Lombardi, Gabriela; Castaño, Eduardo M; Schinder, Alejandro F

    2013-09-01

    Neuronal connectivity and synaptic remodeling are fundamental substrates for higher brain functions. Understanding their dynamics in the mammalian allocortex emerges as a critical step to tackle the cellular basis of cognitive decline that occurs during normal aging and in neurodegenerative disorders. In this work we have designed a novel approach to assess alterations in the dynamics of functional and structural connectivity elicited by chronic cell-autonomous overexpression of the human amyloid precursor protein (hAPP). We have taken advantage of the fact that the hippocampus continuously generates new dentate granule cells (GCs) to probe morphofunctional development of GCs expressing different variants of hAPP in a healthy background. hAPP was expressed together with a fluorescent reporter in neural progenitor cells of the dentate gyrus of juvenile mice by retroviral delivery. Neuronal progeny was analyzed several days post infection (dpi). Amyloidogenic cleavage products of hAPP such as the β-C terminal fragment (β-CTF) induced a substantial reduction in glutamatergic connectivity at 21 dpi, at which time new GCs undergo active growth and synaptogenesis. Interestingly, this effect was transient, since the strength of glutamatergic inputs was normal by 35 dpi. This delay in glutamatergic synaptogenesis was paralleled by a decrease in dendritic length with no changes in spine density, consistent with a protracted dendritic development without alterations in synapse formation. Finally, similar defects in newborn GC development were observed by overexpression of α-CTF, a non-amyloidogenic cleavage product of hAPP. These results indicate that hAPP can elicit protracted dendritic development independently of the amyloidogenic processing pathway.

  7. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed Central

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-01-01

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

  8. Exploring parameter space in detailed single neuron models: simulations of the mitral and granule cells of the olfactory bulb.

    PubMed

    Bhalla, U S; Bower, J M

    1993-06-01

    1. Detailed compartmental computer simulations of single mitral and granule cells of the vertebrate olfactory bulb were constructed using previously published geometric data. Electrophysiological properties were determined by comparing model output to previously published experimental data, mainly current-clamp recordings. 2. The passive electrical properties of each model were explored by comparing model output with intracellular potential data from hyperpolarizing current injection experiments. The results suggest that membrane resistivity in both cells is nonuniform, with somatas having a substantially lower resistivity than the dendrites. 3. The active properties of these cells were explored by incorporating active ion channels into modeled compartments. On the basis of evidence from the literature, the mitral cell model included six channel types: fast sodium, fast delayed rectifier (Kfast), slow delayed rectifier (K), transient outward potassium current (KA), voltage- and calcium-dependent potassium current (KCa), and L-type calcium current. The granule cell model included four channel types: rat brain sodium, K, KA, and the non-inactivating muscarinic potassium current (KM). Modeled channels were based on the Hodgkin-Huxley formalism. 4. Representative kinetics for each of the channel classes above were obtained from the literature. The experimentally unknown spatial distributions of each included channel were obtained by systematic parameter searches. These were conducted in two ways: large-scale simulation series, in which each parameter was varied in turn, and an adaptation of a multidimensional conjugate gradient method. In each case, the simulated results were compared wtih experimental data using a curve-matching function evaluating mean squared differences of several aspects of the simulated and experimental voltage waveforms. 5. Systematic parameter variations revealed a single distinct region of parameter space in which the mitral cell model best

  9. Glomerular and Mitral-Granule Cell Microcircuits Coordinate Temporal and Spatial Information Processing in the Olfactory Bulb

    PubMed Central

    Cavarretta, Francesco; Marasco, Addolorata; Hines, Michael L.; Shepherd, Gordon M.; Migliore, Michele

    2016-01-01

    The olfactory bulb processes inputs from olfactory receptor neurons (ORNs) through two levels: the glomerular layer at the site of input, and the granule cell level at the site of output to the olfactory cortex. The sequence of action of these two levels has not yet been examined. We analyze this issue using a novel computational framework that is scaled up, in three-dimensions (3D), with realistic representations of the interactions between layers, activated by simulated natural odors, and constrained by experimental and theoretical analyses. We suggest that the postulated functions of glomerular circuits have as their primary role transforming a complex and disorganized input into a contrast-enhanced and normalized representation, but cannot provide for synchronization of the distributed glomerular outputs. By contrast, at the granule cell layer, the dendrodendritic interactions mediate temporal decorrelation, which we show is dependent on the preceding contrast enhancement by the glomerular layer. The results provide the first insights into the successive operations in the olfactory bulb, and demonstrate the significance of the modular organization around glomeruli. This layered organization is especially important for natural odor inputs, because they activate many overlapping glomeruli. PMID:27471461

  10. Progressive behavioral changes during the maturation of rats with early radiation-induced hypoplasia of fascia dentata granule cells

    SciTech Connect

    Mickley, G.A.; Ferguson, J.L.; Mulvihill, M.A.; Nemeth, T.J.

    1989-01-01

    Localized exposure of the neonatal rat brain to x rays produces neuronal hypoplasia specific to the granule cell layer of the hippocampal dentate gyrus. This brain damage causes locomotor hyperactivity, slowed acquisition of passive avoidance tasks and long bouts of spontaneous turning (without reversals) in a bowl apparatus. The authors report here how these behavioral deficits change as a function of subject aging and behavioral test replications. Portions of the neonatal rat cerebral hemispheres were X-irradiated in order to selectively damage the granule cells of the dentate gyrus. Rats between the ages of 71-462 days were tested 3 separate times on each of the following 3 behavioral tests: (1) spontaneous locomotion, (2) passive avoidance acquisition, and (3) spontaneous circling in a large plastic hemisphere. Rats with radiation-induced damage to the fascia dentata exhibited long bouts of slow turns without reversals. Once they began, irradiated subjects perseverated in turning to an extent significantly greater than sham-irradiated control subjects. The hyperactivity of the irradiated animals decreased significantly as they matured. These data suggest that radiation-induced damage to the fascia dentata produces task-dependent behavioral deficits that change as a function of subject age and/or behavioral testing.

  11. Granule cell ascending axon excitatory synapses onto Golgi cells implement a potent feedback circuit in the cerebellar granular layer.

    PubMed

    Cesana, Elisabetta; Pietrajtis, Katarzyna; Bidoret, Céline; Isope, Philippe; D'Angelo, Egidio; Dieudonné, Stéphane; Forti, Lia

    2013-07-24

    The function of inhibitory interneurons within brain microcircuits depends critically on the nature and properties of their excitatory synaptic drive. Golgi cells (GoCs) of the cerebellum inhibit cerebellar granule cells (GrCs) and are driven both by feedforward mossy fiber (mf) and feedback GrC excitation. Here, we have characterized GrC inputs to GoCs in rats and mice. We show that, during sustained mf discharge, synapses from local GrCs contribute equivalent charge to GoCs as mf synapses, arguing for the importance of the feedback inhibition. Previous studies predicted that GrC-GoC synapses occur predominantly between parallel fibers (pfs) and apical GoC dendrites in the molecular layer (ML). By combining EM and Ca(2+) imaging, we now demonstrate the presence of functional synaptic contacts between ascending axons (aa) of GrCs and basolateral dendrites of GoCs in the granular layer (GL). Immunohistochemical quantification estimates these contacts to be ∼400 per GoC. Using Ca(2+) imaging to identify synaptic inputs, we show that EPSCs from aa and mf contacts in basolateral dendrites display similarly fast kinetics, whereas pf inputs in the ML exhibit markedly slower kinetics as they undergo strong filtering by apical dendrites. We estimate that approximately half of the local GrC contacts generate fast EPSCs, indicating their basolateral location in the GL. We conclude that GrCs, through their aa contacts onto proximal GoC dendrites, define a powerful feedback inhibitory circuit in the GL.

  12. Apoptosis of cerebellar granule cells induced by organotin compounds found in drinking water: involvement of MAP kinases.

    PubMed

    Mundy, William R; Freudenrich, Theresa M

    2006-01-01

    Mono- and dialkyl organotin compounds are used primarily as heat stabilizers in polyvinyl chloride (PVC) plastics. Recently, monomethyltin (MMT), dimethyltin (DMT), monobutyltin (MBT), and dibutyltin (DBT) have been detected in water from homes and businesses served by PVC pipes. While trialkyl organotins such as trimethyltin (TMT) and triethyltin (TET) are well known neurotoxicants, the toxicity of the mono- and dialkyl organotins is not well described. The present study compared the cytotoxicity of organotins found in drinking water with the known neurotoxicant TMT in primary cultures of cerebellar granule cells, and examined the role of MAP kinase signaling in organotin-induced cell death. Twenty-four hour exposure to TMT resulted in a concentration-dependent decrease in cell viability with an EC(50) of 3 microM. Exposure to MMT, DMT, and MBT at concentrations up to 10 microM had no effect. DBT, however, was very potent, and decreased cell viability with an EC(50) of 0.3 microM. Staining of organotin-treated cerebellar granule cells with the nuclear dye Syto-13 revealed that TMT and DBT, but not MMT, DMT, or MBT, produced condensation and fragmentation of chromatin characteristic of apoptosis. TMT- and DBT-induced apoptosis was confirmed using TUNEL staining and measurement of PARP cleavage. Activation of MAP kinase pathways was examined after 6 h of exposure to the organotins which induced apoptosis. Both TMT and DBT activated ERK1/2, but only TMT activated the JNK/c-Jun and p38 pathways. Pharmacologic blockade of JNK/c-Jun and p38 activation significantly decreased apoptosis produced by TMT, but not by DBT. These results show that DBT is a potent neurotoxicant in vitro, but unlike TMT, does not induce cell death via activation of MAP kinase signaling.

  13. Granule Associated Serine Proteases of Hematopoietic Cells – An Analysis of Their Appearance and Diversification during Vertebrate Evolution

    PubMed Central

    Akula, Srinivas; Thorpe, Michael; Boinapally, Vamsi; Hellman, Lars

    2015-01-01

    Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish. PMID:26569620

  14. An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse

    PubMed Central

    Bertrand, Florie; Müller, Sabina; Roh, Kyung-Ho; Laurent, Camille; Dupré, Loïc; Valitutti, Salvatore

    2013-01-01

    It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide–MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell–cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery. PMID:23536289

  15. New insights into the role of hilar ectopic granule cells in the dentate gyrus based on quantitative anatomic analysis and three-dimensional reconstruction

    PubMed Central

    Scharfman, Helen E.; Pierce, Joseph P.

    2014-01-01

    SUMMARY The dentate gyrus is one of two main areas of the mammalian brain where neurons are born throughout adulthood, a phenomenon called postnatal neurogenesis. Most of the neurons that are generated are granule cells (GCs), the major principal cell type in the dentate gyrus. Some adult-born granule cells develop in ectopic locations, such as the dentate hilus. The generation of hilar ectopic granule cells (HEGCs) is greatly increased in several animal models of epilepsy and has also been demonstrated in surgical specimens from patients with intractable temporal lobe epilepsy (TLE). Herein we review the results of our quantitative neuroanatomic analysis of HEGCs that were filled with Neurobiotin following electrophysiologic characterization in hippocampal slices. The data suggest that two types of HEGCs exist, based on a proximal or distal location of the cell body relative to the granule cell layer, and based on the location of most of the dendrites, in the molecular layer or hilus. Three-dimensional reconstruction revealed that the dendrites of distal HEGCs can extend along the transverse and longitudinal axis of the hippocampus. Analysis of axons demonstrated that HEGCs have projections that contribute to the normal mossy fiber innervation of CA3 as well as the abnormal sprouted fibers in the inner molecular layer of epileptic rodents (mossy fiber sprouting). These data support the idea that HEGCs could function as a “hub” cell in the dentate gyrus and play a critical role in network excitability. PMID:22612815

  16. Lack of Intestinal Epithelial Atg7 Affects Paneth Cell Granule Formation but Does Not Compromise Immune Homeostasis in the Gut

    PubMed Central

    Wittkopf, Nadine; Günther, Claudia; Martini, Eva; Waldner, Maximilian; Amann, Kerstin U.; Neurath, Markus F.; Becker, Christoph

    2012-01-01

    Genetic polymorphisms of autophagy-related genes have been associated with an increased risk to develop inflammatory bowel disease (IBD). Autophagy is an elementary process participating in several cellular events such as cellular clearance and nonapoptotic programmed cell death. Furthermore, autophagy may be involved in intestinal immune homeostasis due to its participation in the digestion of intracellular pathogens and in antigen presentation. In the present study, the role of autophagy in the intestinal epithelial layer was investigated. The intestinal epithelium is essential to maintain gut homeostasis, and defects within this barrier have been associated with the pathogenesis of IBD. Therefore, mice with intestinal epithelial deletion of Atg7 were generated and investigated in different mouse models. Knockout mice showed reduced size of granules and decreased levels of lysozyme in Paneth cells. However, this was dispensable for gut immune homeostasis and had no effect on susceptibility in mouse models of experimentally induced colitis. PMID:22291845

  17. Population imaging at subcellular resolution supports specific and local inhibition by granule cells in the olfactory bulb

    PubMed Central

    Wienisch, Martin; Murthy, Venkatesh N.

    2016-01-01

    Information processing in early sensory regions is modulated by a diverse range of inhibitory interneurons. We sought to elucidate the role of olfactory bulb interneurons called granule cells (GCs) in odor processing by imaging the activity of hundreds of these cells simultaneously in mice. Odor responses in GCs were temporally diverse and spatially disperse, with some degree of non-random, modular organization. The overall sparseness of activation of GCs was highly correlated with the extent of glomerular activation by odor stimuli. Increasing concentrations of single odorants led to proportionately larger population activity, but some individual GCs had non-monotonic relations to concentration due to local inhibitory interactions. Individual dendritic segments could sometimes respond independently to odors, revealing their capacity for compartmentalized signaling in vivo. Collectively, the response properties of GCs point to their role in specific and local processing, rather than global operations such as response normalization proposed for other interneurons. PMID:27388949

  18. Synaptic excitation of individual rat cerebellar granule cells in situ: evidence for the role of NMDA receptors.

    PubMed Central

    D'Angelo, E; De Filippi, G; Rossi, P; Taglietti, V

    1995-01-01

    1. Current-clamp recordings were made in whole-cell patch-clamp configuration from ninety-one granule cells in parasagittal cerebellar slices obtained from 21- to 31-day-old rats. Recordings were performed at 30 degrees C. 2. Resting membrane potential was -58 +/- 6 mV (n = 43). The membrane voltage response to step current injection showed inward rectification consistent with increasing input resistance during membrane depolarization. Over -35 +/- 7 mV (n = 14) repetitive firing with little or no adaptation was activated. Spike frequency increased nearly linearly with injected current. 3. Unitary EPSPs obtained by stimulating the mossy fibre bundle had an amplitude of 11.4 +/- 2.1 mV (n = 22, holding potential = -75 mV). Synchronous activation of greater than one to two mossy fibres was needed to elicit action potentials. Antidromic stimulation elicited antidromic spikes and also EPSPs, presumably through a mossy fibre 'axon reflex'. 4. EPSPs were brought about by NMDA and non-NMDA receptor activation, accounting for about 70 and 30%, respectively, of peak amplitude at the holding potential of -70 mV. The EPSP decay conformed to passive membrane discharge after blocking the NMDA receptors. 5. No appreciable correlation was found between the time-to-peak and decay time constant of the EPSPs, consistent with the compact electrotonic structure of these neurons. 6. During membrane depolarization EPSP amplitude increased transiently, due to both a voltage-dependent increase of the NMDA component and inward rectification. In addition, EPSPs slowed down due to a slowdown of the NMDA component. 7. Temporal summation during high-frequency stimulation was sustained by NMDA receptors, whose contribution to depolarization tended to prevail over that of non-NMDA receptors during the trains. A block of the NMDA receptors resulted in reduced depolarization and output spike frequency. 8. This study, as well as extending previous knowledge to the intracellular level in vivo

  19. The effect of gallium nitride on long-term culture induced aging of neuritic function in cerebellar granule cells.

    PubMed

    Chen, Chi-Ruei; Young, Tai-Horng

    2008-04-01

    Gallium nitride (GaN) has been developed for a variety of microelectronic and optical applications due to its unique electric property and chemical stability. In the present study, n-type and p-type GaN were used as substrates to culture cerebellar granule neurons to examine the effect of GaN on cell response for a long-term culture period. It was found that GaN could rapidly induce cultured neurons to exhibit a high phosphorylated Akt level after 20h of incubation. It was assumed that the anti-apoptotic effect of Akt phosphorylation could be correlated with cell survival, neurite growth and neuronal function for up to 35 days of incubation. Morphological studies showed GaN induced larger neuronal aggregates and neurite fasciculation to exhibit a dense fiber network after 8 days of incubation. Western blot analysis and immunocytochemical characterization showed that GaN still exhibited the expression of neurite growth and function, such as high levels of GAP-43, synapsin I and synaptophysin even after 35 days of incubation. In addition, survival of cerebellar granule neurons on GaN was improved by the analysis of lactate dehydrogenase (LDH) release from damaged cells. These results indicated that neuronal connections were formed on GaN by a gradual process from Akt activation and cell aggregation to develop neurite growth, fasciculation and function. Therefore, GaN offers a good model system to identify a well-characterized pattern of neuronal behavior for a long-term culture period, consistent with the development of a neurochip requiring the integration of biological system and semiconductor material.

  20. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells.

    PubMed Central

    Stevens, R L; Fox, C C; Lichtenstein, L M; Austen, K F

    1988-01-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of 35S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although [35S]heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. We here demonstrate that human lung mast cells of 96% purity incorporate [35S] sulfate into separate heparin and chondroitin sulfate proteoglycans in an approximately equal to 2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin [35S]sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin [35S]sulfate E proteoglycans and the [35S]heparin proteoglycans were exocytosed from the [35S]sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of 35S-labeled proteoglycans reside in the secretory granules of these human lung mast cells. PMID:3353378

  1. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells

    SciTech Connect

    Stevens, R.L.; Austen, K.F. ); Fox, C.C.; Lichtenstein, L.M. )

    1988-04-01

    The predominant subclasses of mast cells in both the rat and the mouse can be distinguished from one another by their preferential synthesis of {sup 35}S-labeled proteoglycans that contain either heparin or oversulfated chondroitin sulfate glycosaminoglycans. Although ({sup 35}S)heparin proteoglycans have been isolated from human lung mast cells of 40-70% purity and from a skin biopsy specimen of a patient with urticaria pigmentosa, no highly sulfated chondroitin sulfate proteoglycan has been isolated from any enriched or highly purified population of human mast cells. The authors demonstrate that human lung mast cells of 96% purity incorporate ({sup 35}S)sulfate into separate heparin and chondroitin sulfate proteoglycans in an {approx}2:1 ratio. As assessed by HPLC of the chondroitinase ABC digests, the chondroitin ({sup 35}S)sulfate proteoglycans isolated from these human lung mast cells contain the same unusual chondroitin sulfate E disaccharide that is present in proteoglycans produced by interleukin 3-dependent mucosal-like mouse mast cells. Both the chondroitin ({sup 35}S)sulfate E proteoglycans and the ({sup 35}S)heparin proteoglycans were exocytosed from the ({sup 35}S)sulfate-labeled cells via perturbation of the IgE receptor, indicating that both types of {sup 35}S-labeled proteoglycans reside in the secretory granules of these human lung mast cells.

  2. Granulation Properties in DOT Images from Solar Maximum to Minimum

    NASA Astrophysics Data System (ADS)

    Pötzi, W.

    DOT granulation filtergrams in the G-Band from solar maximum to solar minimum (1999 to 2007) were investigated for changes of granulation properties like areas, perimeter, fractal dimension, cell sizes, and life times. Granules seem to become larger during solar minimum, whereas the distances between the granule centres stay constant. Nonetheless, the uncertainties are very high.

  3. 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

  4. Environmental lead exposure during early life alters granule cell neurogenesis and morphology in the hippocampus of young adult rats.

    PubMed

    Verina, T; Rohde, C A; Guilarte, T R

    2007-03-30

    Exposure to environmentally relevant levels of lead (Pb(2+)) during early life produces deficits in hippocampal synaptic plasticity in the form of long-term potentiation (LTP) and spatial learning in young adult rats [Nihei MK, Desmond NL, McGlothan JL, Kuhlmann AC, Guilarte TR (2000) N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning. Neuroscience 99:233-242; Guilarte TR, Toscano CD, McGlothan JL, Weaver SA (2003) Environmental enrichment reverses cognitive and molecular deficits induced by developmental lead exposure. Ann Neurol 53:50-56]. Other evidence suggests that the performance of rats in the Morris water maze spatial learning tasks is associated with the level of granule cell neurogenesis in the dentate gyrus (DG) [Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza P-V, Abrous DN (2003) Spatial memory performance of aged rats in the water maze predicts level of hippocampal neurogenesis. Proc Natl Acad Sci U S A 100:14385-14390]. In this study, we examined whether continuous exposure to environmentally relevant levels of Pb(2+) during early life altered granule cell neurogenesis and morphology in the rat hippocampus. Control and Pb(2+)-exposed rats received bromodeoxyuridine (BrdU) injections (100 mg/kg; i.p.) for five consecutive days starting at postnatal day 45 and were killed either 1 day or 4 weeks after the last injection. The total number of newborn cells in the DG of Pb(2+)-exposed rats was significantly decreased (13%; P<0.001) 1 day after BrdU injections relative to controls. Further, the survival of newborn cells in Pb(2+)-exposed rats was significantly decreased by 22.7% (P<0.001) relative to control animals. Co-localization of BrdU with neuronal or astrocytic markers did not reveal a significant effect of Pb(2+) exposure on cellular fate. In Pb(2+)-exposed rats, immature granule cells immunolabeled with doublecortin (DCX) displayed aberrant dendritic morphology

  5. Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.

    PubMed

    Chan, Helen; Zwingmann, Claudia; Pannunzio, Marc; Butterworth, Roger F

    2003-07-01

    Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at

  6. Delayed coupling to feedback inhibition during a critical period for the integration of adult-born granule cells.

    PubMed

    Temprana, Silvio G; Mongiat, Lucas A; Yang, Sung M; Trinchero, Mariela F; Alvarez, Diego D; Kropff, Emilio; Giacomini, Damiana; Beltramone, Natalia; Lanuza, Guillermo M; Schinder, Alejandro F

    2015-01-07

    Developing granule cells (GCs) of the adult dentate gyrus undergo a critical period of enhanced activity and synaptic plasticity before becoming mature. The impact of developing GCs on the activity of preexisting dentate circuits remains unknown. Here we combine optogenetics, acute slice electrophysiology, and in vivo chemogenetics to activate GCs at different stages of maturation to study the recruitment of local target networks. We show that immature (4-week-old) GCs can efficiently drive distal CA3 targets but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition toward maturity, they reliably recruit GABAergic feedback loops that restrict spiking of neighbor GCs, a mechanism that would promote sparse coding. Such inhibitory loop impinges only weakly in new cohorts of young GCs. A computational model reveals that the delayed coupling of new GCs to FBI could be crucial to achieve a fine-grain representation of novel inputs in the dentate gyrus.

  7. Delayed coupling to feedback inhibition during a critical period for the integration of adult-born granule cells

    PubMed Central

    Temprana, Silvio G.; Mongiat, Lucas A.; Yang, Sung M.; Trinchero, Mariela F.; Alvarez, Diego D.; Kropff, Emilio; Giacomini, Damiana; Beltramone, Natalia; Lanuza, Guillermo M.; Schinder, Alejandro F.

    2014-01-01

    SUMMARY Developing granule cells (GCs) of the adult dentate gyrus undergo a critical period of enhanced activity and synaptic plasticity before becoming mature. The impact of developing GCs on the activity of preexisting dentate circuits remains unknown. Here we combine optogenetics, acute slice electrophysiology, and in vivo chemogenetics to activate GCs at different stages of maturation to study the recruitment of local target networks. We show that immature (four-week-old) GCs can efficiently drive distal CA3 targets, but poorly activate proximal interneurons responsible for feedback inhibition (FBI). As new GCs transition towards maturity, they reliably recruit GABAergic feedback loops that restrict spiking of neighbor GCs, a mechanism that would promote sparse coding. Such inhibitory loop impinges only weakly in new cohorts of young GCs. A computational model reveals that the delayed coupling of new GCs to FBI could be crucial to achieve a fine-grain representation of novel inputs in the dentate gyrus. PMID:25533485

  8. Chronic pregabalin treatment decreases excitability of dentate gyrus and accelerates maturation of adult-born granule cells.

    PubMed

    Lempel, Augusto Abel; Coll, Lucia; Schinder, Alejandro F; Uchitel, Osvaldo Daniel; Piriz, Joaquin

    2017-01-01

    Pregabalin (PGB) is extensively prescribed to treat neurological and neuropsychiatrical conditions such as neuropathic pain, anxiety disorders, and epilepsy. Although PGB is known to bind selectively to the α2δ subunit of voltage-gated calcium channels, there is little understanding about how it exerts its therapeutic effects. In this article, we analyzed the effects of an in vivo chronic treatment with PGB over the physiology of dentate gyrus granule cells (DGGCs) using ex vivo electrophysiological and morphological analysis in adult mice. We found that PGB decreases neuronal excitability of DGGCs. In addition, PGB accelerates maturation of adult-born DGGCs, an effect that would modify dentate gyrus plasticity. Together, these findings suggest that PGB reduces activity in the dentate gyrus and modulates overall network plasticity, which might contribute to its therapeutic effects. Cover Image for this issue: doi: 10.1111/jnc.13783.

  9. Intrinsic neurophysiological properties of hilar ectopic and normotopic dentate granule cells in human temporal lobe epilepsy and a rat model

    PubMed Central

    Althaus, A. L.; Sagher, O.; Parent, J. M.

    2014-01-01

    Hilar ectopic dentate granule cells (DGCs) are a salient feature of aberrant plasticity in human temporal lobe epilepsy (TLE) and most rodent models of the disease. Recent evidence from rodent TLE models suggests that hilar ectopic DGCs contribute to hyperexcitability within the epileptic hippocampal network. Here we investigate the intrinsic excitability of DGCs from humans with TLE and the rat pilocarpine TLE model with the objective of comparing the neurophysiology of hilar ectopic DGCs to their normotopic counterparts in the granule cell layer (GCL). We recorded from 36 GCL and 7 hilar DGCs from human TLE tissue. Compared with GCL DGCs, hilar DGCs in patient tissue exhibited lower action potential (AP) firing rates, more depolarized AP threshold, and differed in single AP waveform, consistent with an overall decrease in excitability. To evaluate the intrinsic neurophysiology of hilar ectopic DGCs, we made recordings from retrovirus-birthdated, adult-born DGCs 2–4 mo after pilocarpine-induced status epilepticus or sham treatment in rats. Hilar DGCs from epileptic rats exhibited higher AP firing rates than normotopic DGCs from epileptic or control animals. They also displayed more depolarized resting membrane potential and wider AP waveforms, indicating an overall increase in excitability. The contrasting findings between disease and disease model may reflect differences between the late-stage disease tissue available from human surgical specimens and the earlier disease stage examined in the rat TLE model. These data represent the first neurophysiological characterization of ectopic DGCs from human hippocampus and prospectively birthdated ectopic DGCs in a rodent TLE model. PMID:25429123

  10. Long-Term Potentiation at the Mossy Fiber-Granule Cell Relay Invokes Postsynaptic Second-Messenger Regulation of Kv4 Channels.

    PubMed

    Rizwan, Arsalan P; Zhan, Xiaoqin; Zamponi, Gerald W; Turner, Ray W

    2016-11-02

    Mossy fiber afferents to cerebellar granule cells form the primary synaptic relay into cerebellum, providing an ideal site to process signal inputs differentially. Mossy fiber input is known to exhibit a long-term potentiation (LTP) of synaptic efficacy through a combination of presynaptic and postsynaptic mechanisms. However, the specific postsynaptic mechanisms contributing to LTP of mossy fiber input is unknown. The current study tested the hypothesis that LTP induces a change in intrinsic membrane excitability of rat cerebellar granule cells through modification of Kv4 A-type potassium channels. We found that theta-burst stimulation of mossy fiber input in lobule 9 granule cells lowered the current threshold to spike and increases the gain of spike firing by 2- to 3-fold. The change in postsynaptic excitability was traced to hyperpolarizing shifts in both the half-inactivation and half-activation potentials of Kv4 that occurred upon coactivating NMDAR and group I metabotropic glutamatergic receptors. The effects of theta-burst stimulation on Kv4 channel control of the gain of spike firing depended on a signaling cascade leading to extracellular signal-related kinase activation. Under physiological conditions, LTP of synaptically evoked spike output was expressed preferentially for short bursts characteristic of sensory input, helping to shape signal processing at the mossy fiber-granule cell relay.

  11. Twin screw granulation: steps in granule growth.

    PubMed

    Dhenge, Ranjit M; Cartwright, James J; Hounslow, Michael J; Salman, Agba D

    2012-11-15

    The present work focuses on the study of the progression of granules in different compartments along the length of screws in a twin screw granulator (TSG). The effects of varying powder feed rate; liquid to solid ratio and viscosity of granulation liquid on properties of granules was studied. The bigger granules produced at the start of the process were found to change in terms of size, shape and strength along the screw length at all the conditions investigated. The granules became more spherical and their strength increased along the screw length. Tracer granules were also introduced in order to understand the role of kneading and conveying elements in the TSG. The kneading elements promoted consolidation and breakage while the conveying elements led to coalescence, breakage and some consolidation. The results presented here help to provide a qualitative and quantitative understanding of the twin screw granulation process.

  12. Light and electron microscopic observations of fabrication, release, and fate of biphasic secretion granules produced by epididymal epithelial principal cells of the fan-throated lizard Sitana ponticeriana cuvier.

    PubMed

    Akbarsha, M A; Tamilarasan, V; Kadalmani, B

    2006-06-01

    The epididymis of the fan-throated lizard Sitana ponticeriana was examined with light and transmission electron microscopy to understand the cellular mechanisms of fabrication of secretion granules in epithelial principal cells, granule release into the lumen, and the fate of the dense structured granules after reaching the lumen. Principal cells of the ductus epididymis, except at the cauda, secrete electron-dense biphasic granules copiously, which decrease in abundance from the initial segment to corpus. The principal cell possesses a prominent Golgi apparatus and all versions of endoplasmic reticulum (ER), rough, smooth, and sparsely granulated. The material of the dense portion of the secretion granules, after processing at the Golgi apparatus, appears to accumulate in large ER cisternae in the supranuclear cytoplasm. It undergoes condensation when the cisternae become condensing vacuoles. Mitochondria appear to play a role in dense granule formation. The condensing vacuoles are displaced toward the apical cytoplasm when the material of the less dense portion is added to the condensing vacuoles at the Golgi area. Thus, the less dense and dense portions of the secretion granules are secreted and added to the condensing vacuoles separately. The composite granules are released into the lumen by exocytosis when the less dense portion merges with the luminal content, whereas the dense portion maintains its structured identity. The latter, initially measuring 1-2 microm in diameter, increases in size several times. It is inferred that these granules release their content gradually, resulting in the appearance of vacuoles, and suggesting that the granules have an insoluble matrix in which there is a sparingly soluble material. The substance leaching out of the granules appears to contribute to keeping the sperm quiescent and alive during storage in the male reproductive tract.

  13. Fuling Granule, a Traditional Chinese Medicine Compound, Suppresses Cell Proliferation and TGFβ-Induced EMT in Ovarian Cancer

    PubMed Central

    Ruan, Shanming; Liu, Wenhong; Wang, Libin; Xiong, Yang; Shen, Minhe

    2016-01-01

    The compound fuling granule (CFG) is a traditional Chinese drug which has been used to treat ovarian cancer in China for over twenty years. Nevertheless, the underlying molecular mechanism of its anti-cancer effect remains unclear. In this study, microarray data analysis was performed to search differentially expressed genes in CFG-treated ovarian cancer cells. Several cell cycle and epithelial-mesenchymal transition (EMT) related genes were identified. The microarray analyses also revealed that CFG potentially regulates EMT in ovarian cancer. We also found that, functionally, CFG significantly suppresses ovarian cancer cell proliferation by cell cycle arrest, apoptosis and senescence and the AKT/GSK-3β pathway is possibly involved. Additionally, the invasion and migration ability of ovarian cancer induced by TGFβ is significantly suppressed by CFG. In conclusion, our results demonstrated that CFG suppresses ovarian cancer cell proliferation as well as TGFβ1-induced EMT in vitro. Finally, we discovered that CFG suppresses tumor growth and distant metastasis in vivo. Overall, these findings provide helpful clues to design novel clinical treatments against cancer. PMID:28036353

  14. Casein Kinase 1δ Is an APC/CCdh1 Substrate that Regulates Cerebellar Granule Cell Neurogenesis

    PubMed Central

    Penas, Clara; Govek, Eve-Ellen; Fang, Yin; Ramachandran, Vimal; Daniel, Mark; Wang, Weiping; Maloof, Marie E.; Rahaim, Ronald J.; Bibian, Mathieu; Kawauchi, Daisuke; Finkelstein, David; Han, Jeng-Liang; Long, Jun; Li, Bin; Robbins, David J.; Malumbres, Marcos; Roussel, Martine F.; Roush, William R.; Hatten, Mary E.; Ayad, Nagi G.

    2015-01-01

    SUMMARY Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of central nervous system progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/CCdh1) ubiquitin ligase, and conditional deletion of the APC/CCdh1 activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/CCdh1 also downregulates CK1δ during cell cycle exit. Therefore, we conclude that APC/CCdh1 controls CK1δ levels to balance proliferation and cell cycle exit in the developing central nervous system. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a new therapeutic target. PMID:25843713

  15. Casein kinase 1δ is an APC/C(Cdh1) substrate that regulates cerebellar granule cell neurogenesis.

    PubMed

    Penas, Clara; Govek, Eve-Ellen; Fang, Yin; Ramachandran, Vimal; Daniel, Mark; Wang, Weiping; Maloof, Marie E; Rahaim, Ronald J; Bibian, Mathieu; Kawauchi, Daisuke; Finkelstein, David; Han, Jeng-Liang; Long, Jun; Li, Bin; Robbins, David J; Malumbres, Marcos; Roussel, Martine F; Roush, William R; Hatten, Mary E; Ayad, Nagi G

    2015-04-14

    Although casein kinase 1δ (CK1δ) is at the center of multiple signaling pathways, its role in the expansion of CNS progenitor cells is unknown. Using mouse cerebellar granule cell progenitors (GCPs) as a model for brain neurogenesis, we demonstrate that the loss of CK1δ or treatment of GCPs with a highly selective small molecule inhibits GCP expansion. In contrast, CK1δ overexpression increases GCP proliferation. Thus, CK1δ appears to regulate GCP neurogenesis. CK1δ is targeted for proteolysis via the anaphase-promoting complex/cyclosome (APC/C(Cdh1)) ubiquitin ligase, and conditional deletion of the APC/C(Cdh1) activator Cdh1 in cerebellar GCPs results in higher levels of CK1δ. APC/C(Cdh1) also downregulates CK1δ during cell-cycle exit. Therefore, we conclude that APC/C(Cdh1) controls CK1δ levels to balance proliferation and cell-cycle exit in the developing CNS. Similar studies in medulloblastoma cells showed that CK1δ holds promise as a therapeutic target.

  16. Loss of protohaem IX farnesyltransferase in mature dentate granule cells impairs short‐term facilitation at mossy fibre to CA3 pyramidal cell synapses

    PubMed Central

    Booker, Sam A.; Campbell, Graham R.; Mysiak, Karolina S.; Brophy, Peter J.; Kind, Peter C.

    2017-01-01

    Key points Neurodegenerative disorders can exhibit dysfunctional mitochondrial respiratory chain complex IV activity.Conditional deletion of cytochrome c oxidase, the terminal enzyme in the respiratory electron transport chain of mitochondria, from hippocampal dentate granule cells in mice does not affect low‐frequency dentate to CA3 glutamatergic synaptic transmission.High‐frequency dentate to CA3 glutamatergic synaptic transmission and feedforward inhibition are significantly attenuated in cytochrome c oxidase‐deficient mice.Intact presynaptic mitochondrial function is critical for the short‐term dynamics of mossy fibre to CA3 synaptic function. Abstract Neurodegenerative disorders are characterized by peripheral and central symptoms including cognitive impairments which have been associated with reduced mitochondrial function, in particular mitochondrial respiratory chain complex IV or cytochrome c oxidase activity. In the present study we conditionally removed a key component of complex IV, protohaem IX farnesyltransferase encoded by the COX10 gene, in granule cells of the adult dentate gyrus. Utilizing whole‐cell patch‐clamp recordings from morphologically identified CA3 pyramidal cells from control and complex IV‐deficient mice, we found that reduced mitochondrial function did not result in overt deficits in basal glutamatergic synaptic transmission at the mossy‐fibre synapse because the amplitude, input–output relationship and 50 ms paired‐pulse facilitation were unchanged following COX10 removal from dentate granule cells. However, trains of stimuli given at high frequency (> 20 Hz) resulted in dramatic reductions in short‐term facilitation and, at the highest frequencies (> 50 Hz), also reduced paired‐pulse facilitation, suggesting a requirement for adequate mitochondrial function to maintain glutamate release during physiologically relevant activity patterns. Interestingly, local inhibition was reduced, suggesting the effect

  17. Selective sorting of alpha-granule proteins.

    PubMed

    Italiano, J E; Battinelli, E M

    2009-07-01

    One of the main functions of blood platelets is to secrete a variety of substances that can modify a developing thrombus, regulate the growth of the vasculature, promote wound repair, and contribute to cell-adhesive events. A majority of this vast array of secreted proteins are stored in alpha-granules. Until recently, it was assumed that platelets contained one homogeneous population of alpha-granules that undergo complete de-granulation during platelet activation. This review focuses on the mechanisms of alpha-granule biogenesis and secretion, with a particular emphasis on recent findings that clearly demonstrate that platelets contain distinct subpopulations of alpha-granules that undergo differential release during activation. We consider the implications of this new paradigm of platelet secretion, discuss mechanisms of alpha-granule biogenesis, and review the molecular basis of transport and delivery of alpha-granules to assembling platelets.

  18. Extracts of ECL-cell granules/vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca2+ second messenger response in osteoblastic cells.

    PubMed

    Larsson, B; Gritli-Linde, A; Norlén, P; Lindström, E; Håkanson, R; Linde, A

    2001-03-02

    Surgical removal of the acid-producing part of the stomach (oxyntic mucosa) reduces bone mass through mechanisms not yet fully understood. The existence of an osteotropic hormone produced by the so-called ECL cells has been suggested. These cells, which are numerous in the oxyntic mucosa, operate under the control of circulating gastrin. Both gastrin and an extract of the oxyntic mucosa decrease blood calcium and stimulate Ca2+ uptake into bone. Conceivably, gastrin lowers blood calcium indirectly by releasing a hypothetical hormone from the ECL cells. The present study investigated, by means of fura-2 fluorometry, the effect of extracts of preparations enriched in ECL cell granules/vesicles from rat oxyntic mucosa on mobilization of intracellular Ca2+ in three osteoblast-like cell lines, UMR-106.01, MC3T3-E1 and Saos-2, and of extracts of isolated ECL cells in UMR-106.01 cells. The extracts were found to induce a dose-related rapid increase in intracellular Ca2+ concentrations in the osteoblast-like cells. The response was not due to histamine or pancreastatin, known ECL cell constituents, and could be abolished by pre-digesting the extracts with exo-aminopeptidase. The results show that the increase in [Ca2+](i) reflects a mobilization of Ca2+ from the endoplasmic reticulum. The observation of an increase in [Ca2+](i) also in murine embryonic fibroblasts show that the response is not limited to osteoblastic cells. The finding that the extracts evoked a typical Ca2+ -mediated second messenger response in osteoblastic cells provides evidence for the existence of a novel osteotropic peptide hormone (gastrocalcin), produced in the ECL cells, and supports the view that gastrectomy-induced osteopathy may reflect a lack of this hormone.

  19. CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells

    PubMed Central

    Chen, Xi; Allan, David S. J.; Krzewski, Konrad; Ge, Baoxue; Kopcow, Hernan; Strominger, Jack L.

    2006-01-01

    Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell, followed by actin polymerization, and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here, by using the YTS NK cell line as a model, CD28 is shown to be an activating receptor. It signals cytotoxicity in a process dependent on phosphoinositide-3 kinase activation, leading to sustained extracellular signal-regulated kinase 2 (ERK2) phosphorylation. ERK and phospho-ERK localize to microtubule filaments. Neither conjugation with targets nor actin polymerization is affected by blocking ERK2 activation. However, both polarization of the MTOC and cytolytic granules to the synaptic region and NK cell cytotoxicity are strongly reduced by blocking ERK2 activation. A role for the CD28/CD80 interaction in cytotoxicity of human peripheral NK cells also was established. By contrast, lymphocyte function-associated antigen 1 (LFA-1) ligation transduces only a transient ERK2 activation and fails to induce killing in YTS cells. Thus, in YTS cells, a CD28 signal is used to polarize the MTOC and cytolytic granules to the NK cell immune synapse by stimulating sustained ERK2 activation. PMID:16801532

  20. The brain-specific RasGEF very-KIND is required for normal dendritic growth in cerebellar granule cells and proper motor coordination

    PubMed Central

    Hayashi, Kanehiro; Furuya, Asako; Sakamaki, Yuriko; Akagi, Takumi; Shinoda, Yo; Sadakata, Tetsushi; Hashikawa, Tsutomu; Shimizu, Kazuki; Minami, Haruka; Sano, Yoshitake; Nakayama, Manabu

    2017-01-01

    Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance. PMID:28264072

  1. The neuroendocrine protein VGF is sorted into dense-core granules and is secreted apically by polarized rat thyroid epithelial cells.

    PubMed

    Gentile, Flaviana; Calì, Gaetano; Zurzolo, Chiara; Corteggio, Annunziata; Rosa, Patrizia; Calegari, Federico; Levi, Andrea; Possenti, Roberta; Puri, Claudia; Tacchetti, Carlo; Nitsch, Lucio

    2004-04-15

    We have expressed the neuroendocrine VGF protein in FRT rat thyroid cells to study the molecular mechanisms of its sorting to the regulated and polarized pathways of secretion. By immunoelectron microscopy, we have demonstrated that VGF localizes in dense-core granules. Rapid secretion of VGF is induced by PMA stimulation. Moreover, human chromogranin B, a protein of the regulated pathway, co-localizes in the same granules with VGF. In confluent, FRT monolayers on filters protein secretion occur from the apical cell domain. VGF deletion mutants have been generated. By confocal microscopy, we have found that in transient transfection, all mutant proteins are sorted into granules and co-localize with the full-length VGF. They all retain the apical polarity of secretion. We also found that intracellular VGF and its deletion mutants are largely in an aggregated form. We conclude that FRT thyroid cells correctly decode the sorting information of VGF. The signals present on the protein to enter the granules and to be secreted apically cannot be separated from each other and are not in just one discrete portion of the protein. We propose that selective aggregation might represent the signal for sorting VGF to the regulated, apical route.

  2. Mitochondrial trafficking through Rhot1 is involved in the aggregation of germinal granule components during primordial germ cell formation in Xenopus embryos.

    PubMed

    Tada, Haru; Taira, Yuya; Morichika, Keisuke; Kinoshita, Tsutomu

    2016-10-01

    In many animals, the germ plasm is sufficient and necessary for primordial germ cell (PGC) formation. It contains germinal granules and abundant mitochondria (germline-Mt). However, the role of germline-Mt in germ cell formation remains poorly understood. In Xenopus, the germ plasm is distributed as many small islands at the vegetal pole, which gradually aggregates to form a single large mass in each of the four vegetal pole cells at the early blastula stage. Polymerized microtubules and the adapter protein kinesin are required for the aggregation of germ plasm. However, it remains unknown whether germline-Mt trafficking is important for the cytoplasmic transport of germinal granules during germ plasm aggregation. In this study, we focused on the mitochondrial small GTPase protein Rhot1 to inhibit mitochondrial trafficking during the germ plasm aggregation. Expression of Rhot1ΔC, which lacks the C-terminal mitochondrial transmembrane domain, inhibited the aggregation of germline-Mt during early development. In Rhot1-inhibited embryos, germinal granule components did not aggregate during cleavage stages, which reduced the number of PGCs on the genital ridge at tail-bud stage. These results suggest that mitochondrial trafficking is involved in the aggregation of germinal granule components, which are essential for the formation of PGCs.

  3. Role of the actin-binding protein profilin1 in radial migration and glial cell adhesion of granule neurons in the cerebellum.

    PubMed

    Rust, Marco B; Kullmann, Jan A; Witke, Walter

    2012-01-01

    Profilins are small G-actin-binding proteins essential for cytoskeletal dynamics. Of the four mammalian profilin isoforms, profilin1 shows a broad expression pattern, profilin2 is abundant in the brain, and profilin3 and profilin4 are restricted to the testis. In vitro studies on cancer and epithelial cell lines suggested a role for profilins in cell migration and cell-cell adhesion. Genetic studies in mice revealed the importance of profilin1 in neuronal migration, while profilin2 has apparently acquired a specific function in synaptic physiology. We recently reported a mouse mutant line lacking profilin1 in the brain; animals display morphological defects that are typical for impaired neuronal migration. We found that during cerebellar development, profilin1 is specifically required for radial migration and glial cell adhesion of granule neurons. Profilin1 mutants showed cerebellar hypoplasia and aberrant organization of cerebellar cortex layers, with ectopically arranged granule neurons. In this commentary, we briefly introduce the profilin family and summarize the current knowledge on profilin activity in cell migration and adhesion. Employing cerebellar granule cells as a model, we shed some light on the mechanisms by which profilin1 may control radial migration and glial cell adhesion. Finally, a potential implication of profilin1 in human developmental neuropathies is discussed.

  4. FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons.

    PubMed

    Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2015-01-01

    The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3-4 and 8-9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner.

  5. FIB/SEM technology and high-throughput 3D reconstruction of dendritic spines and synapses in GFP-labeled adult-generated neurons

    PubMed Central

    Bosch, Carles; Martínez, Albert; Masachs, Nuria; Teixeira, Cátia M.; Fernaud, Isabel; Ulloa, Fausto; Pérez-Martínez, Esther; Lois, Carlos; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2015-01-01

    The fine analysis of synaptic contacts is usually performed using transmission electron microscopy (TEM) and its combination with neuronal labeling techniques. However, the complex 3D architecture of neuronal samples calls for their reconstruction from serial sections. Here we show that focused ion beam/scanning electron microscopy (FIB/SEM) allows efficient, complete, and automatic 3D reconstruction of identified dendrites, including their spines and synapses, from GFP/DAB-labeled neurons, with a resolution comparable to that of TEM. We applied this technology to analyze the synaptogenesis of labeled adult-generated granule cells (GCs) in mice. 3D reconstruction of dendritic spines in GCs aged 3–4 and 8–9 weeks revealed two different stages of dendritic spine development and unexpected features of synapse formation, including vacant and branched dendritic spines and presynaptic terminals establishing synapses with up to 10 dendritic spines. Given the reliability, efficiency, and high resolution of FIB/SEM technology and the wide use of DAB in conventional EM, we consider FIB/SEM fundamental for the detailed characterization of identified synaptic contacts in neurons in a high-throughput manner. PMID:26052271

  6. Effects of ECL cell extracts and granule/vesicle-enriched fractions from rat oxyntic mucosa on cAMP and IP(3) in rat osteoblast-like cells.

    PubMed

    Larsson, Birgitta; Norlén, Per; Lindström, Erik; Zhao, Dawei; Håkanson, Rolf; Linde, Anders

    2002-06-15

    The existence of an osteotropic hormone (referred to as gastrocalcin) in the ECL cells of the gastric mucosa has been suggested. Both gastrin and an extract of the oxyntic mucosa lower blood Ca(2+) and stimulate Ca(2+) uptake into bone. The ECL cells are known to operate under gastrin control and, conceivably, gastrin lowers blood Ca(2+) indirectly by releasing the hypothetical ECL cell hormone. We have shown earlier that extracts of isolated ECL cells or of the granule/vesicle fraction of the oxyntic mucosa evoke a typical Ca(2+)-mediated second messenger response in osteoblastic cells. In the present study, we characterize this response further. An increase in intracellular inositol 1,4,5-trisphosphate (IP(3)) concentration was observed after treatment of UMR-106.01 osteoblast-like cells with extracts of ECL cells or granule/vesicle-enriched fractions from oxyntic mucosa. Intracellular cyclic adenosine monophosphate (cAMP) concentrations were not affected. Inhibition of phospholipase C (PLC) by U-73122 abolished the increase in [Ca(2+)](i). Preincubation of UMR-106.01 cells with pertussis toxin, which blocks many G-proteins, did not prevent the increases in IP(3) and [Ca(2+)](i). It was also found that the novel peptide hormone ghrelin, produced in the A-like cells of the oxyntic mucosa, did not evoke any Ca(2+) signal in osteoblastic cells. The results indicate that the extracts mediate their effects through a pertussis toxin-insensitive mechanism, and that binding to a receptor leads to activation of PLC and production of IP(3) resulting in increased [Ca(2+)](i). The putative osteotropic hormone is distinct from ghrelin.

  7. Redistribution of K+ channels into dendrites is unlikely to account for developmental down regulation of A-currents in rat dentate gyrus granule cells.

    PubMed

    Strecker, T; Heinemann, U

    1993-12-24

    The electrical reactions of many central neurons depend on two voltage-activated K+ currents: the fast transient A-current IA and the delayed rectifier current IK. In rat dentate gyrus granule cells, the A-current density decreases during ontogenesis, possibly due to a redistribution of K+ channels from somata into dendrites. We tested this possibility in mechanically isolated granule cells with preserved dendrites of different length. Potassium currents were recorded with the whole-cell patch-clamp technique using prepulse protocols with and without a delay interval to isolate IA. A correlation between the length of the dendrites and the amount of A-current expressed in a given cell could not be demonstrated. Our findings therefore confirm an ontogenetic down regulation of A-currents.

  8. The plastic neurotransmitter phenotype of the hippocampal granule cells and of the moss in their messy fibers.

    PubMed

    Gutiérrez, Rafael

    2016-04-01

    The granule cells (GCs) and their axons, the mossy fibers (MFs), make synapses with interneurons in the hilus and CA3 area of the hippocampus and with pyramidal cells of CA3, each with distinct anatomical and functional characteristics. Many features of synaptic communication observed at the MF synapses are not usually observed in most cortical synapses, and thus have drawn the attention of many groups studying different aspects of the transmission of information. One particular aspect of the GCs, that makes their study unique, is that they express a dual glutamatergic-GABAergic phenotype and several groups have contributed to the understanding of how two neurotransmitters of opposing actions can act on a single target when simultaneously released. Indeed, the GCs somata and their mossy fibers express in a regulated manner glutamate and GABA, GAD, VGlut and VGAT, all markers of both phenotypes. Finally, their activation provokes both glutamate-R-mediated and GABA-R-mediated synaptic responses in the postsynaptic cell targets and even in the MFs themselves. The developmental and activity-dependent expression of these phenotypes seems to follow a "logical" way to maintain an excitation-inhibition balance of the dentate gyrus-to-CA3 communication.

  9. Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

    PubMed Central

    Kissenpfennig, Adrien; Aït-Yahia, Smina; Clair-Moninot, Valérie; Stössel, Hella; Badell, Edgar; Bordat, Yann; Pooley, Joanne L.; Lang, Thierry; Prina, Eric; Coste, Isabelle; Gresser, Olivia; Renno, Toufic; Winter, Nathalie; Milon, Geneviève; Shortman, Ken; Romani, Nikolaus; Lebecque, Serge; Malissen, Bernard; Saeland, Sem; Douillard, Patrice

    2005-01-01

    Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin−/− mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin−/− mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin−/− LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin−/− mice were not impaired in their capacity to process native OVA protein for I-Ab-restricted presentation to CD4+ T lymphocytes or for H-2Kb-restricted cross-presentation to CD8+ T lymphocytes. langerin−/− mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin−/− and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin−/− C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG. PMID:15601833

  10. Endogenous intracellular calcium buffering and the activation/inactivation of HVA calcium currents in rat dentate gyrus granule cells

    PubMed Central

    1991-01-01

    Granule cells acutely dissociated from the dentate gyrus of adult rat brains displayed a single class of high-threshold, voltage-activated (HVA) Ca2+ channels. The kinetics of whole-cell Ca2+ currents recorded with pipette solutions containing an intracellular ATP regenerating system but devoid of exogenous Ca2+ buffers, were fit best by Hodgkin- Huxley kinetics (m2h), and were indistinguishable from those recorded with the nystatin perforated patch method. In the absence of exogenous Ca2+ buffers, inactivation of HVA Ca2+ channels was a predominantly Ca(2+)-dependent process. The contribution of endogenous Ca2+ buffers to the kinetics of inactivation was investigated by comparing currents recorded from control cells to currents recorded from neurons that have lost a specific Ca(2+)-binding protein, Calbindin-D28K (CaBP), after kindling-induced epilepsy. Kindled neurons devoid of CaBP showed faster rates of both activation and inactivation. Adding an exogenous Ca2+ chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), to the intracellular solution largely eliminated inactivation in both control and kindled neurons. The results are consistent with the hypothesis that endogenous intraneuronal CaBP contributes significantly to submembrane Ca2+ sequestration at a concentration range and time domain that regulate Ca2+ channel inactivation. PMID:1662686

  11. Neurotoxicity of the pentabrominated diphenyl ether mixture, DE-71, and hexabromocyclododecane (HBCD) in rat cerebellar granule cells in vitro.

    PubMed

    Reistad, Trine; Fonnum, Frode; Mariussen, Espen

    2006-11-01

    Polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are compounds used as additive flame retardants in plastics, electronic equipment, and textiles. The aim of the present study was to investigate the in vitro effects of the pentabrominated diphenyl ether mixture, DE-71, and HBCD on cerebellar granule cells (CGC). Both DE-71 and HBCD induced death of CGC in low micromolar concentrations. The NMDA receptor antagonist MK801 (3 microM), and the antioxidant alpha-tocopherol (50 microM) significantly reduced the cell death. Incubation of the compounds together with the rat liver post-mitochondrial (S9) fraction reduced cell death by 58 and 64% for DE-71 and HBCD, respectively. No ROS formation and no elevation in intracellular calcium were observed. We further demonstrated apoptotic morphology (Hoechst straining) after exposure to low levels of the two brominated flame retardants and signs of DNA laddering were found after DE-71 exposure. However, other hallmarks of apoptosis, like caspase activity, were absent indicating an atypical form of apoptosis induced by DE-71. After intraperitoneal injection of the two compounds both DE-71 and HBCD were found in significant amounts in brain (559 +/- 194 and 49 +/- 13 microg/kg, respectively) and liver (4,010 +/- 2,437 and 1,248 +/- 505 microg/kg, respectively) 72 h after injection. Our results indicate that the lower brominated PBDEs have a higher potency of bioaccumulation than HBCD, and that both compounds have a neurotoxic potential in vitro.

  12. Starch granule formation and protein deposition in wheat (Triticum aestivum L.) starchy endosperm cells is altered by high temperature during grain fill

    NASA Astrophysics Data System (ADS)

    Hurkman, William J.; Wood, Delilah F.

    2010-06-01

    High temperatures during wheat grain fill decrease starch and protein levels, adversely affecting wheat yield and flour quality. To determine the effect of high temperature on starchy endosperm cell development, grain (Triticum aestivum L. 'Butte 86') was produced under a 24/17°C or 37/28°C day/night regimen imposed from flowering to maturity and starch and protein deposition examined using scanning electron microscopy. The high temperature regimen shortened the duration of grain fill from 40 to 18 days. Under the 37/28°C regimen, A- and B-type starch granules decreased in size. A-type starch granules also exhibited pitting, suggesting enhanced action of starch degradative enzymes. Under both temperature regimens, protein bodies originated early in development and coalesced during mid to late development to form a continuous protein matrix surrounding the starch granules. Under the 37/28°C regimen, the proportion of protein matrix increased in endosperm cells of mature grain. Taken together, the changes in starch granule number and size and in protein matrix amount provide clues for understanding how high temperature during grain fill can affect end use properties of wheat flour.

  13. Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells.

    PubMed

    Zhu, Li; Tatsuke, Tsuneyuki; Xu, Jian; Li, Zhiqing; Mon, Hiroaki; Lee, Jae Man; Kusakabe, Takahiro

    2015-09-01

    The phenomenon of RNA interference (RNAi) has been found in various organisms. However, the proteins implicated in RNAi pathway in different species show distinct roles. Knowledge on the underlying mechanism of lepidopteron RNAi is quite lacking such as the roles of Loquacious (Loqs) and R2D2, the dsRNA-binding proteins in silkworm RNAi pathway. Here, we report that Loqs and R2D2 protein depletion affected efficiency of dsRNA-mediated RNAi pathway. Besides, Loqs was found to co-localize with Dicer2 to some specific cytoplasmic foci, which were looked like D2-bodies marked by R2D2 and Dicer2 in Fly cells, thereby calling the foci as D2 body-like granules. Using RNAi methods, Loqs was found to be the key protein in these granules, although R2D2 determined the localization of Loqs in D2 body-like granules. Interestingly, in the R2D2-depeted silkworm cells, the formation of processing bodies, another cytoplasmic foci, was affected. These data indicated R2D2 regulated these two kinds of cytoplasmic foci. Domain deletion analysis demonstrated that dsRBD 1 and 2 were required for Loqs in D2 body-like granules and dsRBD 2 and 3 were required for Loqs to interact with R2D2 and Ago1, respectively. Altogether, our observations provide important information for further study on D2 body-like granules, the newly found cytoplasmic foci in silkworm cells.

  14. Knockout of Atg5 delays the maturation and reduces the survival of adult-generated neurons in the hippocampus

    PubMed Central

    Xi, Y; Dhaliwal, J S; Ceizar, M; Vaculik, M; Kumar, K L; Lagace, D C

    2016-01-01

    Autophagy is an evolutionarily conserved lysosomal degradation pathway that plays important roles in cell maintenance, expansion and differentiation. Removal of genes essential for autophagy from embryonic neural stem and precursor cells reduces the survival and inhibits neuronal differentiation of adult-generated neurons. No study has modified autophagy within the adult precursor cells, leaving the cell-autonomous role of autophagy in adult neurogenesis unknown. Here we demonstrate that autophagic flux exists in the adult dividing progenitor cells and their progeny in the dentate gyrus. To investigate the role of autophagy in adult hippocampal neurogenesis, we genetically deleted Autophagy-related gene 5 (Atg5) that reduced autophagic flux and the survival of the progeny of dividing progenitor cells. This significant reduction in survival of adult-generated neurons is accompanied by a delay in neuronal maturation, including a transient reduction in spine density in the absence of a change in differentiation. The delay in cell maturation and loss of progeny of the Atg5-null cells was not present in mice that lacked the essential pro-apoptotic protein Bax (Bcl-2-associated X protein), suggesting that Atg5-deficient cells die through a Bax-dependent mechanism. In addition, there was a loss of Atg5-null cells following exposure to running, suggesting that Atg5 is required for running-induced increases in neurogenesis. These findings highlight the cell-autonomous requirement of Atg5 in the survival of adult-generated neurons. PMID:26938300

  15. Causal Evidence for the Role of Specific GABAergic Interneuron Types in Entorhinal Recruitment of Dentate Granule Cells

    PubMed Central

    Lee, Cheng-Ta; Kao, Min-Hua; Hou, Wen-Hsien; Wei, Yu-Ting; Chen, Chin-Lin; Lien, Cheng-Chang

    2016-01-01

    The dentate gyrus (DG) is the primary gate of the hippocampus and controls information flow from the cortex to the hippocampus proper. To maintain normal function, granule cells (GCs), the principal neurons in the DG, receive fine-tuned inhibition from local-circuit GABAergic inhibitory interneurons (INs). Abnormalities of GABAergic circuits in the DG are associated with several brain disorders, including epilepsy, autism, schizophrenia, and Alzheimer disease. Therefore, understanding the network mechanisms of inhibitory control of GCs is of functional and pathophysiological importance. GABAergic inhibitory INs are heterogeneous, but it is unclear how individual subtypes contribute to GC activity. Using cell-type-specific optogenetic perturbation, we investigated whether and how two major IN populations defined by parvalbumin (PV) and somatostatin (SST) expression, regulate GC input transformations. We showed that PV-expressing (PV+) INs, and not SST-expressing (SST+) INs, primarily suppress GC responses to single cortical stimulation. In addition, these two IN classes differentially regulate GC responses to θ and γ frequency inputs from the cortex. Notably, PV+ INs specifically control the onset of the spike series, whereas SST+ INs preferentially regulate the later spikes in the series. Together, PV+ and SST+ GABAergic INs engage differentially in GC input-output transformations in response to various activity patterns. PMID:27830729

  16. Decreased tonic inhibition in cerebellar granule cells causes motor dysfunction in a mouse model of Angelman syndrome.

    PubMed

    Egawa, Kiyoshi; Kitagawa, Kyoko; Inoue, Koichi; Takayama, Masakazu; Takayama, Chitoshi; Saitoh, Shinji; Kishino, Tatsuya; Kitagawa, Masatoshi; Fukuda, Atsuo

    2012-12-05

    Angelman syndrome is a neurodevelopmental disorder caused by loss of function of the UBE3A gene encoding a ubiquitin E3 ligase. Motor dysfunction is a characteristic feature of Angelman syndrome, but neither the mechanisms of action nor effective therapeutic strategies have yet been elucidated. We report that tonic inhibition is specifically decreased in cerebellar granule cells of Ube3a-deficient mice, a model of Angelman syndrome. As a mechanism underlying this decrease in tonic inhibition, we show that Ube3a controls degradation of γ-aminobutyric acid (GABA) transporter 1 (GAT1) and that deficiency of Ube3a induces a surplus of GAT1 that results in a decrease in GABA concentrations in the extrasynaptic space. Administering low doses of 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP), a selective extrasynaptic GABA(A) receptor agonist, improves the abnormal firing properties of a population of Purkinje cells in cerebellar brain slices and reduces cerebellar ataxia in Ube3a-deficient mice in vivo. These results suggest that pharmacologically increasing tonic inhibition may be a useful strategy for alleviating motor dysfunction in Angelman syndrome.

  17. Corruption of the Dentate Gyrus by “Dominant” Granule cells: Implications for Dentate Gyrus Function in Health and Disease

    PubMed Central

    Scharfman, Helen E.; Myers, Catherine E.

    2015-01-01

    The dentate gyrus (DG) and area CA3 of the hippocampus are highly organized lamellar structures which have been implicated in specific cognitive functions such as pattern separation and pattern completion. Here we describe how the anatomical organization and physiology of the DG and CA3 are consistent with structures that perform pattern separation and completion. We then raise a new idea related to the complex circuitry of the DG and CA3 where CA3 pyramidal cell ‘backprojections’ play a potentially important role in the sparse firing of granule cells (GCs), considered important in pattern separation. We also propose that GC axons, the mossy fibers, already known for their highly specialized structure, have a dynamic function that imparts variance – ‘mossy fiber variance’ – which is important to pattern separation and completion. Computational modeling is used to show that when a subset of GCs become ‘dominant,’ one consequence is loss of variance in the activity of mossy fiber axons and a reduction in pattern separation and completion in the model. Empirical data are then provided using an example of ‘dominant’ GCs – subsets of GCs that develop abnormally and have increased excitability. Notably, these abnormal GCs have been identified in animal models of disease where DG-dependent behaviors are impaired. Together these data provide insight into pattern separation and completion, and suggest that behavioral impairment could arise from dominance of a subset of GCs in the DG-CA3 network. PMID:26391451

  18. Corruption of the dentate gyrus by "dominant" granule cells: Implications for dentate gyrus function in health and disease.

    PubMed

    Scharfman, Helen E; Myers, Catherine E

    2016-03-01

    The dentate gyrus (DG) and area CA3 of the hippocampus are highly organized lamellar structures which have been implicated in specific cognitive functions such as pattern separation and pattern completion. Here we describe how the anatomical organization and physiology of the DG and CA3 are consistent with structures that perform pattern separation and completion. We then raise a new idea related to the complex circuitry of the DG and CA3 where CA3 pyramidal cell 'backprojections' play a potentially important role in the sparse firing of granule cells (GCs), considered important in pattern separation. We also propose that GC axons, the mossy fibers, already known for their highly specialized structure, have a dynamic function that imparts variance--'mossy fiber variance'--which is important to pattern separation and completion. Computational modeling is used to show that when a subset of GCs become 'dominant,' one consequence is loss of variance in the activity of mossy fiber axons and a reduction in pattern separation and completion in the model. Empirical data are then provided using an example of 'dominant' GCs--subsets of GCs that develop abnormally and have increased excitability. Notably, these abnormal GCs have been identified in animal models of disease where DG-dependent behaviors are impaired. Together these data provide insight into pattern separation and completion, and suggest that behavioral impairment could arise from dominance of a subset of GCs in the DG-CA3 network.

  19. Tumor necrosis factor (TNF)-receptor 1 and 2 mediate homeostatic synaptic plasticity of denervated mouse dentate granule cells

    PubMed Central

    Becker, Denise; Deller, Thomas; Vlachos, Andreas

    2015-01-01

    Neurological diseases are often accompanied by neuronal cell death and subsequent deafferentation of connected brain regions. To study functional changes after denervation we generated entorhino-hippocampal slice cultures, transected the entorhinal pathway, and denervated dentate granule cells in vitro. Our previous work revealed that partially denervated neurons respond to the loss of input with a compensatory, i.e., homeostatic, increase in their excitatory synaptic strength. TNFα maintains this denervation-induced homeostatic strengthening of excitatory synapses. Here, we used pharmacological approaches and mouse genetics to assess the role of TNF-receptor 1 and 2 in lesion-induced excitatory synaptic strengthening. Our experiments disclose that both TNF-receptors are involved in the regulation of denervation-induced synaptic plasticity. In line with this result TNF-receptor 1 and 2 mRNA-levels were upregulated after deafferentation in vitro. These findings implicate TNF-receptor signaling cascades in the regulation of homeostatic plasticity of denervated networks and suggest an important role for TNFα-signaling in the course of neurological diseases accompanied by deafferentation. PMID:26246237

  20. Gray platelet syndrome. Dissociation between abnormal sorting in megakaryocyte alpha-granules and normal sorting in Weibel-Palade bodies of endothelial cells.

    PubMed Central

    Gebrane-Younès, J; Cramer, E M; Orcel, L; Caen, J P

    1993-01-01

    The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which megakaryocytes and platelets are deficient in alpha-granule secretory proteins. Since the Weibel-Palade bodies (WPB) of endothelial cells as well as the alpha-granules contain the von Willebrand Factor (vWF) and P-selectin, we examined by transmission electron microscopy the dermis capillary network of two patients with GPS. Endothelial cells showed the presence of normal WPB with typical internal tubules. Using single and double immunogold labeling for vWF and P-selectin, we detected vWF within WPB, where it was codistributed with the tubules, whereas P-selectin delineated the outline of WPB. Therefore, the fundamental targeting defect in GPS is specific to the megakaryocytic cell line. Images PMID:7504696

  1. Effects of prenatal protein malnutrition on kindling-induced alterations in dentate granule cell excitability. I. Synaptic transmission measures.

    PubMed

    Bronzino, J D; Austin-LaFrance, R J; Morgane, P J; Galler, J R

    1991-05-01

    The effects of prenatal protein malnutrition upon the efficacy of excitatory synaptic transmission at the level of the perforant path/dentate granule cell synapse were examined during development of perforant path kindling in chronically implanted adults rats. Rats born to dams fed a low protein (6% casein) or control protein (25% casein) diet were fostered to lactating dams fed the 25% casein diet 24 h after birth and were maintained on this diet throughout life following weaning. Beginning at 90-120 days of age, animals received daily kindling stimulations applied to the perforant path. Extracellular field potentials recorded from the granule cell layer of the dentate gyrus in response to single-pulse stimulation of the perforant path were analyzed to determine the effects of prenatal protein malnutrition on the efficacy of synaptic transmission during the kindling process. Measures used for these analyses included the EPSP slope, an indicator of the level of synaptic drive, the population spike amplitude which is a measure of postsynaptic activation and cellular firing, and the ratio of the population spike amplitude relative to the corresponding EPSP slope value, which was used to evaluate the overall efficacy of synaptic transmission. animals of the 6%/25% diet group were found to have significantly lower afterdischarge thresholds, yet required significantly more daily kindling stimulations to develop generalized motor convulsions (stage 5 seizure) than control animals. Examination of dentate field potentials obtained prior to kindling revealed no significant between group differences in measures of EPSP slope or population spike amplitude. Statistically significant increases in measures of both the population EPSP slope and population spike amplitude were observed in both diet groups 24 h after the first kindled afterdischarge. The degree of increase in both of these measures was significantly greater in animals of the 6%/25% group. Evaluation of input

  2. Fluorescent Labeling of Newborn Dentate Granule Cells in GAD67-GFP Transgenic Mice: A Genetic Tool for the Study of Adult Neurogenesis

    PubMed Central

    Zhao, Shengli; Zhou, Yang; Gross, Jimmy; Miao, Pei; Qiu, Li; Wang, Dongqing; Chen, Qian; Feng, Guoping

    2010-01-01

    Neurogenesis in the adult hippocampus is an important form of structural plasticity in the brain. Here we report a line of BAC transgenic mice (GAD67-GFP mice) that selectively and transitorily express GFP in newborn dentate granule cells of the adult hippocampus. These GFP+ cells show a high degree of colocalization with BrdU-labeled nuclei one week after BrdU injection and express the newborn neuron marker doublecortin and PSA-NCAM. Compared to mature dentate granule cells, these newborn neurons show immature morphological features: dendritic beading, fewer dendritic branches and spines. These GFP+ newborn neurons also show immature electrophysiological properties: higher input resistance, more depolarized resting membrane potentials, small and non-typical action potentials. The bright labeling of newborn neurons with GFP makes it possible to visualize the details of dendrites, which reach the outer edge of the molecular layer, and their axon (mossy fiber) terminals, which project to the CA3 region where they form synaptic boutons. GFP expression covers the whole developmental stage of newborn neurons, beginning within the first week of cell division and disappearing as newborn neurons mature, about 4 weeks postmitotic. Thus, the GAD67-GFP transgenic mice provide a useful genetic tool for studying the development and regulation of newborn dentate granule cells. PMID:20824075

  3. 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.

  4. Characterization of muscarinic receptor subtypes in primary cultures of cerebellar granule cells using specific muscarinic receptor antagonists

    SciTech Connect

    McLeskey, S.W.

    1989-01-01

    In cerebellar granule cell cultures, two muscarinic receptor mediated responses were observed: inhibition of adenylate cyclase (M-AC) and stimulation of phosphoinositide hydrolysis (M-PI). These responses were antagonized by three purported specific muscarinic antagonists: pirenzipine and (-)QNX (specific for M-PI) and methoctramine (specific for M-AC). However, the specificity for the three antagonists in blocking these responses is not comparable to the specificity observed in binding studies on these cells or to that quoted in the literature. Two peaks of molecular sizes were found in these cells corresponding to the two molecular sizes of muscarinic receptive proteins reported in the literature. Muscarinic receptive proteins were alkylated with {sup 3}H-propylbenzilylcholine mustard followed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pirenzipine and (-)QNX were able to block alkylation of the high molecular size peak, which corresponds to the receptive protein m{sub 3} reported in the literature. Methoctramine was able to block alkylation of a portion of the lower molecular size peak, possibly corresponding to the m{sub 2} and/or m{sub 4} receptive proteins reported in the literature. Studies attempting to show the presence of receptor reserve for either of the two biochemical responses present in these cells by alkylation of the receptive protein with nonradiolabeled propylbenzilylcholine mustard (PBCM) were confounded by specificity of this agent for the lower molecular weight peak of muscarinic receptive protein. Thus the muscarinic receptive proteins coupled to M-AC were alkylated preferentially over the ones coupled to M-PI.

  5. 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

  6. Isolation of chromaffin granules.

    PubMed

    Creutz, Carl E

    2010-09-01

    Adrenal medullary chromaffin granules (dense core secretory vesicles) have been a valuable model system for the study of the proteins and membrane components involved in the process of exocytosis. Because of the abundance of chromaffin granules in a readily available tissue source, bovine adrenal medullae, and their unique sedimentation properties, it is possible to obtain large quantities of highly purified granules and granule membranes in a short period of time. Two protocols are presented here for the isolation of chromaffin granules: a basic protocol based on differential centrifugation in an iso-osmotic medium that yields intact chromaffin granules, and an alternate protocol based on sedimentation through a density step gradient that provides a greater yield of more highly purified chromaffin granules. Since in the latter case the granules cannot be returned to a medium of physiological osmolarity without lysis after purification on the step gradient, the alternate protocol is more useful to obtain the granule membranes or contents for further study.

  7. Cocultures of GFP(+) -granule cells with GFP(-) -pyramidal cells and interneurons for the study of mossy fiber neurotransmission with paired recordings.

    PubMed

    Osorio, Beatriz; León, Uriel; Galván, Emilio J; Gutiérrez, Rafael

    2013-04-01

    Synaptic transmission of the granule cells (GCs) via their axons, the mossy fibers (MFs), is traditionally studied on acutely prepared or cultured slices. Usually, extracellular, bulk or minimal stimulation is used to evoke transmitter release from MF terminals, while recording from their postsynaptic target cells, the pyramidal cells and interneurons of CA3. However, the ideal method to assess MF neurotransmission, the simultaneous recording of a presynaptic GC and one of its target cells, is extremely difficult to achieve using slices. Alternatively, cultures of GCs establishing autapses have been developed, but in these, GCs do not contact their natural targets. We developed cocultures of GCs, dissociated from transgenic GFP(+) rats, with pyramidal cells and interneurons of CA3, dissociated from wild-type rats, and confirmed the expression of cell-specific markers by immunofluorescence. We conducted recordings of GFP(+) -GCs synaptically connected with their GFP(-) -target cells, and demonstrate that synaptic transmission and its plasticity have the signature of transmission of MF. Besides being strongly depressed by activation of mGluRs, high frequency activation of GC-to-pyramidal cells synapses undergo LTP, while GC-to-interneuron synapses undergo LTD. This coculture method allows a high reproducibility of recording connected pairs of identified cells, constituting a valuable tool to study MF transmission, as well as different combinations of identifiable pre- and postsynaptic cells.

  8. Effects of prenatal stress and exercise on dentate granule cells maturation and spatial memory in adolescent mice.

    PubMed

    Bustamante, Carlos; Bilbao, Pamela; Contreras, William; Martínez, Mauricio; Mendoza, Antonio; Reyes, Alvaro; Pascual, Rodrigo

    2010-11-01

    Exposure to prenatal stress (PS) increases the risk of developing neurobehavioral disturbances later in life. Previous work has shown that exercise can exert beneficial effects on brain damage; however, it is unknown whether voluntary wheel running (VWR) can ameliorate the neurobehavioral impairments induced by PS in adolescent offspring. Pregnant CF-1 mice were randomly assigned to control (n=5) or stressed (n=5) groups. Pregnant dams were subjected to restraint stress between gestational days 14 and 21 (G14-21), whereas controls remained undisturbed in their home cages. On postnatal day 21 (P21), male pups were randomly assigned to the following experimental groups: control (n=5), stressed (n=5), and stressed mice+daily submitted to VWR (n=4). At P52, all groups were behaviorally evaluated in the Morris water maze. Animals were then sacrificed, and Golgi-impregnated granule cells were morphometrically analyzed. The results indicate that PS produced significant behavioral and neuronal impairments in adolescent offspring and that VWR significantly offset these deleterious effects.

  9. 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).

  10. 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

  11. Respiratory Modulation of Spontaneous Subthreshold Synaptic Activity in Olfactory Bulb Granule Cells Recorded in Awake, Head-Fixed Mice

    PubMed Central

    Youngstrom, Isaac A.

    2015-01-01

    Although the firing patterns of principal neurons in the olfactory bulb are known to be modulated strongly by respiration even under basal conditions, less is known about whether inhibitory local circuit activity in the olfactory bulb (OB) is modulated phasically. The diverse phase preferences of principal neurons in the OB and olfactory cortex that innervate granule cells (GCs) may interfere and prevent robust respiratory coupling, as suggested by recent findings. Using whole-cell recording, we examined the spontaneous, subthreshold membrane potential of GCs in the OBs of awake head-fixed mice. We found that, during periods of basal respiration, the synaptic input to GCs was strongly phase modulated, leading to a phase preference in the average, cycle-normalized membrane potential. Subthreshold phase tuning was heterogeneous in both mitral and tufted cells (MTCs) and GCs but relatively constant within each GC during periods of increased respiratory frequency. The timing of individual EPSPs in GC recordings also was phase modulated with the phase preference imparted by large-amplitude EPSPs, with fast kinetics often matching the phase tuning of the average membrane potential. These results suggest that activity in a subset of excitatory afferents to GCs, presumably including cortical feedback projections and other sources of large-amplitude unitary EPSPs, function to provide a timing signal linked to respiration. The phase preference we find in the membrane potential may provide a mechanism to dynamically modulate recurrent and lateral dendrodendritic inhibition of MTCs and to selective engage a subpopulation of interneurons based on the alignment of their phase tuning relative to sensory-driven MTC discharges. PMID:26063910

  12. Beneficial effect of Boswellia serrata gum resin on spatial learning and the dendritic tree of dentate gyrus granule cells in aged rats

    PubMed Central

    Hosseini-Sharifabad, Mohammad; Kamali-Ardakani, Razieh; Hosseini-Sharifabad, Ali

    2016-01-01

    Objective: The hippocampal formation, particularly the dentate gyrus (DG), shows age-related morphological changes that could cause memory decline. It is indicated that Boswellia resins attenuates memory deficits and the major component of Boswellia serrata (Bs) gum resin, beta boswellic acid increased neurite outgrowth and branching in hippocampal neurons. This study was designed to investigate the effect of Boswellia treatment on spatial learning performance and the morphology of dentate granule cells in aged rats. Materials and Methods: Sixteen male Wistar rats (24 months old) were divided into experimental and control groups. Experimental group was intragastrically administered with the aqueous extract of Bs (100 mg/kg/d for 8 weeks) and control group received a similar volume of water. Spatial learning performance of rats was tested using Morris water maze task. At the end of experiment, the brain was removed and the right hippocampus was serially sectioned for morphometric analysis. The Cavalieri principle was employed to estimate the volume of the DG. A quantitative Golgi study was used to analyze the dendritic trees of dentate granule cells. Results: Chronic treatment with Bs improved spatial learning capability during the three acquisition days. Comparisons also revealed that Bs-treated aged rat had greater DG with increased dendritic complexity in the dentate granule cells than control rats. Hippocampal granule cells of Bs-treated aged rats had more dendritic segments, larger arbors, more numerical branching density and more dendritic spines in comparison to control animals. Conclusion: This study provided a neuro-anatomical basis for memory improvement due to chronic treatment with Bs. PMID:27222832

  13. Localized hypoxia within the subgranular zone determines the early survival of newborn hippocampal granule cells

    PubMed Central

    Chatzi, Christina; Schnell, Eric; Westbrook, Gary L

    2015-01-01

    The majority of adult hippocampal newborn cells die during early differentiation from intermediate progenitors (IPCs) to immature neurons. Neural stem cells in vivo are located in a relative hypoxic environment, and hypoxia enhances their survival, proliferation and stemness in vitro. Thus, we hypothesized that migration of IPCs away from hypoxic zones within the SGZ might result in oxidative damage, thus triggering cell death. Hypoxic niches were observed along the SGZ, composed of adult NSCs and early IPCs, and oxidative byproducts were present in adjacent late IPCs and neuroblasts. Stabilizing hypoxia inducible factor-1α with dimethyloxallyl glycine increased early survival, but not proliferation or differentiation, in neurospheres in vitro and in newly born SGZ cells in vivo. Rescue experiments in Baxfl/flmutants supported these results. We propose that localized hypoxia within the SGZ contributes to the neurogenic microenvironment and determines the early, activity-independent survival of adult hippocampal newborn cells. DOI: http://dx.doi.org/10.7554/eLife.08722.001 PMID:26476335

  14. Two-Photon Na(+) Imaging Reports Somatically Evoked Action Potentials in Rat Olfactory Bulb Mitral and Granule Cell Neurites.

    PubMed

    Ona-Jodar, Tiffany; Gerkau, Niklas J; Sara Aghvami, S; Rose, Christine R; Egger, Veronica

    2017-01-01

    Dendrodendritic synaptic interactions are a hallmark of neuronal processing in the vertebrate olfactory bulb. Many classes of olfactory bulb neurons including the principal mitral cells (MCs) and the axonless granule cells (GCs) dispose of highly efficient propagation of action potentials (AP) within their dendrites, from where they can release transmitter onto each other. So far, backpropagation in GC dendrites has been investigated indirectly via Ca(2+) imaging. Here, we used two-photon Na(+) imaging to directly report opening of voltage-gated sodium channels due to AP propagation in both cell types. To this end, neurons in acute slices from juvenile rat bulbs were filled with 1 mM SBFI via whole-cell patch-clamp. Calibration of SBFI signals revealed that a change in fluorescence ΔF/F by 10% corresponded to a Δ[Na(+)]i of ∼22 mM. We then imaged proximal axon segments of MCs during somatically evoked APs (sAP). While single sAPs were detectable in ∼50% of axons, trains of 20 sAPs at 50 Hz always resulted in substantial ΔF/F of ∼15% (∼33 mM Δ[Na(+)]i). ΔF/F was significantly larger for 80 Hz vs. 50 Hz trains, and decayed with half-durations τ1/2 ∼0.6 s for both frequencies. In MC lateral dendrites, AP trains yielded small ΔF/F of ∼3% (∼7 mM Δ[Na(+)]i). In GC apical dendrites and adjacent spines, single sAPs were not detectable. Trains resulted in an average dendritic ΔF/F of 7% (16 mM Δ[Na(+)]i) with τ1/2 ∼1 s, similar for 50 and 80 Hz. Na(+) transients were indistinguishable between large GC spines and their adjacent dendrites. Cell-wise analysis revealed two classes of GCs with the first showing a decrease in ΔF/F along the dendrite with distance from the soma and the second an increase. These classes clustered with morphological parameters. Simulations of Δ[Na(+)]i replicated these behaviors via negative and positive gradients in Na(+) current density, assuming faithful AP backpropagation. Such specializations of dendritic

  15. Two-Photon Na+ Imaging Reports Somatically Evoked Action Potentials in Rat Olfactory Bulb Mitral and Granule Cell Neurites

    PubMed Central

    Ona-Jodar, Tiffany; Gerkau, Niklas J.; Sara Aghvami, S.; Rose, Christine R.; Egger, Veronica

    2017-01-01

    Dendrodendritic synaptic interactions are a hallmark of neuronal processing in the vertebrate olfactory bulb. Many classes of olfactory bulb neurons including the principal mitral cells (MCs) and the axonless granule cells (GCs) dispose of highly efficient propagation of action potentials (AP) within their dendrites, from where they can release transmitter onto each other. So far, backpropagation in GC dendrites has been investigated indirectly via Ca2+ imaging. Here, we used two-photon Na+ imaging to directly report opening of voltage-gated sodium channels due to AP propagation in both cell types. To this end, neurons in acute slices from juvenile rat bulbs were filled with 1 mM SBFI via whole-cell patch-clamp. Calibration of SBFI signals revealed that a change in fluorescence ΔF/F by 10% corresponded to a Δ[Na+]i of ∼22 mM. We then imaged proximal axon segments of MCs during somatically evoked APs (sAP). While single sAPs were detectable in ∼50% of axons, trains of 20 sAPs at 50 Hz always resulted in substantial ΔF/F of ∼15% (∼33 mM Δ[Na+]i). ΔF/F was significantly larger for 80 Hz vs. 50 Hz trains, and decayed with half-durations τ1/2 ∼0.6 s for both frequencies. In MC lateral dendrites, AP trains yielded small ΔF/F of ∼3% (∼7 mM Δ[Na+]i). In GC apical dendrites and adjacent spines, single sAPs were not detectable. Trains resulted in an average dendritic ΔF/F of 7% (16 mM Δ[Na+]i) with τ1/2 ∼1 s, similar for 50 and 80 Hz. Na+ transients were indistinguishable between large GC spines and their adjacent dendrites. Cell-wise analysis revealed two classes of GCs with the first showing a decrease in ΔF/F along the dendrite with distance from the soma and the second an increase. These classes clustered with morphological parameters. Simulations of Δ[Na+]i replicated these behaviors via negative and positive gradients in Na+ current density, assuming faithful AP backpropagation. Such specializations of dendritic excitability might confer

  16. Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na(+) currents through intracellular Ca(2+) release.

    PubMed

    Liu, Dong-Dong; Ren, Zhen; Yang, Guang; Zhao, Qian-Ru; Mei, Yan-Ai

    2014-06-01

    Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa ) densities by 62.5%. MT (5 μM) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca(2+) level, but it significantly elevated the intracellular Ca(2+) level evoked by the high K(+) stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca(2+) influx-induced Ca(2+) release.

  17. Melatonin protects rat cerebellar granule cells against electromagnetic field-induced increases in Na+ currents through intracellular Ca2+ release

    PubMed Central

    Liu, Dong-Dong; Ren, Zhen; Yang, Guang; Zhao, Qian-Ru; Mei, Yan-Ai

    2014-01-01

    Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa) densities by 62.5%. MT (5 μM) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca2+ level, but it significantly elevated the intracellular Ca2+ level evoked by the high K+ stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca2+ influx-induced Ca2+ release. PMID:24548607

  18. The Influence of Ectopic Migration of Granule Cells into the Hilus on Dentate Gyrus-CA3 Function

    PubMed Central

    Myers, Catherine E.; Bermudez-Hernandez, Keria; Scharfman, Helen E.

    2013-01-01

    Postnatal neurogenesis of granule cells (GCs) in the dentate gyrus (DG) produces GCs that normally migrate from the subgranular zone to the GC layer. However, GCs can mismigrate into the hilus, the opposite direction. Previous descriptions of these hilar ectopic GCs (hEGCs) suggest that they are rare unless there are severe seizures. However, it is not clear if severe seizures are required, and it also is unclear if severe seizures are responsible for the abnormalities of hEGCs, which include atypical dendrites and electrophysiological properties. Here we show that large numbers of hEGCs develop in a transgenic mouse without severe seizures. The mice have a deletion of BAX, which normally regulates apoptosis. Surprisingly, we show that hEGCs in the BAX-/- mouse have similar abnormalities as hEGCs that arise after severe seizures. We next asked if there are selective effects of hEGCs, i.e., whether a robust population of hEGCs would have any effect on the DG if they were induced without severe seizures. Indeed, this appears to be true, because it has been reported that BAX-/- mice have defects in a behavior that tests pattern separation, which depends on the DG. However, inferring functional effects of hEGCs is difficult in mice with a constitutive BAX deletion because there is decreased apoptosis in and outside the DG. Therefore, a computational model of the normal DG and hippocampal subfield CA3 was used. Adding a small population of hEGCs (5% of all GCs), with characteristics defined empirically, was sufficient to disrupt a simulation of pattern separation and completion. Modeling results also showed that effects of hEGCs were due primarily to “backprojections” of CA3 pyramidal cell axons to the hilus. The results suggest that hEGCs can develop for diverse reasons, do not depend on severe seizures, and a small population of hEGCs may impair DG-dependent function. PMID:23840835

  19. Noradrenaline blocks potassium conductance in rat dentate granule cells in vitro.

    PubMed

    Haas, H L; Rose, G M

    1987-07-22

    The actions of noradrenaline and the beta-adrenergic agonist, isoproterenol, were studied on the dentate gyrus in hippocampal slices from rats using extra- and intracellular recording. These agents facilitated field EPSPs (excitatory postsynaptic potentials) and population spikes evoked by perforant path stimulation. Intracellular recording revealed an attenuation of the long lasting afterhyperpolarization (AHP) and the accommodation of cell discharge in response to depolarizing current injection. It is suggested that beta-receptor activation blocks a calcium-dependent potassium current.

  20. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells

    PubMed Central

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei

    2016-01-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic–adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition. PMID:27402067

  1. Effect of heart failure on catecholamine granule morphology and storage in chromaffin cells.

    PubMed

    Mahata, Sushil K; Zheng, Hong; Mahata, Sumana; Liu, Xuefei; Patel, Kaushik P

    2016-09-01

    One of the key mechanisms involved in sympathoexcitation in chronic heart failure (HF) is the activation of the adrenal glands. Impact of the elevated catecholamines on the hemodynamic parameters has been previously demonstrated. However, studies linking the structural effects of such overactivation with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not been previously reported. In this study, HF was induced in male Sprague-Dawley rats by ligation of the left coronary artery. Five weeks after surgery, cardiac function was assessed by ventricular hemodynamics. HF rats showed increased adrenal weight and adrenal catecholamine levels (norepinephrine, epinephrine and dopamine) compared with sham-operated rats. Rats with HF demonstrated increased small synaptic and dense core vesicle in splanchnic-adrenal synapses indicating trans-synaptic activation of catecholamine biosynthetic enzymes, increased endoplasmic reticulum and Golgi lumen width to meet the demand of increased catecholamine synthesis and release, and more mitochondria with dilated cristae and glycogen to accommodate for the increased energy demand for the increased biogenesis and exocytosis of catecholamines from the adrenal medulla. These findings suggest that increased trans-synaptic activation of the chromaffin cells within the adrenal medulla may lead to increased catecholamines in the circulation which in turn contributes to the enhanced neurohumoral drive, providing a unique mechanistic insight for enhanced catecholamine levels in plasma commonly observed in chronic HF condition.

  2. Granulophysin is located in the membrane of azurophilic granules in human neutrophils and mobilizes to the plasma membrane following cell stimulation.

    PubMed Central

    Cham, B. P.; Gerrard, J. M.; Bainton, D. F.

    1994-01-01

    Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increased following stimulation of the cells, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding of anti-CD63 antibodies to the cell surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils. Images Figure 1 Figure 2 Figure 3 Figure 6 Figure 9 Figure 10 Figure 11 Figure 12 PMID:8203473

  3. The taurine uptake inhibitor guanidinoethyl sulphonate is an agonist at gamma-aminobutyric acid(A) receptors in cultured murine cerebellar granule cells.

    PubMed

    Mellor, J R; Gunthorpe, M J; Randall, A D

    2000-05-26

    In patch clamp experiments the beta-amino acid uptake inhibitor guanidinoethyl sulphonate (GES) activated currents in intact cultured murine cerebellar granule neurones. These responses could be attenuated by the gamma-aminobutyric acid(A) (GABA(A)) receptor antagonists bicuculline and picrotoxin. With intracellular chloride concentrations of either 20 or 130 mM, GES-induced current responses reversed polarity near the chloride equilibrium potential. When fast applications of agonist were made to excised granule cell macropatches GES responses were dose-dependent and exhibited significant outward rectification. Like taurine (but unlike GABA and beta-alanine) responses, macroscopic desensitisation of GES-induced currents was slow. Our data indicate that care should be exercised when using GES as a taurine uptake inhibitor in systems that also contain GABA(A) receptors.

  4. Fluoro-jade identification of cerebellar granule cell and purkinje cell death in the alpha1A calcium ion channel mutant mouse, leaner.

    PubMed

    Frank, T C; Nunley, M C; Sons, H D; Ramon, R; Abbott, L C

    2003-01-01

    Cell death is a critical component of normal nervous system development; too little or too much results in abnormal development and function of the nervous system. The leaner mouse exhibits excessive, abnormal cerebellar granule cell and Purkinje cell death during postnatal development, which is a consequence of a mutated calcium ion channel subunit, alpha(1A). Previous studies have shown that leaner cerebellar Purkinje cells die in a specific pattern that appears to be influenced by functional and anatomical boundaries of the cerebellum. However, the mechanism of Purkinje cell death and the specific timing of the spatial pattern of cell death remain unclear. By double labeling both leaner and wild-type cerebella with Fluoro-Jade and terminal deoxynucleotide transferase-mediated, deoxyuridine triphosphate nick-end labeling or Fluoro-Jade and tyrosine hydroxylase immunohistochemistry we demonstrated that the relatively new stain, Fluoro-Jade, will label neurons that are dying secondary to a genetic mutation. Then, by staining leaner and wild-type cerebella between postnatal days 20 and 80 with Fluoro-Jade, we were able to show that Purkinje cell death begins at approximately postnatal day 25, peaks in the vermis about postnatal day 40 and in the hemispheres at postnatal day 50 and persists at a low level at postnatal day 80. In addition, we showed that there is a significant difference in the amount of cerebellar Purkinje cell death between rostral and caudal divisions of the leaner cerebellum, and that there is little to no Purkinje cell death in the wild type cerebellum at the ages we examined. This is the first report of the use of Fluoro-Jade to identify dying neurons in a genetic model for neuronal cell death. By using Fluoro-Jade, we have specifically defined the temporospatial pattern of postnatal Purkinje cell death in the leaner mouse. This information can be used to gain insight into the dynamic mechanisms controlling Purkinje cell death in the leaner

  5. Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16.

    PubMed

    Pfeiffer, Daniel; Wahl, Andreas; Jendrossek, Dieter

    2011-11-01

    A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.

  6. Properties of two voltage-activated potassium currents in acutely isolated juvenile rat dentate gyrus granule cells.

    PubMed

    Beck, H; Ficker, E; Heinemann, U

    1992-12-01

    1. The properties of outward currents were investigated in acutely isolated dentate gyrus granule cells at postnatal ages of day 5-7, 10-14, 18-24 (P5-7, P10-14, P18-24) and at adulthood (2-3 mo), with the use of the whole-cell patch-clamp technique. 2. Kinetic analysis and pharmacological properties showed that an A-type K+ current (IA) and a delayed rectifier current (IK) were present in these cells. 3. IA in P10-14 cells activated and inactivated rapidly with a decay time constant of 7.5 +/- 2.1 (SD) ms with command pulses to +30 mV. The removal of inactivation was monoexponential with a time constant of 23.1 ms (holding potential, -50 mV; conditioning voltage steps of varying duration to -110 mV). V 1/2 of the Boltzmann function describing steady-state inactivation was -65.1 +/- 1.8 mV with a slope factor of -6.0. IA was sensitive to 5 mM 4-aminopyridine (4-AP) but not to 10 mM tetraethylammonium (TEA). 4. IK in P10-14 cells displayed a voltage-dependent activation time constant (4.3 +/- 0.8 ms for command pulses to +30 mV and 16.2 +/- 2.4 for command pulses to -10 mV) and a double-exponential decay (time constants 194 +/- 21 and 1,625 +/- 254 ms). The rate constant of removal of inactivation was 332.1 ms. IK showed a reduction by 61.4 +/- 5.3% with 10 mM TEA and was partially blocked by 5 mM 4-AP in a subpopulation of cells. 5. Whereas IA remained stable over time, IK showed a substantial reduction of current amplitude by 67% after 30 min of cell perfusion through the patch pipette. The time course of this reduction was monoexponential with a time constant of 6.9 min and was partly due to a shift in V1/2 of the steady-state inactivation from -79.2 to -99.6 mV. 6. IA and IK remained stable with respect to kinetic properties during ontogenesis. However, the relative contribution and pharmacological properties of the investigated K+ currents varied with age. Although IA dominated in P5-7 cells, IK was prominent in most older cells. Five millimolars 4-AP reduced

  7. A multilamellate body in the granulated hypophysial cells of the teleost, Hemihaplochromis philander.

    PubMed

    Adamson, J H; Rawdon, B B

    1976-10-06

    A multilamellate body (MLB), bearing close resemblance to an array of annulate lamellae, has been observed in several adenohypophysial cell types of the teleost, Hemihaplochromis philander. In longitudinal section, each MLB comprises a ladder-like row of 12-50 sausage-shaped profiles, termed lamellae. A few lamellae in each section show connections with the endoplasmic reticulum. Apposition of paired lamellar membranes at regularly spaced intervals results in a beaded appearance, whereby sites of membrane apposition are probable pore sites. The MLB differs from annulate lamellae in having poorly developed pores and closer packing of lamellae. It is suggested that the MLBs described here, may represent annulate lamellae at a stage of development or break-down when pores are incomplete.

  8. 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.

  9. Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus.

    PubMed

    Kitahara, Yosuke; Ohta, Keisuke; Hasuo, Hiroshi; Shuto, Takahide; Kuroiwa, Mahomi; Sotogaku, Naoki; Togo, Akinobu; Nakamura, Kei-ichiro; Nishi, Akinori

    2016-01-01

    A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission.

  10. Chronic Fluoxetine Induces the Enlargement of Perforant Path-Granule Cell Synapses in the Mouse Dentate Gyrus

    PubMed Central

    Kitahara, Yosuke; Ohta, Keisuke; Hasuo, Hiroshi; Shuto, Takahide; Kuroiwa, Mahomi; Sotogaku, Naoki; Togo, Akinobu; Nakamura, Kei-ichiro; Nishi, Akinori

    2016-01-01

    A selective serotonin reuptake inhibitor is the most commonly prescribed antidepressant for the treatment of major depression. However, the mechanisms underlying the actions of selective serotonin reuptake inhibitors are not fully understood. In the dentate gyrus, chronic fluoxetine treatment induces increased excitability of mature granule cells (GCs) as well as neurogenesis. The major input to the dentate gyrus is the perforant path axons (boutons) from the entorhinal cortex (layer II). Through voltage-sensitive dye imaging, we found that the excitatory neurotransmission of the perforant path synapse onto the GCs in the middle molecular layer of the mouse dentate gyrus (perforant path-GC synapse) is enhanced after chronic fluoxetine treatment (15 mg/kg/day, 14 days). Therefore, we further examined whether chronic fluoxetine treatment affects the morphology of the perforant path-GC synapse, using FIB/SEM (focused ion beam/scanning electron microscopy). A three-dimensional reconstruction of dendritic spines revealed the appearance of extremely large-sized spines after chronic fluoxetine treatment. The large-sized spines had a postsynaptic density with a large volume. However, chronic fluoxetine treatment did not affect spine density. The presynaptic boutons that were in contact with the large-sized spines were large in volume, and the volumes of the mitochondria and synaptic vesicles inside the boutons were correlated with the size of the boutons. Thus, the large-sized perforant path-GC synapse induced by chronic fluoxetine treatment contains synaptic components that correlate with the synapse size and that may be involved in enhanced glutamatergic neurotransmission. PMID:26788851

  11. Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.

    PubMed

    Babot, Zoila; Cristòfol, Rosa; Suñol, Cristina

    2005-01-01

    Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl

  12. THE NUMBER OF CATECHOLAMINE STORAGE GRANULES IN ADRENAL MEDULLA

    DTIC Science & Technology

    A method is described for counting the catecholamine-containing heavy granules of adrenal glands. There are 5.0 ! 0.8 (S. E.) x 10 to the 12th power... granules /gram wet weight of fowl adrenal gland. Individual heavy granules contain about 8 million molecules of catecholamines (1.4 x 10 to the 17th...power mole). Reference to published electron microphotographs of adrenal medulla cells allows estimation of the average volume of heavy granules and

  13. Granulation of fine powder

    DOEpatents

    Chen, Ching-Fong

    2016-08-09

    A mixture of fine powder including thorium oxide was converted to granulated powder by forming a first-green-body and heat treating the first-green-body at a high temperature to strengthen the first-green-body followed by granulation by crushing or milling the heat-treated first-green-body. The granulated powder was achieved by screening through a combination of sieves to achieve the desired granule size distribution. The granulated powder relies on the thermal bonding to maintain its shape and structure. The granulated powder contains no organic binder and can be stored in a radioactive or other extreme environment. The granulated powder was pressed and sintered to form a dense compact with a higher density and more uniform pore size distribution.

  14. In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic β-Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking

    PubMed Central

    Cai, Erica P.; Casimir, Marina; Schroer, Stephanie A.; Luk, Cynthia T.; Shi, Sally Yu; Choi, Diana; Dai, Xiao Qing; Hajmrle, Catherine; Spigelman, Aliya F.; Zhu, Dan; Gaisano, Herbert Y.; MacDonald, Patrick E.; Woo, Minna

    2012-01-01

    Focal adhesion kinase (FAK) acts as an adaptor at the focal contacts serving as a junction between the extracellular matrix and actin cytoskeleton. Actin dynamics is known as a determinant step in insulin secretion. Additionally, FAK has been shown to regulate insulin signaling. To investigate the essential physiological role of FAK in pancreatic β-cells in vivo, we generated a transgenic mouse model using rat insulin promoter (RIP)–driven Cre-loxP recombination system to specifically delete FAK in pancreatic β-cells. These RIPcre+fakfl/fl mice exhibited glucose intolerance without changes in insulin sensitivity. Reduced β-cell viability and proliferation resulting in decreased β-cell mass was observed in these mice, which was associated with attenuated insulin/Akt (also known as protein kinase B) and extracellular signal–related kinase 1/2 signaling and increased caspase 3 activation. FAK-deficient β-cells exhibited impaired insulin secretion with normal glucose sensing and preserved Ca2+ influx in response to glucose, but a reduced number of docked insulin granules and insulin exocytosis were found, which was associated with a decrease in focal proteins, paxillin and talin, and an impairment in actin depolymerization. This study is the first to show in vivo that FAK is critical for pancreatic β-cell viability and function through regulation in insulin signaling, actin dynamics, and granule trafficking. PMID:22498697

  15. Impaired reelin processing and secretion by Cajal-Retzius cells contributes to granule cell dispersion in a mouse model of temporal lobe epilepsy.

    PubMed

    Duveau, Venceslas; Madhusudan, Amrita; Caleo, Matteo; Knuesel, Irene; Fritschy, Jean-Marc

    2011-09-01

    Cajal-Retzius cells play a crucial role during ontogeny in regulating cortical lamination via release of reelin. In adult brain, they comprise small calretinin-positive interneurons located in the marginal zone of the cerebral cortex and in the hippocampal fissure. Alterations of reelin signaling or expression have been involved in major neurological disorders, and they underlie granule cell dispersion (GCD) in mesial temporal lobe epilepsy (TLE). Here, we investigated in a mouse model of TLE the contribution of Cajal-Retzius cells to reelin production in epileptic hippocampus and the molecular mechanisms underlying GCD. Following unilateral intrahippocampal Kainic acid injection in adult mice to induce an epileptic focus, we observed that Cajal-Retzius cells gradually became strongly immunopositive for reelin, due to intracellular accumulation. This phenotype resembled the morphology of Cajal-Retzius cells in reeler Orleans (reln (orl/orl) ) mice, which express a secretion-deficient 310-kDa reelin fragment. The possibility that GCD might result from abnormal reelin processing in Cajal-Retzius cells, leading to a lack of reelin secretion, was confirmed by KA injection in reln (orl/+) mice, which induced severe GCD. Furthermore, Western blot analysis in KA-treated wildtype mice revealed increased production of ∼300-kDa reelin fragments, confirming abnormal proteolytic processing. This effect was not seen upon treatment with Botulinum neurotoxin E (BoNT/E), which prevents GCD in KA-lesioned hippocampus by chronic blockade of synaptic transmission. Furthermore, BoNT/E blocked upregulation of TrkB in Cajal-Retzius cells, suggesting that production of truncated reelin in KA-treated hippocampus is activity-dependent and regulated by BDNF. Altogether, these data reveal that GCD results from abnormal reelin processing in Cajal-Retzius cells under the control of BDNF. Our findings highlight the critical role played by Cajal-Retzius cells for hippocampal neuronal

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

    NASA Astrophysics Data System (ADS)

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

    2016-08-01

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

  17. Impairment of stress granule assembly via inhibition of the eIF2alpha phosphorylation sensitizes glioma cells to chemotherapeutic agents.

    PubMed

    Vilas-Boas, Fabrício de Almeida Souza; da Silva, Aristóbolo Mendes; de Sousa, Lirlândia Pires; Lima, Kátia Maciel; Vago, Juliana Priscila; Bittencourt, Lucas Felipe Fernandes; Dantas, Arthur Estanislau; Gomes, Dawidson Assis; Vilela, Márcia Carvalho; Teixeira, Mauro Martins; Barcelos, Lucíola Silva

    2016-04-01

    Malignant gliomas are a lethal type of brain tumors that poorly respond to chemotherapeutic drugs. Several therapy resistance mechanisms have been characterized. However, the response to stress through mRNA translational control has not been evaluated for this type of tumor. A potential target would involve the alpha subunit of eukaryotic translation initiation factor (eIF2α) that leads to assembly of stress granules (SG) which are cytoplasmic granules mainly composed by RNA binding proteins and untranslated mRNAs. We assessed whether glioma cells are capable of assembling SG after exposure to different classes of chemotherapeutic agents through evaluation of the effects of interfering in this process by impairing the eIF2α signaling. C6 and U87MG cells were exposed to bortezomib, cisplatin, or etoposide. Forced expression of a dominant negative mutant of eIF2α (eIF2α(DN)) was employed to block this pathway. We observed that exposure to drugs stimulated SG assembly. This was reduced in eIF2α(DN)-transfected cells and this strategy enhanced chemotherapeutically-induced cell death for all drugs. Our data suggest that SG assembly occurs in glioma cells in response to chemotherapeutic drugs in an eIF2α-dependent manner and this response is relevant for drug resistance. Interfering with eIF2α signaling pathway may be a potential strategy for new co-adjuvant therapies to treat gliomas.

  18. 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.

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

    PubMed Central

    Dey, Abhinav; Robitaille, Mélanie; Remke, Marc; Maier, Caroline; Malhotra, Anshu; Gregorieff, Alex; Wrana, Jeffrey L; Taylor, Michael D; Angers, Stéphane; Kenney, Anna Marie

    2016-01-01

    Post-natal proliferation of cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for the SHH-associated subgroup of medulloblastoma (MB), 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 up-regulated across human medulloblastoma subclasses as well as in other varieties of pediatric brain tumors. Utilizing the cerebellar progenitor model for the Shh-subgroup of MB 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 medulloblastoma cells and they identify the SHH:YAP:YB1:IGF2 axis as a powerful target for therapeutic intervention in medulloblastomas. PMID:26725322

  20. Statistical Frailty Modeling for Quantitative Analysis of Exocytotic Events Recorded by Live Cell Imaging: Rapid Release of Insulin-Containing Granules Is Impaired in Human Diabetic β-cells

    PubMed Central

    Cortese, Giuliana; Gandasi, Nikhil R.; Barg, Sebastian; Pedersen, Morten Gram

    2016-01-01

    Hormones and neurotransmitters are released when secretory granules or synaptic vesicles fuse with the cell membrane, a process denoted exocytosis. Modern imaging techniques, in particular total internal reflection fluorescence (TIRF) microscopy, allow the investigator to monitor secretory granules at the plasma membrane before and when they undergo exocytosis. However, rigorous statistical approaches for temporal analysis of such exocytosis data are still lacking. We propose here that statistical methods from time-to-event (also known as survival) analysis are well suited for the problem. These methods are typically used in clinical settings when individuals are followed over time to the occurrence of an event such as death, remission or conception. We model the rate of exocytosis in response to pulses of stimuli in insulin-secreting pancreatic β-cell from healthy and diabetic human donors using piecewise-constant hazard modeling. To study heterogeneity in the granule population we exploit frailty modeling, which describe unobserved differences in the propensity to exocytosis. In particular, we insert a discrete frailty in our statistical model to account for the higher rate of exocytosis in an immediately releasable pool (IRP) of insulin-containing granules. Estimates of parameters are obtained from maximum-likelihood methods. Since granules within the same cell are correlated, i.e., the data are clustered, a modified likelihood function is used for log-likelihood ratio tests in order to perform valid inference. Our approach allows us for example to estimate the size of the IRP in the cells, and we find that the IRP is deficient in diabetic cells. This novel application of time-to-event analysis and frailty modeling should be useful also for the study of other well-defined temporal events at the cellular level. PMID:27907065

  1. Time-lapse imaging reveals highly dynamic structural maturation of postnatally born dentate granule cells in organotypic entorhino-hippocampal slice cultures

    PubMed Central

    Radic, Tijana; Jungenitz, Tassilo; Singer, Mathias; Beining, Marcel; Cuntz, Hermann; Vlachos, Andreas; Deller, Thomas; Schwarzacher, Stephan W.

    2017-01-01

    Neurogenesis of hippocampal granule cells (GCs) persists throughout mammalian life and is important for learning and memory. How newborn GCs differentiate and mature into an existing circuit during this time period is not yet fully understood. We established a method to visualize postnatally generated GCs in organotypic entorhino-hippocampal slice cultures (OTCs) using retroviral (RV) GFP-labeling and performed time-lapse imaging to study their morphological development in vitro. Using anterograde tracing we could, furthermore, demonstrate that the postnatally generated GCs in OTCs, similar to adult born GCs, grow into an existing entorhino-dentate circuitry. RV-labeled GCs were identified and individual cells were followed for up to four weeks post injection. Postnatally born GCs exhibited highly dynamic structural changes, including dendritic growth spurts but also retraction of dendrites and phases of dendritic stabilization. In contrast, older, presumably prenatally born GCs labeled with an adeno-associated virus (AAV), were far less dynamic. We propose that the high degree of structural flexibility seen in our preparations is necessary for the integration of newborn granule cells into an already existing neuronal circuit of the dentate gyrus in which they have to compete for entorhinal input with cells generated and integrated earlier. PMID:28256620

  2. Cdc42 and Rac stimulate exocytosis of secretory granules by activating the IP(3)/calcium pathway in RBL-2H3 mast cells.

    PubMed

    Hong-Geller, E; Cerione, R A

    2000-02-07

    We have expressed dominant-active and dominant-negative forms of the Rho GTPases, Cdc42 and Rac, using vaccinia virus to evaluate the effects of these mutants on the signaling pathway leading to the degranulation of secretory granules in RBL-2H3 cells. Dominant-active Cdc42 and Rac enhance antigen-stimulated secretion by about twofold, whereas the dominant-negative mutants significantly inhibit secretion. Interestingly, treatment with the calcium ionophore, A23187, and the PKC activator, PMA, rescues the inhibited levels of secretion in cells expressing the dominant-negative mutants, implying that Cdc42 and Rac act upstream of the calcium influx pathway. Furthermore, cells expressing the dominant-active mutants exhibit elevated levels of antigen-stimulated IP(3) production, an amplified antigen-stimulated calcium response consisting of both calcium release from internal stores and influx from the extracellular medium, and an increase in aggregate formation of the IP(3) receptor. In contrast, cells expressing the dominant-negative mutants display the opposite phenotypes. Finally, we are able to detect an in vitro interaction between Cdc42 and PLCgamma1, the enzyme immediately upstream of IP(3) formation. Taken together, these findings implicate Cdc42 and Rac in regulating the exocytosis of secretory granules by stimulation of IP(3) formation and calcium mobilization upon antigen stimulation.

  3. 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.

  4. The regulation of synaptic vesicle recycling by cGMP-dependent protein kinase type II in cerebellar granule cells under strong and sustained stimulation.

    PubMed

    Collado-Alsina, Andrea; Ramírez-Franco, Jorge; Sánchez-Prieto, José; Torres, Magdalena

    2014-06-25

    From the early periods of neurogenesis and migration, up until synaptogenesis, both nitric oxide (NO) and its downstream messenger, cGMP, are thought to influence the development of neurons. The NO/cGMP/cGMP-dependent protein kinase (cGK) pathway regulates the clustering and recruitment of synaptic proteins and vesicles to the synapse, adjusting the exoendocytic cycle to the intensity of activity and accelerating endocytosis following large-scale exocytosis. Here, we show that blockage of the N-methyl-D-aspartate receptor impairs the cycling of synaptic vesicles in a subset of boutons on cerebellar granule cells, an effect that was reversed by increasing cGMP. Furthermore, we demonstrate that presynaptic cGK type II (cGKII) plays a major role in this process. Using the FM1-43 dye to track vesicle recycling, we found that knockdown of cGKII and/or the application of a cGK inhibitor reduced the efficiency of synaptic vesicle recycling to a similar extent. Likewise, in cerebellar granule cells transfected with vGlut1-pHluorin to follow the exoendocytotic cycle, application of a cGK inhibitor slowed vesicle endocytosis when exocytosis was accelerated through strong and sustained stimulation. Additionally, ultrastructural analysis showed that cGKII knockdown or inhibition favored the formation of endosomal-like structures after strong and sustained stimulation. We conclude that cGKII controls the homeostatic balance of vesicle exocytosis and endocytosis in synaptic boutons of rat cerebellar granule cells.

  5. 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

  6. The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

    PubMed Central

    Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

    2014-01-01

    Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

  7. The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells.

    PubMed

    Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

    2013-08-01

    Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity.

  8. Sniff-Like Patterned Input Results in Long-Term Plasticity at the Rat Olfactory Bulb Mitral and Tufted Cell to Granule Cell Synapse

    PubMed Central

    Perez de los Cobos Pallares, Fernando; Loebel, Alex; Lukas, Michael

    2016-01-01

    During odor sensing the activity of principal neurons of the mammalian olfactory bulb, the mitral and tufted cells (MTCs), occurs in repetitive bursts that are synchronized to respiration, reminiscent of hippocampal theta-gamma coupling. Axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between the excitatory MTCs via reciprocal dendrodendritic synapses. We have explored long-term plasticity at this synapse by using a theta burst stimulation (TBS) protocol and variations thereof. GCs were excited via glomerular stimulation in acute brain slices. We find that TBS induces exclusively long-term depression in the majority of experiments, whereas single bursts (“single-sniff paradigm”) can elicit both long-term potentiation and depression. Statistical analysis predicts that the mechanism underlying this bidirectional plasticity involves the proportional addition or removal of presynaptic release sites. Gamma stimulation with the same number of APs as in TBS was less efficient in inducing plasticity. Both TBS- and “single-sniff paradigm”-induced plasticity depend on NMDA receptor activation. Since the onset of plasticity is very rapid and requires little extra activity, we propose that these forms of plasticity might play a role already during an ongoing search for odor sources. Our results imply that components of both short-term and long-term olfactory memory may be encoded at this synapse. PMID:27747107

  9. 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.

  10. Yizhijiannao Granule and a combination of its effective monomers, icariin and Panax notoginseng saponins, inhibit early PC12 cell apoptosis induced by beta-amyloid (25–35)☆

    PubMed Central

    Zhang, Ting; Zhang, Zhanwei; Dong, Keli; Li, Guangcheng; Zhu, Hong

    2012-01-01

    One of our previous studies showed that Yizhijiannao Granule, a compound Chinese medicine, effectively improved the clinical symptoms of Alzheimer's disease. In the present study, we established a model of Alzheimer's disease using beta-amyloid (25–35) in PC12 cells, and treated the cells with Yizhijiannao Granule and its four monomers, i.e., icariin, catechin, Panax notoginseng saponins, and eleutheroside E. Flow cytometry showed that Yizhijiannao Granule-containing serum, icariin, Panax notoginseng saponins, and icariin + Panax notoginseng saponins were protective against beta-amyloid (25–35)-induced injury in PC12 cells. Icariin in combination with Panax notoginseng saponins significantly inhibited early apoptosis of PC12 cells with beta-amyloid (25–35)-induced injury compared to icariin or Panax notoginseng saponins alone. The effects of icariin + Panax notoginseng saponins were similar to the effects of Yizhijiannao Granule. The findings indicate that two of the effective monomers of Yizhijiannao Granule, icariin and Panax notoginseng saponins, can synergistically inhibit early apoptosis of PC12 cells induced by beta-amyloid (25–35). PMID:25624809

  11. Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

    PubMed Central

    Dvorak, A. M.; Ackerman, S. J.; Furitsu, T.; Estrella, P.; Letourneau, L.; Ishizaka, T.

    1992-01-01

    The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:1562046

  12. The effects of a lengthy period of undernutrition from birth and subsequent nutritional rehabilitation on the granule-to-Purkinje cell ratio in the rat cerebellum.

    PubMed Central

    Warren, M A; Bedi, K S

    1988-01-01

    Male rats were undernourished for various lengths of time between birth and 150 days of age, with some rats being nutritionally rehabilitated between 75 and 150 days of age. Eight control and eight experimental rats were anaesthetised and perfused with 2.5% glutaraldehyde at each of 21, 75 and 150 days of age. Stereological procedures were used to estimate granule-to-Purkinje cell ratios in lobes IV, V and VI, using 0.5 micron thick toluidine blue-stained sections. Undernourished rats had significantly lower body and cerebellar weights than controls at all ages examined. These deficits persisted even after a period of nutritional rehabilitation. The granule-to-Purkinje cells ratio did not differ between control and experimental groups at 21 or 75 days of age. However, at 150 days both undernourished and rehabilitated groups of animals had significant deficits in this ratio compared with age-matched controls. These results suggest that undernutrition can have profound effects on brain development in later life even if the effects are not apparent during the period of undernutrition. PMID:3248962

  13. Selective isolation of ammonia-oxidizing bacteria from autotrophic nitrifying granules by applying cell-sorting and sub-culturing of microcolonies.

    PubMed

    Fujitani, Hirotsugu; Kumagai, Asami; Ushiki, Norisuke; Momiuchi, Kengo; Tsuneda, Satoshi

    2015-01-01

    Nitrification is a key process in the biogeochemical nitrogen cycle and biological wastewater treatment that consists of two stepwise reactions, ammonia oxidation by ammonia-oxidizing bacteria (AOB) or archaea followed by nitrite oxidation by nitrite-oxidizing bacteria. One of the representatives of the AOB group is Nitrosomonas mobilis species. Although a few pure strains of this species have been isolated so far, approaches to their preservation in pure culture have not been established. Here, we report isolation of novel members of the N. mobilis species from autotrophic nitrifying granules used for ammonia-rich wastewater treatment. We developed an isolation method focusing on microcolonies formation of nitrifying bacteria. Two kinds of distinctive light scattering signatures in a cell-sorting system enabled to separate microcolonies from single cells and heterogeneous aggregates within granule samples. Inoculation of a pure microcolony into 96-well microtiter plates led to successful sub-culturing and increased probability of isolation. Obtained strain Ms1 is cultivated in the liquid culture with relatively high ammonia or nitrite concentration, not extremely slow growing. Considering environmental clones that were closely related to N. mobilis and detected in various environments, the availability of this novel strain would facilitate to reveal this member's ecophysiology in a variety of habitats.

  14. Endogenous zinc depresses GABAergic transmission via T-type Ca2+ channels and broadens the time window for integration of glutamatergic inputs in dentate granule cells

    PubMed Central

    Grauert, Antonia; Engel, Dominique; Ruiz, Arnaud J

    2014-01-01

    Abstract Zinc actions on synaptic transmission span the modulation of neurotransmitter receptors, transporters, activation of intracellular cascades and alterations in gene expression. Whether and how zinc affects inhibitory synaptic signalling in the dentate gyrus remains largely unexplored. We found that mono- and di-synaptic GABAergic inputs onto dentate granule cells were reversibly depressed by exogenous zinc application and enhanced by zinc chelation. Blocking T-type Ca2+ channels prevented the effect of zinc chelation. When recording from dentate fast-spiking interneurones, zinc chelation facilitated T-type Ca2+ currents, increased action potential half-width and decreased spike threshold. It also increased the offset of the input–output relation in a manner consistent with enhanced excitability. In granule cells, chelation of zinc reduced the time window for the integration of glutamatergic inputs originating from perforant path synapses, resulting in reduced spike transfer. Thus, zinc-mediated modulation of dentate interneurone excitability and GABA release regulates information flow to local targets and hippocampal networks. PMID:24081159

  15. DAG-sensitive and Ca2+ permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation

    PubMed Central

    Nagy, Gergő A.; Botond, Gergő; Borhegyi, Zsolt; Plummer, Nicholas W.; Freund, Tamás F.; Hájos, Norbert

    2012-01-01

    Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6-expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed-forward and feed-back inhibitory circuits in this brain region. PMID:23193081

  16. Endogenous zinc depresses GABAergic transmission via T-type Ca(2+) channels and broadens the time window for integration of glutamatergic inputs in dentate granule cells.

    PubMed

    Grauert, Antonia; Engel, Dominique; Ruiz, Arnaud J

    2014-01-01

    Zinc actions on synaptic transmission span the modulation of neurotransmitter receptors, transporters, activation of intracellular cascades and alterations in gene expression. Whether and how zinc affects inhibitory synaptic signalling in the dentate gyrus remains largely unexplored. We found that mono- and di-synaptic GABAergic inputs onto dentate granule cells were reversibly depressed by exogenous zinc application and enhanced by zinc chelation. Blocking T-type Ca(2+) channels prevented the effect of zinc chelation. When recording from dentate fast-spiking interneurones, zinc chelation facilitated T-type Ca(2+) currents, increased action potential half-width and decreased spike threshold. It also increased the offset of the input-output relation in a manner consistent with enhanced excitability. In granule cells, chelation of zinc reduced the time window for the integration of glutamatergic inputs originating from perforant path synapses, resulting in reduced spike transfer. Thus, zinc-mediated modulation of dentate interneurone excitability and GABA release regulates information flow to local targets and hippocampal networks.

  17. Anticonvulsive Activity in Audiogenic DBA/2 Mice of 1,4-Benzodiazepines and 1,5-Benzodiazepines with Different Activities at Cerebellar Granule Cell GABAA Receptors.

    PubMed

    Gatta, Elena; Cupello, Aroldo; Di Braccio, Mario; Grossi, Giancarlo; Robello, Mauro; Scicchitano, Francesca; Russo, Emilio; De Sarro, Giovambattista

    2016-12-01

    Herein, we tested in a model of generalized reflex epilepsy in mice different 1,4-benzodiazepines and 1,5-benzodiazepines with agonistic activity at the GABAA receptor population contributing to the peak component of the chloride current elicited by GABA in cerebellar granule cells (CGCs) in culture. The substances have all higher lipophilia than clobazam, an antiepileptic drug well known and used in human therapy. This ensures that they all can pass relatively easily the blood-brain barrier (BBB). The benzodiazepines were administered intraperitoneally (i.p.) and tested for their activity against sound-induced tonic and clonic seizures in a genetic model of experimental epilepsy, the DBA/2 mouse. Our data demonstrates an interesting inverse correlation between the ED50s and the efficacy (E %) of the drugs in increasing the peak chloride current elicited by GABA in cerebellar granule cells in culture. There is indication of the existence of a threshold of E % above which the increase of ED50 with increasing E % becomes linear. This is statistically significant for the clonic phase, whereas it is at the limit of significance for the tonic one. A possible interpretation of these results is that in this epilepsy model, projections from the cerebellum exert a convulsion prevention activity.

  18. EAT-2, a SAP-like adaptor, controls NK cell activation through phospholipase Cγ, Ca++, and Erk, leading to granule polarization.

    PubMed

    Pérez-Quintero, Luis-Alberto; Roncagalli, Romain; Guo, Huaijian; Latour, Sylvain; Davidson, Dominique; Veillette, André

    2014-04-07

    Ewing's sarcoma-associated transcript 2 (EAT-2) is an Src homology 2 domain-containing intracellular adaptor related to signaling lymphocytic activation molecule (SLAM)-associated protein (SAP), the X-linked lymphoproliferative gene product. Both EAT-2 and SAP are expressed in natural killer (NK) cells, and their combined expression is essential for NK cells to kill abnormal hematopoietic cells. SAP mediates this function by coupling SLAM family receptors to the protein tyrosine kinase Fyn and the exchange factor Vav, thereby promoting conjugate formation between NK cells and target cells. We used a variety of genetic, biochemical, and imaging approaches to define the molecular and cellular mechanisms by which EAT-2 controls NK cell activation. We found that EAT-2 mediates its effects in NK cells by linking SLAM family receptors to phospholipase Cγ, calcium fluxes, and Erk kinase. These signals are triggered by one or two tyrosines located in the carboxyl-terminal tail of EAT-2 but not found in SAP. Unlike SAP, EAT-2 does not enhance conjugate formation. Rather, it accelerates polarization and exocytosis of cytotoxic granules toward hematopoietic target cells. Hence, EAT-2 promotes NK cell activation by molecular and cellular mechanisms distinct from those of SAP. These findings explain the cooperative and essential function of these two adaptors in NK cell activation.

  19. Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis.

    PubMed

    Atlante, Anna; de Bari, Lidia; Bobba, Antonella; Marra, Ersilia; Passarella, Salvatore

    2007-11-01

    Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.

  20. Understanding size enlargement and hardening of granules on tabletability of unlubricated granules prepared by dry granulation.

    PubMed

    Patel, Sarsvatkumar; Dahiya, Sandeepkumar; Sun, Changquan Calvin; Bansal, Arvind Kumar

    2011-02-01

    The mechanism of loss of "reworkability" or tabletability of dry granulated microcrystalline cellulose (MCC) was investigated in relation to both granule size enlargement and granule hardness. Slugs of MCC were prepared under three pressures (12.5, 37.5, and 93.8 MPa) and tabletability (tensile strength vs. pressure) of respective granules (three different sizes) was determined. Nominal single granule fracture strength and granule friability were measured. The reduction in tabletability was profound for harder granules, which were obtained from higher slugging pressure. This is consistent with their ability to resist granule fragmentation during tableting. Variation in granule size exhibits negligible effect on tabletability for the lowest slugging pressure and only a small effect for the middle and highest slugging pressure. This observation is again related to different tendency to granule fragmentation during compaction. The results suggest that granule-hardening negatively affects tensile strength more than that of granule size enlargement for MCC.

  1. The type 1 Interleukin 1 receptor is not required for the death of murine hippocampal dentate granule cells and microglia activation

    PubMed Central

    Harry, G. Jean; Funk, Jason; Lefebvre d’Hellencourt, Christian; Aoyama, Mineyoshi

    2008-01-01

    Alterations in the inflammatory process, neuronal death, and glia response have been observed under manipulation of the interleukin-1 (IL-1) cytokine and subsequent signaling through the type 1 IL-1 receptor (IL-1R1). To investigate the influence of IL-1R1 activation in the pathophysiology of a chemical-induced injury to the murine hippocampus, we examined the level and pattern of neuronal death and neuroinflammation in 25-day-old male mice exposed to trimethyltin hydroxide (2.0 mg/kg, i.p.). In IL-1R1 null (IL-1R1−/−) mice, the pattern and severity of dentate granule cell death was similar as compared to wild type mice. In both groups of mice, mRNA levels for TNFα and MIP-1α were elevated and the early activation of microglia, including their ability to progress to a phagocytic phenotype, was maintained. Compared to WT mice, IL-1R1−/− mice displayed a limited glial fibrillary acidic protein (GFAP) astrocytic response, as well as a preferential induction in mRNA levels of Fas signaling components. Cumulatively, these results indicate that IL-1R1 activation is not necessary for TMT-induced death of dentate granule neurons or local activation of microglia; however, IL-1R1 signaling is involved in mediating the structural response of astrocytes to injury and may also regulate apoptotic mechanisms by influencing Fas signaling components. PMID:18191113

  2. High Frequency Burst Firing of Granule Cells Ensures Transmission at the Parallel Fiber to Purkinje Cell Synapse at the Cost of Temporal Coding

    PubMed Central

    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

  3. Phasic boosting of medial perforant path-evoked granule cell output time-locked to spontaneous dentate EEG spikes in awake rats.

    PubMed

    Bramham, C R

    1998-06-01

    Dentate spikes (DSs) are positive-going field potential transients that occur intermittently in the hilar region of the dentate gyrus during alert wakefulness and slow-wave sleep. The function of dentate spikes is unknown; they have been suggested to be triggered by perforant path input and are associated with firing of hilar interneurons and inhibition of CA3 pyramidal cells. Here we investigated the effect of DSs on medial perforant path (MPP)-granule cell-evoked transmission in freely moving rats. The MPP was stimulated selectively in the angular bundle while evoked field potentials and the EEG were recorded with a vertical multielectrode array in the dentate gyrus. DSs were identified readily on the basis of their characteristic voltage-versus-depth profile, amplitude, duration, and state dependency. Using on-line detection of the DS peak, the timing of MPP stimulation relative to single DSs was controlled. DS-triggered evoked responses were compared with conventional, manually evoked responses in still-alert wakefulness (awake immobility) and, in some cases, slow-wave sleep. Input-output curves were obtained with stimulation on the positive DS peak (0 delay) and at delays of 50, 100, and 500 ms. Stimulation on the peak DS was associated with a significant increase in the population spike amplitude, a reduction in population spike latency, and a decrease in the field excitatory postsynaptic potential (fEPSP) slope, relative to manual stimulation. Granule cell excitability was enhanced markedly during DSs, as indicated by a mean 93% increase in the population spike amplitude and a leftward shift in the fEPSP-spike relation. Maximum effects occurred at the DS peak, and lasted between 50 and 100 ms. Paired-pulse inhibition of the population spike was unaffected, indicating intact recurrent inhibition during DSs. The results demonstrate enhancement of perforant path-evoked granule cell output time-locked to DSs. DSs therefore may function to intermittently boost

  4. Transcriptomic and Physiological Insights into the Robustness of Long Filamentous Cells of Methanosaeta harundinacea, Prevalent in Upflow Anaerobic Sludge Blanket Granules

    PubMed Central

    Zhou, Liguang; Yu, Haiying; Ai, Guomin; Zhang, Bo; Hu, Songnian

    2014-01-01

    Methanosaeta spp. are widely distributed in natural environments, and their filamentous cells contribute significantly to sludge granulation and the good performance of anaerobic reactors. A previous study indicated that Methanosaeta harundinacea 6Ac displays a quorum sensing-regulated morphological transition from short to long filaments, and more acetate is channeled into methane production in long filaments, whereas more is channeled into biomass synthesis in short filaments. Here, we performed transcriptomic and physiological analysis to gain insights into active methanogenesis in long filaments of M. harundinacea 6Ac. Both RNA sequencing (RNA-seq) and quantitative reverse transcription-PCR indicated that transcription of the genes involved in aceticlastic methanogenesis and energy metabolism was upregulated 1.2- to 10.3-fold in long filaments, while transcription of the genes for the methyl oxidative shunt was upregulated in short filaments. [2-13C]acetate trace experiments demonstrated that a relatively higher portion of the acetate methyl group was oxidized to CO2 in short filaments than in long filaments. The long filaments exhibited higher catalase activity and oxygen tolerance than the short ones, which is consistent with increased transcription of the oxidant-scavenging genes. Moreover, transcription of genes for cell surface structures was upregulated in the long filaments, and transmission electron microscopy revealed a thicker cell envelope in the filaments. RNA-seq determined a >2-fold upregulation of a variety of antistress genes in short filaments, like those encoding chaperones and DNA repair systems, which implies that the short filaments can be stressed. This study reveals the genetic basis for the prevalence of the long filamentous morphology of M. harundinacea cells in upflow anaerobic sludge blanket granules. PMID:25398869

  5. Transcriptomic and physiological insights into the robustness of long filamentous cells of Methanosaeta harundinacea, prevalent in upflow anaerobic sludge blanket granules.

    PubMed

    Zhou, Liguang; Yu, Haiying; Ai, Guomin; Zhang, Bo; Hu, Songnian; Dong, Xiuzhu

    2015-02-01

    Methanosaeta spp. are widely distributed in natural environments, and their filamentous cells contribute significantly to sludge granulation and the good performance of anaerobic reactors. A previous study indicated that Methanosaeta harundinacea 6Ac displays a quorum sensing-regulated morphological transition from short to long filaments, and more acetate is channeled into methane production in long filaments, whereas more is channeled into biomass synthesis in short filaments. Here, we performed transcriptomic and physiological analysis to gain insights into active methanogenesis in long filaments of M. harundinacea 6Ac. Both RNA sequencing (RNA-seq) and quantitative reverse transcription-PCR indicated that transcription of the genes involved in aceticlastic methanogenesis and energy metabolism was upregulated 1.2- to 10.3-fold in long filaments, while transcription of the genes for the methyl oxidative shunt was upregulated in short filaments. [2-(13)C]acetate trace experiments demonstrated that a relatively higher portion of the acetate methyl group was oxidized to CO2 in short filaments than in long filaments. The long filaments exhibited higher catalase activity and oxygen tolerance than the short ones, which is consistent with increased transcription of the oxidant-scavenging genes. Moreover, transcription of genes for cell surface structures was upregulated in the long filaments, and transmission electron microscopy revealed a thicker cell envelope in the filaments. RNA-seq determined a >2-fold upregulation of a variety of antistress genes in short filaments, like those encoding chaperones and DNA repair systems, which implies that the short filaments can be stressed. This study reveals the genetic basis for the prevalence of the long filamentous morphology of M. harundinacea cells in upflow anaerobic sludge blanket granules.

  6. Cytotoxic effector cell granules recognized by the monoclonal antibody TIA-1 are present in CD8+ lymphocytes in lymph nodes of human immunodeficiency virus-1-infected patients.

    PubMed Central

    Tenner-Racz, K.; Racz, P.; Thomé, C.; Meyer, C. G.; Anderson, P. J.; Schlossman, S. F.; Letvin, N. L.

    1993-01-01

    A novel monoclonal antibody (mAB) TIA-1, which recognizes a 15-kd granule-associated protein of cytotoxic T lymphocytes and natural killer cells, has been applied to sections of lymph nodes with human immunodeficiency virus (HIV)-induced lymphadenopathy (follicular hyperplasia and lymphocyte depletion). The protein recognized by this mAB induces apoptosis in permeabilized lymphocytes in vitro. While this mAB reacted with approximately 46% of paracortical CD8+ cells in control nodes, it reacted with 75% of such cells in HIV-induced follicular hyperplasia. Germinal centers of the control nodes contained few TIA-1 + cells; in follicular hyperplasia caused by HIV-1, almost all germinal center CD8+ cells were TIA-1 +. Both in the control nodes and in HIV-induced follicular hyperplasia the majority of TIA-1 + cells coexpressed CD45R0. A marked loss of CD8+TIA-1+ cells was seen in lymphocyte-depleted nodes of patients with AIDS. The loss of these cytotoxic T lymphocytes may have a significant impact on the progression of the disease. Images Figure 1 Figure 2 Figure 3 PMID:8506945

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

    PubMed Central

    Bradford, Aaron B.; Mancini, Jayme 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 Ca2+ (Ca2+i) are crucial to proper migration, and MeHg is well known to disrupt CGC Ca2+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 Ca2+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 Ca2+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 Ca2+i dysregulation of CGCs. PMID:26514794

  8. Adult-generated hippocampal and neocortical neurons in macaques have a transient existence

    PubMed Central

    Gould, E.; Vail, N.; Wagers, M.; Gross, C. G.

    2001-01-01

    Previously we reported that new neurons are added to the hippocampus and neocortex of adult macaque monkeys. Here we compare the production and survival of adult-generated neurons and glia in the dentate gyrus, prefrontal cortex, and inferior temporal cortex. Twelve adult macaques were injected with the thymidine analogue BrdUrd, and the phenotypes of labeled cells were examined after 2 h, 24 h, 2 wk, 5 wk, 9 wk, and 12 wk by using the following immunocytochemical markers: for immature and mature neurons, class III β-tubulin (TuJ1); for mature neurons, neuronal nuclei; for astrocytes, glial fibrillary acidic protein; and for oligodendrocytes, 2′,3′-cyclic nucleotide 3′ phosphodiesterase. We found that the dentate gyrus had many more BrdUrd-labeled cells than either neocortical area. Furthermore, a greater percentage of BrdUrd-labeled cells expressed a neuronal marker in the dentate gyrus than in either neocortical area. The number of new cells in all three areas declined by 9 wk after BrdUrd labeling, suggesting that some of the new cells have a transient existence. BrdUrd-labeled cells also were found in the subventricular zone and in the white matter between the lateral ventricle and neocortex; some of the latter cells were double-labeled for BrdUrd and TuJ1. Adult neocortical neurogenesis is not restricted to primates. Five adult rats were injected with BrdUrd, and after a 3-wk survival time, there were cells double-labeled for BrdUrd and either TuJ1 or neuronal nuclei in the anterior neocortex as well as the dentate gyrus. PMID:11526209

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

    PubMed

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

    2015-07-21

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

  10. The effect of GABA stimulation on GABAA receptor subunit protein and mRNA expression in rat cultured cerebellar granule cells.

    PubMed Central

    Platt, K. P.; Zwartjes, R. E.; Bristow, D. R.

    1996-01-01

    1. After 8 days in vitro, rat cerebellar granule cells were exposed to 1 mM gamma-aminobutyric acid (GABA) for periods of 1, 2, 4, 6, 8 and 10 days. The effect of the GABA exposure on GABAA receptor alpha 1, alpha 6 and beta 2,3 subunit protein expression and alpha 1 and alpha 6 subunit steady-state mRNA levels, was examined using Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. 2. GABA exposure for 2 days decreased alpha 1 (35 +/- 10%, mean +/- s.e.mean), beta 2,3 (21 +/- 9%) and alpha 6 (28 +/- 10%) subunit protein expression compared to control levels. The GABA-mediated reduction in alpha 1 subunit expression after 2 days treatment was abolished in the presence of the GABAA receptor antagonist, Ru 5135 (10 microM). 3. GABA exposure for 8 days increased alpha 1 (26 +/- 10%, mean +/- s.e.mean) and beta 2,3 (56 +/- 23%) subunit protein expression over control levels, whereas alpha 6 subunit protein expression remained below control levels (by 38 +/- 10%). However, after 10 days GABA exposure, alpha 6 subunit protein expression was also increased over control levels by 65 +/- 29% (mean +/- s.e.mean). 4. GABA exposure did not change the alpha 1 or alpha 6 subunit steady-state mRNA levels over and 8 day period, nor did it alter the expression of cyclophilin mRNA over 1-8 days. 5. These results suggest that chronic GABA exposure of rat cerebellar granule cells has a bi-phasic effect on GABAA receptor subunit expression that is independent of changes to mRNA levels. Therefore, the regulation of the GABAA receptor expression by chronic agonist treatment appears to involve post-transcriptional and/or post-translational processes. Images Figure 1 Figure 3 Figure 4 PMID:8968548

  11. Dopamine D1 and D5 Receptors Modulate Spike Timing-Dependent Plasticity at Medial Perforant Path to Dentate Granule Cell Synapses

    PubMed Central

    Yang, Kechun

    2014-01-01

    Although evidence suggests that DA modulates hippocampal function, the mechanisms underlying that dopaminergic modulation are largely unknown. Using perforated-patch electrophysiological techniques to maintain the intracellular milieu, we investigated how the activation of D1-type DA receptors regulates spike timing-dependent plasticity (STDP) of the medial perforant path (mPP) synapse onto dentate granule cells. When D1-type receptors were inhibited, a relatively mild STDP protocol induced LTP only within a very narrow timing window between presynaptic stimulation and postsynaptic response. The stimulus protocol produced timing-dependent LTP (tLTP) only when the presynaptic stimulation was followed 30 ms later by depolarization-induced postsynaptic action potentials. That is, the time between presynaptic stimulation and postsynaptic response was 30 ms (Δt = +30 ms). When D1-type receptors were activated, however, the same mild STDP protocol induced tLTP over a much broader timing window: tLTP was induced when −30 ms ≤ Δt ≤ +30 ms. The result indicated that D1-type receptor activation enabled synaptic potentiation even when postsynaptic activity preceded presynaptic stimulation within this Δt range. Results with null mice lacking the Kv4.2 potassium channel and with the potassium channel inhibitor, 4-aminopyridine, suggested that D1-type receptors enhanced tLTP induction by suppressing the transient IA-type K+ current. Results obtained with antagonists and DA receptor knock-out mice indicated that endogenous activity of both D1 and D5 receptors modulated plasticity in the mPP. The DA D5 receptors appeared particularly important in regulating plasticity of the mPP onto the dentate granule cells. PMID:25429131

  12. Studies of alpha-granule proteins in cultured human megakaryocytes.

    PubMed

    Veljkovic, Dragoslava Kika; Cramer, Elisabeth M; Alimardani, Gulie; Fichelson, Serge; Massé, Jean-Marc; Hayward, Catherine P M

    2003-11-01

    alpha-Granule protein storage is important for producing platelets with normal haemostatic function. The low to undetectable levels of several megakaryocyte-synthesized alpha-granule proteins in normal plasma suggest megakaryocytes are important to sequester these proteins in vivo. alpha-Granule protein storage in vitro has been studied using other cell types, with differences observed in how some proteins are processed compared to platelets. Human megakaryocytes, cultured from cord blood CD34(+) cells and grown in serum-free media containing thrombopoietin, were investigated to determine if they could be used as a model for studying normal alpha-granule protein processing and storage. ELISA indicated that cultured megakaryocytes contained the alpha-granule proteins multimerin, von Willebrand factor, thrombospondin-1, beta-thromboglobulin and platelet factor 4, but no detectable fibrinogen and factor V. A significant proportion of the alpha-granule protein in megakaryocyte cultures was contained within the cells (averages: 41-71 %), consistent with storage. Detailed analyses of multimerin and von Willebrand factor confirmed that alpha-granule proteins were processed to mature forms and were predominantly located in the alpha-granules of cultured megakaryocytes.Thrombopoietin-stimulated cultured megakaryocytes provide a useful model for studying alpha-granule protein processing and storage.

  13. Effect of oviductal and cumulus cells on zona pellucida and cortical granules of porcine oocytes fertilized in vitro with epididymal spermatozoa.

    PubMed

    Romar, R; Coy, P; Gadea, J; Rath, D

    2005-02-01

    The objective of this study was to evaluate the effects of porcine oviductal epithelial cell (POEC) monolayers and cumulus cells on the zona pellucida (ZP) and cortical granules (CG) of in vitro matured porcine oocytes. Denuded and cumulus-enclosed oocytes were exposed to POEC before or during in vitro fertilization (IVF). The functional effects of the co-culture system were the tested on the ZP resistance, measured by the time necessary to dissolve the ZP with 0.1% pronase, and the distribution and density of the cortical granules. CG density in the equator and cortex of each oocyte was evaluated by confocal microscopy after staining with fluorescein isothiocyanate-labelled peanut agglutinin (FITC-PNA). Both variables were assessed immediately after an in vitro maturation period (IVM group), 3 and 6h after culture with or without (Control) oviductal cells (Experiment 1) and 3h after insemination with frozen-thawed epididymal spermatozoa in the presence or absence (Control) of oviductal cells (Experiment 2). The time to dissolve the ZP of oocytes from IVM group was 440.4 +/- 61.7 s and no difference was observed among groups in Experiment 1. In contrast, the density of CG was affected; oocytes pre-incubated for 6h had a higher density than those pre-incubated for 3 h (P <0.001). Oocytes fertilized in vitro in the presence of POEC (Experiment 2) had a similar ZP digestion time as control oocytes 3 h after insemination. The presence of POEC during IVF as well as the presence of cumulus cells had no effect on the density and distribution of CG. However, a significant decrease in the density of CG was observed in the fertilized oocytes compared to in vitro matured oocytes (P <0.001). It is concluded that under the conditions employed the oviductal and cumulus cells in the perifertilization period had no effect on ZP hardening and CG density. However, an increase in CG density was observed when oocytes were maintained in culture. In addition, no hardening of ZP was

  14. Inferred properties of stellar granulation

    SciTech Connect

    Gray, D.F.; Toner, C.G.

    1985-06-01

    Apparent characteristics of stellar granulation in F and G main-sequence stars are inferred directly from observed spectral-line asymmetries and from comparisons of numerical simulations with the observations: (1) the apparent granulation velocity increases with effective temperature, (2) the dispersion of granule velocities about their mean velocity of rise increases with the apparent granulation velocity, (3) the mean velocity of rise of granules must be less than the total line broadening, (4) the apparent velocity difference between granules and dark lanes corresponds to the granulation velocity deduced from stellar line bisectors, (5) the dark lanes show velocities of fall approximately twice as large as the granule rise velocities, (6) the light contributed to the stellar flux by the granules is four to ten times more than the light from the dark lanes. Stellar rotation is predicted to produce distortions in the line bisectors which may give information on the absolute velocity displacements of the line bisectors. 37 references.

  15. Formation of artificial granules for proving gelation as the main mechanism of aerobic granulation in biological wastewater treatment.

    PubMed

    Li, Yun; Yang, Shu-Fang; Zhang, Jian-Jun; Li, Xiao-Yan

    2014-01-01

    In this study, gelation-facilitated biofilm formation as a new mechanism is proposed for the phenomenon of aerobic granulation in biological wastewater treatment. To obtain an experimental proof for the gelation-based theory, the granulation process was simulated in a chemical system using latex particles for bacterial cells and organic polymers (alginate and peptone) for extracellular polymeric substances (EPS) in a solution with the addition of cations (Ca²⁺, Mg²⁺ and Fe³⁺). The results showed that at a low alginate content (70 mg g⁻¹ mixed liquid suspended solids (MLSS)) flocculation was observed in the suspension with loose flocs. At a higher alginate content (180 mg g⁻¹ MLSS), together with discharge of small flocs, formation of artificial gel granules was successfully achieved leading to granulation. The artificial granules show a morphological property similar to that of actual microbial granules. However, if the protein content increased, granulation became difficult with little gel formation. The experimental work demonstrates the importance of the bonding interactions between EPS functional groups and cations in gel formation and granulation. The laboratory results on the formation of artificial granules provide a sound proof for the theory of gelation-facilitated biofilm formation as the main mechanism for aerobic granulation in sludge suspensions.

  16. Glucose 1-phosphate is efficiently taken up by potato (Solanum tuberosum) tuber parenchyma cells and converted to reserve starch granules.

    PubMed

    Fettke, Joerg; Albrecht, Tanja; Hejazi, Mahdi; Mahlow, Sebastian; Nakamura, Yasunori; Steup, Martin

    2010-02-01

    Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [(14)C]glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.

  17. Coupling of Insulin Secretion and Display of a Granule-resident Zinc Transporter ZnT8 on the Surface of Pancreatic Beta Cells.

    PubMed

    Huang, Qiong; Merriman, Chengfeng; Zhang, Hao; Fu, Dax

    2017-03-10

    The islet-specific zinc transporter ZnT8 mediates zinc enrichment in the insulin secretory granules of the pancreatic beta cell. This granular zinc transporter is also a major self-antigen found in type 1 diabetes patients. It is not clear whether ZnT8 can be displayed on the cell surface and how insulin secretion may regulate the level of ZnT8 exposure to extracellular immune surveillance. Here we report specific antibody binding to the extracellular surface of rat insulinoma INS-1E cells that stably expressed a tagged human zinc transporter ZnT8. Flow cytometry analysis after fluorescent antibody labeling revealed strong correlations among the levels of ZnT8 expression, its display on the cell surface, and glucose-stimulated insulin secretion (GSIS). Glucose stimulation increased the surface display of endogenous ZnT8 from a basal level to 32.5% of the housekeeping Na(+)/K(+) ATPase on the cell surface, thereby providing direct evidence for a GSIS-dependent surface exposure of the ZnT8 self-antigen. Moreover, the variation in tagged-ZnT8 expression and surface labeling enabled sorting of heterogeneous beta cells to subpopulations that exhibited marked differences in GSIS with parallel changes in endogenous ZnT8 expression. The abundant surface display of endogenous ZnT8 and its coupling to GSIS demonstrated the potential of ZnT8 as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations.

  18. Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.

    PubMed

    Ohara-Imaizumi, Mica; Nakamichi, Yoko; Tanaka, Toshiaki; Katsuta, Hidenori; Ishida, Hitoshi; Nagamatsu, Shinya

    2002-04-01

    The dynamics of exocytosis/endocytosis of insulin secretory granules in pancreatic beta-cells remains to be clarified. In the present study, we visualized and analysed the motion of insulin secretory granules in MIN6 cells using pH-sensitive green fluorescent protein (pHluorin) fused to either insulin or the vesicle membrane protein, phogrin. In order to monitor insulin exocytosis, pHluorin, which is brightly fluorescent at approximately pH 7.4, but not at approximately pH 5.0, was attached to the C-terminus of insulin. To monitor the motion of insulin secretory granules throughout exocytosis/endocytosis, pHluorin was inserted between the third and fourth amino acids after the identified signal-peptide cleavage site of rat phogrin cDNA. Using this method of cDNA construction, pHluorin was located in the vesicle lumen, which may enable discrimination of the unfused acidic secretory granules from the fused neutralized ones. In MIN6 cells expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy (5 or 10 s intervals) revealed the appearance of fluorescent spots by depolarization after stimulation with 50 mM KCl and 22 mM glucose. The number of these spots in the image at the indicated times was counted and found to be consistent with the results of insulin release measured by RIA during the time course. In MIN6 cells expressing phogrin-pHluorin, data showed that fluorescent spots appeared following high KCl stimulation and remained stationary for a while, moved on the plasma membrane and then disappeared. Thus we demonstrate the visualized motion of insulin granule exocytosis/endocytosis using the pH-sensitive marker, pHluorin.

  19. Mucous granule exocytosis and CFTR expression in gallbladder epithelium.

    PubMed

    Kuver, R; Klinkspoor, J H; Osborne, W R; Lee, S P

    2000-02-01

    A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.

  20. Granuphilin exclusively mediates functional granule docking to the plasma membrane

    PubMed Central

    Mizuno, Kouichi; Fujita, Takuji; Gomi, Hiroshi; Izumi, Tetsuro

    2016-01-01

    In regulated exocytosis, it is generally assumed that vesicles must stably “dock” at the plasma membrane before they are primed to become fusion-competent. However, recent biophysical analyses in living cells that visualize fluorescent secretory granules have revealed that exocytic behaviors are not necessarily uniform: some granules beneath the plasma membrane are resistant to Ca2+ -triggered release, while others are accelerated to fuse without a pause for stable docking. These findings suggest that stable docking is unnecessary, and can even be inhibitory or nonfunctional, for fusion. Consistently, pancreatic β cells deficient in the Rab27 effector, granuphilin, lack insulin granules directly attached to the plasma membrane in electron micrographs but nevertheless exhibit augmented exocytosis. Here we directly compare the exocytic behaviors between granuphilin-positive and -negative insulin granules. Although granuphilin makes granules immobile and fusion-reluctant beneath the plasma membrane, those granuphilin-positive, docked granules release a portion of granuphilin upon fusion, and fuse at a frequency and time course similar to those of granuphilin-negative undocked granules. Furthermore, granuphilin forms a 180-nm cluster at the site of each docked granule, along with granuphilin-interacting Rab27a and Munc18-1 clusters. These findings indicate that granuphilin is an exclusive component of the functional and fusion-inhibitory docking machinery of secretory granules. PMID:27032672

  1. PLC-dependent intracellular Ca2+ release was associated with C6-ceramide-induced inhibition of Na+ current in rat granule cells.

    PubMed

    Liu, Zheng; Fei, Xiao-Wei; Fang, Yan-Jia; Shi, Wen-Jie; Zhang, Yu-Qiu; Mei, Yan-Ai

    2008-09-01

    In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule

  2. Molecular defects that affect platelet dense granules.

    PubMed

    Gunay-Aygun, Meral; Huizing, Marjan; Gahl, William A

    2004-10-01

    Platelet dense granules form using mechanisms shared by melanosomes in melanocytes and by subsets of lysosomes in more generalized cells. Consequently, disorders of platelet dense granules can reveal how organelles form and move within cells. Models for the study of new vesicle formation include isolated delta-storage pool deficiency, combined alphadelta-storage pool deficiency, Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, thrombocytopenia absent radii syndrome, and Wiskott-Aldrich syndrome. The molecular bases of dense granule deficiency are known for the seven subtypes of HPS, as well as for Chediak-Higashi syndrome, Griscelli syndrome, and Wiskott-Aldrich syndrome. The gene products involved in these disorders help elucidate the generalized process of the formation of vesicles from extant membranes such as the Golgi.

  3. Arsenite-induced stress granule formation is inhibited by elevated levels of reduced glutathione in West Nile virus-infected cells

    PubMed Central

    Basu, Mausumi; Courtney, Sean C.

    2017-01-01

    Oxidative stress activates the cellular kinase HRI, which then phosphorylates eIF2α, resulting in stalled translation initiation and the formation of stress granules (SGs). SG assembly redirects cellular translation to stress response mRNAs and inhibits cap-dependent viral RNA translation. Flavivirus infections were previously reported to induce oxidative stress in infected cells but flavivirus-infected cells paradoxically develop resistance to arsenite (Ars)-induced SG formation with time after infection. This resistance was previously postulated to be due to sequestration of the SG protein Caprin1 by Japanese encephalitis virus capsid protein. However, Caprin1 did not co-localize with West Nile virus (WNV) capsid protein in infected cells. Other stressors induced SGs with equal efficiency in mock- and WNV-infected cells indicating the intrinsic ability of cells to assemble SGs was not disabled. Induction of both reactive oxygen species (ROS) and the antioxidant response was detected at early times after WNV-infection. The transcription factors, Nrf2 and ATF4, which activate antioxidant genes, were upregulated and translocated to the nucleus. Knockdown of Nrf2, ATF4 or apoptosis-inducing factor (AIF), a mitochondrial protein involved in regenerating intracellular reduced glutathione (GSH) levels, with siRNA or treatment of cells with buthionine sulphoximine, which induces oxidative stress by inhibiting GSH synthesis, decreased intracellular GSH levels and increased the number of SG-positive, infected cells. Mitochondria were protected from Ars-induced damage by WNV infection until late times in the infection cycle. The results indicate that the increase in virus-induced ROS levels is counterbalanced by a virus-induced antioxidant response that is sufficient to also overcome the increase in ROS induced by Ars treatment and prevent Ars-induced SG assembly and mitochondrial damage. The virus-induced alterations in the cellular redox status appear to provide benefits

  4. GABAergic hyperinnervation of dentate granule cells in the Ts65Dn mouse model of down syndrome: Exploring the role of App.

    PubMed

    Mojabi, Fatemeh S; Fahimi, Atoossa; Moghadam, Shahrzad; Moghadam, Sarah; Windy McNerneny, M; Ponnusamy, Ravikumar; Kleschevnikov, Alexander; Mobley, William C; Salehi, Ahmad

    2016-12-01

    It has been suggested that increased GABAergic innervation in the hippocampus plays a significant role in cognitive dysfunction in Down syndrome (DS). Bolstering this notion, are studies linking hyper-innervation of the dentate gyrus (DG) by GABAergic terminals to failure in LTP induction in the Ts65Dn mouse model of DS. Here, we used extensive morphometrical methods to assess the status of GABAergic interneurons in the DG of young and old Ts65Dn mice and their 2N controls. We detected an age-dependent increase in GABAergic innervation of dentate granule cells (DGCs) in Ts65Dn mice. The primary source of GABAergic terminals to DGCs somata is basket cells (BCs). For this reason, we assessed the status of these cells and found a significant increase in the number of BCs in Ts65Dn mice compared with controls. Then we aimed to identify the gene/s whose overexpression could be linked to increased number of BCs in Ts65Dn and found that deleting the third copy of App gene in Ts65Dn mice led to normalization of the number of BCs in these mice. Our data suggest that App overexpression plays a major role in the pathophysiology of GABAergic hyperinnervation of the DG in Ts65Dn mice. © 2016 Wiley Periodicals, Inc.

  5. Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly.

    PubMed

    Emara, Mohamed M; Brinton, Margo A

    2007-05-22

    The West Nile virus minus-strand 3' terminal stem loop (SL) RNA was previously shown to bind specifically to cellular stress granule (SG) components, T cell intracellular antigen-1 (TIA-1) and the related protein TIAR. In vitro TIAR binding was 10 times more efficient than TIA-1. The 3'(-)SL functions as the promoter for genomic RNA synthesis. Colocalization of TIAR and TIA-1 with the viral replication complex components dsRNA and NS3 was observed in the perinuclear regions of West Nile virus- and dengue virus-infected cells. The kinetics of accumulation of TIAR in the perinuclear region was similar to those of genomic RNA synthesis. In contrast, relocation of TIA-1 to the perinuclear region began only after maximal levels of RNA synthesis had been achieved, except when TIAR was absent. Virus infection did not induce SGs and progressive resistance to SG induction by arsenite developed coincident with TIAR relocation. A progressive decrease in the number of processing bodies was secondarily observed in infected cells. These data suggest that the interaction of TIAR with viral components facilitates flavivirus genome RNA synthesis and inhibits SG formation, which prevents the shutoff of host translation.

  6. Boric acid induces cytoplasmic stress granule formation, eIF2α phosphorylation, and ATF4 in prostate DU-145 cells.

    PubMed

    Henderson, Kimberly A; Kobylewski, Sarah E; Yamada, Kristin E; Eckhert, Curtis D

    2015-02-01

    Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca(+2)) channel, and lowers endoplasmic reticulum (ER) [Ca(2+)]. Low ER [Ca(2+)] has been reported to induce ER stress and activate the eIF2α/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2α, GRP78/BiP, and ATF4. Mild activation of eIF2α and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2α/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron.

  7. Formation of volutin granules in Corynebacterium glutamicum.

    PubMed

    Pallerla, Srinivas Reddy; Knebel, Sandra; Polen, Tino; Klauth, Peter; Hollender, Juliane; Wendisch, Volker F; Schoberth, Siegfried M

    2005-02-01

    Volutin granules are intracellular storages of complexed inorganic polyphosphate (poly P). Histochemical staining procedures differentiate between pathogenic corynebacteria such as Corynebacterum diphtheriae (containing volutin) and non-pathogenic species, such as C. glutamicum. Here we report that strains ATCC13032 and MH20-22B of the non-pathogenic C. glutamicum also formed subcellular entities (18-37% of the total cell volume) that had the typical characteristics of volutin granules: (i) volutin staining, (ii) green UV fluorescence when stained with 4',6-diamidino-2-phenylindole, (iii) electron-dense and rich in phosphorus when determined with transmission electron microscopy and X-ray microanalysis, and (iv) 31P NMR poly P resonances of isolated granules dissolved in EDTA. MgCl2 addition to the growth medium stimulated granule formation but did not effect expression of genes involved in poly P metabolism. Granular volutin fractions from lysed cells contained polyphosphate glucokinase as detected by SDS-PAGE/MALDI-TOF, indicating that this poly P metabolizing enzyme is present also in intact poly P granules. The results suggest that formation of volutin is a more widespread phenomenon than generally accepted.

  8. Reduction of the immunostainable length of the hippocampal dentate granule cells' primary cilia in 3xAD-transgenic mice producing human A{beta}{sub 1-42} and tau

    SciTech Connect

    Chakravarthy, Balu; Gaudet, Chantal; Menard, Michel; Brown, Leslie; Atkinson, Trevor; LaFerla, Frank M.; Ito, Shingo; Armato, Ubaldo; Dal Pra, Ilaria; Whitfield, James

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer A{beta} and tau-induced neurofibrillary tangles play a key role in Alzheimer's disease. Black-Right-Pointing-Pointer A{beta}{sub 1-42} and mutant tau protein together reduce the primary cilium length. Black-Right-Pointing-Pointer This shortening likely reduces cilium-dependent neurogenesis and memory function. Black-Right-Pointing-Pointer This provides a model of an A{beta}/tau targeting of a neuronal signaling organelle. -- Abstract: The hippocampal dentate gyrus is one of the two sites of continuous neurogenesis in adult rodents and humans. Virtually all dentate granule cells have a single immobile cilium with a microtubule spine or axoneme covered with a specialized cell membrane loaded with receptors such as the somatostatin receptor 3 (SSTR3), and the p75 neurotrophin receptor (p75{sup NTR}). The signals from these receptors have been reported to stimulate neuroprogenitor proliferation and the post-mitotic maturation of newborn granule cells into functioning granule cells. We have found that in 6-24-months-old triple transgenic Alzheimer's disease model mice (3xTg-AD) producing both A{beta}{sub 1-42} and the mutant human tau protein tau{sub P301L,} the dentate granule cells still had immunostainable SSTR3- and p75{sup NTR}-bearing cilia but they were only half the length of the immunostained cilia in the corresponding wild-type mice. However, the immunostainable length of the granule cell cilia was not reduced either in 2xTg-AD mice accumulating large amounts of A{beta}{sub 1-42} or in mice accumulating only a mutant human tau protein. Thus it appears that a combination of A{beta}{sub 1-42} and tau protein accumulation affects the levels of functionally important receptors in 3xTg-AD mice. These observations raise the important possibility that structural and functional changes in granule cell cilia might have a role in AD.

  9. Chromaffin granules in the rat adrenal medulla release their secretory content in a particulate fashion.

    PubMed

    Crivellato, Enrico; Belloni, Anna; Nico, Beatrice; Nussdorfer, Gastone G; Ribatti, Domenico

    2004-03-01

    Exocytosis is considered the main route of granule discharge in chromaffin cells. We recently provided ultrastructural evidence suggesting that piecemeal degranulation (PMD) occurs in mouse adrenal chromaffin cells. In the present study, we processed rat adrenal glands for transmission electron microscopy (TEM), and examined chromaffin cells for changes characteristic of PMD. Both adrenaline (A)- and noradrenaline (NA)-storing cells express ultrastructural features suggestive of a slow and particulate mode of granule discharge. In adrenaline-containing cells, some granules present enlarged dimensions accompanied by eroded or dissolved matrices. Likewise, a number of granules in NA-releasing cells show content reduction with variably expanded granule chambers. Dilated, empty granule containers are recognizable in the cytoplasm of both cell types. Characteristically, altered granules and empty containers are seen intermingled with normal, resting granules. In addition, chromaffin granules often show irregular profiles, with budding or tail-like projections of their limiting membranes. Thirty 150-nm-diameter membrane-bound vesicles with a moderately electron-dense or -lucent internal structure are observable in the cytoplasm of both cell types. These vesicles are seen among the granules and some of them are fused with the perigranule membranes in the process of attachment to or budding from the granules. These data add further support to the concept that PMD may be an alternative secretory pathway in adrenal chromaffin cells.

  10. GRANULES ASSOCIATED WITH THE CHLOROPLAST LAMELLAE OF PORPHYRIDIUM CRUENTUM

    PubMed Central

    Gantt, E.; Conti, S. F.

    1966-01-01

    Small granules with a diameter of approximately 350 A are attached to the chloroplast lamellae of the red alga Porphyridium cruentum. To some extent, their size depends on the culture conditions and the age of the cell. It was possible to preserve the granules only with aldehyde prefixation. It can be seen that fixed or negatively stained granules are comprised of smaller subunits. The granules are arranged regularly on the lamellae in repeating rows with a center-to-center granule distance of 400 to 500 A. Attempts at characterization of these chloroplast granules revealed that they are resistant to hydrolysis by ribonuclease and appear to be structurally unaffected by methanol-acetone extraction. Because of their close association with the chloroplast lamellae, they are considered as possible sites of phycobilin concentration. This possibility is supported by two observations: when the phycobilins are removed, the granules disappear; and, when the chlorophyll and stainable membrane portions are selectively removed, the phycobilins and granules are still present. It was found that all other marine red algae examined had granules which were associated with the chloroplast lamellae. PMID:5962937

  11. Ultrastructural detection of nucleic acids within heat shock-induced perichromatin granules of HeLa cells by cytochemical and immunocytological methods.

    PubMed

    Charlier, Christine; Lamaye, Françoise; Thelen, Nicolas; Thiry, Marc

    2009-06-01

    The perichromatin granules (PGs) are enigmatic structures of the cell nucleus. The major drawbacks for a biological study are their rare occurrence and their small size in normal conditions. As heat shock has been shown to increase their number, we applied a hyperthermal shock on HeLa cells to investigate the nucleic acid content of PGs by means of cytochemical and immunocytological approaches. These heat shock-induced PGs (hsiPGs) appeared as clusters organized in the form of honeycomb structures and were always associated with some blocks of condensed chromatin, such as the perinucleolar chromatin shell. A stalk connecting the hsiPG to the chromatin could be observed. For the detection of RNA, we applied an immunocytological method involving two anti-RNA antibodies and quantified the gold labelling obtained. The results clearly revealed that hsiPGs contained RNA. Regarding to the detection of DNA, we used three different methods followed by quantitative analyses. The results seemed to indicate that a small amount of DNA was present in hsiPGs. Together, these findings suggest that hsiPGs might be RNP structures associated with particular regions of DNA.

  12. Doublecortin-positive newly born granule cells of hippocampus have abnormal apical dendritic morphology in the pilocarpine model of temporal lobe epilepsy.

    PubMed

    Arisi, Gabriel Maisonnave; Garcia-Cairasco, Norberto

    2007-08-24

    Here, we describe dentate gyrus newly born granule cells morphology in rats' temporal lobe epilepsy pilocarpine model. Digital reconstruction of doublecortin-positive neurons revealed that apical dendrites had the same total length and number of nodes in epileptic and control animals. Nonetheless, concentric spheres analyses revealed that apical dendrites spatial distribution was radically altered in epileptic animals. The apical dendrites had more bifurcations inside the granular cell layer and more terminations in the inner molecular layer of epileptic dentate gyrus. Branch order analyses showed that second- and third-order dendrites were shorter in epileptic animals. Apical dendrites were concentrated in regions like the inner molecular layer where granular neuron axons, named mossy fibers, sprout in epileptic animals. The combination of altered dendritic morphology and number enhancement of the new granular neurons suggests a deleterious role of hippocampal neurogenesis in epileptogenesis. Being more numerous and with dendrites concentrated in regions where aberrant axon terminals sprout, the new granular neurons could contribute to the slow epileptogenesis at hippocampal circuits commonly observed in temporal lobe epilepsy.

  13. Abnormal UP/DOWN Membrane Potential Dynamics Coupled with the Neocortical Slow Oscillation in Dentate Granule Cells during the Latent Phase of Temporal Lobe Epilepsy123

    PubMed Central

    Ouedraogo, David W.; Lenck-Santini, Pierre-Pascal; Marti, Geoffrey; Robbe, David; Crépel, Valérie

    2016-01-01

    The dentate gyrus, a major entry point to the hippocampus, gates (or filters) incoming information from the cortex. During sleep or anesthesia, the slow-wave oscillation (SWO) orchestrates hippocampus–neocortex communication, which is important for memory formation. The dentate gate is altered in temporal lobe epilepsy (TLE) early during epileptogenesis, which favors the propagation of pathological activities. Yet, whether the gating of physiological SWO by dentate granule cells (DGCs) is altered in TLE has remained unexplored. We combined intracellular recordings of membrane potential (Vm) of DGCs and local field potential recordings of the SWO in parietal cortex in anesthetized rats early during epileptogenesis [post-status epilepticus (SE) rats]. As expected, in control rats, the Vm of DGCs weakly and rarely oscillated in the SWO frequency range. In contrast, in post-SE rats, the Vm of DGCs displayed strong and long-lasting SWO. In these cells, clear UP and DOWN states, in phase with the neocortical SWO, led to a bimodal Vm distribution. In post-SE rats, the firing of DGCs was increased and more temporally modulated by the neocortical SWO. We conclude that UP/DOWN state dynamics dominate the Vm of DGCs and firing early during epileptogenesis. This abnormally strong neocortical influence on the dynamics of DGCs may profoundly modify the hippocampus–neocortex dialogue during sleep and associated cognitive functions. PMID:27257629

  14. [Effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expression of membrane transport proteins in K562/A02 cell xenografts].

    PubMed

    Li, Dong-Yun; Zheng, Zhi; Hou, Li; Jiang, Miao; Dong, Qing; Tian, Shao-Dan; Ma, Wei; Chen, Ju; Wang, Jing; Chen, Xin-Yi

    2010-02-01

    This study was purposed to investigate the effects of compound Zhe-Bei granule (CZBG) combined with doxorubicin on expressions of P-gp, MRP, LRP in K562/A02 cell xenografts. Tumor xenograft model were established by injecting the multidrug resistant cell line K562/A02 in the axillary flank of BALB/c-nu-nu mice. CZBG-intragastric administration and doxorubicin-intraperitoneal injection in combination were given to the BALB/c-nu nude mice. The tumor xenografts were made into slice after the dissection, and the expression of P-gp, MRP, LRP in K562/A02 tumor xenografts in mice were investigated by immunohistochemical technique. The integral optical density (IOD) of P-gp, MRP, LRP in K562/A02 tumor xenografts were measured by Image Pro Plus 6.0. The results showed that as compared with the doxorubicin alone, the combination of the doxorubicin and CZBG with high, middle and low dosage could decrease IOD of P-gp, MRP in K562/A02 tumor xenografts with statistical significance (p < 0.05). The LRP expression in K562/A02 tumor xenografts was not observed in five groups. It is concluded that the combination of CZBG with doxorubicin decreases the expressions of P-gp, MRP in K562/A02 tumor xenografts of mice.

  15. Down-regulation of CYP1A2 induction during the maturation of mouse cerebellar granule cells in culture: role of nitric oxide accumulation.

    PubMed

    Mulero-Navarro, Sonia; Santiago-Josefat, Belen; Pozo-Guisado, Eulalia; Merino, Jaime M; Fernandez-Salguero, Pedro M

    2003-10-01

    Nitric oxide (NO) is responsible for cytochrome P450 (CYP450) loss during isolation and cytokine treatment of primary rat hepatocytes. As P450s mediate the metabolism of toxic chemicals, their inhibition could compromise the cells competence to eliminate toxins, a condition potentially relevant in neurological diseases involving constitutive activation of nitric oxide synthase (NOS) and NO over-production. Here, we have investigated the correlation between NO accumulation and CYP1A2 down-regulation during maturation of mouse cerebellar granule cells (CGC). As neurons matured in culture, the inducible levels of CYP1A2 protein and catalytic activity decreased to almost undetectable values. In parallel, a significant increase in NO concentration was observed. Neuronal NOS remained constitutively active during maturation, thus contributing to NO accumulation. The NOS inhibitor l-NAME, restored CYP1A2 catalytic activity up to 9 days in vitro, supporting a role for NO in the inhibition process. Maturation was also followed by increased NMDA receptor activity and intracellular Ca2+ concentration. We suggest that maintained NOS activity during CGC maturation could lead to NO accumulation and to decreased CYP1A2 inducibility. Increased NMDA receptor activity and Ca2+ entry could contribute to this process. Thus, neurodegeneration could diminish the induction of specific P450s and impair the metabolism of foreign and/or endogenous chemicals in the CNS.

  16. Adrenaline Stimulates Glucagon Secretion in Pancreatic A-Cells by Increasing the Ca2+ Current and the Number of Granules Close to the L-Type Ca2+ Channels

    PubMed Central

    Gromada, Jesper; Bokvist, Krister; Ding, Wei-Guang; Barg, Sebastian; Buschard, Karsten; Renström, Erik; Rorsman, Patrik

    1997-01-01

    We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through ω-conotoxin-GVIA–sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via β-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels. PMID:9276750

  17. Apparatus for granulating coal

    SciTech Connect

    Ogino, E.; Harada, K.; Yoshii, N.

    1983-08-30

    A granulating apparatus is disclosed comprising a stirring tank or a duct for containing a slurry particulate to granular coal having a binder incorporated therein, a rotary shaft disposed in the tank or duct and at least one agitating blade made of metal netting and attached to the rotary shaft.

  18. Probing platelet factor 4 alpha-granule targeting.

    PubMed

    Briquet-Laugier, V; Lavenu-Bombled, C; Schmitt, A; Leboeuf, M; Uzan, G; Dubart-Kupperschmitt, A; Rosa, J-P

    2004-12-01

    The storage mechanism of endogenous secretory proteins in megakaryocyte alpha-granules is poorly understood. We have elected to study the granule storage of platelet factor 4 (PF4), a well-known platelet alpha-granule protein. The reporter protein green fluorescent protein (GFP), PF4, or PF4 fused to GFP (PF4-GFP), were transfected in the well-characterized mouse pituitary AtT20 cell line, and in the megakaryocytic leukemic DAMI cell line. These proteins were also transduced using a lentiviral vector, in human CD34+ cells differentiated into megakaryocytes in vitro. Intracellular localization of expressed proteins, and colocalization studies were achieved by laser scanning confocal microscopy and immuno-electronmicroscopy. In preliminary experiments, GFP, a non-secretory protein (no signal peptide), localized in the cytoplasm, while PF4-GFP colocalized with adrenocorticotropin hormone (ACTH)-containing granules in AtT20 cells. In the megakaryocytic DAMI cell line and in human megakaryocytes differentiated in vitro, PF4-GFP localized in alpha-granules along with the alpha granular protein von Willebrand factor (VWF). The signal peptide of PF4 was not sufficient to specify alpha-granule storage of PF4, since when PF4 signal peptide was fused to GFP (SP4-GFP), GFP was not stored into granules in spite of its efficient translocation to the ER-Golgi constitutive secretory pathway. We conclude that the PF4 storage pathway in alpha-granules is not a default pathway, but rather a regular granule storage pathway probably requiring specific sorting mechanisms. In addition PF4-GFP appears as an appropriate probe with which to analyze alpha-granule biogenesis and its alterations in the congenital defect gray platelet syndrome.

  19. Toxoplasma secretory granules: one population or more?

    PubMed

    Mercier, Corinne; Cesbron-Delauw, Marie-France

    2015-02-01

    In Toxoplasma gondii, dense granules are known as the storage secretory organelles of the so-called GRA proteins (for dense granule proteins), which are destined to the parasitophorous vacuole (PV) and the PV-derived cyst wall. Recently, newly annotated GRA proteins targeted to the host cell nucleus have enlarged this view. Here we provide an update on the latest developments on the Toxoplasma secreted proteins, which to date have been mainly studied at both the tachyzoite and bradyzoite stages, and we point out that recent discoveries could open the issue of a possible, yet uncharacterized, distinct secretory pathway in Toxoplasma.

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

    PubMed

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

    2004-07-01

    Keishi-bukuryo-gan (Gui-Zhi-Fu-Ling-Wan) (KBG) is a traditional Chinese/Japanese medical (Kampo) formulation that has been administered to patients with "Oketsu" (blood stagnation) syndrome. In the process of neuronal cell death induced by brain ischemia, excessive generation of nitric oxide (NO) free radicals is implicated in the neurotoxicity. In the present study, we examined the protective effects of KBG and its constituent medic