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Sample records for granule cell axon

  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. Cholinergic afferent stimulation induces axonal function plasticity in adult hippocampal granule cells.

    PubMed

    Martinello, Katiuscia; Huang, Zhuo; Lujan, Rafael; Tran, Baouyen; Watanabe, Masahiko; Cooper, Edward C; Brown, David A; Shah, Mala M

    2015-01-21

    Acetylcholine critically influences hippocampal-dependent learning. Cholinergic fibers innervate hippocampal neuron axons, dendrites, and somata. The effects of acetylcholine on axonal information processing, though, remain unknown. By stimulating cholinergic fibers and making electrophysiological recordings from hippocampal dentate gyrus granule cells, we show that synaptically released acetylcholine preferentially lowered the action potential threshold, enhancing intrinsic excitability and synaptic potential-spike coupling. These effects persisted for at least 30 min after the stimulation paradigm and were due to muscarinic receptor activation. This caused sustained elevation of axonal intracellular Ca(2+) via T-type Ca(2+) channels, as indicated by two-photon imaging. The enhanced Ca(2+) levels inhibited an axonal KV7/M current, decreasing the spike threshold. In support, immunohistochemistry revealed muscarinic M1 receptor, CaV3.2, and KV7.2/7.3 subunit localization in granule cell axons. Since alterations in axonal signaling affect neuronal firing patterns and neurotransmitter release, this is an unreported cellular mechanism by which acetylcholine might, at least partly, enhance cognitive processing. PMID:25578363

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  5. Automated kymograph analysis for profiling axonal transport of secretory granules.

    PubMed

    Mukherjee, Amit; Jenkins, Brian; Fang, Cheng; Radke, Richard J; Banker, Gary; Roysam, Badrinath

    2011-06-01

    This paper describes an automated method to profile the velocity patterns of small organelles (BDNF granules) being transported along a selected section of axon of a cultured neuron imaged by time-lapse fluorescence microscopy. Instead of directly detecting the granules as in conventional tracking, the proposed method starts by generating a two-dimensional spatio-temporal map (kymograph) of the granule traffic along an axon segment. Temporal sharpening during the kymograph creation helps to highlight granule movements while suppressing clutter due to stationary granules. A voting algorithm defined over orientation distribution functions is used to refine the locations and velocities of the granules. The refined kymograph is analyzed using an algorithm inspired from the minimum set cover framework to generate multiple motion trajectories of granule transport paths. The proposed method is computationally efficient, robust to significant levels of noise and clutter, and can be used to capture and quantify trends in transport patterns quickly and accurately. When evaluated on a collection of image sequences, the proposed method was found to detect granule movement events with 94% recall rate and 82% precision compared to a time-consuming manual analysis. Further, we present a study to evaluate the efficacy of velocity profiling by analyzing the impact of oxidative stress on granule transport in which the fully automated analysis correctly reproduced the biological conclusion generated by manual analysis. PMID:21330183

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

    PubMed

    Blot, Antonin; Barbour, Boris

    2014-02-01

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

  7. RNA Granules in Germ Cells

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

    Burton, Shawn D.

    2015-01-01

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

  9. Iron granules in plasma cells.

    PubMed Central

    Cook, M K; Madden, M

    1982-01-01

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

  10. Degeneration and regeneration of ganglion cell axons.

    PubMed

    Weise, J; Ankerhold, R; Bähr, M

    2000-01-15

    The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed. PMID:10649506

  11. Mast cell secretory granules: armed for battle.

    PubMed

    Wernersson, Sara; Pejler, Gunnar

    2014-07-01

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

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

    PubMed

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

    2002-01-01

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

  13. How Schwann Cells Sort Axons: New Concepts.

    PubMed

    Feltri, M Laura; Poitelon, Yannick; Previtali, Stefano Carlo

    2016-06-01

    Peripheral nerves contain large myelinated and small unmyelinated (Remak) fibers that perform different functions. The choice to myelinate or not is dictated to Schwann cells by the axon itself, based on the amount of neuregulin I-type III exposed on its membrane. Peripheral axons are more important in determining the final myelination fate than central axons, and the implications for this difference in Schwann cells and oligodendrocytes are discussed. Interestingly, this choice is reversible during pathology, accounting for the remarkable plasticity of Schwann cells, and contributing to the regenerative potential of the peripheral nervous system. Radial sorting is the process by which Schwann cells choose larger axons to myelinate during development. This crucial morphogenetic step is a prerequisite for myelination and for differentiation of Remak fibers, and is arrested in human diseases due to mutations in genes coding for extracellular matrix and linkage molecules. In this review we will summarize progresses made in the last years by a flurry of reverse genetic experiments in mice and fish. This work revealed novel molecules that control radial sorting, and contributed unexpected ideas to our understanding of the cellular and molecular mechanisms that control radial sorting of axons. PMID:25686621

  14. Intraretinal projection of retinal ganglion cell axons as a model system for studying axon navigation

    PubMed Central

    Bao, Zheng-Zheng

    2008-01-01

    The initial step of retinal ganglion cell (RGC) axon pathfinding involves directed growth of RGC axons toward the center of the retina, the optic disc, a process termed “intraretinal guidance”. Due to the accessibility of the system, and with various embryological, molecular, and genetic approaches, significant progress has been made in recent years toward understanding the mechanisms involved in the precise guidance of the RGC axons. As axons are extending from RGCs located throughout the retina, a multitude of factors expressed along with the differentiation wave are important for the guidance of the RGC axons. To ensure that the RGC axons are oriented correctly, restricted to the optic fiber layer (OFL) of the retina, and exit the eye properly, different sets of positive and negative factors cooperate in the process. Fasciculation mediated by a number of cell adhesion molecules (CAMs) and modulation of axonal response to guidance factors provide additional mechanisms to ensure proper guidance of the RGC axons. The intraretinal axon guidance thus serves as an excellent model system for studying how different signals are regulated, modulated and integrated for guiding a large number of axons in three-dimensional space. PMID:17320832

  15. 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.

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

    PubMed

    Yaeger, Daniel B; Trussell, Laurence O

    2015-03-18

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

  17. Calcium transients in cerebellar granule cell presynaptic terminals.

    PubMed Central

    Regehr, W G; Atluri, P P

    1995-01-01

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

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

    PubMed

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

    2015-10-01

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

  19. 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

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

    PubMed

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

    1993-01-01

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

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

    PubMed

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

    2015-10-01

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

  2. Synaptic representation of locomotion in single cerebellar granule cells

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Ishikawa, Taro; Shimuta, Misa; Häusser, Michael

    2015-01-01

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

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

    EPA Science Inventory

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

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

    PubMed Central

    Krzewski, Konrad; Coligan, John E.

    2012-01-01

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

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

    PubMed Central

    Diño, Maria. R.; Mugnaini, Enrico

    2009-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  8. Inhibition of Cerebellar Granule Cell Turning by Alcohol

    PubMed Central

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

    2010-01-01

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

  9. Retinal ganglion cell axons regenerate in the presence of intact sensory fibres.

    PubMed

    King, Carolyn; Bartlett, Carole; Sauvé, Yves; Lund, Ray; Dunlop, Sarah; Beazley, Lyn

    2006-02-01

    A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053+/-223) and conventional grafts (1323+/-881; P>0.05). The results indicate that an intact sensory environment supports central axon regeneration. PMID:16407770

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

    PubMed Central

    Azouz, Nurit Pereg; Hammel, Ilan

    2014-01-01

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

  11. Axonal Control of the Adult Neural Stem Cell Niche

    PubMed Central

    Tong, Cheuk Ka; Chen, Jiadong; Cebrián-Silla, Arantxa; Mirzadeh, Zaman; Obernier, Kirsten; Guinto, Cristina D.; Tecott, Laurence H.; García-Verdugo, Jose Manuel; Kriegstein, Arnold; Alvarez-Buylla, Arturo

    2014-01-01

    SUMMARY The ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSC) in the walls of the lateral ventricles of the adult brain. How the adult brain’s neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C. PMID:24561083

  12. Axonal control of the adult neural stem cell niche.

    PubMed

    Tong, Cheuk Ka; Chen, Jiadong; Cebrián-Silla, Arantxa; Mirzadeh, Zaman; Obernier, Kirsten; Guinto, Cristina D; Tecott, Laurence H; García-Verdugo, Jose Manuel; Kriegstein, Arnold; Alvarez-Buylla, Arturo

    2014-04-01

    The ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain's neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C. PMID:24561083

  13. 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

  14. Retrograde and Wallerian Axonal Degeneration Occur Synchronously after Retinal Ganglion Cell Axotomy

    PubMed Central

    Kanamori, Akiyasu; Catrinescu, Maria-Magdalena; Belisle, Jonathan M.; Costantino, Santiago; Levin, Leonard A.

    2013-01-01

    Axonal injury and degeneration are pivotal pathological events in diseases of the nervous system. In the past decade, it has been recognized that the process of axonal degeneration is distinct from somal degeneration and that axoprotective strategies may be distinct from those that protect the soma. Preserving the cell body via neuroprotection cannot improve function if the axon is damaged, because the soma is still disconnected from its target. Therefore, understanding the mechanisms of axonal degeneration is critical for developing new therapeutic interventions for axonal disease treatment. We combined in vivo imaging with a multilaser confocal scanning laser ophthalmoscope and in vivo axotomy with a diode-pumped solid-state laser to assess the time course of Wallerian and retrograde degeneration of unmyelinated retinal ganglion cell axons in living rats for 4 weeks after intraretinal axotomy. Laser injury resulted in reproducible axon loss both distal and proximal to the site of injury. Longitudinal polarization-sensitive imaging of axons demonstrated that Wallerian and retrograde degeneration occurred synchronously. Neurofilament immunostaining of retinal whole-mounts confirmed axonal loss and demonstrated sparing of adjacent axons to the axotomy site. In vivo fluorescent imaging of axonal transport and photobleaching of labeled axons demonstrated that the laser axotomy model did not affect adjacent axon function. These results are consistent with a shared mechanism for Wallerian and retrograde degeneration. PMID:22642911

  15. Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations

    PubMed Central

    Carrasco, Monica A.; Williams, Luis A.; Winborn, Christina S.; Han, Steve S. W.; Kiskinis, Evangelos; Winborn, Brett; Freibaum, Brian D.; Kanagaraj, Anderson; Clare, Alison J.; Badders, Nisha M.; Bilican, Bilada; Chaum, Edward; Chandran, Siddharthan; Shaw, Christopher E.; Eggan, Kevin C.; Maniatis, Tom; Taylor, J. Paul

    2014-01-01

    Summary The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43. PMID:24507191

  16. RNA-binding protein Hermes/RBPMS inversely affects synapse density and axon arbor formation in retinal ganglion cells in vivo.

    PubMed

    Hörnberg, Hanna; Wollerton-van Horck, Francis; Maurus, Daniel; Zwart, Maarten; Svoboda, Hanno; Harris, William A; Holt, Christine E

    2013-06-19

    The RNA-binding protein Hermes [RNA-binding protein with multiple splicing (RBPMS)] is expressed exclusively in retinal ganglion cells (RGCs) in the CNS, but its function in these cells is not known. Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons. Hermes loss of function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum, and expression of a dominant acting mutant Hermes protein, defective in RNA-granule localization, causes similar defects in arborization. Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability. Surprisingly, Hermes depletion also leads to enhanced early visual behavior and an increase in the density of presynaptic puncta, suggesting that reduced arborization is accompanied by increased synaptogenesis to maintain synapse number. PMID:23785151

  17. RNA-binding protein Hermes/RBPMS inversely affects synapse density and axon arbor formation in retinal ganglion cells in vivo

    PubMed Central

    Hörnberg, Hanna; Horck, Francis Wollerton-van; Maurus, Daniel; Zwart, Maarten; Svoboda, Hanno; Harris, William A.; Holt, Christine E.

    2015-01-01

    The RNA-binding protein, Hermes (RBPMS), is expressed exclusively in retinal ganglion cells (RGCs) in the CNS, but its function in these cells is not known. Here we show that Hermes protein translocates in granules from RGC bodies down the growing axons. Hermes loss-of-function in both Xenopus laevis and zebrafish embryos leads to a significant reduction in retinal axon arbor complexity in the optic tectum, and expression of a dominant acting mutant Hermes protein, defective in RNA-granule localisation, causes similar defects in arborisation. Time-lapse analysis of branch dynamics reveals that the decrease in arbor complexity is caused by a reduction in new branches rather than a decrease in branch stability. Surprisingly, Hermes depletion also leads to enhanced early visual behaviour and an increase in the density of pre-synaptic puncta suggesting that reduced arborisation is accompanied by increased synaptogenesis to maintain synapse number. PMID:23785151

  18. Cellular synthesis and axonal transport of gamma-aminobutyric acid in a photoreceptor cell of the barnacle.

    PubMed Central

    Koike, H; Tsuda, K

    1980-01-01

    1. [3H]glutamate or [3H]gamma-aminobutyric acid (GABA) was injected into the photoreceptor cell of the lateral ocellus of Balanus eburneus, in order to study the transmitter substance of the cell. 2. The photoreceptor cell synthesized [3H]GABA from injected [3H]glutamate. 3. The newly formed [3H]GABA moved inside the photoreceptor axon towards the axon terminal with a velocity of about 0.9 mm/hr. Injected [3H]GABA moved at 0.9 mm/hr and also at 0.4 mm/hr. 4. Axonally transported [3H]GABA reached the axon terminal within several hours following the injection. It did not accumulate at the terminal, but gradually disappeared. 5. Light-microscope and electron-microscope autoradiography following the injection of [3H]GABA revealed that [3H]-reacted silver grains were present in a certain type of axon terminal. The terminal thus identified as that of a photoreceptor cell contains many clear, polymorphic synaptic vesicles about 300-500 A in diameter, some dense-cored vesicles 700-1300 A in diameter, and glycogen granules. The terminal forms many synapses, and each synapse has a synaptic dense body. The terminal always faces two post-synaptic elements at the synapse, forming a triad with a gap distance of about 160-200 A. 6. A GABA analogue, [3H]di-aminobutyric acid, was selectively taken up into the terminals previously identified as those of photoreceptors. 7. These results support the notion that the transmitter substance of the photoreceptor cell of the barnacle is GABA. Images Plate 1 Plate 2 PMID:6160239

  19. Connecting the eye to the brain: the molecular basis of ganglion cell axon guidance

    PubMed Central

    Oster, S F; Sretavan, D W

    2003-01-01

    In the past several years, a great deal has been learnt about the molecular basis through which specific neural pathways in the visual system are established during embryonic development. This review provides a framework for understanding the principles of retinal ganglion cell axon guidance, and introduces some of the families of axon guidance molecules involved. In addition, the potential relevance of retinal axon guidance to human visual developmental disorders, and to retinal axon regeneration, is discussed. PMID:12714414

  20. 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

  1. 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.

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

    PubMed Central

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

    2015-01-01

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

  3. Severely dystrophic axons at amyloid plaques remain continuous and connected to viable cell bodies.

    PubMed

    Adalbert, Robert; Nogradi, Antal; Babetto, Elisabetta; Janeckova, Lucie; Walker, Simon A; Kerschensteiner, Martin; Misgeld, Thomas; Coleman, Michael P

    2009-02-01

    Synapse loss precedes cell death in Alzheimer's disease, but the timing of axon degeneration relative to these events, and the causal relationships remain unclear. Axons become so severely dystrophic near amyloid plaques that their interruption, causing permanent loss of function, extensive synapse loss, and potentially cell death appears imminent. However, it remains unclear whether axons are truly interrupted at plaques and whether cell bodies fail to support their axons and dendrites. We traced TgCRND8 mouse axons longitudinally through, distal to, and proximal from dystrophic regions. The corresponding neurons not only survived but remained morphologically unaltered, indicating absence of axonal damage signalling or a failure to respond to it. Axons, no matter how dystrophic, remained continuous and initially morphologically normal outside the plaque region, reflecting support by metabolically active cell bodies and continued axonal transport. Immunochemical and ultrastructural studies showed dystrophic axons were tightly associated with disruption of presynaptic transmission machinery, suggesting local functional impairment. Thus, we rule out long-range degeneration axons or dendrites as major contributors to early synapse loss in this model, raising the prospect of a therapeutic window for functional rescue of individual neurons lasting months or even years after their axons become highly dystrophic. We propose that multi-focal pathology has an important role in the human disease in bringing about the switch from local, and potentially recoverable, synapse loss into permanent loss of neuronal processes and eventually their cell bodies. PMID:19059977

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

    PubMed Central

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

    2003-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. 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.

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

    PubMed Central

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

    1997-01-01

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

  8. Control of cerebellar granule cell output by sensory-evoked Golgi cell inhibition

    PubMed Central

    Duguid, Ian; Branco, Tiago; Chadderton, Paul; Arlt, Charlotte; Powell, Kate; Häusser, Michael

    2015-01-01

    Classical feed-forward inhibition involves an excitation–inhibition sequence that enhances the temporal precision of neuronal responses by narrowing the window for synaptic integration. In the input layer of the cerebellum, feed-forward inhibition is thought to preserve the temporal fidelity of granule cell spikes during mossy fiber stimulation. Although this classical feed-forward inhibitory circuit has been demonstrated in vitro, the extent to which inhibition shapes granule cell sensory responses in vivo remains unresolved. Here we combined whole-cell patch-clamp recordings in vivo and dynamic clamp recordings in vitro to directly assess the impact of Golgi cell inhibition on sensory information transmission in the granule cell layer of the cerebellum. We show that the majority of granule cells in Crus II of the cerebrocerebellum receive sensory-evoked phasic and spillover inhibition prior to mossy fiber excitation. This preceding inhibition reduces granule cell excitability and sensory-evoked spike precision, but enhances sensory response reproducibility across the granule cell population. Our findings suggest that neighboring granule cells and Golgi cells can receive segregated and functionally distinct mossy fiber inputs, enabling Golgi cells to regulate the size and reproducibility of sensory responses. PMID:26432880

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

    PubMed

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

    2014-01-01

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

  10. The myelinated axon is dependent on the myelinating cell for support and maintenance: molecules involved.

    PubMed

    Edgar, J M; Garbern, J

    2004-06-01

    The myelin-forming cells, oligodendrocytes and Schwann cells, extend processes that spirally wrap axons and provide the insulation that allows rapid saltatory conduction. Recent data suggest a further role for the myelin-forming cells in axonal support and maintenance. This Mini-Review summarises some of the data that support this view and highlights the molecules involved. PMID:15139018

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

    PubMed Central

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

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

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

    PubMed

    Nunes, Daniel; Kuner, Thomas

    2015-01-01

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

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

    PubMed

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

    1999-05-01

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

  14. 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

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

    PubMed

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

    1995-06-27

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

  16. Clustering of voltage-dependent sodium channels on axons depends on Schwann cell contact.

    PubMed

    Joe, E H; Angelides, K

    1992-03-26

    In myelinated nerves, segregation of voltage-dependent sodium channels to nodes of Ranvier is crucial for saltatory conduction along axons. As sodium channels associate and colocalize with ankyrin at nodes of Ranvier, one possibility is that sodium channels are recruited and immobilized at axonal sites which are specified by the subaxolemmal cytoskeleton, independent of glial cell contact. Alternatively, segregation of channels at distinct sites along the axon may depend on glial cell contact. To resolve this question, we have examined the distribution of sodium channels, ankyrin and spectrin in myelination-competent cocultures of sensory neurons and Schwann cells by immunofluorescence, using sodium channel-, ankyrin- and spectrin-specific antibodies. In the absence of Schwann cells, sodium channels, ankyrin and spectrin are homogeneously distributed on sensory axons. When Schwann cells are introduced into these cultures, the distribution of sodium channels dramatically changes so that channel clusters on axons are abundant, but ankyrin and spectrin remain homogeneously distributed. Addition of latex beads or Schwann cell membranes does not induce channel clustering. Our results suggest that segregation of sodium channels on axons is highly dependent on interactions with active Schwann cells and that continuing axon-glial interactions are necessary to organize and maintain channel distribution during differentiation of myelinated axons. PMID:1312680

  17. Tonic inhibition sets the state of excitability in olfactory bulb granule cells

    PubMed Central

    Labarrera, Christina; London, Michael; Angelo, Kamilla

    2013-01-01

    GABAergic granule cells (GCs) regulate, via mitral cells, the final output from the olfactory bulb to piriform cortex and are central for the speed and accuracy of odour discrimination. However, little is known about the local circuits in which GCs are embedded and how GCs respond during functional network activity. We recorded inhibitory and excitatory currents evoked during a single sniff-like odour presentation in GCs in vivo. We found that synaptic excitation was extensively activated across cells, whereas phasic inhibition was rare. Furthermore, our analysis indicates that GCs are innervated by a persistent firing of deep short axon cells that mediated the inhibitory evoked responses. Blockade of GABAergic synaptic input onto GCs revealed a tonic inhibitory current mediated by furosemide-sensitive GABAA receptors. The average current associated with this tonic GABAergic conductance was 3-fold larger than that of phasic inhibitory postsynaptic currents. We show that the pharmacological blockage of tonic inhibition markedly increased the occurrence of supra-threshold responses during an odour-stimulated sniff. Our findings suggest that GCs mediate recurrent or lateral inhibition, depending on the ambient level of extracellular GABA. PMID:23318869

  18. Axon ensheathment and metabolic supply by glial cells in Drosophila.

    PubMed

    Schirmeier, Stefanie; Matzat, Till; Klämbt, Christian

    2016-06-15

    Neuronal function requires constant working conditions and a well-balanced supply of ions and metabolites. The metabolic homeostasis in the nervous system crucially depends on the presence of glial cells, which nurture and isolate neuronal cells. Here we review recent findings on how these tasks are performed by glial cells in the genetically amenable model organism Drosophila melanogaster. Despite the small size of its nervous system, which would allow diffusion of metabolites, a surprising division of labor between glial cells and neurons is evident. Glial cells are glycolytically active and transfer lactate and alanine to neurons. Neurons in turn do not require glycolysis but can use the glially provided compounds for their energy homeostasis. Besides feeding neurons, glial cells also insulate neuronal axons in a way similar to Remak fibers in the mammalian nervous system. The molecular mechanisms orchestrating this insulation require neuregulin signaling and resemble the mechanisms controlling glial differentiation in mammals surprisingly well. We hypothesize that metabolic cross talk and insulation of neurons by glial cells emerged early during evolution as two closely interlinked features in the nervous system. This article is part of a Special Issue entitled SI: Myelin Evolution. PMID:26367447

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

    PubMed

    Hammel, Ilan; Anaby, Debbie

    2007-09-01

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

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

    PubMed

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

    2016-06-01

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

  1. Jab1 regulates Schwann cell proliferation and axonal sorting through p27

    PubMed Central

    Porrello, Emanuela; Rivellini, Cristina; Dina, Giorgia; Triolo, Daniela; Del Carro, Ubaldo; Ungaro, Daniela; Panattoni, Martina; Feltri, Maria Laura; Wrabetz, Lawrence; Pardi, Ruggero; Quattrini, Angelo

    2014-01-01

    Axonal sorting is a crucial event in nerve formation and requires proper Schwann cell proliferation, differentiation, and contact with axons. Any defect in axonal sorting results in dysmyelinating peripheral neuropathies. Evidence from mouse models shows that axonal sorting is regulated by laminin211– and, possibly, neuregulin 1 (Nrg1)–derived signals. However, how these signals are integrated in Schwann cells is largely unknown. We now report that the nuclear Jun activation domain–binding protein 1 (Jab1) may transduce laminin211 signals to regulate Schwann cell number and differentiation during axonal sorting. Mice with inactivation of Jab1 in Schwann cells develop a dysmyelinating neuropathy with axonal sorting defects. Loss of Jab1 increases p27 levels in Schwann cells, which causes defective cell cycle progression and aberrant differentiation. Genetic down-regulation of p27 levels in Jab1-null mice restores Schwann cell number, differentiation, and axonal sorting and rescues the dysmyelinating neuropathy. Thus, Jab1 constitutes a regulatory molecule that integrates laminin211 signals in Schwann cells to govern cell cycle, cell number, and differentiation. Finally, Jab1 may constitute a key molecule in the pathogenesis of dysmyelinating neuropathies. PMID:24344238

  2. Jab1 regulates Schwann cell proliferation and axonal sorting through p27.

    PubMed

    Porrello, Emanuela; Rivellini, Cristina; Dina, Giorgia; Triolo, Daniela; Del Carro, Ubaldo; Ungaro, Daniela; Panattoni, Martina; Feltri, Maria Laura; Wrabetz, Lawrence; Pardi, Ruggero; Quattrini, Angelo; Previtali, Stefano Carlo

    2014-01-13

    Axonal sorting is a crucial event in nerve formation and requires proper Schwann cell proliferation, differentiation, and contact with axons. Any defect in axonal sorting results in dysmyelinating peripheral neuropathies. Evidence from mouse models shows that axonal sorting is regulated by laminin211- and, possibly, neuregulin 1 (Nrg1)-derived signals. However, how these signals are integrated in Schwann cells is largely unknown. We now report that the nuclear Jun activation domain-binding protein 1 (Jab1) may transduce laminin211 signals to regulate Schwann cell number and differentiation during axonal sorting. Mice with inactivation of Jab1 in Schwann cells develop a dysmyelinating neuropathy with axonal sorting defects. Loss of Jab1 increases p27 levels in Schwann cells, which causes defective cell cycle progression and aberrant differentiation. Genetic down-regulation of p27 levels in Jab1-null mice restores Schwann cell number, differentiation, and axonal sorting and rescues the dysmyelinating neuropathy. Thus, Jab1 constitutes a regulatory molecule that integrates laminin211 signals in Schwann cells to govern cell cycle, cell number, and differentiation. Finally, Jab1 may constitute a key molecule in the pathogenesis of dysmyelinating neuropathies. PMID:24344238

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

    PubMed

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

    2015-08-31

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

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

    PubMed

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

    2016-09-01

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

  5. 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

  6. Permissive Schwann cell graft/spinal cord interfaces for axon regeneration.

    PubMed

    Williams, Ryan R; Henao, Martha; Pearse, Damien D; Bunge, Mary Bartlett

    2015-01-01

    The transplantation of autologous Schwann cells (SCs) to repair the injured spinal cord is currently being evaluated in a clinical trial. In support, this study determined properties of spinal cord/SC bridge interfaces that enabled regenerated brainstem axons to cross them, possibly leading to improvement in rat hindlimb movement. Fluid bridges of SCs and Matrigel were placed in complete spinal cord transections. Compared to pregelled bridges of SCs and Matrigel, they improved regeneration of brainstem axons across the rostral interface. The regenerating brainstem axons formed synaptophysin(+) bouton-like terminals and contacted MAP2A(+) dendrites at the caudal interface. Brainstem axon regeneration was directly associated with glial fibrillary acidic protein (GFAP(+)) astrocyte processes that elongated into the SC bridge. Electron microscopy revealed that axons, SCs, and astrocytes were enclosed together within tunnels bounded by a continuous basal lamina. Neuroglycan (NG2) expression was associated with these tunnels. One week after injury, the GFAP(+) processes coexpressed nestin and brain lipid-binding protein, and the tips of GFAP(+)/NG2(+) processes extended into the bridges together with the regenerating brainstem axons. Both brainstem axon regeneration and number of GFAP(+) processes in the bridges correlated with improvement in hindlimb locomotion. Following SCI, astrocytes may enter a reactive state that prohibits axon regeneration. Elongation of astrocyte processes into SC bridges, however, and formation of NG2(+) tunnels enable brainstem axon regeneration and improvement in function. It is important for spinal cord repair to define conditions that favor elongation of astrocytes into lesions/transplants. PMID:24152553

  7. Selective expression of ligand-gated ion channels in L5 pyramidal cell axons

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2009-01-01

    NMDA receptor (NMDAR)-dependent strengthening of neurotransmitter release has been widely observed, including in layer 5 (L5) pyramidal cells of the visual cortex, and is attributed to the axonal expression of NMDARs. However, we failed to detect NMDAR-mediated depolarizations or Ca2+ entry in L5 pyramidal cell axons when focally stimulated with NMDAR agonists. This suggests that NMDARs are excluded from the axon. In contrast, local GABAAR activation alters axonal excitability indicating that exclusion of ligand-gated ion channels from the axon is not absolute. Because NMDARs are restricted to the dendrite, NMDARs must signal to the axon by an indirect mechanism to alter release. Although subthreshold somatic depolarizations were found to spread electrotonically hundreds of micrometers through the axon, the resulting axonal potential was insufficient to open voltage-sensitive Ca2+ channels (VSCCs). Therefore, if NMDAR-mediated facilitation of release is cell-autonomous, it may depend on voltage signaling but apparently is independent of changes in basal Ca2+. Alternatively, this facilitation may be even less direct, requiring a cascade of events that are merely triggered by NMDAR activation. PMID:19759293

  8. Vertebrate epidermal cells are broad-specificity phagocytes that clear sensory axon debris.

    PubMed

    Rasmussen, Jeffrey P; Sack, Georgeann S; Martin, Seanna M; Sagasti, Alvaro

    2015-01-14

    Cellular debris created by developmental processes or injury must be cleared by phagocytic cells to maintain and repair tissues. Cutaneous injuries damage not only epidermal cells but also the axonal endings of somatosensory (touch-sensing) neurons, which must be repaired to restore the sensory function of the skin. Phagocytosis of neuronal debris is usually performed by macrophages or other blood-derived professional phagocytes, but we have found that epidermal cells phagocytose somatosensory axon debris in zebrafish. Live imaging revealed that epidermal cells rapidly internalize debris into dynamic phosphatidylinositol 3-monophosphate-positive phagosomes that mature into phagolysosomes using a pathway similar to that of professional phagocytes. Epidermal cells phagocytosed not only somatosensory axon debris but also debris created by injury to other peripheral axons that were mislocalized to the skin, neighboring skin cells, and macrophages. Together, these results identify vertebrate epidermal cells as broad-specificity phagocytes that likely contribute to neural repair and wound healing. PMID:25589751

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

    PubMed

    McDole, B; Isgor, C; Pare, C; Guthrie, K

    2015-09-24

    Olfactory bulb granule cells (GCs) are axon-less, inhibitory interneurons that regulate the activity of the excitatory output neurons, the mitral and tufted cells, through reciprocal dendrodendritic synapses located on GC spines. These contacts are established in the distal apical dendritic compartment, while GC basal dendrites and more proximal apical segments bear spines that receive glutamatergic inputs from the olfactory cortices. This synaptic connectivity is vital to olfactory circuit function and is remodeled during development, and in response to changes in sensory activity and lifelong GC neurogenesis. Manipulations that alter levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in vivo have significant effects on dendritic spine morphology, maintenance and activity-dependent plasticity for a variety of CNS neurons, yet little is known regarding BDNF effects on bulb GC spine maturation or maintenance. Here we show that, in vivo, sustained bulbar over-expression of BDNF in transgenic mice produces a marked increase in GC spine density that includes an increase in mature spines on their apical dendrites. Morphometric analysis demonstrated that changes in spine density were most notable in the distal and proximal apical domains, indicating that multiple excitatory inputs are potentially modified by BDNF. Our results indicate that increased levels of endogenous BDNF can promote the maturation and/or maintenance of dendritic spines on GCs, suggesting a role for this factor in modulating GC functional connectivity within adult olfactory circuitry. PMID:26211445

  10. NF-Protocadherin Regulates Retinal Ganglion Cell Axon Behaviour in the Developing Visual System

    PubMed Central

    Leung, Louis C.; Harris, William A.; Holt, Christine E.; Piper, Michael

    2015-01-01

    Cell adhesion molecules play a central role in mediating axonal tract development within the nascent nervous system. NF-protocadherin (NFPC), a member of the non-clustered protocadherin family, has been shown to regulate retinal ganglion cell (RGC) axon and dendrite initiation, as well as influencing axonal navigation within the mid-optic tract. However, whether NFPC mediates RGC axonal behaviour at other positions within the optic pathway remains unclear. Here we report that NFPC plays an important role in RGC axonogenesis, but not in intraretinal guidance. Moreover, axons with reduced NFPC levels exhibit insensitivity to Netrin-1, an attractive guidance cue expressed at the optic nerve head. Netrin-1 induces rapid turnover of NFPC localized to RGC growth cones, suggesting that the regulation of NFPC protein levels may underlie Netrin-1-mediated entry of RGC axons into the optic nerve head. At the tectum, we further reveal a function for NFPC in controlling RGC axonal entry into the final target area. Collectively, our results expand our understanding of the role of NFPC in RGC guidance and illustrate that this adhesion molecule contributes to axon behaviour at multiple points in the optic pathway. PMID:26489017

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

    PubMed

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

    2016-05-01

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

  12. 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.

  13. 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

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

    PubMed Central

    Lavut, Anna; Raveh, Dina

    2012-01-01

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

  15. Loss of Modifier of Cell Adhesion Reveals a Pathway Leading to Axonal Degeneration

    PubMed Central

    Chen, Qi; Peto, Charles A.; Shelton, G. Diane; Mizisin, Andrew; Sawchenko, Paul E.; Schubert, David

    2009-01-01

    Axonal dysfunction is the major phenotypic change in many neurodegenerative diseases, but the processes underlying this impairment are not clear. Modifier of cell adhesion (MOCA) is a presenilin binding protein that functions as a guanine nucleotide exchange factor for Rac1. The loss of MOCA in mice leads to axonal degeneration and causes sensorimotor impairments by decreasing cofilin phosphorylation and altering its upstream signaling partners LIM kinase and p21-activated kinase, an enzyme directly downstream of Rac1. The dystrophic axons found in MOCA-deficient mice are associated with abnormal aggregates of neurofilament protein, the disorganization of the axonal cytoskeleton, and the accumulation of autophagic vacuoles and polyubiquitinated proteins. Furthermore, MOCA deficiency causes an alteration in the actin cytoskeleton and the formation of cofilin-containing rod-like structures. The dystrophic axons show functional abnormalities, including impaired axonal transport. These findings demonstrate that MOCA is required for maintaining the functional integrity of axons and define a model for the steps leading to axonal degeneration. PMID:19129390

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

    PubMed

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

    2010-12-01

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

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

    PubMed

    Borodinsky, L N; Fiszman, M L

    1998-04-17

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

  18. Sealing frequency of B104 cells declines exponentially with decreasing transection distance from the axon hillock.

    PubMed

    McGill, Christopher H; Bhupanapadu Sunkesula, Solomon Raju; Poon, Andrew D; Mikesh, Michelle; Bittner, George D

    2016-05-01

    Transection of nerve axons (axotomy) leads to rapid (Wallerian) degeneration of the distal portion of the severed axon whereas the proximal portion and the soma often survive. Clinicians and neuroscientists have known for decades that somal survival is less likely for cells transected nearer to the soma, compared to further from the soma. Calcium ion (Ca(2+)) influx at the cut axonal end increases somal Ca(2+) concentration, which subsequently activates apoptosis and other pathways that lead to cell death. The same Ca(2+) influx activates parallel pathways that seal the plasmalemma, reduce Ca(2+) influx, and thereby enable the soma to survive. In this study, we have examined the ability of transected B104 axons to seal, as measured by uptake or exclusion of fluorescent dye, and quantified the relationship between sealing frequency and transection distance from the axon hillock. We report that sealing frequency is maximal at about 150μm (μm) from the axon hillock and decreases exponentially with decreasing transection distance with a space constant of about 40μm. We also report that after Ca(2+) influx is initiated, the curve of sealing frequency versus time is well-fit by a one-phase, rising exponential model having a time constant of several milliseconds that is longer nearer to, versus further from, the axon hillock. These results could account for the increased frequency of cell death for axotomies nearer to, versus farther from, the soma of many types of neurons. PMID:26851541

  19. Axonal and Schwann Cell BACE1 Is Equally Required for Remyelination of Peripheral Nerves

    PubMed Central

    Hu, Xiangyou; Hu, Jinxuan; Dai, Lu; Trapp, Bruce

    2015-01-01

    Inhibition of β-site APP cleaving enzyme 1 (BACE1) is being pursued as a therapeutic target for treating patients with Alzheimer's disease because BACE1 is the sole β-secretase for generating β-amyloid peptide. Knowledge regarding the other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination in injured sciatic nerves. Since BACE1 is expected to be ubiquitously expressed, we asked whether axonal or Schwann cell BACE1 is required for optimal remyelination. By swapping sciatic nerve segments from BACE1-null mice with the corresponding wild-type nerve segments or vice versa, we tested how a deficiency of BACE1 in Schwann cells or axons affects remyelination. Our results show that BACE1 in axons and Schwann cells is similarly important for remyelination of regenerated axons. Nerve injury induces BACE1 transcription and protein levels are elevated in Schwann cells. Expression of type I neuregulin 1 (Nrg1), rather than type III Nrg1, was induced by Schwann cells, and the abolished Nrg1 cleavage in BACE1-null Schwann cells contributed to decreased remyelination of regenerated axons. Hence, this study is the first to demonstrate the equal importance of axonal and Schwann cell BACE1 for remyelination of injured nerves. PMID:25740511

  20. Axonal and Schwann cell BACE1 is equally required for remyelination of peripheral nerves.

    PubMed

    Hu, Xiangyou; Hu, Jinxuan; Dai, Lu; Trapp, Bruce; Yan, Riqiang

    2015-03-01

    Inhibition of β-site APP cleaving enzyme 1 (BACE1) is being pursued as a therapeutic target for treating patients with Alzheimer's disease because BACE1 is the sole β-secretase for generating β-amyloid peptide. Knowledge regarding the other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination in injured sciatic nerves. Since BACE1 is expected to be ubiquitously expressed, we asked whether axonal or Schwann cell BACE1 is required for optimal remyelination. By swapping sciatic nerve segments from BACE1-null mice with the corresponding wild-type nerve segments or vice versa, we tested how a deficiency of BACE1 in Schwann cells or axons affects remyelination. Our results show that BACE1 in axons and Schwann cells is similarly important for remyelination of regenerated axons. Nerve injury induces BACE1 transcription and protein levels are elevated in Schwann cells. Expression of type I neuregulin 1 (Nrg1), rather than type III Nrg1, was induced by Schwann cells, and the abolished Nrg1 cleavage in BACE1-null Schwann cells contributed to decreased remyelination of regenerated axons. Hence, this study is the first to demonstrate the equal importance of axonal and Schwann cell BACE1 for remyelination of injured nerves. PMID:25740511

  1. c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy

    PubMed Central

    Hantke, Janina; Carty, Lucy; Wagstaff, Laura J.; Turmaine, Mark; Wilton, Daniel K.; Quintes, Susanne; Koltzenburg, Martin; Baas, Frank; Mirsky, Rhona

    2014-01-01

    Charcot–Marie–Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot–Marie–Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot–Marie–Tooth disease type 1A. In line with our previous findings in humans with Charcot–Marie–Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun−/− mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot–Marie–Tooth disease type 1A: on the one hand they are the genetic source of

  2. Retroviral misexpression of cVax disturbs retinal ganglion cell axon fasciculation and intraretinal pathfinding in vivo and guidance of nasal ganglion cell axons in vivo.

    PubMed

    Mühleisen, Thomas W; Agoston, Zsuzsa; Schulte, Dorothea

    2006-09-01

    The transcription factor cVax (Vax2) is expressed in the ventral neural retina and restricted expression is a prerequisite for at least three prominent aspects of retinal dorsal-ventral patterning: polarized expression of EphB/B-ephrin molecules, the retinotectal projection and the distribution of rod photoreceptors across the retina. In the chick retina, the fasciculation pattern of ganglion cell axons also differs between the dorsal and ventral eye. To investigate the molecular mechanisms involved, the nerve fiber layer was analyzed after retroviral misexpression of several factors known to regulate the positional specification of retinal ganglion cells. Forced cVax expression ventralized the fasciculation pattern and caused axon pathfinding errors near the optic disc. Ectopic expression of different ephrin molecules indicated that axon fasciculation is, at least in part, mediated by the EphB system. Finally, we report that retroviral misexpression of cVax increased the pool of EphA4 receptors phosphorylated on tyrosine residues and altered the guidance preference of nasal axons in vitro. These results identify novel functions for cVax in intraretinal axon fasciculation and pathfinding as well as suggest a mechanism to explain how restricted cVax expression may influence map formation along the dorso-ventral and antero-posterior axes of the optic tectum. PMID:16769047

  3. Abnormal Schwann cell/axon interactions in the Trembler-J mouse

    PubMed Central

    ROBERTSON, A. M.; KING, R. H. M.; MUDDLE, J. R.; THOMAS, P. K.

    1997-01-01

    The Trembler-J (TrJ) mouse has a point mutation in the gene coding for peripheral myelin protein 22 (PMP22). Disturbances in PMP22 are associated with abnormal myelination in a range of inherited peripheral neuropathies both in mice and humans. PMP22 is produced mainly by Schwann cells in the peripheral nervous system where it is localised to compact myelin. The function of PMP22 is unclear but its low abundance (∼5% of total myelin protein) means that it is unlikely to play a structural role. Its inclusion in a recently discovered family of proteins suggests a function in cell proliferation/differentiation and possibly in adhesion. Nerves from TrJ and the allelic Trembler (Tr) mouse are characterised by abnormally thin myelin for the size of the axon and an increased number of Schwann cells. We report ultrastructural evidence of abnormal Schwann cell-axon interactions. Schwann cell nuclei have been found adjacent to the nodes of Ranvier whereas in normal animals they are located near the centre of the internodes. In some fibres the terminal myelin loops faced outwards into the extracellular space instead of turning inwards and terminating on the axon. In severely affected nerves many axons were only partially surrounded by Schwann cell cytoplasm. All these features suggest a failure of Schwann cell–axon recognition or interaction. In addition to abnormalities related to abnormal myelination there was significant axonal loss in the dorsal roots. PMID:9147228

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

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2015-01-01

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

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

    PubMed Central

    WANG, BING; CHENG, YING; XU, MIN

    2016-01-01

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

  6. 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. PMID:26391451

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

    PubMed Central

    LaSarge, Candi L.; McAuliffe, John J.

    2015-01-01

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

  8. 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.

  9. 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

  10. Transfer of newly synthesized proteins from Schwann cells to the squid giant axon.

    PubMed

    Lasek, R J; Gainer, H; Przybylski, R J

    1974-04-01

    The squid giant axon is presented as a model for the study of macromolecular interaction between cells in the nervous system. When the isolated giant axon was incubated in sea water containing [(3)H]leucine for 0.5-5 hr, newly synthesized proteins appeared in the sheath and axoplasm as demonstrated by: (i) radioautography, (ii) separation of the sheath and axoplasm by extrusion, and (iii) perfusion of electrically excitable axons. The absence of ribosomal RNA in the axoplasm [Lasek, R. J. et al. (1973) Nature 244, 162-165] coupled with other evidence indicates that the labeled proteins that are found in the axoplasm originate in the Schwann cells surrounding the axon. Approximately 50% of the newly synthesized Schwann cell proteins are transferred to the giant axon. These transferred proteins are soluble for the most part and range in molecular size from 12,000 to greater than 200,000 daltons. It is suggested that proteins transferred from the Schwann cell to the axon have a regulatory role in neuronal function. PMID:4524631

  11. Human bone marrow mesenchymal stem cell transplantation attenuates axonal injury in stroke rats

    PubMed Central

    Xu, Yi; Du, Shiwei; Yu, Xinguang; Han, Xiao; Hou, Jincai; Guo, Hao

    2014-01-01

    Previous studies have shown that transplantation of human bone marrow mesenchymal stem cells promotes neural functional recovery after stroke, but the neurorestorative mechanisms remain largely unknown. We hypothesized that functional recovery of myelinated axons may be one of underlying mechanisms. In this study, an ischemia/reperfusion rat model was established using the middle cerebral artery occlusion method. Rats were used to test the hypothesis that intravenous transplantation of human bone marrow mesenchymal stem cells through the femoral vein could exert neuroprotective effects against cerebral ischemia via a mechanism associated with the ability to attenuate axonal injury. The results of behavioral tests, infarction volume analysis and immunohistochemistry showed that cerebral ischemia caused severe damage to the myelin sheath and axons. After rats were intravenously transplanted with human bone marrow mesenchymal stem cells, the levels of axon and myelin sheath-related proteins, including microtubule-associated protein 2, myelin basic protein, and growth-associated protein 43, were elevated, infarct volume was decreased and neural function was improved in cerebral ischemic rats. These findings suggest that intravenously transplanted human bone marrow mesenchymal stem cells promote neural function. Possible mechanisms underlying these beneficial effects include resistance to demyelination after cerebral ischemia, prevention of axonal degeneration, and promotion of axonal regeneration. PMID:25657721

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

    PubMed Central

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

    2011-01-01

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

  13. 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.

  14. Schwann cell LRP1 regulates Remak bundle ultrastructure and axonal interactions to prevent neuropathic pain

    PubMed Central

    Orita, Sumihisa; Henry, Kenneth; Mantuano, Elisabetta; Yamauchi, Kazuyo; De Corato, Alice; Ishikawa, Tetsuhiro; Feltri, M. Laura; Wrabetz, Lawrence; Gaultier, Alban; Pollack, Melanie; Ellisman, Mark; Takahashi, Kazuhisa; Gonias, Steven L.; Campana, W. Marie

    2013-01-01

    Trophic support and myelination of axons by Schwann cells in the PNS are essential for normal nerve function. Herein, we show that deletion of the LDL receptor-related protein-1 (LRP1) gene in Schwann cells (scLRP1−/−) induces abnormalities in axon myelination and in ensheathment of axons by non-myelinating Schwann cells in Remak bundles. These anatomical changes in the PNS were associated with mechanical allodynia, even in the absence of nerve injury. In response to crush injury, sciatic nerves in scLRP1−/− mice showed accelerated degeneration and Schwann cell death. Remyelinated axons were evident 20 days after crush injury in control mice, yet were largely absent in scLRP1−/− mice. In the partial nerve ligation model, scLRP1−/− mice demonstrated significantly increased and sustained mechanical allodynia and loss of motor function. Evidence for central sensitization in pain processing included increased p38MAPK activation and activation of microglia in the spinal cord. These studies identify LRP1 as an essential mediator of normal Schwann cell-axonal interactions and as a pivotal regulator of the Schwann cell response to PNS injury in vivo. Mice in which LRP1 is deficient in Schwann cells represent a model for studying how abnormalities in Schwann cell physiology may facilitate and sustain chronic pain. PMID:23536074

  15. Axon outgrowth along segmental nerves in the leech. II. Identification of actual guidance cells

    SciTech Connect

    Braun, J.; Stent, G.S.

    1989-04-01

    Some peripheral neurons, previously identified as candidate guidance cells for axonal outgrowth along the segmental nerves in embryos of the glossiphoniid leech Helobdella triserialis, were photoablated by laser illumination to ascertain whether their presence is necessary for generation of the normal axonal growth pattern. These experiments showed that focal photoablation of peripheral neurons nz3 or pz8 prevents normal axonal outgrowth along the ultraposterior nerve path or along the distal sector of the medial-anterior nerve path, respectively, in conformance with the inference that these two neurons do function as guidance cells. However, ablation of these neurons affects axon outgrowth only if the neurons are illuminated prior to the end of a sensitive period in segmental development. By contrast, photoablation of previously identified candidate guidance cells situated on the anterior-anterior and posterior-posterior nerve paths, among them peripheral neurons nz1, nz2, oz1, oz2, pz6, and LD1, does not prevent normal axonal outgrowth. It is possible that the guidance role, if any, of these neurons is facultative rather than necessary, since each of the several neurons that lies on either of these nerve paths may provide an alternative axon guidance cue.

  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. PMID:2519933

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

    PubMed Central

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

    2016-01-01

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

  18. 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.

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

    PubMed

    Fu, Zhanyan; Vicini, Stefano

    2009-09-01

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

  20. Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

    PubMed Central

    Penfold, M E; Armati, P; Cunningham, A L

    1994-01-01

    To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus. Images PMID:7517552

  1. Axon-Schwann cell interactions during peripheral nerve regeneration in zebrafish larvae

    PubMed Central

    2014-01-01

    Background Peripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells. Results Using pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored. Conclusion The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration. PMID:25326036

  2. Neuron-glia signaling and the protection of axon function by Schwann cells.

    PubMed

    Quintes, Susanne; Goebbels, Sandra; Saher, Gesine; Schwab, Markus H; Nave, Klaus-Armin

    2010-03-01

    The interaction between neurons and glial cells is a feature of all higher nervous systems. In the vertebrate peripheral nervous system, Schwann cells ensheath and myelinate axons thereby allowing rapid saltatory conduction and ensuring axonal integrity. Recently, some of the key molecules in neuron-Schwann cell signaling have been identified. Neuregulin-1 (NRG1) type III presented on the axonal surface determines the myelination fate of axons and controls myelin sheath thickness. Recent observations suggest that NRG1 regulates myelination via the control of Schwann cell cholesterol biosynthesis. This concept is supported by the finding that high cholesterol levels in Schwann cells are a rate-limiting factor for myelin protein production and transport of the major myelin protein P0 from the endoplasmic reticulum into the growing myelin sheath. NRG1 type III activates ErbB receptors on the Schwann cell, which leads to an increase in intracellular PIP3 levels via the PI3-kinase pathway. Surprisingly, enforced elevation of PIP3 levels by inactivation of the phosphatase PTEN in developing and mature Schwann cells does not entirely mimic NRG1 type III stimulated myelin growth, but predominantly causes focal hypermyelination starting at Schmidt-Lanterman incisures and nodes of Ranvier. This indicates that the glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve integrity of the myelinated axon. Understanding the cross talk between neurons and Schwann cells will help to further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A. PMID:20433601

  3. TRANSDUCED SCHWANN CELLS PROMOTE AXON GROWTH AND MYELINATION AFTER SPINAL CORD INJURY

    PubMed Central

    Golden, Kevin L.; Pearse, Damien D.; Blits, Bas; Garg, Maneesh S.; Oudega, Martin; Wood, Patrick M.; Bunge, Mary Bartlett

    2007-01-01

    We sought to directly compare growth and myelination of local and supraspinal axons by implanting into the injured spinal cord Schwann cells (SCs) transduced ex vivo with adenoviral (AdV) or lentiviral (LV) vectors encoding a bifunctional neurotrophin molecule (D15A). D15A mimics actions of both neurotrophin-3 and brain-derived neurotrophic factor. Transduced SCs were injected into the injury center one week after a moderate thoracic (T8) adult rat spinal cord contusion. D15A expression and bioactivity in vitro; D15A levels in vivo; and graft volume, SC number, implant axon number and cortico-, reticulo-, raphe-, coerulo-spinal and sensory axon growth were determined for both types of vectors employed to transduce SCs. ELISAs revealed that D15A-secreting SC implants contained significantly higher levels of neurotrophin than non-transduced SC and AdV/GFP and LV/GFP SC controls early after implantation. At 6 wk post-implantation, D15A-secreting SC grafts exhibited 5-fold increases in graft volume, SC number and myelinated axon counts and a 3-fold increase in myelinated to unmyelinated (ensheathed) axon ratios. The total number of axons within grafts of LV/GFP/D15A SCs was estimated to be over 70,000. Also 5-HT, DβH, and CGRP axon length was increased up to 5-fold within D15A grafts. In sum, despite qualitative differences using the two vectors, increased neurotrophin secretion by the implanted D15A SCs led to the presence of a significantly increased number of axons in the contusion site. These results demonstrate the therapeutic potential for utilizing neurotrophin-transduced SCs to repair the injured spinal cord. PMID:17719577

  4. Disrupted axon-glia interactions at the paranode in myelinated nerves cause axonal degeneration and neuronal cell death in the aged Caspr mutant mouse shambling.

    PubMed

    Takagishi, Yoshiko; Katanosaka, Kimiaki; Mizoguchi, Hiroyuki; Murata, Yoshiharu

    2016-07-01

    Emerging evidence suggests that axonal degeneration is a disease mechanism in various neurodegenerative diseases and that the paranodes at the nodes of Ranvier may be the initial site of pathogenesis. We investigated the pathophysiology of the disease process in the central and peripheral nervous systems of a Caspr mutant mouse, shambling (shm), which is affected by disrupted paranodal structures and impaired nerve conduction of myelinated nerves. The shm mice manifest a progressive neurological phenotype as mice age. We found extensive axonal degeneration and a loss of neurons in the central nervous system and peripheral nervous system in aged shm mice. Axonal alteration of myelinated nerves was defined by abnormal distribution and expression of neurofilaments and derangements in the status of phosphorylated and non/de-phosphorylated neurofilaments. Autophagy-related structures were also accumulated in degenerated axons and neurons. In conclusion, our results suggest that disrupted axon-glia interactions at the paranode cause the cytoskeletal alteration in myelinated axons leading to neuronal cell death, and the process involves detrimental autophagy and aging as factors that promote the pathogenesis. PMID:27255813

  5. Rescuing axons from degeneration does not affect retinal ganglion cell death.

    PubMed

    de Lima, S; Mietto, B S; Paula, C; Muniz, T; Martinez, A M B; Gardino, P F

    2016-01-01

    After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage. PMID:27007653

  6. Rescuing axons from degeneration does not affect retinal ganglion cell death

    PubMed Central

    de Lima, S.; Mietto, B.S.; Paula, C.; Muniz, T.; Martinez, A.M.B.; Gardino, P.F.

    2016-01-01

    After a traumatic injury to the central nervous system, the distal stumps of axons undergo Wallerian degeneration (WD), an event that comprises cytoskeleton and myelin breakdown, astrocytic gliosis, and overexpression of proteins that inhibit axonal regrowth. By contrast, injured neuronal cell bodies show features characteristic of attempts to initiate the regenerative process of elongating their axons. The main molecular event that leads to WD is an increase in the intracellular calcium concentration, which activates calpains, calcium-dependent proteases that degrade cytoskeleton proteins. The aim of our study was to investigate whether preventing axonal degeneration would impact the survival of retinal ganglion cells (RGCs) after crushing the optic nerve. We observed that male Wistar rats (weighing 200-400 g; n=18) treated with an exogenous calpain inhibitor (20 mM) administered via direct application of the inhibitor embedded within the copolymer resin Evlax immediately following optic nerve crush showed a delay in the onset of WD. This delayed onset was characterized by a decrease in the number of degenerated fibers (P<0.05) and an increase in the number of preserved fibers (P<0.05) 4 days after injury. Additionally, most preserved fibers showed a normal G-ratio. These results indicated that calpain inhibition prevented the degeneration of optic nerve fibers, rescuing axons from the process of axonal degeneration. However, analysis of retinal ganglion cell survival demonstrated no difference between the calpain inhibitor- and vehicle-treated groups, suggesting that although the calpain inhibitor prevented axonal degeneration, it had no effect on RGC survival after optic nerve damage. PMID:27007653

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

    PubMed

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

    2004-04-14

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

  8. Arrest of Myelination and Reduced Axon Growth when Schwann Cells Lack mTOR

    PubMed Central

    Sherman, Diane L.; Krols, Michiel; Wu, Lai-Man; Grove, Matthew; Nave, Klaus-Armin; Gangloff, Yann-Gaël; Brophy, Peter J.

    2014-01-01

    In developing peripheral nerves differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years there has been an increasing understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination together with a growing appreciation of some of the signalling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal post-natal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signalling in both the longitudinal and radial growth of the myelinating Schwann cell. PMID:22302821

  9. Arrest of myelination and reduced axon growth when Schwann cells lack mTOR.

    PubMed

    Sherman, Diane L; Krols, Michiel; Wu, Lai-Man N; Grove, Matthew; Nave, Klaus-Armin; Gangloff, Yann-Gaël; Brophy, Peter J

    2012-02-01

    In developing peripheral nerves, differentiating Schwann cells sort individual axons from bundles and ensheath them to generate multiple layers of myelin. In recent years, there has been an increased understanding of the extracellular and intracellular factors that initiate and stimulate Schwann cell myelination, together with a growing appreciation of some of the signaling pathways involved. However, our knowledge of how Schwann cell growth is regulated during myelination is still incomplete. The mammalian target of rapamycin (mTOR) is a core kinase in two major complexes, mTORC1 and mTORC2, that regulate cell growth and differentiation in a variety of mammalian cells. Here we show that elimination of mTOR from murine Schwann cells prevented neither radial sorting nor the initiation of myelination. However, normal postnatal growth of myelinating Schwann cells, both radially and longitudinally, was highly retarded. The myelin sheath in the mutant was much thinner than normal; nevertheless, sheath thickness relative to axon diameter (g-ratio) remained constant in both wild-type and mutant nerves from P14 to P90. Although axon diameters were normal in the mutant at the initiation of myelination, further growth as myelination proceeded was retarded, and this was associated with reduced phosphorylation of neurofilaments. Consistent with thinner axonal diameters and internodal lengths, conduction velocities in mutant quadriceps nerves were also reduced. These data establish a critical role for mTOR signaling in both the longitudinal and radial growth of the myelinating Schwann cell. PMID:22302821

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

    PubMed Central

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

    1996-01-01

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

  11. The slow Wallerian degeneration gene in vivo protects motor axons but not their cell bodies after avulsion and neonatal axotomy.

    PubMed

    Adalbert, Robert; Nógrádi, Antal; Szabó, András; Coleman, Michael P

    2006-10-01

    The slow Wallerian degeneration gene (Wld(S)) delays Wallerian degeneration and axon pathology for several weeks in mice and rats. Interestingly, neuronal cell death is also delayed in some in vivo models, most strikingly in the progressive motoneuronopathy mouse. Here, we tested the hypothesis that Wld(S) has a direct protective effect on motoneurone cell bodies in vivo. Cell death was induced in rat L4 motoneurones by intravertebral avulsion of the corresponding ventral roots. This simultaneously removed most of the motor axon, minimizing the possibility that the protective effect toward axons could rescue cell bodies secondarily. There was no significant difference between the survival of motoneurones in control and Wld(S) rats, suggesting that the Wld(S) gene has no direct protective effect on cell bodies. We also tested for any delay in apoptotic motoneurone death following neonatal nerve injury in Wld(S) rats and found that, unlike Wld(S) mice, Wld(S) rats show no delay in cell death. However, the corresponding distal axons were preserved, confirming that motoneurone cell bodies and motor axons die by different mechanisms. Thus, Wld(S) does not directly prevent death of motoneurone cell bodies. It follows that the protection of neuronal cell bodies observed in several disease and injury models where axons or significant axonal stumps remain is most probably secondary to axonal protection. PMID:17074042

  12. 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.

  13. A Self-Assembling Injectable Biomimetic Microenvironment Encourages Retinal Ganglion Cell Axon Extension in Vitro.

    PubMed

    Laughter, Melissa R; Ammar, David A; Bardill, James R; Pena, Brisa; Kahook, Malik Y; Lee, David J; Park, Daewon

    2016-08-17

    Sensory-somatic nervous system neurons, such as retinal ganglion cells (RGCs), are typically thought to be incapable of regenerating. However, it is now known that these cells may be stimulated to regenerate by providing them with a growth permissive environment. We have engineered an injectable microenvironment designed to provide growth-stimulating cues for RGC culture. Upon gelation, this injectable material not only self-assembles into laminar sheets, similar to retinal organization, but also possesses a storage modulus comparable to that of retinal tissue. Primary rat RGCs were grown, stained, and imaged in this three-dimensional scaffold. We were able to show that RGCs grown in this retina-like structure exhibited characteristic long, prominent axons. In addition, RGCs showed a consistent increase in average axon length and neurite-bearing ratio over the 7 day culture period, indicating this scaffold is capable of supporting substantial RGC axon extension. PMID:27434231

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-03-01

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

  17. Assessment of retinal ganglion cell damage in glaucomatous optic neuropathy: Axon transport, injury and soma loss.

    PubMed

    Nuschke, Andrea C; Farrell, Spring R; Levesque, Julie M; Chauhan, Balwantray C

    2015-12-01

    Glaucoma is a disease characterized by progressive axonal pathology and death of retinal ganglion cells (RGCs), which causes structural changes in the optic nerve head and irreversible vision loss. Several experimental models of glaucomatous optic neuropathy (GON) have been developed, primarily in non-human primates and, more recently and commonly, in rodents. These models provide important research tools to study the mechanisms underlying glaucomatous damage. Moreover, experimental GON provides the ability to quantify and monitor risk factors leading to RGC loss such as the level of intraocular pressure, axonal health and the RGC population. Using these experimental models we are able to gain a better understanding of GON, which allows for the development of potential neuroprotective strategies. Here we review the advantages and disadvantages of the relevant and most often utilized methods for evaluating axonal degeneration and RGC loss in GON. Axonal pathology in GON includes functional disruption of axonal transport (AT) and structural degeneration. Horseradish peroxidase (HRP), rhodamine-B-isothiocyanate (RITC) and cholera toxin-B (CTB) fluorescent conjugates have proven to be effective reporters of AT. Also, immunohistochemistry (IHC) for endogenous AT-associated proteins is often used as an indicator of AT function. Similarly, structural degeneration of axons in GON can be investigated via changes in the activity and expression of key axonal enzymes and structural proteins. Assessment of axonal degeneration can be measured by direct quantification of axons, qualitative grading, or a combination of both methods. RGC loss is the most frequently quantified variable in studies of experimental GON. Retrograde tracers can be used to quantify RGC populations in rodents via application to the superior colliculus (SC). In addition, in situ IHC for RGC-specific proteins is a common method of RGC quantification used in many studies. Recently, transgenic mouse models

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

    PubMed

    Wada, Norio; Funabiki, Kazuo; Nakanishi, Shigetada

    2014-04-01

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

  19. Development of microarray device for functional evaluation of PC12D cell axonal extension ability

    NASA Astrophysics Data System (ADS)

    Nakamachi, Eiji; Yanagimoto, Junpei; Murakami, Shinya; Morita, Yusuke

    2014-04-01

    In this study, we developed a microarray bio-MEMS device that could trap PC12D (rat pheochromocytoma cells) cells to examine the intercellular interaction effect on the cell activation and the axonal extension ability. This is needed to assign particular patterns of PC12D cells to establish a cell functional evaluation technique. This experimental observation-based technique can be used for design of the cell sheet and scaffold for peripheral and central nerve regeneration. We have fabricated a micropillar-array bio-MEMS device, whose diameter was approximately 10 μm, by using thick photoresist SU-8 on the glass slide substrate. A maximum trapped PC12D cell ratio, 48.5%, was achieved. Through experimental observation of patterned PC12D "bi-cells" activation, we obtained the following results. Most of the PC12D "bi-cells" which had distances between 40 and 100 μm were connected after 24 h with a high probability. On the other hand, "bi-cells" which had distances between 110 and 200 μm were not connected. In addition, we measured axonal extension velocities in cases where the intercellular distance was between 40 and 100 μm. A maximum axonal extension velocity, 86.4 μm/h, was obtained at the intercellular distance of 40 μm.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-01-01

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

  4. 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

  5. Effects of p-xylene inhalation on axonal transport in the rat retinal ganglion cells

    SciTech Connect

    Padilla, S.S.; Lyerly, D.P. )

    1989-12-01

    Although the solvent xylene is suspected of producing nervous system dysfunction in animals and humans, little is known regarding the neurochemical consequences of xylene inhalation. The intent of this study was to determine the effect of intermittent, acute, and subchronic p-xylene exposure on the axonal transport of proteins and glycoproteins within the rat retinofugal tract. A number of different exposure regimens were tested ranging from 50 ppm for a single 6-hr exposure to 1600 ppm 6 hr/day, 5 days/week, for a total of 8 exposure days. Immediately following removal from the inhalation chambers rats were injected intraocularly with (35S)methionine and (3H)fucose (to label retinal proteins and glycoproteins, respectively) and the axonal transport of labeled macromolecules to axons (optic nerve and optic tract) and nerve endings (lateral geniculate body and superior colliculus) was examined 20 hr after precursor injection. Only relatively severe exposure regimens (i.e., 800 or 1600 ppm 6 hr/day, 5 days/week, for 1.5 weeks) produced significant reductions in axonal transport; there was a moderate reduction in the axonal transport of 35S-labeled proteins in the 800-ppm-treated group which was more widespread in the 1600 ppm-treated group. Transport of 3H-labeled glycoproteins was less affected. Assessment of retinal metabolism immediately after isotope injection indicated that the rate of precursor uptake was not reduced in either treatment group. Furthermore, rapid transport was still substantially reduced in animals exposed to 1600 ppm p-xylene and allowed a 13-day withdrawal period. These data indicate that p-xylene inhalation decreases rapid axonal transport supplied to the projections of the rat retinal ganglion cells immediately after cessation of inhalation exposure and that this decreased transport is still apparent 13 days after the last exposure.

  6. Three-dimensional evaluation of retinal ganglion cell axon regeneration and pathfinding in whole mouse tissue after injury.

    PubMed

    Luo, Xueting; Salgueiro, Yadira; Beckerman, Samuel R; Lemmon, Vance P; Tsoulfas, Pantelis; Park, Kevin K

    2013-09-01

    Injured retinal ganglion cell (RGC) axons do not regenerate spontaneously, causing loss of vision in glaucoma and after trauma. Recent studies have identified several strategies that induce long distance regeneration in the optic nerve. Thus, a pressing question now is whether regenerating RGC axons can find their appropriate targets. Traditional methods of assessing RGC axon regeneration use histological sectioning. However, tissue sections provide fragmentary information about axonal trajectory and termination. To unequivocally evaluate regenerating RGC axons, here we apply tissue clearance and light sheet fluorescence microscopy (LSFM) to image whole optic nerve and brain without physical sectioning. In mice with PTEN/SOCS3 deletion, a condition known to promote robust regeneration, axon growth followed tortuous paths through the optic nerve, with many axons reversing course and extending towards the eye. Such aberrant growth was prevalent in the proximal region of the optic nerve where strong astroglial activation is present. In the optic chiasms of PTEN/SOCS3 deletion mice and PTEN deletion/Zymosan/cAMP mice, many axons project to the opposite optic nerve or to the ipsilateral optic tract. Following bilateral optic nerve crush, similar divergent trajectory is seen at the optic chiasm compared to unilateral crush. Centrally, axonal projection is limited predominantly to the hypothalamus. Together, we demonstrate the applicability of LSFM for comprehensive assessment of optic nerve regeneration, providing in-depth analysis of the axonal trajectory and pathfinding. Our study indicates significant axon misguidance in the optic nerve and brain, and underscores the need for investigation of axon guidance mechanisms during optic nerve regeneration in adults. PMID:23510761

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

    PubMed

    Jiang, Bo; Liu, Yu

    2013-06-01

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

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

    PubMed

    Hammel, Ilan; Meilijson, Isaac

    2016-03-01

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

  9. MRI evaluation of axonal reorganization after bone marrow stromal cell treatment of traumatic brain injury

    PubMed Central

    Jiang, Quan; Qu, Changsheng; Chopp, Michael; Ding, Guang Liang; Nejad-Davarani, Siamak P.; Helpern, Joseph A.; Jensen, Jens H.; Zhang, Zheng Gang; Li, Lian; Lu, Mei; Kaplan, David; Hu, Jiani; Shen, Yimin; Kou, Zhifeng; Li, Qingjiang; Wang, Shiyang; Mahmood, Asim

    2012-01-01

    We treated traumatic brain injury (TBI) with human bone marrow stromal cells (hMSCs) and evaluated the effect of treatment on white matter reorganization using MRI. We subjected male Wistar rats (n = 17) to controlled cortical impact and either withheld treatment (controls; n = 9) or inserted collagen scaffolds containing hMSCs (n = 8). Six weeks later, the rats were sacrificed and MRI revealed selective migration of grafted neural progenitor cells towards the white matter reorganized boundary of the TBI-induced lesion. Histology confirmed that the white matter had been reorganized, associated with increased fractional anisotropy (FA; p <0.01) in the recovery regions relative to the injured core region in both treated and control groups. Treatment with hMSCs increased FA in the recovery regions, lowered T2 in the core region, decreased lesion volume and improved functional recovery relative to untreated controls. Immunoreactive staining showed axonal projections emanating from neurons and extruding from the corpus callosum into the ipsilateral cortex at the boundary of the lesion. Fiber tracking (FT) maps derived from diffusion tensor imaging confirmed the immunohistological data and provided information on axonal rewiring. The apparent kurtosis coefficient (AKC) detected additional axonal remodeling regions with crossing axons, confirmed by immunohistological staining, compared with FA. Our data demonstrate that AKC, FA, FTand T2 can be used to evaluate treatment-induced white matter recovery, which may facilitate restorative therapy in patients with TBI. PMID:21432927

  10. c-Jun in Schwann cells promotes axonal regeneration and motoneuron survival via paracrine signaling

    PubMed Central

    Fontana, Xavier; Hristova, Mariya; Da Costa, Clive; Patodia, Smriti; Thei, Laura; Makwana, Milan; Spencer-Dene, Bradley; Latouche, Morwena; Mirsky, Rhona; Jessen, Kristjan R.; Klein, Rüdiger

    2012-01-01

    The AP-1 transcription factor c-Jun is a master regulator of the axonal response in neurons. c-Jun also functions as a negative regulator of myelination in Schwann cells (SCs) and is strongly reactivated in SCs upon axonal injury. We demonstrate here that, after injury, the absence of c-Jun specifically in SCs caused impaired axonal regeneration and severely increased neuronal cell death. c-Jun deficiency resulted in decreased expression of several neurotrophic factors, and GDNF and Artemin, both of which encode ligands for the Ret receptor tyrosine kinase, were identified as novel direct c-Jun target genes. Genetic inactivation of Ret specifically in neurons resulted in regeneration defects without affecting motoneuron survival and, conversely, administration of recombinant GDNF and Artemin protein substantially ameliorated impaired regeneration caused by c-Jun deficiency. These results reveal an unexpected function for c-Jun in SCs in response to axonal injury, and identify paracrine Ret signaling as an important mediator of c-Jun function in SCs during regeneration. PMID:22753894

  11. 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.

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

    PubMed

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

    2010-01-01

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

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

    PubMed

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

    2012-06-01

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

  14. Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells

    PubMed Central

    Chang, Haigang; Song, Shanshan; Chen, Zhongcan; Wang, Yaxiao; Yang, Lujun; Du, Mouxuan; Ke, Yiquan; Xu, Ruxiang; Jin, Baozhe; Jiang, Xiaodan

    2014-01-01

    Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells. PMID:25206849

  15. Role of primary afferents in the developmental regulation of motor axon synapse numbers on Renshaw cells.

    PubMed

    Siembab, Valerie C; Gomez-Perez, Laura; Rotterman, Travis M; Shneider, Neil A; Alvarez, Francisco J

    2016-06-15

    Motor function in mammalian species depends on the maturation of spinal circuits formed by a large variety of interneurons that regulate motoneuron firing and motor output. Interneuron activity is in turn modulated by the organization of their synaptic inputs, but the principles governing the development of specific synaptic architectures unique to each premotor interneuron are unknown. For example, Renshaw cells receive, at least in the neonate, convergent inputs from sensory afferents (likely Ia) and motor axons, raising the question of whether they interact during Renshaw cell development. In other well-studied neurons, such as Purkinje cells, heterosynaptic competition between inputs from different sources shapes synaptic organization. To examine the possibility that sensory afferents modulate synaptic maturation on developing Renshaw cells, we used three animal models in which afferent inputs in the ventral horn are dramatically reduced (ER81(-/-) knockout), weakened (Egr3(-/-) knockout), or strengthened (mlcNT3(+/-) transgenic). We demonstrate that increasing the strength of sensory inputs on Renshaw cells prevents their deselection and reduces motor axon synaptic density, and, in contrast, absent or diminished sensory afferent inputs correlate with increased densities of motor axons synapses. No effects were observed on other glutamatergic inputs. We conclude that the early strength of Ia synapses influences their maintenance or weakening during later development and that heterosynaptic influences from sensory synapses during early development regulates the density and organization of motor inputs on mature Renshaw cells. J. Comp. Neurol. 524:1892-1919, 2016. © 2016 Wiley Periodicals, Inc. PMID:26660356

  16. Axon growth and guidance genes identify T-dependent germinal centre B cells.

    PubMed

    Yu, Di; Cook, Matthew C; Shin, Dong-Mi; Silva, Diego G; Marshall, Jennifer; Toellner, Kai-Michael; Havran, Wendy L; Caroni, Pico; Cooke, Michael P; Morse, Herbert C; MacLennan, Ian C M; Goodnow, Christopher C; Vinuesa, Carola G

    2008-01-01

    Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms. PMID:17938642

  17. Schwann cell mitochondrial metabolism supports long-term axonal survival and peripheral nerve function

    PubMed Central

    Viader, Andreu; Golden, Judith P.; Baloh, Robert H.; Schmidt, Robert E.; Hunter, Daniel A.; Milbrandt, Jeffrey

    2011-01-01

    Mitochondrial dysfunction is a common cause of peripheral neuropathies. While the role of neuron and axonal mitochondria in peripheral nerve disease is well appreciated, whether Schwann cell (SC) mitochondrial deficits contribute to peripheral neuropathies is unclear. Here we examine how SC mitochondrial dysfunction affects axonal survival and contributes to the decline of peripheral nerve function by generating mice with SC-specific mitochondrial deficits. These mice (Tfam-SCKOs) were produced through the tissue-specific deletion of the mitochondrial transcription factor A gene (Tfam), which is essential for mitochondrial DNA (mtDNA) transcription and maintenance. Tfam-SCKOs were viable but, as they aged, they developed a progressive peripheral neuropathy characterized by nerve conduction abnormalities as well as extensive muscle denervation. Morphological examination of Tfam-SCKO nerves revealed early preferential loss of small unmyelinated fibers followed by prominent demyelination and degeneration of larger-caliber axons. Tfam-SCKOs displayed sensory and motor deficits consistent with this pathology. Remarkably, the severe mtDNA depletion and respiratory chain abnormalities in Tfam-SCKO mice did not affect SC proliferation or survival. Mitochondrial function in SCs is therefore essential for maintenance of axonal survival and normal peripheral nerve function, suggesting that SC mitochondrial dysfunction contributes to human peripheral neuropathies. PMID:21752989

  18. Axonal neuregulin 1 type III activates NF-kappaB in Schwann cells during myelin formation.

    PubMed

    Limpert, Allison S; Carter, Bruce D

    2010-05-28

    The formation of myelin requires a series of complex signaling events initiated by the axon to surrounding glial cells, which ultimately respond by tightly wrapping the axon with layers of specialized plasma membrane thereby allowing for saltatory conduction. Activation of the transcription factor NF-kappaB in Schwann cells has been suggested to be critical for these cells to differentiate into a myelinating phenotype; however, the mechanisms by which it is activated have yet to be elucidated. Here, we demonstrate that axonal membranes are sufficient to promote NF-kappaB activation in cultured Schwann cells and identify neuregulin 1 (NRG1), specifically the membrane-bound type III isoform, as the signal responsible for activating this transcription factor. Surprisingly, neither membrane-bound type I nor the soluble NRG1 EGF domain could activate NF-kappaB, indicating that type III induces a qualitatively unique signal. The transcriptional activity of NF-kappaB was significantly enhanced by treatment with forskolin, indicating these two signals converge for maximal activation. Both ErbB2 and -3 receptors were required for transducing the NRG1 signal, because gene deletion of ErbB3 in Schwann cells or treatment with the ErbB2 selective inhibitor, PKI-166, prevented the stimulation of NF-kappaB by axonal membranes. Finally, PKI-166 blocked the activation of the transcription factor in myelinating neuron/Schwann cell co-cultures and in vivo, in developing sciatic nerves. Taken together, these data establish NRG1 type III as the activator of NF-kappaB during myelin formation. PMID:20360002

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

    SciTech Connect

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

    1989-09-01

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

  20. Combination of Engineered Schwann Cell Grafts to Secrete Neurotrophin and Chondroitinase Promotes Axonal Regeneration and Locomotion after Spinal Cord Injury

    PubMed Central

    Pressman, Yelena; Moody, Alison; Berg, Randall; Muir, Elizabeth M.; Rogers, John H.; Ozawa, Hiroshi; Itoi, Eiji; Pearse, Damien D.; Bunge, Mary Bartlett

    2014-01-01

    Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function. PMID:24478364

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

    PubMed

    Baade, Carolin; Byczkowicz, Niklas; Hallermann, Stefan

    2016-07-01

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

  2. Non-Cell-Autonomous Regulation of Retrograde Motoneuronal Axonal Transport in an SBMA Mouse Model.

    PubMed

    Halievski, Katherine; Kemp, Michael Q; Breedlove, S Marc; Miller, Kyle E; Jordan, Cynthia L

    2016-01-01

    Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor (AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique "myogenic" transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons. PMID:27517091

  3. Non-Cell-Autonomous Regulation of Retrograde Motoneuronal Axonal Transport in an SBMA Mouse Model

    PubMed Central

    Halievski, Katherine; Kemp, Michael Q.; Breedlove, S. Marc; Miller, Kyle E.

    2016-01-01

    Abstract Defects in axonal transport are seen in motoneuronal diseases, but how that impairment comes about is not well understood. In spinal bulbar muscular atrophy (SBMA), a disorder linked to a CAG/polyglutamine repeat expansion in the androgen receptor (AR) gene, the disease-causing AR disrupts axonal transport by acting in both a cell-autonomous fashion in the motoneurons themselves, and in a non-cell-autonomous fashion in muscle. The non-cell-autonomous mechanism is suggested by data from a unique “myogenic” transgenic (TG) mouse model in which an AR transgene expressed exclusively in skeletal muscle fibers triggers an androgen-dependent SBMA phenotype, including defects in retrograde transport. However, motoneurons in this TG model retain the endogenous AR gene, leaving open the possibility that impairments in transport in this model also depend on ARs in the motoneurons themselves. To test whether non-cell-autonomous mechanisms alone can perturb retrograde transport, we generated male TG mice in which the endogenous AR allele has the testicular feminization mutation (Tfm) and, consequently, is nonfunctional. Males carrying the Tfm allele alone show no deficits in motor function or axonal transport, with or without testosterone treatment. However, when Tfm males carrying the myogenic transgene (Tfm/TG) are treated with testosterone, they develop impaired motor function and defects in retrograde transport, having fewer retrogradely labeled motoneurons and deficits in endosomal flux based on time-lapse video microscopy of living axons. These findings demonstrate that non-cell-autonomous disease mechanisms originating in muscle are sufficient to induce defects in retrograde transport in motoneurons. PMID:27517091

  4. NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells.

    PubMed

    Huang, Zhenhui; Gao, Yarong; Sun, Yuhui; Zhang, Chao; Yin, Yue; Shimoda, Yasushi; Watanabe, Kazutada; Liu, Yaobo

    2016-08-15

    Little is known about the molecules mediating the cross-talk between post-traumatic axons and scar-forming cells after spinal cord injury. We found that a sustained NB-3 induction was simultaneously present in the terminations of post-traumatic corticospinal axons and scar-forming cells at the spinal lesion site, where they were in direct contact when axons tried to penetrate the glial scar. The regrowth of corticospinal axons was enhanced in vivo with NB-3 deficiency or interruption of NB-3 trans-homophilic interactions. Biochemical, in vitro and in vivo evidence demonstrated that NB-3 homophilically interacted in trans to initiate a growth inhibitory signal transduction from scar-forming cells to neurons by modulating mTOR activity via CHL1 and PTPσ. NB-3 deficiency promoted BMS scores, electrophysiological transmission, and synapse reformation between regenerative axons and neurons. Our findings demonstrate that NB-3 trans-homophilic interactions mediate the cross-talk between post-traumatic axons and scar-forming cells and impair the intrinsic growth ability of injured axons. PMID:27192985

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

    PubMed

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

    2016-04-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

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

    1991-03-11

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

  8. Exogenous glycosaminoglycans induce complete inversion of retinal ganglion cell bodies and their axons within the retinal neuroepithelium.

    PubMed Central

    Brittis, P A; Silver, J

    1994-01-01

    Prior to forming an axon, retinal ganglion cells retain a primitive radial configuration while maintaining ventricular and vitreal endfeet attachments. During their subsequent differentiation, ganglion cells polarize their cell body and axon only along the vitreal surface. When the ventricular surfaces of intact retinas in organ culture were exposed to free chondroitin sulfate (CS) in solution, both the cell body and nerve fiber layers were repolarized to the opposite side of the neuroepithelium. However, the basal lamina remained in its usual position. Thus, the ability to initiate an axon is not restricted to the vitreal endfoot region of differentiating neurons, and in addition, the radial position at which the axon emerges can be mediated by the location and concentration of the extracellular CS milieu. Images PMID:8052616

  9. 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.

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

    PubMed Central

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

    2014-01-01

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

  11. Proper migration and axon outgrowth of zebrafish cranial motoneuron subpopulations require the cell adhesion molecule MDGA2A

    PubMed Central

    Ingold, Esther; vom Berg-Maurer, Colette M.; Burckhardt, Christoph J.; Lehnherr, André; Rieder, Philip; Keller, Philip J.; Stelzer, Ernst H.; Greber, Urs F.; Neuhauss, Stephan C. F.; Gesemann, Matthias

    2015-01-01

    ABSTRACT The formation of functional neuronal circuits relies on accurate migration and proper axonal outgrowth of neuronal precursors. On the route to their targets migrating cells and growing axons depend on both, directional information from neurotropic cues and adhesive interactions mediated via extracellular matrix molecules or neighbouring cells. The inactivation of guidance cues or the interference with cell adhesion can cause severe defects in neuronal migration and axon guidance. In this study we have analyzed the function of the MAM domain containing glycosylphosphatidylinositol anchor 2A (MDGA2A) protein in zebrafish cranial motoneuron development. MDGA2A is prominently expressed in distinct clusters of cranial motoneurons, especially in the ones of the trigeminal and facial nerves. Analyses of MDGA2A knockdown embryos by light sheet and confocal microscopy revealed impaired migration and aberrant axonal outgrowth of these neurons; suggesting that adhesive interactions mediated by MDGA2A are required for the proper arrangement and outgrowth of cranial motoneuron subtypes. PMID:25572423

  12. Ephrin-B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions

    PubMed Central

    Petros, Timothy J.; Bryson, J. Barney; Mason, Carol

    2010-01-01

    The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin-B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin-B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin-B2, we utilized time-lapse imaging to characterize the effects of ephrin-B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin-B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally-projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally-projecting ventronasal axons are less sensitive to ephrin-B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general. PMID:20629048

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

    PubMed

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

    1995-12-01

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

  14. Axonal degeneration, regeneration and ganglion cell death in a rodent model of anterior ischemic optic neuropathy (rAION).

    PubMed

    Zhang, Cheng; Guo, Yan; Slater, Bernard J; Miller, Neil R; Bernstein, Steven L

    2010-08-01

    Using laser-induced photoactivation of intravenously administered rose Bengal in rats, we generated an ischemic infarction of the intrascleral portion of the optic nerve (ON) comparable to that which occurs in humans to investigate optic nerve axon degenerative events following optic nerve infarct and the potential for axon re-growth. Animals were euthanized at different times post infarct. Axon degeneration was evaluated with SMI312 immunolabeling, and GAP-43 immunostaining was used to identify axon regeneration. Terminal dUTP nick end labeling (TUNEL) was used to evaluate retinal ganglion cell (RGC) death. There was significant axon structural disruptinot ion at the anterior intrascleral portion of the ON by 3d post-infarct, extending to the posterior ON by 7d post-stroke. Destruction of normal axon structure and massive loss of axon fibers occurred by 2 weeks. GAP-43 immunoreactivity occurred in the anterior ON by 7d post-infarct, lasting 3-4 weeks, without extension past the primary ischemic lesion. TUNEL-positive cells in the RGC layer appeared by 7d post-insult. These results indicate that following induction of ischemic optic neuropathy, significant axon damage occurs by 3d post-infarct, with later neuronal death. Post-stroke adult rat retinal ganglion cells attempt to regenerate their axons, but this effort is restricted to the unmyelinated region of the anterior ON. These responses are important in understanding pathologic process that underlies human non-arteritic anterior ischemic optic neuropathy (NAION) and may guide both the appropriate treatment of NAION and the window of opportunity for such treatment. PMID:20621651

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

    PubMed

    Bywalez, Wolfgang G; Patirniche, Dinu; Rupprecht, Vanessa; Stemmler, Martin; Herz, Andreas V M; Pálfi, Dénes; Rózsa, Balázs; Egger, Veronica

    2015-02-01

    Neuronal dendritic spines have been speculated to function as independent computational units, yet evidence for active electrical computation in spines is scarce. Here we show that strictly local voltage-gated sodium channel (Nav) activation can occur during excitatory postsynaptic potentials in the spines of olfactory bulb granule cells, which we mimic and detect via combined two-photon uncaging of glutamate and calcium imaging in conjunction with whole-cell recordings. We find that local Nav activation boosts calcium entry into spines through high-voltage-activated calcium channels and accelerates postsynaptic somatic depolarization, without affecting NMDA receptor-mediated signaling. Hence, Nav-mediated boosting promotes rapid output from the reciprocal granule cell spine onto the lateral mitral cell dendrite and thus can speed up recurrent inhibition. This striking example of electrical compartmentalization both adds to the understanding of olfactory network processing and broadens the general view of spine function. PMID:25619656

  16. Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

    PubMed Central

    Hammel, Ilan; Shoichetman, Tanya; Amihai, Dina; Galli, Stephen J.; Skutelsky, Ehud

    2013-01-01

    We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower adivin gold binding density (by approximately 50%, P<0.001). The latter result provides additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild type mice. In both wild type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild type cells (P < 0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in the bm/bm cells. PMID:20127366

  17. BK Channels Localize to the Paranodal Junction and Regulate Action Potentials in Myelinated Axons of Cerebellar Purkinje Cells

    PubMed Central

    Hirono, Moritoshi; Ogawa, Yasuhiro; Misono, Kaori; Zollinger, Daniel R.; Trimmer, James S.

    2015-01-01

    In myelinated axons, K+ channels are clustered in distinct membrane domains to regulate action potentials (APs). At nodes of Ranvier, Kv7 channels are expressed with Na+ channels, whereas Kv1 channels flank nodes at juxtaparanodes. Regulation of axonal APs by K+ channels would be particularly important in fast-spiking projection neurons such as cerebellar Purkinje cells. Here, we show that BK/Slo1 channels are clustered at the paranodal junctions of myelinated Purkinje cell axons of rat and mouse. The paranodal junction is formed by a set of cell-adhesion molecules, including Caspr, between the node and juxtaparanodes in which it separates nodal from internodal membrane domains. Remarkably, only Purkinje cell axons have detectable paranodal BK channels, whose clustering requires the formation of the paranodal junction via Caspr. Thus, BK channels occupy this unique domain in Purkinje cell axons along with the other K+ channel complexes at nodes and juxtaparanodes. To investigate the physiological role of novel paranodal BK channels, we examined the effect of BK channel blockers on antidromic AP conduction. We found that local application of blockers to the axon resulted in a significant increase in antidromic AP failure at frequencies above 100 Hz. We also found that Ni2+ elicited a similar effect on APs, indicating the involvement of Ni2+-sensitive Ca2+ channels. Furthermore, axonal application of BK channel blockers decreased the inhibitory synaptic response in the deep cerebellar nuclei. Thus, paranodal BK channels uniquely support high-fidelity firing of APs in myelinated Purkinje cell axons, thereby underpinning the output of the cerebellar cortex. PMID:25948259

  18. Spatial convergence and divergence between cutaneous afferent axons and dorsal horn cells are not constant.

    PubMed

    Brown, P B; Harton, P; Millecchia, R; Lawson, J; Kunjara-Na-Ayudhya, T; Stephens, S; Miller, M A; Hicks, L; Culberson, J

    2000-05-01

    We have proposed a quantitative model of the development of dorsal horn cell receptive fields (RFs) and somatotopic organization (Brown et al. [1997] Somatosens. Motor Res. 14:93-106). One component of that model is a hypothesis that convergence and divergence of connections between low-threshold primary afferent mechanoreceptive axons and dorsal horn cells are invariant over skin location and dorsal horn location. The more limited, and more easily tested, hypothesis that spatial convergence and divergence between cutaneous mechanoreceptors and dorsal horn cell are constant was examined. Spatial divergence is the number of dorsal horn cells whose RFs overlap the RF center of a primary afferent, and spatial convergence is the number of afferent RF centers that lie within the RF of a dorsal horn cell. Innervation density was determined as a function of location on the hindlimb by using peripheral nerve recording and axon counting. A descriptive model of dorsal horn cell receptive fields (Brown et al. [1998] J. Neurophysiol. 31:833-848) was used to simulate RFs of the entire dorsal horn cell population in order to estimate RF area and map scale as a function of location on the hindlimb. Previously reported correlations among innervation density, map scale, and RF size were confirmed. However, these correlations were not linear. The hypothesis that spatial convergence and divergence are constant was rejected. The previously proposed model of development of dorsal horn cell somatotopy and RF geometries must be revised to take variable spatial convergence and divergence into account. PMID:10754502

  19. Long-range projection neurons of the mouse ventral tegmental area: a single-cell axon tracing analysis

    PubMed Central

    Aransay, Ana; Rodríguez-López, Claudia; García-Amado, María; Clascá, Francisco; Prensa, Lucía

    2015-01-01

    Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four “subtypes” could be distinguished among the FPN cells based on their projection targets: (1) “Mesocorticolimbic” FPN projecting to both neocortex and basal forebrain; (2) “Mesocortical” FPN innervating the neocortex almost exclusively; (3) “Mesolimbic” FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) “Mesostriatal” FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and

  20. Long-range projection neurons of the mouse ventral tegmental area: a single-cell axon tracing analysis.

    PubMed

    Aransay, Ana; Rodríguez-López, Claudia; García-Amado, María; Clascá, Francisco; Prensa, Lucía

    2015-01-01

    Pathways arising from the ventral tegmental area (VTA) release dopamine and other neurotransmitters during the expectation and achievement of reward, and are regarded as central links of the brain networks that create drive, pleasure, and addiction. While the global pattern of VTA projections is well-known, the actual axonal wiring of individual VTA neurons had never been investigated. Here, we labeled and analyzed the axons of 30 VTA single neurons by means of single-cell transfection with the Sindbis-pal-eGFP vector in mice. These observations were complemented with those obtained by labeling the axons of small populations of VTA cells with iontophoretic microdeposits of biotinylated dextran amine. In the single-cell labeling experiments, each entire axonal tree was reconstructed from serial sections, the length of terminal axonal arbors was estimated by stereology, and the dopaminergic phenotype was tested by double-labeling for tyrosine hydroxylase immunofluorescence. We observed two main, markedly different VTA cell morphologies: neurons with a single main axon targeting only forebrain structures (FPN cells), and neurons with multibranched axons targeting both the forebrain and the brainstem (F + BSPN cells). Dopaminergic phenotype was observed in FPN cells. Moreover, four "subtypes" could be distinguished among the FPN cells based on their projection targets: (1) "Mesocorticolimbic" FPN projecting to both neocortex and basal forebrain; (2) "Mesocortical" FPN innervating the neocortex almost exclusively; (3) "Mesolimbic" FPN projecting to the basal forebrain, accumbens and caudateputamen; and (4) "Mesostriatal" FPN targeting only the caudateputamen. While the F + BSPN cells were scattered within VTA, the mesolimbic neurons were abundant in the paranigral nucleus. The observed diversity in wiring architectures is consistent with the notion that different VTA cell subpopulations modulate the activity of specific sets of prosencephalic and brainstem structures

  1. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

    PubMed Central

    Gao, Fei; Song, Xueying; Zhu, Dexiao; Wang, Xiaochen; Hao, Aijun; Nadler, J. Victor; Zhan, Ren-Zhi

    2015-01-01

    To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells. PMID:26500490

  2. Mature granule cells of the dentate gyrus-Passive bystanders or principal performers in hippocampal function?

    PubMed

    Lopez-Rojas, Jeffrey; Kreutz, Michael R

    2016-05-01

    The dentate gyrus is the main entrance of highly processed information to the hippocampus which derives from associative cortices and it is one of the few privileged areas in the brain where adult neurogenesis occurs. This creates the unique situation that neurons of diverse maturation stages are part of one neuronal network at any given point in life. While recently adult-born cells have a low induction threshold for long-term potentiation several studies suggest that following maturation granule cells are poorly excitable and they exhibit reduced Hebbian synaptic plasticity to an extent that it was even suggested that they functionally retire. Here, we review the functional properties of mature granule cells and discuss how plasticity of intrinsic excitability and alterations in excitation-inhibition balance might impact on their role in hippocampal information processing. PMID:26949226

  3. 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

  4. Rapid erasure of hippocampal memory following inhibition of dentate gyrus granule cells.

    PubMed

    Madroñal, Noelia; Delgado-García, José M; Fernández-Guizán, Azahara; Chatterjee, Jayanta; Köhn, Maja; Mattucci, Camilla; Jain, Apar; Tsetsenis, Theodoros; Illarionova, Anna; Grinevich, Valery; Gross, Cornelius T; Gruart, Agnès

    2016-01-01

    The hippocampus is critical for the acquisition and retrieval of episodic and contextual memories. Lesions of the dentate gyrus, a principal input of the hippocampus, block memory acquisition, but it remains unclear whether this region also plays a role in memory retrieval. Here we combine cell-type specific neural inhibition with electrophysiological measurements of learning-associated plasticity in behaving mice to demonstrate that dentate gyrus granule cells are not required for memory retrieval, but instead have an unexpected role in memory maintenance. Furthermore, we demonstrate the translational potential of our findings by showing that pharmacological activation of an endogenous inhibitory receptor expressed selectively in dentate gyrus granule cells can induce a rapid loss of hippocampal memory. These findings open a new avenue for the targeted erasure of episodic and contextual memories. PMID:26988806

  5. Cytokine signals through STAT3 promote expression of granulocyte secondary granule proteins in 32D cells

    PubMed Central

    Wang, Lei; Arcasoy, Murat O.; Watowich, Stephanie S.; Forget, Bernard G.

    2008-01-01

    Objective In a previous study, we showed that activation of a transfected human erythropoietin receptor (EPOR) in the murine myeloid cell line 32D resulted in the development of morphologic features of granulocytic differentiation and expression of the neutrophil primary granule protein myeloperoxidase. We now studied if EPOR signaling could also mediate secondary granule protein gene expression and investigated the signal transduction requirements for induction of secondary granule gene expression in 32D cells. Materials and Methods Wild-type and variant 32D cells expressing normal or chimeric EPORs or receptors for granulocyte colony-stimulating factor (G-CSFRs) were stimulated with EPO or G-CSF and the expression of granulocyte-specific genes was analyzed by Northern blot analysis. To determine the signaling mechanisms required for secondary granule protein gene induction, the activation of STAT pathways following growth factor stimulation was studied by Western blot analysis. Results We found that EPO treatment of 32D cells engineered to express EPOR did not result in induction of the secondary granule protein genes encoding lactoferrin and 24p3 lipocalin, the mouse homolog of human N-Gal, or the myeloid transcription factor C/EBPε. Replacement of the intracellular domain of EPOR with the intracellular domain of G-CSFR in a chimeric receptor was associated with EPO-mediated induction of lactoferrin, 24p3 lipocalin, and C/EBPε genes. We found that STAT3 phosphorylation was mediated by the intracellular domain of G-CSFR, but not EPOR. Replacement of one or two of the STAT5 binding sites in the intracytoplasmic domain of the EPOR with STAT3 binding sites resulted in EPO-mediated STAT3 activation and a marked increase in the expression of the 24p3 lipocalin gene. Knockdown of STAT3 protein levels with siRNA caused significant decrease in 24p3 lipocalin gene induction. Conclusion These results indicate that EPOR signaling cannot substitute for G-CSFR signaling to

  6. Diffuse Traumatic Axonal Injury in the Optic Nerve Does Not Elicit Retinal Ganglion Cell Loss

    PubMed Central

    Wang, Jiaqiong; Fox, Michael A.; Povlishock, John T.

    2013-01-01

    Much of the morbidity following traumatic brain injury (TBI) is associated with traumatic axonal injury (TAI). Although most TAI studies focus on corpus callosum white matter, the visual system has received increased interest. To assess visual system TAI, we developed a mouse model of optic nerve TAI. It is unknown, however, whether this TAI causes retinal ganglion cell (RGC) death. To address this issue, YFP-16 transgenic mice were subjected to mild TBI and followed from 2 to 28 days. Neither TUNEL-positive or cleaved caspase-3 immunoreactive RGCs were observed from 2 to 28 days post-TBI. Quantification of immunoreactivity of Brn3a, an RGC marker, demonstrated no RGC loss; parallel electron microscopic analysis confirmed RGC viability. Persistent RGC survival was also consistent with the finding of reorganization in the proximal axonal segments following TAI wherein microglia/macrophages remained inactive. In contrast, activated microglia/macrophages closely enveloped the distal disconnected, degenerating axonal segments at 7 to 28 days post-injury, thereby confirming that this model consistently evoked TAI followed by disconnection. Collectively, these data provide novel insight into the evolving pathobiology associated with TAI that will form a foundation for future studies exploring TAI therapy and its downstream consequences. PMID:23860030

  7. Action potential processing in a detailed Purkinje cell model reveals a critical role for axonal compartmentalization

    PubMed Central

    Masoli, Stefano; Solinas, Sergio; D'Angelo, Egidio

    2015-01-01

    The Purkinje cell (PC) is among the most complex neurons in the brain and plays a critical role for cerebellar functioning. PCs operate as fast pacemakers modulated by synaptic inputs but can switch from simple spikes to complex bursts and, in some conditions, show bistability. In contrast to original works emphasizing dendritic Ca-dependent mechanisms, recent experiments have supported a primary role for axonal Na-dependent processing, which could effectively regulate spike generation and transmission to deep cerebellar nuclei (DCN). In order to account for the numerous ionic mechanisms involved (at present including Nav1.6, Cav2.1, Cav3.1, Cav3.2, Cav3.3, Kv1.1, Kv1.5, Kv3.3, Kv3.4, Kv4.3, KCa1.1, KCa2.2, KCa3.1, Kir2.x, HCN1), we have elaborated a multicompartmental model incorporating available knowledge on localization and gating of PC ionic channels. The axon, including initial segment (AIS) and Ranvier nodes (RNs), proved critical to obtain appropriate pacemaking and firing frequency modulation. Simple spikes initiated in the AIS and protracted discharges were stabilized in the soma through Na-dependent mechanisms, while somato-dendritic Ca channels contributed to sustain pacemaking and to generate complex bursting at high discharge regimes. Bistability occurred only following Na and Ca channel down-regulation. In addition, specific properties in RNs K currents were required to limit spike transmission frequency along the axon. The model showed how organized electroresponsive functions could emerge from the molecular complexity of PCs and showed that the axon is fundamental to complement ionic channel compartmentalization enabling action potential processing and transmission of specific spike patterns to DCN. PMID:25759640

  8. Netrin-1 induces local translation of down syndrome cell adhesion molecule in axonal growth cones.

    PubMed

    Jain, Shruti; Welshhans, Kristy

    2016-07-01

    Down syndrome cell adhesion molecule (DSCAM) plays an important role in many neurodevelopmental processes such as axon guidance, dendrite arborization, and synapse formation. DSCAM is located in the Down syndrome trisomic region of human chromosome 21 and may contribute to the Down syndrome brain phenotype, which includes a reduction in the formation of long-distance connectivity. The local translation of a select group of mRNA transcripts within growth cones is necessary for the formation of appropriate neuronal connectivity. Interestingly, we have found that Dscam mRNA is localized to growth cones of mouse hippocampal neurons, and is dynamically regulated in response to the axon guidance molecule, netrin-1. Furthermore, netrin-1 stimulation results in an increase in locally translated DSCAM protein in growth cones. Deleted in colorectal cancer (DCC), a netrin-1 receptor, is required for the netrin-1-induced increase in Dscam mRNA local translation. We also find that two RNA-binding proteins-fragile X mental retardation protein (FMRP) and cytoplasmic polyadenylation element binding protein (CPEB)-colocalize with Dscam mRNA in growth cones, suggesting their regulation of Dscam mRNA localization and translation. Finally, overexpression of DSCAM in mouse cortical neurons results in a severe stunting of axon outgrowth and branching, suggesting that an increase in DSCAM protein results in a structural change having functional consequences. Taken together, these results suggest that netrin-1-induced local translation of Dscam mRNA during embryonic development may be an important mechanism to regulate axon growth and guidance in the developing nervous system. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 799-816, 2016. PMID:26518186

  9. Enterovirus 71 induces anti-viral stress granule-like structures in RD cells.

    PubMed

    Zhu, Yuanmei; Wang, Bei; Huang, He; Zhao, Zhendong

    2016-08-01

    Stress granules (SGs) are dynamic cytoplasmic granules formed in response to a variety of stresses, including viral infection. Several viruses can modulate the formation of SG with different effects, but the relationship between SG formation and EV71 infection is poorly understood. In this study, we report that EV71 inhibits canonical SGs formation in infected cells and induces the formation of novel RNA granules that were distinguished from canonical SGs in composition and morphology, which we termed 'SG like structures'. Our results also demonstrated that EV71 triggered formation of SG-like structures is dependent on PKR and eIF2α phosphorylation and requires ongoing cellular mRNA synthesis. Finally, we found that SG-like structures are antiviral RNA granules that promote cellular apoptosis and suppress EV71 propagation. Taken together, our findings explain the formation mechanism of SG-like structures induced by EV71 and shed light on virus-host interaction and molecular mechanism underlying EV71 pathogenesis. PMID:27216457

  10. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans

    PubMed Central

    Ueki, Shigeharu; Melo, Rossana C. N.; Ghiran, Ionita; Spencer, Lisa A.; Dvorak, Ann M.; Weller, Peter F.

    2013-01-01

    Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane–enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets. PMID:23303825

  11. Auditory cortical axons contact commissural cells throughout the guinea pig inferior colliculus.

    PubMed

    Nakamoto, Kyle T; Sowick, Colleen S; Schofield, Brett R

    2013-12-01

    Projections from auditory cortex (AC) affect how cells in both inferior colliculi (IC) respond to acoustic stimuli. The large projection from the AC to the ipsilateral IC is usually credited with the effects in the ipsilateral IC. The circuitry underlying effects in the contralateral IC is less clear. The direct projection from the AC to the contralateral IC is relatively small. An unexplored possibility is that the large ipsilateral cortical projection contacts the substantial number of cells in the ipsilateral IC that project through the commissure to the contralateral IC. Apparent contacts between cortical boutons and commissural cells were identified in the left IC after injection of different fluorescent tracers into the left AC and the right IC. Commissural cells were labeled throughout the left IC, and many (23-34%) appeared to be contacted by cortical axons. In the central nucleus, both disc-shaped and stellate cells were contacted. Antibodies to glutamic acid decarboxylase (GAD) were used to identify GABAergic commissural cells. The majority (>86%) of labeled commissural cells were GAD-immunonegative. Despite low numbers of GAD-immunopositive commissural cells, some of these cells were contacted by cortical boutons. Nonetheless, most cortically contacted commissural cells were GAD-immunonegative (i.e., presumably glutamatergic). We conclude that auditory cortical axons contact primarily excitatory commissural cells in the ipsilateral IC that project to the contralateral IC. These corticocollicular contacts occur in each subdivision of the ipsilateral IC, suggesting involvement of commissural cells throughout the IC. This pathway - from AC to commissural cells in the ipsilateral IC - is a prime candidate for the excitatory effects of activation of the auditory cortex on responses in the contralateral IC. Overall this suggests that the auditory corticofugal pathway is integrated with midbrain commissural connections. PMID:24140579

  12. Extensive cell migration, axon regeneration and improved function with polysialic acid-modified Schwann cells after spinal cord injury

    PubMed Central

    Ghosh, Mousumi; Tuesta, Luis M.; Puentes, Rocio; Patel, Samik; Melendez, Kiara; Maarouf, Abderrahman El; Rutishauser, Urs; Pearse, Damien Daniel

    2015-01-01

    Schwann cells (SC) implantation after spinal cord injury (SCI) promotes axonal regeneration, remyelination repair and functional recovery. Reparative efficacy, however, may be limited due to the inability of SCs to migrate outward from the lesion-implant site. Altering SC cell surface properties by over-expressing polysialic acid (PSA) has been shown to promote SC migration. In the current study, a SCI contusion was used to evaluate the migration, supraspinal axon growth support and functional recovery associated with polysialyltransferase (PST)-over-expressing SCs (PST-GFP SCs) or controls (GFP SCs). Compared to GFP SCs, which remained confined to the injection site at the injury center, PST-GFP SCs migrated across the lesion:host cord interface for distances of up to 4.4 mm within adjacent host tissue. In addition, with PST-GFP SCs, there was extensive serotonergic and corticospinal axon in-growth within the implants that was limited in the GFP SC controls. The enhanced migration of PST-GFP SCs was accompanied by significant growth of these axons caudal to lesion. Animals receiving PST-GFP SCs exhibited improved functional outcome, both in the open-field and on the gridwalk test, over modest improvements provided by GFP SC controls. The current study for the first time demonstrates that a lack of migration by SC may hinder their reparative benefits and that cell surface overexpression of PSA enhances the ability of implanted SCs to associate with and support the growth of corticospinal axons. These results provide further promise that PSA modified SCs will be a potent reparative approach for SCI. PMID:22460918

  13. Extensive cell migration, axon regeneration, and improved function with polysialic acid-modified Schwann cells after spinal cord injury.

    PubMed

    Ghosh, Mousumi; Tuesta, Luis M; Puentes, Rocio; Patel, Samik; Melendez, Kiara; El Maarouf, Abderrahman; Rutishauser, Urs; Pearse, Damien Daniel

    2012-05-01

    Schwann cell (SC) implantation after spinal cord injury (SCI) promotes axonal regeneration, remyelination repair, and functional recovery. Reparative efficacy, however, may be limited because of the inability of SCs to migrate outward from the lesion-implant site. Altering SC cell surface properties by overexpressing polysialic acid (PSA) has been shown to promote SC migration. In this study, a SCI contusion model was used to evaluate the migration, supraspinal axon growth support, and functional recovery associated with polysialyltransferase (PST)-overexpressing SCs [PST-green fluorescent protein (GFP) SCs] or controls (GFP SCs). Compared with GFP SCs, which remained confined to the injection site at the injury center, PST-GFP SCs migrated across the lesion:host cord interface for distances of up to 4.4 mm within adjacent host tissue. In addition, with PST-GFP SCs, there was extensive serotonergic and corticospinal axon in-growth within the implants that was limited in the GFP SC controls. The enhanced migration of PST-GFP SCs was accompanied by significant growth of these axons caudal to lesion. Animals receiving PST-GFP SCs exhibited improved functional outcome, both in the open-field and on the gridwalk test, beyond the modest improvements provided by GFP SC controls. This study for the first time demonstrates that a lack of migration by SCs may hinder their reparative benefits and that cell surface overexpression of PSA enhances the ability of implanted SCs to associate with and support the growth of corticospinal axons. These results provide further promise that PSA-modified SCs will be a potent reparative approach for SCI. © 2012 Wiley Periodicals, Inc. PMID:22460918

  14. Differential Calcium Signaling Mediated by Voltage-Gated Calcium Channels in Rat Retinal Ganglion Cells and Their Unmyelinated Axons

    PubMed Central

    Sargoy, Allison; Sun, Xiaoping

    2014-01-01

    Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury. PMID:24416240

  15. Recording axonal conduction to evaluate the integration of pluripotent cell-derived neurons into a neuronal network.

    PubMed

    Shimba, Kenta; Sakai, Koji; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

    2015-10-01

    Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network. PMID:26303583

  16. 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.

  17. Adult-born hippocampal dentate granule cells undergoing maturation modulate learning and memory in the brain

    PubMed Central

    Deng, Wei; Saxe, Michael D.; Gallina, Iryna S.; Gage, Fred H.

    2009-01-01

    Adult-born dentate granule cells (DGCs) contribute to learning and memory, yet it remains unknown when adult-born DGCs become involved in the cognitive processes. During neurogenesis, immature dentate granule cells (DGCs) display distinctive physiological characteristics while undergoing morphological maturation before final integration into the neural circuits. The survival and activity of the adult-born DGCs can be influenced by the experience of the animal during a critical period when newborn DGCs are still immature. To assess the temporal importance of adult neurogenesis, we developed a transgenic mouse model that allowed us to transiently reduce the numbers of adult-born DGCs in a temporally regulatable manner. We found that mice with a reduced population of adult-born DGCs at the immature stage were deficient in forming robust, long-term spatial memory and displayed impaired performance in extinction tasks. These results suggest that immature DGCs that undergo maturation make important contributions to learning and memory. PMID:19864566

  18. Effects of zinc chloride on the RNP structures in HEp-2 cells: accumulation of perichromatin granules

    SciTech Connect

    Cervera, J.; Baguena-Cervellera, R.; Martinez, A.

    1985-12-01

    The effects of zinc on the ribonucleoprotein (RNP) constituents of HEp-2 cells have been analyzed. Pulse-chase autoradiographic experiments show a preferential inhibition of nucleolar RNA synthesis and a block in the transport of nucleolar and extranucleolar RNA in zinc-treated cells. Concomitantly with the disturbance in RNA metabolism and in protein synthesis, nucleolar condensation, accumulation of perichromatin granules and fibrils, condensation of interchromatin fibrils, and appearance of dense granular bodies occur. Accumulation of perichromatin fibrils and condensation of interchromatin fibrils appear to be related to the block in the transport of heterogeneous nuclear RNA. Depletion of certain proteins required for the assembly of RNP particles could share in the abnormal behavior of RNA and lead to the accumulation of perichromatin granules and the appearance of dense granular bodies.

  19. Parallel Computational Subunits in Dentate Granule Cells Generate Multiple Place Fields

    PubMed Central

    Ujfalussy, Balázs; Kiss, Tamás; Érdi, Péter

    2009-01-01

    A fundamental question in understanding neuronal computations is how dendritic events influence the output of the neuron. Different forms of integration of neighbouring and distributed synaptic inputs, isolated dendritic spikes and local regulation of synaptic efficacy suggest that individual dendritic branches may function as independent computational subunits. In the present paper, we study how these local computations influence the output of the neuron. Using a simple cascade model, we demonstrate that triggering somatic firing by a relatively small dendritic branch requires the amplification of local events by dendritic spiking and synaptic plasticity. The moderately branching dendritic tree of granule cells seems optimal for this computation since larger dendritic trees favor local plasticity by isolating dendritic compartments, while reliable detection of individual dendritic spikes in the soma requires a low branch number. Finally, we demonstrate that these parallel dendritic computations could contribute to the generation of multiple independent place fields of hippocampal granule cells. PMID:19750211

  20. Tethering of ICAM on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization

    PubMed Central

    Gross, Catharina C.; Brzostowski, Joseph A.; Liu, Dongfang; Long, Eric O.

    2013-01-01

    αLβ2 integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion, but also polarization of cytolytic granules in NK cells. Here we tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adapter molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, while NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes. PMID:20675589

  1. Receptive field properties of bipolar cell axon terminals in direction-selective sublaminas of the mouse retina

    PubMed Central

    Chen, Minggang; Lee, Seunghoon; Park, Silvia J. H.; Looger, Loren L.

    2014-01-01

    Retinal bipolar cells (BCs) transmit visual signals in parallel channels from the outer to the inner retina, where they provide glutamatergic inputs to specific networks of amacrine and ganglion cells. Intricate network computation at BC axon terminals has been proposed as a mechanism for complex network computation, such as direction selectivity, but direct knowledge of the receptive field property and the synaptic connectivity of the axon terminals of various BC types is required in order to understand the role of axonal computation by BCs. The present study tested the essential assumptions of the presynaptic model of direction selectivity at axon terminals of three functionally distinct BC types that ramify in the direction-selective strata of the mouse retina. Results from two-photon Ca2+ imaging, optogenetic stimulation, and dual patch-clamp recording demonstrated that 1) CB5 cells do not receive fast GABAergic synaptic feedback from starburst amacrine cells (SACs); 2) light-evoked and spontaneous Ca2+ responses are well coordinated among various local regions of CB5 axon terminals; 3) CB5 axon terminals are not directionally selective; 4) CB5 cells consist of two novel functional subtypes with distinct receptive field structures; 5) CB7 cells provide direct excitatory synaptic inputs to, but receive no direct GABAergic synaptic feedback from, SACs; and 6) CB7 axon terminals are not directionally selective, either. These findings help to simplify models of direction selectivity by ruling out complex computation at BC terminals. They also show that CB5 comprises two functional subclasses of BCs. PMID:25031256

  2. Model of very fast (>75 Hz) network oscillations generated by electrical coupling between the proximal axons of cerebellar Purkinje cells

    PubMed Central

    Traub, Roger D; Middleton, Steven J; Knöpfel, Thomas; Whittington, Miles A

    2009-01-01

    Very fast oscillations (VFO, >75 Hz) occur transiently in vivo, in the cerebellum of mice genetically modified to model Angelman syndrome, and in a mouse model of fetal alcohol syndrome. We recently reported VFO in slices of mouse cerebellar cortex (Crus I and II of ansiform and paramedian lobules), either in association with gamma oscillations (~40 Hz, evoked by nicotine), or in isolation (evoked by nicotine in combination with GABAA receptor blockade). The experimental data suggest a role for electrical coupling between Purkinje cells (blockade of VFO by drugs reducing gap junction conductance, and spikelets in some Purkinje cells); and the data suggest the specific involvement of Purkinje cell axons (because of field oscillation maxima in the granular layer). We show here that a detailed network model (1,000 multicompartment Purkinje cells) replicates the experimental data, when gap junctions are located on the proximal axons of Purkinje cells, provided sufficient spontaneous firing is present. Unlike other VFO models, most somatic spikelets do not correspond to axonal spikes in the parent axon, but reflect spikes in electrically coupled axons. The model predicts gating of VFO frequency by gNa inactivation, and experiments prolonging this inactivation time constant, with β-pompilidotoxin, are consistent with this prediction. The model also predicts that cerebellar VFO can be explained as an electrically coupled system of axons which are not intrinsic oscillators: the electrically uncoupled cells do not individually oscillate (in the model), and axonal firing rates are much lower in the uncoupled state than in the coupled state. PMID:18973579

  3. Receptive field properties of bipolar cell axon terminals in direction-selective sublaminas of the mouse retina.

    PubMed

    Chen, Minggang; Lee, Seunghoon; Park, Silvia J H; Looger, Loren L; Zhou, Z Jimmy

    2014-10-15

    Retinal bipolar cells (BCs) transmit visual signals in parallel channels from the outer to the inner retina, where they provide glutamatergic inputs to specific networks of amacrine and ganglion cells. Intricate network computation at BC axon terminals has been proposed as a mechanism for complex network computation, such as direction selectivity, but direct knowledge of the receptive field property and the synaptic connectivity of the axon terminals of various BC types is required in order to understand the role of axonal computation by BCs. The present study tested the essential assumptions of the presynaptic model of direction selectivity at axon terminals of three functionally distinct BC types that ramify in the direction-selective strata of the mouse retina. Results from two-photon Ca(2+) imaging, optogenetic stimulation, and dual patch-clamp recording demonstrated that 1) CB5 cells do not receive fast GABAergic synaptic feedback from starburst amacrine cells (SACs); 2) light-evoked and spontaneous Ca(2+) responses are well coordinated among various local regions of CB5 axon terminals; 3) CB5 axon terminals are not directionally selective; 4) CB5 cells consist of two novel functional subtypes with distinct receptive field structures; 5) CB7 cells provide direct excitatory synaptic inputs to, but receive no direct GABAergic synaptic feedback from, SACs; and 6) CB7 axon terminals are not directionally selective, either. These findings help to simplify models of direction selectivity by ruling out complex computation at BC terminals. They also show that CB5 comprises two functional subclasses of BCs. PMID:25031256

  4. Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

    PubMed Central

    Manuel, Martine; Pratt, Thomas; Liu, Min; Jeffery, Glen; Price, David J

    2008-01-01

    Background The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey) and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs). At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+) showed that (1) the total number of RGC axons projected by the retina and (2) the proportions that are sorted into the ipsilateral and contralateral optic tracts at the

  5. Fine Tuning of Synaptic Plasticity and Filtering by GABA Released from Hippocampal Autaptic Granule Cells.

    PubMed

    Valente, Pierluigi; Orlando, Marta; Raimondi, Andrea; Benfenati, Fabio; Baldelli, Pietro

    2016-03-01

    The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ∼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits. PMID:25576534

  6. Final steps in exocytosis observed in a cell with giant secretory granules.

    PubMed Central

    Breckenridge, L J; Almers, W

    1987-01-01

    Secretion by single mast cells was studied in normal and beige mice, a mutant with grossly enlarged secretory vesicles or granules. During degranulation, the membrane capacitance increased in steps, as single secretory vesicles fused with the cell membrane. The average step size was 10 times larger in beige than in normal mice, in agreement with the different granule sizes measured microscopically in the two preparations. Following individual capacitance steps in beige mice, individual granules of the appropriate size were observed to swell rapidly. Capacitance steps are frequently followed by the stepwise loss of a fluorescent dye loaded into the vesicles. Stepwise capacitance increases were occasionally intermittent before they became permanent, indicating the existence of an early, reversible, and incomplete state of vesicle fusion. During such "capacitance flicker," loss of fluorescent dye from vesicles did not occur, suggesting that the earliest aqueous connection between vesicle interior and cell exterior is a narrow channel. Our results support the view that the reversible formation of such a channel, which we term the fusion pore, is an early step in exocytosis. Images PMID:3470768

  7. Anaglyph of retinal stem cells and developing axons: selective volume enhancement in microscopy images.

    PubMed

    Carri, Néstor Gabriel; Bermúdez, Sebastián Noo; Fiore, Luciano; Di Napoli, Jennifer; Scicolone, Gabriel

    2014-04-01

    Retinal stem cell culture has become a powerful research tool, but it requires reliable methods to obtain high-quality images of living and fixed cells. This study describes a procedure for using phase contrast microscopy to obtain three-dimensional (3-D) images for the study of living cells by photographing a living cell in a culture dish from bottom to top, as well as a procedure to increase the quality of scanning electron micrographs and laser confocal images. The procedure may also be used to photograph clusters of neural stem cells, and retinal explants with vigorous axonal growth. In the case of scanning electron microscopy and laser confocal images, a Gaussian procedure is applied to the original images. The methodology allows for the creation of anaglyphs and video reconstructions, and provides high-quality images for characterizing living cells or tissues, fixed cells or tissues, or organs observed with scanning electron and laser confocal microscopy. Its greatest advantage is that it is easy to obtain good results without expensive equipment. The procedure is fast, precise, simple, and offers a strategic tool for obtaining 3-D reconstructions of cells and axons suitable for easily determining the orientation and polarity of a specimen. It also enables video reconstructions to be created, even of specimens parallel to the plastic base of a tissue culture dish, It is also helpful for studying the distribution and organization of living cells in a culture, as it provides the same powerful information as optical tomography, which most confocal microscopes cannot do on sterile living cells. PMID:24510888

  8. Epithelial Cell Transforming 2 and Aurora Kinase B Modulate Formation of Stress Granule-Containing Transcripts from Diverse Cellular Pathways in Astrocytoma Cells.

    PubMed

    Weeks, Adrienne; Agnihotri, Sameer; Lymer, Jennifer; Chalil, Alan; Diaz, Roberto; Isik, Semra; Smith, Christian; Rutka, James T

    2016-06-01

    Stress granules are small RNA-protein granules that modify the translational landscape during cellular stress to promote survival. The RhoGTPase RhoA is implicated in the formation of RNA stress granules. Our data demonstrate that the cytokinetic proteins epithelial cell transforming 2 and Aurora kinase B (AurkB) are localized to stress granules in human astrocytoma cells. AurkB and its downstream target histone-3 are phosphorylated during arsenite-induced stress. Chemical (AZD1152-HQPA) and siRNA inhibition of AurkB results in fewer and smaller stress granules when analyzed using high-throughput fluorescent-based cellomics assays. RNA immunoprecipitation with the known stress granule aggregates TIAR and G3BP1 was performed on astrocytoma cells, and subsequent analysis revealed that astrocytoma stress granules harbor unique mRNAs for various cellular pathways, including cellular migration, metabolism, translation, and transcriptional regulation. Human astrocytoma cell stress granules contain mRNAs that are known to be involved in glioma signaling and the mammalian target of rapamycin pathway. These data provide evidence that RNA stress granules are a novel form of epigenetic regulation in astrocytoma cells, which may be targetable by chemical inhibitors and enhance astrocytoma susceptibility to conventional therapy, such as radiation and chemotherapy. PMID:27106762

  9. Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells.

    PubMed

    Rudolf, Rüdiger; Kögel, Tanja; Kuznetsov, Sergei A; Salm, Thorsten; Schlicker, Oliver; Hellwig, Andrea; Hammer, John A; Gerdes, Hans-Hermann

    2003-04-01

    Neuroendocrine secretory granules, the storage organelles for neuropeptides and hormones, are formed at the trans-Golgi network, stored inside the cell and exocytosed upon stimulation. Previously, we have reported that newly formed secretory granules of PC12 cells are transported in a microtubule-dependent manner from the trans-Golgi network to the F-actin-rich cell cortex, where they undergo short directed movements and exhibit a homogeneous distribution. Here we provide morphological and biochemical evidence that myosin Va is associated with secretory granules. Expression of a dominant-negative tail domain of myosin Va in PC12 cells led to an extensive clustering of secretory granules close to the cell periphery, a loss of their cortical restriction and a strong reduction in their motility in the actin cortex. Based on this data we propose a model that implies a dual transport system for secretory granules: after microtubule-dependent delivery to the cell periphery, secretory granules exhibit a myosin Va-dependent transport leading to their restriction and even dispersal in the F-actin-rich cortex of PC12 cells. PMID:12615975

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

    PubMed

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

    1996-11-01

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

  11. Transcriptional control of cholesterol biosynthesis in Schwann cells by axonal neuregulin 1.

    PubMed

    Pertusa, Maria; Morenilla-Palao, Cruz; Carteron, Christelle; Viana, Felix; Cabedo, Hugo

    2007-09-28

    A characteristic feature of many vertebrate axons is their wrapping by a lamellar stack of glially derived membranes known as the myelin sheath. Myelin is a cholesterol-rich membrane that allows for rapid saltatory nerve impulse conduction. Axonal neuregulins instruct glial cells on when and how much myelin they should produce. However, how neuregulin regulates myelin sheath development and thickness is unknown. Here we show that neuregulin receptors are activated by drops in plasma membrane cholesterol, suggesting that they can sense sterol levels. In Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutarylcoenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. Neuregulin activity is mediated by the phosphatidylinositol 3-kinase pathway and a cAMP-response element located on the reductase promoter. We propose that by activating neuregulin receptors, neurons exploit a cholesterol homeostatic mechanism forcing Schwann cells to produce new membranes for the myelin sheath. We also show that a strong phylogenetic correlation exists between myelination and cholesterol biosynthesis, and we propose that the absence of the sterol branch of the mevalonate pathway in invertebrates precluded the myelination of their nervous system. PMID:17652086

  12. Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier.

    PubMed

    Eshed, Yael; Feinberg, Konstantin; Poliak, Sebastian; Sabanay, Helena; Sarig-Nadir, Offra; Spiegel, Ivo; Bermingham, John R; Peles, Elior

    2005-07-21

    Accumulation of Na(+) channels at the nodes of Ranvier is a prerequisite for saltatory conduction. In peripheral nerves, clustering of these channels along the axolemma is regulated by myelinating Schwann cells through a yet unknown mechanism. We report the identification of gliomedin, a glial ligand for neurofascin and NrCAM, two axonal immunoglobulin cell adhesion molecules that are associated with Na+ channels at the nodes of Ranvier. Gliomedin is expressed by myelinating Schwann cells and accumulates at the edges of each myelin segment during development, where it aligns with the forming nodes. Eliminating the expression of gliomedin by RNAi, or the addition of a soluble extracellular domain of neurofascin to myelinating cultures, which caused the redistribution of gliomedin along the internodes, abolished node formation. Furthermore, a soluble gliomedin induced nodal-like clusters of Na+ channels in the absence of Schwann cells. We propose that gliomedin provides a glial cue for the formation of peripheral nodes of Ranvier. PMID:16039564

  13. 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

  14. 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. PMID:26788851

  15. A comparative study of axon-surrounding cells in the two nasal nerve tracts from mouse olfactory epithelium and vomeronasal organ.

    PubMed

    Nakajima, Mitsunari; Tsuruta, Momoko; Mori, Hisamichi; Nishikawa, Chisa; Okuyama, Satoshi; Furukawa, Yoshiko

    2013-03-29

    The olfactory and vomeronasal systems are the two nasal chemical detectors in mammals. While glial cells in the olfactory nerve tracts have been well-investigated, little is known about cells in the vomeronasal nerve tracts. In the present study, we compared the expression patterns of marker proteins in the cells comprising the two nasal nerve tracts in mice. Neural crest-derived cells surrounded the olfactory nerve axons in the lamina propria of the olfactory epithelium. These cells expressed glial fibrillary acidic protein (GFAP) and p75 glycoprotein, which are markers of olfactory ensheathing cells. Neural crest-derived cells also surrounded the vomeronasal nerve axons in the lamina propria of the vomeronasal epithelium. These nerve axon-surrounding cells, however, did not express GFAP or p75. Rather, the vomeronasal nerve axons expressed GFAP and p75. These results suggest that axon-surrounding cells functionally differ between the olfactory and vomeronasal nerve tracts. PMID:23410787

  16. Study of Mast Cells and Granules from Primo Nodes Using Scanning Ionic Conductance Microscopy.

    PubMed

    Yoo, Yeong-Yung; Jung, Goo-Eun; Kwon, Hee-Min; Bae, Kyoung-Hee; Cho, Sang-Joon; Soh, Kwang-Sup

    2015-12-01

    Acupuncture points have a notable characteristic in that they have a higher density of mast cells (MCs) compared with nonacupoints in the skin, which is consistent with the augmentation of the immune function by acupuncture treatment. The primo vascular system, which was proposed as the anatomical structure of the acupuncture points and meridians, also has a high density of MCs. We isolated the primo nodes from the surfaces of internal abdominal organs, and the harvested primo nodes were stained with toluidine blue. The MCs were easily recognized by their stained color and their characteristic granules. The MCs were classified into four stages according to the degranulation of histamine granules in the MCs. Using conventional optical microscopes details of the degranulation state of MCs in each stage were not observable. However, we were able to investigate the distribution of the granules on the surfaces of the MCs in each stage, and to demonstrate the height profiles and three-dimensional structures of the MCs without disturbance of the cell membrane by using the scanning ion conductance microscopy. PMID:26742911

  17. 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.

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

    PubMed Central

    Santhakumar, Vijayalakshmi; Otis, Thomas S.

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  20. Selective autophagic degradation of maternally-loaded germline P granule components in somatic cells during C. elegans embryogenesis.

    PubMed

    Zhao, Yu; Tian, E; Zhang, Hong

    2009-07-01

    Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development. PMID:19372764

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

    PubMed

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

    2000-07-15

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

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

    PubMed

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

    1994-08-01

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

  3. Nuclear Factor One B regulates neural stem cell differentiation and axonal projection of corticofugal neurons

    PubMed Central

    Betancourt, Jennifer; Katzman, Sol; Chen, Bin

    2014-01-01

    During development of the cerebral cortex, neural stem cells divide to expand the progenitor pool and generate basal progenitors, outer radial glia and cortical neurons. As these newly born neurons differentiate, they must properly migrate toward their final destination in the cortical plate, project axons to appropriate targets, and develop dendrites. However, a complete understanding of the precise genetic mechanisms regulating these steps is lacking. Here we show that a member of the nuclear factor one (NFI) family of transcription factors, NFIB, is essential for many of these processes in mice. We performed a detailed analysis of NFIB expression during cortical development, and investigated defects in cortical neurogenesis, neuronal migration and differentiation in NfiB−/− brains. We found that NFIB is strongly expressed in radial glia and corticofugal neurons throughout cortical development. However, in NfiB−/− cortices, radial glia failed to generate outer radial glia, subsequently resulting in a loss of late basal progenitors. In addition, corticofugal neurons showed a severe loss of axonal projections, while late-born cortical neurons displayed defects in migration and ectopically expressed the early-born neuronal marker, CTIP2. Furthermore, gene expression analysis, by RNA-sequencing, revealed a misexpression of genes that regulate the cell cycle, neuronal differentiation and migration in NfiB−/− brains. Together these results demonstrate the critical functions of NFIB in regulating cortical development. PMID:23749646

  4. The composition of intracellular granules from the metal-accumulating cells of the common garden snail (Helix aspersa).

    PubMed Central

    Howard, B; Mitchell, P C; Ritchie, A; Simkiss, K; Taylor, M

    1981-01-01

    Certain cells in the hepatopancreas of the common garden snail (Helix aspersa) contain intracellular granules that are sites of metal-ion accumulation. These granules have been extracted and investigated by u.v. and i.r. spectroscopy, atomic-absorption spectroscopy, X-ray microanalysis, thermogravimetric analysis, enzymic assay and microanalysis. The deposits contain about 18% (w/w) water, 5% (w/w) organic matter and 76% (w/w) inorganic material of which the main components are Ca2+, Mg2+ and P2O7(4)-. The possible origin of these granules is discussed, as is their role in detoxifying heavy-metal ions. PMID:6272732

  5. Imaging exocytosis of single glucagon-like peptide-1 containing granules in a murine enteroendocrine cell line with total internal reflection fluorescent microscopy

    SciTech Connect

    Ohara-Imaizumi, Mica; Aoyagi, Kyota; Akimoto, Yoshihiro; Nakamichi, Yoko; Nishiwaki, Chiyono; Kawakami, Hayato; Nagamatsu, Shinya

    2009-12-04

    To analyze the exocytosis of glucagon-like peptide-1 (GLP-1) granules, we imaged the motion of GLP-1 granules labeled with enhanced yellow fluorescent protein (Venus) fused to human growth hormone (hGH-Venus) in an enteroendocrine cell line, STC-1 cells, by total internal reflection fluorescent (TIRF) microscopy. We found glucose stimulation caused biphasic GLP-1 granule exocytosis: during the first phase, fusion events occurred from two types of granules (previously docked granules and newcomers), and thereafter continuous fusion was observed mostly from newcomers during the second phase. Closely similar to the insulin granule fusion from pancreatic {beta} cells, the regulated biphasic exocytosis from two types of granules may be a common mechanism in glucose-evoked hormone release from endocrine cells.

  6. Distribution of Mesenchymal Stem Cells and Effects on Neuronal Survival and Axon Regeneration after Optic Nerve Crush and Cell Therapy

    PubMed Central

    Mesentier-Louro, Louise Alessandra; Zaverucha-do-Valle, Camila; da Silva-Junior, Almir Jordão; Nascimento-dos-Santos, Gabriel; Gubert, Fernanda; de Figueirêdo, Ana Beatriz Padilha; Torres, Ana Luiza; Paredes, Bruno D.; Teixeira, Camila; Tovar-Moll, Fernanda; Mendez-Otero, Rosalia; Santiago, Marcelo F.

    2014-01-01

    Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3–5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells in vivo by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1β expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime. PMID:25347773

  7. A double transgenic mouse used to track migrating Schwann cells and regenerating axons following engraftment of injured nerves

    PubMed Central

    Hayashi, Ayato; Koob, Jason W; Liu, Daniel Z; Tong, Alice Y; Hunter, Daniel A.; Parsadanian, Alexander; Mackinnon, Susan E.; Myckatyn, Terence M.

    2007-01-01

    We propose that double transgenic thy1-CFP(23)/S100-GFP mice whose Schwann cells constitutively express green fluorescent protein (GFP) and axons express cyan fluorescent protein (CFP) can be used to serially evaluate the temporal relationship between nerve regeneration and Schwann cell migration through acellular nerve grafts. Thy1-CFP(23)/S100-GFP and S100-GFP mice received non-fluorescing cold preserved nerve allografts from immunologically disparate donors. In vivo fluorescent imaging of these grafts was then performed at multiple points. The transected sciatic nerve was reconstructed with a 1 cm nerve allograft harvested from a Balb-C mouse and acellularized via 7 weeks of cold preservation prior to transplantation. The presence of regenerated axons and migrating Schwann cells was confirmed with confocal and electron microscopy on fixed tissue. Schwann cells migrated into the acellular graft (163 ± 15 intensity units) from both proximal and distal stumps, and bridged the whole graft within 10 days (388 ± 107 intensity units in the central 4-6 mm segment). Nerve regeneration lagged behind Schwann cell migration with 5 or 6 axons imaged traversing the proximal 4 mm of the graft under confocal microcopy within 10 days, and up to 21 labeled axons crossing the distal coaptation site by 15 days. Corroborative electron and light microscopy 5 mm into the graft demonstrated relatively narrow diameter myelinated (431±31) and unmyelinated (64±9) axons by 28 but not 10 days. Live imaging of the double-transgenic thy1-CFP(23)/S100-GFP murine line enabled serial assessment of Schwann cell-axonal relationships in traumatic nerve injuries reconstructed with acellular nerve allografts. PMID:17628544

  8. Axons take a dive

    PubMed Central

    Tong, Cheuk Ka; Cebrián-Silla, Arantxa; Paredes, Mercedes F; Huang, Eric J; García-Verdugo, Jose Manuel; Alvarez-Buylla, Arturo

    2015-01-01

    In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular–subventricular zone (V–SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the function of supraependymal axons in the regulation of E1 cells. PMID:26413556

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

    PubMed Central

    Le, Hoa H.; Belcher, Scott M.

    2010-01-01

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

  10. Axon-Schwann cell interaction in degenerating and regenerating peripheral nerve

    SciTech Connect

    Pellegrino, R.G.

    1984-01-01

    Severance of a peripheral nerve stimulates a characteristic sequence of events in the distal stump, including the dissolution of axons and myelin and the proliferation of Schwann cells within their basal lamina. The first part of this thesis employs the cat tibial nerve to examine the relationship between the spatio-temporal pattern of Schwann cell mitosis, loss of the structural and functional properties of axolemma, synthesis of P/sub 0/, the major myelin glycoprotein, and the clearance of morphological myelin. Induction of S phase was measured by determining the uptake of /sup 3/H thymidine into trichloroacetic acid (TCA) precipitates following a 3 hour in vitro incubation in Krebs-Ringers buffer containing /sup 3/H thymidine. Nerve transection stimulated a monophasic increase in /sup 3/H thymidine uptake that peaked at 4 days post-transection throughout an 80 mm length of distal stump. Light microscope autoradiography revealed prominent incorporation into Schwann cells of myelinated fibers. Nerve transection also produced dramatic changes in the intrafascicular binding of /sup 3/H STX which binds to voltage-sensitive sodium channels STX binding fell precipitously to 20% of normal at 4 days post-transection, concurrent with the peak of /sup 3/H thymidine uptake. In conclusion, these studies suggest: (a) Schwann cells divide more or less contemporaneously throughout the distal stump; (b) changes in axons rather than myelin are likely to stimulate the Schwann cell to divide; (c) mitosis regulates other events during Wallerian degeneration, including myelin degeneration and the clearance of sodium channels from nodal axolemma.

  11. 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

  12. The Stress Granule RNA-Binding Protein TIAR-1 Protects Female Germ Cells from Heat Shock in Caenorhabditis elegans.

    PubMed

    Huelgas-Morales, Gabriela; Silva-García, Carlos Giovanni; Salinas, Laura S; Greenstein, David; Navarro, Rosa E

    2016-01-01

    In response to stressful conditions, eukaryotic cells launch an arsenal of regulatory programs to protect the proteome. One major protective response involves the arrest of protein translation and the formation of stress granules, cytoplasmic ribonucleoprotein complexes containing the conserved RNA-binding proteins TIA-1 and TIAR. The stress granule response is thought to preserve mRNA for translation when conditions improve. For cells of the germline-the immortal cell lineage required for sexual reproduction-protection from stress is critically important for perpetuation of the species, yet how stress granule regulatory mechanisms are deployed in animal reproduction is incompletely understood. Here, we show that the stress granule protein TIAR-1 protects the Caenorhabditis elegans germline from the adverse effects of heat shock. Animals containing strong loss-of-function mutations in tiar-1 exhibit significantly reduced fertility compared to the wild type following heat shock. Analysis of a heat-shock protein promoter indicates that tiar-1 mutants display an impaired heat-shock response. We observed that TIAR-1 was associated with granules in the gonad core and oocytes during several stressful conditions. Both gonad core and oocyte granules are dynamic structures that depend on translation; protein synthesis inhibitors altered their formation. Nonetheless, tiar-1 was required for the formation of gonad core granules only. Interestingly, the gonad core granules did not seem to be needed for the germ cells to develop viable embryos after heat shock. This suggests that TIAR-1 is able to protect the germline from heat stress independently of these structures. PMID:26865701

  13. 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

  14. Diminished Schwann cell repair responses underlie age-associated impaired axonal regeneration

    PubMed Central

    Painter, Michio W.; Brosius Lutz, Amanda; Cheng, Yung-Chih; Latremoliere, Alban; Duong, Kelly; Miller, Christine M.; Posada, Sean; Cobos, Enrique J.; Zhang, Alice X.; Wagers, Amy J.; Havton, Leif A.; Barres, Ben; Omura, Takao

    2014-01-01

    SUMMARY The regenerative capacity of the peripheral nervous system declines with age. Why this occurs, however, is unknown. We demonstrate that 24-month old mice exhibit an impairment of functional recovery after nerve injury compared to 2-month old animals. We find no difference in the intrinsic growth capacity between aged and young sensory neurons in vitro nor in their ability to activate growth-associated transcriptional programs after injury. Instead, using age-mismatched nerve transplants in vivo, we show that the extent of functional recovery depends on the age of the nerve graft, and not the age of the host. Molecular interrogation of the sciatic nerve reveals that aged Schwann cells (SCs) fail to rapidly activate a transcriptional repair program after injury. Functionally, aged SCs exhibit impaired de-differentiation, myelin clearance and macrophage recruitment. These results suggest that the age-associated decline in axonal regeneration results from diminished Schwann cell plasticity, leading to slower myelin clearance. PMID:25033179

  15. Diminished Schwann cell repair responses underlie age-associated impaired axonal regeneration.

    PubMed

    Painter, Michio W; Brosius Lutz, Amanda; Cheng, Yung-Chih; Latremoliere, Alban; Duong, Kelly; Miller, Christine M; Posada, Sean; Cobos, Enrique J; Zhang, Alice X; Wagers, Amy J; Havton, Leif A; Barres, Ben; Omura, Takao; Woolf, Clifford J

    2014-07-16

    The regenerative capacity of the peripheral nervous system declines with age. Why this occurs, however, is unknown. We demonstrate that 24-month-old mice exhibit an impairment of functional recovery after nerve injury compared to 2-month-old animals. We find no difference in the intrinsic growth capacity between aged and young sensory neurons in vitro or in their ability to activate growth-associated transcriptional programs after injury. Instead, using age-mismatched nerve transplants in vivo, we show that the extent of functional recovery depends on the age of the nerve graft, and not the age of the host. Molecular interrogation of the sciatic nerve reveals that aged Schwann cells (SCs) fail to rapidly activate a transcriptional repair program after injury. Functionally, aged SCs exhibit impaired dedifferentiation, myelin clearance, and macrophage recruitment. These results suggest that the age-associated decline in axonal regeneration results from diminished Schwann cell plasticity, leading to slower myelin clearance. PMID:25033179

  16. Mitochondrial alarmins released by degenerating motor axon terminals activate perisynaptic Schwann cells

    PubMed Central

    Duregotti, Elisa; Negro, Samuele; Scorzeto, Michele; Zornetta, Irene; Dickinson, Bryan C.; Chang, Christopher J.; Montecucco, Cesare; Rigoni, Michela

    2015-01-01

    An acute and highly reproducible motor axon terminal degeneration followed by complete regeneration is induced by some animal presynaptic neurotoxins, representing an appropriate and controlled system to dissect the molecular mechanisms underlying degeneration and regeneration of peripheral nerve terminals. We have previously shown that nerve terminals exposed to spider or snake presynaptic neurotoxins degenerate as a result of calcium overload and mitochondrial failure. Here we show that toxin-treated primary neurons release signaling molecules derived from mitochondria: hydrogen peroxide, mitochondrial DNA, and cytochrome c. These molecules activate isolated primary Schwann cells, Schwann cells cocultured with neurons and at neuromuscular junction in vivo through the MAPK pathway. We propose that this inter- and intracellular signaling is involved in triggering the regeneration of peripheral nerve terminals affected by other forms of neurodegenerative diseases. PMID:25605902

  17. Regrowth of transected retinal ganglion cell axons despite persistent astrogliosis in the lizard (Gallotia galloti)

    PubMed Central

    del Mar Romero-Alemán, María; Monzón-Mayor, Maximina; Santos, Elena; Yanes, Carmen M

    2013-01-01

    We analysed the astroglia response that is concurrent with spontaneous axonal regrowth after optic nerve (ON) transection in the lizard Gallotia galloti. At different post-lesional time points (0.5, 1, 3, 6, 9 and 12 months) we used conventional electron microscopy and specific markers for astrocytes [glial fibrillary acidic protein (GFAP), vimentin (Vim), sex-determining region Y-box-9 (Sox9), paired box-2 (Pax2)¸ cluster differentiation-44 (CD44)] and for proliferating cells (PCNA). The experimental retina showed a limited glial response since the increase of gliofilaments was not significant when compared with controls, and proliferating cells were undetectable. Conversely, PCNA+ cells populated the regenerating ON, optic tract (OTr) and ventricular wall of both the hypothalamus and optic tectum (OT). Subpopulations of these PCNA+ cells were identified as GFAP+ and Vim+ reactive astrocytes and radial glia. Reactive astrocytes up-regulated Vim at 1 month post-lesion, and both Vim and GFAP at 12 months post-lesion in the ON-OTr, indicating long-term astrogliosis. They also expressed Pax2, Sox9 and CD44 in the ON, and Sox9 in the OTr. Concomitantly, persistent tissue cavities and disorganised regrowing fibre bundles reaching the OT were observed. Our ultrastructural data confirm abundant gliofilaments in reactive astrocytes joined by desmosomes. Remarkably, they also accumulated myelin debris and lipid droplets until late stages, indicating their participation in myelin removal. These data suggest that persistent mammalian-like astrogliosis in the adult lizard ON contributes to a permissive structural scaffold for long-term axonal regeneration and provides a useful model to study the molecular mechanisms involved in these beneficial neuron–glia interactions. PMID:23656528

  18. Morphology and connections of intratrigeminal cells and axons in the macaque monkey

    PubMed Central

    Warren, Susan; May, Paul J.

    2013-01-01

    Trigeminal primary afferent fibers have small receptive fields and discrete submodalities, but second order trigeminal neurons often display larger receptive fields with complex, multimodal responses. Moreover, while most large caliber afferents terminate exclusively in the principal trigeminal nucleus, and pars caudalis (sVc) of the spinal trigeminal nucleus receives almost exclusively small caliber afferents, the characteristics of second order neurons do not always reflect this dichotomy. These surprising characteristics may be due to a network of intratrigeminal connections modifying primary afferent contributions. This study characterizes the distribution and morphology of intratrigeminal cells and axons in a macaque monkeys. Tracer injections centered in the principal nucleus (pV) and adjacent pars oralis retrogradely labeled neurons bilaterally in pars interpolaris (sVi), but only ipsilaterally, in sVc. Labeled axons terminated contralaterally within sVi and caudalis. Features of the intratrigeminal cells in ipsilateral sVc suggest that both nociceptive and non-nociceptive neurons project to principalis. A commissural projection to contralateral principalis was also revealed. Injections into sVc labeled cells and terminals in pV and pars oralis on both sides, indicating the presence of bilateral reciprocal connections. Labeled terminals and cells were also present bilaterally in sVi and in contralateral sVc. Interpolaris injections produced labeling patterns similar to those of sVc. Thus, the rostral and caudal poles of the macaque trigeminal complex are richly interconnected by ipsilateral ascending and descending connections providing an anatomical substrate for complex analysis of oro-facial stimuli. Sparser reciprocal crossed intratrigeminal connections may be important for conjugate reflex movements, such as the corneal blink reflex. PMID:23754988

  19. Genetically induced adult oligodendrocyte cell death is associated with poor myelin clearance, reduced remyelination, and axonal damage.

    PubMed

    Pohl, Hartmut B F; Porcheri, Cristina; Mueggler, Thomas; Bachmann, Lukas C; Martino, Gianvito; Riethmacher, Dieter; Franklin, Robin J M; Rudin, Markus; Suter, Ueli

    2011-01-19

    Loss of oligodendrocytes is a feature of many demyelinating diseases including multiple sclerosis. Here, we have established and characterized a novel model of genetically induced adult oligodendrocyte death. Specific primary loss of adult oligodendrocytes leads to a well defined and highly reproducible course of disease development that can be followed longitudinally by magnetic resonance imaging. Histological and ultrastructural analyses revealed progressive myelin vacuolation, in parallel to disease development that includes motor deficits, tremor, and ataxia. Myelin damage and clearance were associated with induction of oligodendrocyte precursor cell proliferation, albeit with some regional differences. Remyelination was present in the mildly affected corpus callosum. Consequences of acutely induced cell death of adult oligodendrocytes included secondary axonal damage. Microglia were activated in affected areas but without significant influx of B-cells, T-helper cells, or T-cytotoxic cells. Analysis of the model on a RAG-1 (recombination activating gene-1)-deficient background, lacking functional lymphocytes, did not change the observed disease and pathology compared with immune-competent mice. We conclude that this model provides the opportunity to study the consequences of adult oligodendrocyte death in the absence of primary axonal injury and reactive cells of the adaptive immune system. Our results indicate that if the blood-brain barrier is not disrupted, myelin debris is not removed efficiently, remyelination is impaired, and axonal integrity is compromised, likely as the result of myelin detachment. This model will allow the evaluation of strategies aimed at improving remyelination to foster axon protection. PMID:21248132

  20. Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve

    PubMed Central

    Vajda, F; Jordi, N; Dalkara, D; Joly, S; Christ, F; Tews, B; Schwab, M E; Pernet, V

    2015-01-01

    Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre+/−xRtn4/Nogo-Aflox/flox) and neuron-specific (Thy1-Cretg+xRtn4flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS. PMID:25257170

  1. Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve.

    PubMed

    Vajda, F; Jordi, N; Dalkara, D; Joly, S; Christ, F; Tews, B; Schwab, M E; Pernet, V

    2015-02-01

    Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4(flox/flox) animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS. PMID:25257170

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

    PubMed Central

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

    2014-01-01

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

  3. BACE2 is stored in secretory granules of mouse and rat pancreatic beta cells.

    PubMed

    Finzi, Giovanna; Franzi, Francesca; Placidi, Claudia; Acquati, Francesco; Palumbo, Elisa; Russo, Antonella; Taramelli, Roberto; Sessa, Fausto; La Rosa, Stefano

    2008-01-01

    BACE2 is a protease homologous to BACE1 protein, an enzyme involved in the amyloid formation of Alzheimer disease (AD). However, despite the high homology between these two proteins, the biological role of BACE2 is still controversial, even though a few studies have suggested a pathogenetic role in sporadic inclusion-body myositis and hereditary inclusion-body myopathy, which are characterized by vacuolization of muscular fibers with intracellular deposits of proteins similar to those found in the brain of AD patients. Although BACE2 has also been identified in the pancreas, its function remains unknown and its specific localization in different pancreatic cell types has not been definitively ascertained. For these reasons, the authors have investigated the cellular and subcellular localization of BACE2 in normal rodent pancreases. BACE2 immunoreactivity was found in secretory granules of beta cells, co-stored with insulin and IAPP, while it was lacking in the other endocrine and exocrine cell types. The presence of BACE2 in secretory granules of beta cells suggests that it may play a role in diabetes-associated amyloidogenesis. PMID:19117266

  4. 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.

  5. Entorhinal Denervation Induces Homeostatic Synaptic Scaling of Excitatory Postsynapses of Dentate Granule Cells in Mouse Organotypic Slice Cultures

    PubMed Central

    Vlachos, Andreas; Becker, Denise; Jedlicka, Peter; Winkels, Raphael; Roeper, Jochen; Deller, Thomas

    2012-01-01

    Denervation-induced changes in excitatory synaptic strength were studied following entorhinal deafferentation of hippocampal granule cells in mature (≥3 weeks old) mouse organotypic entorhino-hippocampal slice cultures. Whole-cell patch-clamp recordings revealed an increase in excitatory synaptic strength in response to denervation during the first week after denervation. By the end of the second week synaptic strength had returned to baseline. Because these adaptations occurred in response to the loss of excitatory afferents, they appeared to be in line with a homeostatic adjustment of excitatory synaptic strength. To test whether denervation-induced changes in synaptic strength exploit similar mechanisms as homeostatic synaptic scaling following pharmacological activity blockade, we treated denervated cultures at 2 days post lesion for 2 days with tetrodotoxin. In these cultures, the effects of denervation and activity blockade were not additive, suggesting that similar mechanisms are involved. Finally, we investigated whether entorhinal denervation, which removes afferents from the distal dendrites of granule cells while leaving the associational afferents to the proximal dendrites of granule cells intact, results in a global or a local up-scaling of granule cell synapses. By using computational modeling and local electrical stimulations in Strontium (Sr2+)-containing bath solution, we found evidence for a lamina-specific increase in excitatory synaptic strength in the denervated outer molecular layer at 3–4 days post lesion. Taken together, our data show that entorhinal denervation results in homeostatic functional changes of excitatory postsynapses of denervated dentate granule cells in vitro. PMID:22403720

  6. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    PubMed

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D. PMID:26845357

  7. Large nerve cells with long axons in the granular layer and white matter of the murine cerebellum.

    PubMed Central

    Müller, T

    1994-01-01

    The murine cerebellum was investigated by light microscopy using an improved modification of Ehrlich's methylene blue supravital staining technique. The dye exhibited a special affinity for the perikarya as well as the axons of Purkinje cells. In addition, large fusiform or stellate nerve cells which were characterised by long descending axons were seen to be distributed diffusely within the granular layer and the subcortical white matter. These findings indicate the existence of a 2nd type of projection neuron besides the Purkinje cells and are therefore in full accordance with older neuroanatomical observations based on silver impregnation. When correlated with recent studies on the occurrence of different calcium-binding proteins, the results show that the large perikarya demonstrated immunohistochemically within the granular layer seem to belong to the group of methylene blue positive neurons. Nevertheless, the definitive association of a single neuron with a nerve cell class is only possible if the axon is stained and clearly identifiable. Because of its selectivity for a special type of nerve cell, including its axon, the histological method used in this study may therefore also be suitable for investigating other parts of the brain and the spinal cord. Images Fig. 1 Fig. 2 PMID:7516932

  8. 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.

  9. [Microspectrofluorometric analysis of the effect of centrophenoxine on lipofuscin granules of hybridoma (Retrovirus-transformed) cells].

    PubMed

    Tatariunas, A B

    1990-04-01

    The increase of the own luminescence lipofuscin granules (LG) in the course of single and repeated ultraviolet (UV) excitations (365 nm) in hybridoma (retrovirus transformed) cells cultured with or without 5 x 10(-4) M centrophenoxine (CP) was studied by microspectrofluorometric method. It was shown that CP influences only the rate of photochemical changes of chromophores in LG. Kinetic patterns of the own luminescence intensity of LG at the wavelength of 540 nm during excitation by UV permit one to suggest the occurrence of the cyclic chromophore changes. PMID:2117472

  10. Metazoan Scc4 Homologs Link Sister Chromatid Cohesion to Cell and Axon Migration Guidance

    PubMed Central

    Seitan, Vlad C; Banks, Peter; Laval, Steve; Majid, Nazia A; Dorsett, Dale; Rana, Amer; Smith, Jim; Bateman, Alex; Krpic, Sanja; Hostert, Arnd; Rollins, Robert A; Erdjument-Bromage, Hediye; Tempst, Paul; Benard, Claire Y; Hekimi, Siegfried; Newbury, Sarah F

    2006-01-01

    Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development. PMID:16802858

  11. Short-axon cells in the olfactory bulb: dendrodendritic synaptic interactions.

    PubMed Central

    Getchell, T V; Shepherd, G M

    1975-01-01

    1. In the rabbit olfactory bulb, analysis has been carried out of extracellular unitary responses in the glomerular layer to olfactory nerve volleys. 2. Units in the glomerular layer responded to single volleys with single, double, triple or longer repetitive spike discharges. The shortest initial latencies are consistent with monosynaptic excitation from the olfactory nerves; longer latencies may reflect longer nerve pathways or polysynaptic connexions in the glomerular layer. 3. Like mitral and tufted cells, some glomerular layer units gave evidence of activation by discrete nerve bundles. This correlates with recent anatomical evidence for projections of discrete olfactory nerve bundles to the glomeruli. 4. Facilitation of glomerular layer units took the form of lower spike thresholds and shorter latencies, when testing with paired olfactory nerve volleys of weak strength at relatively short intervals (less than 40 msec). Supression took the form of raised thresholds, longer latencies and briefer repetitive discharges; this was particularly evident with strong volleys at long testing intervals. 5. The early period of facilitation and later period of suppression did not correlate with the recovery cycle of the olfactory nerves; the nerves had an absolute refractory period of approximately 3 msec, relative refractory period of 15-30 msec, and a small supernormal period of several hundred msec or more. 6. The evidence that the facilitation and suppression are mediated by dendrodendritic pathways through the periglomerular short-axon cells is discussed in relation to recent electronmicroscopical studies. The results have implications for similar pathways through short-axon cell dendrites in other parts of the nervous system. PMID:1185673

  12. Metazoan Scc4 homologs link sister chromatid cohesion to cell and axon migration guidance.

    PubMed

    Seitan, Vlad C; Banks, Peter; Laval, Steve; Majid, Nazia A; Dorsett, Dale; Rana, Amer; Smith, Jim; Bateman, Alex; Krpic, Sanja; Hostert, Arnd; Rollins, Robert A; Erdjument-Bromage, Hediye; Tempst, Paul; Benard, Claire Y; Hekimi, Siegfried; Newbury, Sarah F; Strachan, Tom

    2006-07-01

    Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein-protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development. PMID:16802858

  13. In vivo imaging of cell behaviors and F-actin reveals LIM-HD transcription factor regulation of peripheral versus central sensory axon development

    PubMed Central

    2011-01-01

    Background Development of specific neuronal morphology requires precise control over cell motility processes, including axon formation, outgrowth and branching. Dynamic remodeling of the filamentous actin (F-actin) cytoskeleton is critical for these processes; however, little is known about the mechanisms controlling motile axon behaviors and F-actin dynamics in vivo. Neuronal structure is specified in part by intrinsic transcription factor activity, yet the molecular and cellular steps between transcription and axon behavior are not well understood. Zebrafish Rohon-Beard (RB) sensory neurons have a unique morphology, with central axons that extend in the spinal cord and a peripheral axon that innervates the skin. LIM homeodomain (LIM-HD) transcription factor activity is required for formation of peripheral RB axons. To understand how neuronal morphogenesis is controlled in vivo and how LIM-HD transcription factor activity differentially regulates peripheral versus central axons, we used live imaging of axon behavior and F-actin distribution in vivo. Results We used an F-actin biosensor containing the actin-binding domain of utrophin to characterize actin rearrangements during specific developmental processes in vivo, including axon initiation, consolidation and branching. We found that peripheral axons initiate from a specific cellular compartment and that F-actin accumulation and protrusive activity precede peripheral axon initiation. Moreover, disruption of LIM-HD transcriptional activity has different effects on the motility of peripheral versus central axons; it inhibits peripheral axon initiation, growth and branching, while increasing the growth rate of central axons. Our imaging revealed that LIM-HD transcription factor activity is not required for F-actin based protrusive activity or F-actin accumulation during peripheral axon initiation, but can affect positioning of F-actin accumulation and axon formation. Conclusion Our ability to image the dynamics of

  14. Myeloperoxidase and Crystalline Bodies in the Granules of DMBA-Induced Rat Chloroma Cells

    PubMed Central

    Ioachim, Harry L.; Keller, Steven; Sabbath, Marlene; Andersson, Barbro; Dorsett, Brent; Essner, Edward

    1972-01-01

    Chloroma (chloroleukemia) was induced in a splenectomized rat by repeatedly administering dimethylbenz(a)anthracene (DMBA) and was serially transplanted thereafter. Composed of immature myeloid cells, the tumor imparted a green discoloration to the tissues that it infiltrated extensively. Chloroma cells fluoresced red in ultraviolet light, produced a characteristic curve in spectrophotometry, and contained large amounts of myeloperoxidase. They included numerous intracytoplasmic granules of both types A and B, which contained occasional crystalline bars. Permanent lines of chloroma cells were established in tissue culture. These cells, while maintaining their initial morphology, ceased producing myeloperoxidase and subsequently induced white tumors when they were isotransplanted. ImagesFig 12Fig 13Fig 4Fig 5Fig 6Fig 7Fig 8Fig 9Fig 10Fig 11Fig 1Fig 2Fig 3 PMID:4333120

  15. TIA1 oxidation inhibits stress granule assembly and sensitizes cells to stress-induced apoptosis

    PubMed Central

    Arimoto-Matsuzaki, Kyoko; Saito, Haruo; Takekawa, Mutsuhiro

    2016-01-01

    Cytoplasmic stress granules (SGs) are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of misfolded proteins, and that are formed in response to certain types of stress including ER stress. SG formation contributes to cell survival not only by suppressing translation but also by sequestering some apoptosis regulatory factors. Because cells can be exposed to various stresses simultaneously in vivo, the regulation of SG assembly under multiple stress conditions is important but unknown. Here we report that reactive oxygen species (ROS) such as H2O2 oxidize the SG-nucleating protein TIA1, thereby inhibiting SG assembly. Thus, when cells are confronted with a SG-inducing stress such as ER stress caused by protein misfolding, together with ROS-induced oxidative stress, they cannot form SGs, resulting in the promotion of apoptosis. We demonstrate that the suppression of SG formation by oxidative stress may underlie the neuronal cell death seen in neurodegenerative diseases. PMID:26738979

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

    PubMed

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

    1994-10-01

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

  17. Different subsets of newborn granule cells: a possible role in epileptogenesis?

    PubMed

    Bielefeld, Pascal; van Vliet, Erwin A; Gorter, Jan A; Lucassen, Paul J; Fitzsimons, Carlos P

    2014-01-01

    Several factors, including epileptic seizures, can strongly stimulate ongoing neurogenesis in the adult hippocampus. Although adult-born granule cells generated after seizure activity have different physiological properties from their normal counterparts, they integrate into the existing, mature network of the adult hippocampal dentate gyrus. However, the exact role of the neurogenic response during epilepsy and its possible involvement in epileptogenesis have remained elusive. Here, we discuss recent studies shedding new light on the interplay between epilepsy and neurogenesis, and try to explain discrepancies in this literature by proposing seizure severity-dependent induction of two subsets of newborn cells with different properties. We hypothesise that a low seizure intensity would stimulate neurogenesis to a 'physiological plasticity' level and have few pathological consequences. In contrast, a high initial seizure intensity may induce a specific subset of altered and/or ectopically located new granule cells with different electrophysiological properties that could initiate hyperexcitatory recurrent networks that could, in turn, contribute to chronic epilepsy. This hypothesis may clarify previously contradictory data in the literature, and could thereby aid in our understanding of the role of neurogenesis in epileptogenesis, and open up promising avenues for therapeutic intervention. PMID:24387591

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

    PubMed Central

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

    2014-01-01

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

  19. Calcium buffering in rodent olfactory bulb granule cells and mitral cells.

    PubMed

    Egger, Veronica; Stroh, Olga

    2009-09-15

    In the mammalian olfactory bulb, axonless granule cells (GCs) mediate self- and lateral inhibitory interactions between mitral cells (MCs) via reciprocal dendrodendritic synapses. Calcium signals in the GC dendrites and reciprocal spines appear to decay unusually slowly, hence GC calcium handling might contribute to the known asynchronous release at this synapse. By recording fluorescence transients of different Ca(2+)-sensitive dyes at variable concentrations evoked by backpropagating action potentials (APs) and saturating AP trains we extrapolated Ca(2+) dynamics to conditions of zero added buffer for juvenile rat GC apical dendrites and spines and MC lateral dendrites. Resting [Ca(2+)] was at approximately 50 nM in both GC dendrites and spines. The average endogenous GC buffer capacities (kappa(E)) were within a range of 80-90 in the dendrites and 110-140 in the spines. The extrusion rate (gamma) was estimated as 570 s(-1) for dendrites and 870 s(-1) for spines and the decay time constant as approximately 200 ms for both. Single-current-evoked APs resulted in a [Ca(2+)] elevation of approximately 250 nM. Calcium handling in juvenile and adult mouse GCs appeared mostly similar. In MC lateral dendrites, we found AP-mediated [Ca(2+)] elevations of approximately 130 nM with a similar decay to that in GC dendrites, while kappa(E) and gamma were roughly 4-fold higher. In conclusion, the slow GC Ca(2+) dynamics are due mostly to sluggish Ca(2+) extrusion. Under physiological conditions this slow removal may well contribute to delayed release and also feed into other Ca(2+)-dependent mechanisms that foster asynchronous output from the reciprocal spine. PMID:19635818

  20. PLR-1, a putative E3 ubiquitin ligase, controls cell polarity and axonal extensions in C. elegans.

    PubMed

    Bhat, Jaffar M; Pan, Jie; Hutter, Harald

    2015-02-01

    During embryonic development neurons differentiate and extend axons and dendrites that have to reach their appropriate targets. In Caenorhabditis elegans the AVG neuron is the first neuron to extend an axon during the establishment of the ventral nerve cord, the major longitudinal axon tract in the animal. In genetic screens we isolated alleles of plr-1, which caused polarity reversals of the AVG neuron as well as outgrowth and navigation defects of the AVG axon. In addition plr-1 mutants show outgrowth defects in several other classes of neurons as well as the posterior excretory canals. plr-1 is predicted to encode a transmembrane E3 ubiquitin ligase and is widely expressed in the animal including the AVG neuron and the excretory cell. plr-1 has recently been shown to negatively regulate Wnt signalling by removing Wnt receptors from the cell surface. We observed that mutations in a gene reducing Wnt signalling as well as mutations in unc-53/NAV2 and unc-73/Trio suppress the AVG polarity defects in plr-1 mutants, but not the defects seen in other cells. This places plr-1 in a Wnt regulation pathway, but also suggests that plr-1 has Wnt independent functions and interacts with unc-53 and unc-73 to control cell polarity. PMID:25448694

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

    PubMed Central

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

    2008-01-01

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

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

    PubMed Central

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

    1995-01-01

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

  3. The perinuclear factor, a rheumatoid arthritis-specific autoantigen, is not present in keratohyalin granules of cultured buccal mucosa cells.

    PubMed Central

    Hoet, R M; Voorsmit, R A; Van Venrooij, W J

    1991-01-01

    Rheumatoid arthritis patients have antibodies in their serum directed against the perinuclear factor, a protein component present in keratohyalin granules in the cytoplasm of human buccal mucosa cells. The anti-perinuclear factor (APF) can only be detected by an indirect immunofluorescence test performed on fresh buccal mucosa cells from 'selected donors'. To obtain a more reliable antigen source and to gain more insight into the origin and nature of the perinuclear factor we attempted to culture perinuclear factor-containing buccal mucosa cells. Here we describe the successful culturing of such cells, which, however, did not contain keratohyalin granules nor the perinuclear factor. By adding the phorbol ester 12-o-tetradecanoylphorbol-13-acetate (TPA) we were able to induce keratohyalin granules in both cultured primary buccal mucosa cells and a squamous carcinoma cell line of the cheek (SqCC/Y1). These induced keratohyalin granules do contain the protein profilaggrin, which in vivo, in fresh buccal mucosa cells, co-localizes with the perinuclear factor. However, we were not able to demonstrate the presence of the perinuclear factor, not even after induction of terminal differentiation of the cultured cells nor after Epstein-Barr virus infection. Our results suggest that the perinuclear factor, in contrast to profilaggrin, is not an integral component of buccal mucosa cells. Images Fig. 1 PMID:1849807

  4. Interplay of cell-autonomous and non-autonomous mechanisms tailors synaptic connectivity of converging axons in vivo

    PubMed Central

    Okawa, Haruhisa; Santina, Luca Della; Schwartz, Gregory W.; Rieke, Fred; Wong, Rachel O. L.

    2014-01-01

    Summary Neurons receive input from diverse afferents but form stereotypic connections with axons of each type to execute their precise functions. Developmental mechanisms that specify the connectivity of individual axons across populations of converging afferents are not well-understood. Here, we untangled the contributions of activity-dependent and independent interactions that regulate connections of two input types providing major and minor input onto a neuron. Individual transmission-deficient retinal bipolar cells (BCs) reduced synapses with retinal ganglion cells (RGCs), but active BCs of the same type sharing the dendrite surprisingly did not compensate for this loss. Genetic ablation of some BC neighbors resulted in increased synaptogenesis by the remaining axons in a transmission-independent manner. Presence but not transmission of the major BC input also dissuades wiring with the minor input, and with synaptically-compatible but functionally-mismatched afferents. Cell-autonomous, activity-dependent and non-autonomous, activity-independent mechanisms thus together tailor connections of individual axons amongst converging inner retinal afferents. PMID:24698272

  5. Early profiles of axonal growth and astroglial response after spinal cord hemisection and implantation of Schwann cell-seeded guidance channels in adult rats.

    PubMed

    Hsu, Jung-Yu C; Xu, Xiao-Ming

    2005-11-15

    We previously demonstrated that transplantation of Schwann cell-seeded channels promoted the regrowth of injured axons in the adult spinal cord. It is not clear, however, whether injured axons recapitulate the developmental scenarios to accomplish regeneration. In the present study, we investigated the early events associated with axonal regrowth after spinal cord hemisection at the eighth thoracic level and implantation of a Schwann cell-seeded minichannel in adult rats. Animals were sacrificed at postoperative days (PO) 2, 4, 7, and 14. Anterograde tracing with fluoro-ruby showed that regenerating axons grew into the graft prior to PO2 and reached the distal end of the channel at PO7. These axons expressed both embryonic neural cell adhesion molecule (E-NCAM) and growth associated protein-43 (GAP-43). Although the expression of E-NCAM decreased by PO7, that of GAP-43 remained high throughout the first 2 weeks after implantation. A close relation of vimentin-positive astroglia to the growing axons in the host tissue suggested a contact-mediated role of these cells in axon guidance. Aggregation of glial fibrillary acidic protein (GFAP)-positive astrocytes together with the increased expression of chondroitin sulfate proteoglycans (CSPGs) starting at PO7 appeared to inhibit axonal growth at the host-graft interface. Thus, adult regenerating axons and astroglia do express developmentally related molecules that may facilitate axonal growth into a permissive graft at the early phase of injury and regeneration. These results suggest that molecules and astroglia essential to development are both important in influencing axonal regrowth in the adult spinal cord. PMID:16240391

  6. 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.

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

    PubMed

    Meier, E; Drejer, J; Schousboe, A

    1984-12-01

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

  8. Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush

    PubMed Central

    Sharma, T P; Liu, Y; Wordinger, R J; Pang, I-H; Clark, A F

    2015-01-01

    Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6–7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG–hNRN1 prior to ONC promoted RGC survival (450%, n=3–7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG–green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5–8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5–6, P<0.05) expression was observed within the optic nerves of the AAV2–hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases. PMID:25719245

  9. Exosomes mediate cell contact-independent ephrin-Eph signaling during axon guidance.

    PubMed

    Gong, Jingyi; Körner, Roman; Gaitanos, Louise; Klein, Rüdiger

    2016-07-01

    The cellular release of membranous vesicles known as extracellular vesicles (EVs) or exosomes represents a novel mode of intercellular communication. Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands have very important roles in such biologically diverse processes as neuronal development, plasticity, and pathological diseases. Until now, it was thought that ephrin-Eph signaling requires direct cell contact. Although the biological functions of ephrin-Eph signaling are well understood, our mechanistic understanding remains modest. Here we report the release of EVs containing Ephs and ephrins by different cell types, a process requiring endosomal sorting complex required for transport (ESCRT) activity and regulated by neuronal activity. Treatment of cells with purified EphB2(+) EVs induces ephrinB1 reverse signaling and causes neuronal axon repulsion. These results indicate a novel mechanism of ephrin-Eph signaling independent of direct cell contact and proteolytic cleavage and suggest the participation of EphB2(+) EVs in neural development and synapse physiology. PMID:27354374

  10. Telocytes as a Source of Progenitor Cells in Regeneration and Repair Through Granulation Tissue.

    PubMed

    Díaz-Flores, Lucio; Gutiérrez, Ricardo; Pino García, Maria; González, Miriam; Díaz-Flores, Lucio; Francisco Madrid, Juan

    2016-01-01

    This review outlines the role of CD34+ stromal cells/telocytes (CD34+ SC/TCs) in repair and considers the following issues. Firstly, the conceptual aspects of repair, including regeneration and repair through granulation tissue (RTGT) as two types of repair, RTGT stages (inflammatory, proliferative, and remodeling), and tissue in repair as a substrate to assess the in vivo behavior of activated CD34+ SC/TCs. Subsequently, current knowledge of CD34+ SC/TCs, such as identification, characteristics, and functions, as well as possible stages (quiescent and activated) are taken into account. We then consider the role in regeneration of quiescent CD34+ SC/TCs (in unperturbed physiological conditions) as a nurse of stem cells (e.g., in the heart, skin, respiratory tree, gastrointestinal tract, liver, eye, and choroid plexus). Special attention is paid to the characteristics of activated CD34+ SC/TCs and the overlapping steps of activation with and without loss of CD34 expression and with and without gain of αSMA expression. With this contribution, we establish the role of CD34+ SC/TCs as progenitor cells and as a source of fibroblasts and myofibroblasts in repair through granulation tissue, fibrosis, and tumor stroma. Activated CD34+ SC/TCs in encapsulation and other processes (e.g., Reinke's edema, cutaneous myxoid cyst, mixomatous mitral valve degeneration, and fibrous papula of the face) are also outlined. Finally, similarities between modifications of CD34+ SC/TCs during in vivo activation and of multipotent mesenchymal stromal/stem cells in culture are examined in order to correlate the growing literature on CD34+ SC/TCs and the exponential research in cultured mesenchymal stromal/stem cells. PMID:26423297