All Title Author
Keywords Abstract

Lesion-Induced Alterations in Astrocyte Glutamate Transporter Expression and Function in the Hippocampus

DOI: 10.1155/2013/893605

Full-Text   Cite this paper   Add to My Lib


Astrocytes express the sodium-dependent glutamate transporters GLAST and GLT-1, which are critical to maintain low extracellular glutamate concentrations. Here, we analyzed changes in their expression and function following a mechanical lesion in the CA1 area of organotypic hippocampal slices. 6-7 days after lesion, a glial scar had formed along the injury site, containing strongly activated astrocytes with increased GFAP and S100β immunoreactivity, enlarged somata, and reduced capability for uptake of SR101. Astrocytes in the scar’s periphery were swollen as well, but showed only moderate upregulation of GFAP and S100β and efficiently took up SR101. In the scar, clusters of GLT-1 and GLAST immunoreactivity colocalized with GFAP-positive fibers. Apart from these, GLT-1 immunoreactivity declined with increasing distance from the scar, whereas GLAST expression appeared largely uniform. Sodium imaging in reactive astrocytes indicated that glutamate uptake was strongly reduced in the scar but maintained in the periphery. Our results thus show that moderately reactive astrocytes in the lesion periphery maintain overall glutamate transporter expression and function. Strongly reactive astrocytes in the scar, however, display clusters of GLAST and GLT-1 immunoreactivity together with reduced glutamate transport activity. This reduction might contribute to increased extracellular glutamate concentrations and promote excitotoxic cell damage at the lesion site. 1. Introduction Glutamate reuptake represents the principal mechanism for inactivation of synaptically released glutamate [1, 2]. In the rodent hippocampus, it is mainly accomplished by astrocytic glutamate transporters (EAATs: excitatory amino acid transporters), namely, GLAST (glutamate/aspartate transporter) and GLT-1 (glutamate-transporter-1; rodent analogues of EAAT1 and EAAT2, resp.; [3–7]). Glutamate uptake is energized by the concomitant inward transport of three sodium ions and a proton, while one potassium ion is transported outward. Consequently, its activation is accompanied by an increase in the intracellular sodium concentration of astrocytes [8, 9]. Under pathological conditions, astrocytes undergo a complex reaction referred to as reactive astrogliosis, which is seen in diverse preparations and conditions ranging from primary cell culture to the intact brain [10, 11]. The hallmarks of reactive gliosis are a massive upregulation of the expression of the intermediate filament Glial Fibrillary Acidic Protein (GFAP) and a cellular hypertrophy [12, 13]. Reactive astrocytes display several


[1]  N. C. Danbolt, “Glutamate uptake,” Progress in Neurobiology, vol. 65, no. 1, pp. 1–105, 2001.
[2]  A. V. Tzingounis and J. I. Wadiche, “Glutamate transporters: confining runaway excitation by shaping synaptic transmission,” Nature Reviews Neuroscience, vol. 8, no. 12, pp. 935–947, 2007.
[3]  D. E. Bergles, J. S. Diamond, and C. E. Jahr, “Clearance of glutamate inside the synapse and beyond,” Current Opinion in Neurobiology, vol. 9, no. 3, pp. 293–298, 1999.
[4]  C. M. Anderson and R. A. Swanson, “Astrocyte glutamate transport: review of properties, regulation, and physiological functions,” Glia, vol. 32, no. 1, pp. 1–14, 2000.
[5]  P. Marcaggi and D. Attwell, “Role of glial amino acid transporters in synaptic transmission and brain energetics,” Glia, vol. 47, no. 3, pp. 217–225, 2004.
[6]  N. J. Maragakis and J. D. Rothstein, “Glutamate transporters: animal models to neurologic disease,” Neurobiology of Disease, vol. 15, no. 3, pp. 461–473, 2004.
[7]  G. Gegelashvili and A. Schousboe, “Cellular distribution and kinetic properties of high-affinity glutamate transporters,” Brain Research Bulletin, vol. 45, no. 3, pp. 233–238, 1998.
[8]  J. W. Deitmer and C. R. Rose, “Ion changes and signalling in perisynaptic glia,” Brain Research Reviews, vol. 63, no. 1-2, pp. 113–129, 2010.
[9]  S. Kirischuk, V. Parpura, and A. Verkhratsky, “Sodium dynamics: another key to astroglial excitability?” Trends in Neurosciences, vol. 35, no. 8, pp. 497–506, 2012.
[10]  M. V. Sofroniew, “Molecular dissection of reactive astrogliosis and glial scar formation,” Trends in Neurosciences, vol. 32, no. 12, pp. 638–647, 2009.
[11]  J. L. Ridet, S. K. Malhotra, A. Privat, and F. H. Gage, “Reactive astrocytes: cellular and molecular cues to biological function,” Trends in Neurosciences, vol. 20, no. 12, pp. 570–577, 1997.
[12]  J. Middeldorp and E. M. Hol, “GFAP in health and disease,” Progress in Neurobiology, vol. 93, no. 3, pp. 421–443, 2011.
[13]  M. Pekny and M. Nilsson, “Astrocyte activation and reactive gliosis,” Glia, vol. 50, no. 4, pp. 427–434, 2005.
[14]  A. Bordey, S. A. Lyons, J. J. Hablitz, and H. Sontheimer, “Electrophysiological characteristics of reactive astrocytes in experimental cortical dysplasia,” Journal of Neurophysiology, vol. 85, no. 4, pp. 1719–1731, 2001.
[15]  R. Jabs, G. Seifert, and C. Steinh?user, “Astrocytic function and its alteration in the epileptic brain,” Epilepsia, vol. 49, supplement 2, pp. 3–12, 2008.
[16]  A. Buffo, I. Rite, P. Tripathi et al., “Origin and progeny of reactive gliosis: a source of multipotent cells in the injured brain,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 9, pp. 3581–3586, 2008.
[17]  S. Robel, B. Berninger, and M. G?tz, “The stem cell potential of glia: lessons from reactive gliosis,” Nature Reviews Neuroscience, vol. 12, no. 2, pp. 88–104, 2011.
[18]  H. Kawano, J. Kimura-Kuroda, Y. Komuta et al., “Role of the lesion scar in the response to damage and repair of the central nervous system,” Cell and Tissue Research, vol. 349, no. 1, pp. 169–180, 2012.
[19]  G. Yiu and Z. He, “Glial inhibition of CNS axon regeneration,” Nature Reviews Neuroscience, vol. 7, no. 8, pp. 617–627, 2006.
[20]  M. T. Fitch and J. Silver, “CNS injury, glial scars, and inflammation: inhibitory extracellular matrices and regeneration failure,” Experimental Neurology, vol. 209, no. 2, pp. 294–301, 2008.
[21]  L. Li, A. Lundkvist, D. Andersson, et al., “Protective role of reactive astrocytes in brain ischemia,” Journal of Cerebral Blood Flow & Metabolism, vol. 28, no. 3, pp. 468–481, 2008.
[22]  F. K. H. van Landeghem, T. Weiss, M. Oehmichen, and A. Von Deimling, “Decreased expression of glutamate transporters in astrocytes after human traumatic brain injury,” Journal of Neurotrauma, vol. 23, no. 10, pp. 1518–1528, 2006.
[23]  V. L. Raghavendra Rao, M. K. Ba?kaya, A. Do?an, J. D. Rothstein, and R. J. Dempsey, “Traumatic brain injury down-regulates glial glutamate transporter (GLT- 1 and GLAST) proteins in rat brain,” Journal of Neurochemistry, vol. 70, no. 5, pp. 2020–2027, 1998.
[24]  J.-C. Chen, H. Hsu-Chou, J.-L. Lu et al., “Down-regulation of the glial glutamate transporter GLT-1 in rat hippocampus and striatum and its modulation by a group III metabotropic glutamate receptor antagonist following transient global forebrain ischemia,” Neuropharmacology, vol. 49, no. 5, pp. 703–714, 2005.
[25]  S. D. Ginsberg, L. J. Martin, and J. D. Rothstein, “Regional deafferentation down-regulates subtypes of glutamate transporter proteins,” Journal of Neurochemistry, vol. 65, no. 6, pp. 2800–2803, 1995.
[26]  J. E. Springer, R. D. Azbill, R. J. Mark, J. G. Begley, G. Waeg, and M. P. Mattson, “4-Hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake,” Journal of Neurochemistry, vol. 68, no. 6, pp. 2469–2476, 1997.
[27]  M. B. Moretto, N. S. Arteni, D. Lavinsky et al., “Hypoxic-ischemic insult decreases glutamate uptake by hippocampal slices from neonatal rats: Prevention by guanosine,” Experimental Neurology, vol. 195, no. 2, pp. 400–406, 2005.
[28]  L. Stoppini, P.-A. Buchs, and D. Muller, “A simple method for organotypic cultures of nervous tissue,” Journal of Neuroscience Methods, vol. 37, no. 2, pp. 173–182, 1991.
[29]  A. Nimmerjahn, F. Kirchhoff, J. N. D. Kerr, and F. Helmchen, “Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo,” Nat Methods, vol. 1, no. 1, pp. 31–37, 2004.
[30]  K. W. Kafitz, S. D. Meier, J. Stephan, and C. R. Rose, “Developmental profile and properties of sulforhodamine 101-Labeled glial cells in acute brain slices of rat hippocampus,” Journal of Neuroscience Methods, vol. 169, no. 1, pp. 84–92, 2008.
[31]  C. Schnell, Y. Hagos, and S. Hulsmann, “Active sulforhodamine 101 uptake into hippocampal astrocytes,” PLoS One, vol. 7, no. 11, Article ID e49398, 2012.
[32]  S. D. Meier, Y. Kovalchuk, and C. R. Rose, “Properties of the new fluorescent Na+ indicator CoroNa Green: comparison with SBFI and confocal Na+ imaging,” Journal of Neuroscience Methods, vol. 155, no. 2, pp. 251–259, 2006.
[33]  J. Langer and C. R. Rose, “Synaptically induced sodium signals in hippocampal astrocytes in situ,” Journal of Physiology, vol. 587, part 24, pp. 5859–5877, 2009.
[34]  J. Langer, J. Stephan, M. Theis, and C. R. Rose, “Gap junctions mediate intercellular spread of sodium between hippocampal astrocytes in situ,” Glia, vol. 60, no. 2, pp. 239–252, 2012.
[35]  C. R. Rose and B. R. Ransom, “Intracellular sodium homeostasis in rat hippocampal astrocytes,” Journal of Physiology, vol. 491, part 2, no. 2, pp. 291–305, 1996.
[36]  A. M. Benediktsson, G. S. Marrs, J. C. Tu et al., “Neuronal activity regulates glutamate transporter dynamics in developing astrocytes,” Glia, vol. 60, no. 2, pp. 175–188, 2012.
[37]  M. Zhou, G. P. Schools, and H. K. Kimelberg, “Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive,” Journal of Neurophysiology, vol. 95, no. 1, pp. 134–143, 2006.
[38]  I. Suárez, G. Bodega, and B. Fernández, “Modulation of glutamate transporters (GLAST, GLT-1 and EAAC1) in the rat cerebellum following portocaval anastomosis,” Brain Research, vol. 859, no. 2, pp. 293–302, 2000.
[39]  X. Zhu, D. E. Bergles, and A. Nishiyama, “NG2 cells generate both oligodendrocytes and gray matter astrocytes,” Development, vol. 135, no. 1, pp. 145–157, 2008.
[40]  B. Brunne, S. Zhao, A. Derouiche et al., “Origin, maturation, and astroglial transformation of secondary radial glial cells in the developing dentate gyrus,” Glia, vol. 58, no. 13, pp. 1553–1569, 2010.
[41]  Y. Wu, A.-Q. Zhang, and D. T. Yew, “Age related changes of various markers of astrocytes in senescence-accelerated mice hippocampus,” Neurochemistry International, vol. 46, no. 7, pp. 565–574, 2005.
[42]  S. Magavi, D. Friedmann, G. Banks, A. Stolfi, and C. Lois, “Coincident generation of pyramidal neurons and protoplasmic astrocytes in neocortical columns,” Journal of Neuroscience, vol. 32, no. 14, pp. 4762–4772, 2012.
[43]  M. Olabarria, H. N. Noristani, A. Verkhratsky, and J. J. Rodríguez, “Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer's disease,” Glia, vol. 58, no. 7, pp. 831–838, 2010.
[44]  A. M. Benediktsson, S. J. Schachtele, S. H. Green, and M. E. Dailey, “Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures,” Journal of Neuroscience Methods, vol. 141, no. 1, pp. 41–53, 2005.
[45]  I. Lushnikova, G. Skibo, D. Muller, and I. Nikonenko, “Synaptic potentiation induces increased glial coverage of excitatory synapses in CA1 hippocampus,” Hippocampus, vol. 19, no. 8, pp. 753–762, 2009.
[46]  E. Raponi, F. Agenes, C. Delphin et al., “S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage,” Glia, vol. 55, no. 2, pp. 165–177, 2007.
[47]  C. G. Schipke, C. Boucsein, C. Ohlemeyer, F. Kirchhoff, and H. Kettenmann, “Astrocyte Ca2+ waves trigger responses in microglial cells in brain slices,” The FASEB Journal, vol. 16, no. 2, pp. 255–257, 2002.
[48]  J. Zhou and M. L. Sutherland, “Glutamate transporter cluster formation in astrocytic processes regulates glutamate uptake activity,” Journal of Neuroscience, vol. 24, no. 28, pp. 6301–6306, 2004.
[49]  T. Nakagawa, Y. Otsubo, Y. Yatani, H. Shirakawa, and S. Kaneko, “Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial-neuronal cultures,” European Journal of Neuroscience, vol. 28, no. 9, pp. 1719–1730, 2008.
[50]  J.-Y. Chatton, P. Marquet, and P. J. Magistretti, “A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: Implications for cellular bioenergetics,” European Journal of Neuroscience, vol. 12, no. 11, pp. 3843–3853, 2000.
[51]  E. F?rster, S. Zhao, and M. Frotscher, “Laminating the hippocampus,” Nature Reviews Neuroscience, vol. 7, no. 4, pp. 259–267, 2006.
[52]  B. H. G?hwiler, M. Capogna, D. Debanne, R. A. McKinney, and S. M. Thompson, “Organotypic slice cultures: a technique has come of age,” Trends in Neurosciences, vol. 20, no. 10, pp. 471–477, 1997.
[53]  P. E. Kunkler and R. P. Kraig, “Reactive astrocytosis from excitotoxic injury in hippocampal organ culture parallels that seen in vivo,” Journal of Cerebral Blood Flow and Metabolism, vol. 17, no. 1, pp. 26–43, 1997.
[54]  I. E. Holopainen, “Organotypic hippocampal slice cultures: a model system to study basic cellular and molecular mechanisms of neuronal cell death, neuroprotection, and synaptic plasticity,” Neurochemical Research, vol. 30, no. 12, pp. 1521–1528, 2005.
[55]  M. Haber, L. Zhou, and K. K. Murai, “Cooperative astrocyte and dendritic spine dynamics at hippocampal excitatory synapses,” Journal of Neuroscience, vol. 26, no. 35, pp. 8881–8891, 2006.
[56]  H. Nishida and S. Okabe, “Direct astrocytic contacts regulate local maturation of dendritic spines,” Journal of Neuroscience, vol. 27, no. 2, pp. 331–340, 2007.
[57]  A. Buffo, C. Rolando, and S. Ceruti, “Astrocytes in the damaged brain: molecular and cellular insights into their reactive response and healing potential,” Biochemical Pharmacology, vol. 79, no. 2, pp. 77–89, 2010.
[58]  J.-H. Yi and A. S. Hazell, “Excitotoxic mechanisms and the role of astrocytic glutamate transporters in traumatic brain injury,” Neurochemistry International, vol. 48, no. 5, pp. 394–403, 2006.
[59]  C. Werner and K. Engelhard, “Pathophysiology of traumatic brain injury,” British Journal of Anaesthesia, vol. 99, no. 1, pp. 4–9, 2007.
[60]  G. Gegelashvili, M. B. Robinson, D. Trotti, and T. Rauen, “Regulation of glutamate transporters in health and disease,” Progress in Brain Research, vol. 132, pp. 267–286, 2001.
[61]  C. Escartin, E. Brouillet, P. Gubellini et al., “Ciliary neurotrophic factor activates astrocytes, redistributes their glutamate transporters GLAST and GLT-1 to raft microdomains, and improves glutamate handling in vivo,” Journal of Neuroscience, vol. 26, no. 22, pp. 5978–5989, 2006.
[62]  C. Vermeiren, M. Najimi, N. Vanhoutte et al., “Acute up-regulation of glutamate uptake mediated by mGluR5a in reactive astrocytes,” Journal of Neurochemistry, vol. 94, no. 2, pp. 405–416, 2005.
[63]  C. L. Poitry-Yamate, L. Vutskits, and T. Rauen, “Neuronal-induced and glutamate-dependent activation of glial glutamate transporter function,” Journal of Neurochemistry, vol. 82, no. 4, pp. 987–997, 2002.
[64]  P. M. Beart and R. D. O'Shea, “Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement,” British Journal of Pharmacology, vol. 150, no. 1, pp. 5–17, 2007.
[65]  F. D. Lima, M. A. Souza, A. F. Furian et al., “Na+,K+-ATPase activity impairment after experimental traumatic brain injury: relationship to spatial learning deficits and oxidative stress,” Behavioural Brain Research, vol. 193, no. 2, pp. 306–310, 2008.
[66]  G. A. Gusarova, H. E. Trejo, L. A. Dada et al., “Hypoxia leads to Na,K-ATPase downregulation via Ca2+ release-activated Ca2+ channels and AMPK activation,” Molecular and Cellular Biology, vol. 31, no. 17, pp. 3546–3556, 2011.
[67]  S. S. L. Chew, C. S. Johnson, C. R. Green, and H. V. Danesh-Meyer, “Role of connexin43 in central nervous system injury,” Experimental Neurology, vol. 225, no. 2, pp. 250–261, 2010.
[68]  P. Nilsson, L. Hillered, U. Ponten, and U. Ungerstedt, “Changes in cortical extracellular levels of energy-related metabolites and amino acids following concussive brain injury in rats,” Journal of Cerebral Blood Flow and Metabolism, vol. 10, no. 5, pp. 631–637, 1990.
[69]  H. Katoh, K. Sima, H. Nawashiro, K. Wada, and H. Chigasaki, “The effect of MK-801 on extracellular neuroactive amino acids in hippocampus after closed head injury followed by hypoxia in rats,” Brain Research, vol. 758, no. 1-2, pp. 153–162, 1997.
[70]  H. Koizumi, H. Fujisawa, H. Ito, T. Maekawa, X. Di, and R. Bullock, “Effects of mild hypothermia on cerebral blood flow-independent changes in cortical extracellular levels of amino acids following contusion trauma in the rat,” Brain Research, vol. 747, no. 2, pp. 304–312, 1997.


comments powered by Disqus