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Astrocytes pp 283–303Cite as

Connexin-Based Channels in Astrocytes: How to Study Their Properties

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 814))

Abstract

A typical feature of astrocytes is their high level of connexin expression. These membrane proteins constitute the molecular basis of two types of channels: gap junction channels that allow direct cytoplasm-to-cytoplasm communication and hemichannels that provide a pathway for exchanges between the intra- and extracellular media. An unusual property of these channels is their permeability for ions but also for small signaling molecules. They support intercellular communication that contribute to dynamic neuroglial interaction and interplay with neuronal activity and survival. Here, we describe multiple techniques based either on electrophysiological approaches or the monitoring of dye intercellular diffusion and uptake that permits an investigation of the properties of gap junction channels and hemichannels, respectively. These techniques are applied in astrocyte studies using in vitro models, mainly primary cultures and acute brain slices.

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References

  1. Volterra, A., Bezzi, P. (2002) Release of transmitters from glial cells, in The tripartite synapse: glial in synaptic transmission (Volterra A, M., P. and Haydon, P.G., Ed.), pp p 164–182, Oxford University Press, Oxford.

    Google Scholar 

  2. Araque, A., Parpura, V., Sanzgiri, R. P., and Haydon, P. G. (1999) Tripartite synapses: glia, the unacknowledged partner, Trends Neurosci 22, 208–215.

    Article  PubMed  CAS  Google Scholar 

  3. Roerig, B., and Feller, M. B. (2000) Neurotransmitters and gap junctions in developing neural circuits, Brain Res Brain Res Rev 32, 86–114.

    Article  PubMed  CAS  Google Scholar 

  4. Harris, A. L. (2007) Connexin channel permeability to cytoplasmic molecules, Prog Biophys Mol Biol 94, 120–143.

    Article  PubMed  CAS  Google Scholar 

  5. Spray, D. C., Ye, Z. C., and Ransom, B. R. (2006) Functional connexin “hemichannels”: a critical appraisal, Glia 54, 758–773.

    Article  PubMed  Google Scholar 

  6. Scemes, E., Suadicani, S. O., Dahl, G., and Spray, D. C. (2007) Connexin and pannexin mediated cell-cell communication, Neuron Glia Biol 3, 199–208.

    Article  PubMed  Google Scholar 

  7. Baranova, A., Ivanov, D., Petrash, N., Pestova, A., Skoblov, M., Kelmanson, I., Shagin, D., Nazarenko, S., Geraymovych, E., Litvin, O., Tiunova, A., Born, T. L., Usman, N., Staroverov, D., Lukyanov, S., and Panchin, Y. (2004) The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins, Genomics 83, 706–716.

    Article  PubMed  CAS  Google Scholar 

  8. Panchin, Y., Kelmanson, I., Matz, M., Lukyanov, K., Usman, N., and Lukyanov, S. (2000) A ubiquitous family of putative gap junction molecules, Curr Biol 10, R473–474.

    Article  PubMed  CAS  Google Scholar 

  9. Bruzzone, R., Hormuzdi, S. G., Barbe, M. T., Herb, A., and Monyer, H. (2003) Pannexins, a family of gap junction proteins expressed in brain, Proc Natl Acad Sci USA 100, 13644–13649.

    Article  PubMed  CAS  Google Scholar 

  10. Vanden Abeele, F., Bidaux, G., Gordienko, D., Beck, B., Panchin, Y. V., Baranova, A. V., Ivanov, D. V., Skryma, R., and Prevarskaya, N. (2006) Functional implications of calcium permeability of the channel formed by pannexin 1, J Cell Biol 174, 535–546.

    Article  PubMed  CAS  Google Scholar 

  11. Lai, C. P., Bechberger, J. F., Thompson, R. J., MacVicar, B. A., Bruzzone, R., and Naus, C. C. (2007) Tumor-suppressive effects of pannexin 1 in C6 glioma cells, Cancer Res 67, 1545–1554.

    Article  PubMed  CAS  Google Scholar 

  12. Houades, V., Koulakoff, A., Ezan, P., Seif, I., and Giaume, C. (2008) Gap junction-mediated astrocytic networks in the mouse barrel cortex, J Neurosci 28, 5207–5217.

    Article  PubMed  CAS  Google Scholar 

  13. Rouach, N., Koulakoff, A., Abudara, V., Willecke, K., and Giaume, C. (2008) Astroglial metabolic networks sustain hippocampal synaptic transmission, Science 322, 1551–1555.

    Article  PubMed  CAS  Google Scholar 

  14. Giaume, C., Koulakoff, A., Roux, L., Holcman, D., and Rouach, N. (2010) Astroglial networks: a step further in neuroglial and gliovascular interactions, Nat Rev Neurosci 11, 87–99.

    Article  PubMed  CAS  Google Scholar 

  15. Kunze, A., Congreso, M. R., Hartmann, C., Wallraff-Beck, A., Huttmann, K., Bedner, P., Requardt, R., Seifert, G., Redecker, C., Willecke, K., Hofmann, A., Pfeifer, A., Theis, M., and Steinhauser, C. (2009) Connexin expression by radial glia-like cells is required for neurogenesis in the adult dentate gyrus, Proc Natl Acad Sci USA 106, 11336–11341.

    Article  PubMed  CAS  Google Scholar 

  16. Wallraff, A., Kohling, R., Heinemann, U., Theis, M., Willecke, K., and Steinhauser, C. (2006) The impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus, J Neurosci 26, 5438–5447.

    Article  PubMed  CAS  Google Scholar 

  17. Orellana, J. A., Sáez, P. J., Shoji, K. F., Schalper, K. A., Palacios-Prado, N., Velarde, V., Giaume, C., Bennett, M. V., and Sáez, J. C. (2009) Modulation of brain hemichannels and gap junction channels by pro-inflammatory agents and their possible role in neurodegeneration, Antioxid Redox Signal 11, 369–399.

    Article  PubMed  CAS  Google Scholar 

  18. Giaume, C., and Theis, M. (2009) Pharmacological and genetic approaches to study connexin-mediated channels in glial cells of the central nervous system, Brain Res Rev.

    Google Scholar 

  19. Kang, J., Kang, N., Lovatt, D., Torres, A., Zhao, Z., Lin, J., and Nedergaard, M. (2008) Connexin 43 hemichannels are permeable to ATP, J Neurosci 28, 4702–4711.

    Article  PubMed  CAS  Google Scholar 

  20. Neyton, J., and Trautmann, A. (1985) Single-channel currents of an intercellular junction, Nature 317, 331–335.

    Article  PubMed  CAS  Google Scholar 

  21. Spray, D. C., Harris, A. L., and Bennett, M. V. (1979) Voltage dependence of junctional conductance in early amphibian embryos, Science 204, 432–434.

    Article  PubMed  CAS  Google Scholar 

  22. Giaume, C., Fromaget, C., el Aoumari, A., Cordier, J., Glowinski, J., and Gros, D. (1991) Gap junctions in cultured astrocytes: single-channel currents and characterization of channel-forming protein, Neuron 6, 133–143.

    Google Scholar 

  23. Dermietzel, R., Hertberg, E. L., Kessler, J. A., and Spray, D. C. (1991) Gap junctions between cultured astrocytes: immunocytochemical, molecular, and electrophysiological analysis, J Neurosci 11, 1421–1432.

    PubMed  CAS  Google Scholar 

  24. Même, W., Ezan, P., Venance, L., Glowinski, J., and Giaume, C. (2004) ATP-induced inhibition of gap junctional communication is enhanced by interleukin-1 beta treatment in cultured astrocytes, Neuroscience 126, 95–104.

    Article  PubMed  CAS  Google Scholar 

  25. Rouach, N., Pebay, A., Meme, W., Cordier, J., Ezan, P., Etienne, E., Giaume, C., and Tence, M. (2006) S1P inhibits gap junctions in astrocytes: involvement of G and Rho GTPase/ROCK, Eur J Neurosci 23, 1453–1464.

    Article  PubMed  Google Scholar 

  26. Venance, L., Piomelli, D., Glowinski, J., and Giaume, C. (1995) Inhibition by anandamide of gap junctions and intercellular calcium signalling in striatal astrocytes, Nature 376, 590–594.

    Article  PubMed  CAS  Google Scholar 

  27. Meme, W., Vandecasteele, M., Giaume, C., and Venance, L. (2009) Electrical coupling between hippocampal astrocytes in rat brain slices, Neurosci Res 63, 236–243.

    Article  PubMed  Google Scholar 

  28. Xu, G., Wang, W., Kimelberg, H. K., and Zhou, M. Electrical coupling of astrocytes in rat hippocampal slices under physiological and simulated ischemic conditions, Glia 58, 481–493.

    Google Scholar 

  29. Blomstrand, F., Venance, L., Siren, A. L., Ezan, P., Hanse, E., Glowinski, J., Ehrenreich, H., and Giaume, C. (2004) Endothelins regulate astrocyte gap junctions in rat hippocampal slices, Eur J Neurosci 19, 1005–1015.

    Article  PubMed  CAS  Google Scholar 

  30. Maglione, M., Tress, O., Haas, B., Karram, K., Trotter, J., Willecke, K., and Kettenmann, H. Oligodendrocytes in mouse corpus callosum are coupled via gap junction channels formed by connexin47 and connexin32, Glia 58, 1104–1117.

    Google Scholar 

  31. Venance, L., Cordier, J., Monge, M., Zalc, B., Glowinski, J., and Giaume, C. (1995) Homotypic and heterotypic coupling mediated by gap junctions during glial cell differentiation in vitro, Eur J Neurosci 7, 451–461.

    Article  PubMed  CAS  Google Scholar 

  32. Chvatal, A., Pastor, A., Mauch, M., Sykova, E., and Kettenmann, H. (1995) Distinct populations of identified glial cells in the developing rat spinal cord slice: ion channel properties and cell morphology, Eur J Neurosci 7, 129–142.

    Article  PubMed  CAS  Google Scholar 

  33. Zhou, M., Schools, G. P., and Kimelberg, H. K. (2006) Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive, J Neurophysiol 95, 134–143.

    Article  PubMed  CAS  Google Scholar 

  34. Bennett, M. V. (1977) Electrical transmission: a functional analysis and comparison with chemical transmission, in Cellular Biology of Neurons, vol. I, sec. I, Handbook of Physiology. The Nervous System (Kandel, E. R., Ed.), Williams and Wilkins, Baltimore.

    Google Scholar 

  35. Venance, L., Glowinski, J., and Giaume, C. (2004) Electrical and chemical transmission between striatal GABAergic output neurones in rat brain slices, J Physiol 559, 215–230.

    Article  PubMed  CAS  Google Scholar 

  36. Neher, E., and Sakmann, B. (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres, Nature 260, 799–802.

    Article  PubMed  CAS  Google Scholar 

  37. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pflugers Arch 391, 85–100.

    Article  PubMed  CAS  Google Scholar 

  38. Contreras, J. E., Sáez, J. C., Bukauskas, F. F., and Bennett, M. V. (2003) Gating and regulation of connexin 43 (Cx43) hemichannels, Proc Natl Acad Sci USA 100, 11388–11393.

    Article  PubMed  CAS  Google Scholar 

  39. Retamal, M. A., Froger, N., Palacios-Prado, N., Ezan, P., Sáez, P. J., Sáez, J. C., and Giaume, C. (2007) Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia, J Neurosci 27, 13781–13792.

    Article  PubMed  CAS  Google Scholar 

  40. Schalper, K. A., Palacios-Prado, N., Retamal, M.A., Shoji, K.F., Martínez, A.D., Sáez, J.C. (2008) Connexin hemichannels composition determines the FGF-1-induced membrane permeability and free [Ca2+]i responses, Mol Biol Cell, 19, 3501–3513.

    Google Scholar 

  41. Sánchez, H. A., Orellana, J. A., Verselis, V. K., and Sáez, J. C. (2009) Metabolic inhibition increases activity of connexin-32 hemichannels permeable to Ca2+ in transfected HeLa cells, Am J Physiol Cell Physiol 297, C665-678.

    Article  PubMed  CAS  Google Scholar 

  42. Neher, E. (1992) Correction for liquid junction potentials in patch clamp experiments, Methods Enzymol 207, 123–131.

    Article  PubMed  CAS  Google Scholar 

  43. Bao, X., Lee, S. C., Reuss, L., and Altenberg, G. A. (2007) Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC, Proc Natl Acad Sci USA 104, 4919–4924.

    Article  PubMed  CAS  Google Scholar 

  44. Retamal, M. A., Cortés, C. J., Reuss, L., Bennett, M. V., and Sáez, J. C. (2006) S-nitrosylation and permeation through connexin 43 hemichannels in astrocytes: induction by oxidant stress and reversal by reducing agents, Proc Natl Acad Sci USA 103, 4475–4480.

    Article  PubMed  CAS  Google Scholar 

  45. D’Ambrosio, R., Wenzel, J., Schwartzkroin, P. A., McKhann, G. M., 2nd, and Janigro, D. (1998) Functional specialization and topographic segregation of hippocampal astrocytes, J Neurosci 18, 4425–4438.

    PubMed  Google Scholar 

  46. Giaume, C. (1991) Application of the patch-clamp technique to the study of junctional conductance, in Biophysiscs of gap junctions (C, P., Ed.), CRC press.

    Google Scholar 

  47. Cotrina, M. L., Gao, Q., Lin, J. H., and Nedergaard, M. (2001) Expression and function of astrocytic gap junctions in aging, Brain Res 901, 55–61.

    Article  PubMed  CAS  Google Scholar 

  48. Soroceanu, L., Manning, T. J., Jr., and Sontheimer, H. (2001) Reduced expression of connexin-43 and functional gap junction coupling in human gliomas, Glia 33, 107–117.

    Article  PubMed  CAS  Google Scholar 

  49. Goldberg, G. S., Bechberger, J. F., and Naus, C. C. (1995) A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer, Biotechniques 18, 490–497.

    PubMed  CAS  Google Scholar 

  50. Koulakoff, A., Ezan, P., and Giaume, C. (2008) Neurons control the expression of connexin 30 and connexin 43 in mouse cortical astrocytes, Glia 56, 1299–1311.

    Article  PubMed  Google Scholar 

  51. Siller-Jackson, A. J., Burra, S., Gu, S., Xia, X., Bonewald, L. F., Sprague, E., and Jiang, J. X. (2008) Adaptation of connexin 43-hemichannel prostaglandin release to mechanical loading, J Biol Chem 283, 26374–26382.

    Article  PubMed  CAS  Google Scholar 

  52. Muller, T., Moller, T., Neuhaus, J., and Kettenmann, H. (1996) Electrical coupling among Bergmann glial cells and its modulation by glutamate receptor activation, Glia 17, 274–284.

    Article  PubMed  CAS  Google Scholar 

  53. Houades, V., Rouach, N., Ezan, P., Kirchhoff, F., Koulakoff, A., Giaume, C. (2006) Shapes of astrocyte networks in the juvenile brain, in Neuron Glia Biology, pp 3–14.

    Google Scholar 

  54. Nolte, C., Matyash, M., Pivneva, T., Schipke, C. G., Ohlemeyer, C., Hanisch, U. K., Kirchhoff, F., and Kettenmann, H. (2001) GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue, Glia 33, 72–86.

    Article  PubMed  CAS  Google Scholar 

  55. Barres, B. A. (2008) The mystery and magic of glia: a perspective on their roles in health and disease, Neuron 60, 430–440.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by a collaborative grant from ECOS-CONICYT (N° C10S01).

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Authors and Affiliations

  1. CIRB, CNRS UMR7241/INSERM U1050 Collège de France, Paris, France

    Christian Giaume & Verónica Abudara

  2. Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile

    Juan A. Orellana

  3. Departamento de Fisiología, Universidad de la República, Montevideo, Uruguay

    Verónica Abudara

  4. Instituto Milenio, Inmunología e Inmunoterapia, Santiago, Chile

    Juan C. Sáez

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  1. Christian Giaume
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  2. Juan A. Orellana
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  3. Verónica Abudara
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  4. Juan C. Sáez
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Correspondence to Christian Giaume .

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Editors and Affiliations

  1. Dept. Molecular & Experimental, Scripps Research Institute, N. Torrey Pines Road 10550, La Jolla, 92037, California, USA

    Richard Milner

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Giaume, C., Orellana, J.A., Abudara, V., Sáez, J.C. (2012). Connexin-Based Channels in Astrocytes: How to Study Their Properties. In: Milner, R. (eds) Astrocytes. Methods in Molecular Biology, vol 814. Humana Press. https://doi.org/10.1007/978-1-61779-452-0_19

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  • DOI: https://doi.org/10.1007/978-1-61779-452-0_19

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