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Glial calcium signalling

Glial calcium signalling. Alexei Verkhratsky The University of Manchester, UK. How calcium signalling was discovered Principles of calcium signalling Calcium signalling in neuroglia Voltage- and ligand-gated channels Endoplasmic reticulum calcium store Store-operated calcium entry

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Glial calcium signalling

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  1. Glial calcium signalling Alexei Verkhratsky The University of Manchester, UK How calcium signalling was discovered Principles of calcium signalling Calcium signalling in neuroglia Voltage- and ligand-gated channels Endoplasmic reticulum calcium store Store-operated calcium entry Calcium signals in neuronal-glial interactions Glial calcium waves

  2. Calcium - the beginning The scientific story of calcium began in 1808, when Sir Humphry Davy was able to show that lime (which had hitherto been considered to be an inseparable element) was actually a combination of metal and oxygen (hence Calcium - from Latin Calx for chalk). Sir Humphry tried to purify calcium by exposing a mixture of lime and mercuric acid to electric current; he succeeded in obtaining an amalgam of calcium, but further separation of mercury was so difficult that even Davy himself was not certain of whether he had obtained pure metallic calcium. In fact, he never managed to isolate this new metal in a pure form, and metallic calcium remained a laboratory curiosity for another 50 years, until Henry Moissan obtained 99% pure calcium by electrolysing calcium iodide. Professor Hamphry Davy (1778-1829)

  3. Calcium signalling: The Beginning The creator of calcium signalling Sydney Ringer found that Ca2+ is crucially important for - survival of fish Ringer S. (1883) The influence of saline media on fishes. J. Physiol. Lond., 4, vi-viii. - the contraction of the heart and skeletal muscle Ringer S. (1883) A further contribution regarding the influence of different constituents of the blood on the contractions of the heart. J. Physiol. Lond., 4, 29-43. Ringer S. (1886) Further experiments regarding the influence of small quantituies of lime, potassium and other salts on musclular tissue. J. Physiol. Lond., 7, 291-308. Ringer S, Buxton LW. (1887) Concerning the action of calcium, potassium and sodium salts upon the eel's heart and upon skeletal muscles of the frog. J. Physiol. Lond., 8, 15 - 19. - fertilisation of eggs and development of the tadpole Ringer S, Sainsbury H. (1894) The action of potassium, sodium and calcium salts on Tubifex rivulorum. J. Physiol. Lond.,16, 1-9 Several years later Locke and Overton found that Ca2+ is critical for impulse transmission between nerve and muscle. Locke FS. (1894) Notiz uber den Einfluss, physiologisher Kochsalzlosung auf die Eregbarkeit von Muscel and Nerve. Zentralbl. Physiol., 8, 166-167. Overton E. (1904) Beitrage zur allgemeinen Muskel- und Nerven physiologie. III. Mittheilung. Studien uber die Wirkung der Alkali- und Erdkali-salze auf Skeletalmuskeln und Nerven. Pflugers Arch.,105, 176-290

  4. Calcium chloride added to distilled water sustains life much longer than either corresponding quantities of sodium or potassium salts. For instance, with 30 cc of 1 per cent. solution of calcium chloride to the 1000 c.c. of distilled water, six fish died on average in 47 hours; whilst nine were still alive on the 12th day. • Ringer, S., 1883, • The influence of saline media on fishes, • J. Physiol. Lond.4, vi-viii.

  5. Calcium signalling – the idea is born Lewis Victor Heilbrunn 1892-1959 "The sensitivity of protoplasm and its response to stimulation are believed to be due to a sensitivity to free calcium ion and it is believed that the freeing of calcium and the reaction of this calcium with the protoplasm inside the cell is the most basic of all protoplasmic reactions.“ An Outline of General Physiology (1943 – 1952) • 2. Typically (and perhaps always) the outer part of the protoplasm consists of a rigid cortex. • … • 7. When a cell is exposed to stimuli, such as heat, cold, mechanical impact, electric shock, ultraviolet radiation, etc., the cortex is liquefied and calcium is released from the cortex into the cell interior. • The Dynamics of Living Protoplasm (1956)

  6. Calcium signalling - Early discoveries 1947: Heilbrunn and Wiercinski observed rapid and strong contractions after directly injecting minute amounts of Ca2+ into muscle fibres Heilbrunn LV, Wiercinsky FJ. (1947) Action of various cations on muscle protoplasm. J. Cell. Comp. Physiol.,19, 15 - 32. 1948 - 1954: Schwarzenbach and Ackerman synthesized EDTA Schwarzenbach, v. G. & Ackermann, H. (1954) Helv. Chim. Acta, 30, 1798 - 1804. 1954: Bozler found that the removal of Ca2+ by EDTA relaxed muscle fibers. Bozler E. (1954) Relaxation in extracted muscle fibers. J. Gen. Physiol.,38, 149 - 159. 1959: Anne-Marie Weber discovered that Ca2+ ions after binding to myofibrils activate actomyosin Weber A. (1959) On the role of calcium in the activity of adenosine 5'-triphosphate hydrolysis by actomyosin. J. Biol. Chem.,234, 2764 - 2769.

  7. Calcium signalling - Early discoveries Setsuro Ebashi In the 1950s, Setsuro Ebashi, working under the guidance of Professor Kumagai, found that in glycerol-extracted muscle, ATP could induce contraction, which was not followed by relaxation, but subsequent addition of a muscle extract induced relaxation. Ebashi and his colleagues called this extract relaxing factor [1]. This relaxing factor turned out to have characteristics in common with a Mg2+-activated ATPase described earlier by Wayne Kielley and Otto Meyerhof [2]. In Fritz Lipmann’s laboratory, at the Rockefeller Institute in New York, Ebashi was later able to demonstrate that the relaxing effect is due to Ca2+ uptake into sarcoplasmic reticulum vesicles mediated by a Ca2+, Mg2+-activated ATPase [3]; similar results were obtained independently by Hasselbach and Makinose [4]. This finding, although originally related specifically to muscle relaxation, is fundamental to our understanding of Ca2+ signalling generally, since it introduced for the first time the concept of an intracellular membrane-bounded Ca2+ store. In 1968, Setsuro Ebashi and Makoto Endo [5] outlined the theory of muscle contraction as we know it now. They wrote “Ca ion discharged from the sarcoplasmic reticulum under the influence of action potential affects troponin and releases the actin filament from its depressed state, resulting in contraction. The sarcoplasmic reticulum then removes Ca ion from troponin at the expense of ATP and induces relaxation” [5]. Kumagai H, Ebashi S, Takeda F. (1955) Essential relaxing factor in muscle other than myokinase and creatine phosphokinase. Nature, 176, 166. Kielley WW, Meyerhof O. (1948) Studies on adenosintriphosphatase of muscle. II. A new magnesium-activated adenosinetriphosphatase. J. Biol. Chem.,176, 591-601. Ebashi S, Lipmann F. (1962) Adenosine triphophate-linked concentration of calcium ions in a particulate fraction of rabbit muscle. J. Cell. Biol., 14, 389 - 400. Hasselbach W, Makinose M. (1962) ATP and active transport. Biochem. Biophys. Res. Commun., 7, 132-6. Ebashi S, Endo M. (1968) Calcium ion and muscle contraction. Prog. Biophys. Mol. Biol.,18, 123-83.

  8. Patch-clamp and fluorescent Ca2+ indicators Patch-Clamp Calcium probes Roger Tsien Erwin Neher Bert Sakmann Neher E, Sakmann B. (1976) Single-channel currents recorded from membrane of denervated frog muscle fibres. Nature, 260, 799-802. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch, 391, 85-100. Tsien RY. (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. Biochemistry, 19, 2396-2404. Tsien RY. (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature, 290, 527-528. Grynkiewicz G, Poenie M, Tsien RY. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem,260, 3440-3450.

  9. Diversity and versatility of calcium probes

  10. Combination of patch- voltage-clamp with Ca2+ indicators Kano M, Garaschuk O, Verkhratsky A & Konnerth A (1995)Journal of Physiology, London., 487, 1- 16 Solovyova N & Verkhratsky A (2002): J.Neurosci Meth,21: 622-620

  11. Calcium as universal signalling molecule Calcium as a hormone Calcium as multi-level intracellular messenger Fluctuations of extracellular [Ca2+] From: Ward DT (2004) In: Cell Calcium special issue on “Calcium-sensing receptor: physiology, pathology and pharmacological modulation” Ed by D. Riccardi From: Verkhratsky A & Toescu EC (1998) Integrative aspects of calcium signalling, Plenum Press

  12. Calcium signalling in neurones and neuroglia: general principles Nedergaard, M., Rodriguez, JJ & Verkhratsky, A (2010): Cell Calciumv. 47, p. 140-149.

  13. Calcium signalling provides the substrate for glial excitability

  14. Glial calcium signalling: mechanisms of generation 1. Voltage-gated calcium channels Glial cells are non-excitable cells in physiological sense (i.e. they cannot generate action potentials. Nonetheless glial cells express a sevarl types of voltage-gated ion channels including voltage-gated calcium channels. Voltage-gated Ca2+ channels are generally present in immature glial cells or in glial precursors and their expression is down-regulated during development.

  15. Calcium currents in glial cells Oligodendrocyte precursor Immature astrocyte Verkhratsky, AN, Trotter, J & Kettenmann, H, (1990): Neurosci Lett, v. 112, p. 194-198. Akopian, G, Kressin, K, Derouiche, A & Steinhauser, C, (1996): Glia,v. 17, p. 181-194.

  16. Kirischuk, S, Scherer, J, Moller, T, Verkhratsky, A & Kettenmann, H, (1995): Glia,v. 13, p. 1-12.

  17. Glial calcium signalling: mechanisms of generation 2. Ligand-gated calcium channels All types of neuroglial cells express ligand-gated ion channels, generally referred to as ionotropic receptors. The most abundant are glutamate receptors (of AMPA, Kainate and NMDA types) and purinoceptors (or P2X receptors). Many of these channels are permeable to Ca2+ and can generate cytosolic Ca2+ signals.

  18. Verkhratsky, A, Orkand, RK & Kettenmann, H, (1998) : Physiol Rev,v. 78, 99-141.

  19. Glial calcium signalling: mechanisms of generation 3. Endoplasmic reticulum provides a substrate for glial excitability Ca2+ release following stimulation of metabotropic receptors and production of InsP3 forms cytosolic Ca2+ signals, Ca2+ oscillations and propagating Ca2+ waves, which travel along single glial cell and between glial cells, thus allowing integration within glial networks.

  20. Neurotransmitter receptors and InsP3-mediated Ca2+ signalling in glial cells

  21. Visualisation of endoplasmic reticulum by fluorescent thapsigargin in cultured astrocytes Verkhratsky, Solovyeva & Toescu (2002): In: Glia in synaptic transmission, Ed. by Volterra, Haydon & Magistretti, OUP, p. 91 - 109.

  22. Resting Ca2+ concentration within the ER lumen in astroglia Verkhratsky, Solovyeva & Toescu (2002): In: Glia in synaptic transmission, Ed. by Volterra, Haydon & Magistretti, OUP, p. 91 - 109.

  23. Expression of InsP3 receptors in glial cells astroglia oligodendroglia 600 300 100 M M InsP3 R1 InsP3 R2 InsP3 R3 InsP3 R1 InsP3 R2 InsP3 R3 Kirchhoff & Verkhratsky, unpublished

  24. Calcium signalling in neurones and glia: Extracellular vs. intracellular pathway Verkhratsky, Solovyeva & Toescu (2002): In: Glia in synaptic transmission, Ed. by Volterra, Haydon & Magistretti, OUP, p. 91 - 109.

  25. ATP triggers calcium release from InsP3-sensitive stores in Bergmann glial cells in cerebellar slices Kirischuk, S, Moller, T, Voitenko, N, Kettenmann, H & Verkhratsky, A, (1995): J Neurosci,v. 15, p.7861-7871.

  26. InsP3 triggers Ca2+ efflux from astroglial endoplasmic reticulum Solovyova & Verkhratsky, unpublished

  27. Astrocytes express ryanodine receptors, yet their function remains enigmatic Solovyova & Verkhratsky, unpublished

  28. Depletion of ER store triggers store-operated Ca2+ entry that is necessary for ER store refilling ER [Ca2+] Cytoplasmic Ca2+ Solovyova & Verkhratsky, unpublished

  29. Store-operated Ca2+ is universally present in all types of glia: Store-operated Ca2+ entry in Bergmann glial cells in situin cerebellar slice Tuschick, Kirischuk, Kirchhoff, Liefeldt, Paul, Verkhratsky & Kettenmann, (1997): Cell Calcium, v. 21, p. 409-419.

  30. Store-operated Ca2+ is universally present in all types of glia: Activation of purinoceptors triggers both Ca2+ release and store-operated Ca2+ entry in human glioma cells Hartmann & Verkhratsky (1998): J Physiol,v. 513, p. 411-424.

  31. Dissociation between activation of Ca2+ release and store-operated Ca2+ entry in human glioblastoma cells Hartmann & Verkhratsky (1998): J Physiol,v. 513, p. 411-424.

  32. Store-operated Ca2+ entry is controlled by specific portion of the ER possibly in the very vicinity of the plasmalemma Hartmann & Verkhratsky (1998): J Physiol,v. 513, p. 411-424.

  33. Expression of TRP channels in glial cells microglia oligodendrocytes astrocytes 1500 1200 600 430 100 trp 6 5/4 3 2 1 6 5/4 3 2 1 6 5/4 3 2 1 Kirchhoff & Verkhratsky, unpublished

  34. Glial calcium signalling: mechanisms of generation 4. Glial cells generate calcium signals in response to neuronal activity Release of neurotrasnmittters from presynaptic terminals activates metabotropic receptors in astroglial perysinaptic processes, that trigger Ca2+ signals. Similarly release of glutamate and ATP from electrically active axons axons trigger receptor/InsP3-mediated Ca2+ signals in oligodendroglia.

  35. Grosche, Matyash, Moller, Verkhratsky, Reichenbach & Kettenmann (1999): Nat Neurosci, v. 2 p. 139-143.

  36. Inhibition of neuronal excitability abolishes Ca2+ signals in Bergmann glia Grosche, Matyash, Moller, Verkhratsky, Reichenbach & Kettenmann (1999): Nat Neurosci, v. 2 p. 139-143.

  37. Synaptically evoked Ca2+ signalling occurs in the microdomains represented by elementary structures which envelope groups of synapses Grosche, Matyash, Moller, Verkhratsky, Reichenbach & Kettenmann (1999): Nat Neurosci, v. 2 p. 139-143.

  38. Glutamate induces Ca2+ release from thapsigargin-sensitive store Kirischuk, Kirchhoff, Matyash, Kettenmann & Verkhratsky (1999): Neuroscience, v. 92, p. 1051-1059.

  39. Heparine inhibits glutamate-induced Ca2+ signalling Kirischuk, Kirchhoff, Matyash, Kettenmann & Verkhratsky (1999): Neuroscience, v. 92, p. 1051-1059.

  40. Expression of mGluR 5 in Bergmann glia Kirischuk, Kirchhoff, Matyash, Kettenmann & Verkhratsky (1999): Neuroscience, v. 92, p. 1051-1059.

  41. Glial calcium signalling: mechanisms of generation 5. Stimulation of glial cells triggers propagating Ca2+ waves that spread through glial syncytia Mechanisms of propagating Ca2+ waves in astroglia are complex and involve diffusion of InsP3 through gap junctions and Ca2+ dependent release of transmitters (most probably ATP).

  42. Propagating calcium waves in astroglial confluent cultures were discovered by Ann Cornell-Bell and her colleagues in 1990 and are seen in many preparations in vitro and in situ Astroglial calcium waves in retina Newman & Zahs (1997) Science, v.275, p. 844-847 Calcium waves in cultured astrocytes Charles (1998) Glia,v.24, p. 39-49

  43. Mechanisms of glial Ca2+ wave propagation InsP3 diffusion through gap junctions; release of transmitters or combination of both Verkhratsky & Butt (2007): Glial Neurobiology, A Textbook Wiley & Sons

  44. Conclusions Stimulation of neuroglial cells with neurotransmitters and neuromodulators often triggers cytosolic Ca2+ signals. Glial Ca2+ signals are predominantly generated by Ca2+ release from the endoplasmic reticulum though opening of InsP3-gated Ca2+ channels (InsP3 receptors). Endoplasmic reticulum membrane represents an excitable media that allows generation of propagating calcium waves, which integrate astroglial syncytia. Neuroglial Ca2+ signals can be instrumental for integrative processes in neuronal-glial networks.

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