Neuronal-glial chemical transmission mediated by glutamate and ATP Prof. Alexei Verkhratsky

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1 MD, PhD, DSc Professor of Neurophysiology, The University of Manchester, Faculty of Life Sciences, Manchester, UK 1 Experiments on glial cells in tissue culture demonstrated expression of neurotransmitter receptors Verkhratsky & Kettenmann (1996) Trends Neurosci.,19: Bergmann glial cells in situ express restricted receptor pattern which matches that of neuronal partners Experiments on glial cells in situ showed that receptors patterns are restricted to match neurotransmitters released in a particular brain region NT LC CF PF Bergmann glial cell AMPA mglur1,5 PLC P 2Y H 1 PLC H 1 PLC ET B NA, Glu, Hist Glutamate receptors Purinoreceptors Histamine receptors Adrenoreceptors Endotheline receptors AMPA mglur1 PLC P 2Y P 2X PLC H 1 H 1 ET B(?) Purkinje neurone GABA A GABA receptors GABA GABA A CF Verkhratsky, Orkand & Kettenmann (1998) Physiol Rev.,78: BA ST 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Astrocytes form communication channels with nearby neurones Verkhratsky & Kirchhoff (2007) Neuroscientist, 13: Neurotransmission by glutamate: glutamate sensors Neurotransmission by glutamate: glutamate sensors Verkhratsky & Kirchhoff (2007) Neuroscientist, 13: mediated through glutamate Ionotropic glutamate receptors 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Identification of neurotransmitter receptors in cultured astrocytes V m (mv) GABA Aspartate Glutamate Glycine Kettenmann, Backus, Schachner (1984) Neurosci Lett,52: (IF~2) L-glutamate D-glutamate Kainic acid L-aspartate D-aspartate NMDA Quinquialate Bowman, Kimelberg (1984) Nature, 311: (IF ~20) 7 Kainate induces massive calcium influx mediated by AMPA receptors into Bergmann glial cells in cerebellar slice preparations A Control Kainate Patch pipette loaded with Fura-2 Bergmann glial cell Purkinje cell layer F 340/380 Verkhratsky, A., Orkand, RK & Kettenmann., H. (1998) Physiol. Rev.,78: NMDA receptors in astroglial cells The NMDA receptor was, for a long time, believed to have exclusively neuronal localisation The NMDA receptors possess an important property of Mg 2+ block that precludes their opening at hyperpolarised membrane potentials The astroglial resting potential is set at -80 to -90 mv and therefore it was assumed that NMDA receptors can not function in glial cells Vorobjev VS. (1991) J Neurosci. Methods, 38: Pankratov Y, Lalo U, Krishtal O, & Verkhratsky A. (2002) J. Physiol.,542: Akaike N & Moorhouse AJ. (2003) Trends Pharmacol. Sci.,24: I norm V m (mv) The screen versions of these slides have full details of copyright and acknowledgements 3

4 Glutamate induces a complex current response sensitive to inhibitors of AMPA and NMDA receptors and glutamate/na + transporters Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006) Journal of Neuroscience, 26: NMDA induces glycine-sensitive current response in isolated astrocytes I norm N A [NMDA], mm Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006) Journal of Neuroscience,26: Astroglial NMDA receptors are magnesium insensitive D I norm V m (mv) mm Mg 2+ 5 mm Mg mm Mg 2+ Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006) Journal of Neuroscience,26: The screen versions of these slides have full details of copyright and acknowledgements 4

5 mediated through glutamate Ionotropic receptors Metabotropic receptors 13 A Parallel fibers stimulation triggers calcium signals in Bergmann glial cell microdomains Pia PF STIM 200 mm B 1 2 C s PCL D 255 BG 10 mm 4 mm F/F 1.0 F/F 0 2 s Grosche, Matyash, Moller, Verkhratsky, Reichenbach & Kettenmann (1999) Nat. Neurosci., 2: mediated through glutamate Ionotropic receptors Metabotropic receptors Glutamate transporters 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Astrocytes form the main glutamate-uptake system in the CNS 80% 20% Verkhratsky & Butt, (2007) Glial Neurobiology A Textbook, Wiley & Sons 16 Voltage-dependence of GluT- and AMPAR-mediated currents I norm Kirischuk, Kettenmann & Verkhratsky (2007) Pflugers Arch.,454: Application of glutamate triggers inward current and sodium influx Kirischuk, Kettenmann & Verkhratsky (2007) Pflugers Arch.,454: The screen versions of these slides have full details of copyright and acknowledgements 6

7 Different sensitivities of glial glutamate sensors allow precise determination of extracellular glutamate concentration NMDAR K D ~2 mm K D ~50 mm K D ~3-100 mm K D ~130 mm AMPAR GluNa + T mglur1/5 Verkhratsky & Kirchhoff (2007) Neuroscientist, 13: principal transmitter in neuronal-glial networks 20 -mediated transmission: the beginning was discovered in 1929 Extracellular signalling role of purines was suggested in 1929 Geoffrey Burnstock was born on May 10, 1929 Burnstock G., (1972) Purinergic nerves, Pharmacol. Rev., 24: GB: I came up with this concept in Pharmacological Reviews in 1972, and nobody believed it At the first Purines Meeting in Canada way back in the late 70s, probably 95% of the science concentrated on adenosine. And my stuff [on signaling through ] was very much frowned on. There were certain very strong figures very influential figures, and gifted and one of them was Robert Berne, who had early on claimed that adenosine was the physiological regulator of blood flow during hypoxia and ischemia in the heart and other organs. He was charming and he was clever, and although he collected adenosine from the hypoxic heart, he was totally opposed to the fact that it might be originating as. At the opening party of the second Purines Meeting a while later, I was suddenly trapped in the corner by several scientists who were also very opposed to the idea of as an extracellular regulator, and being an ex-boxer, I got very worried because I had the sense that it was going to become physical. People were very passionately against this! Geoffrey Burnstock: most highly cited scientist Molecular Interventions(2004) 4: The screen versions of these slides have full details of copyright and acknowledgements 7

8 : the primordial signalling molecule The purines were a part of very early signalling evolution Indeed the purines in the form of adenine and guanine phosphates occurred in the prebiotic period (as a result of purely thermal synthetic processes) and rapidly acquired high importance Purines and pyrimidines were essential for construction of both RNA and DNA, and hence their intracellular concentration was high Furthermore, was selected very early as the main source of biological energy, and thus became an indispensable feature of life The prominent roles of adenine-phosphates in energy metabolism in the most primitive life forms and ancient origin of adenine-binding sites are widely recognised This was a critical evolutionary choice as it immediately resulted in birth of the universal intracellular signalling system based on calcium ions, as keeping cytosolic Ca 2+ extremely low became vitally important, since otherwise insoluble Ca 2+ -phopshates would preclude the cell energetics Thus even the most primitive ancient cell had high cytosolic concentrations of (or GTP) and upon cell disintegration gradients of both would be present in the surrounding water This may have been the initial form of chemical transmission, which remained throughout evolution Indeed every known cell or single cell organism does display some form of sensitivity to, and moreover the transmission is unique in a sense that it is not confined to a particular tissue or organ of higher organisms it exists everywhere; Indeed it is difficult to find a cell type, which does not show sensitivity to Bursntock& Verkhratsky (2009) Acta Physiologica,195: Mechanisms of release in the central nervous system 23 release and degradation in the CNS Exocytosis in presynaptic terminals Exocytotic and non-exocytotic release in astrocytes Maxi channels Ca 2+ Ca 2+ Transporter-mediated release in axons Damaged cells Ca 2+ Ca 2+ ADP AMP Adenosine NTPDases, NPPs, Alk Phosp, 5 -Nuc, Myok, NDK Abbracchio, Burnstock, Verkhratsky & Zimmermann (2009) TINS, 32: The screen versions of these slides have full details of copyright and acknowledgements 8

9 Exocytotic release from synaptic terminals Dissection of GLUR/P2X-mediated mepscs in cortical neurones P2X antagonists selectively inhibit small amplitude mepscs Pankratov, Lalo, Verkhratsky, North (2007) J. Gen. Physiol., 129: Quantal release of from acutely isolated cortical astrocytes A B TFLLR 10 mm HEK293-P2X 2 cell Astrocyte [Ca 2+ ] i DF/F 0 (100%) HEK293 I m 8 pa Isolated astrocyte PPADS 10 mm TFLLR 10 mm 5 s Astrocyte [Ca 2+ ] i Lalo, Verkhratsky, Pankratov (2011) Semin Cell Dev Biol 22: HEK293 I m 26 Purinoceptors: an overview Abbracchio, Burnstock, Verkhratsky & Zimmermann (2009) TINS, 32: The screen versions of these slides have full details of copyright and acknowledgements 9

10 as a universal transmitter in neuronal-glial circuits Verkhratsky, Krishtal, & Burnstock (2009) Mol. Neurobiol., 39: Iono- and metabotropic receptors expressed in astroglia trigger calcium signals in the optic nerve DF/F 0 Thapsigargin 30s Zero-Ca 2+ Hamilton, Vayro, Kirchhoff, Verkhratsky, Robbins, Gorecki & Butt (2008) Glia,56: as a universal glial transmitter: oligodendrocytes A D B 10 mm 25 mm E C Kirischuk S, Scherer J, Kettenmann H, Verkhratsky A., (1995) J. Physiol., (London) 483: The screen versions of these slides have full details of copyright and acknowledgements 10

11 -induced currents in cortical astrocytes Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008) Journal of Neuroscience, 28: Concentration-dependence of -induced currents Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008) Journal of Neuroscience, 28: A Pharmacology of -induced currents B * TNP-: 2,3 -O-(2,4,6-trinitrophenyl) adenosine 5 -triphosphate Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008) Journal of Neuroscience, 28: The screen versions of these slides have full details of copyright and acknowledgements 11

12 Subunit composition of the astroglial P2X receptor Sensitivity to Sensitivity to abme Kinetics Rebound upon washout Ca 0-dependence Desensitization Inhibition by PPADS TNP- P2X 1/5 receptor expressed in artificial systems Exceptionally High, EC50 ~ 50 nm Yes Biphasic: rapid peak and steady-state Yes No No 100% inhibition Astroglial P2X receptor Exceptionally High, EC50 ~ 40 nm Yes Biphasic: rapid peak and steady-state Yes No No 100% inhibition Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008) Journal of Neuroscience, 28: Cortical astrocytes express P2X 1 and P2X 5 subunits Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008) Journal of Neuroscience, 28: Synaptic stimulation triggers fast astroglial currents mediated by ionotropic receptors and glutamate transporters 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 Glial synaptic currents (GSC) in the neocortex Stimulus/TTX dependence of GSCs and EPSCs Stimulus-frequency dependence of GSCs Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: Ionotropic receptors and glutamate transporter mediate GSC in the neocortex Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: A Spontaneous glial synaptic currents B Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: The screen versions of these slides have full details of copyright and acknowledgements 13

14 Calcium permeability of astroglial NMDA and P2X 1/5 receptors 40 Calcium permeability of NMDA and P2X 1/5 receptors in cortical astrocytes P Ca /P mono ~ 3 P Ca /P mono ~ 2 Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, 28: Stimulation of NMDA and P2X 1/5 receptors triggers Ca 2+ signals in isolated cortical astrocytes Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, 28: The screen versions of these slides have full details of copyright and acknowledgements 14

15 Synaptic stimulation-induced Ca 2+ signals in cortical astrocytes in situ are sensitive to ionotropic receptors antagonists C Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, 28: Age-dependent remodelling of ionotropic receptors expression in cortical astroglia 44 In situ recordings from ageing cortical astroglia B Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: The screen versions of these slides have full details of copyright and acknowledgements 15

16 Ageing changes the relative weight of GSCs components 3 months C Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: months 46 Ageing modifies expression of glutamate and purinergic receptors in astroglia Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: Ageing affects ionotropic Ca 2+ signalling in astroglia in situ Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell10: The screen versions of these slides have full details of copyright and acknowledgements 16

17 Ionotropic receptors in neuronal-astroglial signalling: What is the role of excitable molecules in non-excitable cells? Fast local signalling in astroglial perisynaptic compartments? NCX iglurs P2XRs EAAT Na/K pump Direct mode Reverse mode Lactate shuttle Lalo, Pankratov, Parpura, Verkhratsky (2011) Biochim Biophys Acta 1813: Conclusions Astroglial cells express several classes of fast ionotropic receptors These receptors are activated by ongoing synaptic transmission and mediate local cytoplasmic signalling mediated through [Ca 2+ ] i and [Na + ] i transients Rapid activation of astroglial ionotropic receptors can be instrumental for highly localised neuronal-glial signalling at the synaptic level Age-dependent decrease in synaptic communication in neuronal-glial circuitry may signify the beginning of age-dependent decline in brain functions Alternatively however, the bell-shaped changes in glial synaptic transmission may reflect maturation of the CNS, pruning of functionally irrelevant synaptic contacts and manifest the attainment of wisdom which generally accompanies advanced age The screen versions of these slides have full details of copyright and acknowledgements 17