Neuronal-glial chemical transmission mediated by glutamate and...
Transcript of Neuronal-glial chemical transmission mediated by glutamate and...
Neuronal-glial chemical transmissionmediated by glutamate and ATP
Alexei VerkhratskyThe University of Manchester, UK
1 Experiments on glial cells in tissue culture demonstrated expression of neurotransmitter receptors
Verkhratsky & Kettenmann (1996): Trends Neurosci, v. 19, p. 346-352.
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Glutamate receptors
AMPA AMPA
mGluR1,5 mGluR1
Purinoreceptors
Histamine receptors
Adrenoreceptors
Endothelinereceptors
P2YP2Y
P2X
H1H1
Η1Η1
ETB ET (?)B
Purkinje neurone
Bergmannglial cell
CFPF
NTLC
NA, ATP,Hist
Glu
GABA receptorsGABAA GABAA
GABA
BAST
Bergmann glial cell express restricted receptor pattern which matches that of neuronal partner
in situ
Experiments on glial cells in situ showed that receptors patterns are restricted to match neurotransmitters released in particular brain region
Verkhratsky, Orkand & Kettenmann (1998): Physiol Rev, v. 78, p. 99-141
3 Astrocytes form communication “channels” with nearby neurones
Verkhratsky & Kirchhoff (2007): Neuroscientist 13: 1 - 10.
4
Neurotransmission by glutamate: Glutamate sensors
Verkhratsky & Kirchhoff (2007): Neuroscientist 13: 1 - 10.
5
Neuronal-glial chemical transmission mediated through
glutamate
Ionotropic glutamate receptors
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Identification of neurotransmitter receptors in cultured astrocytes
Kettenmann, Backus, Schachner (1984) Neurosci Lett 52:25-29 (IF~2).
Bowman, Kimelberg (1984) Nature 311:656-659 (IF ~20).
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B C
kainate kainate
30 s0.25
30 s0.25
F 340/380F 340/380
kainate kainate
kainate
30 s100 pA 50 pA
30 s
Ca2+ -free
kainate + CNQXkainate
kainate + CNQX
A
F340/380
control kainate
Purkinje celllayer
Bergmannglial cell
patch pipette loadedwith fura-2
Kainate induces massive calcium influx mediated by AMPA receptors into Bergmann glial cell in cerebellar slice preparation
Verkhratsky, Orkand & Kettenmann (1998): Physiological Reviews, 78, 99 - 141
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Acute isolation of green astrocytes
Acute vibrodissociation of cells from brain slicesVorobjev VS. (1991): J Neurosci Methods 38: 145-150. Pankratov Y, Lalo U, Krishtal O, & Verkhratsky A. (2002): J Physiol 542: 529-536.Akaike N & Moorhouse AJ. (2003): Trends Pharmacol Sci 24: 44-47.
B C
V (mV)m
-80 -40 40
1.0
0.5
-0.5
2.0
I nor
m
-80 mV
60 mV
-120 mV
20 ms
400 pA
10 mμ
A
9
AGlutamate
500 ms
50 pA
1.0
0.8
0.6
0.4
0.2
0.0
1 10
I NBQ
X (n
orm
alis
ed)
[NBQX] ( M)μControl
Washout
NBQX, 30 Mμ
NBQX - sensitive current
B Glutamate
Control
Washout 500 ms
200 pA
D-AP5, 1 MμD-AP5 - sensitive current 1.0
0.8
0.6
0.4
0.2
0.010.1 10
I D-A
P5 (n
orm
alis
ed)
[D-AP5] ( M)μ
C
Glutamate Glutamate Glutamate Glutamate
NBQX + D-AP5
2 s
50 pA
DL-TBOA
TBOA-sensitive current
Glutamate induces 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: 2673-2683
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NMDA
NMDA NMDA/glycine
0.0
0.2
0.2
0.4
0.4
0.6
0.6
0.8
0.8
1.0
1.0
0.01 1 100
[NMDA], Mμ
[glycine], Mμ
A
B
500 ms
100 pA
100 Mμ
10 Mμ
1 Mμ
300 nM
100 nM30 nM
ΔI n
orm
I nor
m
A
N
30 nM
1 Mμ
10 Mμ
30 Mμ
500 ms
100 pA
0.01 0.1 1 10
(4)(4)
(6)
(4)(3) (7)
NMDA induces glycine-sensitive current response in isolated astrocytes
Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006): Journal of Neuroscience, 26: 2673-2683.
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NMDA NMDA
20 mV
-80 mV
150 pA
500 ms
20 mV
-80 mV
100 pA
500 ms
NMDA
20 mV
-80 mV
200 pA
500 ms
Mg 0 mM2+ Mg 5 mM2+
Mg 10 mM2+
A B
C D
-80 -40 40
V (mV)m
-1.0
-0.5
I norm
0 mM Mg2+
10 mM Mg2+5 mM Mg2+
Astroglial NMDA receptors are magnesium insensitive
Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006): Journal of Neuroscience, 26: 2673-2683.
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Neuronal-glial chemical transmission mediated through
glutamate
Ionotropic receptorsMetabotropic receptors
13 Parallel fibers stimulation triggers calcium signalsin Bergmann glial cell microdomains
PCL
PF
Pia
BG
STIM
200 mμ
a b
c
12
10 mμ
1 22 s
d
2 s
1.6
1.0
F/F0
4 mμ
0
255
Grosche, Matyash, Moller, Verkhratsky, Reichenbach & Kettenmann (1999): Nat Neurosci, 2, 139-143.
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Neuronal-glial chemical transmission mediated through
glutamate
Ionotropic receptorsMetabotropic receptorsGlutamate transporters
15Astrocytes form the main glutamate-uptake system in the CNS
Verkhratsky & Butt, 2007, Glial Neurobiology, A TextbookWiley & Sons
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Voltage-dependence of GluT- and AMPAR-mediated currents
Kirischuk, Kettenmann & Verkhratsky (2007): Pflugers Arch, 454, 245-252.
17 Application of glutamate triggers inward current and sodium influx
Kirischuk, Kettenmann & Verkhratsky (2007): Pflugers Arch, 454, 245-252.
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αβγ
αβγ
3Na+ 1Glu-
1H+
1K+
K ~2 MD μ K ~ 3 - 100 MD μ K ~130 MD μK ~50 MD μ
NTNMDAR mGluR1/5 GluNa T +AMPAR
Different sensitivities of glial glutamate sensors allow precise determination of extracellular glutamate concentration
Verkhratsky & Kirchhoff (2007) Neuroscientist13: 1 - 10.
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ATP – principal transmitter in neuronal-glial networks
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ATP-mediated transmission: The Beginning
Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509-581.
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 scienceconcentrated on adenosine. And my stuff [on signaling through ATP] was very much frownedon. There were certain very strong figures—very influential figures, and gifted— and one ofthem was Robert Berne, who had early on claimed that adenosine was the physiologicalregulator of blood flow during hypoxia and ischemia in the heart and other organs. He wascharming and he was clever, and although he collected adenosine from the hypoxic heart, hewas totally opposed to the fact that it might be originating as ATP.
At the opening party of the second Purines Meeting a while later, I was suddenly trapped in thecorner by several scientists who were also very opposed to the idea of ATP as an extracellularregulator, and being an ex-boxer, I got very worried because I had the sense that it was goingto become physical. People were very passionately against this!
GEOFFREY BURNSTOCK: MOST HIGHLY CITED SCIENTIST Molecular Interventions (2004) 4:192-195.
Geoffrey Burnstock was born on May 10, 1929
ATP was discovered in 1929
Extracellular signalling role of purines was suggested in 1929
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The purines were the part of very early signalling evolution. Indeed the purines in aform of adenine and guanine phosphates occurred in prebiotic period (as a result ofpurely thermal synthetic processes) and rapidly acquired high importance. Purines andpyrimidines were essential for construction of both RNA and DNA, and hence theirintracellular concentration was high. Furthermore, ATP was selected very early as themain source of biological energy, and thus became an indispensable feature of life.The prominent roles of adenine-phosphates in energy metabolism in the most primitivelife forms and ancient origin of adenine-binding sites are widely recognised. This was acritical evolutionary choice as it immediately resulted in birth of the universalintracellular signalling system based on calcium ions as keeping cytosolic Ca2+
extremely low became vitally important, since otherwise insoluble Ca2+-phopshateswould preclude the cell energetics. Thus even the most primitive ancient cell had highcytosolic concentrations of ATP (or GTP) and upon cell disintegration gradients of bothwould be present in the surrounding water. This may have been the initial form ofchemical transmission, which remained throughout evolution. Indeed every known cellor single cell organism does display some form of sensitivity to ATP, and moreover theATP transmission is unique in a sense that it is not confined to a particular tissue ororgan of higher organisms – it exists everywhere. Indeed there is difficult to find a celltype, which does not show sensitivity to ATP.
ATP: the primordial signalling molecule
Burstock & Verkhratsky (2009): Acta Physiologica, 195, 415-447
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Mechanisms of ATP release in the central nervous system
23 ATP release and degradation in the CNS
Abbracchio, Burnstock, Verkhratsky & Zimmermann (2009): TINS, 32, 19 - 29
ATP AdenosineADP AMP
NTPDases, NPPs, Alk Phosp, 5‘-Nuc, Myok, NDK
ATP degradation
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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: 257-265
Pankratov, Lalo, Verkhratsky, North (2007)J. Gen. Physiol, 129: 257-265
Exocytotic ATP release from synaptic terminals25
8 pA
ΔF/F0(100%)
A B TFLLR 10 Mμ
TFLLR 10 Mμ
Astrocyte [Ca ]2+
i
Astrocyte [Ca ]2+i
HEK293 Im
HEK293 Im
PPADS 10 Mμ5 s
Quantal release of ATP from acutely isolated cortical astrocytes
Pankratov, Lalo, Verkhratsky, submitted
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Purinoceptors: An overview
Abbracchio, Burstock, Verkhratsky & Zimmermann (2009): TINS, 32, 19 - 29
27 ATP as a universal transmitter in neuronal-glial circuits
Verkhratsky, Krishtal, & Burnstock (2009) Mol Neurobiol 39,190-208.
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Iono- and metabotropic ATP receptors expressed in astroglia trigger calcium signals in the optic nerve
Hamilton, Vayro, Kirchhoff, Verkhratsky, Robbins, Gorecki & Butt (2008): Glia 56:734-749.
29ATP as universal glial transmitter: Oligodendrocytes
Kirischuk S, Scherer J, Kettenmann H, Verkhratsky A (1995) J Physiol (London) 483 :41-57.
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ATP-induced currents in cortical astrocytes
Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008): Journal of Neuroscience, 28: 5473 - 5480.
31 Concentration-dependence of ATP-induced currents
Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008): Journal of Neuroscience, 28: 5473 - 5480.
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Pharmacology of ATP-induced currents
* TNP-ATP: 2′,3′-O-(2,4,6- trinitrophenyl) adenosine 5′-triphosphate
Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008): Journal of Neuroscience, 28: 5473 - 5480.
33 Subunit composition of astroglial P2X receptor
P2X receptorexpressed in artificial systems
1/5 AstroglialP2X receptor
Kinetics
Rebound upon washout
Ca -dependenceo
TNP-ATP
Inhibition by PPADS
Sensitivity to ATP
Desensitization
Sensitivity to meATPαβ
Exceptionally High, EC ~ 50 nM50
Exceptionally High, EC ~ 40 nM50
Yes Yes
Yes Yes
Biphasic: rapid peak and steady-state
Biphasic: rapid peak and steady-state
No No
No No
100% 100%
Inhibition Inhibition
Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008): Journal of Neuroscience, 28: 5473 - 5480.
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Cortical astrocytes express P2X1 and P2X5 subunits
Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008): Journal of Neuroscience, 28: 5473 - 5480.
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Synaptic stimulation triggers fast astroglial currents mediated by
ionotropic receptors and glutamate transporters
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Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
Glial synaptic currents (GSC) in the neocortex
Stimulus/TTX dependence of GSCs and EPSCs
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Stimulus-frequency dependence of GSCs
Glial synaptic currents (GSC) in the neocortex
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
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Ionotropic receptors and glutamate transporter mediate GSC in the neocortex
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
39 Spontaneous glial synaptic currents
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
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Calcium permeability of astroglial NMDA and P2X1/5 receptors
41 Calcium permeability of NMDA and P2X1/5 receptors in cortical astrocytes
PCa/Pmono ~ 3 PCa/Pmono ~ 2
Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, v. 28, p. 225-231
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Stimulation of NMDA and P2X1/5 receptors triggers Ca2+ signals in isolated cortical astrocytes
Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, v. 28, p. 225-231
43 Synaptic stimulation-induced Ca2+ signals in cortical astrocytes in situare sensitive to ionotropic receptors antagonists
Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, v. 28, p. 225-231
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Age-dependent remodelling of ionotropic receptors expression in cortical astroglia
45 In situ recordings from ageing cortical astroglia
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
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Ageing changes the relative weight of GSCs components3 months
9 months
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
47 Ageing modifies expression of glutamate and purinergic receptors in astroglia
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
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Ageing affects ionotropic Ca2+ signalling in astroglia in situ
Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.
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Lalo, Pankratov, Parpura & Verkhratsky (2011) BBA, in press.
Ionotropic receptors in neuronal-astroglial signalling: What is the role of “excitable” molecules in non-excitable cells?Fast local signalling in astroglial perisynaptic compartments?
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Astroglial cells express several classes of fast ionotropicreceptors. These receptors are activated by ongoingsynaptic transmission and mediate local cytoplasmicsignalling mediated through [Ca2+]i and [Na+]i transients.Rapid activation of astroglial ionotropic receptors can beinstrumental for highly localised neuronal-glial signalling atthe synaptic level.
Age-dependent decrease in synaptic communication inneuronal-glial circuitry may signify the beginning of age-dependent decline in brain functions; alternatively however,the bell-shaped changes in glial synaptic transmission mayreflect maturation of the CNS, pruning of functionallyirrelevant synaptic contacts and manifest the “attainment ofwisdom” which generally accompanies advanced age.
Conclusions51