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.

2

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

6

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).

7

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

8

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

10

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.

11

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.

12


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.

14


glutamate

Ionotropic receptorsMetabotropic receptorsGlutamate transporters

15Astrocytes form the main glutamate-uptake system in the CNS

Verkhratsky & Butt, 2007, Glial Neurobiology, A TextbookWiley & Sons

16

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.

18

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.

19

ATP – principal transmitter in neuronal-glial networks

20

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

21

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

22

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

24

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

26

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.

28

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.

30

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


32

Pharmacology of ATP-induced currents

* TNP-ATP: 2′,3′-O-(2,4,6- trinitrophenyl) adenosine 5′-triphosphate


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


34

Cortical astrocytes express P2X1 and P2X5 subunits


35

Synaptic stimulation triggers fast astroglial currents mediated by

ionotropic receptors and glutamate transporters

36

Lalo, North, Verkhratsky & Pankratov, (2011): Aging Cell, in press.

Glial synaptic currents (GSC) in the neocortex

Stimulus/TTX dependence of GSCs and EPSCs

37

Stimulus-frequency dependence of GSCs

Glial synaptic currents (GSC) in the neocortex


38

Ionotropic receptors and glutamate transporter mediate GSC in the neocortex


39 Spontaneous glial synaptic currents


40

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

42

Stimulation of NMDA and P2X1/5 receptors triggers Ca2+ signals in isolated cortical astrocytes


43 Synaptic stimulation-induced Ca2+ signals in cortical astrocytes in situare sensitive to ionotropic receptors antagonists


44

Age-dependent remodelling of ionotropic receptors expression in cortical astroglia

45 In situ recordings from ageing cortical astroglia


46

Ageing changes the relative weight of GSCs components3 months

9 months


47 Ageing modifies expression of glutamate and purinergic receptors in astroglia


48

Ageing affects ionotropic Ca2+ signalling in astroglia in situ


49

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?

50

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

neuronal-glial chemical transmission mediated by glutamate and...

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