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

Neuronal-glial chemical transmission mediated by glutamate and ATP

Prof. Alexei Verkhratsky

The screen versions of these slides have full details of copyright and acknowledgements 1

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Prof. Alexei Verkhratsky MD, PhD, DScProfessor of Neurophysiology, The University of Manchester,

Faculty of Life Sciences, Manchester, UK

Neuronal-glial chemical transmissionmediated by glutamate and ATP

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Experiments on glial cells in tissue culture demonstrated expression of neurotransmitter receptors

Verkhratsky & Kettenmann (1996) Trends Neurosci., 19: 346-352

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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

Verkhratsky, Orkand & Kettenmann (1998) Physiol Rev., 78: 99-141

P2YP2X

H1

H1

ETB(?)

Purkinje neurone

GABAA

AMPA mGluR1

PLC

PLC

GABA receptors

Glutamate receptors

Purinoreceptors

Histamine receptors

Adrenoreceptors

Endotheline receptors

Bergmann glial cell

PLC

PLC

P2Y

H1

H1

ETB GABAA

AMPAmGluR1,5

PLC

CF

NA, ATP, Hist

CFPF

NTLC

Glu

BAST

GABA

BAST




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Astrocytes form communication “channels” with nearby neurones

Verkhratsky & Kirchhoff (2007) Neuroscientist, 13: 1-10

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Neurotransmission by glutamate: glutamate sensors


Neurotransmission by glutamate: glutamate sensors

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Neuronal-glial chemical transmission mediated through glutamate

• Ionotropic glutamate receptors




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Identification of neurotransmitter receptors in cultured astrocytes

Bowman, Kimelberg (1984) Nature, 311: 656-659 (IF ~20)

GABA Aspartate

Glutamate Glycine

Vm (mV)

L-glutamate D-glutamate

L-aspartate D-aspartate NMDA

Kainic acid

Quinquialate

Kettenmann, Backus, Schachner (1984) Neurosci Lett, 52: 25-29 (IF~2)

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Kainate induces massive calcium influxmediated by AMPA receptors into Bergmann glial cells

in cerebellar slice preparations

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

A Control Kainate

F340/380

Purkinje cell layer

Bergmann glial cell

Patch pipette loaded with Fura-2

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• The NMDA receptor was, for a long time, believed to have exclusively neuronal localisation

• The NMDA receptors possess an important property of Mg2+ 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: 145-150Pankratov Y, Lalo U, Krishtal O, & Verkhratsky A.(2002) J. Physiol., 542: 529-536Akaike N & Moorhouse AJ. (2003) Trends Pharmacol. Sci., 24: 44-47

NMDA receptors in astroglial cells

Vm (mV)

-80 -40 40

1.0

0.5

-0.5

2.0

I nor

m




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• 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: 2673-2683

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I nor

m

0.0

0.2

0.4

0.6

0.8

1.0

0.01 1 100[NMDA], mM

NMDA induces glycine-sensitive current response in isolated astrocytes

Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006)Journal of Neuroscience, 26: 2673-2683

A

N

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Astroglial NMDA receptors are magnesium insensitive

Lalo, Pankratov, Kirchhoff, North & Verkhratsky (2006) Journal of Neuroscience, 26: 2673-2683

-1.0

-0.5

D

-80 -40 40

Vm (mV)

0 mM Mg2+

10 mM Mg2+5 mM Mg2+

I nor

m




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• Ionotropic receptors

• Metabotropic receptors


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Parallel fibers stimulation triggers calcium signalsin Bergmann glial cell microdomains

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

PCL

PF

Pia

BG

STIM

200 mm

A B

1 2

10 mmD

4 mm

0

255

C

2 s

1 2

2 s

1.6

1.0F/F0

F/F0

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• Ionotropic receptors

• Metabotropic receptors

• Glutamate transporters





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Astrocytes form the main glutamate-uptake system in the CNS

Verkhratsky & Butt, (2007) Glial Neurobiology A Textbook, Wiley & Sons

80%

20%

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Voltage-dependence of GluT- and AMPAR-mediated currents

Kirischuk, Kettenmann & Verkhratsky(2007) Pflugers Arch., 454: 245-252

Inorm

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Application of glutamate triggers inward current and sodium influx

Kirischuk, Kettenmann & Verkhratsky(2007) Pflugers Arch., 454: 245-252




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Different sensitivities of glial glutamate sensors allow precise determination of extracellular glutamate concentration


AMPAR GluNa+T mGluR1/5

KD ~2 mM KD ~3 - 100 mM KD ~130 mMKD ~50 mM

NMDAR

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ATP – principal transmitter in neuronal-glial networks

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ATP-mediated transmission: the beginning

• 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 ATP] 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 ATP. 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 ATP 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: 192-195

• ATP was discovered in 1929

• Extracellular signalling role of purines was suggested in 1929

• Geoffrey Burnstock was born on May 10, 1929Burnstock G., (1972) Purinergic nerves, Pharmacol. Rev., 24: 509-581




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• 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, ATP 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 signallingsystem based on calcium ions, as keeping cytosolic Ca2+ extremely low became vitally important, since otherwise insoluble Ca2+-phopshates would preclude the cell energetics

• Thus even the most primitive ancient cell had high cytosolic concentrations of ATP (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 ATP, and moreover the ATP 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 ATP

Bursntock& Verkhratsky (2009) Acta Physiologica, 195: 415-447

ATP: the primordial signalling molecule

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Mechanisms of ATP releasein the central nervous system

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ATP release and degradation in the CNSExocytotic and

non-exocytotic release in astrocytes

ATP AdenosineADP AMP

NTPDases, NPPs, Alk Phosp, 5‘-Nuc, Myok, NDK

“Maxi” channels

Exocytosis in presynaptic terminals

Ca2+ Ca2+

Ca2+ Ca2+

Transporter-mediated release in axons

ATP

Damaged cells

Abbracchio, Burnstock, Verkhratsky & Zimmermann

(2009) TINS, 32: 19-29




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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

Exocytotic ATP release from synaptic terminals

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Quantal release of ATP from acutely isolated cortical astrocytes

Lalo, Verkhratsky, Pankratov (2011)Semin Cell Dev Biol 22: 220-228

A

Isolated astrocyte

HEK293-P2X2 cell

B TFLLR 10 mM

Astrocyte [Ca2+]iDF/F0(100%)

8 pA

5 s

HEK293 Im

PPADS 10 mM

Astrocyte [Ca2+]i

HEK293 Im

TFLLR 10 mM

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Purinoceptors: an overview

Abbracchio, Burnstock, Verkhratsky & Zimmermann (2009) TINS, 32: 19-29




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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 expressedin astroglia trigger calcium signals in the optic nerve

Hamilton, Vayro, Kirchhoff, Verkhratsky, Robbins, Gorecki & Butt (2008) Glia, 56: 734-749

ATP ATP

ATP ATP

DF/F0

30s

Thapsigargin

Zero-Ca2+

ATP ATP

ATP ATP

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ATP as a universal glial transmitter: oligodendrocytes

Kirischuk S, Scherer J, Kettenmann H, Verkhratsky A., (1995) J. Physiol., (London) 483: 41-57

A

B

D

C

E

10 mm

25 mm




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ATP-induced currents in cortical astrocytes

Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008)

Journal of Neuroscience, 28: 5473 - 5480

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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

A

B




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Subunit composition of the astroglial P2X receptor

Lalo, Pankratov, Wichert, Rossner, North, Kirchhoff & Verkhratsky (2008)Journal of Neuroscience, 28: 5473 - 5480

P2X1/5 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

Sensitivity to ATP

Sensitivity to abmeATP

Kinetics

Rebound upon washout

Ca0-dependence

Desensitization

Inhibition by PPADS

TNP-ATP

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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 currentsmediated by ionotropic receptors

and glutamate transporters




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• Stimulus/TTX dependence of GSCs and EPSCs

• Stimulus-frequency dependence of GSCs

Glial synaptic currents (GSC) in the neocortex

Lalo, Palygin, North, Verkhratsky, Pankratov, (2011) Aging Cell 10: 392-402

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Ionotropic receptors and glutamate transporter mediate GSC in the neocortex


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Spontaneous glial synaptic currents


A B




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Calcium permeability of astroglial NMDA and P2X1/5 receptors

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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, 28: 225-231

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

Palygin, Lalo, North, Verkhratsky & Pankratov, (2010) Cell Calcium, 28: 225-231




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Synaptic stimulation-induced Ca2+ signals in cortical astrocytes in situ

are sensitive to ionotropic receptors antagonists

C

Palygin, Lalo, North,Verkhratsky & Pankratov, (2010)

Cell Calcium, 28: 225-231

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Age-dependent remodelling of ionotropic receptors expression in cortical astroglia

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In situ recordings from ageing cortical astroglia


B




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Ageing changes the relative weight of GSCs components3 months

9 months

CLalo, Palygin, North, Verkhratsky, Pankratov, (2011)

Aging Cell 10: 392-402

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Ageing modifies expression of glutamateand purinergic receptors in astroglia


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Ageing affects ionotropic Ca2+ signalling in astroglia in situ





49Lalo, Pankratov, Parpura, Verkhratsky (2011) Biochim Biophys Acta 1813: 992-1002

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 iGluRsP2XRs EAAT Na/K

pump

Lactate shuttle

Direct mode

Reverse mode

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• Astroglial cells express several classes of fast ionotropic receptors

• These receptors are activated by ongoing synaptic transmission and mediate local cytoplasmic signalling mediated through [Ca2+]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

Conclusions

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