Synaptic corelease of ATP and GABA in cultured spinal neurons

Young-Hawan Jo and Rémy Schlichter

Laboratoire de Neurophysiologie cellulaire et intégrée, UMR 7519 CNRS, Université Louis Pasteur, 21 rue Descartes, Strasbourg, France

Problem

Introduction Methodology Results Discussion

Sensory

inputs

Touch receptors (skin)

Neuronal

pathway

Somatosensory cortex (SI)

Problem

Introduction Methodology Results Discussion

Roles : Transmission Modulation

Peripheral nociceptive sensory messages

Problem

Introduction Methodology Results Discussion

Inputs

Aδ + C Glutamate + Subtance P (fast and slow)

Local interneurons Glut ou GABA

Electrical stimulation of Iaire afferent ATP release in superficial Dorsal Horn by local interneurons (Sawynok & Downie, 1993, Brain research)

Problem

Introduction Methodology Results Discussion

Expression mRNAs that coding for P2X2, P2X4, P2X6 subunits in Lamina I and II (Collo, 1996, J. Neuroscience)

Problem

Introduction Methodology Results Discussion

ATP may serve as a fast neurotransmitter between superficial

dorsal horn neurons?

Cultured spinal dorsal horn

neurons

10dPerforated

patch-clampIPSP or EPSP

Introduction Methodology

Synaptic transmission

Results Discussion

AP-5 + CNQX + Bicuculine + Strychnine = BSCA

Coreleased ATP-GABA

P2X Rr

GABA Rr

Glutamate Rr

Glycinergique Rr

Pré

Post

Inward currents = EPSPs

-60mV

Interaction ATP-GABA

P2X Rr

GABA Rr

Glutamate Rr

Glycinergique Rr

Pré

Post

Introduction Methodology

Synaptic transmission

Results Discussion

Suramine ou PPADS

Coreleased ATP-GABA

-60mV

Interaction ATP-GABA

Release of ATP acts on P2X Rr

Introduction Methodology

Synaptic transmission

Results Discussion

Differents threshold

Coreleased ATP-GABA

P2X Rr

GABA Rr

Glutamate Rr

Glycinergique Rr

Pré

Post

Reversal potential = 0 mV nonselective cation conductance

Interaction ATP-GABA

Introduction Methodology

Synaptic transmission

Results Discussion

EPSPs

Ca2+ Na+

K+

Reversal potential = 0mV

Coreleased ATP-GABA Interaction ATP-GABA

Pré

Post

Pré

Post

P2X Rr

GABA Rr

Glutamate Rr

Glycinergique Rr

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA

 Wash-out CNQX and

AP-5

Wash-out bicuculine

No EPSPs under BSCA

Yes Yes

EPSPs under BSCA

No Yes

I = BSCAII = Whas-out bicuculine

=> ATP is coreleased with GABA

EPSPc No EPSPc

Interaction ATP-GABA

BSCA

EPSP

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA

Pré

Post

Glutamate

No ATP

Pré

Post

Gaba

ATP

Interaction ATP-GABA

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA

Pré

Post

Gaba

ATP

P2X Rr

GABA Rr

If Em = Ex ions flux = 0

=> 70 % of GABAergic synapse corelease ATP

ECl = -90mVEcations = 0 mV

Interaction ATP-GABA

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA Interaction ATP-GABA

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA

Pré

Post

Gaba

Pré

Post

Gaba

70% 30%

Interaction ATP-GABA

P2X Rr

GABA Rr

GABA

ATP

+ spontaneous liberation of GABA and no ATP

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA Interaction ATP-GABA

Post

P2X Rr

GABA Rr

Isoguvacine = Agonist GABAa

ATP = non hydrolysable analog

ATP

Degradation of ATP (metabolite) inhibs IPSP cause

by isoguvacine

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA Interaction ATP-GABA

Post

Isoguvacine = Agonist GABAa

DCPCPX = antagoniste of adenosine RrAdenosineATP

P2X Rr

GABA Rr

Adenosine Rr ATP Adenosine Inhibs GABA IPSP

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA Interaction ATP-GABA

Introduction Methodology

Synaptic transmission

Results Discussion

Coreleased ATP-GABA Interaction ATP-GABA

Adenosine

ATP Pré

Post

Gaba

GABA

5 nucléotidase

-

Adenosine Rr

P2X Rr

GABA Rr

Introduction Methodology Results Discussion

Result are in accord with the fact that P2X receptor mediated excitatory synaptic transmission in 50% of neurons in DH ATP is a prominent NT

ATP is coreleased with GABA

In vivo they don’t find corealesed of ATP and GABA

Important because it doesn’t imply a circuitry to modulate response

Can have implication in a hyperalgesia (repeated stimulation)

Thank you for your

attention