Post on 10-May-2015
Neurons and Oxidative stress in Brain:
“Claiming for the Right Supply of Blood”
Christian Amatore
Ecole Normale Supérieure, Département de Chimie
UMR CNRS-ENS-UPMC 8640 "PASTEUR"
Paris - France
Adapted from: http://www.abcam/neuroscience/
Imaging Neurotransmission in the Brain by IRMf:
IRMf. Photo: Jeff Miller. University of Wisconsin-Madison.
Ecoute subjective ou analytique d’une même pièce de musique par le même sujet. Dept. Neurology and Radiology, Münster.
IRMf d’un sujet adulte de 24 ans durant une tâche de génération de mots. Dept. Neurology and Radiology, Münster.
Subjective Analytical
d
d+d
Modulation of Complex Biological Assemblies Interrelations
o Control of Blood Supply in Brain by Local Release of NO :o Control of Blood Supply in Brain by Local Release of NO ?
Rat Brain Slice 300µm.
Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
Oxidative Stress and Neurotransmission:
o Control of Blood Supply by Dilatation of Blood Vessels :
muscle cellsendothelialcells
tissue
lumen
o Control of Blood Supply by Dilatation of Blood Vessels :
lumen
guanylate cyclasesguanylate cyclases
phosphokinasesphosphokinases
proteinsphosphorylation
proteinsphosphorylation
Oxidative Stress and Neurotransmission:
NO°
NO Release in Aerobic Cells
o NO Synthases:
NH2
2+
NH2
NH N
NH2
O
NH
H3N+ O
O-
NH
H3N+ O
O-
NH
H3N+ O
O-
HO
O2 + e
O2
NO
2e
L-Arginine N-hydroxy-arginine Citrulline
Modulation of Complex Biological Assemblies Interrelations
o Control of Blood Supply in Brain by Local Release of NO :o Control of Blood Supply in Brain by Local Release of NO :
sagittal cut(300µm; vibratome)
stellate neuroncerebellum
Rat Brain Rat Brain
Modulation of Complex Biological Assemblies Interrelations
20 µm
Modulation of Complex Biological Assemblies Interrelations
Molecular Characterization of NOS in Stellate Neuron
Rat Brain Slice 300µm. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. submitted
Patch Clamp onto Neuron Body:
stellateneuron body
patch-clamp pipette
blood capillary vessel
Molecular Characterization of NOS in Stellate Neuron
Characterization of NOS (RT PCR)
Rat Brain Slice 300µm. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. submitted
Patch Clamp onto Neuron Body:
stellateneuron body
patch-clamp pipette
blood capillary vessel
Platinized Carbon Fiber Ultramicroelectrodes
10-12 µm 1-5 µm
glass cases
insulatingpolymer
platinizedsurfaces
5 µm
0
0.2
0.4
0.6
0.8
1
1.2
200 400 600 800
Cur
rent
(a.
u.)
E / mV vs. SSCE
NO° / 1µM
Platinized Carbon Fiber Ultramicroelectrodes
M ic roé lec tr ode M ic roc ap illa ire 0
1
2
3
4
5
0 50 10 0 15 0 200 250 30 0
C ourant/ pA
Tem p s / s
1m M
30 µC
1m M
120 nM 120 nM
600 nMI) II)
III) IV)
0
2
4
6
8
0 20 40 60 80 100 120
C ourant/ n A
Tem ps / s
0
2
4
6
8
10
0 200 400 600 800 1000
C ourant/ nA
[N O°] / µM
0
5
10
15
0 0.5 1 1.5 2
C ourant/ pA
[N O°] / µM
30 µC
Modulation of Complex Biological Assemblies Interrelations
o Control of Blood Supply in Brain by Local Release of NO :o Control of Blood Supply in Brain by Local Release of NO :
Rat Brain Slice 300µm. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
artificial brain medium based on Ringer buffer
optical microscope
peristalticpump
patch-clamp
ratbrainslice
O2 &
CO
2
potentiostat
potential
CAM
ERA
cam
era
white and IR light sourcefor differential contrast
NO-ultramicroelectrode
Modulation of Complex Biological Assemblies Interrelations
o Test 1: Vessel Stimulation by External Local Chemical Release of NO°
5 µm
time
vess
el d
iam
eter
curre
nt 20 pA
5 µm ( 5%)
5 min
DEANOOate 100 µm
0.5 µM NO°
0.5 µM NO°
0.62 V vs. Ag/AgCl
diazeniumdiolate
-ON
NO
N
-
d
Rat Brain Slice 300µm. 0.65 V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
Modulation of Complex Biological Assemblies Interrelations
o Test 2: Stimulation via Chemical Stimulation of Stellate Neuron by NMDA
0.62 V vs. Ag/AgCl
NMDA NO° Star neurons
NOsynthase
d
curre
nt
NMDA 100 µM
5 pA
0.15 µM NO°
0.15 µM NO°
Controls:TTX (): tetrodotoxin (blocking of ion channels) LNAME (): Nitro L Arginine Methyl Ester
Rat Brain Slice 300µm. 0.65 V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
vess
el d
iam
eter
time
2 µm ( 2%)
5 min
o Electrophysiological Stimulation with Patch-Clamp Pipette
Modulation of Complex Biological Assemblies Interrelations
NO° Release
Rat Brain Slice 300µm. 0.65 V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
~V
Action Potential Firing
L-NAME (1 mM)TTX (1 µM)
0 5 10
-2
0
8
10
6
4
2
Patch-Clamp Stim
-0.04
0.04
0.08
0.12
0.16
0Cur
rent
(pA
)
NO
° flu
x (f
mol
/s)
o Electrophysiological Stimulation with Patch-Clamp Pipette
Modulation of Complex Biological Assemblies Interrelations
Rat Brain Slice 300µm. 0.65 V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
o Electrophysiological Stimulation with Patch-Clamp Pipette
Modulation of Complex Biological Assemblies Interrelations
NO° Release and Vessel Dilatation
Rat Brain Slice 300µm. 0.65 V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
~V
Action Potential Firing
NO°
0.1 µM NO°
Modeling NO° Release in Brain
Modeling NO° Release in Brain
Modeling NO° Release in Brain
Acknowledgements
Dr Stéphane ARBAULT
Dr Yann BOURET
Dr Marie ERARD
Dr Yuehong TONG
Dr Imelda BONIFAS
Dr Manon GUILLE
Dr Frédéric LEMAîTRE
Mr Yann VERCHIER
Miss Cécile CROZATIER
Dr Issa TAPSOBA
ENS:
Prof Jean ROSSIER
Dr Bruno CAULI
Dr Armelle RANCILLAC
ESPCI (Paris):
Prof Irina SVIR
Dr Alexander OLEINICK
Dr Oleksiy KLYMENKO
KNURE (Kharkov, Ukraine):