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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/. Subjective Analytical.
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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
Subjective Analytical IRMf. Photo: Jeff Miller. University of Wisconsin-Madison. • Imaging Neurotransmission in the Brain by IRMf: 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.
Modulation of Complex Biological Assemblies Interrelations • Control of Blood Supply in Brain by Local Release of NO : • Control of Blood Supply in Brain by Local Release of NO ? d d+Dd Rat Brain Slice 300µm. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results. 2004.
Oxidative Stress and Neurotransmission: • Control of Blood Supply by Dilatation of Blood Vessels : tissue lumen muscle cells endothelial cells
NO° • Oxidative Stress and Neurotransmission: • Control of Blood Supply by Dilatation of Blood Vessels : guanylatecyclases lumen phosphokinases proteins phosphorylation
NO Release in Aerobic Cells • NO Synthases: N H N H N H 2 2 2 N O + H N N O 2 N H H O N H N H O 2 2e O + e 2 - - - O O O + + + H N O H N O H N O 3 3 3 L-Arginine N-hydroxy-arginine Citrulline
cerebellum stellate neuron sagittal cut (300µm; vibratome) • Modulation of Complex Biological Assemblies Interrelations • Control of Blood Supply in Brain by Local Release of NO : • Control of Blood Supply in Brain by Local Release of NO : Rat Brain
20 µm • Modulation of Complex Biological Assemblies Interrelations
Patch Clamp onto Neuron Body: stellate neuron body blood capillary vessel patch-clamp pipette • 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: Characterization of NOS (RT PCR) stellate neuron body blood capillary vessel patch-clamp pipette • 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
5 µm 1.2 glass cases 1 0.8 NO° / 1µM Current (a.u.) platinized surfaces insulating polymer 0.6 0.4 1-5 µm 10-12 µm 0.2 E / mV vs. SSCE 0 200 400 600 800 • Platinized Carbon Fiber Ultramicroelectrodes
CAMERA camera potential optical microscope potentiostat patch-clamp NO-ultramicroelectrode peristaltic pump rat brain slice O2 & CO2 artificial brain medium based on Ringer buffer white and IR light source for differential contrast • Modulation of Complex Biological Assemblies Interrelations • Control of Blood Supply in Brain by Local Release of NO : • 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.
0.5 µM NO° current d - vessel diameter DEANOOate 100 µm diazeniumdiolate 0.62 V vs. Ag/AgCl time • Modulation of Complex Biological Assemblies Interrelations • Test 1: Vessel Stimulation by External Local Chemical Release of NO° 20 pA 5 µm 5 min 5 µm ( 5%) 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 • Test 2: Stimulation via Chemical Stimulation of Stellate Neuron by NMDA 5 pA 0.15 µM NO° current d NMDA 100 µM Controls: • TTX (): tetrodotoxin (blocking of ion channels) • LNAME (): Nitro L Arginine Methyl Ester 5 min Star neurons NMDA NO° NOsynthase vessel diameter 2 µm ( 2%) 0.62 V vs. Ag/AgCl time 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.
Action Potential Firing ~V Patch-Clamp Stim 10 0.16 8 0.12 6 NO° flux (fmol/s) 0.08 4 Current (pA) 0.04 2 0 0 -0.04 -2 0 5 10 TTX (1 µM) L-NAME (1 mM) • Modulation of Complex Biological Assemblies Interrelations • Electrophysiological Stimulation with Patch-Clamp Pipette 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.
Modulation of Complex Biological Assemblies Interrelations • Electrophysiological Stimulation with Patch-Clamp Pipette 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.
Action Potential Firing NO° 0.1 µM NO° ~V • Modulation of Complex Biological Assemblies Interrelations • Electrophysiological Stimulation with Patch-Clamp Pipette 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.
ESPCI (Paris): • Prof Jean ROSSIER • Dr Bruno CAULI • Dr Armelle RANCILLAC • KNURE (Kharkov, Ukraine): • Prof Irina SVIR • Dr Alexander OLEINICK • Dr Oleksiy KLYMENKO Acknowledgements • ENS: • 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