Neurons and Oxidative stress in Brain: “Claiming for the Right Supply of Blood”

1. 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

2. Adapted from: http://www.abcam/neuroscience/

3. 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.

4. 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 Unpublished results. 2004.

5. Oxidative Stress and Neurotransmission: • Control of Blood Supply by Dilatation of Blood Vessels : tissue lumen muscle cells endothelial cells

6. NO° • Oxidative Stress and Neurotransmission: • Control of Blood Supply by Dilatation of Blood Vessels : guanylatecyclases lumen phosphokinases proteins phosphorylation

7. 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

8. 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

9. 20 µm • Modulation of Complex Biological Assemblies Interrelations

10. Modulation of Complex Biological Assemblies Interrelations

11. 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

12. 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

13. 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

14. Platinized Carbon Fiber Ultramicroelectrodes

15. 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.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. Modeling NO° Release in Brain

22. Modeling NO° Release in Brain

23. Modeling NO° Release in Brain

24. 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