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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.

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Presentation on theme: "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."— Presentation transcript:

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:

3  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

4 d d+dd+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 Unpublished results

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

6 o Control of Blood Supply by Dilatation of Blood Vessels : lumen guanylate cyclases phosphokinases proteins phosphorylation proteins phosphorylation  Oxidative Stress and Neurotransmission: NO°

7  NO Release in Aerobic Cells o NO Synthases: NH NH 2 NHN NH 2 O NH H 3 N + O O - NH H 3 N + O O - NH H 3 N + O O - HO O 2 + e O 2 NO 2e L-ArginineN-hydroxy-arginineCitrulline

8  Modulation of Complex Biological Assemblies Interrelations o Control of Blood Supply in Brain by Local Release of NO : sagittal cut (300µm; vibratome) stellate neuron cerebellum Rat Brain

9  Modulation of Complex Biological Assemblies Interrelations 20 µm

10  Modulation of Complex Biological Assemblies Interrelations

11  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: stellate neuron body patch-clamp pipette blood capillary vessel

12  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: stellate neuron body patch-clamp pipette blood capillary vessel

13  Platinized Carbon Fiber Ultramicroelectrodes µm 1-5 µm glass cases insulating polymer platinized surfaces 5 µm Current (a.u.) E / mV vs. SSCE NO° / 1µM

14  Platinized Carbon Fiber Ultramicroelectrodes

15  Modulation of Complex Biological Assemblies Interrelations 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 artificial brain medium based on Ringer buffer optical microscope peristaltic pump patch-clamp rat brain slice O 2 & CO 2 potentiostat potential CAMERA camera white and IR light source for differential contrast NO-ultramicroelectrode

16  Modulation of Complex Biological Assemblies Interrelations o Test 1: Vessel Stimulation by External Local Chemical Release of NO° 5 µm time vessel diameter current 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 - d Rat Brain Slice 300µm V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results

17  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 current 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 V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results vessel diameter time 2 µm (  2% ) 5 min

18 o Electrophysiological Stimulation with Patch-Clamp Pipette  Modulation of Complex Biological Assemblies Interrelations NO° Release Rat Brain Slice 300µm V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results ~V~V Action Potential Firing L-NAME (1 mM)TTX (1 µM) Patch-Clamp Stim Current (pA) NO° flux (fmol/s)

19 o Electrophysiological Stimulation with Patch-Clamp Pipette  Modulation of Complex Biological Assemblies Interrelations Rat Brain Slice 300µm V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results

20 o Electrophysiological Stimulation with Patch-Clamp Pipette  Modulation of Complex Biological Assemblies Interrelations NO° Release and Vessel Dilatation Rat Brain Slice 300µm V vs. SSCE. Collaboration with J Rossier, A Rancillac & B Cauli, UMR CNRS 7637, ESPCI, Paris. Unpublished results ~V~V Action Potential Firing NO° 0.1 µM NO°

21  Modeling NO° Release in Brain

22

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24 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):


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