Exocytosis And Iii. Oxidative Stress

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:

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. IRMf. Photo: Jeff Miller.  University of Wisconsin-Madison. Subjective Analytical

d d +  d

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 Unpublished results. 2004.

Oxidative Stress and Neurotransmission:

Control of Blood Supply by Dilatation of Blood Vessels :

muscle cells endothelial cells tissue lumen

Control of Blood Supply by Dilatation of Blood Vessels :

lumen guanylate cyclases phosphokinases proteins phosphorylation

Oxidative Stress and Neurotransmission:

NO°

NO Release in Aerobic Cells

NO Synthases:

N H 2 2 + N H 2 N H N N H 2 O N H H 3 N + O O - N H H 3 N + O O - N H H 3 N + O O - H O O 2 + e O 2 N O 2e L-Arginine N-hydroxy-arginine Citrulline

Rat Brain sagittal cut (300µm; vibratome) stellate neuron cerebellum

20 µm

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

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 Characterization of NOS (RT PCR) Patch Clamp onto Neuron Body: stellate neuron body patch-clamp pipette blood capillary vessel

Platinized Carbon Fiber Ultramicroelectrodes

10-12 µm 1-5 µm glass cases insulating polymer platinized surfaces 5 µm 0 0.2 0.4 0.6 0.8 1 1.2 200 400 600 800 Current (a.u.) E / mV vs. SSCE NO° / 1µM

Platinized Carbon Fiber Ultramicroelectrodes

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 Unpublished results. 2004.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 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

Test 1: Vessel Stimulation by External Local Chemical Release of NO°

5 µm 20 pA 5 µm (  5% ) 5 min 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. time vessel diameter current DEANOOate 100 µm 0.5 µM NO° 0.62 V vs. Ag/AgCl diazeniumdiolate - 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.

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°

Controls:

TTX (  ) : tetrodotoxin (blocking of ion channels)

LNAME (  ) : Nitro L Arginine Methyl Ester

vessel diameter time 2 µm (  2% ) 5 min

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. ~ 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 0 Current (pA) NO° flux (fmol/s)

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.

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°