1. Nitric Oxide: an endogenous neurotoxin? Robert Keynes Neural Signalling

2. NO in the CNS Learning and Memory Neurodegeneration Cerebral blood flow

4. A vicious cycle to cell death? NMDA Receptors Glutamate release brief stimulation Ca 2+ nNOS delayed cell death NO L-Arg cell death

6. Response to NMDA 100  M 300  M 1000  M CA1 DG CA3

7. NMDA antagonist – MK801 (during recovery)

8. Inhibition of NOS (throughout expt)

10. How much NO is released? EC 50 of NO at GC =2 nM cGMP < 50 % of max (< 2 nM NO)

11. How much NO is required for toxicity? non-toxic levels DETA/NO 300  M = 1.2  M NO NOC-12 0.3 mM = 2.8  M NO DETA/NO 3 mM = 4.5  M NO NOC-12 1 mM = 6  M NO 1-2  M NO is toxic in dispersed cultures Bal Price and Brown (2000) J.Neurosci, 21 , 6480-6491 10  M NO is toxic

14. NO is inactivated by cerebellar cells and homogenates in vitro 0 10 30 40 0 100 200 300 400 500 Homogenate Cells Buffer [NO] (nM) Time (min) Data from Dr Charmaine Griffiths

15. Inhibiting NO inactivation Ascorbate Oxidase DTPA – Iron Chelator Trolox – antioxidant 0 5 30 40 50 400 300 200 100 [NO] (nM) Time (min) Buffer Hom + A scorbate O xidase Hom 0 0 5 10 15 0 100 200 300 400 500 Cells Cells + DTPA Buffer [NO] (nM) Time (min)

16. Ascorbate and Iron (Leaks from cells) (contaminant) + NO NO consumed Padmaja and Huie., (1993) Biochem.Biophys.Res.Commun. 195, 539-544 Ascorbate Oxidase DTPA Trolox Peroxidation inhibited Goss et al. (1997) J. Biol. Chem. 272, 21647-21653 Lipid peroxidation LOO ● Ascorbate (Fenton reaction) OH ● Iron + H 2 O 2

17. 0 10 30 40 0 100 200 300 400 500 Homogenate Cells Buffer [NO] (nM) Time (min) NO is inactivated by reaction with a pool of peroxidising lipid Continuing NO release prevents further peroxidation - inactivation finally saturates

19. Peroxidation-independent NO inactivation 0 2 4 6 8 10 0 100 200 300 400 Control [NO] (nM) Time (min) 0.5 x 10 6 / ml 1 x 10 6 / ml 2 x 10 6 / ml