The Functional Role of Zinc in the Central Nervous System Review and Current Research

1. The Functional Role of Zinc in the Central Nervous System Review and Current Research ~Josh Ketterman ~Dr. Yang Li

2. The Li Lab- What is the Functional Significance of Zn2+? • Topics of particular interest: • Zn2+ release during ischemia • Epileptic Models- Is Zn2+ excitatory or inhibitory? • Long Term Potentiation

3. How Do We Study Zinc? • A mixture of electrophysiology and fluorescent imaging • My lab mate Chinta does the imaging! • My current focus is on LTP in the hippocampus

4. Zinc Containing Neurons • Neurons that sequester weakly bound (histochemically active) Zn2+ in the vesicles of their presynaptic boutons • 5%~10% of the total brain zinc is weakly bound in these vesicles • This small amount accounts for nearly 100% of the histochemically active brain zinc.

5. Zinc Containing Neurons-Subset of Glutamatergic Neurons • It appears that all zinc containing neurons are glutamatergic, but only some glutamatergic neurons contain Zn2+

6. Zinc Containing Neurons-Subset of Glutamatergic Neurons • Supporting Evidence- • Presynaptic boutons of GABA sequestering neurons do not seem to sequester Zn2+ • Zn2+ containing boutons are absent in regions where the terminals of GABAergic neurons are densely concentrated • Boutons that are immunoreactive for glutamate include a high concentration of Zn2+ boutons

7. So Where are these Neurons? • A great proportion of “glutazinergic” neurons are found in the cerebral cortex and the amygdala

8. So Where are these Neurons? • Efferent zinc-containing fibers from these regions extend to • The cerebral cortex and the Amygdala • Striatum • Limbic targets • (septum, medial hypothalamus)

9. What About Non-Zinc-Containing Glutamatergic Neurons? • Mostly sub-cortical or spinal • Tempting to hypothesize that the function of Zn2+ in glutamatergic neurons may be related to cognition and/or memory

10. Visualization of Zn2+ Containing Neurons • Zinc is loaded into presynaptic vesicles by the transport protein ZnT-3 • This protein appears highly specific to Zn2+ • Possible to stain ZnT-3 with immunohistochemistry • Pitfall- Possible some neurons express the ZnT-3 gene but the protein undergoes post-translational modification

11. Visualization of Zn2+ Containing Neurons • Also possible to label vesicular Zn2+ by precipitating with intravital selenium • Precipitated Zn2+ then undergoes retrograde transport to the soma • ZnSe precipitate can be then be histochemically stained • Pitfall- A large amount of precipitated Zn2+ remains in the axonal boutons, leading to overexposure.

12. So What’s the Problem? • Using traditional staining methods, it is difficult to quantify the amount of zinc in a given region • Fluorescent imaging may provide new insights • Chintha’s work…more interesting than my presentation

13. So What’s the Problem? • Electrophysiology may also help quantify the amount of Zn2+ in a given region • Recent paper by Brown and Dyck claims to eliminate bouton staining

14. Images of Zn2+ Containing Neurons Figure courtesy of Frederickson et al., 2000

15. Zn2+ Localization in the Hippocampus • The hippocampus appears to have four setsof Zn2+ containing neurons- • Prosubicular neurons • Dentate granule neurons • CA3 neurons • CA1 neurons

16. Zn2+ Localization in the Hippocampus Figure courtesy of Frederickson et al., 2000

17. Zn2+ Localization in the Hippocampus Figure courtesy of Brown and Dyck, 2004

18. Zn2+ in the Hippocampus- Required for LTP in CA3 • Rapid chelation of Zn2+ by 10 mM CaEDTA blocked LTP induction in CA3 • Addition of 100 µM exogenous Zn2+ was sufficient to induce LTP in CA3 • Glutamate enhances Zn2+ induced LTP in CA3

19. Rapid Chelation of Zn2+ by CaEDTA blocks LTP in CA3 LTP in Normal ACSF LTP in 10 mM CaEDTA Figures courtesy of Li, et al. 2001

20. Addition of 100 µM Zn2+ is sufficient to induce LTP in CA3 LTP induced with 100 µM exogenous Zn2+ Figures courtesy of Li, et al. 2001

21. Glutamate Enhances Zn2+ Induction of LTP in CA3 Combined effects of Glutamate and Zinc on the EPSP Figures courtesy of Li, et al. 2001

22. Pitfalls- 10 mM CaEDTA?! That’s a lot! 10 nM CaEDTA is necessary to rapidly chelate Zn2+ Figures courtesy of Li, et al. 2001

23. Pitfalls- What about Ca2+? • CaEDTA actually decreases free Ca2+ • Necessary to compensate ACSF by adding 0.22 mM CaCl2 Figure courtesy of Li, et al. 2001

24. Pitfalls- What about Ca2+? 10 mM CaEDTA has no effect on basal transmission 10 mM CaEDTA has no effect on paired pulse facilliation Figures courtesy of Li, et al. 2001

25. Current Question- What is the Role of Zn2+ in CA1? • The amount of free Zn2+ in the CNS is hard to quantify • Growing consensus- Less Zn2+ in CA1 • New images from Brown and Dyck indicate there may be more!

26. Current Question- What is the Role of Zn2+ in CA1? • May be possible to gain some insight into Zn2+ function in CA1 using CaEDTA

27. Preliminary Investigations- LTP in CA1

28. Where do we go from here? • Get a nice EPSP in CA1 • Add 10 mM CaEDTA and… • ?? See what happens! • Hopefully we’ll do patching soon

29. Special Thanks • Dr. Yang Li • Dr. Colvin and Dr. Holmes • Labmate: Chintha • The Undergraduate Research Assistants

30. References • To be added later

31. Questions?