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

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

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

Zinc Containing Neurons Neurons that sequester weakly bound (histochemically active) Zn 2+ 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.

Zinc Containing Neurons- Subset of Glutamatergic Neurons It appears that all zinc containing neurons are glutamatergic, but only some glutamatergic neurons contain Zn 2+

Zinc Containing Neurons- Subset of Glutamatergic Neurons Supporting Evidence- Presynaptic boutons of GABA sequestering neurons do not seem to sequester Zn 2+ Zn 2+ 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 Zn 2+ boutons

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

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)

What About Non-Zinc-Containing Glutamatergic Neurons? Mostly sub-cortical or spinal Tempting to hypothesize that the function of Zn 2+ in glutamatergic neurons may be related to cognition and/or memory

Visualization of Zn 2+ Containing Neurons Zinc is loaded into presynaptic vesicles by the transport protein ZnT-3 This protein appears highly specific to Zn 2+ Possible to stain ZnT-3 with immunohistochemistry Pitfall- Possible some neurons express the ZnT-3 gene but the protein undergoes post-translational modification

Visualization of Zn 2+ 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 Zn 2+ remains in the axonal boutons, leading to overexposure.

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

So What’s the Problem? Electrophysiology may also help quantify the amount of Zn 2+ in a given region Recent paper by Brown and Dyck claims to eliminate bouton staining

Images of Zn 2+ Containing Neurons Figure courtesy of Frederickson et al., 2000

Zn 2+ Localization in the Hippocampus The hippocampus appears to have four setsof Zn 2+ containing neurons- Prosubicular neurons Dentate granule neurons CA3 neurons CA1 neurons

Zn 2+ Localization in the Hippocampus Figure courtesy of Frederickson et al., 2000

Zn 2+ Localization in the Hippocampus Figure courtesy of Brown and Dyck, 2004

Zn 2+ in the Hippocampus- Required for LTP in CA3 Rapid chelation of Zn 2+ by 10 mM CaEDTA blocked LTP induction in CA3 Addition of 100 µM exogenous Zn 2+ was sufficient to induce LTP in CA3 Glutamate enhances Zn 2+ induced LTP in CA3

Rapid Chelation of Zn 2+ by CaEDTA blocks LTP in CA3 LTP in Normal ACSFLTP in 10 mM CaEDTA Figures courtesy of Li, et al. 2001

Addition of 100 µM Zn 2+ is sufficient to induce LTP in CA3 Figures courtesy of Li, et al LTP induced with 100 µM exogenous Zn 2+

Glutamate Enhances Zn 2+ Induction of LTP in CA3 Figures courtesy of Li, et al Combined effects of Glutamate and Zinc on the EPSP

Pitfalls- 10 mM CaEDTA?! That’s a lot! Figures courtesy of Li, et al nM CaEDTA is necessary to rapidly chelate Zn 2+

Pitfalls- What about Ca 2+ ? CaEDTA actually decreases free Ca 2+ Necessary to compensate ACSF by adding 0.22 mM CaCl 2 Figure courtesy of Li, et al. 2001

Pitfalls- What about Ca 2+ ? Figures courtesy of Li, et al mM CaEDTA has no effect on basal transmission 10 mM CaEDTA has no effect on paired pulse facilliation

Current Question- What is the Role of Zn 2+ in CA1? The amount of free Zn 2+ in the CNS is hard to quantify Growing consensus- Less Zn 2+ in CA1 New images from Brown and Dyck indicate there may be more!

Current Question- What is the Role of Zn 2+ in CA1? May be possible to gain some insight into Zn 2+ function in CA1 using CaEDTA

Preliminary Investigations- LTP in CA1

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

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

References To be added later

Questions?