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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 Zn2+? Topics of particular interest: Zn2+ release during ischemia Epileptic Models- Is Zn2+ 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) 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. Zinc Containing NeuronsSubset of Glutamatergic Neurons It appears that all zinc containing neurons are glutamatergic, but only some glutamatergic neurons contain Zn2+ Zinc Containing NeuronsSubset 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 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 Zn2+ in glutamatergic neurons may be related to cognition and/or memory 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 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. 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 Zn2+ in a given region Recent paper by Brown and Dyck claims to eliminate bouton staining Images of Zn2+ Containing Neurons Figure courtesy of Frederickson et al., 2000 Zn2+ Localization in the Hippocampus The hippocampus appears to have four setsof Zn2+ containing neurons Prosubicular neurons Dentate granule neurons CA3 neurons CA1 neurons Zn2+ Localization in the Hippocampus Figure courtesy of Frederickson et al., 2000 Zn2+ Localization in the Hippocampus Figure courtesy of Brown and Dyck, 2004 Zn2+ in the HippocampusRequired 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 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 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 Glutamate Enhances Zn2+ Induction of LTP in CA3 Combined effects of Glutamate and Zinc on the EPSP Figures courtesy of Li, et al. 2001 Pitfalls- 10 mM CaEDTA?! That’s a lot! 10 nM CaEDTA is necessary to rapidly chelate Zn2+ Figures courtesy of Li, et al. 2001 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 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 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! Current Question- What is the Role of Zn2+ in CA1? May be possible to gain some insight into Zn2+ function in CA1 using CaEDTA Preliminary Investigations- LTP in CA1 CA1 LTP March 15, 2005 Paired Pulses Facilitation 700 0.3 0.25 0.2 0.15 EPSP Amplitudes (mV) 600 500 0.1 0.05 0 -0.1 -0.15 400 -0.2 0 0.01 0.02 0.03 0.04 0.05 0.06 Time (Second) High Frequency Stimulation Example Traces of Excitatory Postsynaptic Membrane Potentials (EPSP) 300 First long-term potentiation (LTP) induction reported by Josh on March 15, 2005 0.4 0.2 Control 200 Amplitude of membrane potentials (mV) Percentage of Baseline (%) -0.05 100 Baseline 0 -0.2 -0.4 -0.6 -0.8 0 EPSP potentiation *the trace was recorded 2 hours after LTP induction -30 20 70 120 170 -1 0 0.005 0.01 0.015 0.02 Time (Second) Time (minutes) 0.025 0.03 0.035 0.04 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?