Transcript Title Goes here

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+?
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Topics of particular interest:
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Zn2+ release during ischemia
Epileptic Models- Is Zn2+ excitatory or
inhibitory?
Long Term Potentiation
How Do We Study Zinc?
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A mixture of electrophysiology and
fluorescent imaging
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My lab mate Chinta does the imaging!
My current focus is on LTP in the
hippocampus
Zinc Containing Neurons
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Neurons that sequester weakly bound
(histochemically active) Zn2+ in the
vesicles of their presynaptic boutons
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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
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It appears that all zinc containing
neurons are glutamatergic, but only
some glutamatergic neurons contain
Zn2+
Zinc Containing NeuronsSubset of Glutamatergic Neurons
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Supporting Evidence
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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?
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A great proportion of “glutazinergic”
neurons are found in the cerebral cortex
and the amygdala
So Where are these Neurons?
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Efferent zinc-containing fibers from
these regions extend to
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The cerebral cortex and the Amygdala
Striatum
Limbic targets
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(septum, medial hypothalamus)
What About Non-Zinc-Containing
Glutamatergic Neurons?
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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
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Zinc is loaded into presynaptic vesicles
by the transport protein ZnT-3
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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
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Also possible to label vesicular Zn2+ by
precipitating with intravital selenium
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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?
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Using traditional staining methods, it is
difficult to quantify the amount of zinc
in a given region
Fluorescent imaging may provide new
insights
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Chintha’s work…more interesting than my
presentation
So What’s the Problem?
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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
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The hippocampus appears to have four
setsof Zn2+ containing neurons
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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
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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+?
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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?
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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?
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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?
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Get a nice EPSP in CA1
Add 10 mM CaEDTA and…
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?? See what happens!
Hopefully we’ll do patching soon
Special Thanks
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Dr. Yang Li
Dr. Colvin and Dr. Holmes
Labmate: Chintha
The Undergraduate Research Assistants
References
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To be added later
Questions?