Fluorescent imaging of Zinc in rat hippocampus

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Fluorescent imaging of Zinc in rat hippocampus

• Chintha Bastian
• Dr. Yang Li

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Zinc in the brain

• Brief review most of which was discussed by Josh
  Ketterman
• Indicators used for imaging

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Overview

• Histochemically reactive Zn2 is found in a
  subset of glutamatergic nerve terminals
  throughout mammalian cortex and limbic region
  (Frederickson et al. 2000)
• Studies have shown most of the histochemically
  staining Zn2 localized within synaptic vesicles
  of glutamatergic neurons. (Frederickson et al.
  1983 Huang 1967 Perez- Clausell and Danscher
  1985)

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Courtesy Frederickson et al, Journal of Nutrition
2000
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Rapid Translocation of Zn2 From Presynaptic
Terminals Into Postsynaptic Hippocampal Neurons
After Physiological Stimulation

• Yang Li, Christopher J. Hough, Sang Won Suh, John
  M. Sarvey and Christopher J. Frederickson

Journal of Neurophysiology VOL 86 NOVEMBER
2001
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Why do we study the role of Zinc in our lab?

• Zinc has been indicated in many pathological
  processes in the brain like
• Epilepsy
• Alzheimer's disease
• Ischemia to the brain
• Also, LTP and Memory!

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Experimental animals

• Adult male Sprague Dawley rats
• Weight gt150 g
• Brain slices prepared using a Vibratome

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Newport green

• Newport green dipotassium salt cell
  impermeable for extracellular imaging
• Dissociation constant Kd 1µM
• Sensitive to Zn2

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Artificial cerebrospinal fluid

• ACSF
• 124 mM NaCl, 1.75 mM KCl, 1.3 mM MgSO4, 2.4 mM
  CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3, and 10
  mM dextrose continuously bubbled with 95 O2 and
  5 CO2 (pH 7.4)

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• Stimulation parameters used
• 0.5-mA pulses at 100 Hz for 5 sec.

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CA1
Dentate Gyrus
Hilar region
CA3
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20µM Newport Green
Electrical stimulation
25 mM KCl
Courtesy Li et al 2000.
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Stimulation induced release of Zinc when perfused
with 20µM Newport Green
Courtesy Li et al 2000
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Courtesy Li et al 2000
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Courtesy Li et al 2000
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2µM TTX
Courtesy Li et al 2000
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Determination of Zinc concentration
Zn2   Kd (F -  F min)/(F max- F)
Grynkiewicz et al. (1985)

• F is the measured fluorescence intensity.
• Fmax obtained by measuring the dye fluorescence
  in the presence of 1 mM ZnCl2 added to the ACSF
  bathing the slice
• Fmin obtained by removing the Zn2 by perfusing
  with Zn2 free ACSF, and then measuring the dye
  fluorescence again during perfusion with Zn2
  free ACSF plus 10 mM Ca-EDTA.

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Intracellular Zinc imaging

• Newport Green diacetate 50µM, 0.1 pluronic acid
  and 0.5 DMSO for one hour
• NG washed out with ACSF

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NG diacetate and stimulation (100Hz for 10sec)
Courtesy Li et al 2000
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10mM
10µM
Courtesy Li et al 2000
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Courtesy Li et al 2000
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Drawbacks of the paper

• Calcium EDTA 10 mM Why?
• Graph obtained after adding KCl to extracellular
  Newport Green solution
• Incubated the slices in Newport Green for half an
  hour

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Current research

• Zeiss LSM 510 laser scanning inverted Confocal
  Microscope
• 10 µM Newport Green (impermeant) shorter
  incubation time
• 100 Hz for 10 sec
• Comparing action of other dyes

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10 µM Newport Green
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Regions of interest!!
ROI 1
ROI 2
ROI 3
LSM Toolbox v2.10c (http//rct-cjs3.physiol.cam.ac
.uk/C4/cjs/AnalysisProgram/program.html)
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Data as reported on April 8 ,2005 by Chintha
Bastian
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Kd 1µM (Molecular probes)
Courtesy Handbook, Molecular probes, Invitrogen
detection technologies
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Zinpyr-4
Kd 1nM (Frederickson et al)
Courtesy Shawn Burdette et al 2003 ZP4, an
Improved Neuronal Zn2 Sensor of the Zinpyr
Family
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Kd 15nM (Molecular probes)
Courtesy Handbook, Molecular probes, Invitrogen
detection technologies
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Future studies

• Role of Zinc in epilepsy
• causative , contributive or curative?
• Recurrent mossy fibers releasing Zinc in the
  molecular layer
• Translocation of Zinc

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Thanks!

• Dr. Yang Li
• Josh Ketterman
• Christian Stork
• Jennifer Martin
• Yanli Ding

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Questions???