fluorescent imaging of zinc in rat hippocampus
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Fluorescent imaging of Zinc in rat hippocampus. Chintha Bastian Dr. Yang Li. Zinc in the brain. Brief review most of which was discussed by Josh Ketterman Indicators used for imaging. Overview.

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zinc in the brain
Zinc in the brain
  • Brief review most of which was discussed by Josh Ketterman
  • Indicators used for imaging
overview
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)
slide5
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

why do we study the role of zinc in our lab
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!
experimental animals
Experimental animals
  • Adult male Sprague Dawley rats
  • Weight >150 g
  • Brain slices prepared using a Vibratome
newport green
Newport green
  • Newport green dipotassium salt cell impermeable for extracellular imaging
  • Dissociation constant Kd = ~1μM
  • Sensitive to Zn2+
artificial cerebrospinal fluid
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)

slide10
Stimulation parameters used

0.5-mA pulses at 100 Hz for 5 sec.

slide11

CA1

Dentate Gyrus

Hilar region

CA3

20 m newport green
20µM Newport Green

Electrical stimulation

25 mM KCl

Courtesy Li et al 2000.

slide16

2µM TTX

Courtesy: Li et al 2000

determination of zinc concentration
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.
intracellular zinc imaging
Intracellular Zinc imaging
  • Newport Green diacetate 50µM, 0.1% pluronic acid and 0.5% DMSO for one hour
  • NG washed out with ACSF
slide20

10mM

10µM

Courtesy: Li et al 2000

drawbacks of the paper
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
current research
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
regions of interest
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)

slide27

Kd ~ 1µM

(Molecular probes)

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

zinpyr 4
Zinpyr-4

Kd ~ 1nM (Frederickson et al)

Courtesy: Shawn Burdette et al 2003; ZP4, an Improved Neuronal Zn2+Sensor of the Zinpyr Family

slide29

Kd ~15nM

(Molecular probes)

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

future studies
Future studies
  • Role of Zinc in epilepsy

causative , contributive or curative?

  • Recurrent mossy fibers releasing Zinc in the molecular layer
  • Translocation of Zinc
thanks
Thanks!
  • Dr. Yang Li
  • Josh Ketterman
  • Christian Stork
  • Jennifer Martin
  • Yanli Ding
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