Fluorescent imaging of Zinc in rat hippocampusChintha Bastian

Dr. Yang Li

Zinc in the brain

Brief review most of which was discussed by Josh Ketterman

Indicators used for imaging

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)

Courtesy Frederickson et al, Journal of Nutrition 2000

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?

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

Adult male Sprague Dawley rats Weight >150 g Brain slices prepared using a Vibratome

Newport green

Newport green dipotassium salt cell impermeable for extracellular imaging

Dissociation constant Kd = ~1μM Sensitive to Zn2+

Artificial cerebrospinal fluid

ACSF124 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)

Stimulation parameters used

0.5-mA pulses at 100 Hz for 5 sec.

Dentate Gyrus

CA1

CA3Hilar region

20µM Newport Green

Electrical stimulation 25 mM KCl

Courtesy Li et al 2000.

Stimulation induced release of Zinc when perfused with 20µM Newport Green

Courtesy: Li et al 2000

Courtesy: Li et al 2000

Courtesy: Li et al 2000

Courtesy: Li et al 2000

2µM TTX

Determination of Zinc concentration

•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.

[Zn2+  = Kd (F -  F min)/(F max- F)

Grynkiewicz et al. (1985)

Intracellular Zinc imaging

Newport Green diacetate 50µM, 0.1% pluronic acid and 0.5% DMSO for one hour

NG washed out with ACSF

NG diacetate and stimulation (100Hz for 10sec)

Courtesy: Li et al 2000

10mM 10µM

Courtesy: Li et al 2000

Courtesy: Li et al 2000

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

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

10 µM Newport Green

Regions of interest!!

LSM Toolbox v2.10c (http://rct-cjs3.physiol.cam.ac.uk/C4/cjs/AnalysisProgram/program.html)

ROI 1

ROI 2

ROI 3

Data as reported on April 8 ,2005 by Chintha Bastian

Electrical Stimulation of Hippocampus

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Time in Seconds

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ROI1 ROI2 ROI3

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

Kd ~ 1µM

(Molecular probes)

Zinpyr-4

Courtesy: Shawn Burdette et al 2003; ZP4, an

Improved Neuronal Zn2+ Sensor of the Zinpyr Family

Kd ~ 1nM (Frederickson et al)

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

Kd ~15nM

(Molecular probes)

Future studies

Role of Zinc in epilepsy

causative , contributive or curative? Recurrent mossy fibers releasing Zinc in

the molecular layer Translocation of Zinc

Thanks!

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

Questions???