Fluorescent imaging of zinc in rat hippocampus l.jpg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 33

Fluorescent imaging of Zinc in rat hippocampus PowerPoint PPT Presentation


  • 141 Views
  • Uploaded on
  • Presentation posted in: General

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.

Download Presentation

Fluorescent imaging of Zinc in rat hippocampus

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Fluorescent imaging of zinc in rat hippocampus l.jpg

Fluorescent imaging of Zinc in rat hippocampus

Chintha Bastian

Dr. Yang Li


Zinc in the brain l.jpg

Zinc in the brain

  • Brief review most of which was discussed by Josh Ketterman

  • Indicators used for imaging


Overview l.jpg

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)


Slide4 l.jpg

Courtesy Frederickson et al, Journal of Nutrition 2000


Slide5 l.jpg

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 l.jpg

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 l.jpg

Experimental animals

  • Adult male Sprague Dawley rats

  • Weight >150 g

  • Brain slices prepared using a Vibratome


Newport green l.jpg

Newport green

  • Newport green dipotassium salt cell impermeable for extracellular imaging

  • Dissociation constant Kd = ~1μM

  • Sensitive to Zn2+


Artificial cerebrospinal fluid l.jpg

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 l.jpg

  • Stimulation parameters used

    0.5-mA pulses at 100 Hz for 5 sec.


Slide11 l.jpg

CA1

Dentate Gyrus

Hilar region

CA3


20 m newport green l.jpg

20µM Newport Green

Electrical stimulation

25 mM KCl

Courtesy Li et al 2000.


Stimulation induced release of zinc when perfused with 20 m newport green l.jpg

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

Courtesy: Li et al 2000


Slide14 l.jpg

Courtesy: Li et al 2000


Slide15 l.jpg

Courtesy: Li et al 2000


Slide16 l.jpg

2µM TTX

Courtesy: Li et al 2000


Determination of zinc concentration l.jpg

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 l.jpg

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 l.jpg

NG diacetate and stimulation (100Hz for 10sec)

Courtesy: Li et al 2000


Slide20 l.jpg

10mM

10µM

Courtesy: Li et al 2000


Slide21 l.jpg

Courtesy: Li et al 2000


Drawbacks of the paper l.jpg

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 l.jpg

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 m newport green l.jpg

10 µM Newport Green


Regions of interest l.jpg

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)


Slide26 l.jpg

Data as reported on April 8 ,2005 by Chintha Bastian


Slide27 l.jpg

Kd ~ 1µM

(Molecular probes)

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies


Zinpyr 4 l.jpg

Zinpyr-4

Kd ~ 1nM (Frederickson et al)

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


Slide29 l.jpg

Kd ~15nM

(Molecular probes)

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies


Future studies l.jpg

Future studies

  • Role of Zinc in epilepsy

    causative , contributive or curative?

  • Recurrent mossy fibers releasing Zinc in the molecular layer

  • Translocation of Zinc


Thanks l.jpg

Thanks!

  • Dr. Yang Li

  • Josh Ketterman

  • Christian Stork

  • Jennifer Martin

  • Yanli Ding


Questions l.jpg

Questions???


  • Login