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Fluorescent imaging of Zinc in rat hippocampus

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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Fluorescent imaging of Zinc in rat hippocampus

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  1. Fluorescent imaging of Zinc in rat hippocampus Chintha Bastian Dr. Yang Li

  2. Zinc in the brain • Brief review most of which was discussed by Josh Ketterman • Indicators used for imaging

  3. 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)

  4. Courtesy Frederickson et al, Journal of Nutrition 2000

  5. 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

  6. 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!

  7. Experimental animals • Adult male Sprague Dawley rats • Weight >150 g • Brain slices prepared using a Vibratome

  8. Newport green • Newport green dipotassium salt cell impermeable for extracellular imaging • Dissociation constant Kd = ~1μM • Sensitive to Zn2+

  9. 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)

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

  11. CA1 Dentate Gyrus Hilar region CA3

  12. 20µM Newport Green Electrical stimulation 25 mM KCl Courtesy Li et al 2000.

  13. Stimulation induced release of Zinc when perfused with 20µM Newport Green Courtesy: Li et al 2000

  14. Courtesy: Li et al 2000

  15. Courtesy: Li et al 2000

  16. 2µM TTX Courtesy: Li et al 2000

  17. 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.

  18. Intracellular Zinc imaging • Newport Green diacetate 50µM, 0.1% pluronic acid and 0.5% DMSO for one hour • NG washed out with ACSF

  19. NG diacetate and stimulation (100Hz for 10sec) Courtesy: Li et al 2000

  20. 10mM 10µM Courtesy: Li et al 2000

  21. Courtesy: Li et al 2000

  22. 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

  23. 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

  24. 10 µM Newport Green

  25. 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)

  26. Data as reported on April 8 ,2005 by Chintha Bastian

  27. Kd ~ 1µM (Molecular probes) Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

  28. Zinpyr-4 Kd ~ 1nM (Frederickson et al) Courtesy: Shawn Burdette et al 2003; ZP4, an Improved Neuronal Zn2+Sensor of the Zinpyr Family

  29. Kd ~15nM (Molecular probes) Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

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

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

  32. Questions???

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