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 Histochemically reactive Zn 2+ 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 Zn 2+ 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 Zn 2+ 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 K d = ~1μM Sensitive to Zn 2+

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% O 2 and 5% CO 2 (pH 7.4)

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

Dentate Gyrus CA1 CA3 Hilar region

20µM Newport Green Electrical stimulation25 mM KCl Courtesy Li et al 2000.

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

2µM TTX

Determination of Zinc concentration F is the measured fluorescence intensity. F max obtained by measuring the dye fluorescence in the presence of 1 mM ZnCl 2 added to the ACSF bathing the slice F min obtained by removing the Zn 2+ by perfusing with Zn 2+ free ACSF, and then measuring the dye fluorescence again during perfusion with Zn 2+ free ACSF plus 10 mM Ca-EDTA. [ Zn 2+ = K d (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

10mM10µM 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 ( ROI 1 ROI 2 ROI 3

Data as reported on April 8,2005 by Chintha Bastian

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies K d ~ 1µM (Molecular probes)

Zinpyr-4 Courtesy: Shawn Burdette et al 2003; ZP4, an Improved Neuronal Zn 2+ Sensor of the Zinpyr Family K d ~ 1nM (Frederickson et al)

Courtesy : Handbook, Molecular probes, Invitrogen detection technologies K d ~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???