Fluorescent imaging of Zinc in rat hippocampus Chintha

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 n Indicators used for imaging n

Overview n Histochemically reactive Zn 2+ is found in a subset of glutamatergic nerve terminals throughout mammalian cortex and limbic region (Frederickson et al. 2000) n 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 n Epilepsy n Alzheimer's disease n Ischemia to the brain n Also, LTP and Memory!

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

Newport green dipotassium salt cell impermeable for extracellular imaging n Dissociation constant Kd = ~1μM n Sensitive to Zn 2+ n

Artificial cerebrospinal fluid ACSF 124 m. M Na. Cl, 1. 75 m. M KCl, 1. 3 m. M Mg. SO 4, 2. 4 m. M Ca. Cl 2, 1. 25 m. M KH 2 PO 4, 26 m. M Na. HCO 3, and 10 m. M dextrose continuously bubbled with 95% O 2 and 5% CO 2 (p. H 7. 4)

Stimulation parameters used 0. 5 -m. A pulses at 100 Hz for 5 sec. n

CA 1 rus Dentate Gy Hilar region CA 3

20µM Newport Green Electrical stimulation Courtesy Li et al 2000. 25 m. M KCl

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

2µM TTX Courtesy: Li et al 2000

Determination of Zinc concentration [Zn 2+ = 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 m. M Zn. Cl 2 added to the ACSF bathing the slice • Fmin 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 m. M Ca-EDTA.

Intracellular Zinc imaging Newport Green diacetate 50µM, 0. 1% pluronic acid and 0. 5% DMSO for one hour n NG washed out with ACSF n

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

10 m. M Courtesy: Li et al 2000 10µM

Courtesy: Li et al 2000

Drawbacks of the paper Calcium EDTA 10 m. M Why? n Graph obtained after adding KCl to extracellular Newport Green solution n Incubated the slices in Newport Green for half an hour n

Current research Zeiss LSM 510 laser scanning inverted Confocal Microscope n 10 µM Newport Green (impermeant) shorter incubation time n 100 Hz for 10 sec n Comparing action of other dyes n

10 µM Newport Green

Regions of interest!! ROI 1 ROI 2 ROI 3 LSM Toolbox v 2. 10 c (http: //rct-cjs 3. physiol. cam. ac. uk/C 4/cjs/Analysis. Program/program. html)

Data as reported on April 8 , 2005 by Chintha Bastian

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

Zinpyr-4 Kd ~ 1 n. M (Frederickson et al) Courtesy: Shawn Burdette et al 2003; ZP 4, an Improved Neuronal Zn 2+ Sensor of the Zinpyr Family

Kd ~15 n. M (Molecular probes) Courtesy : Handbook, Molecular probes, Invitrogen detection technologies

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

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

Questions? ? ?