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MATERIALS & METHODS

 MICROSCOPIC TECHNIQUES SPECIES MORPHOLOGY CELL LINE
638 Measurement of Intracellular Free Zinc Concentrations
Accompanying Zinc-Induced Neuronal Death		 Lorella M. T. Canzoniero, Dorothy M. Turetsky, and Dennis W. Choi voltage-gated calcium channels; calcium; depolarization; kainate; NMDA; neurotoxicity

Neurons for intracellular Zn21 imaging experiments were prepared similarly, using 35 mm glass-bottom dishes (MatTek, Ashland, MA) coated with poly-D-lysine/
laminin (100:4 ng/ml).

 phase-contrast microscopy, inverted microscopy mouse neuronal mixed neocortical
  Toxic zinc influx may contribute to selective neuronal death after transient global ischemia. We previously used the highaffinity (KD 5 27 nM) fluorescent dye mag-fura-5 to detect initial increases in neuronal intracellular free Zn21 ([Zn 21]i ) associated with brief Zn21 exposure. Here we used the specific low-affinity Zn21 indicator Newport Green (KD 5 1 mM) to measure the peak levels of [Zn21]i attained during prolonged, toxic exposures to extracellular Zn21. Murine cortical cell cultures exposed for 5–10 min to 300 mM Zn21 in the presence of kainate or elevated extracellular K1 developed widespread neuronal death over the next 24 hr. Such Zn21 exposure under depolarizing conditions was accompanied by a large increase in [Zn21]i reaching several hundred nanomolar, which gradually recovered over the next 20–40 min after termination of Zn21 exposure. Both the level of [Zn21]i elevation and the extent of subsequent neuronal death depended on the concentration of extracellular Zn21 between 30 mM and 1 mM. In contrast, exposure to 300 mM Zn21 in the presence of 300 mM NMDA resulted in little increase in [Zn21]i and little neuronal death, suggesting that NMDA receptor-gated channels are less important as a route of toxic Zn21 entry than voltage-gated calcium channels.  

 

 

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