Zn2+Influx Is Critical for Some Forms of Spreading Depression in Brain Slices

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Abstract
Spreading depression (SD) is wave of profound depolarization that propagates throughout brain tissue and can contribute to the spread of injury after stroke or traumatic insults. The contribution of Ca2+ influx to SD differs depending on the stimulus, and we show here that Zn2+ can play a critical complementary role in murine hippocampal slices. In initial studies, we used the Na+/K+ ATPase inhibitor ouabain and found conditions in which SD was always prevented by L-type Ca2+ channel blockers; however, Ca2+ influx was not responsible for L-type effects. Cytosolic Ca2+ increases were not detectable in CA1 neurons before SD, and removal of extracellular Ca2+ did not prevent ouabain-SD. In contrast, cytosolic Zn2+ increases were observed in CA1 neurons before ouabain-SD, and L-type channel block prevented the intracellular Zn2+ rises. A slow mitochondrial depolarization observed before ouabain-SD was abolished by L-type channel block, and Zn2+ accumulation contributed substantially to initial mitochondrial depolarizations. Selective chelation of Zn2+ with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) abolished SD, implying that Zn2+ entry can play a critical role in the generation of ouabain-SD. TPEN was most effective when synaptic activity was reduced by adenosine A1 receptor activation, and a combination of Ca2+ and Zn2+ removal was required to prevent ouabain-SD when A1 receptors were blocked. Similarly, Zn2+ chelation could prevent SD triggered by oxygen/glucose deprivation but Zn2+ accumulation did not contribute to SD triggered by localized high K+ exposures. These results identify Zn2+ as a new target for the block of spreading depolarizations after brain injury.