Kainate-stimulated Zn 2+ uptake labels cortical neurons with Ca 2+-permeable AMPA/kainate channels

Abstract

The endogenous cation, Zn 2+, is synaptically released and may trigger neurodegeneration after permeating through NMDA channels, voltage sensitive Ca 2+ channels (VSCC), or Ca 2+ permeable AMPA/kainate channels (Ca-A/K). Neurons expressing Ca-A/K can be identified by a histochemical stain based upon kainate-stimulated Co 2+ uptake (Co 2+(+) neurons). The primary objective of this study was to determine whether a similar approach could be employed to visualize agonist-stimulated intracellular Zn 2+ accumulation, and, thus, to test the hypothesis that Ca-A/K permit particularly rapid Zn 2+ flux. Substituting Zn 2+ for Co 2+ during agonist-stimulated uptake, followed by Timm's sulfide–silver staining to visualize intracellular Zn 2+, resulted in distinct labeling of a subpopulation of cortical neurons (Zn 2+(+) neurons) closely resembling Co 2+(+) neurons, suggesting that, like Co 2+, Zn 2+ may permeate Ca-A/K with particular rapidity. Neither NMDA nor high K + triggered comparable Zn 2+ accumulation, indicating substantially greater permeation through Ca-A/K than through NMDA channels or VSCC. Both fluorescence studies of intracellular Zn 2+ accumulation and double staining studies (using SMI-32 and anti-glutamate decarboxylase antibodies, both markers of cortical neuronal subsets), support the contention that Zn 2+ and Co 2+ labeling identify a common set of neurons characterized by expression of AMPA/kainate channels directly permeable to Zn 2+ and Co 2+ as well as Ca 2+. Furthermore, the preferential destruction of Zn 2+(+) neurons (like Co 2+(+) neurons) after brief kainate exposures in the presence of lower, more physiologic concentrations of Zn 2+ suggests that Zn 2+ permeation through Ca-A/K could contribute to selective neurodegeneration in disease. Finally, the study provides a novel and potentially advantageous histochemical approach for kainate-stimulated Co 2+ or Zn 2+ uptake labeling, using a room temperature technique (Timm's staining) rather than the usual hot AgNO 3 development of the Co 2+ stain.