Kainate-stimulated Zn2+ uptake labels cortical neurons with Ca2+-permeable AMPA/kainate channels.
The endogenous cation, Zn2+, is synaptically released and may trigger neurodegeneration after permeating through NMDA channels, voltage sensitive Ca2+ channels (VSCC), or Ca2+ permeable AMPA/kainate channels (Ca-A/K). Neurons expressing Ca-A/K can be identified by a histochemical stain based upon kainate-stimulated Co2+ uptake (Co2+(+) neurons). The primary objective of this study was to determine whether a similar approach could be employed to visualize agonist-stimulated intracellular Zn2+ accumulation, and, thus, to test the hypothesis that Ca-A/K permit particularly rapid Zn2+ flux. Substituting Zn2+ for Co2+ during agonist-stimulated uptake, followed by Timm's sulfide-silver staining to visualize intracellular Zn2+, resulted in distinct labeling of a subpopulation of cortical neurons (Zn2+(+) neurons) closely resembling Co2+(+) neurons, suggesting that, like Co2+, Zn2+ may permeate Ca-A/K with particular rapidity. Neither NMDA nor high K+ triggered comparable Zn2+ accumulation, indicating substantially greater permeation through Ca-A/K than through NMDA channels or VSCC. Both fluorescence studies of intracellular Zn2+ accumulation and double staining studies (using SMI-32 and anti-glutamate decarboxylase antibodies, both markers of cortical neuronal subsets), support the contention that Zn2+ and Co2+ labeling identify a common set of neurons characterized by expression of AMPA/kainate channels directly permeable to Zn2+ and Co2+ as well as Ca2+. Furthermore, the preferential destruction of Zn2+(+) neurons (like Co2+(+) neurons) after brief kainate exposures in the presence of lower, more physiologic concentrations of Zn2+ suggests that Zn2+ 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 Co2+ or Zn2+ uptake labeling, using a room temperature technique (Timm's staining) rather than the usual hot AgNO3 development of the Co2+ stain.
Yin
H Z
HZ
Department of Neurology, University of California in Irvine, Irvine, CA 92697-4292, USA.
Ha
D H
DH
Carriedo
S G
SG
Weiss
J H
JH
D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011232:Chemical Precipitation; D003035:Cobalt / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects* / Q000378:metabolism; D007608:Kainic Acid / Q000378:metabolism / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D009410:Nerve Degeneration / Q000503:physiopathology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010539:Permeability; D015032:Zinc / Q000378:metabolism*; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000378:metabolism
D007473:Ion Channels; D003035:Cobalt; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; D015032:Zinc; D007608:Kainic Acid; D002118:Calcium