Intracellular Zn2+ increases contribute to the progression of excitotoxic Ca2+ increases in apical dendrites of CA1 pyramidal neurons

Abstract

Sustained intracellular Ca2+ elevation is a well-established contributor to neuronal injury following excessive activation of N-methyl-d-aspartic acid (NMDA)–type glutamate receptors. Zn2+ can also be involved in excitotoxic degeneration, but the relative contributions of these two cations to the initiation and progression of excitotoxic injury is not yet known. We previously concluded that extended NMDA exposure led to sustained Ca2+ increases that originated in apical dendrites of CA1 neurons and then propagated slowly throughout neurons and caused rapid necrotic injury. However the fluorescent indicator used in those studies (Fura-6F) may also respond to Zn2+, and in the present work we examine possible contributions of Zn2+ to indicator signals and to the progression of degenerative signaling along murine CA1 dendrites. Selective chelation of Zn2+ with N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed, but did not prevent the development and progression of sustained high-level Fura-6F signals from dendrites to somata. Rapid indicator loss during the Ca2+ overload response, which corresponds to rapid neuronal injury, was also not prevented by TPEN. The relationship between cytosolic Zn2+ and Ca2+ levels was assessed in single CA1 neurons co-loaded with Fura-6F and the Zn2+-selective indicator FluoZin-3. NMDA exposure resulted in significant initial increases in FluoZin-3 increases that were prevented by TPEN, but not by extracellular Zn2+ chelation with Ca-EDTA. Consistent with this result, Ca-EDTA did not delay the progression of Fura-6F signals during NMDA. Removal of extracellular Ca2+ reduced, but did not prevent FluoZin-3 increases. These results suggest that sustained Ca2+ increases indeed underlie Fura-6F signals that slowly propagate throughout neurons, and that Ca2+ (rather than Zn2+) increases are ultimately responsible for neuronal injury during NMDA. However, mobilization of Zn2+ from endogenous sources leads to significant neuronal Zn2+ increases, that in turn contribute to mechanisms of initiation and progression of progressive Ca2+ deregulation.