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Spreading depolarization (SD) is a feed-forward wave that propagates slowly throughout brain tissue and recovery from SD involves substantial metabolic demand. Presynaptic Zn2+ release and intracellular accumulation occurs with SD, and elevated intracellular Zn2+ ([Zn2+]i) can impair cellular metabolism through multiple pathways. We tested here whether increased [Zn2+]i could exacerbate the metabolic challenge of SD, induced by KCl, and delay recovery in acute murine hippocampal slices. [Zn2+]i loading prior to SD, by transient ZnCl2 application with the Zn2+ ionophore pyrithione (Zn/Pyr), delayed recovery of field excitatory post-synaptic potentials (fEPSPs) in a concentration-dependent manner, prolonged DC shifts, and significantly increased extracellular adenosine accumulation. These effects could be due to metabolic inhibition, occurring downstream of pyruvate utilization. Prolonged [Zn2+]i accumulation prior to SD was required for effects on fEPSP recovery and consistent with this, endogenous synaptic Zn2+ release during SD propagation did not delay recovery from SD. The effects of exogenous [Zn2+]i loading were also lost in slices preconditioned with repetitive SDs, implying a rapid adaptation. Together, these results suggest that [Zn2+]i loading prior to SD can provide significant additional challenge to brain tissue, and could contribute to deleterious effects of [Zn2+]i accumulation in a range of brain injury models.
Spreading depolarizations (SD) are waves of neuronal and glial depolarization that propagate slowly throughout brain tissue (Leao 1944; Somjen 2001). SD is commonly recorded as a large extracellular negative direct current (DC) potential shift and results in a transient suppression of both electrocorticographic activity (Leao 1947; Somjen 2001). Large amounts of energy are required to restore ionic gradients before normal function can return, as demonstrated by large reductions in ATP and glucose levels following SD (Shinohara et al. 1979; Mies and Paschen 1984). Extracellular adenosine accumulation occurs with severe metabolic challenges (Dale et al. 2000; Dunwiddie and Masino 2001), and can contribute to the transient suppression of field excitatory postsynaptic potentials (fEPSPs) in brain slice models of SD (Lindquist and Shuttleworth 2012). In healthy brain tissue SD is not injurious (Nedergaard and Hansen 1988), however, under conditions where metabolism is already compromised, such as in ischemic brain tissue, the additional metabolic challenge of repetitive SD events can lead to irrecoverable damage (Busch et al. 1996; Hartings et al. 2003). A growing body of literature from both animal and human recordings suggests that clusters of SDs can be a significant contributing factor to the enlargement of acute brain injuries (Nakamura et al. 2010; Oliveira-Ferreira et al. 2010; Dreier 2011; Hartings et al. 2011a; Lauritzen et al. 2011).
We have recently demonstrated a significant release of synaptic Zn2+ following SD, and subsequent intracellular accumulation (Carter et al. 2011). Zn2+ is highly regulated and normally kept at very low intracellular levels, however, excessive intracellular Zn2+ ([Zn2+]i) accumulation has been associated with a wide range of brain injuries including ischemia and trauma (Sensi et al. 2011; Shuttleworth and Weiss 2011). It is not yet known whether release and accumulation of Zn2+ following SD may be a significant contributor to Zn2+ toxicity in a variety of brain injury settings. One mechanism for toxic effects of Zn2+ involves disruption of cellular energy production. Zn2+ can directly bind to and inhibit both glyceraldehyde-3-phosphate dehydrogenase [GAPDH, (Sheline et al. 2000)] and the α-ketoglutarate dehydrogenase complex [KGDHC, (Brown et al. 2000)], inhibiting glycolysis and the TCA cycle, respectively. In addition, Zn2+ can inhibit several steps in the mitochondrial electron transport chain (ETC) (Dineley et al. 2003; Sharpley and Hirst 2006). Indirectly, Zn2+ can also inhibit metabolism by activating NAD+-catabolizing enzymes such as poly(ADP-ribose) polymerase 1 (PARP-1) and the sirtuin family of proteins (Sheline et al. 2000; Cai et al. 2006), depleting NAD+ levels and inhibiting glycolysis.
The aim of this study was to determine whether increasing [Zn2+]i can exacerbate the delay in recovery from SD. The results suggest that when [Zn2+]i levels are sufficiently elevated prior to the passage of SD, there is a marked additional metabolic challenge, leading to delayed recovery of synaptic transmission.