Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA
Address correspondence and reprint requests to C. William Shuttleworth, PhD, Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, NM, USA. E-mail: email@example.com
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.
Materials and methods
Slice preparation and recording
All procedures were carried out in accordance with the National Institutes of Health guidelines for the humane treatment of laboratory animals, and the protocol for these procedures was reviewed annually by the Institutional Animal Care and Use Committee (IACUC) at the University of New Mexico School of Medicine. Acute hippocampal brain slices (350 μm) were prepared from wild type C57Bl/6J and ZnT3 KO mice of either sex (4–8 weeks old). After cutting in ice-cold cutting solution and then holding for 1 h at 35°C, artificial cerebrospinal fluid (ACSF) was changed, and slices were held at 25°C until used for recording. Individual slices were then transferred to the recording chamber, and superfused with oxygenated ACSF at 2 mL/min at 32°C. Mice homozygous for deletion of the ZnT3 gene were originally obtained from Dr. Richard Palmiter (Cole et al. 1999) and backcrossed onto C57Bl/6J for > 13 generations (LSUHSC). A homozygous breeding colony of ZnT3 KO animals was then established at the University of New Mexico.
Extracellular measurements of field excitatory postsynaptic potentials (fEPSPs), were made via stimulation of Schaffer collaterals with a concentric bipolar electrode (CBAEC75; FHC, Bowdoin, ME, USA). Stimulation intensity was set to achieve ~ 60% maximum response during recording sessions. Extracellular recordings of fEPSPSs and direct current (DC) shifts were made by glass microelectrodes (~ 2–5 MΩ), filled with ACSF and placed in stratum radiatum of CA1 ~ 50 μm below the surface of the slice. Spreading depolarization (SD) was induced by brief pressure pulses (30 psi, 80–100 ms) of 1 M KCl from a homemade picospritzer (UniBlitz Model D122; Vincent Associates, Rochester, NY, USA; valve: 3-165-900, General Valve Corporation, Fairfield, NY, USA). Onset and propagation of SD was monitored by intrinsic optical signals (IOS), generated by transmission of red light through the tissue (> 575 nm). Propagation rate and IOS analyses were performed in TillVision (Till Photonics, Rochester, NY, USA). Comparison of fEPSP recovery between groups was defined by the time for fEPSPs to return to 50% pre-SD levels (t50), unless stated otherwise.
Intracellular Zn2+ loading
To simulate conditions in which [Zn2+]i levels were increased, we used a transient exposure (20 min) to pyrithione (Pyr, 5 μM) in the presence of 0, 30, or 100 μM ZnCl2 (Zn/Pyr). This was followed by a 10 min washout prior to SD induction to minimize possible contributions of increased extracellular Zn2+ during the passage of SD (Aiba et al. 2012). Histidine (His, 200 μM) was present during all Zn/Pyr exposures to help solubilize Zn2+ and prevent it from precipitating out of solution (Rumschik et al. 2009).
Measurements of extracellular adenosine accumulation were performed as previously described (Lindquist and Shuttleworth 2012). Briefly, enzyme-coupled electrochemical probes (SBS-ADO-05-50, Sarissa Biomedical, Coventry, UK) generate an amperometric signal proportional to the concentration of adenosine. Measurements reported here are estimates of accumulation at the slice surface, and synaptic levels may be different because of transporter activities within the slice. Calibration of the probes with exogenous adenosine (5–50 μM) was performed immediately following individual recording sessions. It is noted that in this study calibration stocks were made daily, and estimates of adenosine levels following SD are ~ 10-fold lower than previously reported (Lindquist and Shuttleworth 2012). Adenosine probes were positioned between the bipolar stimulating and recording electrodes in stratum radiatum of CA1.
Reagents and solutions
Slice cutting solution contained (in mM): 3 KCl, 1.25 NaH2PO4, 6 MgSO4, 26 NaHCO3, 0.2 CaCl2, 10 glucose, 220 sucrose, and 0.43 ketamine. ACSF contained (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 1 MgSO4, 26 NaHCO3, 2 CaCl2, and 10 glucose, equilibrated with 95%O2/5%CO2. N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) was obtained from Intvitrogen (Carlsbad, CA, USA). All other reagents were obtained from Sigma (St. Louis, MO, USA).
Data are reported as mean ± SEM. Statistical analysis was performed in GraphPad Prism (La Jolla, CA, USA) using paired or unpaired Student's t-test, or analysis of variance (anova), with Bonferroni correction for multiple comparisons where appropriate. Differences were considered significant when p < 0.05.
Effects of transient [Zn2+]i loading on fEPSP suppression following SD
Transient loading of [Zn2+]i (using pyrithione, see methods) prior to SD resulted in significant delay in recovery of fEPSPs after SD. In slices not loaded with Zn2+ (5 μM Pyr control), SD resulted in fEPSP amplitude suppression that took 11.22 ± 0.95 min (n = 6) to return to baseline levels (Fig. 1a), similar to previously published data (Lindquist and Shuttleworth 2012). [Zn2+]i loading produced a concentration-dependent increase in the duration of fEPSP suppression following SD (Fig. 1a) and final fEPSP amplitudes remained substantially depressed 25 min following SD with the higher Zn2+ concentrations (30 & 100 μM).
We noted a slight run-down of fEPSP amplitude immediately prior to the onset of SD when slices were loaded with Zn2+. Therefore, a set of time-control studies was completed to test whether progressive run-down of fEPSP amplitude alone (without SD) could have produced the results seen. Thus slices were loaded with 0, 30, or 100 μM Zn/Pyr (20 min followed by wash out) without inducing SD (Fig. 1b). Figure 1c and d compare the effects of [Zn2+]i loading on final fEPSP amplitudes (assessed at the same time point, i.e. 60 min from start of recording) with and without the additional challenge of SD. A concentration-dependent increase in fEPSP run-down was detected even without SD, but the degree of suppression was greatly increased by the combination of Zn2+ with SD.
[Zn2+]i loading increased DC shift duration
We next assessed the characteristics of SDs, generated under the conditions shown in Fig. 1a. In Pyr control slices, the waveform and duration of DC shifts were similar to those previously described (Somjen 2001) and recorded under near-identical conditions without Pyr (Aiba and Shuttleworth 2012) (Fig. 2a). Following [Zn2+]i loading, the duration of DC shifts was markedly increased (Fig. 2b) in a concentration-dependent manner. Propagation rates of SD were assessed using IOS signals (see Methods) and no significant changes in rates were observed (Fig. 2c). The effects of [Zn2+]i loading on DC shift duration are consistent with previous reports that impaired metabolism is associated with increased DC shift duration and worse clinical outcome (Hartings et al. 2011b).
Extracellular adenosine accumulation and A1R activation following SD with [Zn2+]i loading
Impaired metabolism can lead to increased adenosine accumulation (Dunwiddie and Masino 2001). It was recently shown in brain slices that secondary fEPSP suppression after SD is mainly mediated by extracellular adenosine accumulation and activation of adenosine A1 receptors (A1Rs) (Lindquist and Shuttleworth 2012). As shown in Fig. 3a, pre-exposure to a selective A1R antagonist [8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 200 nM], significantly decreased the duration and degree of fEPSP suppression after SD with [Zn2+]i loading, compared to the suppression shown in Fig. 1. The degree of fEPSP suppression under these different conditions is summarized in Fig. 3b. These results are consistent with the effective reversal of secondary fEPSP suppression described previously (Lindquist and Shuttleworth 2012), and imply that the sustained suppression observed in Fig. 1a is largely because of A1 receptor activation rather than neuronal injury.
We next tested whether additional extracellular adenosine accumulation contributed to the A1R-mediated fEPSP suppression seen above. Estimated extracellular adenosine concentration was measured at the slice surface using an enzyme linked probe (see Methods). A test concentration of 30 μM Zn/Pyr in combination with SD was chosen as these conditions resulted in a substantial suppression of fEPSPs (see Fig. 1a) that was completely mediated by A1R activation (see Fig. 3a). Estimated peak extracellular adenosine accumulation during SD was significantly greater when [Zn2+]i loading was combined with SD (Fig. 3c, n = 5 each). To test whether exogenous adenosine could replicate the effects of Zn/Pyr exposure, adenosine (50 μM) was applied immediately following the induction of SD in a separate set of experiments (Fig. 3d). The recovery of fEPSPs following SD was significantly decreased by exogenous adenosine, compared with control slices (Fig. 3d). Together, these findings suggest that the enhanced fEPSP suppression by [Zn2+]i loading with SD was mediated predominantly by increased extracellular adenosine and A1R activation.
Potential intracellular targets for [Zn2+]i loading effects
We next tested potential mechanisms which could link [Zn2+]i loading to metabolic inhibition. Enhanced levels of [Zn2+]i can deplete NAD+ and thereby inhibit glycolysis either by induction of PARP-1 or activation of SIRT protein pathways (Sheline et al. 2000; Cai et al. 2006). These possibilities were tested by using selective inhibitors for either PARP-1 (3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone DPQ, 25 μM) or SIRT (sirtinol, 30 μM). As shown in Fig. 4, DPQ with 30 μM Zn/Pyr did not increase fEPSP recovery compared to vehicle treated slices with 30 μM Zn/Pyr (p = 0.073, n = 8 each). In addition, slices pre-exposed to sirtinol with 30 μM Zn/Pyr did not result in a significant recovery of fEPSPs following SD when compared with preparations exposed to Zn/Pyr alone (p = 0.8, n = 3, data not shown).
In a third set of slices, 5 mM pyruvate was superfused for the entire recording session to provide additional TCA metabolic supplementation. In the presence of 5 mM pyruvate, fEPSPs suppression following SD was not significantly different compared to slices exposed to 30 μM Zn/Pyr alone (Fig. 4b, n = 6 each). In addition, in slices supplemented with lactate (5 mM), fEPSP suppression following 30 μM Zn/Pyr with SD was similar to slices without exogenous lactate (p = 0.6, n = 3, data not shown). Taken together, these results suggest that [Zn2+]i loading with SD may lead to increased extracellular adenosine accumulation via mechanisms downstream of glycolysis and pyruvate utilization.
Intracellular accumulation prior to SD is required for Zn2+ effects
To test whether the enhanced fEPSP suppression (seen in Fig. 1a) was indeed a result of [Zn2+]i accumulation and not a result of an extracellular of Zn2+, a set of slices was exposed to 30 μM ZnCl2 in the absence of Pyr. Figure 5a shows full recovery of fEPSPs to pre-SD levels following 30 μM ZnCl2 application (n = 6), indicating that the enhanced suppression of fEPSPs seen in Fig. 1a was because of intracellular accumulation and action of Zn2+. As seen in Fig. 1, a small but significant decrease in fEPSP amplitude was also seen upon the addition of 30 μM ZnCl2 prior to SD induction (baseline: 101.2 ± 1.62 vs. pre-SD: 89.41 ± 3.05, p < 0.01).
We next tested whether [Zn2+]i loading prior to SD was required for the observed effects on fEPSPs (as seen in Fig. 1a). To test this, Zn/Pyr application was begun only 5 min before SD induction to allow equilibration within the slice prior to the SD challenge. In contrast to the results shown in Fig. 1, this exposure resulted in no combined effect of 30 μM Zn/Pyr with SD (Fig. 5b). Together, these data suggest sufficient time is required (> 5 min) to allow for [Zn2+]i accumulation and activation of intracellular targets to generate a significant effect on fEPSP suppression when combined with SD.
Contributions of endogenous Zn2+
We next evaluated whether intracellular accumulation of endogenous Zn2+ contributes to the metabolic burden of SD, and prolonged fEPSPs recovery. We first tested whether removal of all chelatable Zn2+ with a membrane permeable Zn2+ chelator (TPEN, 20 μM throughout entire recording session) would influence fEPSP recovery from SD. TPEN pre-exposure resulted in a significant increase in the rate of fEPSP recovery after a single SD challenge (Fig. 6a). Interestingly, there also was a significant increase in the fEPSP amplitude following SD in TPEN that was not observed in vehicle slices (TPEN baseline vs. post-SD amplitude: 99.80 ± 1.14 vs. 107.6 ± 1.40, p < 0.01; Vehicle baseline vs. post-SD amplitude: 99.32 ± 0.74 vs. 100.5 ± 1.99, p = 0.58, n = 6 all groups).
We next tested potential sources of endogenous Zn2+ responsible for these effects. Zn2+ can be accumulated to high concentrations in many glutamatergic terminals, as a consequence of loading via the transporter ZnT3 (Cole et al. 1999). As terminals depolarize during the propagation of SD, a significant release of synaptic Zn2+ occurs and can be visualized as a propagating wave of extracellular Zn2+ accumulation (Carter et al. 2011). As significant postsynaptic [Zn2+]i accumulation also occurs after SD (Carter et al. 2011), we investigated here whether endogenous synaptic Zn2+ release during SD could provide an additional metabolic burden to subsequent SDs. To test this, a series of three SDs were induced at 20 min intervals, to allow sufficient time for uptake and activation of intracellular targets following Zn2+ release from the first SD (see section ‘Intracellular accumulation prior to SD is required for Zn2+ effects’ above) to influence recovery from prior SDs. However, as shown in Fig. 6b, there was no stepwise increase in the duration of fEPSP recovery after multiple SDs, suggesting that synaptic Zn2+ release may not be a significant contributor. Figure 6c and d are consistent with this, as there was no difference in fEPSP recovery after SD in slices from ZnT3 knockout animals, when compared with wild-type animals.
The results shown in Fig. 6 were surprising, as we previously showed that there is no evidence of run-down of synaptic Zn2+ release during repetitive SDs (Carter et al. 2011), and the figures above suggest that repetitive [Zn2+]i accumulation in advance of SD should lead to delayed functional recovery. Figure 7 provides an explanation for this apparent discrepancy, showing two consecutive SDs separated by 20 min, with similar recovery rates as shown in Fig. 6d. We next tested whether exogenous [Zn2+]i loading with Zn/Pyr was still effective in suppressing fEPSPs in these slices following repetitive SD. Interestingly, the effects of Zn/Pyr on the enhanced fEPSP suppression described above (in SD-naïve slices, Fig. 1a) were completely lost under conditions following repetitive SD (Fig. 7a, n = 6). A set of time-control experiments showed that exogenous [Zn2+]i loading was still effective in slices where repetitive SDs were not generated (Fig. 7b).
Figure 7c shows extracellular adenosine measurements under these conditions of repetitive SD shown in Fig. 7a and b. In slices pre-exposed to two SDs, subsequent Zn/Pyr addition did not produce any increase in the amplitude of extracellular adenosine following SD. This is consistent with the lack of effect of Zn/Pyr exposure in Fig. 7a. In time-control slices not previously exposed to SDs (matching Fig. 7b), there was still a significant increase in extracellular adenosine accumulation by Zn/Pyr with SD. Exogenous adenosine application (25 μM) following the end of the recording session produced similar decreases in fEPSPs under both SD pre-exposed and time-control conditions (final fEPSP amplitudes: 28.29 ± 3.14 vs. 28.16 ± 2.56% baseline, SD pre-exposed vs. time-control respectively, p = 0.97, n = 5 each). These results suggest that the loss of effectiveness of Zn/Pyr on fEPSP recovery is likely a consequence of decreased extracellular adenosine accumulation, rather than a change in sensitivity of synaptic transmission to adenosine, or other desensitization mechanisms.
This study assessed the contributions of [Zn2+]i accumulation to the recovery from spreading depolarization (SD) in acute hippocampal brain slices. Transient [Zn2+]i loading with pyrithione (Zn/Pyr) appeared enhance the metabolic burden following SD, measured by enhanced fEPSP suppression, prolonged extracellular DC shift durations, and increased extracellular adenosine accumulation. Transient [Zn2+]i loading alone led to a slow run-down of fEPSPs, however, when combined with SD, the cumulative metabolic challenge was significantly enhanced. Endogenous Zn2+ release and accumulation during SD propagation did not lead to a significant delay in fEPSP recovery. This could be explained by the need for sufficient time prior to SD for [Zn2+]i accumulation to have significant additive effects, as well as competition with a rapid adaptive mechanism activated following SD that confers resistance to [Zn2+]i effects during repetitive SDs in the brain slice model.
Additional adenosine accumulation following [Zn2+]i loading together with SD
Previous study has demonstrated accumulation of extracellular adenosine and suppression of evoked fEPSPs under metabolically compromised conditions (Rudolphi et al. 1992; Masino et al. 1999). Such a mechanism appears to explain the additive effect of Zn2+ with SD in this study, as extracellular adenosine accumulation was significantly enhanced by [Zn2+]i loading, and the A1R antagonist DPCPX improved fEPSP recovery (Fig. 3a). The adenosine measurements reported here are estimates of adenosine concentrations at the slice surface, determined by comparison with exogenous adenosine applications (Lindquist and Shuttleworth 2012). Concentrations of endogenous adenosine at synaptic sites within slices may be quite different, but regardless of absolute levels, the results here show significant functional increases in adenosine accumulation after SD in slices pre-loaded with Zn2+.
A significant run-down of fEPSPs was seen with [Zn2+]i loading in the 60 min time-control experiments where SD was not induced (Fig. 1), and suggestions of this run-down could be seen even in the initial 10 min washout period prior to SD challenges. It seems likely that A1R suppression also contributes to this slow run-down, and is additive with the metabolic challenge of SD. Curiously, a small transient increase in fEPSPs was seen upon the addition of Zn/Pyr (seen most easily in Fig. 4). This increase was present during the addition of either pyrithione or histidine alone (data not shown, n = 6 each), which suggests that extracellular or contaminating Zn2+ (see 'Discussion' in Carter et al. 2011) may contribute by effects on either excitatory or inhibitory transmission (Xie et al. 1994; Kim et al. 2002; Huang et al. 2008).
Suppression of excitatory neurotransmission by adenosine acting on A1Rs has been widely considered neuroprotective, and following hypoxic challenges, fEPSP recovery was impaired in the presence of DPCPX or in mice lacking A1Rs (Johansson et al. 2001; Sebastiao et al. 2001). However, in this study of SD in normoxic conditions, we did not detect deleterious effects of DPCPX, and found instead that synaptic recovery was improved under all conditions tested. The majority of fEPSP suppression was mediated by A1R activation following 30 μM Zn/Pyr. While fEPSP recovery was significantly improved by DPCPX with the 100 μM Zn/Pyr challenge, some sustained fEPSP suppression was still seen (Fig. 3a). This suggests that under conditions of greatly increased metabolic inhibition, SD can result in either irrecoverable injury, out competition of DPCPX by excessive adenosine accumulation, or a combination of both. It remains to be determined whether adenosine accumulation following SD can contribute to delayed neuroprotection under other conditions, for example with clusters of SDs in ischemic conditions.
Mechanisms coupling [Zn2+]i accumulation to adenosine accumulation
Several possibilities can be considered for mechanisms coupling [Zn2+]i loading to adenosine accumulation. Sufficient time (many minutes) was required for [Zn2+]i accumulation to have significant additive effects when combined with SD, consistent with a mechanism involving inhibition of metabolism or depletion of metabolic stores prior to SD. Based on the lack of effectiveness of selective inhibitors, PARP-1 or SIRT protein activation (see Introduction) did not appear to be significant contributors to the delayed fEPSP recovery following [Zn2+]i loading together with SD (see Section ’Potential intracellular targets for [Zn2+]i loading effects'). More prolonged Zn2+ exposures were utilized in studies which these pathways were identified in toxic Zn2+ effects (Sheline et al. 2000; Cai et al. 2006) and it is possible that postsynaptic [Zn2+]i accumulation here was not sufficiently high enough, or of long enough duration to activate PARP-1 or SIRT pathways. An additional test of PARP-1 and SIRT pathway involvement could involve NAD+ supplementation (Sheline et al. 2000). However, this possibility was difficult to test experimentally in these slice experiments, as application of NAD+ alone was sufficient to suppress fEPSPs in a DPCPX-sensitive manner (data not shown). This effect may be because of its ability to mimic adenosine binding at A1Rs (Galarreta et al. 1993).
The lack of effect of pyruvate and lactate supplementation (see Section ‘Potential intracellular targets for [Zn2+]i loading effects’) suggest that any metabolic inhibition caused [Zn2+]i loading is likely because of actions downstream of pyruvate utilization, within either the TCA cycle (Brown et al. 2000) or the mitochondrial electron transport chain (ETC) (Sharpley and Hirst 2006). Interestingly, fEPSP recovery was enhanced in the presence of the Dimethyl sulfoxide (DMSO) vehicle used for both the DPQ and sirtinol studies (compare Fig. 4a and b). DMSO has been reported to have antioxidant properties (Sanmartin-Suarez et al. 2011), and it is possible that DMSO reduced reactive oxygen species (ROS) generation by Zn2+. Inhibition of ETC is established to lead to ROS production (Adam-Vizi 2005) and mitochondrial Zn2+ accumulation, impairment of mitochondrial function, and ROS production have all been reported following Zn2+ accumulation in neurons (Sensi et al. 1999; Seo et al. 2001; Dineley et al. 2005).
In addition to accumulation because of metabolic depletion, it is possible that other sources of adenosine accumulation could also contribute to effects of Zn2+ loading. Higher order metabolic precursors of adenosine, such as ATP release from astrocytes (Halassa and Haydon 2010) or cAMP release (Rosenberg and Dichter 1989) can be converted to adenosine extracellularly. It is not yet known whether Zn2+ may play a role in either of these, or other, mechanisms of extracellular adenosine accumulation.
Effects of [Zn2+]i loading on the characteristics of SD
Longer DC shift durations during SD are associated with worse clinical outcomes in a variety of brain injuries that involve impaired metabolism (Dohmen et al. 2008; Hartings et al. 2011b; Lauritzen et al. 2011). Recent study in vitro has also shown that longer DC shifts are associated with longer fEPSP suppression in hippocampal slices (Lindquist and Shuttleworth 2012). We found here that [Zn2+]i loading prior to SD significantly prolonged the duration of DC shifts (Fig. 2), together with the effects on fEPSP recovery and adenosine accumulations discussed above. These results further the notion that longer DC shifts, both in vitro and in vivo, can be an indicator of larger metabolic challenges, and support a metabolic depletion mechanism for the Zn2+ effects described here.
Interestingly, there was no effect of [Zn2+]i loading on SD propagation rate (Fig. 2c), despite the fact that depletion of metabolic substrates can increase SD propagation rates (Seidel and Shuttleworth 2011). This suggests that either the degree of metabolic inhibition with [Zn2+]i loading is mild prior to SD, or the nature of metabolic inhibition (i.e.. substrate removal vs. direct metabolic inhibition) determines effects on propagation rate.
Endogenous Zn2+ accumulation
We previously showed waves of synaptic Zn2+ release and intracellular accumulation following SD propagation (Carter et al. 2011). However, results from this study suggest that synaptic Zn2+ release and subsequent postsynaptic accumulation does not lead to signs of progressive metabolic depletion, even when SDs were generated repetitively within the same slice (see Fig. 6). One explanation for this lack of effect could be that endogenous [Zn2+]i levels achieved following SD are much lower than those achieved during [Zn2+]i loading with pyrithione. However, the results of Fig. 7 also suggest the potential for a significant and rapid adaptation following SD in brain slices, making the tissue entirely resistant to a subsequent metabolic challenge with Zn2+ loading. SD is established to precondition tissues against subsequent ischemic challenges in vivo, although this effect has been tested only at relatively late time points (24 h or greater) following SD induction (Matsushima et al. 1996; Yanamoto et al. 1998). It is not yet known whether the adaptation observed at early time points here shares a similar mechanism with ischemic preconditioning in vivo, or with PKC mechanisms linking intracellular Zn2+ increases to preconditioning in cultured cortical neurons (Aras et al. 2009).
The small increase in fEPSP recovery seen in the presence of TPEN, together with the lack of effect on fEPSP recovery in ZnT3 KO animals (see Fig. 6), suggests that other endogenous, non-synaptic sources of Zn2+ can influence fEPSP recovery following SD. Potential contributors could include liberation of [Zn2+]i from Zn2+-binding proteins (Aizenman et al. 2000) that may occur during conditions generated by SD, or from influx of contaminating Zn2+ present in the superfusate (see 'Intracellular Zn2+ loading' in Carter et al. 2011). However, the effect of TPEN on fEPSP recovery was quite small, and possibly counteracted by adaptive mechanisms following SD as described above.
Implications for injury
The results here emphasize potentially deleterious effects of intracellular Zn2+ accumulation, when combined with SD. Sustained [Zn2+]i increases are known to occur in the days following stroke injury (Koh et al. 1996), and it seems likely that these sustained increases would make neurons more vulnerable to additional metabolic challenges of SDs that occur in the post-stroke brain (Hartings et al. 2003; Selman et al. 2004; Dreier 2011; Lauritzen et al. 2011). However, we have also shown that extracellular actions of Zn2+ can limit the propagation of SD (Aiba et al. 2012). Therefore, selectively targeting intracellular Zn2+ accumulation may be more effective in limiting neuronal injury and improving outcome following several brain injury models, particularly those that involve SD. However, it is important to also emphasize that clusters of repetitive SD have been associated with poor clinical outcomes under some conditions (Dreier 2011; Hartings et al. 2011a; Lauritzen et al. 2011) and a rapid adaptation following SD appears to protect tissues against the additive metabolic burden of intracellular Zn2+ (Fig. 7). Therefore, deleterious metabolic effects of intracellular Zn2+ accumulation may not be significant during repetitive SDs clusters, and under such conditions, extracellular effects of Zn2+ may dominate and limit the spread of these events (Aiba et al. 2012). These and other divergent actions of intracellular and extracellular Zn2+ are likely contributors to different effects of Zn2+ chelation that have been reported in experimental injury settings (Lee et al. 2002; Kitamura et al. 2006).
Supported by NIH grants NS 074584 (R.E.C.), T32 HL007736 (J.L.S.), NS 078805 (B.E.L.), and NS 051288 (C.W.S.). The authors have no conflict of interest to declare.