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The Journal of Neuroscience, February 15, 2002, 22(4):1273-1279

Blockade of Ca²⁺-Permeable AMPA/Kainate Channels Decreases Oxygen-Glucose Deprivation-Induced Zn²⁺ Accumulation and Neuronal Loss in Hippocampal Pyramidal Neurons

Hong Z. Yin¹, Stefano L. Sensi^{1, 4}, Fumio Ogoshi², and John H. Weiss^{1, 2, 3}

Departments of ¹ Neurology, ² Anatomy and Neurobiology, and ³ Neurobiology and Behavior, University of California, Irvine, Irvine, California 92697-4292, and ⁴ Department of Neurology, University G. d'Annunzio, Chieti 66013, Italy

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Synaptic release of Zn²⁺ and its translocation into postsynaptic neurons probably contribute to neuronal injury after ischemia or epilepsy. Studies in cultured neurons have revealed that of the three major routes of divalent cation entry, NMDA channels, voltage-sensitive Ca²⁺ channels (VSCCs), and Ca²⁺-permeable AMPA/kainate (Ca-A/K) channels, Ca-A/K channels exhibit the highest permeability to exogenously applied Zn²⁺. However, routes through which synaptically released Zn²⁺ gains entry to postsynaptic neurons have not been characterized in vivo. To model ischemia-induced Zn²⁺ movement in a system approximating the in vivo situation, we subjected mouse hippocampal slice preparations to controlled periods of oxygen and glucose deprivation (OGD). Timm's staining revealed little reactive Zn²⁺ in CA1 and CA3 pyramidal neurons of slices exposed in the presence of O₂ and glucose. However, 15 min of OGD resulted in marked labeling in both regions. Whereas strong Zn²⁺ labeling persisted if both the NMDA antagonist MK-801 and the VSCC blocker Gd³⁺ were present during OGD, the presence of either the Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) or the extracellular Zn²⁺ chelator Ca²⁺ EDTA substantially decreased Zn²⁺ accumulation in pyramidal neurons of both subregions. In parallel experiments, slices were subjected to 5 min OGD exposures as described above, followed 4 hr later by staining with the cell-death marker propidium iodide. As in the Timm's staining experiments, substantial CA1 or CA3 pyramidal neuronal damage occurred despite the presence of MK-801 and Gd³⁺, whereas injury was decreased by NAS or by Ca²⁺ EDTA (in CA1).

Key words: zinc; ischemia; glutamate; AMPA; naphthyl acetyl spermine; Timm's stain; pyramidal neuron; neurotoxicity; hippocampal slice

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transient global ischemia causes degeneration of certain hippocampal pyramidal neurons, particularly in the CA1 subregion (Pulsinelli et al., 1982). Recent studies implicate Zn²⁺ ions as likely contributors to this injury. Zn²⁺ is sequestered at high concentrations in presynaptic boutons of many excitatory synapses, with particularly high levels in the hippocampus; when released with neuronal activity, it is estimated to achieve peak synaptic concentrations of several hundred micromoles per liter (Frederickson et al., 2000). In vivo, both transient global ischemia and seizure activity have been associated with a depletion of presynaptic Zn²⁺ and concomitant Zn²⁺ accumulation in degenerating postsynaptic neurons ("Zn²⁺ translocation") (Sloviter, 1985; Frederickson et al., 1989; Tonder et al., 1990; Koh et al., 1996). Additional support for a direct injurious role for Zn²⁺ in these conditions is provided by observations that extracellular Zn²⁺ chelators decrease both the appearance of Zn²⁺ in postsynaptic neurons and the resultant selective neuronal loss (Koh et al., 1996; Suh et al., 1996).

Because presynaptic Zn²⁺ is coreleased with glutamate from excitatory terminals and appears to gain direct entry into certain postsynaptic neurons, it is reasonable to consider that Zn²⁺ might permeate postsynaptic glutamate-activated channels. Indeed, in vitro studies have indicated that Zn²⁺ is potently neurotoxic (Choi et al., 1988) and is able to gain entry to neurons through voltage-sensitive Ca²⁺ channels (VSCCs), NMDA channels, or Ca²⁺-permeable AMPA/kainate (Ca-A/K) channels (Weiss et al., 1993; Yin and Weiss, 1995; Sensi et al., 1997). However, neurotoxicity and imaging studies have suggested that of these routes, Ca-A/K channels have the greatest permeability to Zn²⁺ (Yin and Weiss, 1995; Sensi et al., 1999), with intermediate VSCC and minimal NMDA channel permeability (and Zn²⁺ actually being an effective NMDA channel blocker) (Peters et al., 1987; Westbrook and Mayer, 1987).

Although culture studies would favor the possibility that synaptically released Zn²⁺ might preferentially pass through Ca-A/K channels (Yin and Weiss, 1995; Sensi et al., 1999), their presence on pyramidal neurons has not been substantiated by most electrophysiological studies. However, certain histochemical and electrophysiological evidence suggests that Ca-A/K channels might often be present in hippocampal pyramidal neurons, but with preferential localization in the distal dendrites, where they are hard to detect by recording on or near the soma (Pruss et al., 1991; Williams et al., 1992; Toomim and Millington, 1998; Yin et al., 1999; Lerma et al., 1994).

Most models of ischemic neurodegeneration have focused on the putative role of NMDA receptor activation. However, use of NMDA antagonists in animal models of ischemia as well as in human clinical trials has not generally shown the anticipated robust efficacy (Lee et al., 1999). One possible factor is that certain environmental perturbations associated with acute ischemia, specifically synaptic Zn²⁺ elevations and tissue acidosis, each can decrease NMDA channel activity (Peters et al., 1987; Westbrook and Mayer, 1987; Tang et al., 1990; Traynelis and Cull-Candy, 1990). The present study is motivated by the hypothesis that Ca-A/K channels, which share high Ca²⁺ permeability with NMDA channels but are unique in their high permeability to Zn²⁺, contribute to ischemic neurodegeneration by serving as routes through which synaptically released Zn²⁺ gains entry to hippocampal pyramidal neurons. To address this hypothesis, we used acute hippocampal slice preparations from adult mice subjected to brief periods of oxygen and glucose deprivation (OGD) (Kass and Lipton, 1982; Monette et al., 1998) as a model of trans-synaptic Zn²⁺ movement occurring under conditions of ischemia.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and reagents. Propidium iodide (PI) and Newport Green were purchased from Molecular Probes (Eugene, OR). 1-Naphthyl acetyl spermine (NAS) was kindly provided by Daicel Chemical (Tokyo, Japan). MK-801 was purchased from Research Biochemicals (Natick, MA). Tissue culture media and serum were supplied by Invitrogen (Grand Island, NY). Most other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Animal usage and tissue preparations. All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the University of California Irvine Institutional Animal Care and Use Committee. Adult Swiss-Webster mice (8-10 weeks of age; weight 25-30 gm) from Simonsen Laboratories (Gilroy, CA) were deeply anesthetized with halothane and decapitated; their brains were rapidly removed, and coronal slices (400 µm) were cut with a vibratome. (Thus, all slice manipulations were effectively performed in duplicate, with effects on each hemisphere averaged before compilations across experiments.)

Murine forebrain cultures, derived from embryonic day 15 embryos, were plated on previously established astrocytic monolayers and used between 13 and 16 d in vitro (Yin and Weiss, 1995).

Oxygen-glucose deprivation of slices. All slice manipulations (including equilibration) were performed in covered chambers containing 6 ml of buffer, with slices completely submerged and protected from the vigorous bubbling in the chamber by a semipermeable nylon mesh (Millicell CM inserts; Millipore, Bedford, MA) through which small needle holes were made to facilitate solution exchange. All chamber solutions were prebubbled with either O₂/5% CO₂ or N₂/5% CO₂ gas for 30 min before slice immersion to ensure O₂ saturation or O₂ removal as desired. Drugs were all dissolved in water at high concentrations (NAS, MK-801 and Gd³⁺ at 15 mM; Ca²⁺EDTA at 100 mM) and added to buffers immediately before experiments.

Immediately after vibratome sectioning, coronal slices (the most anterior slice was discarded) were transferred to a chamber with 6 ml of cold (4°C) Ca²⁺-free equilibration buffer (in mM: 126 NaCl, 24 NaHCO₃, 1 NaH₂PO₄, 2.5 KCl, 10 MgSO₄, 10 glucose, pH 7.4) bubbled with 95% O₂ and 5% CO₂ and containing the NMDA blocker MK-801 (15 µM), the VSCC blocker Gd³⁺ (20 µM), and the Ca-A/K channel blocker NAS (300 µM) for 25 min. Consistent with a recent report (Suh et al., 2000), longer periods of equilibration were associated with depletion of much of the endogenous Zn²⁺ stores.

At the end of the 25 min of equilibration, slices were transferred to separate chambers, each containing oxygenated equilibration buffer (4°C), but only with antagonists to be used with that slice during the OGD exposure. Specifically, for the primary set of experiments presented, two slices were transferred to buffer alone (for the OGD and the +O₂ conditions) and one each to chambers containing MK-801 (15 µM) with Gd³⁺ (20 µM), NAS (300 µM), or Ca²⁺ EDTA (3 mM). After 2 min, slices were subjected to OGD (for 5 or 15 min, see below) by transfer to chambers containing warmed (37°C) glucose-free artificial CSF (ACSF) (in mM: 126 NaCl, 24 NaHCO₃, 1 NaH₂PO₄, 2.5 KCl, 2 MgCl₂, 1 CaCl₂, 7.0 sucrose, pH 7.4) bubbled with 95% N₂ and 5% CO₂ either alone or with the same antagonists as present in the previous step. In each experiment, one other matched slice was exposed to oxygenated ACSF with glucose (OG-ACSF; 10 mM instead of sucrose; +O₂ condition) in place of the OGD exposure.

To assess Zn²⁺ translocation, OGD exposures occurred for 15 min, followed immediately by Timm's staining as described. To assess injury, 5 min OGD exposures were used, followed by additional incubation of slices at 22°C in OG-ACSF containing the same antagonists as present during the OGD exposure. [This temperature was selected as one that permits evolution of injury without causing the rapid release of Zn²⁺ from slices that has been reported to occur at warmer temperatures (Suh et al., 2000).] After 3.5 hr, the cell-death marker PI (5 µg/ml) was added to the bath for 30 min before fixation in 4% paraformaldehyde (in PBS; 3 hr at room temperature, then 12 hr at 4°C) and visualization of PI staining under confocal microscopy. For all experiments, p < 0.05 was preselected as a cutoff point for significance.

For control experiments examining the effects of NAS on transmitter release after equilibration, slices were loaded with [³H]-D-aspartate (2.5 µCi/ml) in OG-ACSF buffer (37°C, 30 min) with or without NAS (300 µM). (MK-801 was included in each condition to help maintain slice viability during the prolonged loading and wash steps.) After washout of extracellular isotope (20 min, 22°C, in the presence of the same antagonists), slices either were subjected to OGD as above (37°C, 15 min) in the presence of the same antagonists or were identically incubated in OG-ACSF with MK-801. Immediately after OGD, the slice was removed from the buffer and solubilized in 20% HCl; isotope accumulation was then counted in both the slice and the bathing OGD buffer. The percentage of [³H]-D-aspartate released in each condition was calculated as the counts in the buffer divided by the total counts for that slice (buffer + slice).

Timm's staining. Immediately after the OGD exposures, slices were incubated in 0.1% (NH₄)₂S in oxygenated equilibration buffer (containing the same antagonists as present during OGD) for 10 min to precipitate intracellular Zn²⁺, followed by fixation in 4% paraformaldehyde (3 hr at room temperature). Slices were then incubated in the dark in a solution consisting of 1 part solution A (1 M AgNO₃), 20 parts solution B (2% hydroquinone and 5% citric acid in water), and 100 parts solution C (20% gum arabic in water). Development took ~1 hr, was monitored by periodic evaluations under low light, and was terminated by washing in water. Slices were then incubated overnight in 30% sucrose (4°C) before frozen sections (25 µm) were made for dehydration and permanent mount.

To assess the degree of labeling, the top and bottom three sections from each slice were discarded because of frequent nonspecific trauma-induced stain, and the remaining 7-10 sections were kept for examination. In each section, pyramidal neuronal labeling in each of the two hemispheres was visually assessed on a four-point scale (near absence, light, strong, and very strong staining) in two regions of CA3 and three regions of CA1 by careful matching with previously established standards. Thus, in each experiment, values for each condition were derived in 42-60 regions for CA1 and 28-40 regions for CA3. Values for each condition were then averaged within each experiment before averaging across experiments.

Kainate-stimulated Co²⁺ uptake labeling. Kainate-induced Co²⁺ uptake labeling of cultured neurons (used for the experiments summarized in Fig. 1) was performed generally as described previously (Yin et al., 1994, 1999). After Co²⁺ loading (by exposure to 100 µM kainate with 5 mM Co²⁺ for 10 min), intracellular Co²⁺ was precipitated with 0.05% (NH₄)₂S, the cultures were fixed, and the stain silver was enhanced by a modified Timm's stain procedure, largely as described above.

View larger version (24K): [in this window] [in a new window]   Figure 1.   NAS blocks Zn2+ entry through Ca-A/K channels. Cultures were loaded with the Zn2+-sensitive fluorescent probe Newport Green and exposed to kainate (KA; 100 µM) in the presence of Zn2+ (300 µM), MK-801 (MK; 10 µM), and NAS (300 µM) for 5 min. After 20 min, the cultures were identically re-exposed without NAS. After imaging, the subpopulation of neurons possessing large numbers of Ca-A/K(+) neurons was identified by kainate-stimulated Co2+ uptake labeling [n = 3 experiments; 325 total neurons; 28 Ca-A/K(+) neurons; calibrated [Zn2+]i values are ± SEM]. Note the high Zn2+ increases occurring in Ca-A/K(+) neurons after removal of the NAS block. In contrast, the NAS had little effect on Zn2+ increases in other neurons, which result primarily from slower influx through VSCCs.

Imaging studies. Forebrain cultures (plated on coverslips) were loaded with the low-affinity Zn²⁺ selective probe Newport Green diacetate by adding 5 µl of a 1 mM stock (in DMSO) with 0.2% pluronic acid per milliliter of buffer (30 min, room temperature), followed by washing and incubation in the dark for an additional 30 min. Images were obtained (excitation, 490 nm; emission, 530 nm) using a 12 bit digital CCD camera (Roper Scientific, Tucson, AZ) attached to a Nikon Diaphot inverted microscope (Nikon USA, New York, NY) equipped with a 40× (NA, 1.3) epifluorescence oil immersion objective. Experiments were analyzed using Metafluor 4.0 software (Universal Imaging, West Chester, PA), and [Zn²⁺]_i was determined, after background subtraction, as:
using K_d of 1 µM. F_max was obtained at the end of each experiment by adding the Zn²⁺-selective ionophore Na⁺ pyrithione (10 µM) in the presence of 500 µM Zn²⁺ (the fluorescence rapidly approached a maximum) and F_min was obtained (after Zn²⁺ washout) by adding the cell-permeable Zn²⁺ chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (50 µM) (Sensi et al., 1999).

For imaging of slices, we used a Bio-Rad MRC 600 confocal system (Bio-Rad Laboratories, Hercules, CA) attached to an inverted Nikon Diaphot microscope (Nikon USA) equipped with a krypton laser (excitation, 568 nm; emission, >648 nm) and a 20× epifluorescence objective. In each experiment, the OGD condition was initially scanned to determine a depth halfway between the slice surface and loss of fluorescence near midslice, and the identical depth was used for other conditions (between 75 and 100 µm from the slice surface). PI staining intensity in each condition was quantified as the background-subtracted average pixel intensity in the CA1 or CA3 pyramidal cell layers of each image, averaged between the two hemispheres. Because of a moderate degree of experiment to experiment variability intrinsic to this paradigm, values in each condition were first normalized to the OGD condition of that experiment (=100%) before averaging across experiments, and statistical assessment was by a paired t test against the OGD condition.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NAS blocks Zn²⁺ entry through Ca-A/K channels on cultured neurons

To block Ca-A/K channels, we made use of the polyamine Ca-A/K channel pore blocker NAS, a synthetic analog of joro spider toxin (Koike et al., 1997) that has been used previously, much as in this study, to block injury resulting from Ca-A/K channel activation in a hippocampal slice model (Oguro et al., 1999). NAS has been found by electrophysiological studies to block Ca²⁺ entry through Ca-A/K channels in a voltage- and use-dependent manner, with potent (low micromolar) block at resting potentials and decreasing efficacy on depolarized neurons (Koike et al., 1997). Thus, before NAS was used in slice OGD studies, control experiments were performed to characterize its utility as a blocker of Zn²⁺ entry into neurons through Ca-A/K channels.

Because NAS block is highly voltage dependent, we first examined concentrations needed to block Ca-A/K channels on neurons depolarized by ongoing glutamate receptor activation (as would be expected to be the case during OGD). To screen its efficacy, we made use of a histochemical procedure based on kainate-stimulated uptake of Co²⁺ ions that identifies the subpopulation of neurons possessing large numbers of Ca-A/K channels [Ca-A/K(+) neurons] (Pruss et al., 1991; Yin et al., 1994). The specificity of the stain depends on selective permeability of Co²⁺ ions through Ca-A/K channels and has been demonstrated by the inability of NMDA or high-K⁺ depolarization (to activate VSCCs) to trigger comparable Co²⁺ uptake. We found that 300 µM but not 100 µM NAS effectively blocked the Co²⁺ uptake (data not shown); thus, we used this concentration in subsequent studies. Additional control experiments were undertaken to rule out the possibility that NAS chelates extracellular Zn²⁺ and prevents Zn²⁺ entry through that mechanism rather than by blocking channels. Cortical neuronal cultures were loaded with the Zn²⁺-sensitive (and Ca²⁺-insensitive) fluorescent probe Newport Green and exposed for 5 min to 300 µM Zn²⁺ in 60 mM K⁺ buffer to induce neuronal depolarization and consequent entry of Zn²⁺ through VSCCs. With repeat exposures (after 15 min of recovery) in the additional presence of excess NAS (1 mM), there was no decrement in the [Zn²⁺]_i response [peak fluorescence increase of 36 ± 2.1% (SEM) without NAS and 40 ± 3.1% with NAS; n = 44 neurons], indicating that NAS neither appreciably chelates Zn²⁺ nor interferes with Newport Green fluorescence.

Subsequent experiments examined the ability of NAS to specifically block Zn²⁺ entry through Ca-A/K channels. Newport Green-loaded cultures were exposed to kainate (100 µM) in the presence of Zn²⁺ (300 µM), the NMDA channel blocker MK-801 (10 µM), and NAS (300 µM) for 5 min. After 20 min, the cultures were subjected to a second 5 min exposure to an identical solution lacking NAS. After imaging, Ca-A/K(+) neurons were identified by kainate-stimulated Co²⁺ uptake labeling, as described above. In the presence of NAS, all neurons showed moderate increases in [Zn²⁺]_i, indicative of depolarization and Zn²⁺ entry through VSCCs. After removal of the NAS, however, unblocking of Ca-A/K channels resulted in greater [Zn²⁺]_i increases in Ca-A/K(+) neurons (Fig. 1). Thus, NAS appears to be able to effectively block Zn²⁺ entry through Ca-A/K channels of depolarized neurons, while having little effect on influx through VSCCs.

As a final control, we examined possible effects of NAS on presynaptic release, an effect that could also contribute to observed sequelae of OGD in slices. Coronal brain slices were loaded with [³H]-D-aspartate before being subjected to OGD in the presence or absence of NAS (300 µM) or to incubation in the presence of O₂ and glucose (+O₂). We found that both OGD and OGD plus NAS induced significantly more release than +O₂ ([³H]-D-aspartate release of 21.2 ± 6% and 17.1 ± 3.5% in OGD and OGD+NAS, respectively, vs 3.1 ± 0.3% in +O₂), but that NAS had no significant effect on the OGD-induced release (n = 4 experiments; OGD and OGD plus NAS different from +O₂ at p < 0.05 by ANOVA with a Student-Newman-Keuls post hoc test), indicating the paucity of presynaptic action of NAS.

Ca-A/K channels are a major route for OGD-induced Zn²⁺ translocation into hippocampal pyramidal neurons

To model ischemia-induced Zn²⁺ translocation under simplified conditions, acute hippocampal slice preparations have distinct advantages: they can be subjected to well-controlled environmental and pharmacological manipulations while maintaining the synaptic connectivity and presynaptic Zn²⁺ stores present in the animals from which they are derived. Conversely, a potential disadvantage is that Zn²⁺ is rapidly depleted after slice cutting and incubation (Suh et al., 2000). Thus, certain aspects of the experimental protocol were designed to minimize this nonspecific Zn²⁺ loss: the slice-stabilization step (in cold buffer) is of limited duration, followed by rapid transitioning to the OGD treatment (at 37°C), and slices are subsectioned after OGD exposures to visualize Zn²⁺ accumulation deep in the slices, where direct trauma-induced release is minimized.

Coronal slices (400 µm) were obtained from adult (8-10 week old) Swiss-Webster mice and immediately placed in "equilibration chambers" containing cold, Ca²⁺-free equilibration buffer, with the additional presence of NAS (300 µM), MK-801 (15 µM), and the broad-spectrum VSCC antagonist Gd³⁺ (20 µM) (Canzoniero et al., 1993; Sensi et al., 1997) to allow slices to stabilize and equilibrate in the presence of channel blockers. After 25 min, slices were transferred for 2 min to "pre-OGD chambers," allowing each slice to partially equilibrate with the drugs with which it is to be subjected to OGD. Slices were then transferred to "OGD chambers" containing glucose-free, O₂-depleted OGD buffer (37°C) containing no drugs, MK-801 and Gd³⁺, NAS, or Ca²⁺ EDTA (3 mM). One additional slice in each set was handled exactly as the OGD condition but was exposed in the presence of O₂ and glucose (+O₂). Slices were then subjected to a modified Timm's staining procedure to visualize histochemically reactive intracellular Zn²⁺ (Yin et al., 1999) and also cut to 25 µm sections for assessment of labeling intensity, as described previously. [For comparison, in some experiments in which slices were stained immediately after removal from the cold equilibration buffer, labeling was comparable with that seen in +O₂ (data not shown).]

Slices that were incubated in the presence of O₂ and glucose showed characteristic strong Zn²⁺ labeling in the mossy fiber projections from dentate granule cells to CA3 pyramidal neurons (indicative of the high Zn²⁺ content of this pathway) but little or no staining in pyramidal neurons. In contrast, after OGD, distinct Zn²⁺ staining was consistently evident in pyramidal neurons of both CA1 and CA3 subregions. Although strong staining was also seen in the MK-801 and Gd³⁺ condition, slices that were treated with either Ca²⁺ EDTA or NAS showed significantly less Zn²⁺ accumulation in pyramidal neurons of both subregions (Figs. 2, 3). Because it is cell impermeant, the block by Ca²⁺ EDTA strongly supports an extracellular origin for much of the Zn²⁺, consistent with its derivation from presynaptic release. The additional observation of block by NAS suggests that much of this Zn²⁺ enters through Ca-A/K channels.

View larger version (107K): [in this window] [in a new window]   Figure 2.   OGD causes translocation of endogenous Zn2+ through Ca-A/K channels on hippocampal pyramidal neurons in slice. After equilibration, coronal slices of adult mouse hippocampus were exposed for 15 min (37°C) to OGD alone (OGD), with the NMDA antagonist MK-801 (15 µM) and the VSCC antagonist Gd3+ (20 µM) (OGD+MK, Gd3+), with the Ca-A/K channel blocker NAS (300 µM) (OGD+NAS), or with the extracellular Zn2+ chelator Ca2+ EDTA (OGD+CaEDTA). One other slice was exposed in the absence of drugs but in the presence of oxygen and glucose-containing media (+O2). After exposures, intracellular Zn2+ was visualized by Timm's staining; high-magnification photomicrographs show detail of the CA1 (middle column) and CA3 (right column) pyramidal layers. M.F., Mossy fibers; S.O., Stratum oriens; S.P., stratum pyramidale; S.R., stratum radiatum. Scale bar, 500 µm (low-power views) or 50 µm (CA1 and CA3 details). Note the paucity of Zn2+ labeling in pyramidal neurons in the absence of OGD exposure, in contrast to the strong accumulation in both CA1 and CA3 pyramidal neurons after OGD. Note also that whereas strong staining occurred despite the presence of NMDA and VSCC blockers, the presence of either NAS or Ca2+ EDTA substantially decreased Zn2+ labeling in both CA1 and CA3 subfields.

View larger version (30K): [in this window] [in a new window]   Figure 3.   OGD causes translocation of endogenous Zn2+ through Ca-A/K channels on hippocampal pyramidal neurons in slice: quantitative assessment. Hippocampal slices were exposed for 15 min (37°C) to each of the conditions described above. The graph shows mean Timm's staining intensity (mean ± SEM) of neurons in CA1 and CA3 with each exposure (n = 9; * and # indicate difference from staining intensity in same hippocampal region after OGD; p < 0.05 by ANOVA with Student-Newman-Keuls test). A.U., Arbitrary units.

NAS and Ca²⁺ EDTA decrease pyramidal neuronal damage resulting after OGD

Subsequent experiments examined the extent of pyramidal neuronal injury resulting after OGD exposure. These studies used the fluorescent cell-death marker PI to assess injury. PI enters injured neurons in which the plasma membrane is disrupted and preferentially accumulates in the nucleus of dying neurons. After OGD, the slices were incubated for 4 hr (22°C) in oxygenated media containing the same antagonists that were present during the exposures to permit evolution of the injury. PI was added for the last 30 min of the incubation, followed by fixation and subsequent visualization of PI fluorescence under confocal microscopy. Images were obtained deep (75-100 µm) within the slice, where injury resulting from the trauma of slicing is minimized.

Because 15 min OGD exposures resulted in severe neurodegeneration in all conditions, these studies used a shorter (5 min) period of OGD. Paralleling the degree of Zn²⁺ accumulation observed in the translocation experiments, relatively little PI fluorescence was seen in the CA1 and CA3 pyramidal cell layers of the +O₂ condition, but strong signal was present in the OGD condition. Although strong labeling was also seen with OGD in the presence of MK-801 and Gd³⁺, PI labeling was decreased substantially by NAS and to a smaller degree by Ca²⁺ EDTA (significant only in CA1) (Figs. 4, 5). After imaging, in some experiments slices were resectioned to 25 µm and stained with toluidine blue to evaluate structural changes (Fig. 4). Note that the OGD caused extensive distortion, loss, and swelling of CA1 pyramidal neurons and that addition of NAS or Ca²⁺ EDTA resulted in substantial preservation of architecture. Thus, these observations suggest that the Zn²⁺ that enters postsynaptic pyramidal neurons during OGD contributes to the resultant injury.

View larger version (152K): [in this window] [in a new window]   Figure 4.   Ca-A/K channel blockade attenuates OGD-induced pyramidal neuronal damage. After equilibration, coronal slices of adult mouse hippocampus were exposed for 5 min (37°C) to OGD alone, with the NMDA antagonist MK-801 (15 µM) and the VSCC antagonist Gd3+ (20 µM) (OGD+MK, Gd3+), with the Ca-A/K channel blocker NAS (300 µM) (OGD+NAS), or with the extracellular Zn2+ chelator Ca2+ EDTA (OGD+CaEDTA). One other slice was exposed in the absence of drugs but in the presence of oxygen and glucose-containing media (+O2). After exposures, slices were incubated for 4 hr at 22°C, and injury was evaluated by confocal imaging of PI labeling in the CA1 and CA3 pyramidal cell layers. In each hippocampal region, the left column shows a set of confocal images from a single experiment displayed on an eight bit pseudocolor scale. After imaging, matched slices from a single experiment were sectioned to 25 µm and stained with toluidine blue (right columns). Note the disruption of neuronal morphology and loss of neurons (indicated by voids) in the CA1 pyramidal cell layer after OGD alone or with MK-801 and Gd3+ and the relatively preserved morphology in other conditions. Scale bars, 50 µm.

View larger version (34K): [in this window] [in a new window]   Figure 5.   Quantification of neuronal injury. After equilibration, coronal slices of adult mouse hippocampus were exposed for 5 min (37°C) to OGD alone, with the NMDA antagonist MK-801 (15 µM) and the VSCC antagonist Gd3+ (20 µM) (+MK, Gd3+), with the Ca-A/K channel blocker NAS (300 µM) (+NAS), or with the extracellular Zn2+ chelator Ca2+ EDTA (+Ca-EDTA). One other slice was exposed in the absence of drugs but in the presence of oxygen and glucose-containing media (+O2). After exposures, slices were incubated for 4 hr at 22°C, and injury was evaluated by confocal imaging of PI labeling in the CA1 and CA3 pyramidal cell layers. The graph shows mean PI fluorescence (±SEM) in the CA1 (left bars) and CA3 (right bars) pyramidal cell layers with each exposure, scaled to the fluorescence of the same hippocampal subregion in the OGD condition (n = 8-9; * and # indicate difference from OGD in the same subregion; p < 0.05 by paired t test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Despite the considerable evidence supporting a role for Zn²⁺ translocation in the hippocampal pyramidal neuronal injury resulting after transient global ischemia or prolonged seizures, routes through which synaptic Zn²⁺ might enter postsynaptic neurons in vivo have not been examined. Using the system of acute hippocampal slice as an in vitro model of ischemic injury, we found that brief periods of OGD induce substantial accumulation of histochemically detectable Zn²⁺ in hippocampal pyramidal neurons and the subsequent degeneration of these neurons. Strong Zn²⁺ accumulation and pyramidal neuronal injury were still observed if OGD exposures were performed in the presence of combined NMDA channel and VSCC blockade. However, both of these measures were significantly decreased by the presence of either the extracellular Zn²⁺ chelator Ca²⁺ EDTA or the Ca-A/K channel blocker NAS.

The positive findings of the present studies need to be considered in the context of certain ongoing areas of uncertainty regarding ways in which Zn²⁺ contributes to hippocampal neurodegeneration after ischemia or epilepsy. The first concerns the origin of the reactive Zn²⁺ accumulation in pyramidal neurons occurring in these conditions. It is clear that Zn²⁺ is present in vesicles and is released in response to presynaptic activity (Assaf and Chung, 1984; Howell et al., 1984; Thompson et al., 2000). But recent generation of transgenic mice that appear to completely lack vesicular Zn²⁺ (in which the vesicular Zn²⁺ transporter ZnT3 is knocked out) has raised questions about the source of the postsynaptic Zn²⁺ accumulation. In these mice, prolonged seizures still caused the appearance of reactive Zn²⁺ accumulation in and degeneration of hippocampal pyramidal neurons (Cole et al., 2000; Lee et al., 2000). Although the source of the Zn²⁺ is uncertain, it is likely that much of it comes from release from intracellular stores after strong excitotoxic activation. Zn²⁺ is a component of many metalloenzymes (Vallee and Falchuk, 1993), and Zn²⁺ binding proteins such as metallothioneins are hypothesized to be important endogenous Zn²⁺ buffers (Aschner et al., 1997). Indeed, a recent study found that oxidant exposure caused elevation of cytosolic Zn²⁺ levels in cultured forebrain neurons. The additional observation of that study that Zn²⁺ chelators decreased the injury suggested that intracellular Zn²⁺ release might even contribute to neuronal injury (Aizenman et al., 2000).

Thus, one highly relevant question is the degree to which the Zn²⁺ that appears in pyramidal neurons after OGD represents translocation from presynaptic terminals or cytosolic release of Zn²⁺ already present in the neurons. The observation that Ca²⁺ EDTA decreased Zn²⁺ accumulation as assessed immediately after a 15 min period of OGD provides strong evidence that a substantial portion of the Zn²⁺ is of extracellular origin, as would be predicted by the Zn²⁺ translocation model. The additional observation that Ca²⁺ EDTA appeared to mildly decrease injury in the CA1 subfield 4 hr after an OGD exposure also suggests that the Zn²⁺ entering the pyramidal neurons contributes to their injury. The fact that the protection by Ca²⁺ EDTA appears to be relatively mild might be compatible with an increased Ca²⁺-dependent component to the injury. Indeed, because Zn²⁺ is a potent antagonist of NMDA channels (Peters et al., 1987; Westbrook and Mayer, 1987), removal of synaptic Zn²⁺ by Ca²⁺ EDTA (or its absence in ZnT3 knock-out mice) could result in increased Ca²⁺ entry through NMDA channels. Alternatively, previous studies (Vogt et al., 2000; Li et al., 2001) have suggested that Ca²⁺ EDTA may be slow to chelate rapid synaptic Zn²⁺ increases and thus may fail to fully prevent Zn²⁺ interaction with postsynaptic receptors.

A second critical question concerns the route through which Zn²⁺ gains entry to the pyramidal neurons. As discussed in the introductory remarks, we thought it unlikely that NMDA channels, which are poorly Zn²⁺ permeable and are potently blocked by Zn²⁺, could permit much Zn²⁺ entry; thus, we set out to examine the potential role of highly Zn²⁺-permeable Ca-A/K channels. Indeed, observations that NAS attenuates both Zn²⁺ accumulation and the subsequent neuronal cell death to a far greater degree than potent combined blockade of VSCC and NMDA channels implicate Ca-A/K channel activation in both of these events. However, the presence of Ca-A/K channels on pyramidal neurons is controversial; although most electrophysiological studies in slice have failed to detect them, one in which glutamate was locally applied to acutely dissociated pyramidal neurons found evidence for their presence in distal dendrites (Lerma et al., 1994). Other evidence favoring their presence has been based on kainate-stimulated Co²⁺ uptake labeling (Pruss et al., 1991; Williams et al., 1992; Toomim and Millington, 1998; Yin et al., 1999) and on immunostaining for AMPA subunits. Although AMPA channels are made up of combinations of subunits (GluR1-4), the Ca²⁺ permeability of these channels is regulated by the GluR2 subunit, the presence of which in a heteromeric channel blocks Ca²⁺ permeability. Consistent with a preferential dendritic localization of Ca-A/K channels, several studies have reported an apparent gradient in the intensity of the GluR2 label, with strong somatic staining and less in the distal dendrites (Vickers et al., 1993; Ikonomovic et al., 1995; Yin et al., 1999). Thus, our present observations extend these previous studies in providing new evidence for the presence of functional Ca-A/K channels in postsynaptic membranes of hippocampal pyramidal neurons adjacent to sites of Zn²⁺ release. Other recent studies have raised the intriguing possibility that the numbers of Ca-A/K channels on pyramidal neurons might not be constant. Indeed, observations that GluR2 mRNA and protein may be selectively decreased in hippocampal pyramidal neurons after transient global ischemia or prolonged seizures led Zukin and colleagues (Bennett et al., 1996; Pellegrini-Giampietro et al., 1997) to propose the "GluR2 hypothesis," which suggests that these decreases in GluR2 result in increased numbers of Ca-A/K channels, thereby permitting more Ca²⁺ or Zn²⁺ entry and contributing to the delayed neurodegeneration often seen in these conditions.

Although an ischemia-induced increase in the numbers of Ca-A/K channels might be expected to play a late role in injury, the present results suggest that with strong presynaptic activation, basal numbers of Ca-A/K channels permit sufficient Zn²⁺ entry to mediate rapid neuronal damage. Although it is of interest to consider the potential physiological significance of Ca-A/K channel regulation and trans-synaptic Zn²⁺ signaling, the simple demonstration that endogenous Zn²⁺ permeates through Ca-A/K channels of adult brain in an in vitro model of ischemia could have important therapeutic implications. Indeed, neuroprotective trials with NMDA antagonists have been generally disappointing, whereas AMPA/kainate receptor antagonists have demonstrated surprisingly good efficacy in certain ischemia models (Diemer et al., 1992). The poor efficacy of NMDA antagonists could be explained in part if NMDA channels were already substantially blocked by synaptic Zn²⁺, whereas the efficacy of AMPA/kainate antagonists might reflect the presence of functionally significant numbers of Ca-A/K channels on hippocampal pyramidal neurons and their selective high permeability to Zn²⁺. The present observations may thus provide new rationale for neuroprotective strategies targeting Ca-A/K channels and Zn²⁺ passage through them in conditions of ischemia or epilepsy, which are associated with rapid synaptic Zn²⁺ release.

	FOOTNOTES

Received Sept. 28, 2001; revised Nov. 8, 2001; accepted Nov. 21, 2001.

This work was supported by National Institutes of Health Grants NS30884 and AG00836 (J.H.W.), AG00919 (S.L.S.), and 5T32NS07444 (F.O.), and by a grant from the Alzheimer's Association (J.H.W.). We thank Simin Amindari and Dien Ton-That for expert assistance with the cell cultures.

Correspondence should be addressed to John H. Weiss, Departments of Neurology, Anatomy and Neurobiology, and Neurobiology and Behavior, University of California, Irvine, Irvine, CA 92697-4292. E-mail: jweiss{at}uci.edu.

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