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¹ Centre de Recherche Université Laval Robert Giffard and Department of Psychiatry Québec, Québec G1J 2G3, Canada

	Abstract

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Increased levels of intracellular zinc have been implicated in neuronal cell death in ischaemia, epilepsy and traumatic brain damage. However, decreases in zinc levels also lead to increased neuronal death and lowered seizure threshold. In the present study we investigated the physiological role of zinc in neurodegeneration and protection following epileptic seizures. Cells located in the strata oriens and lucidum of the CA3 region accumulated high concentrations of zinc and died. A decrease in zinc level could prevent the death of these neurones after seizures. Most of these cells were GABAergic interneurones. In contrast, neurones in the CA3 pyramidal cell layer accumulated moderate amounts of zinc and survived. Zinc chelation led to an increase in the mortality rate of these cells. Furthermore, in these cells low concentrations of intracellular zinc activated Akt (protein kinase B), thus providing protection against neurodegeneration. These results demonstrate that intracellularly accumulated zinc can be neurotoxic or neuroprotective depending on its concentration. This dual action is cell type specific.

(Received 28 April 2005; accepted after revision 19 May 2005; first published online 26 May 2005)
Corresponding author K. Tóth: Centre de recherche Université Laval Robert Giffard, 2601 chemin de la Canardière, Québec, QC, G1J 2G3 Canada. Email: katalin.toth{at}crulrg.ulaval.ca

	Introduction

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Hippocampal sclerosis is the most common pathology in adult patients with temporal lobe epilepsy. It consists of severe loss of pyramidal cells in the CA1 area, while the CA3 region seems to be more ‘protected’ (Sommer, 1880; Bratz, 1899; Falconer et al. 1964; Margerrison & Corsellis, 1966). More recent studies have demonstrated that some interneurones in the CA3-hilar area are also selectively vulnerable to various insults (Hsu & Buzsaki, 1993; Magloczky & Freund, 1995; Toth et al. 1997). However, the factors underlying selective cell death remain unknown.

Mossy fibres of the dentate granule cells contain unusually high concentrations of zinc in their synaptic terminals (Maske, 1955). Several studies have shown that zinc is released during increased synaptic activity (Assaf & Chung, 1984; Howell et al. 1984; Vogt et al. 2000; Molnar & Nadler, 2001). However, recent work using fluorimetric measurements reached the opposite conclusion, that little zinc is released during synaptic transmission (Kay, 2003). Extracellularly applied zinc can modulate excitatory and inhibitory synaptic responses (Forsythe et al. 1988; Mayer & Vyklicky, 1989; Draguhn et al. 1990; Smart et al. 1991; Chen et al. 1997; Paoletti et al. 1997; Berger et al. 1998; Traynelis et al. 1998; Choi & Lipton, 1999; Vogt et al. 2000; Lin et al. 2001; Molnar & Nadler, 2001). Beside its effect on synaptic transmission, zinc also enters postsynaptic cells during elevated neuronal activity (Frederickson et al. 1989; Sensi et al. 1999), via voltage-sensitive Ca²⁺ channels (Freund & Reddig, 1994) and NMDA receptors (Koh & Choi, 1994). However Ca²⁺-permeable AMPA receptors were shown to be the primary route for zinc entry following increased synaptic activity (Yin & Weiss, 1995). Interneurones possess AMPA receptors that are permeable to Ca²⁺, and Zn²⁺, while pyramidal cells express the GluR2 subunit which renders the receptors much less permeable to calcium and zinc (Isa et al. 1996; Geiger et al. 1997). A major increase in intracellular zinc concentration eventually leads to apoptosis or necrosis (Kim et al. 1999; Jiang et al. 2001). Zinc influx has been shown to play a crucial role in selective neurodegeneration in excitotoxicity and ischaemia (Koh et al. 1996; Weiss & Sensi, 2000). Similarly, following epileptic seizures zinc accumulation was observed in hilar neurones that eventually died (Frederickson et al. 1989).

Interestingly, beside the well documented relation between zinc and neurotoxic cell death (Park et al. 2000), under certain circumstances zinc seems to inhibit apoptosis (Allington et al. 2001; Ganju & Eastman, 2003). The mechanism of this action is not fully understood, but it has been demonstrated to increase the activation of Akt (protein kinase B), a key molecule in cellular survival (Kim et al. 2000; Tang & Shay, 2001). Phosphorylated Akt is known to promote cell survival: it inhibits the apoptotic pathway via the phosphorylation of BAD (Bcl-2-assosiated protein) (Lizcano et al. 2000; Tan et al. 2000; Masters et al. 2001). To determine the role of zinc in neurodegeneration and protection following seizures, we investigated if zinc-containing neurones die and how the absence of zinc influences cellular survival and cell death. Our results shed light on the mechanism underlying the pattern of neuronal damage observed following epileptic seizures.

	Methods

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Animals

Seizure induction.  Male Sprague-Dawley rats weighing 250–300 g were used in all experiments. The protocols were approved by the Animal Protection Committee of Université Laval. Animals were injected intraperitoneally with 10 mg kg^–1 kainic acid (Ocean Produce, Canada) dissolved in 0.9% NaCl. Seizures were stopped 2 h after kainic acid injection with 75 mg kg^–1 sodium phenytoin diluted in 0.9% NaCl solution. Control animals received a single injection of vehicle solution and were also injected with sodium phenytoin. Animals were monitored and their behaviour recorded for several hours after kainic acid injection; only rats that reached status epilepticus were considered. We compared the progression of the seizures and recorded the time when animals reached each stage (Racine, 1972). These data indicated that the maximum seizure severity score and progression of seizures were not significantly different among different test groups. Animals that died before reaching stage 5 or that never reached stage 5 were excluded from the experiments (see the online Supplemental material).

Zinc-chelation and zinc depletion methods

We used two methods to decrease intracellular zinc concentration: diethyldithiocarbamate (DEDTC) injection and zinc-free diet. While both methods reliably reduced seizure-induced intracellular zinc accumulation in cells located outside the str. pyramidale, the zinc-free diet did not alter the concentration of intracellular zinc in neurones situated in the str. pyramidale. Therefore this method was used only to investigate the fate of non-pyramidal cells. Seizures induced in DEDTC-injected animals could not be arrested with sodium phenytoin because the combination of this drug and DEDTC proved to be lethal. We therefore halted seizures with diazepam in this test group. Since benzodiazepines could play a role in neuroprotection, DEDTC-treated animals were used in experiments investigating the neurotoxic but not the neuroprotective effects of zinc chelation.

DEDTC treatment.  The zinc chelator DEDTC was injected intraperitoneally (0.2 g kg^–1, Sigma-Aldrich, Canada) 30 min before the kainic acid injection, and this was repeated every 30 min until the seizures stopped. Seizures were halted with 1–3 mg kg^–1 diazepam. Control animals were injected with the same dose of diazepam.

Zinc-free diet.  Animals were fed with freely accessible zinc-free food pellets (Labdiet, USA) for 85–105 days. The rats with zinc deprivation developed mild to moderate skin lesions. Control rats were kept under the same environment and were fed a zinc-containing diet.

Intraventricular injection of wortmannin.  Wortmannin specifically inhibits phosphatidylinositol 3-kinase (PI-3K), the enzyme responsible for the phosphorylation of Akt (Ui et al. 1995). We injected this drug into the lateral ventricle to block the activation of Akt. Injections were controlled by an Ultra MicroPump II syringe pump (World Precision Instruments): 2 µl of 20 µM solution over 10 min. The needle was retained within the ventricle for 10 min so that the wortmannin could circulate throughout the ventricle. Sham operated animals were anaesthetized and prepared for surgery in the same manner as the wortmannin injected rats, but received a 2 µl injection of 0.1 M phosphate buffered saline (pH 7.4).

Tissue preparation

Brain tissue was harvested using two different methods: perfusion or cryofixation. Visualization of zinc with TSQ (N-(6-methoxy-8-quiolyl)-paratoluenesulphonamide) staining is only possible in unfixed tissue, and therefore all experiments involving TSQ staining were carried out on brain tissue harvested with cryofixation. Immunocytochemical staining was carried out on tissue harvested from transcardially perfused, paraformaldehyde fixed animals.

Perfusion.  Rats were deeply anaesthetized (ketamine, 87 mg kg^–1; xylazine, 15 mg kg^–1) then transcardially perfused with ice-cold 100 ml 0.1 M phosphate-buffered saline containing 30 mM sodium pyrophosphate and 50 mM sodium fluoride (all three chemicals were from Sigma-Aldrich Canada). The perfusion was continued with 100 ml of phosphate-buffered saline with 4% paraformaldehyde and 50 nM calyculin A (Sigma-Aldrich, Canada). Calyculin A is a potent Ser/Thr phosphatase inhibitor; it was used in order to prevent possible dephosphorylation events. Brains were extracted and postfixed overnight at 4°C in the 4% paraformaldehyde/phosphate-buffered saline solution.

Cryofixation.  Animals were deeply anaesthetized (ketamine, 87 mg kg^–1; xylazine, 15 mg kg^–1) and decapitated, brains were harvested and the two hippocampi were quickly frozen in dry ice and 2-methylbutane, then stored at –70°C. Coronal sections 30 µm thick were prepared using a cryostat and mounted on prechilled glass slides coated with poly-L-lysine. TSQ and terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labelling (TUNEL) staining were applied consecutively to the same section; after each staining the results were photographed and later matched using capillaries and the boundaries of the section as landmarks.

Hippocampal slice preparation.  Rats were anaesthetized with isoflurane and decapitated. The brain was quickly removed and transverse slices (400 µm) were prepared in an ice-cold oxygenated (95% O₂–5% CO₂) artificial cerebrospinal fluid (ACSF) containing (mM): NaCl 130, KCl 3.5, NaH₂PO₄ 1.25, MgSO₄ 5, CaCl₂ 1, NaHCO₃ 24 and glucose 10. Slices were transferred to a holding chamber at room temperature containing oxygenated ACSF.

Newport Green staining.  To measure the [Zn²⁺]_i in various cell types in vitro, slices were loaded with 5 µM Newport Green diacetate (NG) in the presence of 0.02% Pluronic F-127 for 30 min at room temperature. Slices were washed in ACSF and images were acquired with a digital camera. The approximate [Zn²⁺]_i was estimated using the following equation (Tsien & Pozzan, 1989):

(1)

F_max was obtained by adding 1 mM ZnCl₂ plus 50 µM sodium pyrithione to the bathing medium, and F_min was estimated in the presence of 200 µM DEDTC. A K_d of 1 µM was assumed.

TSQ staining.  TSQ staining is used to visualize labile, histochemically reactive zinc in cryostat sections (Frederickson et al. 1987). TSQ is specific for Zn²⁺ in the presence of other multivalent cations commonly found in biological systems (Andrews et al. 1995). TSQ forms a fluorescent complex with Zn²⁺, and the relationship between zinc concentration and fluorescence intensity is linear (Frederickson et al. 1987; Reyes et al. 1994). Sections were immersed in a freshly prepared TSQ (Molecular Probes, USA) staining solution for 2.5 min (Frederickson et al. 1987). TSQ-stained sections were examined under a fluorescence microscope (excitation 360–370 nm, dichromatic beamsplitter 400, barrier filter 420 nm) and photographed with a SPOT RT digital camera (Diagnostic Instruments Inc.). TSQ fluorescence intensity is expressed as F/F throughout. Background fluorescence was measured in an area that was not stained, typically in the border of the stratum oriens and alveus of the CA1 region.

TUNEL staining.  Apoptag^® (Serological Corporation, USA) was used to visualize dying cells. Results were viewed with a fluorescence microscope (excitation 545–550 nm, dichromatic beamsplitter 600 nm, barrier filter 610 nm) and photographed.

Immunocytochemistry.  Brains were coronally sectioned at 50 µm (Vibratome, USA) and incubated for 72 h at 4°C with primary antibodies of interest. They included glutamic acid decarboxylase (GAD) (Chemicon), Akt, phospho-Akt (Ser473) (both antibodies from Cell Signalling Technology, USA) or activated caspase-3 (R and D Systems, USA) primary antibodies, diluted, respectively, to 1: 1000, 1: 250, 1: 100 and 1: 500 in 1% blocking solution. Tissue was rinsed and labelled with fluorescent secondary antibodies (Molecular Probes, USA). The results were analysed with a fluorescence microscope. Pictures for Fig. 2 were taken with a Zeiss LM 1500 confocal microscope.

View larger version (29K): [in this window] [in a new window]   Figure 2.  Colocalization of TUNEL and GAD following seizures Double labelling with GAD immunocytochemistry (A) and TUNEL (B) at 10 h after kainic acid injection. Two cells in the stratum oriens of the CA3 region are TUNEL- and GAD-positive (white arrows); the arrowhead indicates a GAD-positive and TUNEL-negative neurone, and the open arrow shows a TUNEL-positive, GAD-negative cell. D and E, bar graphs indicate that the majority of TUNEL-labelled cells are GAD-positive 10 and 18 h after kainic acid injection. Data collected from 8 consecutive sections. Scale bar = 50 µm.

Cell counting.  For quantification, hippocampal sections were chosen at the same anterio-posterior level from each animal (3.3–4.16 mm posterior from the Bregma). Positively labelled cells were counted in the CA3 area of the hippocampus. The surface area of the sections from which data were collected was also measured to ensure that areas of similar size were compared in animals from all test groups. Data were only accepted if this parameter was not significantly (P > 0.05) different among test groups.

TSQ and TUNEL staining could not be visualized simultaneously since staining steps needed to visualize one marker lead to the disappearance of the other. We therefore studied their colocalization on digital images taken sequentially after each staining. Pictures were overlapped using Image Pro software (Media Cybernetics) and the presence or absence of the two dyes in a single cell was determined. Data are presented as means ± S.E.M. Data were analysed using one-way ANOVA (Origin software, OriginLab Corp., USA). Significance was accepted at P < 0.05. Cluster analysis was performed with Euclidean distances using Ward's method which assigns objects to clusters so that the variance within each cluster is minimized. Cluster and discriminant analysis were executed with Systat 11 software.

	Results

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Distribution of zinc-containing neurones

We compared the time course of cell death after hippocampal seizures with the pattern of cellular accumulation of zinc, detected by the TSQ stain. While in control animals TSQ staining was restricted to the mossy fibres, 8 h after kainic acid injection, cells located in the str. oriens of the CA3 region became visible. The highest number of TSQ-positive cells was observed in the str. oriens of the CA3 region between 10 and 12 h after kainic acid injection (6.3 ± 3.8 cells per section, n = 5). Neurones located in the str. pyramidale of the CA3 region became visible only at 18 h post-injection (31.3 ± 13.4 cells per section, n = 6), but the TSQ intensity in these cells was lower than in the cells located outside the pyramidal cell layer (Table 1 and Fig. 1A–C). At 24 h after kainic acid injection, TSQ-staining reverted to its control pattern and neuronal somata were no longer visible. Our data indicate that zinc accumulation in neurones located outside the pyramidal cell layer was faster and more prominent than in cells situated in the str. pyramidale.

View larger version (43K): [in this window] [in a new window]   Figure 1.  Distribution of cells accumulating zinc following seizures. Correlation between zinc accumulation and TUNEL labelling Strongly labelled TSQ-positive neurones were detected 10 h after KA (kainic acid) injection in the stratum oriens of the CA3 area. The vast majority of these cells were TUNEL-positive (B, arrow), while in non-KA-treated animals neither TSQ- nor TUNEL-positive cells were visible (A). Faintly stained cells appeared 18 h after seizure induction in the str. pyramidale of the CA3 region. Most of these cells were TUNEL-negative (C, arrows). D and E, spatial distribution and TSQ intensity of TUNEL-positive (red dots) and TUNEL-negative (black dots) cells are shown at 10 (D) and 18 h (E) after kainic acid injection. The majority of TUNEL-labelled cells were located in the str. lucidum and oriens and accumulated a high concentration of zinc, while cells situated in the str. pyramidale showed faint TSQ staining and survived. F, Newport Green diacetate staining of in vitro slices prepared from animals 18 h after kainic acid injection. Slices were loaded in 5 µM NG for 30 min. Blue arrows indicate strongly labelled cells in the stratum oriens, and red arrows point to faintly labelled cells in the stratum pyramidale. Fluorescence signals were calibrated by obtaining Fmax values in the presence of 1 mM ZnCl2 plus 50 µM sodium pyrithione, and Fmin was estimated in the presence of 200 µM DEDTC. The dashed line indicates the border between str. pyramidale and oriens. G, NG fluorescent intensity measurement from 216 cells located in the str. pyramidale (blue bars) and in the str. oriens and lucidum (red bars), binning at F/F = 0.01. H, the staining intensity was statistically different between cells located in the str. pyramidale and str. oriens/lucidum (P < 0.0001). Scale bar = 25 µm in A–C, 40 µm in F.

We measured the distance of each cell from the midline of the str. pyramidale and the intensity of TSQ staining (Fig. 1D and E). Cluster analysis was performed to identify cell populations that differed significantly in these parameters. While distinct groups did not emerge at 10 h after kainic acid injection, at 18 h post-injection two significantly different cell groups appeared. The best discrimination was observed when we used two clusters for the analysis. The first cluster was formed by neurones located in the str. pyramidale (average distance from str. pyramidale midline = 24 µm) and these cells accumulated moderate amounts of zinc (F/F = 0.28 ± 0.13). The second cluster was formed by neurones located in str. oriens and lucidum (average distance from str. pyramidale midline = 144 µm), and they accumulated significantly higher (P < 0.001) concentrations of zinc (F/F = 0.52 ± 0.13).

Next, we aimed to estimate zinc concentrations in these two cell types (Canzoniero et al. 1999). Since TSQ staining is performed on cryofixed tissue, signals cannot be calibrated. Therefore we visualized zinc accumulation in in vitro slices with Newport Green diacetate (NG). In vitro slices were prepared from kainic acid injected animals at 18 h post-injection (n = 3). Similarly to TSQ staining, faintly stained cells were observed in the str. pyramidale of the CA3 region and strongly labelled cells in the str. oriens and lucidum (Fig. 1F–H). Using eqn (1), the intracellular zinc concentration in neurones located in str. pyramidale was estimated to be 250 ± 24 nM, and 621 ± 65 nM in cells situated in str. oriens and lucidum.

Colocalization of TSQ staining and TUNEL labelling

We next asked if zinc-containing neurones die and if cell death occurs exclusively in zinc-positive cells. TSQ staining was used to visualize zinc accumulation in combination with TUNEL staining to label dying neurones. Ten hours after injection the vast majority of zinc-containing neurones (84.2 ± 2.04%, n = 6) were labelled with TUNEL, and only a small population of dying cells were TSQ negative (15.8 ± 3.4%, n = 6). These data indicate that cells accumulating zinc will almost certainly die and that zinc accumulation occurs only in vulnerable neurones (Fig. 1D). Most (88%) TUNEL-positive cells were located in the CA3 region, although small numbers of neurones in the stratum oriens of the CA1 region were also labelled with TUNEL (3.2 ± 2.1 cells/section; n = 5). At 18 h after kainic acid injection the majority of TSQ-labelled cells were concentrated in the CA3 region (Table 1); only 30.2 ± 4.8% of these cells were TUNEL-positive. Two distinct clusters emerged: one cluster located outside the pyramidal cell layer with a high percentage of TUNEL labelling and one concentrated in the str. pyramidale with a much lower percentage of dying neurones (Fig. 1E).

We used discriminant analysis to determine whether cell fate could be predicted from the concentration of the intracellularly accumulated zinc. The discriminant function was calculated using TSQ-staining intensity as a discriminant variable and TUNEL labelling as grouping variable. At 10 h, only a few (16%) TUNEL-negative TSQ-labelled cells were observed and there was no significant difference in the intracellular zinc concentration of these cells and their TUNEL-positive counterparts (F/F = 0.51 ± 0.08 and 0.5 ± 0.06, respectively; P = 0.871) (Fig. 1D). However, 18 h after kainic acid injection TSQ-staining intensity was significantly different (P < 0.001) between TUNEL-negative and TUNEL-positive cells (F/F = 0.29 ± 0.12 and 0.49 ± 0.11, respectively) (Fig. 1E). Calculating the discriminant function on this cell population revealed that intracellular zinc concentration correctly predicted the fate of the cell in 89% of the cases; this was significantly higher than expected by chance (60%). This result suggests that moderate elevations of intracellular zinc may be correlated with cell survival and high levels of zinc with cell death.

Hippocampal pyramidal cells are situated almost exclusively in the str. pyramidale. Therefore, the strong correlation between cell location and the concentration of intracellularly accumulated zinc indicates that cell type-specific differences might exist in neuronal vulnerability following seizures. We therefore asked whether the dying cells located outside str. pyramidale correspond to GABAergic interneurones. Interneurones were identified on the basis of their GAD content (Seress & Ribak, 1983). Double labelling with GAD and TUNEL showed that at both 10 and 18 h after kainic acid injection, the majority of TUNEL-positive cells were GABAergic (79.8%, 75.5%, respectively, n = 8 and 4) (Fig. 2). The use of this method may underestimate the percentage of GABAergic TUNEL-labelled cells since while TUNEL labelling is visible even when the cell is almost completely disintegrated, GAD immunoreactivity disappears at earlier phases of cell degradation (data not shown).

Zinc depletion decreases the number of TUNEL-positive cells in the strata oriens and radiatum

While our results show a large overlap between TSQ-positive cells and cell death, they do not prove that the increase in intracellular zinc causes cell death. Zinc entry could be a secondary effect triggered by apoptotic processes rather than the cause of cellular vulnerability. In order to differentiate between these two possibilities, we maintained animals on a zinc-free diet for 85–105 days, in an attempt to reduce brain zinc levels (Lu et al. 2000). The zinc-free diet did not deplete zinc completely from the mossy fibres (see Fig. 3C), but the number of TSQ-labelled neurones was significantly decreased 10 h after the kainic acid injection (number of TSQ-positive cells in 10 sections = 73.2 ± 10.8 in control animals versus 25.5 ± 3.4 in animals kept on a zinc-free diet; n = 5 each test groups). Moreover, the intracellular levels of accumulated zinc in those few cells that were labelled with TSQ were significantly reduced to 48% of control values (F/F = 0.54 ± 0.02 in control versus 0.26 ± 0.02 in animals maintained on a zinc-free diet). Next we compared the number of neurones labelled with TSQ and TUNEL at 10 h after kainic acid injection in animals maintained on either a regular or a zinc-free diet (Fig. 3C). This time point was chosen for analysis since it corresponded to the highest number of TSQ-labelled neurones outside the str. pyramidale. The number of double-labelled neurones in the CA3 region in animals kept on a zinc-free diet was dramatically decreased (60.6 ± 11.6 in control versus 13.2 ± 1.6 in zinc-free diet per 10 sections). Furthermore, the total number of dying neurones was also decreased by 83% (77.8 ± 4.8 in control versus 17.2 ± 1.9 in zinc-free diet per 10 sections) (Fig. 3D). This reduction in intracellular zinc concentration after seizures was associated with a significantly reduced mortality rate of interneurones indicating that zinc underlies their vulnerability.

View larger version (29K): [in this window] [in a new window]   Figure 3.  TSQ and TUNEL-staining 10 h after kainate injection in animals fed zinc-free diet A and B, intracellular zinc accumulation following seizures was significantly less in animals kept on zinc-free diet compared to control rats (P < 0.05). TSQ intensity measurements in cells from control animals (A1) and those fed on a zinc-free diet (A2). Both cells are situated in the stratum oriens of the CA3 region. Quantitative analysis indicated that in animals kept on zinc-free diet, the TSQ intensity was decreased by 52% compared to controls. Data collected from 10 consecutive sections from each animal. In non-KA-injected animals following zinc-free diet, no TSQ- or TUNEL-labelled cells were observed (C, zinc-free diet). Ten hours post-injection only very few TSQ and TUNEL-positive interneurones were observed in rats following zinc-free diet (C, zinc-free diet + KA) compared to control KA-injected animals (C, KA). D, statistical analysis of the number of double and TSQ or TUNEL-labelled neurones in the CA3 region, showing the number of labelled cells in 10 consecutive sections. Dashed lines indicate the borders of str. pyramidale. Scale bars = 50 µm on C.

We have described a drastic decrease in intracellular zinc level in TSQ-positive neurones located outside the principal cell layer of animals fed a zinc-free diet. However, after KA injection, the zinc-free diet did not significantly change zinc accumulation in cells located in the str. pyramidale (F/F = 0.28 ± 0.13 in control versus 0.18 ± 0.04 in animals kept on zinc-free diet; P = 0.079). We therefore used another method to deplete zinc in order to determine how pyramidal cell survival changes in the absence of zinc. Animals were injected with DEDTC, a zinc chelator (Lees et al. 1998) before and during seizure activity. In two animals killed at 30 min after the kainic acid injection, TSQ staining revealed no detectable zinc, indicating that it was effectively chelated by this method (Fig. 4B). DEDTC injections were terminated after seizure activity ceased following diazepam injection. DEDTC has several other effects than zinc chelation, such as removal of zinc from vital proteins (Hunziker, 1991). It was thus essential to first examine how DEDTC treatment alone affects neuronal injury and cell death. Our data showed that DEDTC injection alone did not result in the appearance of TUNEL-positive cells (Fig. 4C, n = 3). Since pyramidal cells become visible with TSQ staining at 18 h after KA injection, we studied the fate of these cells at this time point. The number of TSQ-labelled cells was decreased by 65%, but the number of dying cells was increased by 392% in DEDTC-treated animals (63.7 ± 11.1 versus 249.75 ± 55.7 cells in 5 sections, n = 4). Most (92%) TUNEL-positive neurones were located in the str. pyramidale of the CA3 region. Thus our data indicate that eliminating zinc drastically increases the mortality rate of neurones located in the str. pyramidale after epileptic seizures. (Fig. 4)

View larger version (30K): [in this window] [in a new window]   Figure 4.  TSQ and TUNEL staining 18 h after kainate injection in DEDTC-treated animals A, the experimental design of zinc-chelation experiments. B, CA3 region of the hippocampus with no detectable zinc in the mossy fibres revealed by TSQ staining at 30 min after the first DEDTC injection. In non-KA-injected DEDTC-treated animals, no TSQ- or TUNEL-labelled cells were observed (C, DEDTC). Zinc chelation significantly decreased the number of TSQ-positive pyramidal cells in the CA3 region, but the number of TUNEL-labelled cells was dramatically increased (C, DEDTC + KA) compared to control KA-injected animals (C, KA). D, statistical analysis of the number of double- and TSQ- or TUNEL-labelled neurones in the CA3 region showing the number of labelled cells in 5 consecutive sections. Dashed line indicates the border of str. lucidum and pyramidale. Scale bars = 100 µm in B; 50 µm in C.

We next sought to identify the intracellular mechanism by which zinc protects pyramidal cells against cell death. Zinc activates the phosphorylation of Akt, which has a neuroprotective role in cell cultures (Kim et al. 2000; Tang & Shay, 2001). We asked whether it fulfils a similar function in vivo. After kainic acid injection, we noted a dramatic increase in Akt levels in the CA3 region. Several pyramidal cells and interneurones were Akt-positive in each section (n = 5). Neither zinc chelation with DEDTC (data not shown) nor its depletion with zinc-free diet altered Akt up-regulation as detected by immunostaining (Fig. 5A–D, n = 5). Since Akt only promotes cell survival when it is phosphorylated, we examined levels of phosphorylated Akt (P-Akt) in different layers of the hippocampus of control and KA-injected animals, and asked how this distribution is modified in animals kept on a zinc-free diet. P-Akt immunostaining was not apparent in control, non-KA-injected animals (n = 5) (Fig. 5E). Following kainic acid injection (n = 5) P-Akt was detected in the CA3 str. pyramidale but rarely in other layers and areas. It was observed between 5 and 9 h post-injection in the vast majority of cell bodies in this layer and reached a maximum at 6 h after kainic acid injection. In KA-injected animals that had been maintained on a zinc-free diet (n = 5), P-Akt labelling in str. pyramidale was dramatically decreased, but in contrast several immunopositive neurones were observed in the str. oriens and str. radiatum (Fig. 5E–H). Statistical analysis was used to investigate whether a zinc-free diet significantly changed the spatial distribution of P-Akt-positive cells. The locations of 100 P-Akt-labelled neurones were recorded from KA-injected control animals and from KA-injected animals kept on a zinc-free diet. The distance of the soma from the midline of the str. pyramidale was measured (Fig. 5I and J) and the Kolmogorov-Smirnov test was used to investigate the difference in the cumulative probability distribution of this parameter (Fig. 5K). Our analysis showed that the two distributions are significantly different (P < 0.001), indicating that the zinc-free diet was associated with a marked change in the spatial distribution of P-Akt-positive cells. In control KA-injected animals, the majority of CA3 pyramidal cells expressed P-Akt and the staining was restricted to the str. pyramidale. In contrast, in animals fed the zinc-free diet, cells located inside and outside the pyramidal cell layer expressed P-Akt. Since pyramidal cells are located in the str. pyramidale while interneurone somata are located in all layers of the hippocampus, this change in spatial distribution suggests that the type of neurones (i.e. interneurone versus pyramidal cell) expressing P-Akt also differs significantly between control animals and those maintained on a zinc-free diet.

View larger version (34K): [in this window] [in a new window]   Figure 5.  Expression of Akt and P-Akt in normal and zinc-deficient animals following kainic acid-induced epilepsy While Akt immunostaining could not be detected in control animals (A, CA3 region), at 6 h after epileptic seizures, a dramatic increase in Akt level in the CA3-hilar region was observed (B). Zinc-free diet alone caused a minimal increase in Akt-level (C, CA3 region); KA injection in animals kept on zinc-free diet resulted in Akt staining similar to control KA-treated animals (D). E–H, P-Akt immunostaining from control animals and from rats following zinc-free diet. Photomicrographs were taken from the CA3 region of the hippocampus. P-Akt was not detected in non-epileptic control animals (E) or in animals fed a zinc-free diet (F). Following KA-induced seizures, in control animals P-Akt level increased in the CA3 stratum pyramidale but not in other layers (G). In rats kept on zinc-free diet, significantly fewer cells in the stratum pyramidale were stained, but the number of P-Akt-positive cells in other layers increased (arrows) (H). Dashed lines indicate the borders of str. pyramidale. I and J, differences in the spatial distribution of 100 P-Akt cells in the CA3 region from control rats (I) and animals kept on zinc-free diet (J). Data collected from 4 different animals. P-Akt-positive cells are concentrated in the stratum pyramidale in control rats, while in animals kept on zinc-free diet P-Akt-labelled cells were found in every layer. K, the Kolmogorov-Smirnov test showed significant differences (P < 0.001) in the cumulative probability distribution of the two groups. Scale bar = 100 µm in A, C and D, 400 µm in B and 200 µm in E–H.

We next asked how zinc protects CA3 pyramidal cells, by looking for differences in the activation of the apoptotic pathway in control and zinc-depleted animals. During apoptosis, caspase-9 activates caspase-3, and the presence of cleaved caspase-3 indicates cell degeneration. Activated caspase-3 expression is increased 2–7 days following seizures (Narkilahti et al. 2003). Therefore we compared activated caspase-3 staining and TUNEL staining in control and DEDTC-treated animals at 3 days after seizure induction (n = 3 and 4; Fig. 6). DEDTC treatment was used in these experiments since it depletes zinc more completely than the zinc-free diet. In isolation, zinc chelation by DEDTC did not activate caspase-3, and TUNEL-positive cells were not apparent (Fig. 6A1 and 2, n = 3). The level of activated caspase-3 in control KA-injected animals was somewhat increased, but only a few pyramidal cells were positively labelled after kainic acid injection (Fig. 6A3). However, there was a dramatic increase in the number of activated caspase-3-positive neurones in DEDTC-treated KA-injected rats (Fig. 6A5). Furthermore, in these animals, the number of TUNEL-positive neurones was increased by 425% compared to control (data collected from 15 consecutive sections from each animals, P < 0.001) (Fig. 6A4 and 6). Thus significantly more cells enter apoptotic pathways when the zinc level is decreased.

View larger version (35K): [in this window] [in a new window]   Figure 6.  Activated caspase3 and TUNEL staining 3 days after kainate injection Comparison of activated caspase-3 and TUNEL staining in the CA3 region of normal and DEDTC-treated animals following KA injection. DEDTC treatment alone did not increase the number of activated caspase-3 or TUNEL stained cells (A and B). Three days after the kainic acid injection the number of activated caspase-3 and TUNEL-labelled cells in DEDTC-treated animals was increased when compared to control epileptic animals (C–F). Dashed lines indicate the borders of str. pyramidale. Scale bar = 100 µm.

Finally, we used wortmannin (Yano et al. 2001), a potent and selective inhibitor of the phosphatidylinositide 3-kinase that activates Akt, to examine the role of Akt phosphorylation in seizure-induced cell death (Fig. 7). In these experiments wortmannin was injected into the lateral ventricle and 2 h later, when animals had recovered from anaesthesia, they were injected with kainic acid. The effect of wortmannin on P-Akt expression was investigated 6 h after kainic acid injection (n = 3), at a time corresponding to the maximal number of P-Akt-positive cells in control animals. The effect on cell death was investigated 3 days post-injection as in the previous experiments (n = 3). Wortmannin injection alone did not lead to the appearance of P-Akt-positive or TUNEL labelled cells (Fig. 7A1 and 2, n = 3). The distribution of P-Akt-positive neurones in wortmannin-treated KA-injected animals was similar to control KA-injected animals; immunopositive neurones were located in the str. pyramidale. In order to determine if wortmannin caused any quantitative difference in the appearance of P-Akt-positive cells or affected the fate of hippocampal cells after seizures, we counted the number of neurones positively labelled for P-Akt and TUNEL in the CA3 area of consecutive hippocampal sections. We found that intraventricular injection of wortmannin caused a significant 70% decrease (P < 0.05) in the number of P-Akt-positive cells in kainic acid-injected animals compared to animals injected with vehicle (Fig. 7A3 and 4 and B). The number of TUNEL-positive, degenerating cells increased by 307% compared to sham-operated animals (P < 0.001) (Fig. 7A4 and 6 and C). These results point to a specific role for the zinc-related phosphorylation of Akt in the survival of CA3 pyramidal cells after epileptic seizures.

View larger version (13K): [in this window] [in a new window]   Figure 7.  The effect of wortmannin injection on CA3 pyramidal cell survival P-Akt immunostaining and TUNEL labelling in the CA3 region of wortmannin-injected epileptic and non-epileptic animals and in vehicle-injected epileptic rats. For P-Akt immunostaining, animals were killed 6 h after kainic acid injection, while TUNEL labelling was investigated 3 days after kainic acid injection. Intraventricular injection of wortmannin alone did not cause the appearance of P-Akt or TUNEL-positive neurones (A1 and 2). In KA-injected animals, it reduced significantly the number of P-Akt-positive cells (A5), compared to control KA-treated animals injected with vehicle (A3 and B). The number of TUNEL-positive, dying neurones was significantly increased in wortmannin-injected rats (A4 and 6 and C). Data collected from 15 consecutive sections from each animal. Scale bars = 100 µm.

	Discussion

Top Abstract Introduction Methods Results Discussion Supplemental material References

Methodological considerations

Our results depend strongly on an accurate resolution of differences in zinc concentrations in interneurones and pyramidal cells. We used two fluorescent probes, TSQ and Newport Green, to measure zinc. Staining with both dyes indicated that [Zn²⁺]_i is significantly different between these two cell groups. The intracellular level of zinc in unstimulated cells was shown to be below the detection level of both dyes, as previously described (Sensi et al. 1997; Canzoniero et al. 1999). Presumably this low level derives from the high-affinity binding of zinc by metallothioneins, which are abundant in every cell (Maret, 1994). After seizures induced by kainic acid, the intracellular zinc concentrations increased to about 250 nM in pyramidal cells and to about 600 nM in interneurones. These values depend largely on intracellular concentration of the dye (Dineley et al. 2002), a parameter we could not accurately measure. Therefore it is important to note that while this technique can accurately measure the relative ratio of [Zn²⁺]_i between different types of cells, the absolute values should be considered as estimates.

We examined the role of elevated zinc in cell death and neuroprotection, using two different approaches to reduce both intra- and extracellular zinc: a zinc-free diet and the chelator DEDTC. These approaches suggested that reducing zinc levels enhances interneurone survival, but increases pyramidal cell mortality. For the validity of these results, it is important that seizure severity is similar between control animals injected with kainate and those subject to the diet or the chelator. While some work suggests that zinc chelation may intensify seizures over a population, we selected animals on the basis of their behavioural manifestations of seizures ensuring that they experienced comparable seizure intensities. Our data suggest that status epilepticus is needed to induce significant zinc elevation in both interneurones and pyramidal cells. The relation between zinc elevation and cell death was followed using TUNEL staining, which indicates DNA degradation during irreversible, apoptotic cell death (Nagata, 2000). However, some studies indicate that TUNEL staining does not differentiate reliably between apoptotic and necrotic cell death (Charriaut-Marlangue & Ben-Ari, 1995; Grasl-Kraupp et al. 1995; De Torres et al. 1997). In the light of these studies our data cannot determine unequivocally the exact mechanism by which TSQ-positive cells die.

Is zinc neurotoxic or neuroprotective?

There has been a long debate on the role of zinc in neurodegeneration and protection. Strong data suggest that zinc is an ionic mediator of selective neuronal injury (Frederickson et al. 1989; Yin & Weiss, 1995; Choi, 1996; Koh et al. 1996; Kim et al. 1999; Sensi et al. 1999, 2000; Weiss & Sensi, 2000; Weiss et al. 2000). Yet, zinc chelation or depletion not only fails to prevent epileptic cell death, but even increases excitotoxic damage and susceptibility to epileptogenic agents (Lees et al. 1998; Cole et al. 2000; Dominguez et al. 2003; Ganju & Eastman, 2003; Takeda et al. 2003). Dominguez et al. (2003) have suggested that synaptically released zinc is neuroprotective due to its inhibitory action on NMDA receptors. While their data agree well with our findings on the increased neuronal damage caused by zinc chelation, they do not explain the contradiction between studies showing zinc as a mediator of neuronal injury and as an effective player in neuroprotection.

Our data suggest instead that the dual affects of zinc may depend on its intracellular concentration. After epileptic seizures, zinc increases substantially more in interneurones than in pyramidal cells. It seems probable that high zinc concentrations are neurotoxic by causing mitochondrial dysfunction which leads to interneurone death (Sensi et al. 1999, 2000). This difference between interneurones and pyramidal cells could result from cell type-specific expression of zinc, or calcium, signalling pathways (Sik et al. 1998). Some hippocampal interneurones express Ca²⁺-permeable AMPA receptors (Jonas et al. 1994; Racca et al. 1996; Vissavajjhala et al. 1996; Sensi et al. 1999; Topolnik et al. 2005). The elevated zinc levels in interneurones might result from its entry via these receptors (Sensi et al. 1999). Alternatively, calcium entry might trigger zinc release from intracellular stores such as the mitochondria (Sensi et al. 2003). Our data indicate that zinc reaches higher intracellular levels in some types of interneurones than in others. The cells that exhibited high zinc levels in our study were largely located in str. oriens and lucidum of the CA3 region and rarely in str. pyramidale or radiatum. Possibly the differences might derive from differential expression of Ca²⁺-permeable AMPA receptors, intracellular Ca²⁺ buffering systems or differences in mitochondrial metabolism (Freund & Buzsaki, 1996; Racca et al. 1996; Pisani et al. 2002).

Our data suggest that smaller elevations of zinc trigger antiapoptotic processes in pyramidal cells, which are mediated in part by Akt phosphorylation. Biochemical assays show this process is active even at resting [Zn²⁺]_i levels in the hippocampus and is hugely augmented after seizures or ischaemia (Yano et al. 2001; Henshall et al. 2002). While 100 µM zinc is typically used to trigger Akt phosphorylation in vitro, our data raise the possibility that much smaller concentrations may suffice in intact cells. Although few data exist on a differential expression or phosphorylation of Akt in distinct cell types, our results confirm its presence in interneurones, although the phosphorylated form was only reliably detected in animals fed a zinc-free diet. It seems probable that other intracellular pathways, including the heat shock protein-70 which is expressed in zinc-accumulating neurones following seizures, also contribute to the attenuation of excitotoxic cell death (Lee et al. 2000).

Intra- and extracellular zinc pools

The origin of the accumulated zinc has been much debated. Several studies suggest that zinc is released from synaptic vesicles (Assaf & Chung, 1984; Howell et al. 1984; Frederickson et al. 1989; Vogt et al. 2000; Lin et al. 2001; Molnar & Nadler, 2001). Our observation that degenerating, zinc-positive interneurones were spatially restricted to the CA3 and hilar regions could be explained by synaptic release of zinc from the mossy fibres since the somata or dendrites of these cells are located in the zone where they terminate. However, other work finds no evidence for synaptic release of zinc (Lee et al. 2000; Kay, 2003). If zinc is not released from synapses, it might be released from intracellular stores (Berendji et al. 1997; Aizenman et al. 2000; Bossy-Wetzel et al. 2004). Our data showing a long delay of 8–10 h before an increase in intracellular zinc was detected argue against an immediate entry via ion channels or receptors. If so, calcium entry via distinct AMPA receptors might trigger a slow chain of events that leads to zinc release from mitochondria or metallothioneins. Alternatively, zinc may be translocated from the extracellular space (Kay, 2003). In this case the observed spatial distribution of vulnerable neurones would result from the pattern of synaptic inputs to these cells rather than their direct contact with zinc-containing terminals.

A surprising finding of the present study is the differential influence of zinc-free diet on seizure-induced zinc accumulation in interneurones and pyramidal cells. While zinc accumulation was effectively suppressed in interneurones, [Zn²⁺]_i in pyramidal cells was largely unaffected. Possibly one can speculate that, in these two types of cells, zinc is released from different intracellular stores which are differentially affected by the diet. In metallothionein III knock-out animals, seizure-induced cell death was increased among CA3 pyramidal cells (Erickson et al. 1997). The increase in intracellular calcium evoked by NMDA application triggered zinc release from mitochondria (Sensi et al. 2003). Therefore, it is possible that while zinc released from metallothioneins is involved in the antiapoptotic pathway, release from mitochondria would trigger cell death.

Zinc effect on synaptic transmission

Postsynaptic accumulation is not the only known effect of released zinc. It also inhibits NMDA receptors (Paoletti et al. 1997; Vogt et al. 2000; Molnar & Nadler, 2001) and GABA_A receptors (Hosie et al. 2003), modulates AMPA receptor-mediated events (Mayer et al. 1989; Lin et al. 2001) and decreases the augmented inhibition in an epilepsy model (Buhl et al. 1996). Zinc also can inhibit GAT4, a GABA transporter responsible for clearing synaptically released GABA, and hence during increased synaptic activity elevate GABAergic inhibition (Cohen-Kfir et al. 2005). Presumably these effects all contribute to the pathological changes occurring during epileptic seizures. However, the global effect of synaptically released zinc on excitatory neurotransmission remains unclear. Finally, we should consider briefly the intersection between the physiological and pathological roles of zinc. During physiological activities including learning and memorization, mossy fibres may transmit activity to CA3 neurones at unusually high frequencies. During these periods, synaptically released zinc might act to protect pyramidal cells against potential excitotoxic actions of enhanced glutamatergic transmission, firstly by inhibiting NMDA receptors via a high-affinity binding site (Paoletti et al. 1997) and secondly by activating antiapoptotic mechanisms.

	Supplemental material

Top Abstract Introduction Methods Results Discussion Supplemental material References

 
The online version of this paper can be accessed at: DOI: 10.1113/jphysiol.2005.089458
http://jp.physoc.org/cgi/content/full/jphysiol.2005.089458/DC1
and contains supplemental material consisting of a figure entitled: Seizure progression after kainite injection.

This material can also be found as part of the full-text HTML version available from http://www.blackwell-synergy.com

	Footnotes

 
A. Côté and M. Chiasson contributed equally to this work.

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