INTRODUCTION

N-methyl-d-aspartate (NMDA) exposure can lead to the activation of a cation current called the postexposure current (I_pe) in hippocampal neurons (Chen et al. 1997). The conductance underlying I_pe shows a high Ca²⁺ permeability. I_pe is triggered by the increase in intracellular Ca²⁺ concentration caused by Ca²⁺ influx through the NMDA receptor channel. Once triggered, I_pe continues to increase in amplitude (in the absence of NMDA) until death of the neuron occurs. Procedures that prevent the induction of I_pe or suppress it after induction also reduce neuronal death (Chen et al. 1997). This paper identifies Zn²⁺ as an effective blocker of I_pe. The availability of a blocker should facilitate further studies into the intracellular activation pathway of I_pe and the role of I_pe in NMDA toxicity.

METHODS

Acutely isolated hippocampal CA1 neurons from adult guinea pigs were prepared according to the Kay and Wong (1986) method with several modifications to increase the harvest of healthy neurons and preserve NMDA responses (see Chen et al. 1997). Healthy neurons were selected by choosing those that were uniformly bright under phase contrast microscopy. These neurons have a normal (around −60 mV) and stable resting potential within the first hour of recording, have an ability to fire action potentials, and show reversible receptor-channel modulation by second messenger systems (Chen and Wong 1995a,b; Chen et al. 1990).

Whole cell voltage-clamp was performed following the procedure described by Hamill et al. (1981) with the use of a List EPC-7 patch-clamp amplifier and pClamp software (Axon Instruments). Access resistances were ∼10 MΩ. Good recordings were ensured by discarding cells that had seal resistances <20 GΩ, which took more than four gentle sucks to break the membrane, or which did not maintain a stable input resistance during the first 5 min of recording before NMDA exposure. The holding potential was −50 or −55 mV.

Cells were in a 1-ml bath that was perfused continuously with extracellular control solution at a rate of 1–2 ml per min. Extracellular control solution contained (in mM) 140 NaCl, 2 KCl, 2 CaCl₂, 10 N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 0.01 glycine, 25 glucose, pH adjusted to 7.4 with NaOH. Applications of NMDA and divalent cations were achieved with a seven-barrel flow tube (Celentano and Wong 1994) or a three-barrel continuous flow system (Chen and Wong 1995b). NMDA solution was made by dissolving NMDA (100 μM) in extracellular control solution. The divalent cation solutions were made by adding the chloride salt of the divalent cation to extracellular control solution.

In one set of experiments intracellular perfusion was used to switch from control intracellular solution to high calcium intracellular solution while recording from a single cell (Chen et al. 1990). Control intracellular solution contained (in mM) 20 CsCl, 100 CsOH, 0.5 bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid (BAPTA), 10 HEPES, pH adjusted to 7.2 with methanesulfonic acid. High calcium intracellular solution was made by adding 1 mM Ca²⁺ to the control intracellular solution (Chen et al. 1990; Chen et al. 1997). The dose-response curve fitting was performed with Sigmaplot (Jandel Scientific).

RESULTS

As shown previously (Chen et al. 1997), prolonged application of NMDA (10 min, 100 μM) triggers I_pe, which is associated with an increase in conductance (Fig. 1A). Figure 1A shows that I_pe was blocked during short applications of Zn²⁺ (25 s, 500 μM) but otherwise continued to grow in amplitude once it was triggered. The conductance associated with I_pe was greatly reduced during the Zn²⁺ block (Fig. 2B). A shorter (2 min) application of NMDA could also activate I_pe, but there was a delay after the end of the NMDA application before I_pe was evident (Fig. 1B). The I_pe triggered by these shorter applications of NMDA was also blocked during short applications of Zn²⁺ (Fig. 1B).

All twelve cells tested with a 10 min NMDA exposure developed I_pe. In all twelve cases, the I_pe became evident during the NMDA exposure. Eight of 10 cells tested with a 2-min NMDA exposure developed I_pe. In these eight cells, the membrane current returned to baseline after NMDA exposure and I_pe became evident after a 7 ± 3 min (mean ± SE) delay. The proportion of cells developing I_pe further fell to 4 of 12 cells and 1 of 10 cells exposed to 40 s and 10 s of NMDA, respectively. In the four cells that developed I_pe after a 40 s NMDA exposure, I_pe became evident after a10 ± 4 min delay. In the one cell that developed I_pe after a 10 s exposure, I_pe became evident after a 12-min delay. In all cases in which I_pe developed, it was blocked during short pulses of Zn²⁺ (20–25 s, 100–500 μM).

In the earlier study (Chen et al. 1997) we showed that a cation current with the properties of I_pe could also be triggered by intracellular perfusion of Ca²⁺. Figure 1C shows that the current triggered by intracellular perfusion of high calcium solution was also blocked during short applications of Zn²⁺ (500 μM, n = 8).

Additional experiments were done to test the ability of various divalent cations to block I_pe (Fig. 2A). Cd²⁺(1 mM) and Zn²⁺(100 μM) reduced I_pe by 90 ± 6% (mean ± SD, n = 25) and 88 ± 5% (n = 21), respectively. Co²⁺ (1 mM) and Mn²⁺ (1 mM) reduced I_pe by 17 ± 4% (n = 7) and5 ± 3% (n = 4), respectively. Mg²⁺ (1 mM, n = 8) and Ba²⁺ (1 mM, n = 6) had no effect. Figure 2C illustrates the dose-response curve for Zn²⁺. Some suppression of I_pe was apparent at 5 μM and the effect saturated at 500 μM. Between these two concentrations the effect of Zn²⁺ showed a steep concentration dependence. The IC₅₀ of Zn²⁺(the concentration of Zn²⁺ at which 50% of maximal blockwas achieved, see Fig. 2) was 64 μM. Cd²⁺ had an IC₅₀ of∼350 μM.

Because I_pe is triggered by Ca²⁺ and partially carried by Ca²⁺ (Chen et al. 1997), we suspected that I_pe-mediated Ca²⁺ influx might be responsible for the continuous growth of I_pe. Figure 3B illustrates a prolonged block of I_pe with 100 μM Zn²⁺. Brief pulses of Zn²⁺-free solution were applied once every min to test the development of I_pe during the prolonged Zn²⁺ block. If the hypothesis were incorrect and cation influx through I_pe were not required for the further growth of I_pe, one would expect the Zn²⁺-free pulses to reveal a continuous growth in the underlying I_pe similar to that seen in the control (Fig. 3A). Instead, in support of the hypothesis, the I_pe, which was continuously growing before Zn²⁺ block, was maintained at nearly a constant amplitude during the 10-min Zn²⁺ block. The experiment was performed 10 times with either 100 μM Zn²⁺ or 1 mM Cd²⁺. Six of 10 cells showed a ≤5% change in the amplitude of the underlying I_pe during the prolonged block and 4 of 10 showed a 75% or greater reduction in the rate of increase of I_pe as compared with the rate at which it was increasing before the Zn²⁺ (or Cd²⁺) block. When the Zn²⁺ (or Cd²⁺) was removed, the growth in I_pe resumed.

DISCUSSION

Ca²⁺ and Na⁺ influx associated with I_pe may be the cause of NMDA-triggered neuronal toxicity in acutely isolated hippocampal neurons (Chen et al. 1997). In fact, in our studies the development of I_pe is diagnostic for whether or not a cell will die because of NMDA exposure: those cells that develop I_pe (and are allowed to express I_pe) die in <1 h; those that do not develop I_pe die at the same rate as cells that were not exposed to NMDA (2–4 h).

This paper establishes Zn²⁺ as an effective blocker of I_pe. The saturation of the Zn²⁺ effect at a submillimolar concentration supports the hypothesis that Zn²⁺ is binding to a specific receptor site. Having a blocker of I_pe available allowed us to confirm and extend findings made in the previous study. Our earlier results showed that extracellular Ca²⁺ is required for the continuous growth of I_pe but not for the maintenance of a steady I_pe (Chen et al. 1997). These earlier results combined with the finding that I_pe remained at a steady level during prolonged Zn²⁺ block indicate that once triggered, the maintenance of a steady I_pe is not dependent on I_pe-mediated Ca²⁺ influx but that the continuous growth in I_pe is dependent on I_pe-mediated Ca²⁺ influx.

Zn²⁺ is present in many axon terminals and can be released during neuronal activity (Assaf and Chung 1984; Howell et al. 1984). During intense neuronal activity, which may be expected to be associated with a large level of glutamate release, the Zn²⁺ concentration in the extracellular space of rat hippocampus was estimated to peak at 300 μM (Assaf and Chung 1984), which would be a sufficient concentration to suppress I_pe. Earlier studies have shown that extracellular Zn²⁺ can attenuate NMDA toxicity by blocking the NMDA receptor channel (Peters et al. 1987) but, on the other hand, can kill cells at higher concentrations (Choi et al. 1988). I_pe block is another avenue by which low concentrations of extracellular Zn²⁺ might help protect neurons from cell death.