Zn2+ Blocks the NMDA- and Ca2+-Triggered Postexposure Current Ipe in Hippocampal Pyramidal Cells

Qiang X. Chen*, Katherine L. Perkins, and Robert K. S. Wong

Department of Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Chen, Qiang X., Katherine L. Perkins, and Robert K. S. Wong. Zn2+ blocks the NMDA- and Ca2+-triggered postexposure current Ipe in hippocampal pyramidal cells. J. Neurophysiol. 79: 1124-1126, 1998. Whole cell voltage-clamp recordings from acutely isolated hippocampal CA1 pyramidal cells from adult guinea pigs were used to evaluate divalent cations as possible blockers of the postexposure current (Ipe). Ipe is a cation current that is triggered by the rise in intracellular Ca2+ concentration that occurs after the application of a toxic level of N-methyl-D-aspartate (NMDA). Once triggered, Ipe continues to grow until death of the neuron occurs. Ipe may be a critical link between transient NMDA exposure and cell death. Ipe was blocked by micromolar concentrations of Zn2+. The Zn2+ effect had an IC50 of 64 µM and saturated at 500 µM. Prolonged Zn2+ block of Ipe revealed that the maintenance of a steady Ipe is not dependent on Ipe-mediated Ca2+ influx but that the continuous growth in Ipe is dependent on Ipe-mediated Ca2+ influx. The availability of an effective blocker of Ipe should facilitate the investigation of the intracellular activation pathway of Ipe and the role of Ipe in neuronal death.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

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

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega . Good recordings were ensured by discarding cells that had seal resistances <20 GOmega , 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
Abstract
Introduction
Methods
Results
Discussion
References

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


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FIG. 1. Postexposure current (Ipe) activated by NMDA exposure or intracellular calcium perfusion is blocked by brief pulses of Zn2+. A: Ipe triggered by a 10 min N-methyl-D-aspartate (NMDA) exposure was blocked during brief (25 s) pulses of Zn2+ (500 µM). Zn2+ application had no effect on holding current before NMDA exposure. bullet , Zn2+ pulses. Voltage steps (-10 mV, 25 s) demonstrate increase in membrane conductance associated with Ipe. B: Ipe triggered by a 2 min NMDA exposure. Note delay between NMDA exposure and development of a noticeable Ipe. Ipe was again blocked during brief (20 s) pulses of Zn2+ (500 µM). C: Ipe triggered by intracellular perfusion of high calcium solution was blocked during brief (20 s) pulses of Zn2+ (500 µM). Holding potential was -50 mV.


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FIG. 2. Effect of various extracellular divalent cations on Ipe. A: Ipe was triggered with a 30 s NMDA application. Recordings shown are from a single cell beginning at 20 min after NMDA application. Pulses (4 s) of 1 mM Cd2+, 100 µM Zn2+, and 1 mM Co2+, Mn2+, Mg2+, and Ba2+ were applied in turn with a 1-min interval between applications. Same applications of divalent cations had no effect on holding current when applied before NMDA application. B: example of effect of Zn2+ application on Ipe. Zn2+ (500 µM) was applied for 4 s (horizontal line). Membrane conductance was tested before and during Zn2+ application with use of a -10 mV, 1-s voltage step. Zn2+ blocked Ipe and reduced membrane conductance. C: Dose-response curve for Zn2+. I/Imax is ratio of amplitude of Ipe during Zn2+ application to amplitude of Ipe directly before Zn2+ application. (Ipe amplitude was determined by subtracting holding current amplitude before NMDA exposure from holding current amplitude after NMDA exposure.) Each data point is average of data from 9 or more cells. Error bars represent SE. Line through data points was obtained by fitting data with modified Hill equation y = ICn50/(ICn50 + xn), which generated an IC50 of 64 µM and an n of 1.9. Data for graph were obtained from recordings in which Ipe was <800 pA immediately before Zn2+ application. Holding potential was -50 mV.

All twelve cells tested with a 10 min NMDA exposure developed Ipe. In all twelve cases, the Ipe became evident during the NMDA exposure. Eight of 10 cells tested with a 2-min NMDA exposure developed Ipe. In these eight cells, the membrane current returned to baseline after NMDA exposure and Ipe became evident after a 7 ± 3 min (mean ± SE) delay. The proportion of cells developing Ipe 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 Ipe after a 40 s NMDA exposure, Ipe became evident after a10 ± 4 min delay. In the one cell that developed Ipe after a 10 s exposure, Ipe became evident after a 12-min delay. In all cases in which Ipe 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 Ipe 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 Ipe (Fig. 2A). Cd2+(1 mM) and Zn2+(100 µM) reduced Ipe by 90 ± 6% (mean ± SD, n = 25) and 88 ± 5% (n = 21), respectively. Co2+ (1 mM) and Mn2+ (1 mM) reduced Ipe 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 Ipe 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 Ipe is triggered by Ca2+ and partially carried by Ca2+ (Chen et al. 1997), we suspected that Ipe-mediated Ca2+ influx might be responsible for the continuous growth of Ipe. Figure 3B illustrates a prolonged block of Ipe with 100 µM Zn2+. Brief pulses of Zn2+-free solution were applied once every min to test the development of Ipe during the prolonged Zn2+ block. If the hypothesis were incorrect and cation influx through Ipe were not required for the further growth of Ipe, one would expect the Zn2+-free pulses to reveal a continuous growth in the underlying Ipe similar to that seen in the control (Fig. 3A). Instead, in support of the hypothesis, the Ipe, 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 Ipe during the prolonged block and 4 of 10 showed a 75% or greater reduction in the rate of increase of Ipe 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 Ipe resumed.


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FIG. 3. A prolonged application of Zn2+ halts continuous increase in amplitude of underlying Ipe. A: control Ipe recorded 22 min after a 35-s NMDA application. Note that Ipe continuously increased in amplitude. Ipe was blocked during brief (4 s) pulses of Zn2+ (100 µM), which were applied once every 5 min. B: Ipe recorded 20 min after a 35-s NMDA application. Ipe was initially gradually increasing in amplitude. Then Ipe was blocked by a 10-min application of Zn2+. Brief (4 s) pulses of Zn2+-free solution applied once every minute during period of prolonged Zn2+ application revealed that underlying Ipe neither grew nor shrank while being blocked by Zn2+. Growth of Ipe resumed after removal of Zn2+. Holding potential was -50 mV.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

Ca2+ and Na+ influx associated with Ipe 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 Ipe is diagnostic for whether or not a cell will die because of NMDA exposure: those cells that develop Ipe (and are allowed to express Ipe) die in <1 h; those that do not develop Ipe 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 Ipe. 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 Ipe 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 Ipe but not for the maintenance of a steady Ipe (Chen et al. 1997). These earlier results combined with the finding that Ipe remained at a steady level during prolonged Zn2+ block indicate that once triggered, the maintenance of a steady Ipe is not dependent on Ipe-mediated Ca2+ influx but that the continuous growth in Ipe is dependent on Ipe-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 Ipe. 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). Ipe block is another avenue by which low concentrations of extracellular Zn2+ might help protect neurons from cell death.

    ACKNOWLEDGEMENTS

  The authors thank R. Bianchi, J. Celentano, L. Merlin, H. Michelson, and S. Young for helpful discussion.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-24682.

    FOOTNOTES

*   Deceased December 1995.

  Address for reprint requests: K. L. Perkins, SUNY HSCB, Box 29, 450 Clarkson Ave., Brooklyn, NY 11203.

  Received 20 August 1997; accepted in final form 19 September 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society