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Volume 17, Number 11, Issue of June 1, 1997 pp. 4032-4036
Copyright ©1997 Society for Neuroscience

Secondary Activation of a Cation Conductance Is Responsible for NMDA Toxicity in Acutely Isolated Hippocampal Neurons

Qiang X. Chen¹, Katherine L. Perkins¹, Dennis W. Choi², and Robert K. S. Wong¹

¹ Department of Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203, and ² Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

FOOTNOTES

REFERENCES

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ABSTRACT

One of the key questions concerning glutamate toxicity is how a transient NMDA exposure can lead to a delayed death of neurons. To address this issue, we performed whole-cell recording on acutely isolated hippocampal CA1 neurons to monitor the membrane response after NMDA exposure. Transient NMDA exposure (100 µM, 10 min) induced an inward current (postexposure current; I_pe) which was associated with a Ca²⁺- and Na⁺-permeable cation conductance. I_pe continuously increased (in the absence of NMDA) until death of the neuron occurred. Application of NMDA in the absence of extracellular calcium failed to trigger I_pe and neuronal death. Postexposure suppression of I_pe protected against NMDA toxicity. These results indicate that a cation current, which is induced by an increase in intracellular calcium concentration ([Ca²⁺]_i) and is itself partly carried by Ca²⁺, links the initial NMDA exposure to neuronal death.

Key words: NMDA; neurotoxicity; postexposure current; I_pe; excitotoxicity; calcium; hippocampus; glutamate; toxicity; cell death; neuronal death

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INTRODUCTION

Excess levels of the excitatory neurotransmitter glutamate can induce neuronal degeneration, primarily through NMDA receptor channel-mediated Ca²⁺ influx (Choi, 1988). Neuronal death associated with ischemia, hypoglycemia, trauma, and epilepsy can be reduced with antagonists of the NMDA type of glutamate receptor, suggesting the involvement of NMDA toxicity in these clinical conditions (Simon et al., 1984; Wieloch, 1985; Faden et al., 1989; Meldrum, 1994). However, the clinical utility of NMDA receptor antagonists faces a key impediment: the timing of treatment. The neuroprotective effect of NMDA receptor antagonists is significant only when administered before the insult (ischemia or transient NMDA exposure) or within a short window after the insult (Hartley and Choi, 1989; Graham et al., 1993). This short therapeutic window of NMDA receptor antagonists limits their usefulness and prompts the study of downstream mechanisms in the postexposure phase, the period between the initial transient NMDA exposure (exposure phase) and the death of neurons. During the postexposure phase, there is a secondary rise in intracellular calcium concentration ([Ca²⁺]_i), which is not reversed by NMDA antagonists (Randall and Thayer, 1992; Tymianski et al., 1993). This secondary rise in [Ca²⁺]_i is correlated with subsequent cell death (Randall and Thayer, 1992; Tymianski et al., 1993). Neither the secondary rise in [Ca²⁺]_i nor the cell death occurs if the NMDA is applied in Ca²⁺-free solution (Randall and Thayer, 1992), suggesting that Ca²⁺ influx through NMDA receptor channels somehow triggers a process that causes the secondary rise in [Ca²⁺]_i and subsequent cell death. Suggested mechanisms for the secondary rise in [Ca²⁺]_i include an increase in Ca²⁺ conductance, derangement of Ca²⁺ transport, and release of Ca²⁺ from intracellular stores (de Erausquin et al., 1990; Randall and Thayer, 1992; Tymianski et al., 1993). In this paper, we have done electrophysiological recordings to monitor the membrane response after NMDA exposure and have determined that Ca²⁺ influx through NMDA receptors leads to the activation of a persistent Ca²⁺- and Na⁺-permeable cation conductance that is responsible for the subsequent neuronal death.

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MATERIALS AND METHODS

Whole-cell voltage-clamp recording was performed on acutely isolated hippocampal CA1 neurons. Neurons were prepared according to the Kay and Wong (1986) method, with the following modifications to increase the harvest of healthy neurons and preserve NMDA responses. Hippocampal slices were prepared by vibratome instead of tissue chopper, the [Ca²⁺] in the incubation solution was reduced to 0.5 mM and the [Mg²⁺] was increased to 7 mM, and 12-15 µM CPP (Tocris Cookson, St. Louis) was added during trypsin incubation of tissue slices to preserve the NMDA response (Chen and Wong, 1995a). Healthy neurons were selected for our experiments by choosing those that were uniformly bright under phase-contrast microscopy. These neurons have a normal (approximately 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 et al., 1990; Chen and Wong, 1995a,b).

Whole-cell voltage-clamp recording was performed using the procedure described by Hamill et al. (1981) with the use of a List EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany) and pClamp software (Axon Instruments, Foster City, CA). Access resistances were ~10 M and were compensated only in the experiments shown in Figure 2. Liquid junction potentials (V_lj) were measured using the procedure of Neher (1992). All potentials have been corrected for V_lj, which ranged from 9 to 14 mV. Good recordings were ensured by discarding cells that had seal resistances <20 G, that took more than three gentle sucks to break the membrane, or that did not maintain a stable input resistance during the first 5 min of recording before NMDA exposure. The holding potential was 55 mV.

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Fig. 2. I_pe is carried by Ca²⁺ and Na⁺. A-D, I_pe was triggered by NMDA exposure (10 min, 100 µM) or intracellular perfusion of high-calcium solution. A, Responses of I_pe to changes of extracellular ionic composition. Line above current traces indicates period of perfusion of 20 mM NaCl solution, 20 mM KCl solution, or 10 mM CaCl₂ solution. Changes of I_pe reached a plateau (indication of complete solution change around cell) within 2 sec. These responses are not from the same cell; because I_pe grows over time, we chose instead to show responses in which the I_pe amplitudes (directly before the change to test solution) matched. B, C, Voltage ramps (90 mV to +90 mV, 600 msec duration) performed 3-6 sec after changing to the indicated solutions. B, I_pe during the voltage ramp in the presence of extracellular control solution (continuous trace) and low-Cl solution (dotted trace). (Traces essentially overlap.) The reversal potentials of I_pe were 0.8 ± 0.6 mV (control solution) and 0.8 ± 0.7 mV (low-Cl solution, mean ± SD, n = 5). C, I_pe during the voltage ramp in the presence of extracellular control (a), 10 mM CaCl₂ (b), 20 mM KCl (c), and 20 mM NaCl (d) solutions. During the voltage ramps, TTX (5 µM) and Co²⁺ (200 µM) were applied to block voltage-dependent Na⁺ and Ca²⁺ currents. Baseline leak current before NMDA exposure was not subtracted from current traces shown in B and C, because the subtraction made no significant difference in the reversal potential or conductance measured. D, Neither MK-801 (20 µM) nor Co²⁺ (200 µM) suppressed I_pe. Voltage steps (+10 mV) were applied every 25 sec. Cells examined had an I_pe smaller than 1 nA.
[View Larger Version of this Image (19K GIF file)]

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In one set of experiments, the intracellular perfusion technique was used to switch from control intracellular solution to high-calcium intracellular solution while recording from a single cell. For details of the intracellular perfusion method, see Chen et al. (1990).

Cells were in a 1 ml bath that was perfused continuously with extracellular solution at a rate of 1-2 ml/min. Extracellular control solution contained (in mM): 140 NaCl, 2 KCl, 2 CaCl₂, 10 HEPES, 0.01 glycine, 25 glucose, pH adjusted to 7.4 with NaOH (which raised the [Na⁺] to 145 mM). Zero-calcium solution was the same as control solution, except with 0.5 BAPTA and no CaCl₂. Application of NMDA and changes of ionic concentration around the recorded cells were achieved by a seven-barrel flow tube (Celentano and Wong, 1994). NMDA (100 µM) was dissolved in control solution or zero-calcium solution as indicated. The 20 mM NaCl, 20 mM KCl, and 10 mM CaCl₂ solutions contained only the indicated ions, 10 mM HEPES, 0.01 mM glycine, 25 mM glucose, and sucrose added to preserve osmolarity. Intracellular control solution contained (in mM): 20 CsCl, 100 CsOH, 0.5 BAPTA, 10 HEPES, pH adjusted to 7.2 with methane sulfonic acid. High-calcium intracellular solution was made by adding 1 mM Ca²⁺ to the control intracellular solution. The estimate of free Ca²⁺ in the high-calcium intracellular solution is 0.5 mM (Chen et al., 1990). The actual [Ca²⁺]_i reached because of perfusion of this solution, although likely to be <0.5 mM, is unknown. Chemicals were obtained from Sigma (St. Louis, MO) unless otherwise indicated.

Ionic activities were used for the calculation of relative permeabilities. To simplify the calculation of relative permeabilities, we assumed no significant effect from the surface charge on the permeation of cations. We also assumed that both the ionic composition around the intracellular side of the membrane and the relative permeability of the membrane were unchanged during the experiment. We used cells in which the I_pe was less than 1 nA in obtaining data for the evaluation of the relative permeability of the I_pe channel, because as I_pe increased in amplitude, extracellular ions accumulated around the intracellular side of the membrane as indicated by the gradual negative shift in the reversal potential when the amplitude of I_pe exceeded 1 nA.

Trypan blue stain was used to assess cell death. Staining was performed by a 5 min bath perfusion of dye solution (0.4% final concentration, made up in extracellular control solution). Neurons were examined for stain under bright-field microscope 5 min after changing back to control bath perfusion.

In some experiments, cell death was assessed in nonrecorded cells. For these experiments, cells were isolated from a single animal and put in several dishes called "sister dishes." Dishes were prescored into square fields with a 5 mm × 5 mm grid. For each dish, one or two fields, each containing 10-30 healthy neurons, were selected before treatment, and the healthy neurons in the selected fields were identified. Neurons were perfused continuously with extracellular solution at a rate of 1-2 ml/min. Trypan blue stain was used to assess neuronal death after treatment.

Data are reported as mean ± SD.

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RESULTS

Prolonged NMDA application triggers I_pe and cell death

We have shown that with a short (2-8 sec, 100 µM) NMDA application, the holding current returns to baseline after termination of the NMDA application (Chen and Wong, 1995a,b). Figure 1A shows the result of a long (10 min, 100 µM) NMDA application (exposure). The NMDA response changed kinetics at 5.6 ± 1.4 min (n = 8) of NMDA application, with the appearance of a superimposing and continuously increasing inward current accompanied by an increase in membrane conductance. This inward current, which we call the postexposure current (I_pe), persisted after termination of NMDA application and increased at a rate of ~62 pA/min throughout the recordings, which ended at 30-40 min postexposure (n = 8 cells). The input resistance and holding current at 55 mV were 2.9 ± 1.1 G and 22.5 ± 10.3 pA before NMDA exposure and 23 ± 3 M and 2.4 ± 0.3 nA (n = 8) at 30 min after exposure. Cell death was assessed at 30 min postexposure; all eight cells showed positive trypan blue stain. In four of these eight cells, trypan blue stain was also performed at 0, 10, and 20 min postexposure; only one neuron showed positive stain at 20 min postexposure.

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Fig. 1. NMDA exposure and rises in [Ca²⁺]_i trigger I_pe. A, Response to NMDA exposure (10 min, 100 µM) in the presence of Ca²⁺_o. B, Response to NMDA exposure (10 min, 100 µM) in zero-calcium extracellular solution. C, Response to intracellular perfusion of high-Ca²⁺solution. To measure input resistance, voltage steps (+10 mV, 30 sec) were applied once every 5 min at the times indicated by the circles. These voltage steps have no effect on the development of I_pe. Horizontal lines on top of each current trace indicate periods of solution application.
[View Larger Version of this Image (17K GIF file)]

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A rise in [Ca²⁺]_i induces I_pe

When cells were bathed in zero-calcium solution during NMDA exposure, no continuously increasing holding current was observed during the postexposure phase (Fig. 1B). The input resistance and holding current were 2.8 ± 1.2 G and 24 ± 12 pA before exposure and 2.5 ± 1 G and 29 ± 19 pA (n = 6) after exposure. None of the six neurons stained with trypan blue at 30 min postexposure. The possibility that a rise in [Ca²⁺]_i triggers I_pe was tested further by using the intracellular perfusion technique. Figure 1C shows that an increase in [Ca²⁺]_i caused by an 18 min intracellular perfusion of high-calcium intracellular solution induced an inward current (n = 7). This inward current was indistinguishable from I_pe in its reversal potential and ionic selectivity (see below).

I_pe is a cation current

To test the ionic basis of I_pe, we performed several ion substitution experiments. In the first, replacing 120 mM extracellular Cl with the presumably less-permeant anion gluconate (120 mM) changed neither the reversal potential of nor the conductance associated with I_pe (Fig. 2B). In another set of experiments, we created a significant salt gradient across the membrane by applying one of three extracellular isotonic low-salt solutions (20 mM NaCl, 20 mM KCl, or 10 mM CaCl₂). Reducing the extracellular salt concentration caused a reduction in the amplitude of the inward I_pe (Fig. 2A) and a negative shift in the reversal potential (Fig. 2C), which is the opposite result of what would be expected if I_pe were an anion current, indicating that the contribution of anion effluxes to I_pe is negligible. The inward I_pe could be obtained with any single cation tested (Na⁺, K⁺, or Ca²⁺) alone in the extracellular solution (Fig. 2A,C), indicating that Na⁺, K⁺, and Ca²⁺ can all carry the inward I_pe. With Cs⁺(120 mM) on the intracellular side of the membrane and either Na⁺ (20 mM), K⁺ (20 mM), or Ca²⁺ (10 mM) on the extracellular side of the membrane, the reversal potentials of I_pe were 45 ± 0.6, 43 ± 0.8, and 5 ± 0.7 mV, respectively (mean ± SD, n = 5). With these data, we estimated the relative permeabilities P_Cs/P_Na/P_K/P_Ca to be 1:0.9:0.9:6.3. The high-Ca²⁺ permeability and the lack of discrimination among different monovalent cations are comparable with the properties of the NMDA receptor channel (Mayer and Westbrook, 1987). However, blockers for the NMDA receptor channel, MK-801 (20 µM, 1 min exposure) and ketamine (20 µM, 1 min exposure), had no effect on I_pe (n = 4; Fig. 2D). In addition, voltage-dependent Ca²⁺ channel blockers Co²⁺ (200 µM, 20 sec exposure; Fig. 2D) and nimodipine 10 µM (20 sec exposure), and the Na⁺ channel blocker tetrodotoxin (5 µM, 10 sec exposure) all failed to affect the I_pe (n = 4).

I_pe is responsible for NMDA toxicity

We have demonstrated that I_pe was carried by Ca²⁺ and Na⁺ (Fig. 2) and persisted and increased continuously in amplitude during the postexposure phase (Fig. 1A). These properties give I_pe the potential of causing substantial accumulation of Na⁺ and Ca²⁺ in the intracellular space. Accumulation of both ions, particularly Ca²⁺, inside a neuron is cytotoxic (Choi, 1988). As presented above, a normally toxic exposure to NMDA does not kill the cells when I_pe is not activated (zero-calcium extracellular solution; Fig. 1B). To further address the causal connection between I_pe and cell death, we tested whether postexposure suppression of I_pe would reduce neuronal death. Without a specific blocker for I_pe, we took advantage of the fact that I_pe can be suppressed by the 20 mM NaCl solution (see Fig. 2A). Extracellular perfusion with the 20 mM NaCl solution after the NMDA exposure caused an immediate suppression of I_pe of 86 ± 1% (n = 8; Fig. 3A). In addition, perfusion with the 20 mM NaCl solution during the postexposure phase prevented the growth of I_pe (Fig. 3A); the change in I_pe amplitude during the 30 min postexposure phase was 18% (n = 8) compared with a 100% change in I_pe amplitude in control extracellular solution (n = 8). None of the cells exposed to the 20 mM NaCl solution during the postexposure phase stained with trypan blue at 30 min postexposure (n = 8), indicating that suppression of I_pe in the postexposure phase was neuroprotective.

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Fig. 3. I_pe-mediated Ca²⁺ and Na⁺ influx leads to the death of NMDA-exposed neurons. A-D, I_pe was triggered by NMDA application (10 min, 100 µM). A, Response of I_pe to simultaneous removal of Ca²⁺_o and reduction of [Na⁺]_o for the postexposure phase (started 1 min postexposure). B, Response of I_pe to removal of Ca²⁺_o for the postexposure phase. C, NMDA exposure triggers a gradually developing I_pe in nonrecorded cells. Healthy pyramidal cells within a field were identified before NMDA exposure and randomly selected at different times postexposure for point recording. Zero time indicates beginning of postexposure phase. Each circle represents a point recording from a different cell in the dish. D, Percentage of survival of neurons in sister dishes with the same treatment as those applied to recorded cells. NMDA application and manipulation of extracellular ionic composition were performed by bath perfusion. Healthy neurons in two fields per dish were identified before treatment. To evaluate the percentage of neuronal death after different treatments, trypan blue staining was performed at 60 min postexposure. The five bars represent average survival values obtained from (left to right) the control group (not exposed to NMDA), the NMDA group (exposed to NMDA), the NMDA + 0 Ca²⁺ group (exposed to NMDA in the presence of zero Ca²⁺_o), the low-Na⁺ + 0 Ca²⁺ postexposure group (exposed to NMDA and then bathed in the 20 mM NaCl solution during postexposure), and the 0 Ca²⁺ postexposure group (exposed to NMDA and then bathed in zero-calcium solution during postexposure). Consistent with data obtained from recorded cells, postexposure removal of Ca²⁺_o reduced neuronal death caused by NMDA exposure (0 Ca²⁺ postexposure group vs NMDA group, p < 0.01). Postexposure reduction of [Na⁺]_o in addition to removing Ca²⁺_o provided additional protection (low-Na⁺ + 0 Ca²⁺ postexposure group vs 0 Ca²⁺ postexposure group, p < 0.01) and was not significantly different from control (p > 0.05). Error bars represent SD (n = 12 fields, 2 fields from each of 6 animals). Student's two-tailed t test was used to obtain p values.
[View Larger Version of this Image (14K GIF file)]

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To determine whether Ca²⁺ and/or Na⁺ influx led to the growth of I_pe and the death of neurons, we removed only Ca²⁺ from the control extracellular solution during the postexposure phase. Extracellular perfusion with the zero-Ca²⁺ solution during the postexposure phase prevented the continuous increase in I_pe amplitude (Fig. 3B; compare Fig. 1A). In addition, we observed an immediate 32 ± 6% (n = 6) increase in I_pe, possibly attributable to removing the suppressive effect of Ca²⁺_o on Na⁺ influx, as observed for other Ca²⁺-permeable conductances (Mayer and Westbrook, 1987; Lux et al., 1990). Only neurons with I_pe amplitudes larger than 1 nA immediately after Ca²⁺ removal (2 of 6 neurons tested) stained with trypan blue at 30 min postexposure.

Because whole-cell recording disturbs the metabolism of the recorded cell, we tested to make sure that whole-cell recording itself is not necessary for the activation of I_pe and the resulting cell death. Nonrecorded cells (both NMDA-exposed and nonexposed cells in sister dishes) were examined by short-duration whole-cell recording (point recording) performed at different times on different cells in the dish during the postexposure phase. Point recording showed that NMDA-exposed cells had a smaller input resistance and a larger inward current immediately after disruption of the patch membrane than nonexposed cells. This inward current in NMDA-exposed cells was indistinguishable from I_pe. In agreement with data from recorded cells, point recordings also revealed that I_pe grew over time during the postexposure phase in nonrecorded cells (Fig. 3C). The seal resistance of point recordings was the same regardless of whether I_pe was present, indicating that the development of I_pe after NMDA exposure in recorded cells (see Fig. 1A) is not attributable to a gradual deterioration of the seal. Figure 3D shows results from trypan blue staining performed at 60 min postexposure on sister dishes of nonrecorded cells that were exposed to the same treatment as recorded cells. Consistent with the results in recorded cells, only protocols shown previously to trigger I_pe caused cell death (i.e., NMDA exposure but not NMDA exposure in zero-calcium solution). In addition, protocols shown to suppress I_pe postexposure (i.e., postexposure removal of Ca²⁺_o and simultaneous reduction in Na⁺_o) or the growth of I_pe during the postexposure (i.e., postexposure removal of Ca²⁺_o) caused a significant reduction in the percentage of dead cells. The solution containing zero-calcium and low-Na⁺ (the 20 mM NaCl) solution provided the greatest postexposure protection, producing a survival rate indistinguishable from control (Fig. 3D).

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DISCUSSION

I_pe

NMDA application caused the appearance of the cation current I_pe, which persisted and increased in size after the removal of NMDA. The strict cation selectivity and differential permeability of I_pe indicate that I_pe is not simply the result of a breakdown of membrane integrity, but is rather associated with a well-behaved membrane conductance. The fact that induction of I_pe required the presence of extracellular Ca²⁺ during NMDA exposure indicates that I_pe is triggered by Ca²⁺ entry through NMDA receptor channels, a conclusion that is supported by the experiments that showed that intracellular perfusion with Ca²⁺ could also lead to the activation of I_pe. Because I_pe is itself partly carried by Ca²⁺, Ca²⁺ entry associated with I_pe could induce additional I_pe. In fact, removing the Ca²⁺ from the extracellular solution during postexposure prevented the gradual growth in I_pe (Fig. 3A,B), indicating that continued I_pe-mediated Ca²⁺ influx is responsible for the continuous increase in the size of I_pe. On the other hand, a large I_pe persisted (but did not increase in amplitude) when zero-Ca²⁺ solution was perfused during the postexposure phase (Fig. 3B), indicating that continued Ca²⁺ influx is not required for the maintenance of I_pe. The activation requirements and properties of I_pe indicate that it is probably responsible for the secondary [Ca²⁺]_i increase which has been recorded in response to glutamate exposure (Randall and Thayer, 1992; Tymianski et al., 1993). Additional studies are needed to address the microscopic conductance and modulatory mechanisms associated with I_pe.

I_pe and neuronal death

Earlier investigations in cultured neurons have shown that cell death from NMDA application is markedly reduced if the NMDA is applied in zero-calcium solution (Choi, 1987). In addition, earlier studies in cultured neurons have shown that removal of extracellular calcium (Ca²⁺_o) in the postexposure phase reduces neuronal death (Hartley and Choi, 1989; Manev et al., 1989), and that simultaneous removal of Ca²⁺_o and Na⁺_o during the postexposure phase produces essentially complete blockade of NMDA neurotoxicity (Hartley and Choi, 1989). Our study has replicated these results in a new system and, more importantly, has shown that these two procedures block NMDA toxicity in acutely isolated hippocampal cells, because the first prevents the induction of I_pe and the second suppresses I_pe in the postexposure phase. Our results indicate that NMDA exposure causes an initial Ca²⁺ influx that triggers the onset of I_pe and that it is the large and persistent secondary influx of Ca²⁺ and Na⁺ underlying the I_pe that is the downstream event associated with cell death.

Previous studies (Choi, 1987) have measured "delayed" cell death, in which cultured neurons exposed to glutamate or NMDA die several hours later. Our experiments cannot be said to measure delayed cell death, because the NMDA-exposed cells died within 1 hr; in fact, acutely isolated hippocampal cells will die in 2-4 hr without NMDA exposure. Regardless, it is possible that I_pe may contribute to NMDA-induced delayed cell death in other systems. The existence of an I_pe in other systems could account for the NMDA antagonist-insensitive sustained increase in [Ca²⁺]_i, which has been correlated with cell death (de Erausquin et al., 1990; Randall and Thayer, 1992; Tymianski et al., 1993) and for the protection of neurons seen with the simultaneous removal of Na⁺ and Ca²⁺ in the postexposure phase (Hartley and Choi, 1989). Because the Na⁺ and Ca²⁺ influx associated with I_pe may be the downstream event that causes glutamate neurotoxicity, pharmacological agents that could suppress I_pe or retard its growth may reduce cell death.

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FOOTNOTES

Received Jan. 15, 1997; revised March 6, 1997; accepted March 12, 1997.

  

This work was supported by National Institutes of Health Grant NS 24682.

Correspondence should be addressed to Dr. Katherine L. Perkins, SUNY Health Science Center, Box 29, 450 Clarkson Avenue, Brooklyn, NY 11203.

We are saddened by the passing of our colleague, Dr. Qiang X. Chen, and dedicate this article to his memory.

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Copyright ©1997 Society for Neuroscience   0270-6474/1997/174032-05$05.00/0

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