Factors That Reverse the Persistent Depolarization Produced by Deprivation of Oxygen and Glucose in Rat Hippocampal CA1 Neurons In Vitro
S. Yamamoto1,
E. Tanaka1,
Y. Shoji1,
Y. Kudo2,
H. Inokuchi1, and
H. Higashi1
1 Department of Physiology, Kurume University School of Medicine, Kurume 830; and 2 Laboratory of Cellular Neurobiology, Tokyo University of Pharmacy and School of Life Science, Hachioji, Tokyo 192-03, Japan
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ABSTRACT |
Yamamoto, S., E. Tanaka, Y. Shoji, Y. Kudo, H. Inokuchi, and H. Higashi. Factors that reverse the persistent depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 78: 903-911, 1997. In CA1 pyramidal neurons in rat hippocampal tissue slices, superfusion with ischemia-simulating medium produced a rapid depolarization after 6 min of exposure. The membrane potential eventually reached 0 after 5 min (a persistent depolarization), even when oxygen and glucose were reintroduced. The role of various ions in the reversal of this persistent depolarization after reintroduction of oxygen and glucose was investigated. The peak of the persistent depolarization was decreased in solutions containing reduced Na+ or Ca2+ and in solutions containing Co2+ or Ni2+. In contrast, the depolarization was not affected by reduction of external K+ or Cl
or by addition of tetrodotoxin (TTX), flunarizine, or nifedipine. These results suggest that sustained Na+ and Ca2+ influxes produce the persistent depolarization. The membrane potential recovered after reintroduction of oxygen and glucose in low Ca2+, low Cl
, or K+-rich medium and in TTX- or tetraethylammonium-containing medium, but not in low Na+ or low K+ medium and in flunarizine- or nifedipine-containing medium. Either reduction in extracellular Ca2+ or addition of Co2+ was the most effective in promoting recovery from the persistent depolarization, suggesting that Ca2+ influx has a key role in causing the membrane dysfunction. The peak of the persistent depolarization was reduced by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-5-phosphonopentanoic acid (AP5), DL-amino-3-phosphonopropionic acid (AP3), or DL-amino-4-phosphonobutyric acid, suggesting that activation of non-N-methyl-D-aspartate (non-NMDA), NMDA, and metabotropic glutamate (Glu) receptors is involved in the generation and maintenance of the persistent depolarization. Among these Glu receptor antagonists, only CNQX or AP5 was able to reduce dose dependently the level of depolarization, suggesting that Ca2+ influx via both
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate type II receptors and NMDA receptors contributes to the membrane dysfunction. trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) did not affect the peak potential of the persistent depolarization, but it dose-dependently restored the membrane potential. AP3 antagonized the protective action of t-ACPD. The membrane potential also recovered after reintroduction when the slice was pretreated by 1,2-bis(2-aminophenoxy)ethane-N, N,N
,N
-tetraacetic acid tetraacetoxymethyl ester, ryanodol 3-(1H-pyrrole-2-carboxylate), 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride, and procaine, suggesting that raised [Ca2+]i from Ca2+-induced Ca2+ release pool contributes to the membrane dysfunction. It, therefore, is concluded that raised [Ca2+]i has a dominant role in causing irreversible changes. The increase in [Ca2+]i during the persistent depolarization may be the result of Ca2+ entry via both a leaky membrane and Glu-activated receptor channels as well as Ca2+ released from internal stores.
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INTRODUCTION |
In CA1 neurons of the rat hippocampal slice preparation, superfusion with an ischemia-simulating medium (deprived of oxygen and glucose) produces a rapid depolarization after ~6 min of exposure. Whether or not oxygen and glucose are reintroduced immediately after the onset of the rapid depolarization, the membrane potential becomes persistently depolarized, reaching at 0 mV after 5 min (Tanaka et al. 1997
). Rader and Lanthorn (1989)
have reported that the rapid depolarization recorded intracellularly in hippocampal CA1 neurons corresponds to the extracellularly recorded anoxic depolarization (a rapid negative-going shift of the DC potential). The intracellularly recorded depolarization consists of two pharmacologically distinct components, a rapid depolarization and a subsequent, persistent depolarization. The persistent depolarization is selectively blocked by reduction in extracellular Ca2+ and elevation of extracellular Mg2+ or by the N-methyl-D-aspartic acid (NMDA) receptor antagonists, dizocilpine malate and 3-([+]-2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid, suggesting that the membrane dysfunction may be due to activation of NMDA receptor channels by interstitial glutamate (Glu) accumulation. On the other hand, Tanaka et al. (1997)
have demonstrated that the rapid depolarization may be due to a nonselective increase in permeability to all participating ions (Na+, Ca2+, K+, and Cl
), which may occur only in pathological conditions. Thus the rapid depolarization leads to massive influxes of Na+, Ca2+, and Cl
and a large efflux of K+. Consequently, neurons would become hyperosmotic. The difference in osmolarity across the cell membrane would give rise to an inflow of water and cause cell swelling, which may destroy physically the structure of the cell membrane. The roles of other Glu receptor subtypes and participating ions except for NMDA receptors and Ca2+ in the membrane dysfunction are still unclear.
The present study concerns the mechanism for the transition to the irreversible membrane dysfunction during the persistent depolarization induced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in the slice preparation. The experiments consist of testing the effects of application of Glu agonists and antagonists and changes in extracellular ion concentrations on the persistent depolarization. In addition, we have examined recovery of the membrane potential and the apparent input resistance as indices of the ability of a given experimental condition to prevent irreversible changes in the neuron. It may be possible to deduce the applicability of some of the results to the clinical situation from such a study. Preliminary accounts of some data have been presented previously (Higashi et al. 1990a
; Yamamoto et al. 1993a
,b
).
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METHODS |
The preparation and recording techniques employed were similar to those described in the preceding paper (Tanaka et al. 1997
). The preparation was submerged completely in the superfusing solution, which was maintained at 36.5 ± 0.5°C, and was flowing continuously at a rate of 6-8 ml/min. Intracellular recordings from CA1 pyramidal cells were made with glass micropipettes (tip resistance 40-80 M
) filled with K acetate (2 M). Cs acetate (2 M)-filled electrodes were used for blocking K+ channels in some experiments. The apparent input resistance was monitored by passing hyperpolarizing currents (200 ms, 0.1-0.3 nA, 0.33 Hz) through the recording electrode. The bridge balance was continually monitored and adjusted. The slices were made "ischemic" by superfusing them with medium equilibrated with 95% N2-5% CO2 and deprived of glucose, which was replaced with NaCl isoosmotically. When switching the superfusing media, there was a delay of 15-20 s before the new medium reached the chamber due to the volume of the connecting tubing. The chamber was filled completely with test solution within 30 s after switching the three-way cock. When testing for the effects of various solutions on the peak potential of the persistent depolarization, we have chosen arbitrarily to measure the potential 1 min after generating the rapid depolarization as the peak, because the persistent depolarization was maintained for 1-2 min when the membrane potential was recovered after reintroduction of oxygen and glucose in test media (see RESULTS). Recovery after reintroduction of oxygen and glucose is defined as follows: no recovery, 30-60 min after reintroduction the membrane potential lay between 0 and
19 mV; complete recovery, the membrane potential was more negative than
60 mV; partial recovery, membrane potential repolarized to a value between
20 and
59 mV. In most neurons with complete recovery, action potentials and fast excitatory postsynaptic potentials similar to those in the initial control period could be elicited by direct and focal stimulation, respectively, when the test solution was replaced by the normal medium. At the end of experiments, the membrane potential was referenced to 0 mV, which was measured after drawing the electrode out of the cell. The recovery ratio is expressed as the percentage of the total number of neurons exhibiting complete and partial recovery in a given group.
Low Na+ (28.6 mM) medium, low K+ (0.36 mM) medium, low Cl
(43 mM) medium, and low Ca2+ (0.25 mM) medium were made, as described in the preceding paper (Tanaka et al. 1997
). The drugs used were 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX), trans-(1S,3R)-amino-1,3-cyclopentane-dicarboxylicacid (t-ACPD, from Tocris Neuramin); DL
2-amino-3-phosphonopropionic acid (AP3), DL
2-amino-4-phosphonobutyric acid (AP4), DL
2-amino-5-phosphonopentanoic acid (AP5), procaine, N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid (all from Sigma Chemical); ryanodol 3-(1H-pyrrole-2-carboxylate) (ryanodine), 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8, all from Research Biochemicals International); tetrodotoxin (TTX, from Sankyo), 1,2-bis(2-aminophenoxy)e t h a n e - N , N , N
,N
- t e t r a a c e t i c a c i d t e t r a a c e t o x y m e t h y l e s t e r(BAPTA/AM, from Wako); tetraethylammonium chloride (TEACl, from Tokyo Kasei Organic Chemicals); flunarizine (gift from Kyowa Hakko); nifedipine (gift from Bayer). All drugs were dissolved in the perfusate and applied by bath application. All quantitative results are expressed as means ± SD. The number of neurons examined is given in parentheses. The analysis of variance test was used to compare data, with P < 0.05 considered significant.
 |
RESULTS |
This study was based on intracellular recordings from >200 CA1 pyramidal neurons of adult rats with stable membrane potentials more negative than
60 mV in control superfusing solution. The resting membrane potential and the apparent input resistance were
71 ± 6 mV and 45 ± 16 M
(n = 130), respectively.
Persistent depolarization produced by ischemia-simulating medium
In response to oxygen and glucose deprivation, hippocampal CA1 neurons showed a rapid depolarization after 5.8 ± 1.1 min (n = 65) at a temperature of 36.5 ± 0.5°C. When oxygen and glucose were reintroduced after the onset of the rapid depolarization, the neuron did not repolarize, but depolarized further at a much slower rate. The membrane potential eventually approached 0 mV at 5.2 ± 1.0 min (n = 65) after the onset of the rapid depolarization and remained at this level for as long as the impalement was maintained (>1 h), as described in the preceding paper (Tanaka et al. 1997
). Such a sustained depolarization hereafter is referred to as the persistent depolarization, as in the report by Rader and Lanthorn (1989)
.
A typical potential change produced by superfusion with medium deprived of oxygen and glucose (ischemia-simulating medium) is shown in Fig. 1A (top). The slope during the first minute of the persistent depolarization was 0.16 ± 0.07 mV/s (n = 65). The peak potential 1 min after generating the rapid depolarization was
5 ± 2 mV (n = 65). When testing for the effects of various solution on the peak potential of the persistent depolarization, we have chosen arbitrarily to measure the potential 1 min after generating the rapid depolarization as the peak.

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| FIG. 1.
Effects of glutamate (Glu) antagonists and an agonist on persistent depolarization. A: ischemia-simulating medium was applied between and . ···, pre-exposure level in this and subsequent figures. Slice would have been re-exposed to oxygen and glucose 20 s after . Tissue slices were without any pretreatment (top) or were pretreated with DL-2-amino-5-phosphonopentanoic acid (AP5; second trace), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; third trace), DL-amino-3-phosphonopropionic acid (AP3; fourth trace), or DL-amino-4-phosphonobutyric acid (AP4; bottom) for 10 min before deprivation of oxygen and glucose. Downward deflections are hyperpolarizing electrotonic potentials elicited by anodal current pulses (in range 0.1-0.3 nA for 200 ms every 3 s) in this and subsequent figures. Pre-exposure level was 76, 60, 73, 63, or 73 mV from top to bottom. Note that in AP5 or CNQX, membrane potential completely recovered. B: effects of Glu antagonists and an agonist on the percent of neurons exhibiting recovery. , , and , complete, partial, and no recovery, respectively. CNQX and AP5 had a dose-dependent protective action.
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Effects of glutamate receptor antagonists and metabotropic glutamate receptor agonists
If the step leading to irreversibility is the increase in cation permeability from activation of excitatory amino acid receptors by accumulation of Glu and/or aspartate in the interstitial space, Glu receptor antagonists may be expected to promote reversibility. Figure 1A illustrates typical responses induced by a period of ischemia-simulating medium in the presence of the NMDA receptor antagonist, AP5 (250 µM), non-NMDA receptor antagonist, CNQX (10 µM), metabotropic Glu receptor antagonist, AP3 (1 mM), or metabotropic Glu receptor antagonist and presynaptic AP4 receptor (autoreceptor) agonist, AP4 (1 mM). Application of AP5 or CNQX 10 min before superfusion with ischemia-simulating medium did not prevent the rapid depolarization but depressed the persistent depolarization. As a result, in the presence of these drugs the membrane potential of the neuron was repolarized partially or completely toward the pre-exposure level after reintroduction of oxygen and glucose in most cells. In contrast, AP3 did not restore the membrane potential in all neurons tested, and AP4 restored the membrane potential in only a few neurons. Figure 1B illustrates the recovery (percent) of the neurons tested in the absence or presence of CNQX, AP5, AP3, or AP4. CNQX or AP5 concentration dependently restored the membrane potential to pre-exposure levels, whereas AP3 or AP4 did not restore the membrane potential. Table 1 summarizes the characteristics, under various conditions, of the peak potential of the persistent depolarization. The peak potential was depressed significantly in the presence of each of the Glu receptor antagonists.
Figure 2A illustrates typical responses induced by a period of ischemia-simulating medium in the presence of the metabotropic Glu receptor agonist t-ACPD (20 µM) or t-ACPD (20 µM) combined with the metabotropic Glu receptor antagonist AP3 (1 mM). The membrane potential fully or partially recovered after reintroduction of oxygen and glucose in most cases where t-ACPD was contained in the superfusing medium. In addition, the protective action of t-ACPD was antagonized by AP3. Figure 2B illustrates the recovery (percent) of the neurons tested in the presence of t-ACPD (1-40 µM) or t-ACPD (5, 20 µM) combined with AP3 (1 mM). The peak potential was not affected significantly by t-ACPD (1-40 µM, Table 1).

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| FIG. 2.
Effect of a metabotropic Glu agonist on persistent depolarization. A: tissue slices were pretreated with trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD; top) or t-ACPD and AP3 (bottom) for 10 min. In most cells, both t-ACPD alone and t-ACPD and presence of AP3 induced a 5-10 mV depolarization (not illustrated) concomitant with an increase in input resistance. Appropriate hyperpolarizing DC currents were passed through recording electrodes to restore membrane potential to initial level before application of ischemia-simulating medium. Pre-exposure levels were 60 (top) and 73 mV (bottom). Note that membrane potential was restored after reintroduction of oxygen and glucose in t-ACPD containing medium, whereas the protective action of t-ACPD is antagonized by AP3. B: effect of t-ACPD on percent of neurons exhibiting recovery.
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Effects of various ionic media
Figure 3A illustrates typical changes in the membrane potential produced by oxygen and glucose deprivation in various ionic media. As outlined previously superfusion with ischemia-simulating medium produced a small hyperpolarization and/or depolarization and then a rapid, large depolarization. The membrane potential fully or partially recovered after reintroduction of oxygen and glucose in solutions containing either 43 mM Cl
, 0.25 mM Ca2+, or 10 mM K+. On the other hand, 28.6 mM Na+ or 0.36 mM K+ medium could not restore the membrane potential. Figure 3B illustrates the recovery (percent) of the neurons after superfusing with the ischemia-simulating media and reintroducing oxygen and glucose in various ionic media. The membrane potential of all neurons tested completely recovered in 0.25 mM Ca2+ medium. Some neurons showed complete recovery in 43 mM Cl
medium and 10 mM K+ medium. Very few neurons showed any recovery in 28.6 mM Na+ or 0.36 mM K+ medium. The result suggests that influx of Ca2+ and Cl
may be involved in the membrane dysfunction produced by oxygen and glucose deprivation. Table 1 summarizes the result of the peak of the persistent depolarization. The peak potential was shifted in the hyperpolarizing direction in 28.6 mM Na+ or 0.25 mM Ca2+ medium but depolarized in 10 mM K+. K+ (0.36 mM) or Cl
(43 mM) did not affect the peak potential. The result suggests that Na+ and Ca2+ are more important than K+ and Cl
in determining the peak potential of persistent depolarization.

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| FIG. 3.
Responses in hippocampal CA1 neurons produced by oxygen and glucose deprivation in various ionic media. A: tissue slices were pretreated with various ionic media (from top to bottom) for 10 min before, as well as during and after, deprivation of oxygen and glucose. During pretreatment, either low Ca2+- or high K+-containing media induced a 3- to 8-mV depolarization with a reduction of input resistance and low K+-containing medium induced a 3- to 5-mV hyperpolarization with an increase in input resistance. Thus appropriate hyperpolarizing DC currents or depolarizing DC currents were passed through recording electrodes to restore membrane potential to initial level. In low Cl -containing medium, membrane potential was shifted by 5-10 mV in depolarizing direction without a change in input resistance. After recordings, electrode was placed into a chamber and same depolarization was observed after changing to low Cl medium, indicating that this was simply a Cl junction potential. Note that membrane potential recovered after reintroduction of oxygen and glucose in low Cl or low Ca2+ medium and high K+ medium, whereas low Na+ or K+ medium had no effect. Pre-exposure level was either 60, 73, 64, 74, or 75 mV from top to bottom, respectively. B: percent recovery of membrane potential in various ionic media. Note that low Ca2+ medium gave complete recovery in all neurons.
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Effects of cation channel blockers
To elucidate the contribution of voltage-dependent cation channels to the persistent depolarization, the actions of the Na+ channel blocker TTX (0.3 µM), the Ca2+ channel blockers Co2+ (2 mM), Ni2+ (2 mM), flunarizine (50 µM), and nifedipine (50 µM), and the K+ channel blocker TEA (20 mM, using Cs acetate-filled electrodes) were examined. Figure 4A illustrates typical responses caused by a period of oxygen and glucose deprivation in the presence of these various constituents. Co2+, Ni2+, and TTX completely, whereas TEA using Cs acetate electrode partially, restored the membrane potential after reintroducing oxygen and glucose; flunarizine or nifedipine had no effect. Figure 4B illustrates recovery (percent). Nonspecific Ca2+ channel blockers produced the greatest recovery: complete recovery of all neurons in Co2+ and in most of the neurons in Ni2+. On the other hand, the voltage-dependent Ca2+ channel blockers, flunarizine (50 µM) and nifedipine (50 µM), had much smaller protective effect than Co2+ and Ni2+, with complete recovery in only a few neurons. Table 1 summarizes the results for the peak potential of the persistent depolarization. The peak potential was shifted to hyperpolarized levels in Co2+ or Ni2+ but did not alter significantly in flunarizine, nifedipine, or TTX. In contrast, the peak potential was shifted to a more depolarized level in TEA.

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| FIG. 4.
Effects of voltage-dependent channel blockers on persistent depolarization. A: tissue slices were pretreated with each channel blocker for 10 min before oxygen- and glucose-deprivation period. K acetate electrodes were used in all but second trace. Note that presence of tetrodotoxin or Co2+ blocks persistent depolarization and results in recovery of membrane potential. Pre-exposure level was either 60, 65, 71, 63, 77, or 60 mV from top to bottom, respectively. B: percent recovery of membrane potential by voltage-dependent channel blockers. Note that membrane potential of all neurons completely recovered in Co2+-containing medium and that of most neurons recovered in Ni2+-containing medium.
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Effects of intracellular Ca2+-chelation and inhibition of Ca2+-release from intracellular stores
If the step leading to irreversibility is an excessive increase in [Ca2+]i, limiting or preventing it may be expected to promote reversibility. Increased Ca2+ influx would be expected to increase Ca2+ release from intracellular stores. Figure 5 illustrates effects of the membrane-permeable Ca2+-chelator BAPTA-AM (50 µM), the intracellular Ca2+-release inhibitor procaine (0.3 mM), the intracellular ryanodine receptor agonist ryanodine (20 µM), and the intracellular Ca2+-release inhibitor TMB-8 (20 µM) (Chiou and Malagodi 1975
; Fujiwara et al. 1994
), on responses induced by a period of oxygen and glucose deprivation. All these drugs prolonged the latency of the rapid depolarization and partially or completely restored the membrane potential after reintroduction of oxygen and glucose. Average latencies for generating the rapid depolarization in the presence of these drugs is shown in Figure 6A. The latency was prolonged significantly by BAPTA-AM (50 µM), ryanodine (40 µM), TMB-8 (20 µM), and procaine (0.3 mM and 1 mM). In these drugs, procaine (0.3 mM) slightly depressed TTX-sensitive spike amplitudes, which were elicited by depolarizing current pulses (1.5-2 nA, 2 ms, 0.2 Hz), by 10-20% of the control (n = 5).

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| FIG. 5.
Effects of a Ca2+ chelator and inhibitors of Ca2+ release from intracellular stores on persistent depolarization. Tissue slices were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM; top), procaine (second), ryanodol 3-(1H-pyrrole-2-carboxylate) (ryanodine; third), or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoatehydrochloride (TMB-8; bottom) medium for 10 min before deprivation of oxygen and glucose. All of drugs enabled membrane potential to recover partially or completely after reintroduction of oxygen and glucose. Pre-exposure level was either 67, 60, 60, or 70 mV from top to bottom, respectively.
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| FIG. 6.
Effects of a Ca2+ chelator and inhibitors of Ca2+ release from intracellular stores on latency of rapid depolarization and on proportion of neurons showing recovery. A: BAPTA-AM, ryanodine, TMB-8, and procaine prolong latency of rapid depolarization. Each column shows mean ± SD B: BAPTA-AM, ryanodine, TMB-8, and procaine have dose-dependent protective actions against membrane dysfunction induced by oxygen- and glucose-free media.
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Figure 6B illustrates the recovery (percent) of neurons in the presence of BAPTA-AM, ryanodine, TMB-8, or procaine. All the agents had some protective effect against the membrane dysfunction, and the effects were dose dependent. Table 1 summarizes the results for the peak potential of the persistent depolarization. The peak potential was shifted in a hyperpolarizing direction by ryanodine but was not significantly altered by BAPTA-AM, TMB-8, or procaine.
Changes in the apparent input resistance of the neurons with complete recovery
In the majority of CA1 pyramidal neurons, pretreatment with a reduction in [Ca2+]o or addition of Co2+, AP5, or TMB-8 restored the membrane potential to the control level after reintroduction of oxygen and glucose. We, therefore, compared the apparent input resistance before deprivation of oxygen and glucose, during the persistent depolarization and after reintroduction of oxygen and glucose in the control condition (no pretreatment), with those of the pretreatment in the neurons with complete recovery. Table 2 summarizes the result. The pre-exposure input resistance was not significantly changed in each solution (P > 0.05). In the control medium, the apparent input resistance rapidly decreased after generation of the rapid depolarization; the resistance during the persistent depolarization or after reintroduction was extremely low (<3 M
) (also see Fig. 1A, top). In contrast, the corresponding values in low Ca2+ (0.25 mM), Co2+ (2 mM), AP5 (250 µM), or TMB-8 (20 µM) medium were much larger than those of the control (also see the traces Fig. 3A in low Ca2+, Fig. 4A in Co2+, and Fig. 5 in TMB-8). Moreover, the apparent input resistance during the persistent depolarization in Co2+ medium was significantly higher than those in low Ca2+, AP5, or TMB-8 medium (P < 0.01). In AP5 or TMB-8 medium, only one out of 10 or 18 neurons tested, respectively, showed partial recovery. The apparent resistance after reintroduction was 22 M
(42% of the preexposure value) at a membrane potential of
33 mV in AP5 medium and 25 M
(44% of the pre-exposure value) at
36 mV in TMB-8 medium.
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TABLE 2.
Changes in the apparent input resistance before deprivation, during persistent depolarization, and after reintroduction
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DISCUSSION |
In response to superfusion with ischemia-simulating medium, CA1 pyramidal neurons showed a rapid large depolarization after ~6 min and then a slow drift to 0 membrane voltage (a persistent depolarization). When the slice was returned to normal medium immediately after the transition from the rapid depolarization to the persistent depolarization, no recovery of the membrane potential occurred in any of the neurons examined in normal medium. In contrast, complete or partial recovery of the membrane potential occurred in a proportion of neurons (
100%) when various drugs, which limited or prevented rises in intracellular Ca2+, were present in the medium or when the ionic composition of the medium was modified. The factors that contribute toward the reversal of the persistent depolarization are discussed in the following sections.
Role of excitatory amino acids
Application of CNQX, AP5, AP3, AP4, or t-ACPD did not prevent the rapid depolarization, but the peak potential of the persistent depolarization was depressed by CNQX, AP5, AP3, and AP4. The results suggest that Glu receptor activation is involved in the persistent depolarization. In contrast to AP3 and AP4, CNQX and AP5 dose dependently enabled the membrane potential to recover, suggesting that Ca2+ influx via both
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate type II receptors and NMDA receptors contributes to the membrane dysfunction.
The present study showed that various drugs that limited or prevented rises in intracellular Ca2+ limit the extent of membrane dysfunction. Activation of metabotropic Glu receptor (mGluR) 1 and 5 stimulates inositol triphosphate (IP3) formation and intracellular Ca2+ mobilization. In fact, in acutely dissociated rat hippocampal CA1 pyramidal neurons, t-ACPD (10-300 µM) produces a transient outward current after the activation of the mGluR, which is insensitive to both AP3 and AP4, coupled to a pertussis-toxin-insensitive G protein (possibly Gq) that stimulates IP3 formation through the activation of phospholipase C (Shirasaki et al. 1994
). The present study showed that the protective action of t-ACPD was antagonized completely by AP3. It is, therefore, unlikely that mGluR5 is involved in the protective action of t-ACPD. AP4 itself had only a small protective action against the membrane dysfunction, suggesting that mGluR4, 6-8 are not involved in the neuroprotection by t-ACPD. In hippocampal cultured neurons, t-ACPD reversibly reduced high-threshold Ca2+ currents (both N type and L type) to 70% of the control, and AP3 blocks the t-ACPD-induced Ca2+ current reduction (Sahara and Westbrook 1993
), whereas AP4 cannot antagonize the inhibition of the high-threshold Ca2+ currents by t-ACPD (Swartz and Bean 1992
). In rat hippocampal CA1 and CA3 neurons of the slice preparation, however, t-ACPD shortens the duration of the Ca2+-dependent spike, but AP3 cannot antagonize the spike shortening (Yamamoto et al. 1993a
). It has been reported that AP3 and AP4 act as agonists to the reductionof intracellular adenosine 3
,5
-cyclic monophosphate(Schoepp and Johnson 1993
), which presumably is induced by activation of mGluR2-4. Poncer et al. (1995)
have demonstrated that, in hippocampal CA3 neurons, t-ACPD (<5 µM) increases the frequency of spontaneous inhibitory postsynaptic currents; this is probably due to enhancement of the excitability of inhibitory interneurons via mGluR1 or 5 activation. The activation of mGluR1
is antagonized by AP3 at a relatively low concentration. It is, therefore, likely that the neuroprotection induced by t-ACPD is probably due to the activation of mGluR1
. Our preceding study showed, however, that bicuculline (20 µM) did not alter the configurations of both the rapid depolarization and the persistent depolarization (Tanaka et al. 1997
). From the present results, combined with the preceding studies, we have considered that AP3-sensitive mGluRs that contribute to the protective action of t-ACPD and the site of the neuroprotection are still unclear. The neuroprotection by t-ACPD against the membrane dysfunction induced by oxygen and glucose deprivation is compatible with reports that activation of the mGluR by t-ACPD attenuates NMDA neurotoxicity in cultured cortical neurons (Koh et al. 1991a
,b
).
Roles of external ions
The peak potential of the persistent depolarization was decreased significantly in low Na+, low Ca2+ and Co2+- or Ni2+-containing medium, whereas the peak was not affected in low K+, low Cl
and TTX-, flunarizine-, or nifedipine-containing medium. L- and T-type Ca2+ channels do not appear to be involved in the peak level of the persistent depolarization because the high concentration (50 µM) of flunarizine or nifedipine, which completely abolishes the N-type Ca2+-dependent spike, L-type slow depolarization, and T-type transient depolarization in hippocampal CA1 neurons (Higashi et al. 1990b
), did not significantly attenuate the persistent depolarization. The concentration of nifedipine is well above the Ki for this voltage-dependent Ca2+ channel blocker (Goldfraind et al. 1986
). Reduction in extracellular Na+, by replacement with tris(hydroxymethyl)aminomethane, depressed the peak potential whereas addition of TTX had no effect, suggesting that the Na+ influx involved in the peak potential is not due to either the regenerative Na+ conductance responsible for spike generation or the TTX-sensitive, persistent Na+-Ca2+ conductance (Hotson et al. 1979
). The preceding study suggested that the rapid depolarization is presumably due to a nonselective increase in permeability to all participating ions rather than the activation of both nonselective cation channels and Ca2+-dependent Cl
channels or the activation of cation channels and passive Cl
influx (Tanaka et al. 1997
). Thus it seems likely that the persistent depolarization is probably due to a nonselective increase in permeability to all participating ions in the leaky membrane. The lack of effects of low K+ medium on the peak of the persistent depolarization would be due to the fact that [K+]o after the rapid depolarization is increased to 45 mM (Pérez-Pinzón et al. 1995
; see Tanaka et al. 1997
).
The membrane potential fully or partially recovered after reintroduction of oxygen and glucose in solutions containing low Cl
, low Ca2+, Co2+, Ni2+, high K+, TTX, TEA, flunarizine, or nifedipine, but not in low Na+ or low K+. Co2+ and Ni2+ were the most effective in promoting recovery from the persistent depolarization. Our previous study has demonstrated that [Ca2+]i during the persistent depolarization is elevated markedly (Tanaka et al. 1997
). These results strongly suggest that Ca2+ has a dominant role in causing the irreversible changes that lead to neuron death. The voltage-dependent Ca2+ channel blockers, flunarizine (50 µM) and nifedipine (50 µM), had much smaller protective effects than Co2+ and Ni2+, with complete recovery in only a few neurons. This finding suggests that the effect of Co2+ and Ni2+ is not the result of blocking L-, N-, and T-type Ca2+ channels, but rather of blocking of Ca2+ entry through P-, Q-, and R-type Ca2+ channels, nonspecific cation channels such as voltage-independent leaky Ca2+ channels and the Glu receptor-operated channels (Mayer and Westbrook 1987
). The present study showed that any manipulations that reduce Ca2+ influx (i.e., reduction in [Ca2+]o or addition of Co2+ or AP5) had neuroprotective effects against the membrane dysfunction induced by oxygen and glucose deprivation. Thus the apparent input resistance was well maintained during the persistent depolarization in these media. These results suggest that the Ca2+ entry through voltage-independent leaky Ca2+ channels and the Glu receptor-operated channels may play a role in the membrane dysfunction.
Reduction in [Na+]o did not even partially prevent the irreversible change. This result is in striking contrast to data on anoxic neuron death in dissociated hippocampal neurons (Friedman and Haddad 1993
; Rothman 1985
). Replacement of Na+ with impermeant cations also has prevented or postponed glutamate toxicity (Choi 1987
; Dubinsky and Rothman 1991
), although this is not a universally accepted idea (Mattson et al. 1989
). On the other hand, the membrane potential partially or completely recovered in the presence of TTX. In rat hippocampal CA1 neurons in tissue slices, TTX has been shown to have a protective effect on the postanoxic recovery of the population spike (Boening et al. 1989
). TTX also reduces the damage of CA1 neurons in situ caused by 20 min ischemia in a limited, but dose-dependent, manner. (Yamasaki et al. 1991
). It is unlikely that this protective effect can be attributed to TTX-induced block of Glu release from presynaptic nerve terminals, because vesicular release of Glu is inhibited when ATP levels fall after a few minutes anoxia (Kauppinen et al. 1988
; Sánchez-Prieto and González 1988
). Voltage-dependent, TTX-sensitive Ca2+ channels exist predominantly in isolated CA1 neurons of the temporal hippocampus (Akaike and Takahashi 1992
; Akaike et al. 1991
). Because this area of the hippocampus is most vulnerable to ischemic attack, activation of TTX-sensitive Ca2+ channels may be involved in the neuronal damage.
Elevation of [K+]o enabled the membrane potential to partially or completely recover and TEA medium (using Cs acetate electrodes) also partially restored the membrane potential. This result seems to be contradictory, because TEA and Cs+ block various K+ channels. It is possible that both acceleration of the Na+ pump activity in the K+-rich medium after reintroduction of oxygen and glucose and the prevention of an excessive drop in membrane resistance during the persistent depolarization by TEA and Cs+ probably result in protective actions against the membrane dysfunction, although there is no direct evidence. Similarly, the absence of recovery when [K+]o was reduced may be due to sustained depression of the Na+ pump activity in low K+ medium.
Reduction in [Cl
]o enabled the membrane potential to recover completely in ~60% of the neurons tested. Cl
would be in a passive Donnan equilibrium under conditions of hypoxia or ischemia, because a marked decrease in [Cl
]o and a resultant increase in [Cl
]i occur during the rapid negative-going DC shift in cortical neurons (Hansen 1985
). Rothman (1985)
has proposed that in low Cl
medium, the passive Cl
influx during the persistent depolarization would be much smaller than that in normal Cl
medium. This may explain the protective effect of low Cl
medium, because under such circumstances, the entry of cations (especially Ca2+) into the cell would be inhibited and therefore reduce intracellular hyperosmolarity and swelling. Alternatively, the Cl
substitute isethionate also might be expected to decrease the external Ca2+ activity (Morita et al. 1980
).
Role of intracellular Ca2+ ions
The increase in [Ca2+]i after the superfusion of ischemia-simulating medium reached a peak during the persistent depolarization (Tanaka et al. 1997
). In addition to activation of nonselective cation channels (see above), there are several possible mechanisms for the increase in [Ca2+]i, such as opening of excitatory amino acid-operated channels (Clark and Rothman 1987
; Iino et al. 1990
; Lobner and Lipton 1987
, 1993
; MacDermott et al. 1986
), inhibition of the Ca2+ pump (Ca2+-ATPase) in the neuron membrane, a net increase in Ca2+ influx via the Na+/Ca2+ exchanger (Lipton 1988
) and Ca2+-induced Ca2+ release (Yamamoto et al. 1993b
) or IP3-induced Ca2+ release from the store sites. Ebine et al. (1994)
have reported, however, that even in Ca2+-free solution containing ethylene glycol-bis(
-aminoethyl ether)-N,N,N
,N
-tetraacetic acid (0.5 mM), deprivation of oxygen and glucose causes an excessive increase in [Ca2+]i. This result suggests that the increase in [Ca2+]i is not only due to Ca2+ influx from the extracellular space but can be caused by inhibition of intracellular Ca2+ buffering systems.
BAPTA-AM, ryanodine, TMB-8, or procaine dose dependently enabled the membrane potential to recover after reintroduction of oxygen and glucose. Among these drugs, only ryanodine significantly depressed the peak potential of the persistent depolarization, suggesting that Ca2+-induced Ca2+ release from ryanodine receptor channels has an important role in both the generation and maintenance of the persistent depolarization and the sustained increase in [Ca2+]i.
From all of these results, it is concluded that the sustained increase in [Ca2+]i during the persistent depolarization has a dominant role in causing irreversible changes in hypoxic, glucose-deprived CA1 pyramidal neurons that lead to neuron death. The raised [Ca2+]i may be the result of Ca2+ entry via both a leaky membrane and Glu-activated receptor channels as well as Ca2+-induced Ca2+ release from ryanodine receptor channels.
 |
ACKNOWLEDGEMENTS |
We thank Profs. C. Polosa and E. M. McLachlan and Dr. S.M.C. Cunningham for valuable comments and suggestions on the manuscript.
This work was supported in part by a Grant-in-Aid for Scientific Research of Japan and an Ishibashi Foundation Grant.
 |
FOOTNOTES |
Address for reprint requests: S. Yamamoto, Dept. of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan.
Received 11 January 1997; accepted in final form 1 May 1997.
 |
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