The Journal of Neurophysiology Vol. 78 No. 2 August 1997, pp. 891-902
Copyright ©1997 by the American Physiological Society

Mechanisms Underlying the Rapid Depolarization Produced by Deprivation of Oxygen and Glucose in Rat Hippocampal CA1 Neurons In Vitro

E. Tanaka¹, S. Yamamoto¹, Y. Kudo², S. Mihara¹, 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

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Tanaka, E., S. Yamamoto, Y. Kudo, S. Mihara, and H. Higashi. Mechanisms underlying the rapid depolarization produced by deprivation of oxygen and glucose in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 78: 891-902, 1997. Intracellular recordings were made to investigate the mechanism, site, and ionic basis of generation of the rapid depolarization induced by superfusion with ischemia-simulating medium in hippocampal CA1 pyramidal neurons of rat tissue slices. Superfusion with ischemia-simulating medium produced a rapid depolarization after ~6 min of exposure. When oxygen and glucose were reintroduced, the membrane potential did not repolarize but depolarized further, reaching 0 mV ~5 min after reintroduction. Simultaneous recordings of changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and membrane potential recorded from 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy] - 2 - ( 2 - amino - 5 - methylphenoxy ) - ethane - N, N, N, Ntetraacetic acid pentaacetoxymethyl ester (Fura-2/AM) loaded slices revealed a rapid increase in [Ca²⁺]_i in all CA1 layers corresponding to the rapid depolarization of the soma membrane. The result suggests that the rapid depolarization is generated not only in the soma but also in the apical and basal dendrites. Application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), DL-2-amino-4-phosphonobutyric acid, and DL-2-amino-3-phosphonopropionic acid or bicuculline did not affect the amplitude and the maximal slope. Reduction in the concentration of extracellular Ca²⁺ or addition of CNQX or DL-2-amino-5-phosphonopentanoic acid delayed the onset of the rapid depolarization. The amplitude of the rapid depolarization recorded with Cs acetate electrodes in tetraethylammonium-containing medium had a linear relationship to the membrane potential between 50 and 20 mV. The reversal potential was shifted in the hyperpolarizing direction by a decrease in either [Na⁺]_o or [Ca²⁺]_o, whereas the reversal potential was shifted in the depolarizing direction by a decrease in [Cl]_o or using CsCl electrodes. An increase or decrease in [K⁺]_o did not affect the reversal potential. These results indicate that the rapid depolarization is Na⁺, Ca²⁺, and Cl dependent. The lack of effects of changes in [K⁺]_o is probably due to the accumulation of interstitial K⁺ before generating the rapid depolarization. Prolonged application of ouabain (30 µM) caused an initial small hyperpolarization, a subsequent slow depolarization, and a rapid depolarization. In summary, the present study has demonstrated that the rapid depolarization is voltage-independent and is probably due to a nonselective increase in permeability to all participating ions, which may occur only in pathological conditions. The underlying conductance change is primarily the result of inhibition of Na,K-ATPase activity in the recorded neuron.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Hippocampal CA1 pyramidal neurons are particularly vulnerable to brief episodes of ischemia or hypoxia in vivo (Brierley and Graham 1984). These neurons in vitro are, however, resistant to 20-40 min deprivation of either oxygen or glucose (Fujiwara et al. 1987; Hansen et al. 1982; Okada 1988; Reid et al. 1984; Schurr et al. 1989) but not to deprivation of both (Higashi et al. 1988; Okada 1988; Schurr et al. 1989). In response to oxygen and glucose deprivation, hippocampal CA1 neurons show a stereotyped response characterized by an initial hyperpolarization followed by a slow depolarization, which leads to a rapid, massive depolarization after ~6 min of exposure to the oxygen- and glucose-free medium. When the rapid depolarization occurs, the condition of the neuron becomes irreversible. There is no recovery, even if oxygen and glucose are reintroduced. On the contrary, the membrane depolarizes further and approaches 0 mV (the persistent depolarization) (Rader and Lanthorn 1989). Thus the neuron shows no functional recovery (Higashi 1990; Higashi et al. 1990; Kudo et al. 1989; Rader and Lanthorn 1989; also see Martin et al. 1994). These changes in the membrane potential produced in CA1 neurons by oxygen and glucose deprivation are a mirror-image of the DC potential changes produced by ischemia or asphyxia in the rat brain cortex (Hansen 1978), suggesting that this in vitro experimental system reflects the events occurring in the whole brain in situ.

There is general agreement that the initial hyperpolarization is generated by an increase in K⁺ conductance (Ben-Ari 1990; Fujimura et al. 1997; Fujiwara et al. 1987; Hansen et al. 1982; Leblond and Krnjevi 1989; Yamamoto et al. 1997a). In hippocampal CA1 neurons, the slow depolarization is probably due to the depression of electrogenic Na⁺ pump activity, and the resultant elevation of [K⁺]_o and the interstitial accumulation of glutamate (Glu) are involved in the slow depolarization (Ben-Ari 1990; Fujiwara et al. 1987; Martin et al. 1994). The mechanism underlying the rapid depolarization is, however, not clear. In in situ experiments, the rapid negative-going shift of the cortical DC potential during ischemia is accompanied by a marked decrease in [Na⁺]_o, [Ca²⁺]_o, and [Cl]_o and a rapid increase in [K⁺]_o (Hansen 1985), suggesting that the rapid depolarization may be due to a nonselective increase in permeability to all participating ions (Tanaka et al. 1994). Nevertheless, the routes for the ionic movements are not firmly established. Activation of Ca²⁺-activated nonselective cation channels (Crépel et al. 1994; Partridge and Swandulla 1988) and/or transmitter-operated channels may be involved in the rapid depolarization but their significance remains unknown. Moreover, the site and mechanism of generation of the rapid depolarization have not yet been addressed. Clinically, the changes occurring in the neuron during and after the rapid depolarization are of interest because they may represent the trigger for the irreversible change that leads to neuron death.

The present study concerns the mechanism for generation, the site, and the ionic basis of the rapid depolarization of rat hippocampal CA1 neurons in the slice preparation. The experiments consist of testing the effects of changes in extracellular ion concentrations and application of exogenous glutamate (Glu) and Glu antagonists or a -aminobutyric acid (GABA) antagonist on the rapid depolarization. Preliminary accounts of some data have been presented previously (Higashi et al. 1990; Kudo et al. 1989; Tanaka et al. 1994).

	METHODS

Abstract Introduction Methods Results Discussion References

The forebrains of adult Wistar rats (male 200-250 g) were removed quickly under ether anesthesia and placed in chilled(4-6°C) Krebs solution, which was aerated with 95% O₂-5% CO₂. The composition of the solution was (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11 glucose. The hippocampus was dissected and then sliced with a Vibratome (Oxford) at a thickness of ~400 µm. A slice was placed on a nylon net in a recording chamber (volume, 500 µl) and immobilized with a titanium grid placed on the upper surface of the section. The preparation was completely submerged in the superfusing solution. The temperature in the recording chamber was continuously monitored and maintained at 36.5 ± 0.5°C, and the solution flowed at a rate of 6-8 ml/min.

Intracellular recordings from CA1 pyramidal cells were made with glass micropipettes filled with K acetate (2 M) or KCl(2 M). CsCl (2 M)- or Cs-acetate (2 M)-filled electrodes were used to record the reversal potential of the rapid depolarization. The electrode resistance was 40-80 M. The membrane potential was usually stable 30 min after the impalement of the neuron, and then recording was started.

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 (ischemia-simulating medium). Low Na⁺ medium, low K⁺ medium, low Cl medium, and low Ca²⁺ medium were made by replacement of NaCl with tris-(hydroxymethyl)-aminomethane chloride (TrisCl), by replacement of KCl with NaCl, by replacement of NaCl with sodium isethionate, and by replacement of CaCl₂ with MgCl₂, respectively. For experiments using low Na⁺ or low Cl media, HCO3 was omitted, and N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid (HEPES, 10 mM) was used as the buffer (pH = 7.4, with Tris); solutions were bubbled with 100% O₂.

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. Thus the chamber was filled with the test solution ~30 s after switching the three-way cock.

[Ca²⁺]_i was measured by incubating the tissue slice with the fluorescent Ca²⁺ indicator 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5b e n z o f u r a n y l o x y ] - 2 - ( 2 - a m i n o - 5 - m e t h y l p h e n o x y ) - e t h a n e - N , N , N  ,N-tetraacetric acid pentaacetoxymethyl ester (Fura-2/AM). Fluoroprobe loadingwas performed by soaking the slice in a solution containing Fura-2/AM (10 µM) for 30-40 min at 36-37°C. Then the slice was placed in the recording chamber, mounted on an inverted epifluorescence microscope (Nikon TMD) equipped with a xenon lamp and band-pass filters of 340 ± 5 nm (wave length that allows activation of a Ca²⁺-dependent increase in signal) and 380 ± 5 nm (giving a Ca²⁺-dependent decrease in signal). The time course of the change in the fluorescence intensities from dendritic and somatic layers of the CA1 region was measured simultaneously by a video-camera/digital frame memory device using alternative excitation by 340 and 380 nm (Kudo and Ogura 1986; Kudo et al. 1986, 1987).

The drugs used were 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; from Tocris Neuramin); DL2-amino-4-phosphonobutyric acid (AP4), DL2-amino-3-phosphonopropionic acid (AP3), DL2-amino-5-phosphonopentanoic acid (AP5), HEPES, ouabain, bicuculline (all from Sigma Chemical); Fura-2/AM (from Dojin); tetraethylammonium chloride (TEACl), 2,4-dinitrophenol, (from Tokyo Kasei Organic Chemical); sodium L-glutamate, sodium cyanide (NaCN), (from Wako).

The response to deprivation of oxygen and glucose mainly constitutes an initial hyperpolarization, a slow depolarization, a rapid depolarization, and a persistent depolarization (Fig. 1A). As indicated in Fig. 1B, the latency of the rapid depolarization wasmeasured from the onset of superfusion to onset of the rapid depolarization, estimated by extrapolating the slope of the rapid depolarization to the slope of the slow depolarization. The onset potential of the rapid depolarization was measured at the membrane potential crossing the extrapolated slopes of the slow depolarization and the rapid depolarization. The peak potential was measured as the membrane potential deflection from the rapid depolarization to the persistent depolarization. The amplitude of the rapid depolarization was measured between the peak potential and the onset potential. The slope of the early phase of the persistent depolarization was measured as the slope between 20 s and 1 min after generating the rapid depolarization. The duration of the persistent depolarization was measured from the onset of the persistent depolarization to the time when the membrane potential became 0 mV. All quantitative results are expressed as means ± SD. The number of neurons examined is given in parentheses. The analysis of variance(ANOVA) test was used to compare data, with P < 0.05 considered significant.

View larger version (16K): [in this window] [in a new window]   FIG. 1. Responses produced by oxygen and glucose deprivation in hippocampal CA1 neurons. In this and subsequent figures, ischemia-simulating medium was applied between  and  and, in each trace the dotted line indicates pre-exposure level of membrane potential, unless specified otherwise. A: changes in membrane potential and apparent input resistance induced by oxygen and glucose deprivation. Pre-exposure level was 70 mV. 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. B: measured parameters of rapid and persistent depolarizations. Onset of rapid depolarization, estimated by extrapolating slope of rapid depolarization (3) to slope of slow depolarization (1). Onset potential of rapid depolarization was measured at membrane potential crossing extrapolating slopes of slow depolarization (1) and rapid depolarization (3). Peak potential was measured as membrane potential deflection from rapid depolarization to persistent depolarization (2). Amplitude of rapid depolarization was measured between peak potential and onset potential (4). Slope of early phase of persistent depolarization was measured as slope between 20 s and 1 min after generating rapid depolarization (5). Duration of persistent depolarization was measured from onset of persistent depolarization to time when membrane potential became 0 mV (6). Bottom dotted line, membrane potential level before oxygen and glucose deprivation.

	RESULTS

Abstract Introduction Methods Results Discussion References

This study was based on intracellular recordings from >250 CA1 pyramidal neurons of adult rats with stable membrane potentials more negative than 60 mV. The resting membrane potential and the apparent input resistance were 71 ± 6 mV and 45 ± 16 M (n = 130), respectively.

Neuron response to perfusion with ischemia-simulating medium

As described previously, deprivation of oxygen and glucose produced a sequence of potential changes consisting of an initial hyperpolarization, a slow depolarization, a rapid depolarization, and a persistent depolarization (Higashi et al. 1990). All responses were accompanied by decreases in apparent input resistance (Fig. 1A). The peak amplitude and duration of the initial hyperpolarization was 4.8 ± 2.4 mV and 2.7 ± 1.0 min (n = 65), respectively. The peak amplitude, slope, and duration of the slow depolarization was7 ± 7 mV, 0.13 ± 0.06 mV/s, and 1.9 ± 0.9 min (n = 65), respectively. Table 1 shows the latency, amplitude, peak potential, and maximal slope of the rapid depolarization in various media. At a temperature of 36.5 ± 0.5°C, the latency, amplitude, and peak potential of the control was 5.8 ± 1.1 min, 49 ± 6 mV, and 15 ± 4 mV (n = 65), respectively. When oxygen and glucose were reintroduced to the slice immediately after generating the rapid depolarization, the neuron did not repolarize and the membrane potential finally became 0 mV after ~5 min. Such sustained depolarizations were referred to as the persistent depolarization by Rader and Lanthorn (1989). The slope of the early phase of the persistent depolarization was 0.16 ± 0.07 mV/s (n = 65), and the duration of the persistent depolarization was 5.2 ± 1.0 min (n = 65).

Changes in intracellular Ca²⁺ concentration induced by ischemia-simulating medium

Several in vivo studies have shown that the extracellular Ca²⁺ concentration ([Ca²⁺]_o) is reduced markedly (Hansen and Zeuthen 1981; Silver and Erecinska 1990) and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is increased rapidly (Silver and Erecinska 1990; Uematsu et al. 1988) during the rapid negative-going shift of the cortical DC potential after ischemic conditions.

To examine the relationship between the membrane potential change and the [Ca²⁺]_i in the stratum pyramidale, simultaneous recordings of changes in the [Ca²⁺]_i and in membrane potential were obtained in 17 Fura-2 loaded slices, as shown in Fig. 2A. The [Ca²⁺]_i began to increase slowly 1 min after starting superfusion of ischemia-simulating medium and markedly increased during generation of the rapid depolarization. The [Ca²⁺]_i reached a peak ~1 min after reintroduction of oxygen and glucose. From faster tracings of these data, we measured the difference of the onset time between the rapid increase in [Ca²⁺]_i and the rapid depolarization. At a temperature of 36.2 ± 0.3°C (n = 17), the average latencies to onset of the rapid depolarization and the rapid increase in [Ca²⁺]_i were 6.0 ± 0.8 min (n = 17) and 6.1 ± 0.6 min (n = 17), respectively; these were not significantly different. In 8 of these 17 neurons, the onset of rapid depolarization was much earlier than the onset of rapid increase in [Ca²⁺]_i. In 6 out of 17 neurons, the rapid depolarization occurred later. In the remaining three neurons, the rapid depolarization and the rapid increase in [Ca²⁺]_i occurred at the same time. A histogram of the difference in the onset time could be fitted by a Gaussian curve (² = 3.69, df = 2, and P < 0.05), and the value at the peak of the Gaussian curve was 0 s, indicating that the rapid increase in [Ca²⁺]_i is correlated with the rapid depolarization.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Changes in ratio of Fura-2/AM fluorescence intensities and simultaneous recorded membrane potentials. A: changes in ratio of Fura-2/AM fluorescence intensities at 340 and 380 nm wavelengths, recorded simultaneously from stratum pyramidale (top, area of measurement was 60 × 400 µm) and membrane potential of a CA1 pyramidal neuron (bottom) were obtained from same slice. B: simultaneous recordings of [Ca2+]i were obtained from stratum oriens (O), stratum pyramidale (P), proximal part of stratum radiatum (R-P), and distal part of stratum radiatum (R-D) of CA1 region. Note that exposure to ischemia-simulating medium caused a slow and a subsequent rapid increase in [Ca2+]i in all layers but oriens, in which onset was somewhat slower.

Figure 2B illustrates the fluorescence ratio increments from the following hippocampal CA1 layers: stratum oriens (O), stratum pyramidale (P), proximal part of the stratum radiatum (R-P), and distal part of the stratum radiatum (R-D). Elevation of the [Ca²⁺]_i occurred in all layers of the CA1 region. The rapid increase was most pronounced in the proximal and distal parts of the stratum radiatum, whereas the elevation of the [Ca²⁺]_i was relatively slow in the stratum oriens in some slices. The onset of the rapid increase in [Ca²⁺]_i in distal parts of the stratum radiatum was 6 ± 3 s (n = 5) earlier than that in the stratum pyramidale. When oxygen and glucose were reintroduced, the increased [Ca²⁺]_i began to reduce gradually but remained at much higher concentrations than the control 15 min after the reintroduction. These results suggest that the rapid depolarization occurs in all layers of the CA1 region.

Effects of glutamate and glutamate antagonists on the rapid depolarization

It generally is thought that neuronal death caused by a reduction in oxygen and glucose supply occurs as a result of massive increases in the extracellular Glu concentration (cf. Martin et al. 1994). Hershkowitz et al. (1993) demonstrated that in rat CA1 pyramidal cells, hypoxia markedly increased spontaneous vesicular release of Glu before generating a rapid depolarization. In response to oxygen and glucose deprivation, 40 out of 65 neurons tested showed an increase in a frequency of spontaneous excitatory postsynaptic potentials (EPSPs) during the slow depolarization at the membrane potential between 70 and 60 mV (Fig. 3A). In the majority of neurons, spontaneous EPSPs were not observed in the presence of CNQX (20 µM, 6 out of 8 neurons) or AP5 (250 µM, 7 out of 11 neurons). Moreover, application of exogenous Glu (3 mM for 20 s) ~4 min after starting oxygen and glucose deprivation triggered a rapid depolarization in all six neurons (Fig. 3B). The latency of the rapid depolarization triggered by the Glu application was 4.7 ± 0.4 min (n = 6) and was significantly shorter than the control (P < 0.05), indicating that accumulation of extracellular Glu could accelerate the generation of the rapid depolarization.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Spontaneous excitatory postsynaptic potentials (EPSPs) during slow depolarization and rapid depolarization triggered by exogenous glutamate (Glu) application. A: superfusion with ischemia-simulating medium produces spontaneous EPSPs during slow depolarization. B: exogenous Glu (3 mM for 20 s) was applied before (a) and 4 min after (b) starting superfusion with ischemia-simulating medium. Note that latter application triggers action potentials followed by a rapid depolarization. Pre-exposure level was 74 mV in A and 67 mV in B.

Next, we examined the effects of CNQX (10-20 µM) and AP5 (50-250 µM) on the potential changes during oxygen and glucose deprivation (Fig. 4A). CNQX (20 µM) and AP5 (250 µM) completely abolished depolarizations induced by application of exogenous Glu (3 mM). Neither CNQX (n = 18) nor AP5 (n = 29) significantly affected the initial hyperpolarization (control: 4.8 ± 2.4 mV; CNQX: 5.9 ± 3.0 mV; AP5: 4.2 ± 3.2 mV). Both CNQX and AP5 significantly depressed the slow depolarization; the control amplitude of 7 ± 7 mV (n = 65) was much greater than the amplitudes of 4 ± 5 mV (n = 18, P < 0.05) in the presence of CNQX and of 4 ± 6 mV(n = 29, P < 0.05) in the presence of AP5. As a result, the onset of the rapid depolarization occurred from a more hyperpolarized potential; the onset voltage in the presence of CNQX was 67 ± 5 mV (n = 18), and that in the presence of AP5 was 72 ± 5 mV (n = 29; P < 0.01).

View larger version (40K): [in this window] [in a new window]   FIG. 4. Effects of Glu antagonists on rapid depolarization. A: tissue slices were without any pretreatment (top) or were pretreated with medium containing 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; middle) or DL-2-amino-5-phosphonopentanoic acid (AP5; bottom) for 10 min before oxygen and glucose deprivation. Note that CNQX and AP5 each prolonged onset of rapid depolarization, and membrane potential recovered after reintroduction of oxygen and glucose. B: effects of Glu antagonists on latency of rapid depolarization. Each column shows mean ± SD. Pre-exposure levels were 61, 61, or 65 mV from top to bottom in A, respectively.

Pretreatment of the slice preparation with CNQX or AP5 concentration dependently prolonged the latency of the rapid depolarization (Fig. 4B), and the membrane potential completely recovered after reintroduction of oxygen and glucose (Fig. 4A). Table 1 summarizes the effects on the latency, amplitude, peak potential, and maximal slope of the rapid depolarization. The amplitude and maximal slope were not significantly affected by CNQX (10, 20 µM) or AP5 (50, 100 µM). AP5 at high concentrations (100, 250 µM) prolonged the latency and reduced the peak potential and the maximal slope. AP3 (1 mM) or AP4 (1 mM) did not affect all the parameters of the rapid depolarization. Moreover, simultaneous application of CNQX (20 µM), AP5 (250 µM), and AP3 (1 mM) prolonged the latency to 7.7 ± 1.9 min (n = 8, P < 0.05), but did not affect the amplitude (51.9 ± 5.7 mV), the peak potential (15 ± 3 mV), and maximal slope (8.7 ± 4.9 mV/s).

Effects of a GABA_A receptor antagonist on the rapid depolarization

Globus et al. (1991) have demonstrated that in rat hippocampal region, ischemia markedly increases the extracellular concentration of GABA (also see Andiné et al.1991). Elevated GABA concentration in the interstitial space may alter the rapid depolarization. We, therefore, examined the effect of a GABA_A receptor antagonist, bicuculline (20 µM), on the rapid depolarization recorded by K acetate-filled electrodes. Bicuculline (20 µM) shortened the latency of the rapid depolarization but did not alter the configurations of both the rapid depolarization (Table 1) and the persistent depolarization (n = 8). Six out of 8 neurons showed an increase in the frequency of spontaneous EPSPs in the presence of bicuculline. Similarly, the latency, amplitude, peak potential, and maximal slope of the rapid depolarization recorded by KCl-filledelectrodes were not significantly different in the presence (n = 7) or absence (n = 7) of bicuculline (20 µM).In the absence of bicuculline, only the peak potential(15 ± 4 mV, n = 65) recorded by K acetate-filled electrodes was more negative than that (11 ± 4 mV, n = 7) recorded by KCl-filled electrodes(P < 0.05).

Effects of various ionic media on the rapid depolarization

To further elucidate the mechanism underlying the rapid depolarization, we examined the effects of changes in extracellular ionic concentrations on the rapid depolarization. Figure 5A illustrates typical changes in the membrane potential produced by oxygen and glucose deprivation in various ionic media. After 10 min of pretreatment of the slice with each medium, superfusion of ischemia-simulating medium produced a transient hyperpolarization, a slow depolarization, and then a rapid depolarization as in the control solution, although the membrane potential completely or partially recovered after reintroduction of oxygen and glucose after reduction in [Cl]_o (from 128 to 43 mM) or [Ca²⁺]_o (from 2.5 to 0.25 mM) and elevation of [K⁺]_o (from 3.6 to 10 mM).

View larger version (37K): [in this window] [in a new window]   FIG. 5. Effects of various ionic media on rapid depolarization. A: tissue slices were without any pretreatment (top) or were pretreated with various ionic media (from second to bottom) for 10 min before oxygen and glucose deprivation. Note that reduction in [Na+]o or [Ca2+]o prolongs latency of rapid depolarization and decreases its slope. B: effects of various ionic media on latency of rapid depolarization. Each column shows mean ± SD. Pre-exposure potential levels were 68, 73, 72, 70, 75, or 68 mV from top to bottom in A, respectively.

Table 1 summarizes the results of the latency, amplitude, peak potential, and maximal slope of the rapid depolarizationin various ionic media. The latency of the rapid depolarization was prolonged in 0.25 mM Ca²⁺ medium, whereas the latency in 10 mM K⁺ medium was shortened (also see Fig. 5B). The amplitude and maximal slope were decreased significantly in 28.6 mM Na⁺, 0.25 mM Ca²⁺, or 10 mM K⁺ medium. The peak potential was shifted in the negative direction by 28.6 mM Na⁺ or 0.25 mM Ca²⁺ medium, and shifted in the positive direction by 10 mM K⁺ medium. Because 10 mM K⁺ medium itself produced a depolarization of 10-15 mV, the membrane potential was restored to the control level by passing hyperpolarizing DC current injection through the electrode before superfusion of ischemia-simulating medium. The decrease in amplitude observed in the 10 mM K⁺ medium was due to elevation of the onset voltage of the rapid depolarization. In 0.36 mM K⁺ medium, the amplitude, peak potential, and maximal slope were not affected. In 43 mM Cl medium, the amplitude was increased significantly without affecting the peak potential and maximal slope. To confirm the lack of effects of activation of GABA_A receptors on the rapid depolarization, we examined the effect of low Cl medium in the presence of bicuculline (20 µM). The latency, amplitude, peak potential, and maximal slope were 6.1 ± 0.9 min, 56 ± 2 mV, 13 ± 2 mV, and 10.5 ± 4.8 mV/s (n = 10), respectively. Similar observations in the absence of bicuculline, only the amplitude was significantly increased (P < 0.01) compared with controls. In addition, there was no significant difference in all the parameters in the low Cl medium, irrespective of the absence or presence of bicuculline.

To examine more accurately the ion dependency of the rapid depolarization, we tried to obtain the reversal potential for the rapid depolarization. The membrane potential was, however, difficult to depolarize effectively more positive than 40 mV by passing depolarizing DC currents through K acetate electrodes, because there is an intrinsic outward rectification at membrane potentials less negative than 60 mV in CA1 pyramidal cells (Higashi et al. 1990). Thus the rapid depolarization was recorded at different levels of the membrane potential between 45 and 80 mV. In this membrane potential range, the amplitude of the rapid depolarization almost linearly was related to the membrane potential, and the reversal potential could be estimated using an extrapolation method. The estimated reversal potential of the rapid depolarization was 13 mV (n = 10, not shown).

To obtain the relationship between the amplitude and polarity of the rapid depolarization to the membrane potentials more positive than 45 mV, we used Cs acetate (2 M)- or CsCl (2 M)-filled electrodes and replaced NaCl (20 mM) with TEACl (20 mM) in the medium. This procedure reduced the outward rectification and improved space clamp, as published previously (Colino and Halliwell 1993; Crépel et al. 1994). Individual amplitudes of the rapid depolarization at different membrane potentials were obtained in various ionic media. Figure 6A illustrates potential changes induced by oxygen and glucose deprivation at five different membrane potentials in the control TEA medium, using Cs acetate-filled electrodes. The amplitude of the rapid depolarization was reduced as the membrane potential was held at a more depolarized level, and was reversed at positive potentials. Figure 6B shows the plot of amplitudes versus membrane potential in all 26 neurons tested. The reversal potential estimated from the regression line was 13.8 mV.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Effects of membrane potential on rapid depolarization. A: responses were obtained in presence of tetraethylammonium (TEA; 20 mM) at different membrane potentials by using Cs acetate electrodes. Pre-exposure level is indicated at far left of each trace. In each trace the dotted line indicates membrane potential of pre-exposure level. B: from experiments such as those shown in A, reversal potential of rapid depolarization was estimated by plot of amplitudes vs. membrane potential in 26 individual neurons. Line of best fit was obtained using least-squares method.

Figure 7A illustrates typical membrane potential recordings at ~0 mV (indicated as dashed line) in 14.3 mM Na⁺, 0.36 mM K⁺, 43 mM Cl, and 0.25 mM Ca²⁺ medium, which contained TEA (20 mM). Figure 7B illustrates the reversal potentials estimated from regression lines in these media. The reversal potential was 26.6 mV in 14.3 mM Na⁺ medium, 12.9 mV in 0.36 mM K⁺ medium, 7.4 mV in 43 mM Cl medium, and 17.9 mV in 0.25 mM Ca²⁺ medium. The difference between regression lines in control and each different ionic medium was tested by analysis of covariance. The reversal potential was significantly shifted in the depolarizing direction in 43 mM Cl medium (P < 0.01), whereas the reversal potential was significantly shifted in the hyperpolarizing direction in 14.3 mM Na⁺ medium (P < 0.01) or in 0.25 mM Ca²⁺ medium (P < 0.05). In addition, the reversal potential obtained by CsCl-filled electrodes in the control medium was 7.0 mV, which was depolarized significantly relative to that recorded with Cs acetate electrodes (P < 0.05).

View larger version (25K): [in this window] [in a new window]   FIG. 7. Reversal potentials for rapid depolarization in various ionic media. Responses were obtained in presence of TEA (20 mM) at different membrane potentials by using Cs acetate electrodes. A: tissue slices were pretreated with various ionic media (from top to bottom) for 10 min before oxygen and glucosedeprivation. Each trace shows a typical record in 14.3 mM Na+-,0.36 mM K+-, 43 mM Cl-, and 0.25 mM Ca2+-containing media, respectively. In each trace the dotted line is the membrane potential of 0 mV. B: summary of experiments such as those shown in A. Reversal potential of rapid depolarization was estimated by plots of amplitudes vs. membrane potential in each solution. Lines of best fit were obtained using least-squares method.

Metabolic inhibitors and a Na,K-ATPase inhibitor mimic the responses produced by ischemia-simulating medium

It is well known that the intracellular ATP concentration ([ATP]_i) is reduced markedly during ischemia (Siesjö 1981). To examine whether reduction of both [ATP]_i and Na,K-ATPase activity causes a similar response to superfusion with ischemia-simulating medium, the effects of metabolic inhibitors and ouabain on the membrane potential were studied in the normoxic condition.

Figure 8A illustrates a typical response to a cytochrome c oxidase complex inhibitor, cyanide. In response to cyanide (0.8-1 mM), 7 out of 10 neurons showed a transient depolarization after 0.3 ± 0.05 min (n = 7) of superfusion, a subsequent hyperpolarization after 1.9 ± 2.8 min (n = 7), followed by a slow depolarization after 8.2 ± 3.8 min (n = 7). After 20 min, the slow depolarization reached a plateau level, which was 23.2 ± 16.4 mV (n = 7) more positive than the control resting potential. The remaining three neurons showed an initial depolarization, which was followed by a subsequent slow depolarization without any hyperpolarization. When superfusion with normal medium was restored, the membrane potential fully recovered in all the neurons tested. Similarly, an uncoupler of oxidative phosphorylation, dinitrophenol (500 µM) caused an initial depolarization after 0.5 ± 0.03 min (n = 8) of superfusion, a transient hyperpolarization after 1.1 ± 0.3 min (n = 8), and a subsequent slow depolarization after 4.4 ± 0.8 min (n = 8) in all tested neurons (Fig. 8B). The amplitude of the slow depolarization was 62 ± 5 mV (n = 8) after 20 min. The membrane potential partially recovered after washing out dinitrophenol. In contrast to these agents, a Na,K-ATPase inhibitor, ouabain produced a rapid depolarization (Fig. 8C). In response to ouabain (30 µM), six out of nine neurons showed a transient hyperpolarization after 1.4 ± 1.3 min (n = 6) of superfusion and a subsequent slow depolarization after3.2 ± 3.6 min (n = 6) that was followed by a rapid depolarization after 8.6 ± 1.7 min (n = 6). The remaining three neurons showed a simple slow depolarization and a subsequent rapid depolarization without any prehyperpolarization. When superfusion with normal medium was restored, the membrane potential did not repolarize and became 0 mV ~3 min after washing out. The latency, onset voltage, amplitude, and maximal slope of the rapid depolarization were 8.6 ± 1.7 min, 67 ± 6.2 mV, 50 ± 7.8 mV, and 15.5 ± 5 mV/s (n = 9), respectively. The latency was significantly longer, and the slope was significantly faster than in oxygen and glucose deprivation. In addition, the amplitude of the hyperpolarization produced by ouabain was significantly smaller than those of the hyperpolarizations elicited by cyanide and dinitrophenol; the ouabain-induced, cyanide-induced, and dinitrophenol-induced hyperpolarizations being 2.8 ± 2.0 mV (n = 6), 6.5 ± 4.1 mV (n = 7) and 5.5 ± 1.5 mV (n = 8), respectively.

View larger version (15K): [in this window] [in a new window]   FIG. 8. Responses induced by metabolic inhibitors and a Na,K-ATPase inhibitor in normoxic medium. Drugs were applied between  and  in normoxic, glucose-containing medium. Note that NaCN and dinitrophenol caused a transient depolarization, a large hyperpolarization followed by a slow depolarization, whereas ouabain caused a small hyperpolarization, a subsequent very slow depolarization and a rapid depolarization. Pre-exposure levels were 70, 78, and 78 mV in A-C, respectively.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The site of the generation of the rapid depolarization, the mechanism for the generation, and the ionic basis will be discussed in the following sections.

Site for the generation of rapid depolarization

The rapid increase in [Ca²⁺]_i in stratum pyramidale was correlated with the rapid depolarization. The rapid increase in [Ca²⁺]_i occurred in all layers of the hippocampal CA1 region, but the onset of the rapid increase in [Ca²⁺]_i in the distal parts of the stratum radiatum was earlier than that in the stratum pyramidale. These results suggest that the rapid depolarization is generated not only at the soma membrane but also at the membrane of apical and basal dendrites.

Mechanisms underlying the generation of slow and rapid depolarizations

In general, cyanide, dinitrophenol, and ouabain are thought to be an inhibitor of cytochrome oxidase, an uncoupler of oxidative phosphorylation and an electrogenic Na, K-ATPase blocker, respectively (Hoffman and Bigger 1990; Klaassen 1990). Our previous study has shown that in hippocampal CA1 neurons in tissue slices, the hypoxia-induced slow depolarization is mimicked by superfusion of normoxicouabain (1 µM)-containing medium or K⁺-rich medium (Fujiwara et al. 1987), suggesting that depression of the electrogenic Na,K-ATPase activity and the resultant elevation of [K⁺]_o are involved in the slow depolarization. The present results that both CNQX and AP5 depressed the amplitude of the slow depolarization and the occurrence of spontaneous EPSPs suggest that activation of non-N-methyl-D-aspartate (NMDA) and NMDA receptors by Glu released into the interstitial space is involved, at least partially, in the slow depolarization. Furthermore, addition of cyanide (0.8-1 mM) or dinitrophenol (500 µM) in normoxic medium produced an initial hyperpolarization followed by a slow depolarization. The results suggest that the reduction in intracellular ATP caused by cyanide or dinitrophenol mimics the initial hyperpolarization and the slow depolarization. In contrast, ouabain (30 µM) produced a transient small hyperpolarization and a subsequent slow depolarization, which was followed by a rapid depolarization after ~8 min. Thus the rapid depolarization may be primarily the result of inhibition of Na,K-ATPase activities in the recording neuron (also see Balestrino 1995 on the anoxic depolarization).

Oxygen and glucose deprivation increased the frequency of spontaneous EPSPs during the slow depolarization. In addition, application of exogenous Glu triggered the rapid depolarization. It is, therefore, possible that the elevation of [K⁺]_o caused by inactivation of Na,K-ATPase and the resultant Glu release from excitatory synaptic terminals would trigger the generation of the rapid depolarization. It has been suggested that the Glu accumulation in the interstitial space results from the reverse operation of the Glu transporter when the transmembrane Na⁺, K⁺ and voltage-gradients are greatly reduced by ischemia or anoxia (Katayama et al. 1991; Madl and Burgesser 1993; Nicholls and Attwell1990; Penning et al. 1993). The present study, however, showed that the membrane potential was depolarized by only 7 mV before the onset of the rapid depolarization. Assuming that the membrane potential depends on [K⁺]_o, [K⁺]_o would be 5 mM according to the Nernst equation. In fact, in the hippocampal tissue slice, severe hypoxia has been shown to increase [K⁺]_o to 5-10 mM during the slow negative-going shift of the DC potential; this corresponds to the slow depolarization (Lehmenkühler et al. 1988; Lipinski and Bingmann 1986). From the expression for the carrier reversal potential (Barbour et al. 1988; also see Nicholls and Attwell 1990), extracellular Glu concentration ([Glu]_o) is estimated to increase from 0.3 µM in control to 0.6 µM before generating the rapid depolarization. This elevation of [Glu]_o is much smaller than that of the [Glu]_o immediately after the rapid depolarization, the latter being 75-250 µM. AP5 (100 and 250 µM) dose-dependently depressed the peak potential. The maximal slope was reduced significantly in the presence of 250 µM AP5. Thus it is likely that activation of NMDA receptor channels is involved in the generation of the rapid depolarization. Nevertheless, application of CNQX (20 µM), AP5 (250 µM), and AP3 (1 mM) did not affect the amplitude, peak potential, and the maximal slope, whereas CNQX (20 µM) and AP5 (250 µM) completely abolished the exogenous Glu (3 mM)-induced depolarization. It, therefore, is concluded that the Glu accumulation accelerates the generation of the rapid depolarization but that the involvement of the activation of NMDA receptor channels in the ionic bases may be small.

The present study showed that both CNQX and AP5 depressed the slow depolarization and prolonged the onset of the rapid depolarization. Similarly, slow depolarizations induced by hypoxia are depressed by kynurenate, dizocilpine malate (MK-801), or AP5 (Ben-Ari 1990; Grigg and Anderson 1990). It is, therefore, possible that the slow rise in [Ca²⁺]_i during deprivation of oxygen and glucose is due to Ca²⁺ influx via both non-NMDA receptor and NMDA receptor. Ebine et al. (1994), however, have reported that even in Ca²⁺ free combined with ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (0.5 mM) medium, oxygen and glucose deprivation causes an excessive increase in [Ca²⁺]i, suggesting that intracellular Ca²⁺-regulating systems, rather than Ca²⁺ influx into the cell, are important in increasing [Ca²⁺]_i. The latency for generating the rapid depolarization also was delayed significantly bythe intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetraacetoxymethyl ester (BAPTA/AM), the intracellular Ca²⁺ depleter ryanodine and Ca²⁺-induced Ca²⁺ release blockers, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and procaine, as described in the accompanying report (Yamamoto et al. 1997b). Moreover, L-type and possibly low threshold transient Ca²⁺ current are suppressed by anoxia (Krnjevi and Leblond 1989). Thus an excessive [Ca²⁺]_i increase caused by both the Ca²⁺ influx via Glu receptors, and the Ca²⁺ release from store sites might be associated with the rapid depolarization.

A GABA_A receptor antagonist, bicuculline (20 µM), which completely blocks fast inhibitory postsynaptic potentials in hippocampal CA1 neurons, did not significantly alter the peak potential, amplitude, and maximal slope of the rapid depolarization but shortened the latency of the rapid depolarization. These results suggest that the activation of GABA_A receptors are not directly involved in the rapid depolarization but may attenuate the action of Glu, which triggers the onset of the rapid depolarization.

Ionic basis of the rapid depolarization

The rapid depolarization obtained with Cs acetate-filled electrodes in the control TEA medium was nullified at a membrane potential of 14 mV. At membrane potentials positive to this value, the response was a rapid hyperpolarization with a latency comparable with that of the rapid depolarization at the resting potential. In addition, the amplitude of the response had a linear relationship to the membrane potential between 55 and 20 mV, as shown in Fig. 6B. These results indicate that the conductances underlying the rapid depolarization can be activated whether the membrane was depolarized or hyperpolarized; i.e., its activation is voltage-independent. Reduction in [Na⁺]_o or [Ca²⁺]_o shifted the reversal potential and peak potential of the rapid depolarization in the hyperpolarizing direction and decreased the maximal slope and amplitude. Reduction in [Cl]_o shifted the reversal potential in the depolarizing direction and increased its amplitude. In addition, intracellular Cl injection from KCl-filled electrodes or CsCl-filled electrodes also shifted the peak potential and the reversal potential in the depolarizing direction. These results suggest that the rapid depolarization is Na⁺, Ca²⁺, and Cl dependent. Elevation of [K⁺]_o decreased the amplitude and maximal slope of the rapid depolarization, whereas reduction in [K⁺]_o did not significantly affect all the parameters. The peak amplitude was shifted significantly in a positive direction by 10 mM K⁺ medium. As expected in the presence of TEA extracellularly and Cs⁺ intracellularly, the reversal potential was unaffected by 0.36 mM K⁺ medium, because TEA and Cs⁺ block voltage-dependent K⁺ channels, such as the delayed rectifier, Ca²⁺-activated, and inward rectifier K⁺ channels. In hippocampal CA1 pyramidal layer, oxygen and glucose deprivation induces an elevation of [K⁺]_o that is elevated from 5 to 16 mM before the rapid depolarization and to 45 mM at the rapid depolarization (Pérez-Pinzón et al. 1995). The increase in [K⁺]_o produced by hypoxia in the inner layer of hippocampal tissue slices is much higher than that in the surface layer (Lipinski and Bingmann 1986). Thus the lack of effects of 0.36 mM K⁺ medium on the amplitude and maximal slope, respectively, of the rapid depolarization in the present study is probably due to the fact that the [K⁺]_o in the immediate extracellular environment of the recorded neuron is influenced primarily by the increase in K⁺ efflux consequent on depression of the electrogenic Na,K-ATPase activity and on the initial hyperpolarization.

Rothman (1985) has suggested that the neurotoxicity of Glu is due to a resultant influx of Cl after the Glu-induced depolarization. In mouse dorsal root ganglion, a convulsant barbiturate, 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid may inhibit mitochondrial respiration, which results in activation of nonselective cation channels (Pearce and Duchen 1995; also see Partridge and Swandulla 1988). Crépel et al. (1994) have suggested that in hippocampal CA1 neurons, a metabotropic Glu receptor agonist [trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD)] probably increases Ca²⁺-activated nonspecific cationic currents in addition to reducing several K⁺ currents. The rapid depolarization, however, was unaffected by either metabotropic Glu receptor antagonists, AP3 and AP4 (this study) or t-ACPD [the following paper (Yamamoto et al. 1997b)]. Nevertheless, it is still possible that the rapid depolarization may be due to either activation of Ca²⁺-activated nonselective cation channels and Ca²⁺-activated Cl channels or activation of the nonselective cation channels and passive Cl influx during the rapid depolarization. Alternatively, it is likely that the rapid depolarization may be due to a nonselective increase in permeability to all participating ions in pathological conditions. Balestrino (1995) has suggested that in hippocampal CA1 neurons, the rapid negative-going DC shift corresponding to the rapid depolarization is due to Na⁺ and Cl influx into the cell, which probably is triggered by cell swelling. Fujiwara et al. (1992) have demonstrated that, in hippocampal CA1 neurons, the pH-sensitive fluorescent dye, 2,7-bis(carboxyethyl)-carboxyfluorescein, is leaked rapidly by a 5- to 7-min application of ischemia-simulating medium, and the rapid depolarization presumably occurs at this period. These results support indirectly the latter possibility.

Numerous studies have shown, in in vivo experiments, that hypoxia or ischemia causes a small initial rise in [K⁺]_o followed, 2-4 min later, by a massive increase in [K⁺]_o and a concomitant decrease in [Na⁺]_o, [Cl]_o, and [Ca²⁺]_o (Astrup et al. 1977; Hansen and Zeuthen 1981; Harris et al. 1981; Siemkowicz and Hansen 1981). Moreover, Silver and Erecinska (1990) have demonstrated that, in rat CA1 hippocampal neurons in vivo, interruption of blood flow causes an initial small hyperpolarization and small increases in [K⁺]_o and [Ca²⁺]_i, and 2-4 min later there is a sudden, large rise in [K⁺]_o, a fall in [Ca²⁺]_o, and a rapid elevation of [Ca²⁺]_i, which are consistent with the rapid depolarization.

It should be noted that one consideration that may constrain extrapolation of results obtained in the slice superfused with ischemia-simulating medium to the situation of ischemic nervous tissue in situ is that the extracellular ion concentrations probably are maintained closer to normal in the slice, so that the role of Na⁺, Ca²⁺, and Cl ions in the rapid depolarization may be overestimated. Similarly, the sudden increase in [K⁺]_o that occurs in ischemic brain tissue is likely to be attenuated in the slice by its removal in the superfusion medium; the role of this ion would, therefore, be underestimated. In addition, it is possible that in the superfused slice release of excitatory amino acids may occur, but their concentration may not be as high as in ischemic tissue in vivo, and hence the role of excitatory amino acids may be underestimated.

In summary, the present results demonstrate the rapid depolarization is voltage-independent and suggest that the depolarization is probably due to a nonselective increase in permeability to all participating ions; this may occur only in pathological conditions. The underlying conductance change is primarily the result of inhibition of Na,K-ATPase activity in the recorded neuron.

	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: E. Tanaka, Dept. of Physiology, Kurume University School of Medicine, 67 Asani-machi, Kurume 830, Japan.

  Received 11 January 1997; accepted in final form 1 May 1997.

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