Na+ Dependence and the Role of Glutamate Receptors and Na+ Channels in Ion Fluxes During Hypoxia of Rat Hippocampal Slices

Michael Müller and George G. Somjen

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Müller, Michael and George G. Somjen. Na+ Dependence and the Role of Glutamate Receptors and Na+ Channels in Ion Fluxes During Hypoxia of Rat Hippocampal Slices. J. Neurophysiol. 84: 1869-1880, 2000. Spreading depression (SD) as well as hypoxia-induced SD-like depolarization in forebrain gray matter are characterized by near complete depolarization of neurons. The biophysical mechanism of the depolarization is not known. Earlier we reported that simultaneous pharmacological blockade of all known major Na+ and Ca2+ channels prevents hypoxic SD. We now recorded extracellular voltage, Na+, and K+ concentrations and the intracellular potential of individual CA1 pyramidal neurons during hypoxia of rat hippocampal tissue slices after substituting Na+ in the bath by an impermeant cation, or in the presence of channel blocking drugs applied individually and in combination. Reducing extracellular Na+ concentration [Na+]o to 90 mM postponed the hypoxia-induced extracellular DC-potential deflection (Delta Vo) and reduced its amplitude, and it also postponed the SD-like depolarization of neurons. After lowering [Na+]o to 25 mM, SD-like Delta Vo became very small, indicating that an influx of Na+ is required for SD; influx of Ca2+ ions alone is not sufficient. We then asked whether the SD-related Na+ current flows through glutamate-controlled and/or through voltage-gated Na+ channels. Administration of either the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the NMDA receptor antagonist (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) postponed the hypoxic Delta Vo and depressed its amplitude but the effect of the combined administration of these two drugs was not greater than that of either alone. During the early phase of hypoxia, before SD onset, [K+]o increased faster and reached a much higher level in the presence of glutamate antagonists than in their absence. The [K+]o level reached at the height of hypoxic SD was, however, not affected. When TTX was added to DNQX and CPP, SD was prevented in half the trials. When SD did occur, it was greatly delayed, yet eventually neurons depolarized to the same extent as in normal solution. The SD-related sudden drop in [Na+]o was depressed by only 19% in the presence of the three drugs, indicating that Na+ can flow into cells through pathways other than ionotropic glutamate receptors and TTX-sensitive Na+ channels. We conclude that, when they are functional, glutamate-receptor-mediated and voltage-gated Na+ currents are the major generators of the self-regenerative rapid depolarization, but in their absence other pathways can sometimes take their place. The final level of SD-like depolarization is determined by positive feedback and not by the number of channels available. A schematic flow chart of the events generating hypoxic SD is discussed.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In forebrain gray matter, spreading depression (SD) is associated with nearly complete depolarization of neurons and glial cells as well as a massive disturbance of extracellular ion concentrations. Ischemia and severe hypoxia trigger a similar depolarization (Bures et al. 1974; Leão 1947; Marshall 1959; Müller and Somjen 2000; Nicholson 1984; Somjen et al. 1992). Despite their magnitude, all these changes are reversible, and up to four repetitive hypoxic SD-like episodes can be induced in hippocampal slices in vitro without necessarily reducing electrophysiological responsiveness, i.e., causing acute neuronal damage (Müller and Somjen 1998).

The first to attempt to explain the nature of SD was Grafstein (1956), who proposed that K+ released by a group of neurons can raise extracellular K+ and thus depolarize nearby cells, providing the feedback responsible for the propagation of SD. Subsequent research has amply confirmed the massive release of K+ during SD (Bures et al. 1974), but other observations suggested that K+ increase alone cannot satisfactorily explain the complicated nature of SD, although it undoubtedly does play a crucial role (Somjen et al. 1992; van Harreveld 1959). As of today, SD is understood as a cascade of various events, but the precise mechanism of its self-regenerative nature remains obscure.

Against the potassium hypothesis, van Harreveld (1959, 1978) proposed that glutamate is the agent, or one of two agents, mediating the generation and the propagation of SD. van Harrelveld (1959) demonstrated the release of glutamate during SD and also that cortical application of glutamate is capable of inducing SD. Indeed, in some models, glutamate receptors seem critically involved in neuronal loss as a consequence of excitotoxic, ischemic, or hypoxic insults (Choi 1987; Garthwaite et al. 1986; Tanaka et al. 1997; Yamamoto et al. 1997). The contribution of glutamate-mediated inward currents to the immediate electrophysiological consequences of oxygen and glucose deprivation is, however, not yet clear. Glutamate's postulated role in the generation and propagation of SD could be important, since recurrent waves of SD have been blamed for the extension of cerebral infarcts into marginal areas (Busch et al. 1996), and during hypoxia, glutamate is apparently released not only by synaptic but also extrasynaptic mechanisms (Drejer et al. 1985). Among others, its release can be induced by elevated [K+]o (Fujikawa et al. 1996; Szerb 1991) and reversal of Na+-dependent glutamate uptake as a consequence of a steadily increasing intracellular Na+ concentration ([Na+]i) (Attwell et al. 1993). Glutamate is also released from glial cells via swelling-activated anion channels (Basarsky et al. 1999; Kimelberg et al. 1990). Earlier tests of the ability of glutamate antagonists to suppress SD unveiled an important difference between normoxic SD and hypoxic SD-like depolarization. Normoxic SD can be successfully depressed by the application of N-methyl-D-aspartate (NMDA) antagonists; hypoxia-induced SD cannot. Hypoxic SD was sometimes delayed, but in some tests its onset was actually accelerated and the associated Delta Vo was also only in some cases reduced (Aitken et al. 1991; Hernández-Cáceres et al. 1987; Jing et al. 1993; Lauritzen and Hansen 1992; Marrannes et al. 1988).

Recently we examined in a computer model the conditions for the generation of SD-like depolarization (Kager et al. 2000; Somjen et al. 2000). In the model either a simulated NMDA-mediated current or a persistent voltage-gated Na+ current could generate this phenomenon. The results validate the dual hypothesis of van Harreveld (1978), who suggested that in the retina two kinds of SD can occur, one mediated by glutamate and the other by K+.

In the study presented here we first tested whether Na+ is a major component of the inward current causing SD-like depolarization. We found that hypoxic SD is indeed Na+ dependent. Then, to define more precisely the channels generating the SD-like depolarization, the effects of glutamate antagonists and TTX were tested on membrane potential and input resistance changes of single pyramidal neurons, and on extracellular voltage and Na+ and K+ concentrations during severe hypoxia. We confirmed that hypoxic SD does occur when both NMDA and non-NMDA glutamate receptors are blocked, but its onset is much delayed. Adding TTX to the glutamate antagonists halved the incidence of SD, but did not completely prevent its occurrence. We conclude that besides channels controlled by ionotropic glutamate receptors and TTX-sensitive Na+ channels, other yet to be identified pathways can mediate SD-like depolarization. At the end of the paper we present a schematic flow chart of the cascade of events generating hypoxic SD.

Some of the results are being published as an abstract (Somjen and Müller 2000).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Preparation

Hippocampal tissue slices were prepared from ether anesthetized, male Sprague-Dawley rats of 120-260 g body wt (4-7 wk old). Following decapitation, the brain was rapidly removed from the skull and placed in chilled artificial cerebrospinal fluid (ACSF) for 1-2 min. The two hemispheres were separated, one hippocampus was isolated, and transverse slices of 400-µm thickness were cut using a tissue chopper. Slices were transferred to an interface recording chamber of the Oslo style and were left undisturbed for at least 90 min. The recording chamber was kept at a temperature of 34.5-35.5°C. It was continuously aerated with 95% O2-5% CO2 (400 ml/min) and perfused with oxygenated ACSF (1.5 ml/min). Hypoxia was induced by switching the chamber's gas supply to 95% N2-5% CO2. Exchange of the bathing solution took about 5 min and diffusion of applied drugs into the slice was completed in about 30 min.

Solutions

The ACSF had the following composition (in mM): 130 NaCl, 3.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 1.2 CaCl2, 1.2 MgSO4, and 10 dextrose; aerated with 95% O2-5% CO2 to adjust pH to 7.4. In reduced Na+ solutions, Na+ was substituted by NMDG+ (N-methyl-D-glucamine, Sigma). TTX (citrate-buffered; Sigma) was prepared as 1-mM stock solution in distilled water and kept frozen. DNQX (6,7-dinitroquinoxaline-2,3-dione, Research Biochemicals International) and CPP [(±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid; RBI and Tocris] were dissolved in ACSF and kept frozen as 1 and 2 mM stock solutions, respectively.

Microelectrodes

Single-barreled glass microelectrodes for extracellular recordings were pulled from thin-walled borosilicate glass [TW150F-4, World Precision Instruments (WPI)] using a horizontal puller (P-80/PC, Flaming Brown). They were filled with ACSF and their tips were broken to a final resistance of 5-10 MOmega . Sharp microelectrodes for current-clamp recordings were made from thick-walled borosilicate glass (1B150F-4, WPI) and filled with 2 M K-Acetate + 5 mM KCl + 10 mM HEPES (Sigma); pH 7.4; their resistances were 60-80 MOmega .

Extracellular Na+ and K+ concentrations were simultaneously measured using triple-barreled Na+/K+-sensitive microelectrodes of the twisted type, as has been described in detail earlier (Müller and Somjen 2000). In brief, a single-barreled capillary (1B150F-4, WPI) was glued to the double-barreled theta-type capillary (GCT 200-10, Clark Electromedical Instruments), and this capillary assembly was then pulled on a vertical puller (Narishige PE-2). In a first step, it was pulled by only 2-4 mm simultaneously twisting the attached reference barrel by 180° around the centered theta capillary. After cooling, the capillaries were pulled apart in a second pulling step. Both barrels of the theta capillary were silanized by 60-min exposure to HMDS vapors (hexamethyldisilazane, 98%, Fluka; vaporized at 40°C) and subsequent baking in the oven (200°C, 2 h). Silanization of the attached reference barrel was prevented by filling it with distilled water.

The reference barrel contained 150 mM NaCl + 10 mM HEPES, pH 7.4, while the tip of the K+-sensitive barrel was filled with the valinomycin-based K+ ion neutral carrier (Potassium Ionophore I, Cocktail A, Fluka 60031) and backfilled with 150 mM KCl + 10 mM HEPES, pH 7.4. The tip of the Na+-sensitive barrel was filled with a Na+ cocktail based on the Na+ ionophore VI (Deitmer and Munsch 1995; Müller and Somjen 2000) and backfilled with 150 mM NaCl + 10 mM HEPES, pH 7.4. Electrode resistances of the Na+-sensitive, K+-sensitive, and reference barrel were 200, 130, and 40 MOmega , respectively. There was no noticeable interference between adjacent barrels.

Ion-sensitive electrodes were calibrated before and after each experiment by detecting their response generated in standard solutions (0, 1, 5, 10, 50, and 100 mM K+; 150, 100, 50, 10, 5, 0 mM Na+). To maintain constant ionic strength similar to that in interstitial fluid, Na+ in calibration solutions was replaced by K+ and vice versa (reciprocal calibration method). The reciprocal adjustment of ion concentrations in the calibrating solutions discounts the small co-ion interference and further correction of the data were not required. Average slopes of the K+- and Na+-sensitive barrels were 53.1 ± 1.9 mV/decade K+ and 54.2 ± 2.0 mV/decade Na+; their detection limits were 1.0 ± 0.4 mM K+ and 4.4 ± 0.5 mM Na+ (n = 9).

Electrical recordings

Ion-sensitive electrode signals were referred to an Ag/AgCl bridge electrode embedded in 2% agar in 3 M KCl. They were recorded by a DC amplifier (constructed locally) and digitized by a TL-1/Lab Master acquisition system at sampling rates of 25 Hz. Since electrodes were calibrated to Na+ and K+ concentrations and the activity coefficient of the measured ion was held constant, changes in [Na+]o and [K+]o could directly be calculated from the electrode responses using the electrodes' averaged slope of pre- and postexperiment calibration. All signal amplitudes were measured between the prehypoxia baseline and the maximal change. Only rapid negative extracellular DC potential changes (Delta Vo) of at least 10 mV amplitude were considered as SD. SD onset was defined as occurrence of the sudden Delta Vo.

Current-clamp recordings from CA1 pyramidal neurons were performed with an intracellular recording amplifier (Neuro Data, IR-283) as described earlier (Müller and Somjen 2000). CA1 pyramidal neurons were identified by their location in st. pyramidale, membrane potential, spontaneous activity, action-potential shape, and input resistance (Morin et al. 1996). Only pyramidal neurons with a stable membrane potential of at least -55 mV were accepted. Their input resistance was determined every 10 s by injecting a hyperpolarizing current of 400-pA amplitude and 200-ms duration. Data were sampled at 1 kHz using the TL-1/Lab Master acquisition system and the Axotape V2 software (Axon Instruments). Input resistances were measured at the steady-state level of the voltage deflections and changes in input resistance were expressed as a percent of pretreatment value.

Antidromically and orthodromically evoked responses were elicited by delivering 10-150 µA stimuli of 0.1-ms duration via monopolar stimulation electrodes (for details see Müller and Somjen 1998).

Statistics

The data were obtained from 30 rats, and since most experiments did not last longer than 2 h, up to four slices could be used from each brain, but, to insure independence of treatments, each group of experiments was performed on three to six different rats. In 17 of the 30 rats, two slices were used from the same brain for the same experimental treatment (low external Na+ or drug), and only one slice was used for a given experimental condition from the 13 other brains. Each slice was used for only one experimental condition. For the extracellular experiments with drugs, to reduce variability of data, two hypoxic SD episodes were induced in every slice, first a control SD, and, after at least 30 min, a second SD in the treated condition. For the low-Na+ experiments, there were four hypoxic episodes, one in control ACSF, by 90 mM and 25 mM Na+, and finally a repeat to check recovery in normal ACSF. The changes in SD parameters (amplitude, onset, ion changes) observed during low Na+ and drug treatments were normalized to the control SD recorded in a given slice; each experiment therefore had its own control. Significance of these averaged changes was tested in a one-sample t-test, comparing the mean of the observed changes to the known standard, i.e., the control conditions defined either as unity or as 100% (Bailey 1992; Sachs 1999).

Technical reasons required that, for intracellular recordings from individual neurons, control and drug effects be recorded in different slices. Significance of these unpaired observations was tested in two-tailed, unpaired Student's t-tests at a significance level of 5%. Multiple comparisons were not done, since each group of drug-treated slices was only compared with the control group of slices. None of the drug-treated groups was compared with any other group than control and none of the slices was treated with more than a single drug or drug combination. All numerical values are represented as mean ± standard deviation (StD) and the number of experiments (n) refers to the number of slices investigated. In the diagrams, significant changes are marked by asterisks (* P < 0.05; ** P < 0.01).


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Na+ dependence of hypoxic SD

To assess the effect of a reduced extracellular Na+ concentration ([Na+]o) on hypoxia-induced SD, either 65 or 130 mM of extracellular Na+ were substituted by the membrane impermeable NMDG+, thereby reducing [Na+]o to 90 and 25 mM, respectively. First, a control SD was recorded in every slice followed by two hypoxic SD episodes in low [Na+]o and one more during recovery in normal solution (Fig. 1). Under control conditions, the extracellular DC potential deflection (Delta Vo) signaling SD onset occurred within 2.1 ± 0.6 min of hypoxia and its amplitude averaged -19.8 ± 3.9 mV (n = 30; Delta Vo amplitude referred to prehypoxic baseline). Reducing extracellular Na+ to 90 and 25 mM increased the time of SD onset more than twofold and the Delta Vo amplitude decreased by 58 ± 9 and 85 ± 7%, respectively (n = 6 each; Table 1). The residual Delta Vo seen with the lowest [Na+]o would, ordinarily, by definition, be considered too small for the diagnosis of SD. The changes measured in the somatic layer [stratum (st.) pyramidale] did not noticeably differ from those in the dendritic layer (st. radiatum). Restoring normal extracellular Na+ completely reversed these depressant effects (Fig. 1).



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Fig. 1. Effects of Na+ substitution on hypoxia-induced spreading depression (SD). Reducing [Na+]o to 90 and 25 mM markedly postponed the onset of hypoxic SD and decreased the amplitude of the extracellular DC potential shift (Delta Vo). Following restoration of normal [Na+]o, the hypoxic Delta Vo regained its control amplitude and the time to onset decreased again. DC potentials were simultaneously recorded in st. radiatum and st. pyramidale of the same slice; the effects were similar at the two sites. Hatched bar, duration of hypoxia.


                              
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Table 1. Effects of reduced extracellular Na+ concentration and reduced Na+ conductance on the hypoxia-induced extracellular DC potential shift (Delta V0) in st. radiatum

As expected, synaptic transmission and axonal conductance were also depressed in low Na+ solution. In 90 mM Na+ solution, the amplitudes of orthodromically evoked focal excitatory postsynaptic potentials (fEPSPs) and antidromically evoked population spikes reversibly decreased by 48.8 ± 17.6 and 50.0 ± 7.2%, respectively (n = 6 each, measured at stimulus intensities of 100-150 µA), while they were completely suppressed in 25 mM Na+ solution (n = 6).

Sensitivity of [Na+]o and [K+]o changes to glutamate antagonists

To investigate the involvement of ionotropic glutamate receptors in the massive extracellular Na+ and K+ changes during SD, we simultaneously recorded [Na+]o and [K+]o during hypoxic SD in control solutions and induced a second hypoxic SD after 35-45 min treatment with the respective glutamate antagonists. The glutamate inhibitors DNQX and CPP were applied one by one as well as simultaneously and also in combination with TTX. In a previous study, we already ascertained that hypoxic SD can repeatedly be induced in a given slice kept in control solutions, without causing significant changes in the characteristic SD parameters for the first three SD episodes (Müller and Somjen 1998).

The extracellular Na+ and K+ concentrations showed characteristic and clearly different time courses (Fig. 2, A and C). In control solutions, during the initial phase of hypoxia, before SD onset, [K+]o already increased slightly at an average linear rate of 3.4 ± 1.5 mM/min, reaching a level of 8.4 ± 1.8 mM immediately before SD onset; [Na+]o still remained unchanged. As soon as the rapid Delta Vo occurred, [K+] rapidly increased to 77.3 ± 20.8 mM (n = 24; measured in st. radiatum). Simultaneously, [Na+]o sharply dropped to 61.8 ± 14.3 mM and then immediately started to increase again, reaching a plateau of 92.5 ± 12.6 mM (n = 23). When reoxygenation was started 100 s after SD onset, the extracellular ion concentrations recovered to their prehypoxia levels. During the recovery phase, [K+]o consistently undershot its prehypoxic baseline (Heinemann and Lux 1975; Pérez-Pinzón et al. 1995), reaching a minimum of 1.3 ± 0.4 mM (Figs. 2 and 3; see also Müller and Somjen 2000).



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Fig. 2. Changes in [K+]o and [Na+]o during hypoxia in normal solution and following glutamate receptor and Na+ channel inhibition. A and C: examples of the increase in [K+]o and the drop in [Na+]o during hypoxia in normal solution. Note the initial [K+]o increase before SD onset and the undershoot of the baseline following reoxygenation (A). By contrast, [Na+]o does not change until SD occurs and it starts to recover again to a plateau level already before reoxygenation (C). A and C were recorded simultaneously from the same slice. B and D: averaged normalized changes of extracellular ion concentrations during hypoxia under different treatments. All data shown as experimental/control; error bars represent standard deviation. The [K+]o level reached before SD onset increased greatly with each treatment, but the maximum (peak) during SD, and the post-SD undershoot were barely affected (B). The peak of the drop of [Na+]o during SD was moderately decreased by the treatments but the Na+ plateau level was not significantly affected. The drug concentrations were 6,7-dinitroquinoxaline-2,3-dione (DNQX) and (±)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) 10 µM; TTX 1 µM. For the combined application of DNQX, CPP, and TTX, only those experiments were included in which SD occurred (** P < 0.01; * P < 0.05; n = 7 for DNQX, n = 5 for CPP, n = 6 for DNQX + CPP, n = 3 for DNQX + CPP + TTX).



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Fig. 3. Hypoxic-SD associated changes in extracellular DC potential (Delta Vo) and [Na+]o and [K+]o in control solutions and following glutamate receptor inhibition. At the break in the traces DNQX and CPP (10 µM each) were administered in the bath; the break represents 30 min. After blockade of glutamate receptors, Delta Vo was smaller and was followed by an increased posthypoxic overshoot. The changes in ion concentration illustrate some of the effects of which Fig. 2, B and D, are statistical summaries.

As already reported by others, inhibition of glutamate receptors does not prevent the occurrence of hypoxia-induced SD (Aitken et al. 1991; Hernández-Cáceres et al. 1987; Jing et al. 1993; Lauritzen and Hansen 1992; Marrannes et al. 1988). Application of either the non-NMDA antagonist DNQX (10 µM) or the NMDA inhibitor CPP (10 µM) postponed the onset of hypoxic SD by 18 ± 18 and 25 ± 21% and reduced the Delta Vo amplitude by 24 ± 22 and 17 ± 9%, respectively (Table 1). In the presence of either DNQX or CPP during the initial phase of hypoxia, before the onset of SD, [K+]o increased by more than twice the normal value, but at the height of SD, the maximal level of [K+]o was about the same as in normal solution (Fig. 2B). It could be thought that the high [K+]o was reached in the initial phase because more time has elapsed before SD onset allowing more K+ to accumulate, but in fact in the presence of the glutamate antagonists [K+]o increased by a substantially higher rate than in normal solution (Table 2). By contrast, neither the K+ peak at the height of hypoxic SD nor the undershoot of the K+ baseline following reoxygenation were significantly different from control (Fig. 2B). The maximal level of the hypoxia-induced [Na+]o decrease was slightly reduced by 19 ± 14% in the presence of DNQX and by 10 ± 5% following administration of CPP, but the Na+ plateau level was not significantly affected (Fig. 2D).


                              
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Table 2. Initial (pre-SD) rate of rise in the extracellular K+ concentration in the presence of glutamate antagonists and TTX

Combined application of DNQX and CPP did not prevent hypoxic SD. Remarkably, the inhibitory effects of DNQX and CPP did not sum up (Table 1). The initial pre-SD K+ level increased to threefold, and the initial rate of increase of [K+]o rose at a 116 ± 88% higher rate. The K+ peak at the height of SD was also slightly enhanced (Figs. 2A and 3, Table 2). However, no significant changes in the hypoxic SD-associated [Na+]o drop were observed (Fig. 2D). Unlike in normal solution, in the presence of the two glutamate antagonists, a small decrease in [Na+]o could be observed in some recordings even before SD occurred (Fig. 3).

Since our previous study (Müller and Somjen 2000) suggested a role of voltage-gated Na+ channels in the triggering of SD, we simultaneously applied DNQX (10 µM), CPP (10 µM), and TTX (1 µM). Under these conditions, there was no sign of SD-like depolarization in three of six slices during severe 20-min hypoxia; in the remaining three slices, the onset of hypoxic SD was even more delayed than with the two glutamate antagonists without TTX, and the Delta Vo amplitude decreased by 38 ± 18% (Table 1). Due to the small number of observations, this amplitude reduction is, however, not significantly different from control. The initial, pre-SD [K+]o increase became more than threefold, slowly approaching a steady-state level of 16.7 ± 2.1 mM (n = 3, Fig. 4B). Once SD occurred, however, the K+ peak did not differ from control conditions (Fig. 2B). The Na+ peak was reduced by 18 ± 5%, but the Na+ plateau level was not significantly affected (Fig. 2D). In those slices which did not respond with a hypoxic SD during 20-min hypoxia, [K+]o reached a steady-state level of 16.0 ± 5.7 mM that was maintained until reoxygenation was started (Fig. 4C). Thus the [K+]o increase amounted to only 22.7 ± 10.6% of the change observed during the control SD in the same slice (n = 3). If no SD occurred, [Na+]o decreased to 125.6 ± 1.9 mM, the decrease being 32.6 ± 1.5% of control. After reoxygenation, [K+]o decreased and, following the characteristic undershoot which did not differ from control conditions, it recovered to its prehypoxic baseline.



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Fig. 4. Extracellular K+ changes associated with SD in normal solution and in the presence of DNQX, CPP, and TTX. A: hypoxic SD-associated [K+]o increase in normal solution. B: example of a slice that responded with SD during the combined application of DNQX, CPP, and TTX. C: an example in which the presence of DNQX, CPP, and TTX prevented the SD-like event during 20 min of severe hypoxia.

Effects of glutamate antagonists on hypoxia-induced membrane and input resistance changes in single pyramidal neurons

In single CA1 pyramidal neurons, hypoxia causes first a hyperpolarization, then a slow depolarization and eventually a near-complete, self-regenerative depolarization coinciding with the SD-like Delta Vo, and it dramatically decreases the input resistance (Hansen 1985; Hansen et al. 1982; Müller and Somjen 1998, 2000). To investigate the contribution of glutamate receptors to the hypoxia-induced changes in pyramidal neurons, we performed current-clamp experiments in control solution and following drug administration of at least 30 min. Since stable cell impalement could not be maintained for hours, and in most cells impalement was lost during the recovery phase from hypoxia following reoxygenation, control and drug effects had to be recorded in different cells. Each slice underwent only one hypoxic episode.

In the control group of slices bathed in normal ACSF, CA1 pyramidal neurons had an average membrane potential of -62.6 mV and an input resistance of 39.0 MOmega (Table 3). The detailed effects of hypoxia on the electrical properties of CA1 pyramidal neurons have been described in detail in our previous study (Müller and Somjen 2000). The initial hyperpolarization was associated with a 37.8 ± 14.8% decrease in input resistance, that reversed into a gradual, slow depolarization. Within 1.8 ± 0.5 min of hypoxia, the rapid SD-like depolarization occurred. It was triggered at an apparent threshold potential of -51.6 ± 4.3 mV and in most cells it was preceded by a spontaneous discharge of action potentials. The rapid depolarization brought the intracellular potential to -23.2 ± 4.9 mV and it slowly continued to shift toward its peak of -7.8 ± 5.0 mV. The input resistance decreased further, in total by 88.5 ± 11.9% (Fig. 5A).


                              
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Table 3. Resting intracellular potential (Vi) and input resistance in CA1 pyramidal neurons in normal ACSF and in low [Na+]o or after the administration of glutamate antagonists and TTX



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Fig. 5. Intracellular potential of current-clamped CA1 pyramidal neurons during hypoxic SD in control solution (A), low [Na+]o solutions (B), following glutamate receptor inhibition (C), and the combined inhibition of glutamate receptors and voltage-gated Na+ channels (D). Note the marked delay in SD onset in low [Na+]o solutions and in the presence of DNQX + CPP + TTX. Downward deflections were elicited by hyperpolarizing current pulses of 400 pA amplitude and 200-ms duration. Upward deflections in the trace from the control cell are spontaneous action potentials truncated by slow sampling. The potential levels labeled a-d in A indicate 1) intracellular potential (Vi) before hypoxia; 2) the apparent threshold from which the SD-like accelerating depolarization takes off; 3) maximum of the fast segment of the depolarization; and 4) the maximum Vi reached.

Reducing [Na+]o to 90 mM as well as application of the glutamate inhibitors and TTX resulted in somewhat more negative resting membrane potentials and a decreased resting input resistance (Table 3). In 90 mM Na+ solution, the onset of the hypoxia-induced SD-like depolarization was markedly delayed (Figs. 5B and 6C). The threshold potential at which the rapid depolarization was triggered, the amplitude of the rapid depolarization itself as well as the peak potential reached at the height of hypoxic SD were, however, not different from control conditions (Fig. 6; n = 7). DNQX acted in a similar way, postponing the onset of the SD-like depolarization but not affecting the amplitude of the rapid depolarization or its absolute peak (Fig. 6; n = 7). The NMDA-receptor antagonist CPP postponed the SD-like depolarization and shifted its apparent threshold by 15 mV to a more positive level. As a result, the amplitude of the rapid depolarization, the b-c segment of Fig. 6A, was reduced to 52% of the magnitude observed in control slices (Fig. 6, A and B). Surprisingly, the effects of CPP were less pronounced when DNQX was added to CPP (n = 9; Figs. 5C and 6).



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Fig. 6. Statistical summary of the hypoxic SD-like depolarization in pyramidal neurons. A: average (±StD) voltages at the points a-d as illustrated in the top trace of Fig. 5A. Note that voltages are not corrected for the extracellular Delta Vo; the true membrane potential (Vm) at the height of the rapid phase (c) is closer to 0. Moreover, the slowly continuing apparent depolarization (c-d) is, in part or whole, due to contamination of the Vi trace by Vo (Müller and Somjen 2000). In the presence of CPP, alone or in combination, the threshold (b) shifted to a more positive potential, thereby reducing the amplitude of the rapid depolarization (b-c segment). Asterisks indicate significant differences from control. B: mean amplitudes of the rapid depolarization (b-c segment). The depressant effect of CPP suggests that activation of NMDA receptors contributes to the rapid depolarization. C: time from the start of hypoxia until the onset of the SD-like depolarization. D: input resistance during the SD-like depolarization, as percent of the resistance measured in each cell in normal solution. Only the combined application of DNQX, CPP, and TTX resulted in a reduced depression of the input resistance.

When slices were pretreated with the triple combination of DNQX, CPP, and TTX, 20 min of hypoxia still induced a SD-like depolarization in five of eight pyramidal neurons, coincidentally with a characteristic Delta Vo. The onset of the hypoxic SD was in these cases delayed more than sixfold and the threshold potential of the SD-like depolarization shifted to more positive potentials, again, however, without significantly decreasing the final amplitude of the depolarization (Figs. 5D and 6A). The combined administration of DNQX, CPP, and TTX did diminish the reduction of the input resistance during the rapid depolarization, which was 32% smaller than control (n = 5; Fig. 6D).

Those three pyramidal neurons which did not respond with a SD-like depolarization in the presence of DNQX, CPP, and TTX showed a slow and incomplete depolarization, lacking the characteristic self-regenerative character observed in normal solution. During 20-min hypoxia, their intracellular potential slowly rose to a peak of -17.3 ± 21.6 mV, and the input resistance decreased by only 35.8 ± 10.6% (n = 3). In these cases, the typical Delta Vo was also absent.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Three main conclusions stand out. 1) Blocking NMDA receptors plus voltage-controlled channels reduces the likelihood and greatly delays the onset of hypoxic SD, but does not always prevent it. 2) Nonetheless, the depolarization associated with SD is dependent on external Na+. 3) Once the SD-like depolarization is started, it continues to completion, even if its onset has been delayed by low [Na+]o or by channel-blocking drugs.

Hypoxic SD-like depolarization is a Na+-dependent all-or-none process

The generation of hypoxic SD is clearly dependent on the presence of extracellular Na+ because substituting Na+ by an impermeant cation dose-dependently reduced the SD-related Delta Vo. With only 25 mM of [Na+]o remaining, the residual Delta Vo was so weak that it could not be classified as SD. Yet, with 90 mM [Na+]o, even though Delta Vo was reduced to 42 ± 9% of normal after a prolonged delay, individual neurons eventually still depolarized fully. Several factors could have contributed to the discrepancy between extracellular and intracellular voltage changes. It should be borne in mind that the extracellular voltage is an average signal from a large population of units. In low [Na+]o with the delay of the onset of depolarization, very probably the individual responses became more dispersed in time. Asynchrony among the responding units depresses the extracellularly recorded signal. Another source for the discrepancy could be if the depolarization would be distributed more evenly over the surface of the cells than is usually the case. Less-steep voltage gradients draw less extracellular current. It is also possible that some neurons did and others did not respond with SD-like depolarization within the same population. Indication for this may be found in the reports by Sugaya et al. (1975) and Czéh et al. (1993), who found a few anomalous cells that retained nearly normal membrane characteristics in the midst of the usual SD-like Delta Vo. In low [Na+]o, the proportion of units that did not participate in SD may have increased. The theoretical possibility of liquid junction or diffusion potentials generated by ion gradients in interstitial fluid needs to be considered. Such potentials are, however, negligibly small compared with the Delta Vo associated with SD (Somjen 1973). Finally, the puncture of the membrane by the intracellular electrode may have exaggerated the hypoxiainduced depolarization of neurons, but this effect need not be stronger in low [Na+]o than in normal ACSF.

The hypoxic responses of extracellular and intracellular voltages differed also in the combined presence of the three drugs, CPP, DNQX, and TTX. Hypoxic Delta Vo was absent in half the cases when this drug combination was used, and its amplitude was on average only 62 ± 18% of normal in the others. It seems that in the presence of the triple drug combination the "ignition point" for starting SD was not always reached. Yet when individual neurons underwent SD, they depolarized, after a very prolonged delay, to the same degree as those in the drug-free condition (Fig. 5). Similarly, if SD occurred, [K+]o eventually increased to the same level in the presence of the three drugs as in their absence (Fig. 4).

The unalterable final level of depolarization of neurons underscores the, already suspected, all-or-none character of SD. As long as any pathway for a persistent inward current is available, the potential to which the membrane tends is not governed by the number of channels that are still open but by the self-regenerating feedback. Lack of Na+ or partially blocked pathways for inward current may postpone SD and even prevent depolarization of some of the neurons but, once the process is set into motion, it will proceed to completion. The fact that the combined administration of DNQX and CPP had no more effect than either drug alone pointed to the same conclusion. This also indicated that as long as there was a remaining pathway for the influx of Na+, the ultimate level of the depolarization as well as of the ion concentration changes were governed by positive feedback.

It should be pointed out that the intracellular potential, Vi, was referred to a "bath" ground and it was not corrected for shifts of Vo. While at rest, Vi correctly represents the membrane potential, Vm; during SD, the large Delta Vo sums with the intracellular signal. Therefore during SD, Vm approaches 0 mV closer than is suggested by Vi shown in Figs. 5 and 6. In comparing the effects of various treatments on the fast depolarization (b-c segment of Figs. 5 and 6A), the error is, however, small.

Alternative conductances that might mediate SD-like depolarization

We have previously reported that hypoxic SD was consistently prevented by the combined application of glutamate antagonists plus drugs inhibiting voltage-gated Ca2+ and Na+ channels, namely DNQX + CPP + TTX + Ni2+ (Müller and Somjen 1998). In the experiments reported here, during the combined application of DNQX, CPP, and TTX, only Ni2+ was the missing ingredient. In the presence of the remaining three agents, which should markedly decrease Na+ conductance, SD failed in about half of the trials but it still occurred in the others. It appears that, in the absence of the main voltage-gated Na+ channels and ionotropic glutamate receptors, other conductances can, if with difficulty, generate hypoxic SD. Ni2+ is considered to be an antagonist of calcium channels (Hille 1992), although it has other actions as well (Hille 1968; Hille et al. 1975). It could therefore be suspected that after blockade of glutamate- and voltage-controlled Na+ channels, Ca2+ currents may have mediated SD. Yet when [Na+]o was reduced to 25 mM, SD was effectively suppressed, indicating that, by themselves, Ca2+ ions are not capable of carrying the necessary current. The minimal contribution of the influx of Ca2+ to the depolarization is also evident from the fact that SD can occur in the absence of external Ca2+ (Basarsky et al. 1998). Nonetheless, when in the presence of the triple combination of inhibitors SD did occur, the SD-related drop of [Na+]o was depressed by only 18 ± 5%. This indicates ample influx of Na+ into cells, bypassing glutamate- and voltage-controlled Na+ channels. We therefore conclude that, in the presence of DNQX, CPP, and TTX, either the influx of Ca2+ triggers the process that, eventually, leads to the influx of Na+, or else, that the residual conductance that is available for Na+ is sensitive to blockade by Ni2+.

The identity of the Na+ conductances that mediate SD after blockade of glutamate- and voltage-controlled channels remains to be discovered. The commonly observed persistent Na+ current is excluded, because it is highly sensitive to TTX (Crill 1996). The existence of TTX-insensitive Na+ currents in neurons has been reported by Hoehn et al. (1993) and Cummins et al. (1999) but deemed to be a laboratory artifact by Chao and Alzheimer (1995). It is not certain whether theirs will be the last word in this dispute. An inward K+ current through open K+ channels could not contribute to the depolarization because, in spite of the very large increase of [K+]o during hypoxic SD, the transmembrane K+ gradient does not reverse (Müller and Somjen 2000) and during the rapid phase of simulated SD-like depolarization, the computed K+ equilibrium potential remains negative relative to the prevailing membrane potential (Kager et al. 2000). Other possible candidates include the nonspecific cation current described by Alzheimer (1994), Ca2+-activated group I metabotropic glutamate receptor-controlled nonselective cation currents characterized by Congar et al. (1997), or an acetylcholine and Ca2+-dependent current reported by Fraser and MacVicar (1996) and Kawasaki et al. (1999).

Changes in extracellular ion levels

Ion currents across membranes of neurons and glial cells are the major source of the massive ionic changes that occur during hypoxic SD. The unusual magnitude of these responses is made possible by the narrow extracellular space, which is restricted even more during hypoxia. Diffusional ion flow can only slightly counteract these changes by blunting the increase in [K+]o and the drop in [Na+]o. Therefore the analysis and interpretation of our data concentrates on ionic currents.

Once SD occurred, ionic homeostasis was not markedly improved by the presence of the channel blocking drugs (Figs. 2 and 3). Glutamate-receptor inhibition curtailed the maximal decrease in [Na+]o by only 19%, but the Na+ plateau level was not at all affected by any of the treatments (Fig. 2). The origin of the Na+ plateau level is not clear. It could be thought that the initial surge of Na+ represents the rapidly inactivating "classical" Na+ current, INa,T, while the plateau is maintained by the persistent Na+ current, INa,P. Against this idea stands the fact that the plateau in the [Na+]o trace was not changed by TTX. The plateau may simply reflect the fact that most of the Na+ that has left interstitial space during the initial inward surge cannot be restored while hypoxia prevails, due to the impaired extrusion of Na+ from cells by the Na+-K+ pump.

The considerable and consistent acceleration of the initial rise of [K+]o under the influence of glutamate receptor antagonists (Table 2) was surprising. The reason is not clear, but a factor could be impaired clearance of excess K+ from interstitial space by glial cells. Glial cells do have glutamate receptors (Barres et al. 1990); whether these could be involved in "K-siphoning" is not known. In the presence of blocking drugs, [K+]o rose before the onset of SD to levels that greatly exceeded the so-called K+ "ceiling" of 10-11 mM defined by Heinemann and Lux (1977) (Fig. 2B). When SD failed to occur under the influence of DNQX, CPP, and TTX during prolonged hypoxia, [K+]o reached a plateau level of 16 mM (Fig. 4C) and neurons progressively depolarized to about -17 mV. In the absence of a large Delta Vo, this value of Vi is probably close to the real Vm. In computer simulation, the ignition point of SD appeared to be a joint function of neuron membrane voltage and [K+]o (Kager et al. 2000; Somjen et al. 2000). With some of the available channels blocked, this ignition point has apparently shifted to higher levels.

Administration of the drugs or drug combinations under normoxic conditions did not induce any detectable changes in the baseline concentrations of extracellular Na+ and K+ (see e.g., Fig. 3). Since drug penetration into interfaced slices is rather slow, minor drug-induced ion changes may have been masked by equilibration with the bath.

The cascade of events generating hypoxic SD: a hypothetical flow chart

The diagram of Fig. 7 attempts to organize the most salient published findings into a coherent flow chart of the events that generate hypoxic SD. Its intention is to clarify both the parallel and the sequential organization of the numerous processes that interact in the triggering and generation of hypoxic SD. Soon after oxygen withdrawal and much before the onset of SD, K+ channels are activated (Erdemli et al. 1998; Fujimura et al. 1997; Hansen et al. 1982; Leblond and Krnjevic' 1989), causing hyperpolarization of neurons (Fig. 5). Extrusion of K+ from neurons into the restricted interstitial space (Mazel et al. 1998; McBain et al. 1990; Pérez-Pinzón et al. 1995) raises [K+]o (Fig. 2), gradually turning the initial hyperpolarization into a slow depolarization (Fig. 5). At a critical point, the initially gradual depolarization starts to accelerate and becomes self-regenerating under the influence of the following factors. 1) Persistent Na+ current is activated by depolarization and it is reinforced by rising [K+]o and decreasing pO2 (Crill 1996; French et al. 1990; Hammarström and Gage 1998; Somjen 2000; Somjen and Müller, unpublished observations). 2) Glutamate receptors, especially NMDA receptors, are activated. Glutamate is released by both Ca2+-dependent (vesicular) and Ca2+-independent (nonvesicular) processes from presynaptic terminals and from glial cells (Attwell et al. 1993; Basarsky et al. 1999; Drejer et al. 1985; Fujikawa et al. 1996; Kimelberg et al. 1990; Szerb 1991). 3) Slowly inactivating voltage-gated Ca2+ channels are also activated. The influx of Ca2+ itself carries some current and, more importantly, elevated [Ca2+]i, then activates additional conductances. The effect of ion fluxes is further amplified and interaction and intercellular crosstalk is intensified due to shrinkage of interstitial volume and increase in electrical tissue resistance caused by cell swelling (Bures et al. 1974; Hansen and Olsen 1980; Jing et al. 1994; Müller 2000; Müller and Somjen 1999). The importance of cell swelling is demonstrated by the fact that hypertonic cell shrinkage prevents hypoxic SD and improves the recovery of function following hypoxia (Huang et al. 1996). Glial cells are known to have stretch-activated channels (e.g., Kimelberg and Kettenmann 1990; Kimelberg et al. 1990) but their presence in neurons has not been demonstrated. Neuron swelling does, however, influence such functions as the release of stored calcium (Borgdorf et al. 2000) and synaptic currents (Huang et al. 1997). The positive feedback generated by this multitude of interacting variables stops when electrochemical driving forces decrease to near zero. Recovery does not start until and unless reoxygenation restores cell metabolism.



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Fig. 7. Schematic flow chart of the cascade of events involved in the triggering of hypoxia-induced SD. The different contributing events are arranged in their sequential time course from top to bottom and grouped into the main three phases of hypoxia: initial phase, hypoxic SD, and recovery phase. For further description, see DISCUSSION.

The simultaneous activation of numerous processes acting in parallel explains why blocking just some of the feedback loops will postpone but not prevent SD, while inhibiting all of them does suppress it. Influx of Na+ through parallel pathways is the main player because, when [Na+]o was reduced to 24 mM, the Delta Vo amplitude became so small that it no longer met the diagnostic criteria for an SD-related extracellular potential shift.


    ACKNOWLEDGMENTS

This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-18670.

Present address of M. Müller: Zentrum Physiologie und Pathophysiologie, Abteilung Neuro- und Sinnesphysiologie, Georg-August-Universität Göttingen, D-37073 Gottingen, Germany.


    FOOTNOTES

Address for reprint requests: G. G. Somjen, Dept. of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710 (E-mail: g.somjen{at}cellbio.duke.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1 March 2000; accepted in final form 26 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society