Endogenous pH Shifts Facilitate Spreading Depression by Effect on NMDA Receptors

C. K. Tong and M. Chesler

Department of Physiology and Neuroscience and Department of Neurosurgery, New York University Medical Center, New York City, New York 10016


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Tong, C. K. and M. Chesler. Endogenous pH shifts facilitate spreading depression by effect on NMDA receptors. Rapid extracellular alkalinizations accompany normal neuronal activity and have been implicated in the modulation of N-methyl-D-aspartate (NMDA) receptors. Particularly large alkaline transients also occur at the onset of spreading depression (SD). To test whether these endogenous pH shifts can modulate SD, the alkaline shift was amplified using benzolamide, a poorly permeant inhibitor of interstitial carbonic anhydrase. SD was evoked by microinjection of 1.2 M KCl into the CA1 stratum radiatum of rat hippocampal slices and recorded by a proximal double-barreled pH microelectrode and a distal potential electrode. In Ringer solution of pH 7.1 containing picrotoxin (but not at a bath pH of 7.4), addition of 10 µM benzolamide increased the SD alkaline shift from 0.20 ± 0.07 to 0.38 ± 0.17 unit pH (means ± SE). This was correlated with a significant shortening of the latency and an increase in the conduction velocity by 26 ± 16%. In the presence of the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), benzolamide still amplified the alkaline transient, however, its effect on the SD latency and propagation velocity was abolished. The intrinsic modulation of SD by its alkaline transient may play an important role under focal ischemic conditions by removing the proton block of NMDA receptors where interstitial acidosis would otherwise limit NMDA receptor activity.


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INTRODUCTION
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Spreading depression (SD) is a propagating electrochemical disturbance associated with dramatic changes in the distribution of ions across neuronal membranes (Kraig and Nicholson 1978). The large influx of Ca2+ associated with SD (Nicholson et al. 1977) has suggested that these events play a role in hypoxic-ischemic injury. In brain slices, outcome after severe hypoxia has been linked to the frequency of SD-like events (Balestrino et al. 1989). In rat models of stroke, repetitive, spontaneous SDs occur in the ischemic penumbral region (Nedergaard and Astrup 1986) and have been correlated with the severity of injury (Iijima et al. 1992). Moreover, antagonists of the N-methyl-D-aspartate (NMDA) receptor reduce infarct size and also decrease the frequency of spontaneous SD in the ischemic penumbra (Iijima et al. 1992).

Although the involvement of NMDA receptors in ischemic injury is well established, this role has not been reconciled with the acid-base status of the tissue. In a rat stroke model, extracellular pH can fall below 6.7 (Nedergaard et al. 1991). The resulting proton block of the NMDA receptor would be expected to limit the opening of these channels (Tang et al. 1990; Traynelis and Cull-Candy 1990; Vyklicky et al. 1990).

At the onset of SD, however, a brief, but pronounced, extracellular alkaline shift occurs, which can attain a magnitude of several tenths of a unit pH (Kraig et al. 1983). This alkalosis may transiently remove the proton block of NMDA receptors, enabling or enhancing SD and neuronal injury. In this report, we used a hippocampal slice model to evaluate whether the alkaline transient can facilitate SD in this manner. Using an extracellular carbonic anhydrase inhibitor to selectively reduce the buffering of rapid pH changes, the alkaline shift of SD was amplified (Chen and Chesler 1992; Kaila et al. 1992). At low extracellular pH, enhancement of the alkaline shift was associated with a significant decrease in SD latency and increase in conduction velocity that were dependent on NMDA receptors. An earlier account has appeared as an abstract (Tong and Chesler 1998).


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Hippocampal slices, prepared from anesthetized, adult, Sprague-Dawley rats (4-5 wk old) were kept in Ringer solution at room temperature (>= 90 min), then placed in an interface slice chamber at 35°C for 30-60 min before recording. Ringer solution contained (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 3 CaCl2, 1.5 MgCl2, 1 NaH2PO4, and 10 glucose equilibrated with 95% O2-5% CO2 to achieve a nominal pH of 7.4. To lower pHo to levels characteristic of ischemia, the NaHCO3 was reduced to 13 mM (with NaCl increased accordingly) to produce a Ringer solution of pH 7.1. Benzolamide (10 µM) was added to increase the amplitude of the SD alkaline shift. This inhibitor of the extracellular carbonic anhydrase will amplify alkaline shifts mediated by proton sinks but will inhibit alkaline shifts generated by bicarbonate efflux across GABAA anion channels (Chen and Chesler 1992; Kaila et al. 1990, 1992). To ensure against confounding effects due to such bicarbonate efflux, 100 µM picrotoxin (Sigma Chemical) was included in all Ringer solutions. DL-2-amino-5-phosphonovaleric acid (DL-APV) was obtained from Tocris Cookson. Benzolamide was a gift of Lederle Laboratories.

Construction of double-barreled pH-sensitive microelectrodes (tip diameter of 3-5 µm) and the method of recording and calibrating extracellular pH have been described (Chesler and Chan 1988). Records of voltages were monitored on a chart recorder and archived to video tape for later analysis. Electrodes were lowered vertically into the tissue to a depth of 150-250 µm and left in place for the duration of the experiment (Fig. 1A). SD was triggered by brief (20-100 ms) ejection of 1.2 M KCl into the CA1 stratum radiatum via a micropipette (tip ~5 µm) connected to a Picospritzer (General Valve). Duration of the KCl pulse was not changed during the course of an experiment. Propagation of SD toward the CA3 region was monitored by a double-barreled pH microelectrode placed ~500 µm from the KCl injection pipette, allowing a simultaneous recording of extracellular potential (V1) and pHo. A single-barreled micropipette monitored extracellular potential (V2) closer to the CA3 region, at ~1,000 µm from the ejection site (Fig. 1A). Interelectrode distances were measured using established markers within the slice chamber with an accuracy of ±5%.



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Fig. 1. Method of recording and analysis. A: electrode placement for inititation and recording of spreading depression (SD) in a hippocampal slice. B: onset of SD was considered to be the sharp negative inflection on the DC shift (left-arrow ), which coincided with the start of the alkaline shift.

The onset of SD was defined as the point of inflection on V1 or V2 that commenced the rapid negative phase of the DC shift. This inflection always coincided with the sudden extracellular alkaline shift (Fig. 1B). The SD latency was defined as the time interval from the instant of KCl injection until the onset of SD at V1. Because of variability in the distance between the KCl injection pipette and the V1 electrode, the measured distances were divided by the latencies (providing a measure of "initial velocity") when averaging results across slices. Thus initial velocity was determined by the time required for initiation of SD around the injection pipette and the time for propagation to the V1 electrode. The SD "propagation velocity" was calculated from the time interval between onset of SD at V1 and V2 and from the measured V1-V2 electrode spacing. Only those slices in which SD could be repeatedly evoked under control conditions were included in the analysis. Unless otherwise noted, statistics were expressed as means ± SD. Paired trials refer to measurements made on the same slice (e.g., before and after addition of benzolamide). Comparisons were made with a paired t-test. Values of n refer to the number of slices.


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In 13 mM bicarbonate Ringer solution (pH 7.1), the pHo ranged from 6.8 to 6.9. The SD initial velocity (n = 16) and propagation velocity (n = 14) averaged 8.7 ± 3.1 and 5.2 ± 1.4 mm/min, respectively. The mean initial alkaline shift was 0.20 ± 0.07 unit pH (n = 18). In 16 paired trials, addition of benzolamide markedly increased the magnitude (P < 0.001) of the extracellular alkaline transient (Fig. 2A) to a mean value of 0.38 ± 0.17 unit pH. By contrast, the slow, late acid shift was not significantly altered (P = 0.89), averaging 0.27 ± 0.12 versus 0.27 ± 0.11 unit pH, before and after benzolamide, respectively (n = 13).



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Fig. 2. Effects of benzolamide on SD in 13 mM bicarbonate Ringer solution. A: benzolamide augmented the initiation and propagation of SD. Top trace: amplification of interstitial alkaline shift after addition of 10 µM benzolamide. Initial latency of SD at V1 (interval a) and the V1-V2 latency (interval b) were shortened markedly in benzolamide. B: in the presence of DL-2-amino-5-phosphonovaleric acid (APV), benzolamide did not significantly affect SD propagation. Time base in B was shortened to make intervals a and c the same length on the page, enabling comparison of percent changes. In APV, the initial latency of SD at V1 (interval c) and the V1-V2 latency (interval d) were slightly less after addition of benzolamide. Percentage changes in c and d were markedly less than the corresponding changes in a and b of A. C: overall effect of benzolamide. Percentage increase in propagation velocity caused by benzolamide was significantly greater in the absence vs. the presence of APV (P < 0.05). By contrast, the augmentation of the alkaline shift caused by benzolamide was similar in the presence and absence of APV. Error bars are the standard error of the mean.

The effect of benzolamide on the latency and the propagation velocity of SD can be seen in Fig. 2, A and C. In paired trials, addition of benzolamide increased the initial velocity by 35 ± 24% (n = 16, P < 0.001). This corresponded to an increase in the mean initial velocity from 8.7 ± 3.1 to 11.6 ± 3.9 mm/min. In 12 paired trials, benzolamide increased the propagation velocity by 26 ± 16% (P < 0.01), corresponding to an increase in the mean value from 5.0 ± 1.4 to 6.4 ± 2.0 mm/min (Fig. 2C).

In Ringer solution of pH 7.4 (26 mM bicarbonate), pHo ranged from 7.1 to 7.2, and the SD initial velocity and propagation velocity averaged 8.2 ± 3.2 and 8.4 ± 1.7 mm/min, respectively. In paired trials, addition of benzolamide significantly increased the SD alkalosis (n = 6, P = 0.026) with an increase in the mean value from 0.11 ± 0.05 to 0.20 ± 0.12 unit pH. However, benzolamide did not significantly increase the initial velocity (n = 6, P = 0.18, mean = 7.9 ± 3.4 mm/min) or the propagation velocity (n = 5, P = 0.21, mean = 7.8 ± 1.0 mm/min).

To test whether the effects of benzolamide on the latency and velocity of SD were related to NMDA receptors, a series of experiments was carried out in Ringer solution containing the NMDA receptor antagonist APV (50 µM). In 13 mM bicarbonate Ringer solution containing APV, the initial velocity and propagation velocity averaged 6.4 ± 1.8 and 5.5 ± 1.4 mm/min, respectively. In paired trials with APV, addition of benzolamide to 13 mM bicarbonate Ringer solution significantly amplified the alkaline shift (Fig. 2B, top) by an average of 75 ± 51% (n = 8, P < 0.01; Fig. 2C) with an increase in the mean alkalinization from 0.23 ± 0.09 to 0.39 ± 0.15 unit pH. However, in the same paired trials with APV, benzolamide did not significantly increase the initial velocity (n = 8, P = 0.14, mean = 7.2 ± 3.0 mm/min) or the propagation velocity (n = 7, P = 0.07, mean = 6.1 ± 1.2 mm/min). In the example shown in Fig. 2B, a small increase in these parameters occurred after addition of benzolamide. Figure 2C illustrates the overall effect of benzolamide in 13 mM bicarbonate Ringer. The percentage change in propagation velocity associated with addition of benzolamide was significantly less in the presence of APV (P < 0.05). By contrast, the amplification of the alkaline shift caused by benzolamide was similar with or without APV.


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The occurrence of SD in rat models of focal ischemia (Nedergaard and Astrup 1986), and the associated role of NMDA receptors in the extension of focal infarcts, is well established (Iijima et al. 1992). However, the involvement of NMDA receptors has not been reconciled with the marked proton block expected during ischemic acidosis. In this context, it is notable that the alkaline transient, which occurs at the onset of SD, may coincide with the release of glutamate. Therefore this pH shift could enable NMDA receptor function at the moment the tissue is most vulnerable to excitotoxic injury.

To test the basis of this concept, we increased the amplitude of the alkaline transient using benzolamide, a poorly permeant inhibitor of the interstitial carbonic anhydrase (Chen and Chesler 1992; Kaila et al. 1992). Augmentation of the alkalosis was associated with a significant shortening of the latency and an increase in the propagation velocity of SD. In the presence of APV, the alkaline shift was enhanced similarly by benzolamide, however, the latency and propagation velocity were not significantly changed.

These data strongly suggest that the effect of benzolamide was mediated by the increased alkalosis, which would be expected to reduce the proton block of the NMDA receptor. Indeed, in previous studies, amplification of the alkalosis by benzolamide was found to selectively enhance synaptic currents mediated by NMDA receptors (Gottfried and Chesler 1994) and could facilitate the induction of long-term potentiation (Taira et al. 1993). Given the pronounced alkalosis associated with SD, and the considerable proton block at the interstitial pH of 6.8-6.9, a similar augmentation of NMDA receptor function may be expected.

In the present study, benzolamide did not affect the latency or propagation velocity of SD in Ringer solution of normal pH (7.4). Because there was less proton block at the onset, the effect of the alkaline shift on NMDA receptors may have been partly occluded. As a result, secondary effects on the initiation and propagation of SD would have been more difficult to resolve.

The alkalosis that occurs at the onset of SD may be generated by the neuronal plasmalemmal calcium ATPase, which extrudes calcium in exchange for protons (Paalasmaa et al. 1994; Schwiening et al. 1993; Smith et al. 1994). This mechanism could add to the regenerative character of SD, as calcium entry would give rise to an extracellular alkaline shift, in turn favoring greater calcium influx via NMDA receptors. However, it should be noted that the timing of these rapid events cannot be discerned with ion-selective microelectrodes, which have response times of several seconds. To resolve the precise timing of the alkalosis in relation to the electrical properties of SD is going to require alternate means of recording fast extracellular ionic shifts.

In summary, our data suggest that the propagation of SD can be enhanced by the initial extracellular alkaline transient and that this facilitation is due to a specific action on NMDA receptors. The occurrence of this effect at low pH makes the results particularly relevant to ischemic pathophysiology, where a pronounced proton block of the NMDA receptor could be relieved by the SD alkalosis. In this regard, further studies on the dynamics of pH regulation in the ischemic setting would be important.


    ACKNOWLEDGMENTS

We thank Dr. Margaret Rice for critical reading of the manuscript.

This work was supported by National Institute of Neurological Disorders and Stroke Grant P50 NS-34115.


    FOOTNOTES

Address for reprint requests: M. Chesler, Dept. of Physiology and Neuroscience, NYU Medical Ctr., 550 First Ave., New York, NY 10016.

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 17 November 1998; accepted in final form 7 January 1999.


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