Department of Physiology and Neuroscience and Department of Neurosurgery, New York University School of Medicine, New York, New York 10016
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ABSTRACT |
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Menna, G., C. K. Tong, and M. Chesler. Extracellular pH Changes and Accompanying Cation Shifts During Ouabain-Induced Spreading Depression. J. Neurophysiol. 83: 1338-1345, 2000. Interstitial ionic shifts that accompany ouabain-induced spreading depression (SD) were studied in rat hippocampal and cortical slices in the presence and absence of extracellular Ca2+. A double-barreled ion-selective microelectrode specific for H+, K+, Na+, or Ca2+ was placed in the CA1 stratum radiatum or midcortical layer. Superfusion of 100 µM ouabain caused a rapid, negative, interstitial voltage shift (2-10 mV) after 3-5 min. The negativity was accompanied by a rapid alkaline transient followed by prolonged acidosis. In media containing 3 mM Ca2+, the alkalosis induced by ouabain averaged 0.07 ± 0.01 unit pH. In media with no added Ca2+ and 2 mM EGTA, the alkaline shift was not significantly different (0.09 ± 0.02 unit pH). The alkaline transient was unaffected by inhibiting Na+-H+ exchange with ethylisopropylamiloride (EIPA) or by blocking endoplasmic reticulum Ca2+ uptake with thapsigargin or cyclopiazonic acid. Alkaline transients were also observed in Ca2+-free media when SD was induced by microinjecting high K+. The late acidification accompanying ouabain-induced SD was significantly reduced in Ca2+-free media and in solutions containing EIPA. The ouabain-induced SD was associated with a rapid but relatively modest increase in [K+]o. In the presence of 3 mM external Ca2+, the mean peak elevation of [K+]o was 12 ± 0.62 mM. In Ca2+-free media, the elevation of [K+]o had a more gradual onset and reached a significantly larger peak value, which averaged 22 ± 1.1 mM. The decrease in [Na+]o that accompanied ouabain-induced SD was somewhat greater. The [Na+]o decreased by averages of 40 ± 7 and 33 ± 3 mM in Ca2+ and Ca2+-free media, respectively. In media containing 1.2 mM Ca2+, ouabain-induced SD was associated with a substantial decrease in [Ca2+]o that averaged 0.73 ± 0.07 mM. These data demonstrate that in comparison with conventional SD, ouabain-induced SD exhibits ion shifts that are qualitatively similar but quantitatively diminished. The presence of external Ca2+ can modulate the phenomenon but is irrelevant to the generation of the SD and its accompanying alkaline pH transient. Significance of these results is discussed in reference to the propagation of SD and the generation of interstitial pH changes.
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INTRODUCTION |
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Extracellular ionic shifts associated with
spreading depression (SD) have been described in a variety of
preparations across several species (Kraig and Nicholson
1978; Kraig et al. 1983
; Mutch and Hansen
1984
; Nicholson et al. 1977
; Somjen
1984
; Vyskocil et al. 1972
). Increasing
significance has been attributed to ionic changes during SD in view of
their putative role in ischemic brain injury (Balestrino et al.
1989
; Iijima et al. 1992
; Nedergaard and
Astrup 1986
). The hallmarks of these phenomena include a rapid, propagating increase in interstitial potassium concentration
([K+]o, etc.) to
50 mM,
associated with a negative shift in interstitial electric potential.
Abrupt [Na+]o and
[Cl
]o decreases of
similar magnitude occur with the
[K+]o change, along with
a decrease in [Ca2+]o
from 1 to ~0.1 mM. The ionic displacements are typically accompanied by pH changes consisting of a rapid interstitial alkaline shift followed by prolonged acidosis (Kraig et al. 1983
;
Mutch and Hansen 1984
; Nicholson et al.
1984
; Somjen 1984
). These endogenous
pHo shifts can significantly influence the
initiation and propagation of SD (Tong and Chesler
1999
).
The mechanisms that give rise to these pHo
shifts, and their relationship to the processes responsible for SD, are
not understood. Related factors implicated in the generation of SD
include the elevation of
[K+]o and the release of
glutamate (Grafstein 1956; Van Harreveld 1959
). Evidence suggests that the accompanying influx of
Ca2+ plays a significant role in SD (Jing
et al. 1993
). Elevation of internal Ca2+
may serve to activate vesicular release of glutamate, furthering the
regenerative process responsible for SD propagation. In addition, it
has been suggested that resulting waves of intracellular
Ca2+ are linked to the mechanism of SD
(Cornell-Bell et al. 1990
; Kunkler and Kraig
1998
; Nedergaard 1994
) .
The interstitial pH shifts tied to SD could also be related to the
influx of Ca2+. Synchronous neural activity
generates an alkaline pHo transient with a marked
dependence on external Ca2+ (Grichtchenko and
Chesler 1996; Paalasmaa and Kaila 1996
;
Paalasmaa et al. 1994
; Smith et al.
1994
). It has been suggested that such alkalinizations arise
from the Ca2+-H+ exchange property of the
plasmalemmal CaATPase (Schwiening et al. 1993
). Thus it
is compelling to speculate that the rapid alkalinization at the onset
of SD may also be triggered by the large initial influx of
Ca2+ (Nicholson et al. 1977
).
Although Ca2+ ions may play a significant role in SD, the
fundamental properties of this phenomenon may not be reliant on the influx of Ca2+. In brain slices, SD has been reported in
low Ca2+ solutions (Snow et al. 1983;
Yaari et al. 1986
). In addition, a form of SD can be
triggered in the absence of external Ca2+ by the
application of ouabain and, on occasion, by focal elevation of
extracellular K+. This Ca2+-independent event
was recorded as an increase in light transmittance associated with a
small negative shift of extracellular potential that propagated more
slowly than conventional SD (Basarsky et al. 1998
).
It is unknown whether the ionic shifts that accompany the
ouabain-induced SD variant are qualitatively or quantitatively similar to those associated with conventional SD. This information would help
place such events within the established context of SD and could
provide insights into the mechanisms of propagation. Moreover, investigating the interstitial pH changes accompanying this form of SD
could directly address their relationship to Ca2+ ions. We
therefore studied the ionic changes that accompany ouabain-induced SD
in the presence and absence of external Ca2+. Some of the
results appeared in an abstract (Menna et al. 1999).
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METHODS |
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Slices of hippocampus and overlying cortical regions (300-400
µM thick) were prepared from anesthetized adult Sprague-Dawley rats
(4-5 wk old). Cortical slices were used in only one set of experiments
in which ethylisopropyl amiloride was used to block Na+-H+ exchange (see
RESULTS). Slices were incubated for 90 min in artificial
cerebral spinal fluid (ACSF) at room temperature and then transferred
to a submersion-style slice chamber maintained at 32°C. The ACSF
contained (in mM) NaCl 124, NaHCO3 26, KCl 3, CaCl2 3, MgCl2 1.5, NaH2PO4 1, glucose 10, equilibrated with 95% O2-5% CO2 to achieve a nominal pH of 7.4. Because the voltage change on an ion-selective microelectrode depends
on the logarithm of the ion change (Eisenman 1961
), a
lower baseline [Ca2+]o
allows for an increased voltage response per absolute change in
[Ca2+]o. For the
recordings of [Ca2+]o,
the concentration of CaCl2 was therefore reduced
to 1.2 mM. In HEPES-buffered ACSF, NaHCO3 was
omitted and 26 mM HEPES was titrated with NaOH to a pH of 7.4. NaCl was
adjusted to maintain a constant sodium concentration. Cyclopiazonic
acid and thapsigargin were obtained from Sigma and Alomone
Laboratories, respectively. Benzolamide was a gift from Lederle Laboratories.
Solutions without added CaCl2 contained 2 mM EGTA. NaHCO3 was increased to 28 mM to compensate for acidity caused by EGTA. In three hippocampal slices, Ca2+-selective microelectrodes were used to record the decrease in [Ca2+]o during transition to Ca2+-free ACSF with EGTA. The [Ca2+]o decreased to ~1 µM within 5 min. In experiments in which SD was studied in the absence of Ca2+, slices were superfused with 0 Ca2+/EGTA solution for 15-20 min before the addition of ouabain.
Investigating the Ca2+-dependence of SD-related
alkaline shifts was a particular focus of the present study. A
bicarbonate-dependent alkalinization that is not dependent on
Ca2+ entry can be triggered by
GABAA receptors (Chesler and Kaila 1992). Because the occurrence of such pH changes would have
confounded the interpretation of results, 100 µM picrotoxin was added
to the ACSF to block GABAA receptors. To ensure
similar conditions during the
[K+]o,
[Na+]o, and
[Ca2+]o studies,
picrotoxin was added during these experiments as well. Studies of
[K+]o and
pHo in the absence of picrotoxin suggested that
ionic changes during SD were similar whether or not this drug was
present (see RESULTS).
Construction of double-barreled ion-sensitive microelectrodes (tip
diameter 3-5 µm) and the method of recording and calibrating extracellular ion shifts have been described in Chesler and Chan (1988). Ion-sensitive barrels responsive to
K+, Na+,
Ca2+, and pH contained Fluka ion-selective
cocktails 60398, 71178, 21048, and 95291, respectively. The ion
electrodes were calibrated with appropriate reference solutions
spanning the ion concentration range encountered during the
experiments. Because the Na+-specific cocktail
(Fluka 71178) does not have negligible sensitivity to
K+, the response was tested in calibration
solutions containing 30 mM K+, which was near the
upper range of [K+]o
encountered in these experiments. With this background
[K+], the Na+-selective
microelectrodes displayed a slope response of 50-54 mV per decade
change in [Na+] between 150 and 75 mM, which
was the concentration range relevant for these experiments. Potentials
on the reference barrels were subtracted continuously from the ion
barrel potentials to yield the ion signal, which was monitored on a
chart recorder.
Electrodes were lowered into the stratum radiatum of hippocampal slices or the midcortical layer of cortex slices to a tissue depth of ~150 µm. In most experiments, superfusion of 100 µM ouabain was used to induce SD. In some cases, slices were studied in an interface chamber and SD was initiated by a brief (20-100 ms) ejection of 1.2 M KCl into the CA1 stratum radiatum via a micropipette connected to a Picospritzer (General Valve Corp.). In Ca2+-free media, however, SD could not be reliably induced by KCl injection, limiting the use of this protocol to just a few experiments. Measurements of potential changes were obtained directly from the chart record. Statistics were expressed as means ± SE. Comparisons were made with a two-tailed t-test. Values of n refer to the number of slices.
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RESULTS |
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SD induced by ouabain
In hippocampal slices, superfusion of 100 µM ouabain
induced SD after 3-5 min, as evidenced by a sudden negative DC shift between 2 and 10 mV (Basarsky et al. 1998). Among 84 slices from all experiments, the mean DC shift was
3.6 ± 0.2 mV. There was no significant difference in the amplitude of the DC
shift in 3 mM Ca2+ versus
Ca2+-free solution (see DISCUSSION).
Shifts in extracellular pH during ouabain-induced SD
In ACSF containing 3 mM Ca2+, the negative
DC shift was accompanied by rapid alkalinization (Fig.
1A). The alkaline transient was followed by slow acidosis, as has been noted for SD in vivo (Kraig et al. 1983; Mutch and Hansen
1984
; Somjen 1984
). In media containing 100 µM
picrotoxin, the initial alkalinization averaged 0.07 ± 0.01 unit
pH and was followed by a mean net acid shift of 0.21 ± 0.04 unit
pH (n = 5). When picrotoxin was omitted, the alkaline
and acid shifts were not significantly altered, averaging 0.08 ± 0.01 and 0.24 ± 0.03, respectively (n = 6).
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In a number of studies, extracellular alkaline shifts evoked by neural
activity were inhibited after external Ca2+ was
removed (Grichtchenko and Chesler 1996; Paalasmaa
and Kaila 1996
; Paalasmaa et al. 1994
;
Smith et al. 1994
). However, the alkalinization
accompanying the ouabain-evoked SD was unaffected by withdrawal of
Ca2+ (Fig. 1B). In 0 Ca2+ ACSF containing 2 mM EGTA, the mean
alkalosis at the onset of SD was 0.09 ± 0.02 unit pH
(n = 6), which was not significantly different from the
alkalosis in media containing Ca2+. The
subsequent acidosis averaged 0.11 ± 0.02 unit pH, which was
significantly diminished (P < 0.05) compared with the
mean acid shift in the presence of Ca2+.
To determine whether or not the Ca2+-independent
alkaline shift was a feature limited to SD induced by ouabain, we
conducted experiments in an interface chamber, using microinjection of
1.2 M KCl to trigger SD. The resulting responses were recorded by a
proximal double-barreled pH microelectrode (pHo
and V1) and a more distal single- barreled
voltage electrode (V2) as illustrated in Fig.
2A. In 0 Ca2+ ACSF containing picrotoxin and no ouabain,
ejection of high K+ did not readily induce SD. A
clear SD was evoked in three slices, where the negative DC shift was
associated with a triphasic pH change. This consisted of a rapidly
developing acidification that was interrupted by an alkaline transient
(Fig. 2B). The alkaline transient had a peak amplitude of
0.054 ± 0.023 unit pH. These SDs propagated between electrodes at
a velocity of 1-2 mm per minute, similar to reported velocities of
ouabain-induced SD in the absence of Ca2+
(Basarsky et al. 1998). The triphasic acid-alkaline-acid
response noted in these Ca2+-free experiments
differed from the response in media containing Ca2+, where K+-evoked SD
was associated with a typical biphasic alkaline-acid sequence
(Tong and Chesler 1999
).
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Does bicarbonate efflux cause the alkalosis during ouabain-induced SD?
Efflux of bicarbonate ions across anion channels can cause a rapid
extracellular alkaline shift (Kaila and Voipio 1987).
The inclusion of picrotoxin in the ACSF precluded the generation of such responses via GABAA anion channels; however,
alternate anion pathways could not be excluded. Benzolamide is a poorly
permeant drug that blocks the dehydration of carbonic acid catalyzed by interstitial carbonic anhydrase, and thereby inhibits
bicarbonate-mediated alkaline shifts. In contrast, external alkalosis
that arises from a bicarbonate-independent net proton sink is amplified
by this carbonic anhydrase inhibitor (Chesler and Kaila
1992
). To distinguish these two forms of alkalosis, we observed
the pHo shifts during ouabain-evoked SD in 0 Ca2+ ACSF containing 10 µM benzolamide. Under
these conditions, the SD was associated with an alkaline shift (Fig.
3A) of 0.19 ± 0.04 unit
pH (n = 6) that was significantly greater than the mean
alkalosis in the absence of benzolamide (P < 0.05).
The slow acid shift was not significantly affected by benzolamide
(P = 0.6), averaging 0.12 ± 0.02 unit pH.
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To more directly address whether or not bicarbonate was necessary for the alkaline shift, ouabain-induced SDs were studied in bicarbonate-free ACSF buffered with 26 mM HEPES. In this solution, an alkaline-acid sequence still occurred. The alkaline transient averaged 0.05 ± 0.01 (n = 4) and was followed by a mean acid shift of 0.04 ± 0.01 unit pH (Fig. 3B).
Pharmacological studies of pH shifts during ouabain-induced SD
In view of the Na+ influx accompanying SD,
it is plausible that slowing or reversal of
Na+-H+ exchange could
underlie interstitial alkalosis. To test this hypothesis,
ouabain-induced SD was studied in the presence and absence of the
Na+-H+ exchange blocker
ethylisopropylamiloride (EIPA), a derivative of amiloride. Because
Na+-H+ exchange in
hippocampal CA1 pyramidal neurons is insensitive to amiloride
(Raley-Susman et al. 1991; Schwiening and Boron
1994
), recordings were made in cortical slices. In cortical
neurons the Na+-H+
exchanger is inhibited by amiloride (Ou-yang et al.
1993
).
In control cortical slices, superfusion of ouabain caused a negative DC shift and typical alkaline-acid transients that averaged 0.08 ± 0.02 and 0.26 ± 0.05 unit pH, respectively (n = 4) (Fig. 4A). In the presence of 100 µM EIPA, superfusion of ouabain did not readily induce SD. In five slices in which SD was evoked, an alkaline-acid sequence occurred that averaged 0.08 ± 0.02 and 0.10 ± 0.04 unit pH, respectively (Fig. 4B). The mean acid shift in EIPA was smaller than the control value (P < 0.05); however, the alkalosis was not significantly different from the control.
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The persistence of the alkaline shift in the nominal absence of
extracellular Ca2+ indicates that influx of
Ca2+ ions is not necessary for the generation of
this pH change. However, this observation does not exclude the
involvement of Ca2+ release from internal stores.
To test whether or not Ca2+ liberated from
endoplasmic reticulum (ER) played a role in generating the alkalosis,
ouabain-induced SD was induced after slices were superfused for 20-30
min with 0 Ca2+ ACSF containing cyclopiazonic
acid or thapsigargin, which are inhibitors of
Ca2+ uptake into the endoplasmic reticulum. In
ACSF containing 10 µM cyclopiazonic acid, the ouabain-induced SD in
hippocampal slices was associated with an early alkaline shift of
0.07 ± 0.13 unit pH (n = 5), which was not
significantly different from controls in the absence of the drug
(P = 0.6). A similar response occurred in 5 µM
thapsigargin, where the mean alkaline shift was 0.09 ± 0.01 unit
pH (n = 4). Basarsky and colleagues (1998) reported no
increase in [Ca2+]i in hippocampal slices
during ouabain-induced SD in Ca2+-free media,
which is consistent with the lack of effect of thapsigargin and
cyclopiazonic acid.
Shifts in [K+]o, [Na+]o and [Ca2+]o during ouabain-induced SD
In normal Ca2+-containing ACSF, superfusion of ouabain had little or no effect on [K+]o until the onset of a negative DC shift, whereupon [K+]o underwent a sudden increase to a mean value of 12 ± 0.62 mM (n = 6) (Fig. 5A). In the absence of picrotoxin, the elevation of [K+]o was similar, averaging 10.9 ± 1.08 mM (n = 7).
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In 0 Ca2+ ACSF, the elevation of
[K+]o began with a
gradual increase as ouabain was washed into the slice (Fig.
5B). The negative DC shift was accompanied by a second, more
rapid [K+]o elevation to
a peak value of 22 ± 1.1 mM (n = 7). The
[K+]o elevation in 0 Ca2+ ACSF was significantly greater than the
level attained in the presence of external Ca2+
(P < 0.001), although both values were considerably
smaller than the [K+]o
increase reported for conventional SD in hippocampal slices (Yaari et al. 1986).
Recordings of [Na+]o also revealed a relatively small ionic shift (Fig. 6). In normal ACSF containing Ca2+, the ouabain-induced SD caused a mean decrease in [Na+]o of 40 ± 7 mM (n = 6). In 0 Ca2+ ACSF, the change in [Na+]o was not significantly different (P = 0.5), decreasing by 33 ± 3 mM (n = 7).
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Recordings of interstitial Ca2+ were obtained in ACSF containing 1.2 mM Ca2+. A lower baseline [Ca2+]o was used in these experiments to maximize the millivolt response per change in [Ca2+]o. In this solution, the ouabain-evoked SD was accompanied by a rapid decrease of [Ca2+]o as shown in Fig. 7. The average decrease in [Ca2+]o was 0.73 ± 0.07 mM to a mean value of 0.47 ± 0.07 mM.
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DISCUSSION |
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The events contributing to conventional SD, although rare in
normal tissue, are likely to play an important role in the way the
brain responds to hypoxic and ischemic insults. Complete brain ischemia
is shortly followed by an SD-like event (Hansen 1985), and focal ischemic lesions can be the source of multiple propagating SDs of long duration (Iijima et al. 1992
;
Nedergaard and Astrup 1986
). In addition to the probable
pathological importance of SD-like mechanisms, the study of such
phenomena is useful for understanding brain ion homeostasis. The ionic
shifts associated with SD-like events have a qualitative resemblance to
those of normal activity but are far greater in magnitude and likely
involve a commensurately greater role of channel- and
transporter-mediated ion pathways. The SD variant elicited by
superfusion of ouabain may be particularly useful in the dissection of
such events because it can be reliably evoked in the absence of
external Ca2+ in a submersion-style brain slice chamber
(Basarsky et al. 1998
). By contrast, SD elicited by
focal injection of K+, although more faithful to
established models of SD, could not be reliably elicited in
Ca2+-free media. Thus ouabain-induced SD was useful as a
trigger of extracellular pH shifts, thereby providing a novel means by
which the Ca2+ dependence of these acid-base changes could
be addressed. A second goal of this study was to measure the ionic
shifts of ouabain-induced SD so that this event could be understood in
relation to conventional SD.
In the adult brain, synchronous neuronal activity is associated with
elevation of [K+]o that does not breach a
ceiling level of ~12 mM (Heinemann and Lux 1977). In
contrast, SD has been characterized by an increase of
[K+]o that can exceed 50 mM (Kraig and
Nicholson 1978
; Nicholson et al. 1977
;
Vyskocil et al. 1972
). In the present study, SD
initiated by ouabain resulted in a relatively small elevation of
[K+]o. In media containing extracellular
calcium, the mean elevation was only 12 mM and never exceeded 14 mM.
This was surprising because inhibition of the
Na+-K+ ATPase by ouabain might be expected to
cause a higher elevation of [K+]o, as was
noted by Kraig and coworkers (1983)
in the cerebellar cortex. It is
likely that the small elevation in K+ was partly caused by
egress of this ion from the tissue into the extracellular medium.
However, there was a significantly greater increase of
[K+]o in the absence of extracellular
Ca2+, where the mean elevation reached 22 mM and the
maximum was 28 mM. The tendency of extracellular Ca2+ to
limit the increase of [K+]o was also evident
during the introduction of ouabain. Before the onset of SD,
[K+]o gradually increased in the absence of
Ca2+ whereas in the presence of Ca2+, the
baseline [K+]o remained stable until the
occurrence of the negative voltage shift. It is unclear why the
baseline [K+]o did not increase in media
containing Ca2+ as would be expected with
inhibition of the Na+-K+ pump. A partial
explanation may be the stabilizing effect of Ca2+-dependent
K+ conductances, which would limit neuronal depolarization
and cause a smaller efflux of K+ ions. In this respect,
effective depolarization brought about by loss of surface charge
screening in Ca2+-free media may also play a role.
Although the elevation of [K+]o was greater in Ca2+-free media, the presence or absence of Ca2+ did not affect the mean extracellular DC shift. There was, however, considerable variability in the amplitude of the DC shift for similar elevations of [K+]o. These observations may stem from a variable site of initiation and a variable wave front of [K+]o elevation when the SD-like event is initiated in different slice preparations. In addition, to the extent that standing extracellular currents contributed to the negative DC shifts, differences in interstitial volume or in the fluid level within the submersion chamber could have led to a variable shunting of such currents.
Ouabain-induced SD was associated with a shift in
[Na+]o of ~30 mM. This observation
distinguishes this SD from repetitive neuronal activity where the
decrease in [Na+]o is an order of magnitude
smaller (Dietzel et al. 1982). However, in conventional
SD, the decrease in [Na+]o is far greater,
typically exceeding 60 mM (Hansen and Zeuthen 1981
;
Kraig and Nicholson 1978
). The relatively small decrease in [Na+]o during ouabain-induced SD is
consistent with the limited increase in
[K+]o. Because net ionic shifts during SD
will be determined by the sum of energetically uphill ion transport and
downhill ionic fluxes, in the face of Na+-K+
pump blockade, the modest net ion shifts probably reflect a limited channel-mediated flux of Na+ and K+ compared
with conventional SD. It is possible that ouabain caused a slow
depolarization and early inactivation of voltage-gated channels,
curtailing the movement of Na+ and K+.
Despite the modest size of the [K+]o and
[Na+]o transients, ouabain-induced SD has
been shown to propagate. In hippocampal slices, Basarsky and colleagues
(1998) measured a conduction velocity of ~1 mm per minute. This is
considerably slower than other forms of SD that spread at >3 mm per
minute in mammals (Nicholson 1984
). This difference in
velocity might be partly caused by the relative magnitude of the ionic
shifts. For example, a greater elevation of
[K+]o and decrease of
[Na+]o could cause a decrease, or possibly a
reversal, of glutamate uptake (Billups et al. 1998
),
which favors the generation of SD.
In conventional SD, [Ca2+]o will typically
decrease by ~1 mM (Nicholson et al. 1977). In
ouabain-induced SD, the decrease in [Ca2+]o
was somewhat less, averaging 0.73 mM. Thus compared with the observed
changes in [K+]o and
[Na+]o, the behavior of
[Ca2+]o seemed to be least altered in this
form of SD. However, this shared behavior of Ca2+ does not
imply a shared importance in the underlying mechanism because
ouabain-induced SD could occur without external Ca2+. With
Ca2+ present, features of the SD may be modulated as was
discussed in the case of [K+]o elevation. It
might also be expected that the magnitude of the Ca2+
influx would govern the extracellular alkaline shift
(Grichtchenko and Chesler 1996
; Paalasmaa and
Kaila 1996
; Paalasmaa et al. 1994
; Smith
et al. 1994
). However, the occurrence of a large
Ca2+ influx did not appear to affect the generation of the
alkaline transient.
The pHo transients that accompanied ouabain-induced SD were
characterized by an early alkalosis followed by a prolonged acid shift.
A similar pattern was noted whether or not Ca2+ was present
in the interstitial fluid. This alkaline-acid pattern has been reported
for conventional SD in vivo (Kraig et al. 1983) and in
vitro (Martins-Ferreira and Do Carmo 1987
; Taira
et al. 1992
) and is also characteristic of synchronous neural
activity in a variety of brain regions (Chesler 1990
;
Chesler and Kaila 1992
).
The late acid shift is thought to arise from the generation of carbon
dioxide and lactic acid because it can be blocked by metabolic
inhibitors (Kraig et al. 1983) and is accompanied by a
large increase in interstitial carbon dioxide concentration (Taira et al. 1992
). The appearance of late acidosis in
the presence of ouabain indicates that an energetic load other than
Na+-K+ ATPase was responsible for the
generation of acid, as was noted by Kraig and colleagues (1983)
in
cerebellar cortex. Because acidosis was larger in the presence of
external Ca2+, a component of the acid response may be
caused by mitochondrial Ca2+-H+ exchange
(Meech and Thomas 1980
). Suppression of late acidosis in
the presence of EIPA suggests that Na+-H+
exchange played a role in bringing cellular acid into the interstitial compartment. In some preparations, stimulus-evoked acid shifts appeared
to be similarly dependent on Na+-H+ exchange
(Rose and Deitmer 1995
; Walz 1989
).
However, in all of these instances, it remains uncertain whether or not
the electrical responses that gave rise to the alkaline shifts were
diminished by the Na+-H+ exchange inhibitor. In
this context it is notable that in hippocampal slices, acid shifts
associated with electrical stimulation (Voipio and Kaila
1993
) and SD (Taira et al. 1992
) could be
explained solely by the accumulation of interstitial CO2.
The onset of ouabain-induced SD was marked by a rapid interstitial alkaline shift. In ACSF containing bicarbonate, these experiments were performed in the presence of picrotoxin to eliminate possible contributions of GABAergic bicarbonate fluxes. It is plausible that GABAergic bicarbonate fluxes contribute to the pHo shifts during SD; however, this topic was not addressed.
The picrotoxin-resistant alkalosis that occurs during repetitive neural
activity has several features in common with the SD alkaline transient.
Both responses were unaffected by Na+-H+
exchange inhibitors (Chesler and Chan 1988), were
increased by benzolamide, and persisted in HEPES-buffered media
(Chesler and Kaila 1992
). Unlike stimulus-evoked
alkaline shifts, however, SD-associated alkalosis was unaffected by the
removal of extracellular Ca2+. It is possible that a
Ca2+-dependent component of the alkaline transient was
obscured by masking effects of Ca2+-dependent acidosis.
Nonetheless, the persistence of a robust alkaline shift after removal
of Ca2+ stands in marked contrast to previous studies of
stimulus-evoked alkalinizations (Grichtchenko and Chesler
1996
; Paalasmaa and Kaila 1996
; Paalasmaa
et al. 1994
; Smith et al. 1994
). Moreover, the
generation of a Ca2+-independent alkaline shift was not a
feature unique to the use of ouabain because a
Ca2+-independent alkaline transient also accompanied SD
evoked by high K+ (Fig. 2).
The alkaline transient was also unaffected by thapsigargin and
cyclopiazonic acid, which would be expected to deplete Ca2+
stores of the endoplasmic reticulum. These observations all suggest that the Ca2+-H+ exchange property of the
plasmalemmal Ca ATPase is not the basis for this pH change. An issue
that arises is whether or not the same bicarbonate-independent
mechanism accounts for the alkaline shifts during SD and during
repetitive neuronal activity. The observed difference in
Ca2+-dependence is an argument for different mechanisms.
Indeed, it was recently reported that a component of activity-dependent
alkalosis can persist in the absence of external Ca2+
(Smith and Chesler 1999), which is consistent with more
than one process. The relationship between the influx of
Ca2+ and accompanying acid-base fluxes remains unclear.
In summary, these results demonstrate that ouabain-induced SD is associated with shifts in pHo, [K+]o, [Na+]o, and [Ca2+]o that are qualitatively similar to those accompanying conventional SD but that display marked quantitative differences. The changes in [K+]o and [Na+]o are comparatively modest, which may be in part caused by exchange with the superfusion fluid. The decrease in [Ca2+]o accompanying this form of SD can be substantial and may influence aspects of the SD but is not a necessary feature. The acid-base shifts show a mixed dependence on Ca2+. The early alkaline transient, coinciding with the negative interstitial DC shift, appears to be independent of Ca2+ whereas the slow acidosis that follows is markedly diminished in the absence of Ca2+. Whether or not these pHo shifts share the same mechanisms with the pHo changes accompanying normal synchronous neural activity remains to be determined.
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ACKNOWLEDGMENTS |
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This work was supported by National Institute of Neurological Disorders and Stroke Grants P50 NS-34115 and NS-34906.
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FOOTNOTES |
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Address for reprint requests: M. Chesler, Dept. of Physiology and Neuroscience, New York University Medical Center, 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 20 September 1999; accepted in final form 19 November 1999.
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REFERENCES |
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