Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Xiong, Zhi-Qi and Janet L. Stringer. Extracellular pH Responses in CA1 and the Dentate Gyrus During Electrical Stimulation, Seizure Discharges, and Spreading Depression. J. Neurophysiol. 83: 3519-3524, 2000. Since neuronal excitability is sensitive to changes in extracellular pH and there is regional diversity in the changes in extracellular pH during neuronal activity, we examined the activity-dependent extracellular pH changes in the CA1 region and the dentate gyrus. In vivo, in the CA1 region, recurrent epileptiform activity induced by stimulus trains, bicuculline, and kainic acid resulted in biphasic pH shifts, consisting of an initial extracellular alkalinization followed by a slower acidification. In vitro, stimulus trains also evoked biphasic pH shifts in the CA1 region. However, in CA1, seizure activity in vitro induced in the absence of synaptic transmission, by perfusing with 0 Ca2+/5 mM K+ medium, was only associated with extracellular acidification. In the dentate gyrus in vivo, seizure activity induced by stimulation to the angular bundle or by injection of either bicuculline or kainic acid was only associated with extracellular acidification. In vitro, stimulus trains evoked only acidification. In the dentate gyrus in vitro, recurrent epileptiform activity induced in the absence of synaptic transmission by perfusion with 0 Ca2+/8 mM K+ medium was associated with extracellular acidification. To test whether glial cell depolarization plays a role in the regulation of the extracellular pH, slices were perfused with 1 mM barium. Barium increased the amplitude of the initial alkalinization in CA1 and caused the appearance of alkalinization in the dentate gyrus. In both CA1 and the dentate gyrus in vitro, spreading depression was associated with biphasic pH shifts. These results demonstrate that activity-dependent extracellular pH shifts differ between CA1 and dentate gyrus both in vivo and in vitro. The differences in pH fluctuations with neuronal activity might be a marker for the basis of the regional differences in seizure susceptibility between CA1 and the dentate gyrus.
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INTRODUCTION |
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Seizure activity is accompanied by rapid changes
in the ionic composition of the extracellular space, including rises in
extracellular potassium concentration and changes in the extracellular
pH. In contrast to the consistent increases in extracellular potassium during seizure activity, activity-dependent pH changes are notable for
their regional diversity (Chesler and Kaila 1992;
Deitmer and Rose 1996
). In some structures, such as
adult spinal cord (Sykova and Svoboda 1990
) and optic
nerve (Davis et al. 1987
), electrical stimulation
induces a predominant extracellular acidification. In other regions,
neuronal activity is accompanied by an initial extracellular
alkalinization, followed by a slower acidification. Such early alkaline
shifts have been reported in the cerebellum (Chesler and Chan
1988
; Kraig et al. 1983
), cortex
(Urbanics et al. 1978
), and CA1 and CA3 regions of
hippocampus (Jarolimek et al. 1989
; Walz
1989
). The functional significance and mechanisms underlying
these regional dependant pH shifts are not well understood.
There is evidence that neuronal excitability can be influenced by
extracellular pH. The conductances of a variety of ion channels are
altered by shifts in pH (Baukrowitz et al. 1999;
Kiss and Korn 1999
; Tombaugh and Somjen
1996
), and these changes in conductance may modulate neuronal
excitability. In addition, both GABAA receptor current and N-methyl-D-aspartate (NMDA) receptor
current are sensitive to extracellular pH. Extracellular alkalosis
decreases the conductance through GABAA receptor
channels (Pasternack et al. 1992
) and increases the NMDA
receptor-mediated current (Tang et al. 1990
;
Traynelis and Cull-Candy 1990
). Therefore
activity-dependent extracellular pH shifts may be sufficient to affect
neuronal excitability and the regional differences in
activity-dependent pH changes may underlie regional differences in
seizure susceptibility. In view of the pH sensitivity of neuronal
excitability and regional diversity in the activity-dependent pH
changes, we compared the extracellular pH response during seizure
activity in the CA1 region and the dentate gyrus in vivo and in vitro.
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METHODS |
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In vivo recording
Adult Sprague-Dawley rats (150-280 g, both sexes) were
anesthetized with urethane (1.2-1.5 g/kg ip) and placed in a
stereotaxic frame. The skull was exposed and burr holes drilled for
electrode placement. A concentric bipolar electrode was placed in the
CA3 region of the left hippocampus at an angle of 5° (AP, 3, ML, 3.5, depth 2.5-3.0 mm). Another stimulating electrode, fashioned from
0.01-in diam Teflon-coated stainless steel wire, was placed in the
right angular bundle (AP,
8, ML, 4.4, depth 2.5-3.5 mm). A
double-barrel hydrogen-sensitive microelectrode was placed in CA1 or
dentate gyrus (lateral 1.2-1.8 mm) on the right side, in the same AP
plane as the CA3 stimulating electrode. The animals were grounded
through a subcutaneous Ag/AgCl wire in the scapular region.
The hydrogen-sensitive microelectrodes were made according to
established techniques (de Curtis et al. 1998). One
barrel of the double-barreled electrode was silanized with 15%
tri-N-butylchlorosilane (Alfrebro, Monroe, OH) in
chloroform, and the tip was filled with the hydrogen ionophore II
(Fluka Cocktail A). The electrode was then backfilled with a buffer
solution [(in mM): 100 NaCl, 10 HEPES, and 10 NaOH, pH 7.5]. The
reference barrel was filled with 2 M NaCl. The electrode was calibrated
before each experiment in a series of standard solutions in artificial
cerebrospinal fluid (ACSF; pH 6.0-8.0). The calibration solutions were
similar to the ACSF, with NaHCO3 substituted for
the corresponding moles of NaCl. The pH-sensitive microelectrodes had a
response of 55-60 mV for a unit pH change. The extracellular field
activity and pH signal were amplified and displayed on a chart recorder.
Administration of 20-Hz (300-600 µA, 0.3-ms biphasic pulses) stimulus trains every 15 min to the CA3 region or the angular bundle were used to initiate seizure activity in CA1 or the dentate gyrus, respectively. At least five seizures were elicited in each recording location, and the peak alkalinization and peak acidification were measured for each stimulus train. These measurements were averaged to produce a mean alkaline or acid peak change for that animal and that stimulus/recording pair. Chemical convulsants, bicuculline and kainic acid, were used to initiate spontaneous epileptiform activity. Kainic acid (Sigma, St. Louis, MO) was dissolved in normal saline (0.9%) at 6 mg/ml and administrated intraperitoneally at a single dose of 12 mg/kg. (+)Bicuculline (Sigma) was dissolved in 1 N HCl at 5 mg/ml and was diluted to 0.5 mg/ml with normal saline just prior to injection. Bicuculline was administrated intravenously at a dose of 0.3-0.5 mg/kg, and the animal was monitored for 10-20 min. Repeated doses (up to 5) of bicuculline were administered to each animal at 1-h intervals. Each animal received either bicuculline or kainic acid, not both. For each animal receiving a chemical convulsant, recordings were obtained from both CA1 and the dentate gyrus.
In vitro recording
Hippocampal slices were prepared by conventional methods from Sprague-Dawley rats (100-200 g, both sexes). After anesthetizing the rats (ketamine 25 mg/kg, xylazine 5 mg/kg, acepromazine 0.8 mg/kg ip), the brains were removed. Transverse slices (400-500 µm) through the hippocampus were cut with a Vibratome (Technical Products International). Slices were placed in an interface-type chamber and continuously perfused with ACSF at 32°C under a stream of humidified 95% O2-5% CO2. Composition of the ACSF was (in mM) 127 NaCl, 2 KCl, 1.5 MgCl, 1.1 KH2PO4, 26 NaHCO3, 2 CaCl2, and 10 glucose. All solutions were bubbled constantly with 95% O2-5% CO2. Slices were allowed to equilibrate for 1 h before electrophysiological recording was begun. A hydrogen-sensitive microelectrode was placed in the cell body layer of CA1 or the dentate gyrus. A bipolar tungsten stimulating electrode was positioned in the Schaffer collaterals to stimulate CA1 or the perforant path to stimulate the dentate gyrus. Stimulus trains (600-800 µA, 0.3-ms biphasic pulses at 15 Hz) were used to initiate synchronized neuronal activity in both CA1 and the dentate gyrus.
Nonsynaptic epileptiform activity was induced in the hippocampus by
changing to ACSF containing 0-added calcium and high potassium. The
potassium was raised to 5 mM to induce epileptiform activity in the CA1
region and was raised to 8 mM to induce epileptiform activity in the
dentate gyrus. In both regions, this nonsynaptic epileptiform activity
can take more than 1 h to appear, but once it appears, the
interval between field bursts and the burst duration remains stable for
many hours (Bikson et al. 1999; Pan and Stringer 1996
).
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RESULTS |
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pH changes in CA1 and dentate gyrus in vivo during synchronized neuronal activity
In urethan-anesthetized rats, 20-Hz stimulation of the CA3 region or the angular bundle caused reproducible changes in the extracellular field potential and extracellular pH in CA1 and dentate gyrus (Fig. 1, n = 12 animals). In the CA1 pyramidal cell layer, stimulation was associated with a biphasic pH response. At the start of the stimulus train, the pH of the extracellular space became alkaline (0.05-0.1 pH units). This was followed by an acidification (0.1-0.25 pH units). Above and below the pyramidal cell layer, the pH shifts were similar in shape to those recorded in the cell body layer. The magnitude of the pH changes was maximal in the pyramidal cell layer and proximal dendrites and then gradually declined as the recording electrode was moved into stratum oriens or distal s. radiatum and s. lacunosum-moleculare. Throughout the CA1 region, the acidification peaked after termination of the stimulus train and after termination of the neuronal activity (Fig. 1 and see Fig. 2A). In the dentate gyrus, stimulus trains to the angular bundle induced only acidification (Fig. 1, n = 12 animals) in the granule cell layer. The magnitude of the pH shift ranged from 0.2 to 0.3 pH units and was fairly constant from s. moleculare through the hilus. In contrast to CA1, in the dentate gyrus, the acidification peaked with the termination of the neuronal discharge (Fig. 2A).
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To rule out the possibility that the difference in pH changes between CA1 and the dentate gyrus depended on the electrical stimulation, the pH changes during spontaneous seizures evoked by chemical convulsants were recorded. The pH responses during spontaneous seizure activity induced by bicuculline (0.3 mg/kg, n = 6 animals) or kainic acid (12 mg/kg, n = 3 animals) were similar to those evoked by stimulus trains. In CA1, the pH responses showed a biphasic pattern. At the onset of synchronized neuronal activity, marked by the negative shift of the extracellular DC potential, there was an initial alkalinization similar to that recorded during stimulus trains. This alkalinization was followed by an acidification. The amplitudes of the acidification and alkalinization were more variable than those seen during stimulation. In the dentate gyrus, only acidification was recorded (Fig. 2, B and C). The acidification was only observed when there was synchronous discharge of the granule cells associated with a negative shift of the DC potential.
pH responses in vitro
To further compare the pH responses in CA1 and the dentate gyrus,
we measured the extracellular pH changes in slices (Fig. 3). The pH of the extracellular space in
the slices was more acidic than the perfusing solution (7.15-7.18 in
the center of the slice compared with 7.35-7.40 in the perfusing
solution) and there was a pH gradient across the thickness of the
slice. This gradient was first reported in 1987 (Schiff and
Somjen 1987) and is now thought to be due to a gradient in
pCO2 (Chesler et al. 1994
; Voipio and Kaila 1993
). All the recordings for
the present study were obtained at the center of the slice (a depth of
~150 µm). Stimulation of Schaffer collateral fibers (15 Hz, 30 s) evoked biphasic pH shifts in the CA1 region similar to those
recorded in vivo during stimulation (n = 6 slices).
Stimulation of synaptic inputs evoked only acid shifts in the dentate
gyrus (n = 6 slices). In both CA1 and the dentate
gyrus, the extracellular space continued to acidify after the
termination of the stimulus trains. But in the dentate gyrus this
poststimulus acidification was smaller than in CA1. The similarity of
the in vitro pH changes to those observed in vivo suggests that
differences in blood flow between CA1 and the dentate gyrus are not an
important contributor to the different activity-dependent pH patterns
measured in vivo (Chesler and Kaila 1992
). However, the
rate of recovery of the extracellular pH in vitro after the stimulus
trains was significantly slower in both CA1 and the dentate gyrus. For
both CA1 and the dentate gyrus, the mean half-time of recovery in vitro
was 31.4 ± 1.7 (SE) s (n = 6 for both CA1 and
dentate gyrus), while in vivo the mean half-time of recovery was
9.7 ± 0.6 s (n = 6 for both CA1 and dentate
gyrus).
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To test whether activity-dependent depolarization of glial cells may
contribute to the extracellular pH changes in the hippocampus, extracellular pH was measured during stimulus trains in the presence of
1 mM barium (Fig. 3, n = 6 slices, no more than 1 slice
from one animal). This concentration of barium has been shown to block glial depolarization (Chesler and Kraig 1989). In the
presence of barium, the extracellular alkaline shifts were increased in CA1 from 0.04 ± 0.01 pH units before to 0.11 ± 0.02 pH
units after perfusion with barium. In the dentate gyrus, barium caused
the appearance of an initial alkalinization (up to 0.09 pH units, range
between 0.06 and 0.13 pH units), so that the pH changes in the dentate
gyrus now resembled the pattern of changes normally recorded in CA1.
The effects of barium were reversible.
To determine whether the extracellular pH changes during synchronized neuronal activity are due to synaptic transmission, we recorded the pH shifts during seizure activity induced in nonsynaptic conditions (0-added calcium). Field bursts in CA1, induced by perfusion with 0-added Ca2+ and 5 mM K+ ACSF, were associated with acidification (Fig. 4A, n = 6 slices). At the onset of the extracellular DC shift, indicating the onset of the synchronized neuronal burst, there was no alkalinization. After termination of the field burst, the extracellular pH began to recover toward baseline, but the onset of the next field burst caused acidification again. Field bursts in the dentate gyrus, induced by perfusion with 0-added Ca2+ and 8 mM K+ ACSF, were associated only with acidification of the extracellular space (Fig. 4B, n = 10 slices). As in CA1, the onset of the extracellular DC shift was associated with acidification. During the field burst, the extracellular pH appears to reach a plateau value and then it recovers after termination of the field burst.
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In some slices, spreading depression appeared spontaneously during perfusion with the 0-added calcium solutions (Fig. 5). In both CA1 and the dentate gyrus, the pH increased at the onset of the spreading depression. This initial alkalinization was followed by a more sustained acidification that recovered with the extracellular DC potential. To rule out the possibility that residual calcium in the bath or in the extracellular space within the slice underlies the initial alkalinization, EGTA (1 mM, n = 3 slices) or the calcium channel blocker cadmium chloride (0.2 mM, n = 3) was added to the 0-calcium ACSF. Neither EGTA nor cadmium chloride blocked the alkaline shift in either CA1 or the dentate gyrus during spreading depression (data not shown).
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DISCUSSION |
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This study demonstrated that activity-dependent extracellular pH shifts differ between CA1 and the dentate gyrus. In the CA1 region, synchronized neuronal activity, induced by stimulus trains or chemical convulsants, was associated with biphasic pH shifts consisting of an extracellular alkalinization followed by a slower acidification. In the dentate gyrus, synchronized neuronal activity was associated only with acidification. In the absence of synaptic transmission, spontaneous epileptiform activity was associated with extracellular acidification in both CA1 and dentate gyrus. Initial alkalinization followed by acidification was measured during spontaneous spreading depression in both CA1 and the dentate gyrus.
What is the cellular basis for the difference in extracellular pH
changes between CA1 and the dentate gyrus? The main difference between
the two regions is the presence of the initial alkalinization in CA1,
which is absent in the dentate gyrus. Activity-dependent extracellular
alkaline shifts have been linked to calcium influx into neurons. In the
absence of external calcium, the alkaline shifts are decreased or
completely blocked (Paalasmaa and Kaila 1996;
Paalasmaa et al. 1994
; Smith et al. 1994
;
Tong and Chesler 1999
). Consistent with this, the
extracellular pH response in CA1 during epileptiform activity in
nonsynaptic conditions displayed only an acidification, suggesting that
calcium influx during synaptic transmission may underlie the
alkalinization in CA1 (Chesler and Kaila 1992
;
Deitmer and Rose 1996
). But this does not explain the
lack of alkaline shift in the dentate gyrus. Certainly during the
stimulus trains in vivo and in vitro there is synaptic activity, which,
presumably, is linked to calcium influx into neurons. Either the
mechanism linking calcium influx to extracellular alkalinization is not
present in the dentate gyrus or the alkalinization is being neutralized
by an overlapping acidification through another mechanism.
Alkalinization of the extracellular space can occur in the dentate
gyrus in vivo, at least during spreading depression (Somjen 1984). In vitro (present study), alkalinization associated with spreading depression was present, even in the absence of external Ca2+. Several questions remain to be answered.
First, why are extracellular pH shifts similar in CA1 and the dentate
gyrus during spreading depression, but not during synchronized neuronal
activity? Why are the alkaline shifts abolished in 0 Ca2+ medium during synchronized neuronal activity
but not during spreading depression? So far we have no explanations.
Previous studies have indicated that alkaline shifts can, in some
instances, persist in the absence of external
Ca2+ (Smith and Chesler 1999
).
Interestingly, it has recently been shown that the alkaline shift
persists in the absence of Ca2+ during spreading
depression induced by ouabain and high-potassium (Menna et al.
1999
). The data suggest that the mechanisms underlying the pH
shifts during spreading depression are different from those active
during synchronous neuronal activity.
Regional differences in glial function may explain the regional
difference in pH regulation during neuronal activity. During neuronal
activity, potassium released from active neurons will depolarize glial
cells, resulting in the net secretion of acid from the glia
(Chesler and Kaila 1992). In the dentate gyrus, this
acid released from the glia may neutralize the initial extracellular alkaline shift. Blockade of the glial depolarization by barium (Ballanyi et al. 1987
) would thus block the resulting
secretion of acid (Chesler and Kraig 1989
;
Grichtchenko and Chesler 1994
). This may "unmask" an
alkalinization in the dentate gyrus, as has been shown in the dorsal
horn of the spinal cord (Sykova et al. 1992
). For this
hypothesis to be true, one must propose regional differences in glial
function between CA1 and the dentate gyrus. The density of the inward
rectifying potassium channels, which account for the activity-dependent
glial depolarization, has been shown to be lower in CA1 then in CA3
(D'Ambrosio et al. 1998
; McKhann et al.
1997
), but glia in the dentate gyrus have not been studied.
Alternatively, recent work has suggested that the effect of barium may
not be entirely due to the blockade of glial acid secretion. Barium may
substitute for calcium and move through calcium channels and in this
way also contribute to the alkaline shifts (Smith and Chesler
1999
)
The regional difference in the activity-dependent pH patterns between
CA1 and the dentate gyrus during synchronized neuronal activity may
have important physiological significance. It has been shown that
extracellular pH affects synaptic transmission and neuronal
excitability. Since extracellular alkalosis decreases the conductance
of GABAA receptor channels (Pasternack et
al. 1992) and increases the NMDA receptor current (Tang
et al. 1990
; Traynelis and Cull-Candy 1990
), the
early extracellular alkaline shift might increase (or sustain)
excitability in the CA1 region prolonging the neuronal activity. The
later acid shift might decrease the neuronal excitability and may be
involved in seizure termination in both CA1 and the dentate gyrus
(Xiong et al. 2000
). Therefore the different
extracellular pH patterns in CA1 and dentate gyrus may contribute
to the difference in their propensity for epileptic activity.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-39941 to J. L. Stringer.
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FOOTNOTES |
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Address for reprint requests: J. L. Stringer, Dept. of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.
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 6 January 2000; accepted in final form 6 March 2000.
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REFERENCES |
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