Extracellular pH Responses in CA1 and the Dentate Gyrus During Electrical Stimulation, Seizure Discharges, and Spreading Depression

Zhi-Qi Xiong and Janet L. Stringer

Department of Pharmacology and Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030


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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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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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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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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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Fig. 1. Laminar profile of extracellular pH shifts in the hippocampus in vivo. Stimulus trains were administered to the contralateral CA3 while advancing a dual barrel (pH-sensitive and DC potential) electrode through the hippocampus. Shown are the extracellular pH changes at different laminae of hippocampus, indicated to the left. The vertical dashed lines mark the onset and termination of the 20-Hz, 30-s stimulus trains. Horizontal dashed lines mark the extracellular pH baselines. Alkalinization is a downward change of the pH tracing, while acidification is an upward change.



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Fig. 2. Extracellular pH shifts during seizure activity. Field potential (f.p.) and extracellular pH changes (Delta pHo) in CA1 (left) and dentate gyrus (DG, right) were recorded during synchronized neuronal activity induced by 20-Hz stimulation (30 s, A) to CA3 for CA1 or angular bundle for dentate gyrus. Similar recordings are shown after intravenous injection of 0.3 mg/kg (+)bicuculline (B). Chart recordings presented start seconds after the bicuculline injection. C: recordings 2 h after intraperitoneal administration of 12 mg/kg kainic acid. After kainic acid, there are nearly continuous interictal discharges that are not associated with detectable changes in pH. Changes in extracellular pH corresponded with synchronized neuronal discharges, marked by the negative shift of the DC potential. As in Fig. 1, alkalinization is represented by a negative change, while acidification is a positive change.

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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Fig. 3. Stimulus evoked changes in extracellular pH in the hippocampal slice. Stimulus trains (15 Hz, 30 s) were alternately administrated to the CA3 Schaffer collateral pathway or perforant path while measuring changes in pH in the pyramidal cell layer or granule cell layer, respectively. Top: stimulation-evoked pH changes in CA1 and the dentate gyrus in normal artificial cerebrospinal fluid (ACSF). After addition 1 mM barium (middle), the alkaline shift in CA1 increased in amplitude and early alkaline response appeared in the dentate gyrus. The effect of barium was reversible (wash). The vertical dashed lines mark the onset and termination of the 15-Hz, 30-s stimulus trains. The horizontal dashed lines mark the extracellular pH baseline. As in Fig. 1, alkalinization is represented by a negative change, while acidification is a positive change.

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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Fig. 4. pH responses during 0 Ca2+/high K+-induced nonsynaptic field bursts. A: simultaneous recordings of the f.p. and Delta pHo in CA1 perfused with 0 Ca2+/5 mM K+ medium. B: simultaneous recordings of the f.p. and Delta pHo in the dentate gyrus perfused with 0-Ca2+/8 mM K+ medium. Dashed lines mark the extracellular pH baseline (The baseline level of extracellular pH of slices is 7.15-7.20. The pH of perfusing ACSF is 7.35). As in Fig. 1, alkalinization is represented by a negative change, while acidification is a positive change.

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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Fig. 5. pH responses during spreading depression in 0 Ca2+/high K+ media. A: simultaneous recordings of the f.p. and Delta pHo during a single spreading depression episode in CA1 perfused with 0 Ca2+/5 mM K+ medium. B: simultaneous recordings of the f.p. and Delta pHo during a single spreading depression episode in the dentate gyrus perfused with 0 Ca2+/8 mM K+ medium. As in Fig. 1, alkalinization is represented by a negative change, while acidification is a positive change.


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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.


    ACKNOWLEDGMENTS

This study was supported by National Institute of Neurological Disorders and Stroke Grant NS-39941 to J. L. Stringer.


    FOOTNOTES

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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