Effect of Divalent Cations on AMPA-Evoked Extracellular Alkaline Shifts in Rat Hippocampal Slices

S. E. Smith and M. Chesler

Department of Physiology and Neuroscience and Department of Neurosurgery, New York University School of Medicine, New York, New York 10016


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Smith, S. E. and M. Chesler. Effect of Divalent Cations on AMPA-Evoked Extracellular Alkaline Shifts in Rat Hippocampal Slices. J. Neurophysiol. 82: 1902-1908, 1999. The generation of activity-evoked extracellular alkaline shifts has been linked to the presence of external Ca2+ or Ba2+. We further investigated this dependence using pH- and Ca2+-selective microelectrodes in the CA1 area of juvenile, rat hippocampal slices. In HEPES-buffered media, alkaline transients evoked by pressure ejection of RS-alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) averaged ~0.07 unit pH and were calculated to arise from the equivalent net addition of ~1 mM strong base to the interstitial space. These alkaline responses were correlated with a mean decrease in [Ca2+]o of ~300 µM. The alkalinizations were abolished reversibly in zero-Ca2+ media, becoming indiscernible at a [Ca2+]o of 117 ± 29 µM. Addition of as little as 30-50 µM Ba2+ caused the reappearance of an alkaline response. In approximately one-fourth of slices, a persistent alkaline shift of ~0.03 unit pH was observed in zero-Ca2+ saline containing EGTA. In HEPES media, addition of 300 µM Cd2+, 100 µM Ni2+, or 100 µM nimodipine inhibited the alkaline shifts by roughly one-half, one-third, and one-third, respectively, whereas Cd+ and Ni2+ in combination fully blocked the response. In bicarbonate media, by contrast, Cd+ and Ni2+ blocked only two-thirds of the response. In the presence of bicarbonate, Ni2+ caused an unexpected enhancement of the alkalinization by ~150%. However, when the extracellular carbonic anhydrase was blocked by benzolamide, addition of Ni2+ reduced the alkaline shift. These results suggested that Ni2+ partially inhibited the interstitial carbonic anhydrase and thereby increased the alkaline responses. These data indicate that an activity-dependent alkaline shift is largely dependent on the entry of Ca2+ or Ba2+ via voltage-gated calcium channels. However, sizable alkaline transients still can be generated with little or no external presence of these ions. Implications for the mechanism of the activity-dependent alkaline shift are discussed.


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Neuronal activity is accompanied by rapid extracellular alkalinizations that can arise from at least two sources (Chesler and Kaila 1992). Activation of GABA-A receptors leads to the efflux of bicarbonate across the anion channel and a consequent rise in extracellular pH (pHo) (Chen and Chesler 1990, 1992a; Kaila and Voipio 1987; Kaila et al. 1990). In addition, there is a bicarbonate-independent alkaline shift that can be elicited by activation of ionotropic glutamate receptors (Chen and Chesler 1992a; Chesler and Chan 1988) or by direct electrical stimulation of a neuronal population (Chen and Chesler 1992b; Grichtchenko and Chesler 1996; Paalasmaa and Kaila 1996; Tong and Chesler 1999). Speculation on the mechanism of the bicarbonate-independent alkaline transient has centered on the role of calcium ions.

In giant nerve cells of the snail, alkaline shifts in surface pH have been linked to Ca2+ entry and its subsequent extrusion by a plasmalemmal CaATPase (Schwiening et al. 1993). This transporter imports two protons per extruded Ca2+ (Niggli et al. 1982) and therefore may account for the bicarbonate-independent alkaline shift in vertebrate preparations. Observations consistent with this hypothesis have been noted in mammalian brain slices. Extracellular alkaline shifts evoked by glutamate agonists or by direct electrical stimulation were abolished in the absence of external Ca2+ (Grichtchenko and Chesler 1996; Paalasmaa and Kaila 1996; Paalasmaa et al. 1994; Smith et al. 1994). In addition, cytoplasmic acid shifts sensitive to CaATPase inhibitors have been linked to neuronal Ca2+ entry (Trapp et al. 1996). On the other hand, Ba2+, which is thought to be poorly extruded by this transporter (Kwan et al. 1990; Zhao and Dhalla 1988), also was able to support these alkaline shifts (Grichtchenko and Chesler 1996; Tong and Chesler 1999).

The relationship between extracellular Ca2+ shifts and the alkaline transient, and the dependence of this pH response on the external concentration of Ca2+ and Ba2+, has not been investigated. In this study, we used pH- and Ca2+-selective microelectrodes, and discrete concentrations of external Ba2+, to more fully examine these relationships, employing focal injections of AMPA to elicit extracellular alkaline shifts. In this context, the effect of divalent Ca2+ channel blockers on the alkaline shift also was studied. Ion substitution and divalent blocker experiments confirmed a large dependence on Ca2+ or Ba2+ ions. However, our data indicate that significant alkaline shifts can be elicited reliably in the presence of micromolar external Ba2+ and often can be evoked in the absence of either Ba2+ or Ca2+. Some of these results have appeared in an abstract (Smith et al. 1996).


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Procedures were carried out with approval of the NYU Medical Center Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering and reduce the number of animals used to the minimum required for our study. Long Evans rat pups, bred in-house (P12-18 of either sex), were anesthetized and rapidly decapitated. Hippocampal slices (300-400 µm) were prepared on a Vibratome in ice-cold Ringer, then incubated at room temperature until use. Experiments were conducted at 28-32°C in a submersion-style chamber with constant superfusion of the Ringer solution at 3-5 ml per min.

Normal Ringer solution contained (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 3 CaCl2, 1.5 MgCl2, 1 NaH2PO4, and 10 glucose, gassed with 95% O2-5% CO2 (pH 7.4). Ringer solution buffered with 26 mM N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) had NaHCO3 omitted and was titrated to pH 7.4 with NaOH. In some experiments, the Ca2+ was reduced to 1 mM to allow for greater resolution of Ca2+ shifts detected with ion-selective microelectrodes. Zero-Ca2+ Ringer had CaCl2 omitted and contained 1 or 5 mM ethylene glycol bis (beta-aminoethyl ether)-N, N,N',N'-tetraacetic acid (EGTA). Solutions containing 5 mM EGTA were prepared with 35 mM NaHCO3 to titrate the acidity of the EGTA and had NaCl reduced accordingly. All experiments were conducted in saline containing 100 µM picrotoxin (PiTX; Sigma Chemical). Solutions containing CdCl2 or NiCl2 had NaH2PO4 omitted. The pressure ejection pipettes had tip diameters of ~5 µm and contained 100 µM RS-alpha -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) dissolved in 150 mM NaCl. In a small number of experiments, the ejection pipette was filled with 10 mM sodium glutamate, and the Ringer contained 50 µM DL-2-amino-5-phosphonovaleric acid (APV) to block activation of N-methyl-D-aspartate receptors. APV and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Tocris Cookson. Benzolamide was a gift of Dr. T. H. Maren and Lederle Laboratories, Pearl River, NY.

Double-barreled pH electrodes were fabricated as previously described (Chesler and Chan 1988). Reference barrels were back-filled with 1 M NaCl. The silanized pH-sensitive barrels were back-filled with 150 mM NaCl plus phosphate buffer (pH 6.87) and front-filled with a pH-sensitive liquid cocktail (Fluka 95291). The pH microlectrodes, calibrated in standard phosphate buffers, exhibited a 55- to 60-mV response per unit pH. Ion and reference signals were recorded via high-impedance (>1013 Omega ) operational amplifiers wired for unity gain. Extracellular DC potential was monitored and subtracted from the signal on the ion-sensitive barrel.

The pH electrode and the pressure ejection pipette were mounted on a Narashige dual micromanipulator with their tips ~50 µm apart. The array was lowered to a tissue depth of ~150 µm in the CA1 stratum pyramidale or s. radiatum. The choice of region was governed only by the ability of the tissue to generate sizable, repeatable alkaline responses. As no consistent differences were noted between the behavior of pHo responses in s. pyramidale versus s. radiatum, the results from these regions were pooled. To avoid movement of the electrode tips relative to one another, the array was not moved after being positioned in the tissue. Therefore baseline pHo was checked only at the start and at the end of experiments when the array was moved between tissue and bath. The pH of the extracellular space was more acid than the bath, typically ranging between 7.0 and 7.20.

To elicit pHo shifts, a Picospritzer (General Valve) was used to deliver pressure pulses (40 psi) of AMPA at regular intervals (1-5 min.). The duration of the agonist pulse was adjusted until a consistent alkaline shift >0.05 unit pH was obtained in normal Ringer. In a few experiments, pHo shifts were evoked by antidromic stimulation (50 Hz, 5 s) of the CA1 pyramidal neurons using a bipolar stimulating electrode placed in the alveus. The unfiltered signals were measured as a change in voltage on a strip chart recorder and archived to video tape. Means were presented with standard errors. Values of n referred to the number of slices. Amplitudes of the alkaline shifts were compared using a paired t-test.

In bicarbonate Ringer, the interstitial buffering of alkaline shifts depends on the extracellular carbonic anhydrase activity and assumptions about the CO2 partial pressure (Chesler 1990). Because these factors are uncertain, experiments requiring estimates of interstitial buffering capacity were conducted in HEPES Ringer. The buffering capacity (beta ) of the extracellular fluid was estimated as
&bgr;=<FR><NU>2.3[H<SUP>+</SUP>][<IT>A</IT><SUB><IT>tot</IT></SUB>][<IT>K</IT><SUB><IT>a</IT></SUB>]</NU><DE>([<IT>H</IT><SUP><IT>+</IT></SUP>]<IT>+</IT><IT>K</IT><SUB><IT>a</IT></SUB>)<SUP><IT>2</IT></SUP></DE></FR> (1)
where Atot is the total concentration of HEPES and Ka is the dissociation constant (Roos and Boron 1981). The equivalent amount of strong base added to the extracellular fluid during an alkaline shift was referred to as the change in strong ion difference (Delta SID) and was estimated as
&Dgr;SID=&bgr;×&Dgr;pH (2)
where Delta pH is the mean amplitude of the alkalinization (Chesler 1990).


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Baseline pHo was similar in 26 mM bicarbonate verses 26 mM HEPES-buffered media as has been previously reported (Gottfried and Chesler 1994). In a sample of 10 slices in bicarbonate saline, the baseline pHo was 7.15 ± 0.019 (range 7.08-7.23). In another 10 slices studied in 26 mM HEPES Ringer, the baseline pHo was 7.13 ± 0.015 (range 7.06-7.20).

Simultaneous measurement of pHo and [Ca2+]o

In seven slices, we studied evoked pH shifts in 26 mM HEPES-buffered Ringer. A pH- and a Ca2+-selective microelectrode were used to record simultaneous ion shifts (Fig. 1, A and B) evoked by AMPA (n = 5) or glutamate (n = 2, in 50 µM APV). The bath Ca2+ was reduced from 3 to 1 mM in these experiments to increase the sensitivity of the Ca2+-selective microelectrode. For all seven slices, the peak alkaline pHo shifts averaged 0.072 ± 0.015 unit pH and were associated with a peak fall in [Ca2+]o of 295 ± 47 µM. Assuming an extracellular pH buffering capacity of 14.7 mM (see METHODS), the alkaline shifts were associated with a change in the extracellular strong ion difference (Delta SID) equivalent to the addition of 1.06 ± 0.22 mM strong base. Over the seven slices, the ratio of the Delta SID:Delta [Ca2+]o ranged from 2.1-5.5 and averaged 3.65 ± 0.44. 



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Fig. 1. Calcium dependence of the AMPA-evoked alkaline shift. A: simultaneous recording of extracellular Ca2+ and pH transients evoked by pressure ejection of 100 µM AMPA. B: pressure ejection of 10 mM glutamate elicited similar ion transients in media containing 50 µM DL-2-amino-5-phosphonovaleric acid (APV). C: alkaline response evoked by pressure ejection of AMPA was inhibited reversibly in 0-Ca2+ media containing 1 mM EGTA. A rapid acid transient was unmasked in the 0-Ca2+ media. All solutions were buffered with 26 mM HEPES.

Alkaline shifts during washout of extracellular Ca2+

In solutions containing EGTA and no added Ca2+, the agonist-evoked alkaline shift was abolished reversibly in 41 slices (34 slices bathed in bicarbonate saline and 7 slices bathed in HEPES saline). In many cases, transition to the zero-Ca2+/EGTA Ringer caused the alkaline shift to be superseded by a rapid acid shift (Fig. 1C) as noted previously (Smith et al. 1994). This acid transient could be abolished by CNQX (not shown), indicating that it was triggered by the activation of AMPA receptors and was not a mechanical or electrical artifact. The mechanism of this acid transient was not explored.

To better resolve the relationship between the pHo changes and [Ca2+]o, adjacent recordings of the ion transients were made in slices during the washout and return of extracellular Ca2+. Superfusion of zero-Ca2+, HEPES-buffered Ringer (with 1 mM EGTA) caused a fall in extracellular [Ca2+]o to micromolar levels within a few minutes (Fig. 2). During the Ca2+ washout, AMPA was intermittently pressure-ejected from a nearby micropipette to follow the effect on the evoked alkaline shift. As shown in Fig. 2, the reduction of [Ca2+]o caused a decrease in the amplitude of the alkaline shifts, accompanied by the appearance of an initial acid-going transient that eventually superseded the alkalinization. The acid transient remained during much of the return of extracellular Ca2+. These observations suggested that a persistent component of the alkaline shift could have been obscured by the early acid transient (see following text). In eight slices, the alkaline shifts became indiscernible at an average [Ca2+]o of 117 ± 29 µM, over a [Ca2+]o range from 1 to 281 µM.



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Fig. 2. Simultaneous measurement of [Ca2+]o and pHo transients during washout and return of bath calcium. Calcium-free solution contained 5 mM EGTA. Solution buffered with 26 mM HEPES.

Evoked alkaline shifts in zero-Ca2+ media

In a subset of experiments (15 of 59 slices), a component of the alkaline shift persisted in the nominal absence of extracellular calcium. These extracellular Ca2+-independent alkalinizations occurred in both bicarbonate (Fig. 3A) and HEPES-buffered Ringer (Fig. 3B) in which the measured [Ca2+]o was <1 µM. In bicarbonate buffer, these alkaline shifts averaged 0.032 ± 0.004 unit pH (n = 9) and were 42 ± 8% of the paired control responses in normal [Ca2+]o. In six slices studied in HEPES Ringer, alkaline shifts noted in the absence of external Ca2+ averaged 0.027 ± 0.006 unit pH and were 28 ± 3% of the paired controls. These persistent responses were abolished by CNQX (not shown), indicating that they were not electrical or mechanical artifacts.



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Fig. 3. Persistence of the AMPA-evoked alkaline shift in the absence of extracellular Ca2+. A: alkaline response evoked in 0-Ca Ringer (1 mM EGTA) buffered with 26 mM bicarbonate. B: alkaline response evoked in 0-Ca Ringer (1 mM EGTA) buffered with 26 mM HEPES.

Effect of Ca2+ channel blockers in bicarbonate-free Ringer

In HEPES-buffered Ringer, addition of 300 µM Cd2+ was able to reversibly reduce the AMPA-evoked alkaline shift. The mean decrease of the response was 55 ± 2% (n = 5, P < 0.001; Fig. 4A). Addition of 100 µM Ni2+ alone reduced the alkaline shift by 36 ± 4% (n = 7, P < 0.001; Fig. 4B). When 300 µM Cd2+ and 100 µM Ni2+ were applied together, the AMPA-evoked alkalinization was blocked completely in four slices (Fig. 4C).



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Fig. 4. Block of the alkaline shift by Cd2+ and Ni2+ in HEPES-buffered Ringer. A: addition of 300 µM Cd2+ reversibly reduced the magnitude of the alkaline shift by approximately two-thirds. B: addition of 100 µM Ni2+ reduced the alkaline shift by about one-third. C: addition of 300 µM Cd2+ plus 100 µM Ni2+ abolished the alkaline shift. All solutions were buffered with 26 mM HEPES.

To better define the Ca2+ entry pathway associated with activation of the alkaline shift, we tested the effect of nimodipine, an L-type calcium channel antagonist. In seven slices, addition of 10 µM nimodipine to HEPES- buffered Ringer inhibited the AMPA-evoked alkaline shift by 30 ± 7% (P < 0.05). When 100 µM Ni2+ was added to the nimodipine-containing Ringer, the alkaline shifts were further reduced to 51 ± 3% of control (n = 5, P < 0.001; not shown).

Effect of Cd2+ and Ni2+ in bicarbonate Ringer

In bicarbonate-buffered Ringer, addition of 300 µM Cd2+ alone (Fig. 5A) reduced the AMPA-evoked alkaline shift by 67 ± 4% (n = 4, P < 0.05). Whereas addition of Cd2+ plus Ni2+ abolished the response in HEPES Ringer, Ni2+ produced no additional block in bicarbonate Ringer (Fig. 5A). In the presence of 300 µM Cd2+ plus 100 µM Ni2+, the AMPA-evoked alkaline shift was reduced 65 ± 5% (n = 4, P < 0.02), which was not significantly different from the effect of Cd2+ alone (n = 4, P = 0.7). When 10 µM benzolamide was added in addition to the Cd2+ and Ni2+, the remaining alkaline shift was amplified three- to sevenfold (Fig. 5B). The residual response therefore originated from a proton sink, typical of alkaline shifts activated by ionotropic glutamate receptors (Chen and Chesler 1992c).



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Fig. 5. Effect of Cd2+ and Ni2+ on the alkaline shift in bicarbonate Ringer. A: addition of 300 µM Cd2+ partly blocked the AMPA-evoked alkaline shift. Subsequent addition of 100 µM Ni2+ did not reduce the response further. B: in bicarbonate Ringer containing 300 µM Cd2+ plus 100 µM Ni2+, the residual alkaline shift was amplified by addition of 10 µM benzolamide.

Further dissection of these effects revealed a paradoxical action of Ni2+ when applied alone. In 11 slices, addition of 50-100 µM Ni2+ increased the AMPA-evoked alkaline shift by 154 ± 9% (P < 0.001; Fig. 6A). The enhancement by Ni2+ was not specific to the response evoked by AMPA receptors. In four slices, alkaline transients were evoked in the absence of synaptic transmission (in 20 µM CNQX, 50 µM APV, and 100 µM PiTX) by repetitive (50 Hz) antidromic activation of the CA1 pyramidal neurons. After addition of 100 µM Ni2+, the antidromic alkaline shifts were increased by 171 ± 12% (n = 4, P < 0.05; Fig. 6B).



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Fig. 6. Paradoxical effect of Ni2+ in bicarbonate Ringer. A: addition of 100 µM Ni2+ consistently increased the AMPA-evoked alkaline shift. B: alkaline shifts evoked by repetitive antidromic stimulation were enhanced similarly after addition of Ni2+. C: in the presence of 10 µM benzolamide, Ni2+ reduced the alkaline shift.

This enhancement of the alkaline shift would be expected if Ni2+ had an inhibitory effect on the interstitial carbonic anhydrase. To test this hypothesis, we prevented the possibility of such inhibition by bathing slices in benzolamide (10-20 µM). With the extracellular carbonic anhydrase inhibited, a comparatively small AMPA ejection pulse was used to elicit an average alkaline shift of 0.10 ± 0.02 unit pH (n = 7). Under these conditions, Ni2+ inhibited the alkaline responses. After addition of 50-100 µM Ni2+ to the benzolamide-containing saline, the AMPA-evoked alkaline shifts were reduced to 73 ± 11% of controls, (P < 0.05) averaging 0.078 ± 0.02 unit pH (Fig. 6C).

Replacement of Ca2+ with Ba2+

Although the AMPA-evoked alkaline shift was blocked in media containing 1-5 mM EGTA and no added Ca2+, a component of the response could be restored by adding Ba2+. In these experiments, Ca2+ first was removed from the interstitial space by superfusion with Ringer containing 1 mM EGTA and no added Ca2+. Before addition of Ba2+, the EGTA was washed out of the slice. Periodic ejection of AMPA confirmed that the alkaline shift remained blocked. With subsequent addition of Ba2+, a significant component of the alkalinization returned. In HEPES (n = 12; Fig. 7A) and bicarbonate media (n = 13; Fig. 7B), the alkaline shift elicited by 500 µM Ba2+ was, respectively, 40 ± 3 and 51 ± 5% of the responses obtained in 3 mM Ca2+.



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Fig. 7. AMPA-evoked alkaline shift is supported by Ba2+. A: barium-dependent alkaline shift in HEPES Ringer. After block of the response in 0-Ca2+/EGTA, the EGTA was washed out. Alkaline shift remained inhibited in the 0-Ca2+ HEPES Ringer. After addition of 500 µM Ba2+, a smaller AMPA-evoked alkaline shift could be elicited. B: barium-dependent alkaline shift in bicarbonate Ringer. After superfusion of 0-Ca2+/EGTA Ringer, followed by 0-Ca2+ Ringer, the alkaline shift was abolished. Response returned after addition of 500 µM Ba2+. Barium-dependent alkaline shift was amplified by addition of 10 µM benzolamide.

Like the control responses in normal Ca2+, the alkalinizations elicited in 500 µM Ba2+ were amplified by benzolamide (273 ± 34%, n = 4; Fig. 7B), indicating that they also arose from a net proton sink (Chen and Chesler 1992c). In addition, the control and the Ba2+ responses were similarly affected by solutions containing Cd2+ (300-500 µM) and Ni2+ (100 µM). Addition of both Cd2+ and Ni2+ only partially blocked the Ba2+-dependent alkaline shift in bicarbonate Ringer (50 ± 4%, n = 3; Fig. 8A) but completely abolished the responses when added to HEPES-buffered solutions (n = 2; Fig. 8B).



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Fig. 8. Effect of Cd2+ and Ni2+ on the barium-dependent alkaline shift. A: in bicarbonate Ringer, addition of 300 µM Cd2+ plus 100 µM Ni2+ partially inhibited the alkaline shift. B: in HEPES Ringer, 300 µM Cd2+ plus 100 µM Ni2+ completely blocked the response.

In a previous study, Ba2+-dependent alkaline shifts evoked by field stimulation required millimolar concentrations of this cation (Grichtchenko and Chesler 1996). By contrast, the AMPA responses obtained in 500 µM Ba2+ were blocked consistently (n = 6) when the Ba2+ concentration was raised to 2 mM (not shown) and sizable AMPA-evoked alkaline shifts could be elicited by Ba2+ at concentrations of <= 50 µM (Fig. 9). Combining data from bicarbonate (n = 4; Fig. 9A) and HEPES-buffered media (n = 3), the alkaline shift evoked in 50 µM Ba2+ averaged 0.030 ± 0.004 unit pH, which was 32 ± 4% of the control responses in 3 mM Ca2+. To further ensure that these responses were unrelated to extracellular Ca2+, we used a Ca2+-free bicarbonate Ringer that contained 2 mM Ba2+ plus 5 mM EGTA to provide a calculated free-Ba2+ concentration of 37 µM. In five slices bathed in this solution, the AMPA-evoked alkaline shift was 0.025 ± 0.01 unit pH and averaged 41 ± 10% of the control responses in 3 mM Ca2+ (Fig. 9B).



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Fig. 9. Alkaline shifts supported by low concentrations of Ba2+. A: after superfusion of 0-Ca2+/EGTA, then 0-Ca2+ Ringer, the AMPA-evoked alkaline shift was abolished. Superfusion of 50 µM Ba2+ restored part of the response. B: to ensure against residual extracellular Ca2+, slices were superfused with a cocktail containing 2 mM Ba2+ and 5 mM EGTA, calculated to contain 37 µM free Ba2+. After block of the response in 0-Ca2+/EGTA, alkaline shifts could be elicited by AMPA in the barium-EGTA cocktail.


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Relationship between divalent cation shifts and the alkaline transient

Simultaneous recording of the alkaline shift and the [Ca2+]o transient allowed comparisons of the net ionic changes after AMPA ejection. If the measured fall in [Ca2+]o represented a cellular Ca2+ load, which subsequently was extruded in entirety by a plasmalemmal CaATPase, a Delta SID:Delta [Ca2+]o ratio of 2:1 would be anticipated (Niggli et al. 1982). Using a HEPES buffering power of 15 mM, the mean AMPA-evoked alkaline shift (0.072 unit pH) corresponded to a change in the strong ion difference (Delta SID) of ~1 mM (equivalent to the addition of this concentration of strong base to the extracellular fluid). Because the corresponding measured fall in [Ca2+]o averaged ~300 µM, the Delta SID:Delta [Ca2+]o ratio would range between 3 and 4. However, the limited response times of the pH and Ca2+ electrodes and the diffusion time between pressure ejection and ion electrodes sites would have distorted these measurements. It is therefore likely that both the Delta SID and the cellular Ca2+ load were greater. Thus, order of magnitude comparisons of Ca2+ and pH responses are probably the best that can be expected using these techniques. However, it should be noted that that evoked intracellular Ca2+ load was likely handled by mechanisms in addition to the CaATPase, such as Na+-Ca2+ exchange (Blaustein 1988), uptake into endoplasmic reticulum (Markram et al. 1995), or sequestration by mitochondria (Wang and Thayer 1996). To the extent that a plasmalemmal CaATPase did not fully handle the intracelluar Ca2+ load, the calculated ratios would have been greater.

In a previous study, electrically evoked alkaline shifts could be supported by Ba2+ but required millimolar concentrations of this ion (Grichtchenko and Chesler 1996). Using AMPA microejection to evoke the pH response, micromolar concentrations of Ba2+ were sufficient. The ability of 50 µM Ba2+ to support the alkaline shift can be considered in the context of H+ antiport. If the entire extracellular concentration of 50 µM Ba2+ had entered cells and was extruded in exchange for protons (in a ratio of 2 H+:1Ba2+), then the expected extracellular Delta SID would have been 100 µM. With a mean measured alkaline shift of ~0.03 unit pH and a calculated buffering capacity of 15 mM, the experimental Delta SID would be ~450 µM. In view of the limited response time of the pH electrodes, the actual Delta SID is likely to have been larger. To generate the observed alkaline shifts using the plasmalemmal CaATPase therefore would have required efficient extrusion of Ba2+ ions by this transporter and cycling of Ba2+, whereby ions entered cells and were extruded multiple times in exchange for protons.

Alkaline shifts in the absence of extracellular Ca2+

In one-fourth of the slices, alkaline shifts were elicited in the nominal absence of external Ca2+ or Ba2+. These shifts averaged ~0.03 unit pH, corresponding to a Delta SID of 450 µM. Although these alkalinizations were not noted in the majority of slices, the appearance of an acid transient at low [Ca2+]o may have obscured such changes, as suggested by Fig. 2. For the plasmalemmal CaATPase hypothesis to be considered in this context, the extruded Ca2+ would have had to originate from internal stores. This possibility has not been excluded by the present experiments.

Effect of Ca2+ channel blockers on the alkaline shift

In HEPES-buffered solution, simultaneous addition of Cd2+ and Ni2+ completely blocked the AMPA-evoked alkaline shift, consistent with a requirement for Ca2+ entry via voltage-gated channels. In isolation, these blockers may roughly distinguish between low- and high-voltage-activated Ca2+ channels (Bean 1989; Fox et al. 1987). The block of roughly one-third of the alkaline shift by Ni2+ was similar to the effect of this cation on spike-induced Ca2+ entry in postnatal CA1 pyramidal cells (Christie et al. 1995) and indicates that both low- and high-voltage-activated Ca2+ channels can trigger the pH response. Nimodipine was about half as effective as Cd2+ alone, suggesting that L-type Ca2+ channels were responsible for triggering approximately one-fourth of the alkaline shift. Nimodipine blocked a similar fraction of somatic Ca2+ entry in postnatal CA1 pyramidal neurons (Christie et al. 1995). Neuronal entry of Ca2+ via AMPA receptors is likely to have played a minor role because the AMPA receptor subtype with significant Ca2+ permeability is confined to the less prominent GABAergic interneurons (Geige et al. 1995; Racca et al. 1996).

In bicarbonate Ringer, Cd2+ and Ni2+ did not fully abolish the alkaline shift. Partial binding of these cations by bicarbonate ions might have contributed to the incomplete inhibition. In addition, because Ni2+ alone had a paradoxical enhancing effect, the combination of Ni2+ and Cd2+ would be less effective than anticipated. The Ni2+-mediated enhancement of the alkaline shift was dependent on bicarbonate and was occluded by benzolamide. These observations are consistent with a partial reduction of extracellular carbonic anhydrase activity mediated by Ni2+. Inhibition of this enzyme by a variety of divalent cations has been reported (Maren 1967; Puscas et al. 1989). These effects of Ni2+ on activity-dependent pHo shifts may warrant consideration in other studies where this divalent is used in brain slices.

In summary, the present data indicate that the influx of Ca2+ or Ba2+ across voltage-gated Ca2+ channels is an important component in the steps that lead to extracellular alkalinization after activation of AMPA receptors. The results are in agreement with other studies of activity-dependent alkaline shifts and can be interpreted to support the involvement of a plasmalemmal CaATPase. However, alkaline responses could be obtained at extremely low concentrations of external Ba2+ and in many instances occurred in the absence of extracellular Ca2+. These results indicate that the entry of Ca2+ and its subsequent extrusion by the plasmalemmal CaATPase cannot explain all of the properties of activity-dependent alkaline shifts. Involvement of internal Ca2+ stores or alternative pathways for transmembrane H+ movement are possibilities that have not been excluded.


    ACKNOWLEDGMENTS

We thank C. K. Tong for assistance.

This research was supported by National Institute of Neurological Disorders and Stroke Grant NS-32123. S. E. Smith was supported by the Medical Scientist Training Program under National Institute of General Medical Sciences Grant 5 T32 GM-07308.


    FOOTNOTES

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

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Received 2 March 1999; accepted in final form 18 May 1999.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society