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Regulation and Modulation of pH in the Brain

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

ABSTRACT

I. INTRODUCTION

II. BUFFERING POWER

III. REGULATION OF INTRACELLULAR pH IN VERTEBRATE NEURONS

A. Neurons From Hippocampus

B. Neurons From Cerebral Cortex

C. Neurons From Cerebellum

D. Neurons From Spinal Cord

E. Medullary Neurons

F. Retinal Neurons and Photoreceptors

G. Sympathetic Neurons

H. Neoplastic Neural Cells

I. Synaptosomes

IV. REGULATION OF INTRACELLULAR pH IN GLIAL CELLS

A. pH Regulation in Nonmammalian Glial Cells

B. pH Regulation in Mammalian Astrocytes

1. Principal acid extrusion mechanisms in mammalian astrocytes

2. The astrocyte Na+-HCO3- cotransporter

3. Na+-independent acid extrusion in astrocytes

4. Cl-/HCO3- exchange in astrocytes

C. pH Regulation in Oligodendroglia

D. pH Regulation in Schwann Cells

E. pH Regulation in Glial Tumor Cells

F. pH Regulation and Volume Control in Glia

V. MOLECULAR IDENTIFICATION OF BRAIN ACID TRANSPORTERS

A. Na+/H+ Exchangers in Brain

B. HCO3- Transporters in Brain

1. The Cl-/HCO3- exchanger in the CNS

2. Na+-HCO3- cotransporters in the CNS

3. Na+-driven Cl-/HCO3- exchange in the CNS

VI. MODULATION OF INTRACELLULAR pH BY NEURONAL ACTIVITY

A. Modulation of pHi in Neurons

1. pHi shifts associated with depolarizing stimuli

2. pHi shifts associated with GABAA and glycine receptors

3. Additional modulators of neuronal pHi

B. Modulation of pHi in Glial Cells

VII. REGULATION AND MODULATION OF BRAIN EXTRACELLULAR pH

A. The pH of Interstitial Fluid

B. Changes in Interstitial pH Caused by Neural Activity

1. Buffering of brain interstitial fluid

2. Interstitial pH transients and CA

3. Nature of interstitial CA activity in brain

4. Bicarbonate-independent alkaline shifts

5. Bicarbonate-dependent alkaline shifts

6. Mechanisms of activity-dependent acid shifts

VIII. EFFECTS OF ACTIVITY-DEPENDENT pH SHIFTS ON NEURAL FUNCTION

A. Modulation of Function by Extracellular Alkaline Shifts

B. Modulation of Function by Activity-Dependent Acid Shifts

IX. CONCLUSION

	ABSTRACT

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	I. INTRODUCTION

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The importance of pH regulation to the viability of cells is widely recognized. The production of intracellular acid is a normal consequence of respiration and, if unchecked, can lead to a marked fall in intracellular pH, compromising vital functions. In this broad respect, the cells of the central nervous system (CNS) do not differ from those found in other tissues. Indeed, many of the mechanisms responsible for long-term "housekeeping" of hydrogen ions are similar.

The cellular diversity of the CNS is unique, however, and significant differences can be found in the complement of acid transport mechanisms expressed among subtypes of neurons and glial cells. This has been made apparent by physiological experiments on particular cells and by molecular studies that have revealed regional diversity in the expression of transporter subtypes.

The study of pH in the CNS is also distinguished by the occurrence of rapid increases or decreases in H⁺ that arise from electrical activity. These changes take place in time frames from milliseconds to minutes, involve neurons as well as glia, and occur in both the intracellular and extracellular compartments. Given numerous enzymes and ion channels that can be influenced by pH, a possible modulatory role of these pH transients has often been considered, particularly in relation to membrane potential and excitability. In this respect, the mechanisms that generate and regulate these pH changes are of considerable neurobiological interest.

The regulation of brain pH, the generation of activity-dependent pH changes, and their functional consequences have been the subject of earlier reviews (76, 80, 94). A comprehensive treatment of pH and brain function has also appeared in an excellent volume edited by Kaila and Ransom (152). The present contribution aims to provide an accessible, updated summary of this field and is consequently more circumscribed. To maintain reasonable limits, certain subjects have not received full coverage. Historical aspects and methodological issues have been described elsewhere (76, 149). The effect of pH on numerous enzymes and channels is an extensive subject that is also beyond the scope of this summary. With the exception of particularly notable studies, the reader is directed to various reviews of this topic (22, 211, 288, 298, 307, 319). Important pioneering work on pH regulation in the nervous system of invertebrates has been covered by several earlier contributions (76, 94, 259, 303). This review emphasizes pH studies in the mammalian CNS and ventures into invertebrate studies only when they are particularly relevant to the topic at hand.

	II. BUFFERING POWER

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The study of pH regulation requires the establishment of fundamental concepts of pH buffering and a consistent nomenclature. The definition and measurement of pH, the mechanisms of extracellular and intracellular buffering, and the chemical equilibria involved have been the subject of previous comprehensive reviews (55, 76, 259, 303). The purpose of this section is to highlight the basic concepts of buffering power relevant to physiological studies of pH, before addressing the means of intracellular and extracellular pH regulation in the nervous system.

The buffering power of aqueous solutions can be defined in a number of ways (157, 259). The most extensively used definition comes from the early work of Koppel and Spiro (172) and Van Slyke (323), where buffering power () is conceptualized as the concentration of added strong acid (or base) divided by the resulting change in pH. Thus

(1)

or

(2)

where A and B are the concentrations of strong acid or base, respectively. Accordingly, the buffering power (or buffering capacity) has units of concentration.

If one could analyze cytosol in the absence of organellar or metabolic influences, the buffering capacity would be determined by the summed contributions of the separate buffer species. In physiological studies, it is convenient to divide the total intracellular buffering capacity into the bicarbonate-dependent component and the nonbicarbonate or "intrinsic" buffering capacity, given the symbols _b and _i, respectively. The total intracellular buffering capacity (_T) is simply the sum of _b and _i.

The contribution to buffering provided by the CO₂-HCO₃^- equilibria (_b) can be approximated as

(3)

where [HCO₃^-]_i is the intracellular HCO₃^- concentration. The derivation of this formula assumes that cytosolic CO₂ is constant, due to its equilibration with extracellular CO₂, which serves as a fixed, infinite source. Such a system is said to be "open" with respect to CO₂. If one knows the system PCO₂ and the intracellular pH (pH_i), then [HCO₃^-]_i and _b can be calculated from rearrangement of the Henderson-Hasselbalch equation as

(4)

where PCO₂ is expressed in Torr and S is the solubility coefficient of CO₂ in mM/torr. It should be noted that the value of _b increases steeply as pH_i rises, being 2.5-fold greater at pH_i 7.4 versus 7.0.

In one study of cultured mammalian neurons, intracellular HCO₃^-, while present in significant concentration, was found to contribute little to _T (7). The basis of this observation was not clear. Faced with a rapid acid-base challenge, the rate of CO₂-HCO₃^- buffering would depend on the presence of carbonic anhydrase (see sect. VIIB, 1-3). It is plausible that insufficient activity of this enzyme limited the contribution of _b under these circumstances.

The major components of _i are associated with the buffering contribution of imidazole residues and phosphate (55). Determination of _i for a cell is not always straightforward. To measure _i, one might apply a known concentration of acid or base (in the absence of CO₂ and HCO₃^-) and measure the resulting change in pH_i. This procedure, however, would rarely give an accurate value, since the imposed pH_i change would be countered by plasma membrane acid transport in addition to cytosolic chemical buffering. Acid or base challenges are therefore typically made under conditions in which these transporters are inactive (e.g., under pharmacological blockade or ion substitution) or are minimally active (e.g., using a series of small base challenges). Under these conditions, the ratio of the acid (or base) load to the pH_i (Eq. 1 or 2) is then considered to be a measure of _i. Although values of _i obtained with such methods are useful in analyzing pH regulatory processes, they should be considered as no better than convenient operational measures, since they ignore the probable contributions of metabolism and organellar transport to the cytosolic H⁺ balance. _i has been operationally determined in this way for a number of neurons and glial cells and tends to falls in a range from 5 to 30 mM (76, 159), but may be as high as 40-50 mM in cells of neonatal brain (256). The value of _i can vary with pH_i and typically increases as pH_i falls (for examples, see Refs. 26, 35, 159).

Determination of the intracellular buffering capacity becomes critical when analyzing acid-base transport across the plasma membrane. The rate at which pH_i is changed by a transporter has quantitative relevance only in view of the buffering capacity that opposes the pH change. The molar rate of acid efflux from a cell (J) would therefore be given by

(5)

where J may be expressed as mM H⁺ extruded per unit time. A common mistake in studies of pH regulation is to compare values of dpH_i/dt instead of calculating J from

Equation 5. For example, if the rate of pH_i recovery from an acid load were the same in the presence and absence of CO₂-HCO₃^-, one could not assert that HCO₃^--dependent acid extrusion played no role. On the contrary, since _T is greater in the CO₂-HCO₃^--buffered media, one should conclude that the acid efflux rate (J) had increased and that HCO₃^--dependent acid extrusion was indeed present. In such studies, comparisons of J are best performed at the same baseline pH_i, since _b rises, and _i typically falls with elevation of pH_i.

	III. REGULATION OF INTRACELLULAR pH IN VERTEBRATE NEURONS

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The plasma membrane transport systems responsible for neuronal pH_i regulation were first investigated in large invertebrate cells. The modern period of this work was initiated by Roos, Boron, and Thomas, who have reviewed their classic studies (259, 303). The essential features of acid transport systems described in invertebrate neurons can also be found in vertebrate nerve cells. In the first investigation of a vertebrate neuron, the giant reticulospinal cells of the lamprey were found to eliminate acid loads by an amiloride-sensitive Na⁺/H⁺ exchanger and an Na⁺-, HCO₃^--, and Cl^--dependent acid extruder (75, 83). In subsequent studies of mammalian neurons, Na⁺/H⁺ exchange and Na⁺-dependent bicarbonate transport were frequently noted. In addition, some nerve cells were found to express a passive Cl^-/HCO₃^- exchanger, which served as an acid-loading mechanism during recovery from cytosolic alkalization. Significant differences in the function, expression, and pharmacological sensitivity of these transporters have been noted in the investigation of mammalian neurons. Details of these studies are provided below and are summarized in Tables 1 and 2.

A. Neurons From Hippocampus

Neurons derived from the hippocampus have been the subject of the most extensive studies of intracellular pH (pH_i) regulation in the mammalian nervous system. Cultured, fetal, rat hippocampal neurons were investigated by Raley-Susman et al. (250). A key finding was the presence of an Na⁺/H⁺ exchanger insensitive to amiloride and its derivatives. This transporter was the principal mechanism for maintenance of resting pH_i and for recovery from acid loads. Evidence for Na⁺/H⁺ exchange in these cells was based on the ion dependence of pH_i regulation in HCO₃^--free media. Thus an acidification was noted upon removal of external Na⁺, and acid extrusion was blocked in the absence of Na⁺, but could be supported by Li⁺. In HCO₃^--containing solutions, pH_i recovery from acid loads was slightly inhibited by the stilbene derivative DIDS and was Na⁺ dependent, suggesting that a form of Na⁺-linked HCO₃^- influx also contributed to net acid extrusion. Whether this mechanism was Cl^- dependent was not addressed.

Baseline pH_i of the fetal hippocampal neurons was not affected by removal of Cl^- or by application of DIDS, indicating that the fetal cells did not utilize a passive Cl^-/HCO₃^- exchanger as an acid-loading mechanism (250). This feature was confirmed in a later study of acutely isolated, fetal hippocampal neurons (251). In contrast, acutely isolated adult hippocampal neurons exhibited a pronounced alkalization upon removal of external Cl^-. In situ hybridization studies targeting the brain isoform of the Cl^-/HCO₃^- exchanger (see sect. VB3) were in agreement with these findings. Although the message could be identified in fetal brain (171, 251), there was a greater predominance in mature tissue (251).

Later studies of the acid extrusion mechanisms in fetal hippocampal neurons were undertaken by Baxter and Church (20). An Na⁺/H⁺ exchanger insensitive to ethylisopropylamiloride (EIPA) was noted, consistent with earlier data (250). However, in contrast to the previous studies, removal of Cl^- produced a DIDS-sensitive, Na⁺-independent alkalization, suggesting the presence of a passive Cl^-/HCO₃^- exchanger in the fetal-derived neuronal culture. Transition from HCO₃^--free (i.e., HEPES-buffered) to HCO₃^--containing solution induced a robust alkalization that was Na⁺ dependent and was blocked by DIDS, or by depletion of internal Cl^-. These observations suggested that fetal hippocampal neurons can extrude acid using both Na⁺/H⁺ exchange and an Na⁺-driven, Cl^-/HCO₃^- exchanger. The latter process appeared to play a greater role in pH_i regulation at room temperature. Thus transition from HEPES to HCO₃^- buffer induced an alkalization, or speeded recovery from acid loading, only at 18-22°C, and application of DIDS caused a fall in pH_i at this low temperature, but not at 37°C. At the warmer, physiological temperature, acid extrusion appeared to be dominated by Na⁺/H⁺ exchange.

Noncultured hippocampal neurons were investigated by Schwiening and Boron (273). These studies utilized a specific population of pyramidal neurons, acutely isolated from the hippocampal CA1 region of 4- to 14-day-old rats. In the absence of HCO₃^-, the cells exhibited a low baseline pH_i (a mean of 6.81) and sluggish recovery from acid loads. In agreement with previous studies of fetal, hippocampal neurons (250), this pH_i recovery was inhibited by removal of Na⁺ (suggesting the presence of Na⁺/H⁺ exchange) and showed little sensitivity to amiloride. Unlike fetal hippocampal cultures, where Na⁺/H⁺ exchange was predominant (20), this mechanism played a minor role in acutely dissociated pyramidal neurons (in both the establishment of baseline pH_i and the extrusion of acid at 37°C). With addition of external HCO₃^-, a marked increase in baseline pH_i occurred. This HCO₃^--dependent acid extrusion required external Na⁺ as well as internal Cl^- and was sensitive to DIDS, indicating the operation of an Na⁺-driven Cl^-/HCO₃^- exchanger. It was notable that the demonstration of Cl^- dependence could not be achieved by incubation in Cl^--free media for up to 3 h. To adequately deplete intracellular Cl^-, repeated activation of the transporter in Cl^--free solution was required. These observations urge caution in the interpretation of experiments in which exposure to Cl^--free solution fails to alter pH_i regulation.

A study of pH_i regulation in both postnatal immature and mature CA1 neurons from rat was undertaken by Bevensee et al. (24). The basic properties of pH_i regulation were similar in the two age groups and featured an EIPA-insensitive Na⁺/H⁺ exchanger and a robust stimulation of acid extrusion by HCO₃^-, attributed to Na⁺-driven Cl^-/HCO₃^- exchange. A fraction of neurons exhibited a slow recovery from acid loads in the absence of external Na⁺, suggesting the presence of an H⁺ pump. Similar Na⁺-independent pH_i recovery was later noted in CA1 neurons acutely isolated from mouse (358). The principal difference between immature and mature CA1 neurons was related to the distribution of baseline pH_i. Acutely dissociated neurons derived from both age groups were found to have a wide range of initial pH_i values (6.3-7.7) in HCO₃^--free, HEPES-buffered media. Mature neurons displayed a bimodal distribution, falling into low pH_i (mean of 6.68) and high pH_i (mean of 7.32) categories. For high pH_i neurons, the relationship between pH_i and the acid extrusion rate was shifted in the alkaline direction and had a greater slope, both in the absence and presence of HCO₃^-.

The presence of a predominant Na⁺/H⁺ exchanger with low sensitivity to amiloride distinguishes hippocampal neurons from the great majority of cells. The nature of this amiloride resistance and its relationship to transporter isoforms remains unclear (see sect. VA). It is notable that acutely isolated CA1 neurons from mice displayed an Na⁺/H⁺ exchanger inhibited by the agent HOE 694 (358). However, the Na⁺/H⁺ exchanger of cultured fetal hippocampal neurons was insensitive to this drug (20).

B. Neurons From Cerebral Cortex

The regulation of pH_i in cortical neurons has been studied in cultured fetal cells obtained from rat (236) and mouse (245). In cultured fetal rat neurons, recovery from intracellular acid loads was partially inhibited by amiloride (or 5-N-methyl-N-isobutylamiloride) and was dependent on external Na⁺, indicating the operation of an Na⁺/H⁺ exchanger (236) [inhibition of acid extrusion by amiloride was also noted by Amos and Richards (8) in cultures of rat neocortical neurons]. Ou-Yang et al. (236) suggested the absence of HCO₃^--dependent acid extrusion in fetal neurons, based on the following: 1) the similarity of acid extrusion rates in HCO₃^--free and HCO₃^--containing solutions, 2) the failure of DIDS or Cl^- removal to inhibit recovery from acid loading, and 3) similar acid extrusion rates in the presence of amiloride versus amiloride plus DIDS. In contrast, recovery from alkaline loads was inhibited by DIDS and by the absence of Cl^-, suggesting the presence of a passive Cl^-/HCO₃^- exchanger.

The putative absence of HCO₃^--dependent acid extrusion in the cortical neurons studied by Ou-yang et al. (236) warrants comment. Although calculated acid efflux rates were similar in the presence and absence of HCO₃^-, analysis was highly dependent on accurate determination of the _i. It should also be noted that the method of acid loading in these studies was unorthodox, consisting of a transition from 5 to 15% CO₂ with compensatory increases in solution HCO₃^- (and decrease in Cl^-) to maintain a constant bath pH. Given the presence of a Cl^-/HCO₃^- exchanger in these cells, the changes in HCO₃^- and Cl^- could have confounded interpretation of the pH_i recoveries. This study also reported that addition of DIDS to amiloride-containing solution did not further reduce acid extrusion. How this experiment was carried out is uncertain, as a precipitate commonly forms when DIDS is combined with amiloride or one of its derivatives (unpublished observation). It is worth noting that like hippocampal neurons (273), the steady-state pH_i of the cultured cortical cells was higher in HCO₃^--containing media, which would be consistent with the presence of a HCO₃^--dependent acid extrusion mechanism.

In a later study of cultured fetal neurons from mouse cortex, it was suggested that cells utilized both Na⁺/H⁺ exchange and an HCO₃^--dependent acid extruder (245). After acid loading with the NH₄⁺ prepulse method (38) in HCO₃^--containing media, the pH_i recovery was markedly reduced by EIPA, indicating the operation of an Na⁺/H⁺ exchanger. The rates of pH_i recovery in the presence and absence of HCO₃^- (and during exposure to DIDS) were consistent with a HCO₃^--dependent component of acid efflux. However, the dpH_i/dt values were not converted to acid extrusion rates (Eq. 5), and therefore the relative contribution of Na⁺/H⁺ exchange versus HCO₃^--dependent processes remained unclear. The authors suggested that recovery from alkaline loads occurred by a DIDS-insensitive Cl^-/HCO₃^- exchanger. In addition, a passive H⁺ influx through voltage-gated H⁺ channels was proposed, since exposure to 1 mM Zn²⁺ slowed the recovery from alkaline loads. Such channels, however, would not be open near the resting membrane potential, as they typically activate near 0 mV. Moreover, there is no compelling evidence for their presence in vertebrate neurons (92).

C. Neurons From Cerebellum

Two studies of nerve cells cultured from rat cerebellum have been performed. Gaillard and Dupont (113) investigated Purkinje cells from neonatal (P0-P1) rats after 6-7 days in culture. In HCO₃^--free media, recovery from acid loading was abolished by withdrawal of external Na⁺ or by exposure to amiloride, indicating the operation of an Na⁺/H⁺ exchanger. In media containing HCO₃^-, recovery from acid loading was also completely blocked by amiloride, which suggested the absence of HCO₃^--dependent acid extrusion. The presence of passive Cl^-/HCO₃^- exchange was indicated by the presence of a DIDS-sensitive, Na⁺-independent alkalization upon removal of external Cl^-. The steady-state pH_i of these cells was consistent with this complement of transporters. Unlike hippocampal (273) and cortical neurons (236), the steady-state pH_i of the cultured Purkinje cells was lower in HCO₃^--containing (7.06) versus HCO₃^--free media (7.40). This finding is consistent with steady-state acid loading via Cl^-/HCO₃^- exchange and the absence of HCO₃^--dependent acid extrusion.

Studies of rat cerebellar granule cells (cultured from 7-day-old pups) revealed a similar behavior of steady-state pH_i (248). In HCO₃^--containing solution, pH_i was lower (7.27) compared with HCO₃^--free media (7.49). Although completely dependent on external Na⁺, the recovery of pH_i from acid loads was only partially blocked by amiloride. In roughly half the cells, this recovery exhibited a dependence on HCO₃^- (but was not blocked by stilbenes). Recovery from acid loading persisted after prolonged exposure to Cl^--free media, suggesting the possible operation of Na⁺-HCO₃^- cotransport in these cells. However, as noted by Schwiening and Boron (273), internal Cl^- is not readily depleted from neurons and may require repetitive acid loading in Cl^--free solution to be sufficiently eliminated from the cytoplasm. The presence of a passive Cl^-/HCO₃^- exchanger in a fraction of the granule cells was suggested by the observation of a reversible alkalization upon withdrawal of external Cl^- (however, this effect was not blocked by stilbenes). The lower steady-state pH_i noted in HCO₃^- media was consistent with the presence of a Cl^-/HCO₃^- exchanger that contributed to steady-state acid loading.

D. Neurons From Spinal Cord

Recently, pH_i regulation was studied in neurons cultured from ventral spinal cord of 14- to 15-day rat embryos (46). In HCO₃^--free media, acid extrusion was mediated by amiloride-sensitive Na⁺/H⁺ exchange. In the presence of HCO₃^-, recovery from acid loading was also significantly slowed by 1 mM amiloride (but was not blocked by 40 µM EIPA). Steady-state pH_i was reduced by DIDS, suggesting that a HCO₃^--dependent acid extruder contributed to the maintenance of baseline pH_i. Recovery from acid loads in HCO₃^--containing media was slowed by DIDS and abolished in Na⁺-free solutions. It was suggested that this recovery was mediated by an electrogenic Na⁺-HCO₃^- cotransporter, since a reversible alkalization was observed when external K⁺ was raised to 30 mM. Whether this alkalization was dependent on Na⁺ or HCO₃^- or could be evoked by other means of depolarization was not addressed, however. Thus experiments to more fully distinguish between Na⁺-driven Cl^-/HCO₃^- exchange and electrogenic Na⁺-HCO₃^- cotransport appear warranted. Removal of external Cl^- was found to elicit a reversible alkaline shift in the spinal cord neurons, suggesting the operation of a passive Cl^-/HCO₃^- exchanger. Baseline pH was lower in HCO₃^--containing media compared with HCO₃^--free, HEPES-buffered solution, consistent with steady-state acid loading via this mechanism.

E. Medullary Neurons

The neurons of the medulla oblongata are heterogeneous in form and function. Of particular interest in this region are the cells of the chemosensitive areas, which depolarize and fire in response to elevation of CO₂ or H⁺. The degree to which this response is triggered by a fall in pH_i, a fall in extracellular pH (pH_o), or elevation of CO₂ per se has been the subject of debate (218). Several recent studies have suggested that a fall in pH_i is the principal trigger for the chemosensitive response (110a, 342, 348). For such cells, therefore, the control of pH_i is a subject of great interest. The regulation of pH_i in chemosensitive neurons has recently been reviewed (249).

Putnam and colleagues (256-258) imaged pH_i from neurons of rat neonatal, medullary brain slices which had been incubated in BCECF. Neurons from two chemosensitive areas, nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), were compared with cells from the nonchemosensitive inferior olive (IO) and hypoglossal nucleus (HYP). In all cells, recovery from acid loading was completely abolished by amiloride but was insensitive to DIDS, suggesting that brain stem neurons utilize Na⁺/H⁺ exchange but not HCO₃^--dependent acid extrusion (257). In chemosensitive neurons, elevation of CO₂ induced a fall in pH_i that recovered only when bath pH was maintained at 7.48 (by simultaneous elevation of external HCO₃^- to 52 mM). In contrast, neurons from nonchemosensitive areas recovered when CO₂ was elevated, despite a fall in bath pH to 7.15 (at constant bath HCO₃^- of 26 mM). This difference was attributed to an Na⁺/H⁺ exchange mechanism in the chemosensitive neurons that was more readily inhibited by external H⁺ (256). Immunocytochemical and pharmacological data from chemosensitive neurons in organotypic culture have implicated the type 3 Na⁺/H⁺ exchanger isoform in these cells (348) (i.e., NHE-3; see sect. VA).

The presence of Cl^-/HCO₃^- exchange was suggested in VLM, HYP, and IO neurons, as a DIDS-sensitive alkalization was noted upon withdrawal of external Cl^-. This exchanger could mediate recovery from alkaline loads in neurons from all three of these regions; however, the Cl^-/HCO₃^- exchanger in VLM cells was inhibited by high bath pH (7.9), unlike the nonchemosensitive cells. In contrast, there was no apparent Cl^-/HCO₃^- exchanger in the chemosensitive NTS neurons, which accordingly, could not recover from alkaline loading (256).

F. Retinal Neurons and Photoreceptors

Early evidence for acid extrusion via Na⁺/H⁺ exchange was provided by Oakley and colleagues (160, 231) in studies of rod photoreceptors from toad. A characterization of pH_i regulatory mechanisms was later carried out in rods from frog (155) and salamander retina (264). Both amphibian rods utilized an amiloride-sensitive Na⁺/H⁺ exchanger. In addition, Na⁺-dependent acid extrusion was accelerated by HCO₃^- and inhibited by DIDS, suggesting the presence of an Na⁺-driven HCO₃^- transporter. Whether this mechanism was linked to Cl^- was not clear. Evidence for passive Cl^-/HCO₃^- exchange was found in both frog and salamander rods, as the withdrawal of Cl^- produced a rise in pH_i, which persisted in the absence of external Na⁺ in the case of frog rods (155). A study of retinal horizontal neurons from the skate also demonstrated the presence of amiloride-sensitive Na⁺/H⁺ exchange, and apparent HCO₃^--dependent acid extrusion, based on the inhibition of acid extrusion by DIDS (135).

Removal of external Cl^- had no effect on pH_i in the horizontal cells, suggesting that these neurons did not possess a passive Cl^-/HCO₃^- exchanger.

G. Sympathetic Neurons

One of the first studies of pH_i regulation in mammalian nerve cells utilized cultured rat sympathetic neurons (306). Acid extrusion was dependent on external Na⁺ and was inhibited by amiloride, providing clear evidence for Na⁺/H⁺ exchange. The role of HCO₃^--dependent acid extrusion in these cultured neurons was less certain. While addition of stilbenes did not affect recovery from acid loads, in some cases, pH_i recovery was stimulated by addition of 5-10 mM HCO₃^-.

H. Neoplastic Neural Cells

Neuroblastoma cells were among the first neural preparations in which Na⁺/H⁺ exchange was identified (21, 212). Acid extrusion via Na⁺/H⁺ exchange was subsequently reported in other studies of neural tumor cells (111, 204). Dickens et al. (100), studying neuroblastoma and pheochromocytoma (PC12) cells, noted the presence of Na⁺/H⁺ as well as Cl^-/HCO₃^- exchange. This paper also reported regional heterogeneity of pH_i, as the tips of extending neurites were more alkaline (by 0.2-0.3 pH units) than the cell body.

I. Synaptosomes

There is wide consensus that acid extrusion from brain synaptosomes is mediated by amiloride-sensitive Na⁺/H⁺ exchange (146, 216, 265, 268). It has been reported that there is no HCO₃^--dependent acid extrusion in synaptosomes (216, 265). This finding was based on similar rates of recovery from acidosis in the presence and absence of HCO₃^- and lack of sensitivity to stilbene derivatives. However, an identical dpH/dt in the presence of HCO₃^- would imply a greater rate of acid efflux, since intracellular buffering capacity should be elevated in the presence of CO₂ and HCO₃^-. Therefore, the possibility of HCO₃^--dependent acid extrusion in these preparations cannot be excluded. A rise in pH_i was noted in synaptosomes exposed to elevated external K⁺ (265) [although in an earlier study (254), elevated external K⁺ did not produce a similar rise in pH_i]. This apparent depolarization-induced alkalization (DIA) persisted in the absence of HCO₃^- and Na⁺, suggesting that it was not attributable to electrogenic Na⁺-HCO₃^- cotransport. The cause of the DIA was not elucidated.

Synaptosomal acid transport linked to a Cl^- flux was suggested by one study in which withdrawal of external Cl^- caused a DIDS-sensitive rise in pH_i. This alkalization, however, was not dependent on HCO₃^-, suggesting the presence of a Cl^--H⁺ cotransport mechanism (195). A similar putative Cl^--H⁺ cotransporter (or equivalently, a Cl^-/OH^- exchanger) has been noted in cardiac myocytes (183).

	IV. REGULATION OF INTRACELLULAR pH IN GLIAL CELLS

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The pH regulatory mechanisms of glial cells are comprised of transport systems very similar to those found in neurons. Physiological studies suggest that glia differ from neurons and from one another in their expression of Na⁺-linked bicarbonate transporters. Electrogenic Na⁺-HCO₃^- cotransporters appear to be especially prevalent among glial cells. Details elaborated below are summarized for glial cells according to cell type in Table 3.

A. pH Regulation in Nonmammalian Glial Cells

The first detailed investigations of glial pH_i were conducted on invertebrate cells which could withstand long-term penetration with pH-sensitive microelectrodes. Pioneering work on the giant glial cells of the leech demonstrated the presence of at least three acid extrusion mechanisms: an Na⁺/H⁺ exchanger, an Na⁺-driven Cl^-/HCO₃^- exchanger, and an electrogenic Na⁺-HCO₃^- co-transporter (95, 96, 98, 293). Later work indicated the additional presence of a passive Cl^-/HCO₃^- exchanger in these cells (294). Evidence for Na⁺/H⁺ exchange (11) and electrogenic Na⁺-HCO₃^- cotransport (12) was also reported for glial cells of the mudpuppy optic nerve. This work is well covered by the review of Deitmer and Rose (94).

B. pH Regulation in Mammalian Astrocytes

Studies of intracellular pH in mammalian glia have focused mainly on astrocytes. With a few exceptions, these studies have made use of pH-sensitive fluorescent dyes rather than pH microelectrodes. Almost all investigations have been performed on cultured astrocytes. Generally, the mechanisms of acid transport appear similar to those found in the invertebrate glia; however, some notable differences exist among mammalian astrocyte studies.

1. Principal acid extrusion mechanisms in mammalian astrocytes

The presence of Na⁺/H⁺ exchange in mammalian astrocytes is well established and uniformly supported. Kimelberg and colleagues (163, 166) provided early evidence for Na⁺/H⁺ exchange in primary astrocyte cultures based on detection of Na⁺-dependent extracellular pH shifts. The use of amiloride or its derivatives to acidify baseline pH_i or inhibit pH_i recovery from acid loads served as the principal evidence for Na⁺/H⁺ exchange in later astrocyte studies (26, 53, 87, 205, 247, 277). The presence of an Na⁺/H⁺ exchanger in mammalian astrocytes has also been confirmed by immunocytochemical studies which revealed the presence of the amiloride-sensitive NHE-1 isoform of this transporter (247) (see sect. VA).

In some studies, the amiloride analog EIPA appeared ineffective against the astrocyte Na⁺/H⁺ exchanger (44) or caused a paradoxical increase in pH_i (45, 247). Bevensee et al. (26) reported that while amiloride and EIPA could inhibit acid extrusion with similar efficacy at low pH_i, EIPA appeared less effective near baseline pH_i. Moreover, a fall in pH_i was consistently produced by amiloride but not by EIPA. The basis of these observations remains unclear.

Inhibition of Na⁺/H⁺ exchange by low external pH was described by Aronson et al. (10) in their studies of the renal transporter. A similar property was noted by Mellergard and Siesjo (205) who found that cultured astrocytes failed to extrude acid in bicarbonate-free media when the external pH was <6.9. This feature has significant implications for the role of astrocyte Na⁺/H⁺ exchange in normal versus pathological settings. While culture studies show a significant contribution of this transporter when astrocyte pH_i approaches 6.0 (26), such a low cytosolic pH would probably only arise in vivo under ischemic conditions, where it would be accompanied by a prominent fall in pH_o, in the range of 6.2-6.9 (174-176, 215, 220, 332). Given the inhibition of this transporter by external H⁺, it seems that the astrocyte Na⁺/H⁺ exchanger would never operate within the pH_i range that caused its maximum activation in tissue culture. In fact, under conditions which simulated the external ion shifts, acidosis and hypoxia of ischemic brain, the pH_i of cultured astrocytes fell to 6.7-6.8 and exhibited little or no recovery of pH_i over a 20- to 30-min period (28).

Virtually all studies agree on the presence of Na⁺-and HCO₃^--linked acid transport in astrocytes. Differences exist regarding the role of Na⁺-driven Cl^-/HCO₃^- exchange and electrogenic Na⁺-HCO₃^- cotransport. Using mouse cortical astrocytes, Chow et al. (87) reported that recovery from acid loads was not Cl^- dependent, and therefore suggested that a form of Na⁺-HCO₃^- cotransport was involved. In contrast, Mellergard et al. (203) noted diminished acid extrusion after withdrawal of Cl^- and suggested that their cultured rat cortical astrocytes utilized Na⁺-driven Cl^-/HCO₃^- exchange, but not Na⁺-HCO₃^- cotransport. Curiously, they reported a DIA, a phenomenon typically associated with an electrogenic Na⁺-HCO₃^- cotransporter (see below). Shrode and Putnam (277) provided strong support for both Na⁺-driven Cl^-/HCO₃^- exchange and electrogenic Na⁺-HCO₃^- cotransport in their cultured rat cortical astrocytes. A later study of cultured cerebellar astrocytes also found evidence for both exchangers (167). A detailed examination of pH_i regulation in cultured rat hippocampal astrocytes was conducted by Bevensee et al. (23, 26). Using a fluorescent Cl^--sensitive dye to confirm the depletion of cytosolic Cl^-, these authors were able to convincingly demonstrate that HCO₃^--linked acid extrusion did not depend upon Cl^- and, accordingly, was attributed to Na⁺-HCO₃^- cotransport.

Differences in the identification of acid extrusion mechanisms among astrocytes could be attributed to a number of factors. Discrepancies may arise due to use of low Cl^- media to test for Cl^--dependent transport. Unless depletion of internal Cl^- can be assured, failure to inhibit acid transport after Cl^- removal cannot be considered convincing (whereas inhibition of transport after Cl^- withdrawal clearly supports Cl^- dependence) (277). Differences in data may also originate from altered expression of transporters, arising from varied culture conditions, species of origin (e.g., mouse vs. rat) or brain region (e.g., cortex, vs. cerebellum vs. hippocampus).

2. The astrocyte Na⁺-HCO₃^- cotransporter

The Na⁺-HCO₃^- cotransporter of astrocytes has received considerable attention due to its electrogenic character and the resulting dependence on membrane potential. A form of this transport mechanism was first described in renal proximal tubule (37), and with the exception of one study (203), its expression in mammalian astrocytes is agreed upon. Among glia, the presence of this transporter was first reported in invertebrates, where Na⁺-HCO₃^--dependent, electrogenic acid extrusion (13, 96), and a membrane potential-driven shifts in pH_i were described (98).

The first clue to the presence of this transporter in mammalian astrocytes came from the observation of a depolarization-induced alkalization (DIA) in rat cortical astrocytes studied in vivo. During stimulation of surrounding cortex, a rapid rise in astrocyte pH_i was correlated with a depolarization of the astrocyte membrane, attributed to elevation of extracellular K⁺ concentration ([K⁺]_o) (81). Addition of Ba²⁺ prevented both the activity-evoked depolarization (17) and the cytosolic alkalization, despite a similar evoked rise in [K⁺]_o (82).

Data consistent with electrogenic Na⁺-HCO₃^- cotransport was subsequently reported in cultured astrocytes (23, 50, 53, 232, 277), reactive astrocytes in gliotic brain slices (128, 129), and acutely isolated astrocytes from retina (221, 223, 224). This body of evidence is formed by two related observations. The first is the occurrence of a hyperpolarization (or an outward current) upon transition from bicarbonate-free to bicarbonate-containing media, which may be inhibited by DIDS or related stilbene derivatives (53, 221, 224, 232). A potential diffi-culty with this criterion is that the presence of a bicarbonate conductance could give rise to a similar shift in membrane potential (or membrane current under voltage clamp). In a novel, alternative approach, Bevensee et al. (23) withdrew external sodium to reverse the transporter and demonstrated a stilbene-inhibited depolarization.

The second observation favoring electrogenic Na⁺-HCO₃^- cotransport has been the presence of a DIA in astrocytes. This rise in pH_i is typically Na⁺ and HCO₃^- dependent and is often blocked by stilbenes (50, 129, 222, 223, 240). In gliotic brain slices, however, an Na⁺-independent component of the DIA and an insensitivity to stilbenes were noted in reactive astrocytes (129).

The dependence of the transport direction upon voltage is governed by the difference between membrane potential and the equilibrium potential for a Na⁺-HCO₃^- cotransporter (E_NBC). The general relation for E_NBC is given by

(6)

where n is the net HCO₃^--Na⁺ transport ratio (37, 96). The preponderance of evidence suggests that the astrocyte mechanism has a stoichiometry of 2:1 (23, 50, 232) and that the net change in transport elicited by membrane depolarization is an influx of Na⁺ and HCO₃^-.

For most astrocytes, values of E_NBC would be on the order of -80 mV (76), and given a membrane potential of similar magnitude, the transporter would normally be close to equilibrium. Some astrocytes have membrane potentials of -50 mV or less, however (32, 287). For these cells, the energetics of transport may favor a large, steady-state influx of Na⁺ and HCO₃^- (provided E_NBC was maintained at a more negative value). However, little is known about how E_NBC or transporter stoichiometry varies among astrocyte subtypes.

A notable exception comes from the work of Newman (221, 222, 224) who investigated Na⁺-HCO₃^- transport in acutely dissociated Muller cells of the salamander retina. These specialized glia are sometimes considered to be astrocyte variants due to their expression of glial fibrillary acidic protein (27). Muller cells were found to have a transporter with a reversal potential near 0 mV, consistent with a stoichiometry of one Na⁺ per three HCO₃^-. As such, the energetics would favor the efflux of these ions at normal negative membrane potentials. Physiological studies found the transporter predominantly localized to the distal end foot process which normally abuts the vitreous humor. This led to the suggestion that the transporter is specialized to shuttle metabolically produced CO₂/HCO₃^- out of the retina (221). In later studies, evidence for Na⁺-HCO₃^- cotransport was also found in Muller cells and astrocytes of rat retina (223).

Numerous questions about localization, function, and role of astrocytic Na⁺-HCO₃^- cotransporters remain unsettled. One issue is whether a DIA can always be completely attributed to this mechanism, since this alkalizing response, albeit diminished, can sometimes occur in HCO₃^--free media (42, 44, 50, 98, 204, 239). These observations might be explained by a mechanism with a high affinity for HCO₃^-, capable of utilizing the small concentrations of this ion generated from metabolically derived CO₂ (97). Whether this carrier actually utilizes HCO₃^- or CO₃^2- in the transport mechanism has not been settled, however (see sect. VIIB6).

From the functional standpoint, Na⁺-HCO₃^- cotrans-port appears capable of net acid extrusion in response to cytosolic acidification, as well as the modulation of transmembrane pH, in response to changes in membrane potential. The latter property may be a normal and unique feature of astrocytes, since neural activity rapidly depolarizes glial cells as a result of [K⁺]_o elevation (132, 161, 234, 253, 284). The astrocyte Na⁺-HCO₃^- cotransporter may therefore play a role in the modulation of both intracellular and interstitial pH (see sects. VIB and VIIB6).

3. Na⁺-independent acid extrusion in astrocytes

While the ability of astrocytes to extrude acid loads appears to be mainly due to a combination of Na⁺/H⁺ exchange and Na⁺-linked HCO₃^- transport, reports of Na⁺-independent transport have appeared. Recording with pH microelectrodes from cultured mouse astrocytes in HCO₃^--free media, Walz (353) noted a recovery from acute acid loads that was unaffected by either amiloride or withdrawal of external Na⁺. This result stands in marked contrast to a large body of evidence indicating that amiloride-sensitive Na⁺/H⁺ exchange is the principal acid extrusion mechanism of astrocytes in the absence of HCO₃^- (see above). However, it may be noted that penetration of the mouse astrocytes with microelectrodes was made possible after rounding the cells by several hours of exposure to 1 mM dibutyryl cAMP (339). In response to this treatment, it is plausible that changes in transporter expression occurred. Downregulation of the Na⁺/H⁺ exchanger and the expression of a plasmalemmal H⁺-ATPase could account for these results.

Pappas and Ransom (239) were able to uncover a plasmalemmal vacuolar H⁺-ATPase in cultured rat hippocampal astrocytes that made a small contribution to acid extrusion. This transporter was identified in HCO₃^--free media by a small, bafilomycin-sensitive component of acid extrusion that remained in the absence of external Na⁺ or the presence of amiloride. In agreement with these findings, V-type ATPase mRNA has been isolated from cultured astrocytes (246). However, in view of the small component of acid extrusion attributed to this mechanism, its normal function is unclear. In the hippocampal astrocyte study, addition of bafilomycin caused a small intracellular acidification, suggesting a role in the maintenance of steady-state pH_i (239). In cultured cortical astrocytes, however, the H⁺-ATPase inhibitors bafilomycin and concanamycin did not affect baseline pH_i (5).

4. Cl^-/HCO₃^- exchange in astrocytes

The presence of a Cl^-/HCO₃^- exchanger in astrocytes was suggested in early studies of astrocyte Cl^- fluxes by Kimelberg et al. (163). In most subsequent reports, the expression of this transporter was noted by the observation of a cytosolic alkalization upon removal of external Cl^-. This alkalosis was reported in cerebellar (53) and cortical astrocytes (203), where it was inhibited by DIDS. In contrast, in hippocampal astrocytes, withdrawal of Cl^- did not alter pH_i notably, and it was suggested that this transporter was not prominent in these cells (23). Shrode and Putnam (277) cautioned that alkalization upon withdrawal of external Cl^- could be mediated by reversal of a Na⁺-coupled Cl^-/HCO₃^- exchanger. To convincingly demonstrate the presence of passive Cl^-/HCO₃^- exchange in cortical astrocytes, these investigators showed that removal of external Cl^- produced a DIDS-sensitive intracellular alkalization that persisted in the absence of Na⁺. This process appeared to be active only at an elevated baseline pH_i, suggesting a role in the recovery from alkaline loads.

C. pH Regulation in Oligodendroglia

A detailed study of pH_i regulation in oligodendrocytes from mouse spinal cord was conducted by Kettenmann and Schlue (162). These cells displayed a high baseline pH_i, ranging from 7.2-7.9 in HCO₃^--containing media of pH 7.4. In the absence of HCO₃^-, pH_i was lower, and recovery from acid loading occurred via a typical amiloride-sensitive Na⁺/H⁺ exchange mechanism. In HCO₃^--containing media, an additional acid extruder was identified that was Na⁺ dependent and apparently not linked to Cl^-. The observation of a DIA (upon elevation of [K⁺]_o) suggested that this mechanism could be an electrogenic Na⁺-HCO₃^- cotransporter; however, this process was unaffected by stilbenes or furosemide. The presence of a Cl^-/HCO₃^- exchanger in these oligodendrocytes was uncertain. Removal of external Cl^- resulted in a higher baseline pH_i, consistent with passive Cl^-/HCO₃^- exchange; however, the removal of HCO₃^- had no complementary effect on Cl^- transport (138).

Further studies of oligodendroglia were carried out by Gaillard and colleagues (41, 42) using cultured cerebellar cells. In mature oligodendrocytes, baseline pH_i was 7.04 in HCO₃^--containing media (a value considerably lower than that of cultured spinal oligodendrocytes) and was unaffected by stilbenes or Cl^- removal, suggesting the absence of a Cl^-/HCO₃^- exchanger. In oligodendrocyte precursors, however, a DIDS-sensitive, passive Cl^-/HCO₃^- exchanger was identified. Steady-state acid loading by Cl^-/HCO₃^- exchange accounted for a lower baseline pH_i of the precursor cells, which averaged 6.88 (41). The differences in pH_i noted during oligodendrocyte development may be related to the events leading to termination of cell division, as has been suggested for rat astrocytes (241).

In HCO₃^--free media, both mature cerebellar oligodendrocytes and precursor cells extruded acid via an amiloride-sensitive Na⁺/H⁺ exchanger (41, 42). In the presence of HCO₃^-, an electrogenic Na⁺-HCO₃^- cotransporter was also identified. This transporter was unaffected by DIDS as was noted earlier for spinal cord oligodendrocytes (162). Analysis of the rate of alkalization and the calculated HCO₃^- influx induced by graded elevations of external K⁺ suggested a reversal potential close to -60 mV, consistent with an Na⁺-HCO₃^- transport stoichiometry of 1:3 (42). Since the resting potential of these cells was also near -60 mV, it was suggested that this transporter was normally close to equilibrium.

D. pH Regulation in Schwann Cells

Primary cultures of Schwann cells from rat sciatic nerve were studied by Nakhoul et al. (217). In addition to a classic, amiloride-sensitive Na⁺/H⁺ exchanger, these cells exhibited a passive Cl^-/HCO₃^- exchanger, revealed by the presence of an Na⁺-independent alkalization upon removal of external Cl^-. The Cl^-/HCO₃^- exchanger of the Schwann cells was unaffected by DIDS. These experiments did not address whether an Na⁺-linked HCO₃^--dependent acid extrusion mechanism was present.

A role for pH_i in the proliferation of Schwann cells was suggested in an early study in which mitogenic stimulation resulted in an apparent rise in pH_i (267). This alkaline shift was associated with the Na⁺/H⁺ exchanger. Inhibition of Na⁺/H⁺ exchange after addition of a mitogen significantly reduced the degree of subsequent mitosis.

E. pH Regulation in Glial Tumor Cells

The regulation of pH_i was studied in C6 glioma cells by Shrode and Putnam (277) in conjunction with their investigation of rat cortical astrocytes. The glioma cells shared two acid extrusion mechanisms with the astrocytes: a conventional, amiloride-sensitive Na⁺/H⁺ exchanger and a DIDS-sensitive, Na⁺-driven Cl^-/HCO₃^- exchanger. Unlike the astrocytes, the glioma cells did not have an electrogenic Na⁺-HCO₃^- cotransporter, as they displayed no depolarization-induced alkalization upon elevation of external K⁺. A second study of C6 glioma cells (utilizing NMR spectroscopy) also presented evidence for Na⁺/H⁺ exchange and HCO₃^- transport (111). A comparison of pH_i regulation in glioma cell lines (C6 and human U-251, U-118, and U-87) versus primary cortical astrocyte cultures (199) reported a higher steady-state pH_i in the gliomas (also noted by Shrode and Putnam for C6 cells) (277) and a higher rate of recovery from acid loads. These effects were attributed to enhanced activity of the Na⁺/H⁺ exchanger in the glioma cell lines, which was not attributed to genetic alterations. Disparity in the efficacy or expression of the HCO₃^--dependent acid transporters was also noted, as the astrocytes rapidly alkalinized upon introduction of CO₂-HCO₃^-, whereas the glioma cells underwent a marked acidification.

A study by Volk et al. (331) provided evidence for a vacuolar H⁺-ATPase in C6 glioma cells, based on a fall in pH_i observed after application of H⁺-ATPase inhibitors. A voltage-clamp study of bafilomycin-sensitive currents from C6 glioma cells provided additional data supporting the presence of an electrogenic plasmalemmal H⁺-ATPase in these cells (246). In a comparison of vacuolar ATPase mRNA in C6 cells, primary astrocyte cultures, and immortalized astrocytes, no differences in levels were noted (246). However, an equivalence in H⁺-ATPase message may not imply similar activity of the transporter in glioma cells and astrocytes. Indeed, significant differences between these cells have been noted in this regard. For example, in the study of Volk et al. (331), the pronounced fall in the steady-state pH_i during application of H⁺-ATPase inhibitors suggested a particularly prominent role for this mechanism in C6 cells. In contrast, H⁺-ATPase inhibitors had little or no effect on baseline pH_i in cultured astrocytes (5, 239).

F. pH Regulation and Volume Control in Glia

The activation of acid transport mechanisms can lead to a net transmembrane flux of osmoles, resulting in changes in cell volume. Linkages between volume regulation of glial cells and the regulation of cytosolic pH can therefore be anticipated. For example, activation of Na⁺/H⁺ exchange can lead to glial swelling, owing to the accumulation of Na⁺ (143a). In addition, the regulatory response to changes in osmolarity involving taurine transport can have immediate, direct effects on pH_i (234a). Due to the role of glial swelling in clinical brain edema, this subject has received considerable attention in literature beyond the scope of the present review. For an overview of this topic, the contribution of Kempski et al. (161a) may be consulted.

	V. MOLECULAR IDENTIFICATION OF BRAIN ACID TRANSPORTERS

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Molecular correlates of several acid transport mechanisms have been identified in the mammalian CNS. Functional characterization in expression systems has served to confirm the physiological identity of these transporters. Localization of particular mRNAs and immunostaining of specific proteins have provided insights into possible regional functions of these molecules. Details provided below are summarized in Table 4.

A. Na⁺/H⁺ Exchangers in Brain

The gene family of Na⁺/H⁺ exchangers identified in vertebrate tissues consists of at least seven members, commonly denoted NHE-1 through NHE-7. Deduced amino acid sequences have indicated similarity in membrane-spanning domains and distinct putative regulatory domains in the cytosolic regions (54, 226, 336). The first member cloned was the human growth factor-activated NHE-1 (266), an abundant isoform thought to play a housekeeping role in maintenance of steady-state pH_i.

NHE-1 mRNA is widespread in the mammalian CNS (191, 235). Immunocytochemical analysis has also identified the protein in assays of whole tissue (105, 247) and astrocyte cultures (247). Transcripts, localized by Northern analysis and in situ hybridization, revealed a marked presence in hippocampus, periamygdaloid cortex, and cerebellum (191). A general increase in message (191) and protein (105) was noted in postnatal cortex. This observation may account for the lower resting pH_i and the slower recovery from acid loading reported in fetal versus adult hippocampal neurons (24). However, NHE-1 is typically sensitive to amiloride (or its analogs) (226), a feature at odds with its predominance in hippocampus, where neuronal Na⁺/H⁺ exchange has little or no sensitivity to these agents (250, 273). It is undetermined whether this characteristic of CA1 pyramidal neurons is due to cell-specific alterations of NHE-1 or the presence of an alternative NHE isoform.

mRNA for other NHE isoforms has been reported in the mammalian CNS. NHE-2 message was noted in whole brain (343), and later localized to cerebral cortex and brain stem (105, 191), where the mRNA level was apparently one-tenth that of NHE-1 (191). Message for NHE-3, however, was limited to Purkinje cells of the cerebellum (191) (but has been noted in medullary chemosensitive neurons in organotypic cultures, Ref. 348).

An in situ hybridization study of NHE-4 reported a predominance of this isoform in hippocampus (30). Compared with NHE-1, -2 and -3, the sensitivity of NHE-4 to amiloride was low. NHE-4 may therefore appear to be a candidate for the neuronal transporter in hippocampus. However, concentrations of amiloride that had no effect on acid extrusion in hippocampal neurons (250, 273) could inhibit NHE-4 activity by 50-60% when expressed in fibroblasts (30). Moreover, NHE-4 could not be activated by acid loading under isosmolar conditions. An increase in acid extrusion required hyperosmolar media, suggesting its activation was linked to changes in cytoskeletal elements brought about by cell shrinkage (31). If NHE-4 displayed similar properties in hippocampal neurons, it could not be responsible for the normal extrusion of H⁺ by these cells. Later studies raise additional questions about the role of NHE-4 in hippocampus, as it was found to be principally located in cerebral cortex and brain stem-diencephalon (105, 191).

Cloning and expression of the NHE-5 isoform in PS120 cells revealed a functional Na⁺/H⁺ exchanger with a sensitivity to EIPA. In situ hybridization demonstrated strong localization to the hippocampal dentate gyrus, with lower levels noted in the CA1 fields and cerebral cortex (14). In separate studies, cloning of NHE-5 and expression in fibroblasts (15), or Chinese hamster ovary cells (292), also yielded a functional Na⁺/H⁺ exchanger. Less specific localization to hippocampus and other regions was noted by Northern analysis (15).

The NHE-6 isoform has a high degree of sequence identity to a mitochondrial Na⁺/H⁺ exchanger of yeast, termed NHA2. Its abundant expression in mitochondria-rich tissues, including brain, increased suspicion that it also was a mitochondrial Na⁺/H⁺ exchanger (230). Recent studies have not confirmed a mitochondrial localization, however, and have provided evidence that the transporter is targeted to endoplasmic reticulum (209), or endosomes (47). NHE-7 is also in organelles, localized to the trans-Golgi network (229a).

The function of individual NHE isoforms in brain is unclear, with the possible exception of NHE-1. It is likely that the principal role of NHE-1 in the CNS is the maintenance of steady-state pH_i and the recovery from cytosolic acid loads. This is supported by recent studies of a spontaneous slow-wave epilepsy mutant mouse (89, 358). The autosomal recessive defect of these animals arises from a null allele of NHE-1 localized to chromosome four and is associated with ataxic gait, absence and tonic-clonic seizures, and selective neuronal death in brain stem and cerebellum (89). In a study of pH_i regulation in HEPES-buffered media, acutely dissociated CA1 pyramidal neurons from mutant mice displayed a more acidic state pH_i and a lower rate of acid extrusion compared with cells from wild-type animals. In some instances, recovery from acid loading was virtually absent in the neurons from mutant animals (358).

The roles of NHE-2 through NHE-7 are more subject to conjecture. Given the well-established utilization of Na⁺/H⁺ exchange in cell volume regulation (336), this function appears plausible, particularly for NHE-4 (30). Regulation of pH within organellar compartments appears likely in the case of NHE-6 (47, 209, 230) and NHE-7 (229a).

B. HCO₃^- Transporters in Brain

The transport processes that regulate pH_i through the transmembrane flux of bicarbonate fall within the bicarbonate transporter superfamily. Identified and cloned species correspond to the physiologically well-characterized categories of Cl^-/HCO₃^- exchange, Na⁺-HCO₃^- cotransport, and Na⁺-driven Cl^-/HCO₃^- exchange. Subspecies of all three transporter classes have been identified in the CNS.

1. The Cl^-/HCO₃^- exchanger in the CNS

This transporter falls within the anion exchanger (AE) gene family, which consists of at least four members (170). AE3 is expressed in both brain and heart (171), with polypeptide subtypes that differ in their NH₂-terminal sequences (185). The AE3 transporter has been localized to CNS neurons (171, 251), and two polypeptide isoforms of the AE3 gene have been separately localized to Muller cells and horizontal neurons within retina (168). While AE3 mRNA was found in both fetal and adult hippocampal neurons, physiological Cl^-/HCO₃^- exchange appeared nearly absent in the younger cells (251). This suggests that inferences regarding AE3 function made on the basis of message localization should be regarded with caution. In one study, AE3 expression was altered in response to environmental changes. Experiments on cultured hippocampal neurons demonstrated increased AE3 immunoreactivity in response to exposure to ammonia (142). This effect may be related to the depolarizing shift in the GABA_A receptor equilibrium potential that has been associated with long-term exposure to ammonium ions (188, 190).

2. Na⁺-HCO₃^- cotransporters in the CNS

Studies of the Na⁺-HCO₃^- cotransporter (NBC) in mammalian brain have indicated a wide distribution in the CNS and the presence of more than one isoform. Using nucleotide probes derived from the electrogenic Na⁺-HCO₃^- cotransporter of rat kidney, in situ hybridization revealed NBC mRNA in olfactory bulb, hippocampal dentate gyrus, and cerebellum, with localization to both glial cells and neurons (270). Immunostaining with polyclonal antibodies indicated that NBC protein was distributed diffusely in cell bodies and processes, as well as in epithelial cells of the choroid plexus, ependyma, and meninges. Colocalization with glial fibrillary acidic protein, microtubule-associated protein, or 2',3'-cyclic mononucleotide 3'-phosphodiesterase demonstrated expression in particular subpopulations of astrocytes, neurons, and oligodendrocytes, respectively. Screening of cDNA libraries resulted in the cloning of two rat brain NBC isoforms, rb1NBC and rb2NBC, which were identical except for a unique 61-amino acid COOH terminus of the latter (25). Generation of polyclonal antibodies to distinguish between the carboxy ends of the isoforms revealed predominant rb1NBC protein in astrocytes compared with neurons, whereas rb2NBC antibody revealed the opposite pattern. When expressed in oocytes, rb2NBC was found to mediate Cl^--independent, DIDS-sensitive, electrogenic Na⁺-HCO₃^- cotransport. An NBC isoform cloned by Giffard et al. (120) was similarly found to generate DIDS-sensitive, HCO₃^--dependent currents when expressed in oocytes, and in situ hybridization showed widespread presence in rat CNS, and colocalization with glial acidic fibrillary protein. NBC mRNA was detected in brain at P0 with an increase at P15 and persistence into adulthood. In spinal cord, in contrast, expression was noted by embryonic day 17. Western blots from microsomal preparations of rat brain indicated NBC protein expression as early as embryonic day 16 in cortex and brain stem-diencephalon (105).

3. Na⁺-driven Cl^-/HCO₃^- exchange in the CNS

The Na⁺-driven Cl^-/HCO₃^- exchanger (NDCBE) was the first acid extrusion mechanism identified in early studies of snail neurons and squid axon (259, 303). In hippocampal neurons, where mammalian neuronal pH_i regulation was extensively dissected, NDCBE played a major role in the extrusion of acid (273). An NDCBE cloned from the insulin-secreting cell line MIN6 cDNA library was found to have high mRNA levels in brain, and when expressed in oocytes, it mediated an Na⁺-HCO₃^- influx in exchange for internal Cl^- (340). This isoform is likely related to the NDCBE1 cloned and extensively characterized by Grichtchenko et al. (131). Northern blots revealed robust presence of the isoform in all major regions of human brain. When expressed in oocytes, it mediated an Na⁺-dependent, DIDS-sensitive increase in pH_i associated with a rise in [Na⁺]_i with a 1:2 stoichiometry of Na⁺ to HCO₃^-. In addition, isotopic Cl^- efflux from oocytes was Na⁺ and HCO₃^- dependent, blocked by DIDS, and required external Cl^- to run in reverse mode.

	VI. MODULATION OF INTRACELLULAR pH BY NEURONAL ACTIVITY

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Both neurons and glia cells can undergo substantial shifts in pH_i in response to electrical stimulation or application of specific membrane ligands. For both classes of cells, these pH changes can occur within seconds, in either the acidifying or alkalizing direction, and can attain magnitudes of several tenths of a pH unit.

A. Modulation of pH_i in Neurons

1. pH_i shifts associated with depolarizing stimuli

A number of investigators have demonstrated a fall in neuronal pH_i associated with membrane depolarization or repetitive spike activity. Many of these studies were first performed on large invertebrate cells. In barnacle photoreceptors, a fall in pH_i was correlated with the depolarizing receptor potential (51). Repetitive firing was associated with a cytosolic acidification in molluscan (1) and leech (261) neurons. In the former study it was found that the acidification induced by repetitive spike activity was dependent on the entry of Ca²⁺. Depolarization of snail neurons also elicited a Ca²⁺-dependent acidification (202). Meech and Thomas (200, 201) had reported that injection of Ca²⁺ into snail neurons caused cytosolic acidification and found that this response was partly mediated by mitochondrial Ca²⁺/H⁺ exchange, but was largely unexplained. It was later demonstrated that the Ca²⁺-linked acidification of snail neurons was sensitive to vanadate. This finding implicated the Ca²⁺/H⁺ exchange property of the plasmalemmal Ca²⁺-H⁺-ATPase (reviewed in Refs. 61, 62) in the origin of the intracellular acid shift (274). It should be mentioned that snail neurons can also generate transmembrane H⁺ fluxes though a specific voltage-gated H⁺ conductance (57, 305). Because this conductive pathway activates at potentials positive to the H⁺ equilibrium potential, and exhibits pronounced outward rectification, its main role appears to be the extrusion of H⁺ (reviewed in Ref. 92).

Depolarizing stimuli have also been associated with a fall in pH_i in vertebrate neurons. In studies of lamprey reticulospinal cells (74) and frog motoneurons (107), intracellular acidosis was associated with depolarization, due to elevation of [K⁺]_o or exposure to excitatory amino acids, respectively. In catfish horizontal cells, application of glutamate elicited a fall in pH_i that was responsible for suppression of voltage-gated Ca²⁺ channels (102). Cultured mammalian neurons have also been found to acidify in response to excitatory amino acids, and these intracellular acid shifts were linked to entry of Ca²⁺ (60, 134, 143, 341, 346). In a brain stem slice preparation, acid shifts in vagal neurons were noted in response to spike activity or depolarization under voltage clamp. These acid shifts appeared to have a dependence on Ca²⁺ entry as they were blocked by Cd²⁺ and Ni²⁺ but persisted in the presence of tetrodotoxin or HCO₃^--free saline (316). In addition, activity-dependent, Ni²⁺-sensitive intracellular acid shifts have been reported in rat thalamic neurons (207).

In contrast, in hippocampal CA1 neurons studied in acute slices, depolarizing agents [including N-methyl-D-aspartate (NMDA)] evoked intracellular acidifications that displayed a minor dependence on Ca²⁺ and were largely attributed to production of metabolic acid (361).

An interesting, rapidly spreading acidification of cerebellar cortex (in vivo) was reported by Ebner and colleagues (66, 67), using neutral red as an epifluorescent pH_i indicator. These responses were elicited by synaptic activation of the cerebellar Purkinje cells through stimulation of the parallel fibers (66, 67). The basis of these acid responses is unclear. They may be related to a recently reported, rapid, activity-linked, Ca²⁺-related acidification of Purkinje cell dendrites studied in acute cerebellar slices (349).

The causes of Ca²⁺-dependent cytosolic acidosis may be multiple. Intracellular acidosis arising from elevated [K⁺]_o or glutamate has been linked to the generation of metabolic acid (341) as well as mitochondrial Ca²⁺/H⁺ exchange (346). In a voltage-clamp study of hippocampal CA1 pyramidal cells (317), Ca²⁺ entry through voltage-gated channels caused a cytosolic acid shift that appeared unrelated to mitochondrial Ca²⁺ transport, as it was not significantly reduced when ruthenium red was included in the patch pipettes. The acid shifts were significantly smaller when the electrodes contained 1-5 mM vanadate, or 100 µM eosin. These data were consistent with cytosolic acidification mediated by the Ca²⁺/H⁺ exchange property of the plasmalemmal Ca²⁺-ATPase, as was noted for snail neurons (274).

In evaluating data from such studies it should be borne in mind that the available tools are still rather poor. The use of BCECF to measure acid shifts may confuse the quantitative interpretation of results, as this agent is itself an inhibitor of the plasma membrane Ca²⁺-ATPase (115). For the human erythrocyte pump, the inhibition constant (K_i) of BCECF is 100 µM (115), and only 30 µM BCECF was able to cause half-inhibition of the plasmalemmal Ca²⁺-ATPase in snail neurons (275).

Qualitative difficulties in interpretation can also arise because of the poor specificity of the transport inhibitors. In addition to blocking the plasmalemmal Ca²⁺-ATPase, vanadate has been reported to inhibit mitochondrial Ca²⁺/H⁺ exchange (with a K_i of 0.6 mM) (173) and is well-established as a blocker of sarcoplasmic/endoplasmic reticulum Ca²⁺ (SERCA) pumps (233). SERCA pumps are also inhibited by low concentrations of eosin (with a reported K_i of 0.8 µM) (173). Moreover, like the plasmalemmal Ca²⁺-ATPase, the SERCA pumps exchange Ca²⁺for H⁺ (85, 359). Thus the effects of eosin and vanadate on pH_i may not be solely attributable to their inhibition of the plasmalemmal transporter.

These considerations underscore the challenges faced when seeking to establish the basis of an intracellular acid shift. Cytosolic acidosis may arise from a wide variety of organellar and metabolic sources in addition to the movement of acid equivalents across the plasma membrane. To distinguish transmembrane movements of acid, it is therefore advantageous if associated pH transients can be recorded in the extracellular domain, either as surface pH from a single cell, or as interstitial pH, when studying responses from a tissue.

Although depolarizing stimuli are most often associated with a fall in pH_i, alkaline responses have occasionally been noted. Gaillard et al. (114) found that elevation of [K⁺]_o elicited a Cd²⁺-sensitive alkalization of cultured cerebellar Purkinje cells, consistent with Ca²⁺ dependence (however, depolarization evoked only acid responses in Purkinje cells studied in acute slices) (349). Recording pH_i from the CA1 pyramidal cell layer of hippocampal slices, Zhan et al. (362) found that application of NMDA caused an initial intracellular alkalization followed by an acid shift. The alkaline response was inhibited in low Ca²⁺ media, while the late acid shift appeared to have a metabolic origin. An alkalization of spinal cord neurons was reported in response to elevation of external K⁺ and was speculated to arise via an electogenic Na⁺-HCO₃^- cotransporter (46).

With prolonged neural activity, cytosolic acidification may occur due to an increase in the production of metabolic acids such as CO₂ and lactate. These products could be generated from the energetic demands associated with the influx of Ca²⁺ (341), but could also arise from a host of metabolic needs including the ATP requirements of the Na⁺-K⁺ pump (186). Metabolic processes are likely to underlie the considerable acidosis associated with seizure and spreading depression. Because a metabolically derived acidosis is expected to lower both intracellular and interstitial pH, the acid changes associated with prolonged stimulation, seizure, and spreading depression are considered further below, in context with extracellular acid shifts (see sects. VIIB6 and VIII).

2. pH_i shifts associated with GABA_A and glycine receptors

In characterizing the properties of GABA_A receptors, Bormann et al. (34) noted a significant permeability to bicarbonate ions. The equilibrium potential for HCO₃^-(which is the same as that of H⁺) typically lies between 0 and -20 mV. Thus, at more negative membrane potentials, the opening of a GABA_A anion channel will result in an HCO₃^- efflux, leading to intracellular acidification and extracellular alkalization. This was first reported by Kaila and Voipio (154), who demonstrated that the opening of GABA_A receptors on crayfish muscle produced a bicarbonate-dependent, intracellular acidification accompanied by a surface alkalization. Kaila and colleagues also demonstrated a GABA_A receptor-mediated acidosis in crayfish stretch receptors (330), cultured astrocytes (151), and acutely isolated hippocampal CA1 pyramidal neurons (244). In situ evidence for similar intracellular acid shifts was provided by Ballanyi and colleagues (316), who demonstrated an HCO₃^--dependent, bicuculline-sensitive fall in pH_i in vagal neurons that was evoked by GABA. Brain stem medullary neurons also exhibited a GABAergic acidosis. In addition, these cells could be acidified by activation of strychnine-sensitive glycine receptors (189), which have an HCO₃^- permeability comparable to that of GABA_A receptors (34).

3. Additional modulators of neuronal pH_i

A variety of endogenous agents have been found capable of altering cytosolic pH. Connor and Hockberger (88) injected molluscan neurons with cAMP or cGMP and noted either a slow acidification or a biphasic alkaline-acid response. In cultured mammalian neurons, an acid shift was elicited by activation of type I metabotropic glutamate receptors (6). The mechanisms underlying these pH_i changes have not been determined.

An interesting alkalization of acutely isolated hippocampal CA1 neurons was reported by Church and colleagues (280). Norepinephrine, acting through -adrenergic receptors, elicited a concentration-dependent, sustained rise in steady-state pH_i and increased the rate of pH_i recovery from imposed acid loads. This alkalosis was Na⁺ linked, but was not dependent on HCO₃^- or the elevation of [Ca²⁺]_i, and appeared to be mediated by increased activity of the amiloride-insensitive Na⁺/H⁺ exchanger. The effect was linked to G protein-coupled stimulation of adenylate cyclase with subsequent activation of the cAMP-dependent protein kinase. These observations suggest a means by which pH_i could vary as a function of the metabolic and electrophysiological history of a set of neurons. In this regard it may be noted that CA1 pyramidal cells have been found to fall into distinct populations with relatively low or high baseline pH_i values (24).

B. Modulation of pH_i in Glial Cells

Neuronal activity has long been associated with depolarizing responses of astrocytes, which have been mainly attributed to the transient elevation of [K⁺]_o (132, 234, 253, 284). A consequence of astrocyte depolarization is the activation of electrogenic Na⁺-HCO₃^- cotransport, which causes a rapid cytosolic alkaline shift. In the great majority of studies, these alkaline responses were evoked in vitro by the elevation of [K⁺]_o (44, 50, 129). The recording of similar alkalizations which followed the elevation and fall of [K⁺]_o was reported for cortical astrocytes in vivo by Chesler and Kraig (81, 82) using double-barreled pH microelectrodes. Stimulation of the cortical surface elicited typical slow depolarizing responses that were accompanied by a rapid rise in pH_i of several tenths of a unit pH. After tetanic stimulation, the pH_i recovered and was sometimes followed by a prolonged intracellular acidosis. During passage of a cortical spreading depression, the alkaline-acid sequence was repeated but was exaggerated in amplitude, with some alkaline transients approaching 0.8 pH units. In recordings from medullary glial cells in vivo, Ballanyi et al. (18) reported similar intracellular alkaline shifts evoked by stimulation of spinal pathways.

Intracellular alkaline shifts in response to neural activity have also been reported in the giant glial cells of the leech (an annelid worm) (94). It therefore appears that this glial property has been conserved through evolution. An activity-dependent alkalization of glial cells may have functional significance in several respects. A rise in cytosolic pH can elevate glycolytic rate (321), increase gap junctional conductance (289), and favor the uptake of glutamate (43), or the formation of glutamine (48). On the other hand, when viewed from an extracellular perspective, the Na⁺-HCO₃^- cotransporter would appear to function as an acid secretory mechanism, as discussed in section VIIB6.

Another form of astrocyte alkalization has been associated with activation of metabotropic glutamate receptors. Amos and colleagues (5, 6) described a group I mGluR-mediated rise in pH_i of cultured cortical and cerebellar astrocytes. The response was independent of Na⁺ but required HCO₃^- (5). The alkaline shift was apparently Ca²⁺ dependent, as it could be triggered by caffeine and ionomycin (5), and was mitigated by intracellular BAPTA (6). Elevation of [Ca²⁺]_i appeared to be dependent on its release from intracellular stores, as the response persisted in the absence of external Ca²⁺. Acutely dissociated astrocytes also exhibited an mGluR-mediated alkalization, suggesting that similar responses might occur in vivo (5).

As discussed for neurons, glial cell acidosis may be expected to arise from a variety of sources, which include transmembrane acid transport, organellar acid fluxes, and metabolic production of CO₂ and lactic acid. Late acidifications of astroctyes studied in vivo were noted by Chesler and Kraig following repetitive neural activity (81, 82). Application of excitatory amino acids was also found to acidify astrocytes in a number of studies (49, 52, 263). Acidosis of glial cells induced by glutamate has been largely attributed to its inward transport (52, 263), as the uptake of this amino acid is coupled to the influx of H⁺ (43, 344, 360). Accordingly, superfusion of hippocampal slices with transportable glutamate analogs was found to cause a fall in tissue pH_i (3).

Acidification of astrocytes has also been noted in response to the excitatory amino acid kainate, which is not taken up by the glutamate carrier (165). In CO₂/HCO₃^--containing media, Rose and Ransom (263) found that the kainate-evoked acidification of hippocampal astrocytes was preceded by an alkaline shift. It was proposed that these pH_i shifts were governed by the Na⁺-HCO₃^- cotransporter responding to sequential depolarization and hyperpolarization of the membrane. In support of this contention, they found little or no kainate-induced acidification in CO₂/HCO₃^--free media. In contrast, in cerebellar astrocytes, kainate-evoked acid shifts were prominent in the absence of CO₂ and HCO₃^- (52), suggesting an alternate mechanism. The kainate responses of the cerebellar astrocytes did not require external Ca²⁺, unlike acidifications evoked by excitatory amino acid receptors in neurons (60, 134, 143, 341, 346).

	VII. REGULATION AND MODULATION OF BRAIN EXTRACELLULAR pH

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A. The pH of Interstitial Fluid

pH microelectrode studies have indicated that brain interstitial fluid is slightly more acidic than blood, with a pH of 7.3 (90, 145, 174). In brain slices, however, the interstitial pH is usually lower, despite the flow of saline buffered to pH 7.4. At the center of a brain slice, baseline pH_o values of 7.1-7.2 have been reported (16, 71, 144, 180, 338). Low pH_o has also been noted in other isolated CNS preparations including lamprey brain stem (75), frog spinal cord (107), and turtle cerebellum (139).

The cause of baseline acidosis in hippocampal slices was studied by Voipio and Kaila (328) using a unique, dual CO₂-pH microelectrode. It was demonstrated that the low pH of the interstitial fluid was entirely due to elevation of tissue PCO₂. In their interface-style recording chamber, the slice PCO₂ ranged from 40 to 50 Torr. In a later study, PCO₂ was calculated in submerged hippocampal slices using pH and CO₃^2--sensitive microelectrodes (78). The submerged slices had a considerably higher baseline CO₂ (75 Torr), which again accounted entirely for the difference in pH between bath and interstitial fluid. The high concentration of CO₂ in brain slices is likely due to its generation by aerobic metabolism, and poor clearance, owing to lack of blood flow. In a slice, the egress of CO₂ would be reliant on diffusion across the tissue and its unstirred layer of surface fluid. CO₂ clearance can therefore be expected to be greater (and thus the PCO₂ lower) in an interface chamber, where CO₂ is able to diffuse across both the upper and lower surfaces (225).

In vivo, CO₂ is cleared by blood flow, and accordingly, large steady-state accumulation of CO₂ is not expected. With a physiological rise in perfusion, due to elevated neural activity, increased clearance of CO₂ and local increases in brain pH could arise, in principle. Indeed, observations of rapid, activity-dependent increases in brain interstitial pH were at first attributed to augmented blood flow (322). The identification of similar pH shifts in numerous in vitro studies (where blood flow was absent) indicated that alternate mechanisms must account for these rapid pH changes. Candidate processes are the subject of detailed discussion in the sections which follow. Alterations in brain pH due to physiological shifts in blood flow remain plausible, however, and evidence for slow extracellular alkaline shifts arising from postactivity hyperemia has been published recently by Venton et al. (325a).

B. Changes in Interstitial pH Caused by Neural Activity

Synchronous activation of nerve cells has been shown to cause changes of interstitial pH in a variety of preparations. Reports of activity-dependent pH_o shifts go back over 60 years. Many of the earlier measurements are suspect, as extracellular potential shifts were not subtracted from the pH recordings, and the spatial and temporal resolutions were poor (76). The first reliable recordings of pH_o transients following synchronous cortical activity were performed in vivo, using pH microelectrodes in combination with an extracellular reference microelectrode to enable subtraction of interstitial potential changes. Stimulated activity, seizure, or spreading depression were associated with an initial interstitial alkaline shift followed by an acidosis that could persist for minutes. This pattern was first noted in cortex (65, 322) and cerebellum (174, 253a) and has been commonly found throughout the CNS. In some instances, however, neural activity has been associated with predominant early acid shifts (see sect. VIIB6).

The processes responsible for the generation of activity-dependent pH_o shifts have not been fully elucidated. In principle, a large number of transport mechanisms could contribute to the dynamic H⁺ balance of the brain interstitial space. To understand whether a flux of given magnitude can account for an observed pH_o transient, however, one must have a quantitative appreciation of the interstitial buffering capacity. Therefore, before addressing the origin of the activity-dependent pH_o transients, the nature of pH buffering in the brain interstitial space will be considered.

1. Buffering of brain interstitial fluid

The acid-base status of extracellular fluid is governed by the chemical buffering properties of the solution and by the transmembrane flux of acid equivalents. In the adoption of nomenclature, it is formally correct as well as heuristically useful to distinguish between these components. Buffering is a term best reserved for the chemical equilibria within the interstitial fluid which act to mitigate the change in pH arising from an imposed acid or base load. Transmembrane fluxes, in contrast, constitute the source of acid-base loads imposed upon the interstitial fluid. In some instances, a pH_o shift caused by one transmembrane flux may be masked by a second flux of opposite direction. The term pH muffling has sometimes been used to distinguish such a process from the ever-present chemical buffering (304).

The buffering capacity of a fluid at equilibrium is governed by the concentrations of the individual buffers and their respective pK_a values. In brain interstitial fluid, the principal buffer is CO₂/HCO₃^- with a total combined concentration of 27 mM. In comparison, the contributions of other buffers appear negligible (109).

Quantification of the CO₂/HCO₃^- buffering capacity in the physiological setting typically relies on two assumptions. First, the buffer components are considered to be at equilibrium, and second, the system is treated as "open" with respect to CO₂ (i.e., the PCO₂ is constant). When these conditions are met, then the extracellular buffering capacity (_e) is reliant solely on bicarbonate concentration, and is given by Equation 3. Thus, for an [HCO₃^-]_o of 26 mM, _e would be 60 mM. However, as discussed below, neither fixed PCO₂ nor its equilibrium with HCO₃^- appear to occur under all conditions in which activity-dependent pH_o shifts are generated. Accordingly, the effective buffering capacity can be considerably less than 60 mM.

In rat hippocampal slices it has been shown that synchronous neuronal activity can cause interstitial PCO₂ to rise by 20-30 Torr, accounting entirely for the prolonged extracellular acidosis (328). Since the PCO₂ is not fixed, interstitial buffering would be less than maximal during these pH_o transients. Acid shifts also occur after synchronous activity in vivo (174, 278, 285), but unlike the slice preparation, blood flow is intact and the contribution of CO₂ to these phenomena is therefore likely to be less. Over periods of minutes, PCO₂ in vivo is governed by the blood flow and pulmonary ventilation, and long-term buffering can be considered to occur at a roughly fixed PCO₂.

A second important constraint on CO₂/HCO₃^--dependent buffering arises from the kinetics of the carbonic acid reactions

(7)

Within the physiological time frame, formation of HCO₃^- and H⁺ from H₂CO₃ occurs virtually instantly. The hydration of CO₂ to form carbonic acid (H₂CO₃) and its dehydration back to CO₂ and water are extremely slow, however, with time constants on the order of 20 s and 1 s, respectively (192). Accordingly, the attainment of equilibrium following an acid or base challenge can require minutes. Biological systems typically overcome these slow kinetics using various forms of the enzyme carbonic anhydrase (CA), which catalyzes both the hydration and dehydration steps. In the brain interstitial space, CA activity is the principal regulatory factor governing the manifestation of pH transients. Indeed, the characterization of activity-dependent pH_o shifts and the function of interstitial CA are inseparable issues and therefore will be considered together.

2. Interstitial pH transients and CA

The first evidence that CA played a role in the regulation of interstitial pH came from the work of Kraig et al. (174). These investigators reported alkaline-acid pH_o transients in rat cerebellum in response to stimulation of the parallel fibers, or the passage of a spreading depression. When the cerebellar cortex was superfused with 5 mM acetazolamide, the amplitude of the alkaline and acid shifts increased two- to threefold. Enhancement of evoked alkaline shifts by millimolar concentrations of acetazolamide was also noted in rat cortex (215) and in brain slices (63, 338). Comparable effects, however, could be obtained in hippocampal slices using as little as 1 µM acetazolamide (71).

At the time of the earliest studies of pH_o transients, it was known that brain CA resided in glia (119, 164, 228), within both oligodendrocytes (58) and astrocytes (59). In view of the permeability of cell membranes to acetazolamide (192), and the high concentrations used in the initial experiments, it appeared likely that glial CA would have been inhibited. However, the means by which inhibition of glial intracellular CA could so profoundly affect rapid pH_o transients was not obvious, and Kraig et al. (174) were careful to stress that the site of action of acetazolamide was uncertain.

Clarification came from a series of experiments using CA inhibitors that do not readily cross cell membranes. Activity-dependent pH_o shifts were profoundly affected by these agents. It was therefore reasoned that the site of enzyme activity had to reside in the extracellular space (71, 72, 150). These CA inhibitors included benzolamide, which is relatively impermeant due to its low pK_a (318), as well as a set of similarly potent sulfonamides conjugated to high-molecular-weight dextrans (117, 156).

In one series of experiments alkaline shifts were evoked in area CA1 of hippocampal slices by either stimulation of the Schaffer collaterals, or by local microejection of glutamate (71, 72). Superfusion of benzolamide (71) or a dextran-conjugated sulfonamide (72) increased the amplitude of these transients severalfold. This increase in the amplitude of an alkaline transient is expected to occur when the pH_o shift is generated by the loss of protons from (or addition of OH^- to) the interstitial compartment (Fig. 1A). Since CO₂-HCO₃^- is the principal extracellular buffer, the ability to replenish extracellular H⁺ hinges on the CA-dependent rate of CO₂ hydration. With inhibition of interstitial CA, the generation of H⁺ is reliant on the slow, uncatalyzed hydration of CO₂, which leads to a poorly buffered, or "amplified" alkalosis. CA inhibitors will similarly amplify alkaline shifts elicited by any extracellular "proton sink" including alkalizations imposed by local microejection of NaOH (72). Properties of this activity-evoked, HCO₃^--independent alkalosis are detailed in section VIIB4.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Effect of extracellular carbonic anhydrase (CA) inhibition on activity-dependent alkaline shifts. A, left: extracellular alkalosis generated by an H+ sink (or equivalently an efflux of OH-) would be buffered via the CA-catalyzed hydration of CO2. Right: inhibition of extracellular CA by benzolamide would diminish the rate of hydration, permitting the development of a larger alkalosis. B, left: extracellular alkalosis generated by an HCO3- source is generated by the CA-catalyzed dehydration of H2CO3. Right: inhibition of extracellular CA by benzolamide slows the dehydration step, thereby limiting development of the alkalosis.

In another set of experiments, inhibitors of extracellular CA were used to study extracellular alkaline shifts evoked by activation of GABA_A receptors (71, 150, 153, 154). Work on crayfish muscle demonstrated that the efflux of HCO₃^- across GABA_A anion channels gives rise to a surface alkalosis (153, 154). In this circumstance, rather than reduce an alkalosis, CA was required to generate the alkaline shift, since the fall in surface H⁺ required the rapid dehydration of H₂CO₃ (Fig. 1B). Thus, when CA on the surface of the muscle was inhibited with benzolamide, the dehydration of H₂CO₃ was slowed, and the GABA-evoked surface alkalosis was prevented (153). Similar effects were later noted when benzolamide and dextran-conjugated inhibitors of CA were used to study GABA_A receptor-mediated extracellular alkalosis in the CA1 region of hippocampal slices (71, 150). Studies of this HCO₃^--dependent alkalosis are detailed in section VIIB5.

The contrasting effects of CA inhibitors on alkaline transients demonstrated that there are two classes of interstitial alkalization that may accompany normal sequences of excitation and inhibition. Excitation appears to generate a rapid proton sink. Because glial cells secrete acid into the interstitial space, and undergo an intracellular alkalization during neural activity, it has been speculated that this proton sink is not a glial response, but rather, represents the movement of H⁺ equivalents into a neuronal compartment (76, 94, 252). Possible mechanisms for the proton sink are considered in section VIIB4. GABA_A receptor-mediated inhibition, on the other hand, appears to generate a local, extracellular "bicarbonate source."

A second implication of these studies was that a form of CA was present in the interstitial space of the brain slice preparation. Given the similar effects of acetazolamide in vivo (174, 215), it appeared likely that this extracellular CA activity was also present in the intact brain. This was confirmed when benzolamide was found to amplify AMPA-evoked alkaline shifts in vivo (140). Similar amplification of interstitial alkaline transients was later reported in nonmammalian preparations exposed to CA inhibitors, including isolated turtle cerebellum (121) and leech segmental ganglia (262).

3. Nature of interstitial CA activity in brain

Extracellular CA activity has been found in tissues outside the CNS associated with the membrane-tethered isoform CA IV (64). Examples include the luminal brush border of renal tubules (350), the surface of muscle fibers (335), and the luminal endothelial surface in lung (334). In the nervous system, in contrast, there has been little morphological evidence of extracellular CA. Histochemical studies suggested the presence of interstitial CA in dorsal root ganglia (106); however, such methods did not allow definitive extracellular localization. The physiological demonstration of extracellular CA in hippocampal slices (71, 72, 150) nearly coincided with immunocytochemical studies of brain using antibodies against CA type IV. In the latter study, CA IV was found on the luminal membrane of brain endothelial cells, but CA IV staining was not observed in brain tissue proper (118). More recently, immunostaining for CA IV was described on scattered glial cells and neurons in mammalian brain (342).

The consistency of the physiological evidence for extracellular CA is at apparent odds with the variability of the morphological studies. This might be explained if interstitial CA activity detected by physiological means in brain slices was an artifact of cellular injury. Indeed, because CA is abundant in glial cells (58, 59, 119, 164, 221a, 228), and there is physiological (207, 244) and morphological evidence (2, 158, 227, 229, 351, 352) for its presence in neurons, it is reasonable to assume that release of intracellular CA would occur when tissue is cut in the preparation of brain slices. Yet, amplification of alkaline transients by acetazolamide (174) and benzolamide (140) was also noted in vivo, suggesting that damage due to slicing was not a primary factor. Indeed, because exogenous CA was found to readily diffuse to and from the interstitial space of brain slices (140), the enzyme would be expected to rapidly wash out of the tissue when released from injured cells.

Additional evidence that simple spillage of the enzyme could not account for interstitial CA came from a study of hippocampal slices prepared from mutant mice lacking CA II. In a comparison of mutant versus wild-type animals, activity-dependent interstitial alkaline shifts were comparable in amplitude and were amplified in a similar manner by benzolamide (311). Moreover, histochemical studies in brains of CA II-deficient mice detected the presence of membrane-associated CA (255) near neuronal processes.

The inability to detect interstitial CA with antisera against CA IV could have several explanations. Extracellular isoforms other than CA IV may account for brain interstitial CA activity. Alternatively, the concentration of CA IV in the interstitial space could be below the detection limit of the immunocytochemical assay. This would not be unprecedented, as early immunocytochemical studies did not detect the CA II isoform in astrocytes (58). These possibilities are not mutually exclusive, and evidence now exists for both a low concentration of interstitial CA IV and the presence of an additional extracellular CA isoform.

A suggestion that interstitial CA was present in low concentration came from experiments in which pH_o, [HCO₃^-]_o, and PCO₂ were monitored during repetitive stimulation of rat hippocampal slices. The rise in [HCO₃^-]_o associated with the extracellular alkaline transient was sometimes less than the value predicted by the Henderson-Hasselbalch equation, suggesting lack of equilibrium between CO₂, HCO₃^-, and H⁺ (79). This result is consistent with an insufficient rate of CO₂ hydration owing to low enzyme activity (Fig. 1A). In a later study, addition of exogenous bovine CA to physiological saline decreased evoked alkaline shifts in rat cortical slices by two- to threefold (140). A similar increase in interstitial buffering capacity was also noted when exogenous CA was superfused over rat cortex in vivo (140). The augmentation of buffering occurred when the exogenous CA concentration was 300 nM but was not observed at 30 nM. After addition of 300 nM enzyme, increasing its level to 3,000 nM had no additional effect on the pH transients. Assuming a molar activity comparable to the added CA, these data were consistent with an overall interstitial CA concentration on the order of 30-300 nM.

Tong et al. (310) provided evidence that interstitial CA activity in brain is attributable to CA IV. The type IV CA isoform is tethered to the extracellular face of membranes by a phosphatidylinositol-glycan linkage. Accordingly, treatment with phosphatidlyinositol-specific phospholipase C (PIPLC) can liberate the enzyme and enable its washout (335, 364). When hippocampal slices were incubated with PIPLC, interstitial alkaline shifts were increased markedly compared with controls, and the amplifying effect of benzolamide was largely occluded. The incubation fluid from PIPLC-treated slices contained a low concentration of CA that was insensitive to SDS, a property consistent with CA IV (197, 347). Western blots confirmed the presence of CA IV in this fluid. The immunocytochemical detection of CA IV required the pooled samples from several slices, consistent with a low interstitial CA IV activity. Based on the liberated SDS-insensitive CA IV activity, the interstitial concentration was estimated to be on the order of 100 nM, a figure consistent with the earlier estimate of 30-300 nM (140).

It is notable that treatment with PIPLC never eliminated all interstitial CA activity in hippocampal slices, since addition of benzolamide always caused some enhancement of alkaline transients (310). Incomplete cleavage of phosphatidylinositol-glycan linkages may explain this result. Alternatively, an additional membrane-associated CA could account for the persistence of CA activity. One candidate would be the CA XIV isoform, a membrane-spanning protein with an apparent extracellular catalytic domain. Transcripts of CA XIV have been detected in brain Northern blots (112, 213), and immunocytochemical studies have indicated its presence in medulla, pons, hippocampus, cerebellar granular layer, and major white matter tracts (242). The extent to which CA XIV catalyzes interstitial buffering is not known.

Interstitial CA in brain is likely due to surface enzyme located on both glial cells and neurons. An association with glia was noted in two early reports. Physiological studies demonstrated interstitial CA activity in gliotic hippocampal slices, where neuronal elements were virtually absent (128) (see Fig. 5A and sect. VIIB6). Surface CA activity on individual, acutely isolated retinal Muller cells (astrocyte variants) was also detected by Newman (222) (see sect. VIIB6). In a recent study, the reliance of lactate influx on surface CA was used to demonstrate the presence of this enzyme on both astrocytes and neurons (289a). The type of surface CA on the astrocytes and neurons cells was not addressed; however, the immunocytochemical study of CA XIV has suggested that neurons specifically express this isoform (242).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Acid shifts evoked by glial acid secretion modified by inhibition of extracellular carbonic anhydrase. A: extracellular acid shifts elicited by depolarization (elevation of [K+]o) in a gliotic hippocampal slice were reversibly amplified by benzolamide. [From Grichtchenko and Chesler (127), copyright 1994, with permission from Elsevier.] B: uptake of Na+ and two HCO3- would elicit a rise in H+ via the hydration of CO2 (left). Addition of benzolamide would be expected to reduce the acid transient (right). C: uptake of Na+ and CO32- would elicit a rise in H+ via the dissociation of HCO3- into CO32- and H+. The elevation of H+ would be buffered by the dehydration of H2CO3, and addition of benzolamide would therefore be expected to increase the acid transient.

4. Bicarbonate-independent alkaline shifts

Although its features have been studied in some detail, the mechanism underlying the activity-evoked, bicarbonate-independent, "net proton sink" (Fig. 1A) has not been established. This alkalosis has been extensively characterized in rat hippocampal slices (63, 71, 72, 124, 130, 144, 177, 237, 238, 282, 283, 329, 338). In this preparation, optical recordings of interstitial pH performed with phenol red (177) or dextran-conjugated fluorescein (124) revealed an onset within 10 ms. Significant characterization of this alkalosis has also been performed in turtle cerebellum (69, 77, 84) and rat lateral geniculate nucleus (312), where the basic features appeared similar to the hippocampal response.

Differentiation from the GABA_A receptor-mediated alkaline shift became apparent in early experiments, since the response persisted in HEPES-buffered Ringer solution (77, 84, 122) and in the presence of picrotoxin (69, 71). These results also indicated that an increase of interstitial HCO₃^- concentration, presumed to arise from activity-dependent shrinkage of the extracellular space (101), could not be responsible for the alkalosis (174). Amplification of the responses by extracellular CA inhibitors (Fig. 2A) provided a further means of differentiation from the GABAergic response (71, 72) (discussed in sect.VIIB2).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Extracellular alkalosis generated by proton sinks and bicarbonate sources in rat hippocampal slices. A: alternate microejection of GABA or glutamate elicited interstitial alkaline shifts of comparable amplitude and time course. Superfusion of increasing concentrations of benzolamide caused inhibition of the GABA response (revealing an acid shift) and amplification of the glutamate response, consistent with generation of the alkalosis via an HCO3- efflux and a proton sink, respectively. [From Chen and Chesler (71).] B: in a rat hippocampal slice, block of excitatory synaptic transmission (with CNQX and APV) permitted selective, high-frequency stimulation of GABAergic inhibitory interneurons. The resulting rapid alkaline transient was largely blocked in the presence of benzolamide. pHo, extracellular pH. [From Kaila et al. (150).]

1. IS THE ACTIVITY-DEPENDENT PROTON SINK DUE TO GLUTAMATE UPTAKE? In principle, a number of transport mechanisms could provide a pathway for an HCO₃^--independent, net proton sink, but might not generate an interstitial base load of sufficient magnitude to account for the observed extracellular pH change. One example is glutamate transport, where the electrogenic, Na⁺-coupled uptake of this amino acid is coupled to an HCO₃^--independent, influx of H⁺ (43, 344, 360). Under conditions of low extracellular buffering capacity, this mechanism can generate a surface alkalosis on retinal glial cells (43). The carrier can also cause a prolonged intracellular acidosis of brain slices when transported amino acids are superfused over the tissue (3). Yet, glutamate uptake does not appear to be responsible for the Schaffer collateral-evoked interstitial alkaline shift in hippocampal slices. This contention is supported by the fact that the synaptically evoked alkalosis can be blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovalerate (APV). In the presence of these ionotropic glutamate receptor antagonists, postsynaptic currents are abolished; however, the presynaptic release of glutamate and its subsequent uptake should be unaffected (70). It is also notable that a similar interstitial alkalosis can be evoked by antidromic activation of the CA1 pyramidal neurons in the presence of CNQX, APV, and picrotoxin (70, 130, 237). These data suggest that in the hippocampal CA1 region, postsynaptic electrical activity is responsible for generation of the extracellular proton sink (see below).
   Given the influx of an acid equivalent linked to glutamate uptake, this process should contribute in some measure to the interstitial alkalosis during excitatory synaptic transmission. Yet, when transportable glutamate analogs (e.g., D-aspartate) were injected into hippocampal slices, extracellular alkaline shifts could not be detected unless the buffering capacity was reduced with benzolamide (281). Thus, under normal buffering conditions, alkaline shifts generated by glutamate transport do not have a measurable impact on the broad interstitial space in which the carriers reside. It is plausible, however, that pH shifts associated with glutamate uptake attain a significant magnitude in microdomains close to the sites of transport.
   The ability of ionotropic glutamate antagonists to prevent the synaptic generation of the proton sink in cerebellum (77) and hippocampus (70) suggested that the response was triggered by excitation of the postsynaptic elements. Indeed, in turtle cerebellum, direct activation of glutamate receptors induced an alkaline shift that persisted after synaptic transmission was blocked by Mn²⁺ or Cd²⁺ (84). In hippocampus, confirmation of the postsynaptic origin came from experiments in which picrotoxin-resistant alkaline shifts were elicited by repetitive antidromic activation of the CA1 pyramidal neurons (70, 237). The antidromically induced alkalization was far smaller than that evoked by orthodromic trains of identical frequency and duration (70). Large alkaline responses could be elicited only by high-frequency antidromic stimulus trains of 50-200 Hz (130, 237). Curiously, in experiments using phenol red as an extracellular pH indicator, antidromic stimulation of hippocampal CA1 or dentate neurons did not produce any detectable pH_o change (177). An insufficient rate or duration of antidromic stimulation may account for this result.
   
2. IS THE ACTIVITY-DEPENDENT PROTON SINK DUE TO THE PLASMALEMMAL CA2+-ATPASE? A mechanism consistent with a postsynaptic origin was suggested by Schwiening et al. (274) in their studies of surface pH on snail neurons. Calcium entry (elicited by voltage-clamp depolarization) triggered a rise in surface pH that was blocked by vanadate, an inhibitor of the plasmalemmal Ca²⁺-ATPase. This transporter is activated by elevation of cytosolic Ca²⁺ and extrudes this ion in exchange for extracellular H⁺ (61, 62). Accordingly, vanadate also slowed the recovery of the cytosolic Ca²⁺ transients.
   These experiments demonstrated the ability of the plasmalemmal Ca²⁺-ATPase to elicit a surface alkalosis, but it remained unclear whether this transporter was responsible for the activity-dependent proton sink in the vertebrate CNS. Preliminary support for this hypothesis came from experiments in which alkaline shifts were found to be dependent on external Ca²⁺. In the CA1 region of hippocampal slices, alkalizations evoked by ionotropic glutamate agonists (238, 283), or by antidromic stimulation (130, 237), were blocked in media containing 0 Ca²⁺ and EGTA. However, subsequent studies revealed that sizable interstitial alkaline shifts could often be obtained in the nominal absence of external Ca²⁺ following interstitial AMPA ejection (282), or during the onset of spreading depression (206). In addition, when alkaline shifts were reduced or abolished in 0 Ca²⁺, addition of Ba²⁺ restored the responses (130, 282, 312). While Ba²⁺ can substitute for Ca²⁺ in carrying charge across voltage-gated Ca²⁺ channels (137), the Ca²⁺-ATPase is not known to utilize Ba²⁺ effectively (181, 363).
   Additional arguments against the Ca²⁺-ATPase mechanism have come from the use of inhibitors. Cd²⁺ is a potent blocker of the plasmalemmal Ca²⁺-ATPase with a K_i near 2 nM (326, 327). At an external concentration of 100 µM, Cd²⁺ did not affect the glutamate-evoked alkaline shift in turtle cerebellum (84), and caused just a partial block of the AMPA-induced alkalosis in rat hippocampal slices (282). Carboxyeosin, with a K_i of 20 nM (115), is a more widely used inhibitor of the plasmalemmal Ca²⁺-ATPase (86, 110, 126, 276). Instead of blocking this alkalosis, carboxyeosin augmented the interstitial alkaline shifts elicited by antidromic stimulation in hippocampal slices (133).
   To date, the reduction of the alkaline shift in zero Ca²⁺ solutions is the best evidence favoring the Ca²⁺-ATPase hypothesis. In view of the myriad processes influenced by a Ca²⁺ influx, however, a specific link to this Ca²⁺ transporter cannot yet be claimed. Nevertheless, because the plasmalemmal Ca²⁺-ATPse is an obligatory Ca²⁺/H⁺ exchanger, it must contribute to interstitial alkalosis to some degree. Such a ubiquitous mechanism might account for the small extracellular alkaline transients associated with electrical activity in muscle (116) and isolated nerve (91, 108). In the CNS, however, it remains unclear whether the H⁺ flux contributed by this process is sufficient to account for large, rapid, stimulus-evoked alkaline shifts. In this regard, it is notable that in cortical pyramidal cells (193), and cerebellar Purkinje neurons (110), cytosolic Ca²⁺ loads are handled mainly by uptake into endoplasmic reticulum and mitochondria, and the fraction of Ca²⁺ entry extruded by the plasmalemmal Ca²⁺-ATPase is accordingly small.
   These observations, coupled with quantitative consideration of the extracellular alkalosis, place significant restrictions on the Ca²⁺-ATPase hypothesis. For example, if an activity-dependent fall in [Ca²⁺]_o was on the order of 200 µM (248a) and the entire Ca²⁺ load was rapidly extruded by the plasmalemmal Ca²⁺-ATPase (with a Ca²⁺/H⁺ exchange ratio of 1:2), then the interstitial space would be subjected to a base load of 400 µM. To produce an alkaline shift of 0.05-0.1 pH units, the extracellular buffering capacity would have to be very small, on the order of 4-8 mM. Because only a small fraction of the cytosolic Ca²⁺ load is handled by the plasmalemmal Ca²⁺-ATPase, a far lower extracellular buffering capacity would be required in order for Ca²⁺/H⁺ exchange to plausibly account for the extracellular proton sink.
   
3. IS THE ACTIVITY-DEPENDENT PROTON SINK DUE TO A CHANNEL-MEDIATED H⁺ FLUX? In principle, an extracellular proton sink could be generated by a conductive pathway for H⁺ or OH^-, since the electrochemical gradients for these ions are inward and outward, respectively (76, 259). Voltage-gated proton conductances were first characterized in snail neurons (57, 305). While they have also been described in vertebrate cells, there is no evidence for such a conductance in vertebrate neurons. Moreover, voltage-gated H⁺ channels typically activate at membrane potentials close to zero, where the electrochemical gradient for H⁺ is outward (reviewed in Ref. 92). This feature is consistent with their presumed role as a rapid H⁺ extrusion mechanism.
   Channels that are permeant to other ions may conduct H⁺ (or OH^-) to a limited degree (92). However, several observations make it doubtful that alkaline shifts arise from nonspecific H⁺ conductances of this sort. A variety of channel blockers do not inhibit alkaline shifts (evoked by either glutamate agonists or electrical stimulation), including tetrodotoxin (73), amiloride (77), Ba²⁺ (130), and stilbene derivatives (215, 338). Ca²⁺ channel antagonists have sometimes inhibited alkaline shifts (130, 237), but in many instances the alkalosis was unaffected or only partially reduced by these agents (84, 282). It has been argued that the low activity of H⁺ and OH^- (on the order of 10^-7 M) is inconsistent with a large, channel-mediated flux of these ions, unless the channels involved are present in exceptionally high density, as has been suggested for cells that exhibit specific proton conductances (92).
   
4. BICARBONATE-INDEPENDENT ALKALINE SHIFTS IN RETINA. The forms of alkalosis discussed above are considered to arise from mechanisms that remove acid or add base to the interstitial fluid. In the retina, a different form of interstitial alkalization can occur in response to light. In this tissue, the high metabolic rate in the dark leads to a sustained interstitial acidosis (33, 357), which varies in a circadian rhythm (103, 104). When the photoreceptor dark current is transiently turned off during light stimulation, a slow, transient increase in pH_o occurs, owing to a decrease in metabolic rate. These slow alkaline shifts have been noted in frog (33) and cat (357) and are most prominent in the proximal retina adjacent to the rods.
   

5. Bicarbonate-dependent alkaline shifts

The means by which the efflux of HCO₃^- generates an external alkalosis is well established. The ability of CO₂ to rapidly equilibrate across cell membranes results in an identical equilibrium potential for H⁺ and HCO₃^- which is typically close to -20 mV in vertebrate neurons and glia (76). Thus, at normal negative membrane potentials, there exists an outward electrochemical gradient for HCO₃^- that can drive the efflux of this base to generate an external alkaline shift. A likely pathway for such an efflux became evident when Bormann et al. (34) demonstrated that GABA_A and glycine receptor channels had a significant permeability to HCO₃^-. Subsequently, Kaila and Voipio (154) showed that agonist gating of GABA_A receptors on crayfish muscle led to an HCO₃^--dependent alkalization of the surface membrane. This external alkalosis had the features expected of a pH shift generated by an HCO₃^- efflux as it was blocked when surface CA was inhibited by benzolamide (153) and could be mimicked by formate (196), an anion that can also pass the GABA_A receptor channel (34).

In the vertebrate CNS, extracellular alkaline shifts generated by GABA_A receptors were first noted in turtle cerebellum. Superfusion of GABA or GABA_A agonists produced a rapid increase in pH_o. This alkalosis was HCO₃^--dependent (68, 69), mimicked by formate (69), and inhibited by benzolamide (125). A similar interstitial alkalosis mediated by GABA_A receptor agonists was noted in the CA1 stratum pyramidale of rat hippocampal slices (71, 150). Pressure ejection of GABA resulted in a picrotoxin-sensitive, bicarbonate-dependent alkalosis that was blocked by extracellular CA inhibitors (Fig. 2A). A more physiological demonstration was provided by Kaila et al. (150) who showed that selective activation of inhibitory interneurons produced an alkaline shift in stratum pyramidale (Fig. 2B) that was augmented by pentobarbital, blocked by picrotoxin, and inhibited by an extracellular, dextran-conjugated CA inhibitor.

These studies indicated that the activation of interneurons can give rise to extracellular alkaline shifts localized around GABA_A receptors. The significance of these pH_o shifts remains to be determined, and the size and speed of these responses during typical sequences of synaptic excitation and inhibition are still unclear. Kaila and colleagues (295) proposed that with prolonged, repetitive stimulation of the Schaffer collaterals, early fatigue of the GABAergic synaptic transmission will limit the contribution of the HCO₃^- efflux to the net alkalosis. Accordingly, alkaline shifts elicited in this manner were amplified by benzolamide (295), signifying prevalence of the excitation-associated proton sink through most of the stimulus train. However, it was noted that high-frequency, short-duration stimulus trains elicited alkaline shifts that were amplified modestly by benzolamide (when compared with the amplification of alkaline transients evoked by low-frequency trains containing the same number of stimuli). Moreover, the alkalosis produced by sufficiently short, high-frequency trains could be inhibited by benzolamide (295). These data suggested that the GABAergic, HCO₃^--dependent alkalosis is predominant early in a stimulus train to the Schaffer collaterals. The speed of this early GABAergic response remains unclear, however. Although the ligand-gated activation of the HCO₃^- conductance would suggest rapid generation of the alkalosis, the rate-limiting step in the generation of this response is the catalyzed dehydration of carbonic acid (Fig. 1B) (71, 150), rather than channel gating.

Since the GABA_A receptor and the strychnine-sensitive glycine receptor have a similar permeability to HCO₃^- (34), it is plausible that glycinergic inhibitory transmission is also associated with HCO₃^--dependent alkaline pH_o shifts. Accordingly, the glycinergic fall in pH_i noted in brain stem medullary neurons (189) might be accompanied by a concomitant external alkalosis. This topic remains largely unexplored, however. In the dorsal horn of neonatal rat spinal cord, Sykova and colleagues (147) noted a small glycine-evoked alkaline shift; however, this response persisted in the HEPES-buffered media, suggesting it was not mediated by an HCO₃^- efflux.

6. Mechanisms of activity-dependent acid shifts

1. LATE ACID SHIFTS: EVIDENCE FOR A METABOLIC ORIGIN. Slow, activity-dependent acid shifts were noted in the earliest pH microelectrode studies (65, 322). Kraig et al. (174) made the first attempt to elucidate the cause of these phenomena. In the in vivo rat cerebellum, stimulation of the parallel fibers, or induction of spreading depression, elicited a small, brief alkaline transient, followed by a pronounced acidification lasting minutes. In the presence of acetazolamide, the acid shift was increased in size. This observation suggests that the acidosis was due to extrusion of H⁺ rather than the generation of CO₂. A persistent acidification was also induced by elevation of [K⁺]_o, which could be abolished by fluoride (a glycolytic inhibitor). A similar acidosis was noted in vivo following spreading depression (174, 215) and could be correlated with an increase in brain lactate (215, 269). Taken together, these observations argue that the metabolic production of acid and its subsequent extrusion were responsible for these interstitial acid shifts.
   Although brain cells express a lactic acid transporter (219), there is no evidence that this mechanism directly contributes to interstitial acidosis. Indeed, in hippocampal slices, lactate transport inhibitors did not affect the extracellular acidosis induced by hypoxia (180), or by elevated K⁺ (128). Given the ability of lactate to serve as an energy source in brain (272), it has been suggested that lactic acid efflux from glial cells is followed by uptake and utilization by neurons (245a, 271, 345). As such, there may be no net effect on interstitial pH. Assuming that prolonged stimulation, spreading depression, and hypoxia cause a fall in pH_i, it seems likely that classic acid extrusion mechanisms would be activated, (e.g., Na⁺/H⁺ exchange, Na⁺-driven Cl^-/HCO₃^- exchange, or electrogenic Na⁺-HCO₃^- cotransport), which could be responsible for the ensuing extracellular acidification. Inhibition of activity-dependent acidosis by amiloride has implicated Na⁺/H⁺ exchange in this regard (338, 262); however, effects of this drug on excitability (301) could not be ruled out.
   In hippocampal slices there is strong evidence that CO₂ generation, rather than acid extrusion, is responsible for slow activity-dependent acid shifts. Voipio and Kaila (328) demonstrated that repetitive activation of the Schaffer collaterals was associated with a slow rise of tissue PCO₂ that fully accounted for a concomitant delayed interstitial acidosis. The late acid shift of spreading depression was similarly attributed to CO₂ generation in hippocampal slices (297). The hippocampal acid shifts were reduced by benzolamide (328), consistent with a CO₂-generated acidosis. These results seem to differ from the observations in vivo, where cerebellar (174) and cortical (215) acid shifts were increased by acetazolamide. The ability of blood flow to rapidly clear CO₂ in vivo may partly explain these differences.
   
2. EARLY ACID SHIFTS: EVIDENCE FOR A GLIAL ORIGIN. In some instances, neural activity induces a rapid interstitial acidification that can precede or preclude early alkaline transients. For example, initial acid shifts have sometimes been noted at the onset of spreading depression (215, 314). Sykova and Svoboda (291) reported robust, early acid shifts in the dorsal horn of the adult rat spinal cord following peripheral nerve stimulation. These acidifications were inhibited by stilbenes and amiloride, consistent with the involvement of several acid extrusion mechanisms. In contrast, in isolated neonatal spinal cord, a prominent early alkalosis occurred, which could be elicited with as little as a single shock to the dorsal root (148). Over the first two postnatal weeks, this response converted to a rapid acid shift that increased in prominence over the next three months (Fig. 3A). A similar developmental shift from stimulus-evoked alkaline to acid shift was noted in rat optic nerve (91) (Fig. 3B) and in the CA3 region of hippocampal slices (356). It was suggested that the alkaline to acid transformation had a glial origin, as the time course of the change roughly paralleled glial cell development (91, 148).
   The idea that glial cells actively secrete acid in response to neural activity has been proposed on many occasions. The observation of a rapid, activity-dependent rise in the cytosolic pH of astrocytes suggested that these cells extrude acid in response to membrane depolarization (81, 82). This hypothesis was tested using Ba²⁺ to inhibit the astrocyte K⁺ conductance and thereby prevent the depolarization that normally results from elevation of [K⁺]_o (17). When rat cortex was superfused with Ba²⁺, the stimulus-evoked depolarization and the cytosolic alkalization of astrocytes were both blocked (82). In the cortical interstitial space, Ba²⁺ unmasked or amplified an early stimulus-evoked alkalosis, consistent with the loss of a rapid acid source (82). In neonatal rat spinal cord, Ba²⁺ had a similar effect. After the developmental shift from stimulus-evoked alkalosis to acidosis (148), addition of Ba²⁺ appeared to inhibit the acidification and unmask the underlying alkaline response (290). Moreover, in the spinal cord of irradiated animals, where astrocyte development was prevented, the early stimulus-evoked acid shift did not develop, and addition of Ba²⁺ had no effect on the prevailing early alkalosis (290).
   Taken together, these observations suggest that the effect of Ba²⁺ on activity-dependent pH_o transients is due to the arrest of glial acid secretion. Nonspecific actions of Ba²⁺ cannot be discounted, however, particularly when this ion is superfused over a complex preparation. Use of simpler preparations to study glial acid secretion has reduced some of these concerns. Direct evidence for depolarization-induced acid secretion of astrocytes came from studies of gliotic hippocampal slices in which the CA3 neuronal population was replaced by a dense proliferation of reactive astrocytes (128, 129). K⁺-evoked depolarization of the astrocyte population elicited an intracellular alkaline shift (129), and a corresponding interstitial acidosis (128), that were both blocked in the presence of Ba²⁺. The acidosis was dependent on external Na⁺ and HCO₃^-, consistent with the operation of electrogenic Na⁺-HCO₃^- cotransport. Similar characteristics of K⁺-evoked intracellular and surface pH shifts were noted in studies of retinal Muller cells from mudpuppy retina (222). External acidification was recorded with a novel method in which the fluorescent pH indicator BCECF was fixed to a coverslip and imaged below the cell. The external acidification was localized mainly to the end foot process and was reduced in HCO₃^--free media or by addition of DIDS.
   The best evidence that glial cells actively secrete acid (via inward electrogenic Na⁺-HCO₃^- cotransport) has come from studies of the leech, where the extracellular fluid of a ganglion is controlled by a single giant glial cell (Fig. 4). Preventing activity-dependent depolarization of the cell under voltage clamp turned the stimulus-induced pH_o shift from an acid to an alkaline response (260). This result convincingly demonstrated that the stimulus-evoked alkalosis in this preparation was normally masked by the action of the glial electrogenic Na⁺-HCO₃^- cotransporter.
   The study of extracellular acid shifts evoked by glial cells has led to further insights into the nature of their Na⁺-HCO₃^- cotransporter. In gliotic slices, and in retinal Muller cells, depolarization-induced external acidification was increased after inhibition of extracellular CA (128, 222; Fig. 5A). This suggested that the mechanism of acidification was not solely dependent on the uptake of HCO₃^-, since an acid shift involving loss of interstitial HCO₃^- alone should have been diminished when external CA was blocked (128) (Fig. 5B). It has been suggested that CO₃^2- is the ion species utilized to accomplish net HCO₃^- flux by the renal Na⁺-HCO₃^- cotransporter as well as other HCO₃^- carriers (36, 39, 40, 283a). Such a mechanism could explain the effects of CA inhibitors on the extracellular acid shifts produced by glial cell depolarization. Grichtchenko and Chesler (128) proposed that the uptake of Na⁺ and CO₃^2- by astrocytes (thermodynamically equivalent to 1:2 Na⁺-HCO₃^- cotransport) would acidify the extracellular compartment by driving the dissociation of HCO₃^-, thereby producing H⁺ and replenishing CO₃^2- (Fig. 5C). The fractional increase in [H⁺]_o would be greater than the fractional fall in [HCO₃^-]_o, driving the dehydration of H₂CO₃, and thereby buffering the increase in H⁺. If external CA were blocked, the rate of buffering would be reduced, leading to a greater acid shift (128). Additional evidence consistent with this idea was provided in a study of the surface pH of oocytes expressing both an Na⁺-HCO₃^- cotransporter and extracellular CA IV. Voltage-clamp depolarization produced a surface acidification that was amplified following inhibition of the CA, consistent with the uptake of carbonate ions (127).
   It should be noted, however, that the same results would be anticipated in the above studies, if a Na⁺-HCO₃^- pair was taken up in exchange for H⁺, as this would result in similar fractional changes in the interstitial ion concentrations. Thus, while the data of the above studies indicate that HCO₃^- is not the sole acid equivalent carried by the Na⁺-HCO₃^- cotransporter, the results can be explained with or without the transport of CO₃^2-.
   It has been suggested that glial acid secretion is a means of regulating interstitial pH at the onset of neural activity, and serves to "muffle" activity-dependent alkaline shifts (76, 94, 252). Because activity-evoked alkalosis can arise within 10 ms (124, 177), glial acid secretion might be expected to occur with similar speed. The rate of this process is not clear, however. In spinal cord, where early acid shifts are presumed to have a glial origin, pH microelectrodes demonstrated an onset within seconds of stimulation (148, 290, 291). Faster changes could not have been detected, however, as the best response time of pH microelectrodes is on the order of 1 s. Studies of glial acid secretion would accordingly benefit from the use of optical recording methods.
   
3. DO SYNAPTIC VESICLE CONTENTS CONTRIBUTE TO EXTRACELLULAR ACIDIFICATION? Extremely rapid, apparent acid transients were reported in hippocampal slices after stimulation of the Schaffer collaterals (177). These changes, detected by phenol red absorbance, peaked 10 ms after an orthodromic stimulus and were followed by slower transients that corresponded to typical alkaline-acid shifts. It was suggested that such an early acidification could be due to release of the acidic contents of synaptic vesicles (187, 198, 208). These putative fast acid responses were not altered by acetazolamide, however, and were not otherwise confirmed to be pH changes by manipulation of the buffering power. In a study using a fluorescein-dextran probe to record Schaffer collateral evoked pH_o shifts, only an early alkaline shift was detected within this time frame (124), and these fluorescent responses were amplified by addition of the CA inhibitor benzolamide (315).
   The release of vesicular contents into the synaptic cleft should nonetheless alter the pH in this microdomain. DeVries (99) has recently provided evidence that acid vesicular contents released from mammalian cone photoreceptors produce a significant local pH change that has a feedback inhibitory action on the cone calcium channels. The onset of inward cone calcium currents was interrupted by a transient outward current that decayed with a 1- to 2-ms time constant. This brief suppression of the Ca²⁺ current was correlated with the onset of transmitter release, and the effect was diminished when the extracellular buffering power was augmented with HEPES. These data are the best evidence to date that a physiologically relevant acid transient may occur in the synaptic cleft due to release of acidic vesicular contents.
   

View larger version (11K): [in this window] [in a new window]   FIG. 3. Developmental onset of rapid, stimulus-evoked acid shifts. A: repetitive stimulation of the dorsal roots elicited an alkaline-acid sequence of pHo transients in the dorsal horn of 4-day-old rats. In older animals, identical dorsal root stimulation elicited a predominant acid shift. [From Sykova et al. (290), copyright 1992, with permission from Elsevier.] B: repetitive stimulation of optic nerve from a 1-day-old rat elicited an initial, small alkalinization, followed by a modest, slow acid shift. In older animals, pure acid shifts were observed. [From Davis et al. (91), copyright 1987, with permission from Elsevier.]

View larger version (10K): [in this window] [in a new window]   FIG. 4. Extracellular pH transients in leech ganglion regulated by glial cell membrane potential (Em). Left traces: afferent stimulation evoked a slow glial depolarization and an extracellular acid transient under current (Im) clamp. Middle traces: afferent stimulation during voltage clamp of glial cell unmasked a rapid extracellular alkaline shift. Right traces: an acid shift was again elicited upon return to afferent stimulation under current clamp. [From Rose and Deitmer (260).]

	VIII. EFFECTS OF ACTIVITY-DEPENDENT pH SHIFTS ON NEURAL FUNCTION

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Processes that could be modulated by activity-dependent pH shifts would be expected to have a steep sensitivity to H⁺ near its physiological range. In this category fall a number of membrane conductances including those associated with NMDA receptors (300, 320, 333), GABA_A receptors (243, 279, 299), voltage-gated Ca²⁺ channels (19, 141, 308, 309) and proton-gated channels (169, 178; reviewed in Refs. 22, 307, 319, 337).

Although channels may have appropriate sensitivities to changes in pH, in most experiments, pH shifts have been elicited by application of neurotransmitters or by artificial, synchronous activation of thousands of neurons. One may therefore question whether these responses are relevant to the in vivo setting. Synchronous or repetitive neural activity, however, is a normal feature of brain function. In hippocampus, for example, large populations of neurons fire together during the theta rhythm at frequencies close to 10 Hz (reviewed in Ref. 56). Stimulation of hippocampal afferents at comparable rates can elicit extracellular alkaline transients on the order of 0.1-0.2 pH units (63, 71, 144, 295, 355). While pH_o shifts have not been recorded in association with normal hippocampal or cortical rhythms, they are known to accompany aberrant neural activity associated with seizure. In vivo, seizure activity has been associated with pronounced interstitial acid shifts (278, 285). In addition, sizable alkaline shifts have been noted at the onset of spontaneous epileptiform bursts in isolated guinea pig brain (93) and hippocampal slices (355). Interstitial pH transients have also been noted in response to physiological stimuli. In the dorsal horn of the rat spinal cord, Sykova and Svoboda (291) reported a prolonged fall in pH_o (of 0.05-0.10 pH units) in response to noxious stimulation of the skin.

A. Modulation of Function by Extracellular Alkaline Shifts

The speculation that channel function is modulated by activity-dependent alkaline pH_o shifts has received some experimental support. Enhanced excitability has long been associated with a rise in pH_o, while acidosis has been observed to diminish neural activity (9, 16, 144, 182, 194, 324) (for review, see Ref. 286). NMDA receptors were implicated in these effects when the increased excitability of hippocampal slices induced by alkaline saline was prevented by specific antagonists (9). A likely biophysical basis for this observation was provided when it was discovered that NMDA receptors are blocked by external protons, with a steep sensitivity in the physiological range of extracellular pH (300, 320, 333).

To implicate a pH_o shift in the modulation of a channel, one can alter the buffering capacity and note whether shifts in the amplitude of the pH change elicit expected changes in channel behavior. This may be done by adding or removing major buffers such as sodium bicarbonate or HEPES; however, this approach necessarily entails large changes in the concentration of these substances, which could have numerous unanticipated effects. Inhibition of the interstitial CA with benzolamide is an attractive alternate means of reducing extracellular buffering power, since only micromolar amounts can markedly amplify HCO₃^--independent, interstitial alkaline shifts. Similar concentrations of benzolamide can also be used to reduce alkaline transients that are generated by a GABA_A receptor-mediated HCO₃^- efflux (153). Because this agent can have this dual effect, it is essential to understand the nature of the pH change one is attempting to modulate. In addition, possible nonspecific effects of CA inhibitors should be considered when conducting such experiments. For example, in neonatal hippocampal CA1 neurons, benzolamide partially inhibited low-threshold Ca²⁺ channels (123), which could diminish excitability. Therefore, to correctly interpret data, it is best that both pH_o transients and excitability be monitored in such experiments.

Taira et al. (296) first attempted to modify excitability using benzolamide in hippocampal slices. An increase in the amplitude (but no change in time course) of Schaffer collateral-evoked excitatory postsynaptic field potentials was noted, which began 5 min after exposure to benzolamide and progressed over the next 35 min. This enhancement was not seen in the presence of APV, indicating the involvement of NMDA receptors. In the presence of CNQX and the absence of external Mg²⁺, benzolamide caused a 20-30% increase in the amplitude of the NMDA receptor-mediated field potential, which did not occur in the presence of APV and Mg²⁺ (without CNQX). Although the pH_o transients were not recorded, and GABA_A receptors were not blocked, these observations implicated NMDA receptors in a benzolamide-mediated enhancement of excitability and suggested a buffering-mediated effect of the drug. If it were assumed that benzolamide amplified the stimulus-evoked alkaline transients, the results would be consistent with an associated augmentation of NMDA receptor currents, leading perhaps to an enhancement of AMPA receptor-mediated synaptic field potentials through long-term potentiation.

In a subsequent study, the effect of benzolamide on NMDA receptor-mediated synaptic transmission was resolved in conjunction with pH_o recordings (122). Excitatory postsynaptic currents (EPSCs) were recorded from CA1 pyramidal neurons under whole cell voltage clamp during single shocks to the Schaffer collaterals, while corresponding interstitial alkaline shifts were monitored with a nearby pH microelectrode. Application of benzolamide (in the presence of picrotoxin) increased the alkaline transients, as expected for a net proton sink (72). Benzolamide caused no significant change in the mean amplitude of the EPSCs. However, the duration of the EPSCs was markedly prolonged, and the onset of this effect coincided with the first observed amplification of the alkaline transient. This prolongation was not seen when benzolamide was added in the presence of APV (although the alkaline transients were still amplified), indicating that the effect was mediated via NMDA receptors. Because neither the prolongation of the synaptic response nor the increase in the alkaline transient occurred in HEPES-buffered media, the synaptic enhancement was not due to a direct action of benzolamide on these receptors. It is notable that when optical recordings of Schaffer collateral-evoked alkaline shifts were made under comparable conditions (with picrotoxin and benzolamide), the alkalosis peaked in 20-200 ms (124). These synaptically evoked alkaline shifts therefore appear well-timed to modulate the decay of the NMDA receptor-mediated synaptic responses, which occur within a similar period (136).

Augmentation of NMDA receptors by benzolamide has also been demonstrated in a pathological setting. Spreading depression is associated with a large alkaline transient that is increased by CA inhibitors (174, 215). In hippocampal slices, the latency and propagation velocity of spreading depression were increased by benzolamide, in conjunction with an amplification of the initial alkalosis. When APV was present, the alkalosis was still amplified by benzolamide, but the propagation of spreading depression was no longer enhanced (313, 314).

Although these data suggest that endogenous alkaline shifts can have a neuromodulatory role, several caveats remain. Assuming the effects of benzolamide on synaptic transmission and spreading depression were due to the pH sensitivity of NMDA receptors, it still remains unclear whether smaller alkaline shifts, elicited under normal buffering conditions (i.e., in the absence of benzolamide), would have a significant modulatory effect. In addition, while benzolamide did not appear to directly affect NMDA receptors, alternative actions of this drug on pre- or postsynaptic processes remain possible.

Similar putative modulation of other channels by endogenous alkaline shifts has not been demonstrated. Enhancement of voltage-gated Ca²⁺ conductances by an alkaline transient appears plausible, particularly for high-threshold channels (307). The GABA_A receptor-evoked alkalosis, being mediated by a channel-gated HCO₃^- efflux, may be particularly suited to influence GABA_A receptors in a feedback manner (243). The nature of this modulation is unlikely to be uniform, however, since the pH_o dependence of GABA_A receptors varies with subunit composition (319). Moreover, because generation of this alkalosis relies on interstitial CA, the localization of this enzyme with respect to GABA_A receptors may be a critical factor.

B. Modulation of Function by Activity-Dependent Acid Shifts

Given the decrease in excitability associated with an imposed acidosis, endogenous interstitial acid shifts could be expected to reduce neural activity. However, unlike an extrinsically imposed acidosis, which is unlocalized and slow, endogenous acid shifts show regional differences and can display a range of onset and decay times. Intracellular acid shifts generated by transmembrane H⁺ fluxes may be rapid and localized to a given cell, whereas metabolic acidosis is likely to affect intracellular and extracellular pH across a wide region. Potential effects of these pH changes are therefore considered according to their time course and location.

The fastest extracellular acid shifts may be those speculated to occur following the release of the acidic contents of synaptic vesicles (177). The strongest evidence for such modulation comes from a study of retinal cones in which depolarization-evoked Ca²⁺ currents underwent a brief inhibition, coincident with the expected release of synaptic vesicles from these cells (99). Detection of pH transients of appropriate time course, localized to the synaptic cleft, is still awaited.

Acid secretion by the astrocyte Na⁺-HCO₃^- cotransporter would be expected to operate on a somewhat slower time scale, commensurate with the time course of membrane depolarization. The onset of external acidification may also rely on the activity of interstitial CA (128). It has often been speculated that this process serves to mask or supercede endogenous, activity-dependent alkaline shifts. Because extracellular alkalization may be expected to increase neural activity, muffling of the alkalosis by glial acid secretion may serve to stabilize neuronal excitability (76, 94, 252) (see sect. VIIB6).

Rapid intracellular acidifications, beginning within a second of repetitive spike activity, have recently been reported in somata and dendrites of cerebellar Purkinje cells (349). In addition to their speed, these phenomenon are notable because they can be generated by the repetitive activity of a single neuron and are therefore likely to occur in vivo. The basis of these phenomena remains to be established. Given the inhibitory effect of internal acid on Ca²⁺ channel activity (307), and the preponderance of dendritic Ca²⁺ spikes in Purkinje neurons (302), one may speculate that these cytosolic acid transients act to diminish dendritic electroresponsiveness.

Delayed interstitial acid shifts can last for minutes following persistent, synchronous neural activity. These phenomena are the likely result of respiratory by-products such as CO₂ and lactic acid (see sect. VIIB6). The association of such responses with in vivo seizure activity (278), and the fall in excitability often associated with external acidosis (9, 16, 144, 182, 194), leads to the obvious speculation that epileptiform events become inhibited, and are possibly terminated, as a result of accruing interstitial acid. Thus, in hippocampal slices, low Mg²⁺-induced seizures in hippocampal slices were suppressed by superfusion with Ringer solution of low pH (324). NMDA receptors are an obvious focus of interest in this context, given their inhibition by external H⁺ (300, 320, 333). However, it should be noted that if these forms of acidosis are metabolic in origin, they would be associated with a fall in intracellular as well as interstitial pH. In addition, most experimental manipulations that sought to reduce pH_o would be likely to acidify the intracellular compartment as well. Concomitant intracellular acidosis may be expected to limit excitability through actions on a variety of channels (211, 288, 298, 307). It can therefore be difficult to pinpoint the manner in which an observed or imposed extracellular acidosis affects excitability.

A number of studies have provided evidence that a fall in intracellular, rather than extracellular, pH is important in arresting seizure activity. Use of weak acids to lower pH_i of hippocampal slices was able to suppress epileptiform events (29, 354), and the spontaneous termination of seizure was correlated with intracellular acidosis (354). The use of acetazolamide as an antiepileptic agent may be similarly related to its effects on brain pH. A fall in pH_i in hippocampal neurons was noted after application of acetazolamide and was proposed as a basis for the antiepileptic action of this drug (184). However, acetazolamide may have complex and diverse effects on baseline pH in the brain as it has been reported to alkalinize the interstitial space in vivo (174), acidify the interstitial space in brain slices (71, 338), and alkalinize the cytoplasm of thalamic neurons (214).

Although acidosis has typically been linked to a fall in excitability, under particular circumstances, a fall in pH may have the opposite effect. Velisek et al. (325) noted that microinfusions with fluid of pH 6.7 had proconvulsant effects in the posterior substantia nigra pars reticulata of rats, but no such effect in the anterior part of the nucleus. Differentiation between extracellular and intracellular sites of action could be difficult in such cases, since application of an acidic fluid is likely to reduce both pH_o and pH_i. An intracellular acidosis could increase excitability if membrane resistance were increased. This was the case in crayfish muscle where Moody (210) was able to attribute acid-induced hyperexcitability to block of an inward rectifying K⁺ conductance. Augmentation of inward currents by acidosis can also occur in the case of proton-gated channels. As these responses inactivate with sustained acidosis, extracellular acid shifts of sufficient size and speed would be required to increase excitability through this mechanism (22, 179, 337).

	IX. CONCLUSION

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A great deal has been learned about the regulation of pH_i in cells derived from the vertebrate CNS. The majority of studies have been performed in tissue culture. From the experimental perspective, culture systems are practical, and doubtless will continue to provide important clues to transporter function, particularly as studies focus on molecular sites responsible for the modulation of transporter activity. Issues concerning the biological relevance of culture systems should not be overlooked, however. The degree to which a cultured neuron or glial cell will express its in situ complement of transporters is not always known. Studies using brain slices or acutely dissociated cells, while more technically challenging, have appeal in this regard.

Many of the mechanisms responsible for activity-dependent modulation of brain pH remain unclear. The study of intracellular pH shifts can benefit from in vitro studies in tissue culture where individual cells are amenable to manipulation. The development of better pharmacological tools to more selectively target H⁺ and Ca²⁺ transport mechanisms would be beneficial for such studies. The adequate stimulus for the generation of a pH_i shift in culture may not be biologically relevant, however. In situ studies may be particularly useful in determining how changes in neural activity modulate acid transport systems and how normal shifts in the composition of the interstitial fluid impact on pH regulation. In this respect, both acute brain slices and organotypic culture systems have appeal, as these preparations maintain an intact interstitial space. In addition, activity-dependent changes in pH may be most relevant when studied in a preparation in which the activation and monitoring of discrete afferent pathways is feasible.

Interstitial pH shifts, by definition, require the use of an intact tissue. In some instances, surface pH transients may be studied on individual cells. However, the geometric constraints and particular buffering properties of the intact interstitium are needed to understand how these phenomena arise and are regulated in situ. Studies of interstitial pH shifts have benefited greatly from the advent of liquid membrane pH microelectrodes (4). These devices provide an outstanding signal-to-noise ratio with a response time as fast as 1-2 s. Nonetheless, it is increasingly clear that faster recording methods are required to fully understand the processes that govern interstitial pH. A few studies using optical probes have indicated that activity-dependent pH_o shifts can occur within milliseconds. Since these pH transients might be generated in discrete locales, improvements in both spatial and temporal resolution would be beneficial. Further identification and localization of interstitial CA would also add important insights into the microphysiology of brain pH, as the proximity and concentration of this enzyme could be the principal determinant of whether or how a pH_o transient is generated.

Address for reprint requests and other correspondence: M. Chesler, Dept. of Physiology & Neuroscience, NYU School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: mitch.chesler{at}med.nyu.edu).

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