Progressin NeurobiologyVol. 34, pp. 401 tO 427, 1990 Printed in Great Britain. All rights reserved

0301-0082/90/$0.00 + 0.50 ~ 1990 Pergamon Press pie

THE REGULATION AND MODULATION OF pH IN THE NERVOUS SYSTEM M.

CHESLER

Department of Neurosurgery and Department of Physiology and Biophysics, New York University Medical Center, 550 First Avenue, New York, NY 10016, U.S.A.

(Received 15 November 1989)

CONTENTS Abbreviations 1. Introduction 1.1, Determinants of pH 1.2. Physicochemical buffering power 1.3. Interstitial buffering 1.4. Intracellular buffering 2. Intracellular pH 2.1. The distribution of H ÷ across cell membranes 2.2. Average intracellular pH of brain 2.3. Intracellular pH of invertebrate neurons 2.4. lntracellular pH of vertebrate neurons 2.5. Intracellular pH of 81ial cells 2.6, Heterogeneity of intracellular pH 3. Regulation of intracellular pH via acid extrusion 3.1. Acid extrusion mechanisms of invertebrate neurons 3.2. Acid extrusion mechanisms of vertebrate neurons 3.3. Acid extrusion mechanisms of glial cells 3.4. The influence of acid extrusion on resting interstitial pH 4. Modulation of brain extraceUular pH by electrical activity 4.1. Technical considerations 4.2. Historical perspective 4.3. Extracellular alkaline shifts 4.4. Extracellular acid shifts 5. Modulation of intracellular pH 5.1. Neuronal phi transients 5.2. Glial pHi transients 6. Relationship of intracellular and extracellular pH shifts 7. Significance of pH modulation 8. Concluding remarks Acknowledgments References

ABBREVIATIONS

alOl B BCECF

blot DIDS DMO DNDS GABA

HEPES

Sum of concentrations of a weak acid and its conjugate base Concentration of strong base 2' 7'-Biscarboxyethyl-5,6 earboxy fluorescein Sum of concentrations of a weak base and its conjugate acid 4,4'-Diisothiocyanostilbene-2,2"-disulphonic acid 5,5-Dimethyloxazolidine-2,4-dione 4,4'-Dinitrostilbene-2,2'-disulfonate 7-Amino Butyric acid

Ka LIX NMDA Pco2 PHi PIle pKo S" SD

SID All correspondence to: Dr M, Chesler, Dept. of Physiology and Biophysics, NYU Medical Ctr, 550 First Ave., New York, NY 10016, U.S.A. 401

SITS TRIS

401 402 402 403 4O4 405 4O5 4O6 4O6 407 407 408 408 4O9 409 410 411 412 412 412 412 413 417 418 419 419 420 421 422 422 422

4-(2-Hydroxyethyl)= 1-piperazineethanesulfonic acid Weak acid dissociation constant Liquid-membrane ion exchanger N-methyl-D-aspartic acid Partial pressure of carbon dioxide in torr (ram Hg) lntracellular pH Extracellular or interstitial pH - l o g K, Solubility coefficient of COs in water in millimoles/liter/nun Hg Spreading depression Strong ion difference 4-Acetamido-4'-isothiocyanato-stilbene-2,2'disulfonic acid Tris-(hydroxymethyl)-aminomethane

402

fit 2

M. CHESLER Buffering power in millimoles per liter Bicarbonate-derived buffering power Intrinsic (non-bicarbonate-derived) intracellular buffering power Total buffering power Buffering capacity against CO.

1. INTRODUCTION The regulation of pH and its modulation by external stimuli have been studied in a wide variety of animal cells (Roos and Boron, 1981; Busa and Nucitelli, 1984; Thomas, 1984; Moolenaar, 1986; Frelin, 1988). In the nervous system, the majority of acid-base studies have dealt with the long term regulation of the cerebral spinal fluid and the effect of pathophysiological disturbances on brain pH (Nattie, 1983; Fencl, 1986; Kazemi and Johnson, 1986; Siesjo, 1985). With continuous, rapid measurements of pH now feasible in single nerve and glial cells, as well as the interstitial space of brain, details of local acid-base dynamics have become available. Although brain pH homeostasis has often been emphasized, it is clear that the acid-base status of neural tissue does not remain stable. Recent studies have established that electrical activity results in significant pH shifts in neurons, gila and the interstitial space. These activity-dependent pH transients occur rapidly, in both the acid and alkaline direction. In addition, the local pH change can be large enough to influence the activity of enzymes and channels, raising the intriguing possibility that pH transients could serve a modulatory role in brain function. This article will primarily address acid-base dynamics in the nervous system, emphasizing activitydependent shifts in brain pH. This subject is preceded by a brief synopsis of acid-base fundamentals, and the regulation ofintracellular pH in neurons and gila. Stimulus-dependent shifts of extracellular and intracellular pH are reviewed in detail, focusing on unresolved mechanistic issues, and intercellular relationships. Beyond the scope of this review are details of methodology and the numerous effects of experimentally altering pH. These subjects have been addressed in earlier reviews (Roos and Boron, 1981; Moody, 1984). 1. I. DETERMINANTSOF pH Before considering the dynamics of pH in the nervous system, it is useful to discuss some formalisms of acid-base physiology and address their relevance in neurobiological experiments. The terminology in this area has frequently been a source of confusion. The notion of buffering power, for example, has been defined in a myriad of ways (cf. Karmann and Held, 1972; Burton, 1973; Roos and Boron, 1981). By grouping variables into dependent and independent categories, Stewart (1978, 1981) sought to clarify acid-base terminology. Since a number of neurobiologists have found merit in Stewart's approach (Kraig et aL, 1983; Nattie, 1983; Carlini and Ransom, 1986; Fend, 1986; Kazemi and Johnson, 1986), his nomenclature will be briefly reviewed and related to classical terminology.

Stewart emphasized that the equilibrium acid-base status of a physiological solution is defined by a few independent variables. These include the strong ion difference (SID), the total concentration of each individual weak acid or base (a,o, or b~o, respectively) and the partial pressure of CO:(Pco:). The activities of other acid-base species (e.g. H~, OH-, HCO/, C O i - , conjugate weak acids and bases) are considered dependent variables, completely determined once the Pco:, % , b,o,, and SID are established. The SID is defined as the sum of the concentrations of fully dissociated cation species (Z [c + 1) minus the sum of dissociated anions (Y [a-]). Thus, SID = "; [c + ] _v" [a-].

(I)

A change in the SID of a solution reflects the net addition of strong acid or strong base. For instance, addition of NaOH or HCI would respectively raise or lower the SID. At physiological pH, addition of a species such as lactic acid (pK, = 3.86, Dean, 1979), would lower the SID, since it would be more than 99% dissociated. The treatment of Pco2 as an independent variable rests on the assumption that the system in question is in equilibrium with an infinite CO: source (i.e. is "open" with respect to CO,). Since the Pc~ is imposed on the system, it is independent of changes in SID, a,°, or b,ot. This describes many in vivo and in vitro experimental settings, where CO: can exchange with a relatively infinite source, such as blood or a superfused solution. Situations where the Pco., cannot be considered an independent variable are discussed below. The ato, or b,ot for an unchanged weak acid (Ha) or weak base (b) is defined as, a,o~= [Ha] +

[a-]

(2)

and bto, = [hi + [bH + 1,

(3)

where a- and bH + are the conjugate base and conjugate acid respectively. Note that in a complex solution, the total concentration of each species of weak acid or weak base would constitute a separate independent variable. For the sake of clarity, only a single ato, will be considered in this discussion. In a system with acid-base status determined by the SID, Pco: and atot, the dependent variables include [H+], [OH-], [HCO~-], [CO~-] and [a-]. Physiologic pH shifts in such a system can be classified according to the altered independent variable. Thus, systemic acidosis can arise with an increase in plasma Pco2 (respiratory acidosis) or a fall in the plasma SID (metabolic acidosis). Similarly, intracellular pH shifts may occur in association with a transmembrane change in the SID. For example, the operation of Na +/H ÷ antiport results in the net extrusion of H + in exchange for Na +. This process results in an increase in the cytoplasmic SID and a rise in intracellular pH. Note that Stewart's approach only describes how equilibrium states differ. As such, it cannot formally address the mechanism of a pH shift. Thus, strictly speaking, it would be erroneous to state that pH rises because the SID increases. For instance, an identical

Tax REGULATION AND MODULATION OF pH IN THE N E R v o u s

rise in the cytoplasmic SID could arise via electroneutral Na÷/H + exchange or electroneutral Na+/ H C O ; contransport, processes which are equivalent thermodynamically, but differ mechanistically. Nonetheless, although the insights which can be gained from an equilibrium description are limited, mechanisms may be broadly classified based on whether they impact on the final SID, Pco, or a,o,. This approach has proven useful in the analysis of activitydependent pH shifts in brain (Kraig et al., 1983). !.2.

PHYSICOCHEMICAL BUFFERING

POWER

dB dSID f = dpH = dpH

(4)

where B is the concentration of added strong base (or the increase in SID). Since pH has no unit, /~ is expressed as a concentration, typically in millimoles per liter. This approach was used by Koppel and Spiro (1914) in the first quantitative account of the buffering power of weak acid-base solutions (their classic work has been translated by Roos and Boron 0980)). These investigators defined the buffering power of a monovalent weak acid with respect to the SID, using a variable "S", where " S " = - S I D . Several years later, Van Slyke (1922) published a similar account of aqueous buffering power, adopting the more commonly employed variable B. Ignoring the self-buffering of water (significant only at pH extremes) Koppel and Spiro's expression for the buffering power of a weak acid in a closed system is = dSID = 2"3[H + ][a'°d[Ka] dpH ([H + ] + K,)-'

(5)

where K, is the weak acid dissociation constant. When pH = pkg, buffering power is maximum and Eqn. (5) reduces to B = 0.575 [a,o,].

(6)

Koppel and Spiro further showed that in a complex solution containing several weak acids (or weak

403

bases), the total power (/~r)is the algebraic sum of the contribution of each component. Thus, for a solution containing weak acids a-j, /~r is given by

~T=tk+~b+'-'+~j,

(7)

where /~:/~j are the respective buffering powers of each weak acid-conjugate base pair. In many biological settings, bicarbonate and CO: constitute the major buffer system. These species participate in buffering through the reaction CO2+

Although a shift in equilibrium acid-base status can be described by a change in SID, a~o~or Pco~, one typically measures only the pH. As a dependent variable, knowledge of the H ÷ activity alone provides limited mechanistic insight. In a poorly buffered solution, small changes in the SID, atot o r eco2 can be associated with large shifts in pH, while in a well buffered solution, underlying acid-base shifts of considerable magnitude can remain masked. Clearly, to quantify an acid-base shift, the pH buffering power must be accounted for. For a simple solution in a closed system, the buffering power is readily defined. However, in many experimental settings, the system is neither simple nor closed, and analysis invariably requires a number of assumptions about the buffering power. Given the three independent acid-base variables outlined by Stewart, the pH buffering power of a solution may be defined with respect to a change in the SID, the Pco: or the atot (i.e. dSID/dpH, dPcoJdpH or datot/dpH). Historically, buffering power (fl) has most often been expressed as

SYSTEM

Ki gz K3 ILl t'~--...~l..l" t ' t " l ----~LIt~t'~---...~t"t"~2 .tm.2x..,~----.tl2",-,'~3~-----tL,,.,x./3 -e-----,,.,x.~3- "4- H ÷

+ H+

(8)

where K,,/(2 and K3 are the equilibrium constants for the respective hydration and dissociation reactions (K t incorporates the constant concentration of water). Van Slyke (1922) noted that when a system is open with respect to CO2, the buffering power provided by CO:/HCO~" (fib) is far greater than that predicted by Eqn. (5), and is given by /~b = 2.3[HCO~" ] + 4.6[CO~- ].

(9)

At physiological pH, the contribution of CO~- is negligible and the buffering power can be reasonably approximated by fib = 2.3[HCOf ].

(10)

Comparison of Eqns (6) and (10) indicates that on a molar basis, a CO:/HCO~" buffer at fixed Pco~ provides four times the maximum buffering power of a monobasic weak acid in a closed system. The relationship between pH, Pco2 and [HCO; ] is given by the Henderson-Hasselbach equation, [HCO;] pH = pKa + log S' Pco----'~. x '

(1 I)

where Pco~ is expressed in mm Hg and S' is the solubility coefficient of CO: in raM/ram Hg. Ka is the overall equilibrium constant for the hydrationdehydration of CO, and the reversible dissociation of H2CO 3 (referring to the reversible equilibria in Eqn. (8), K, = KI.K,). Solving Eqn. (ll) for [HCO~- ] and substituting into Eqn. (10) yields fb = 2.3[HCO; ] =

2.3 x S' x Pco~ x 10OH. (12) 100x`

Note that since [HCO~"] increases exponentially with pH, ~b is highly pH dependent. For a solution with a Pco2 of 40 mm Hg (S' = 0.032 raM/ram Hg and pK, = 6.13), ~b would rise exponentially from 22 mM tO 69 mM as pH increased from 7.0 to 7.5. Thus, any physiologic process which raises pH will be hindered by increasingly effective buffering. Where buffering is provided by a weak acid or base, and the pH is near the pK, ( + 0.5 pH), fl is not markedly pH dependent. Under these circumstances, the ASID underlying a pH change is often approximated by treating/} as a constant. However, a constant fl is sometimes employed because its pH-dependence is unknown. This often occurs in

404

M. CHESLER

the treatment of intracellular buffering power (see below). In such instances, A S I D is approximated as A S I D = ]3 x ApH.

(13)

Where the pH-dependence of /~ is defined, the A S I D underlying a pH shift is more accurately deter-

mined by integrating/~ with respect to pH. In general, for a shift from pH~ to pH,., ASID =

fp'"" /~ dpH.

(14)

H)

In a system buffered exclusively by CO2/HCOj', (e.g. brain interstitial space), Eqns (12) and (14) can be combined to yield A S I D = (2.3)(S')(Pco~)(e z'3*x')

f

pH2

e"3pHdpH

JpH)

= C[e2~PHz--e''3pH'] (15) where C = (S')(Pco2)(e2~Px'). It should be stressed that the equations relevant to a CO,/HCOj- buffer are valid only when the system is open with respect to CO2. In a closed system, the Pco: would not behave as an independent variable. For instance, in ischemia, where gas exchange with blood ceases, the Pco: becomes a dependent variable. This is apparent in ischemic brain, where the generation of lactic acid (a decrease in S I D ) titrates bicarbonate, resulting in a large rise in tissue Pco2 (Kraig et al., 1986; von Hanwehr et aL, 1986) Under closed conditions, the buffering power provided by CO2/HCO~- would be given by Eqn. (5), where a,o, = (S' Pco,, + [HCOj" ]). Note further that the equations for a CO2/HCO~buffer system apply only at equilibrium. In the presence of the enzyme carbonic anhydrase, which catalyzes the hydration of CO2, equilibration occurs rapidly. However, the uncatalyzed reaction has a time constant of nearly 30see (reviewed by Maren, 1967; Carter, 1972). Thus, where this enzyme is not present, rapid pH transients could be poorly buffered. The consideration of buffering kinetics may he particularly relevant to interstitial acid-base dynamics, discussed below. 1.3. INTERSTITIALBUFFERING From the standpoint of acid-base balance, the interstitial fluid of brain contains negligible concentrations of proteins and organic acids (Fencl, 1986). Accordingly, its equilibrium acid-base status is determined mainly by the S I D and Pco2. In such a system, [ H C O f ] - SID, and interstital buffering power is given by Eqn. (10). Since extraccllular bicarbonate concentration ([HCO~']o) varies greatly among animals, the equilibrium buffering power of interstitial fluid ranges accordingly. In mammalian brain, assuming a pHo of 7.30 (Cragg et al., 1977; Javaheri et al., 1983; Kraig et al., 1983), and a /)co, of 46 torr (Ponten and Siesjo, 1966) calculated [HCO~"]o would be 22 raM, providing 50 mM of interstitial buffering power. By comparison, aquatic species do not retain CO2 and consequently have a [HCOf ]0 of approximately 5 mM (Holmes and Donaldson, 1969). Thus, the interstitial buffering power in the brain is gener-

ally four-fold greater in terrestrial species. Consequently, extracellular pH shifts associated vdth neuronal activity (Section 4) are expected to be larger in aquatic animals. Recent in v/vo studies of pH 0 transients in the brain of the skate support this concept. Stimulus-evoked alkaline shifts in the skate cerebellum (Rice and Nicholson, 1988) were 5-10 times greater than comparably-evoked pH transients in rat cerebellum (Kraig et al., 1983). As noted above, the presence of carbonic anhydrase is critical for the CO: hydration reaction to rapidly attain equilibrium. Thus, in the absence of this enzyme, and in the face of rapid interstitial pH transients, the assumptions inherent in the calculation of CO:/HCOj- buffering values may not apply. However, carbonic anhydrase is widely found in the mammalian nervous system. The lowest regional activity is in peripheral nerve (Trachtenberg and Sapirstein. 1980), where levels are sufficient to speed the CO, hydration 100-fold over the uncatalyzed reaction (Cammer, 1979). Thus, at the macroscopic level, all regions of the nervous system would appear to contain significant levels of carbonic anhydrase. At the microscopic level, however, carbonic anhydrase is heterogeneously distributed, localized principally within glial cells (Giacobini, 1962). While significant levels may be found in the peripheral nervous system (Kerhonen and Hyppa, 1967; Cammer, 1979; Trachtenberg and Sapirstein, 1980; Riley et al.. 1984; Peyronnard et al., 1988) it is the central oligodendrocyte that is particularly endowed with carbonic anydrase activity (Cammer, 1984; Kumpulainen, 1984). Central myelin is accordingly rich in this enzyme (Cammer et al., 1976; Yandrasitz et al., 1976; Trachtenberg et al., 1977). In addition, there have been reports of localization within astrocytes (Roussel et al., 1979; Anderson et al., 1980; Church et al.., 1980; Parthe, 1981) and particular groups of neurons (Kerhonen and Hypa, 1967; Wong et al., 1983; Riley et al., 1984; Sommer et al., 1985; Kazimierczak et al., 1986; Rogers and Hunt, 1987; Wong et al., 1987). Since carbonic anhydrase is localized mainly within glial cells, it is unclear whether it can play a significant role in buffering interstitial pH transients. This issue is underscored by recent studies of surface pH transients of snail neurons. Despite a high equilibrium buffering capacity of CO2/HCOj- solutions, rapid pH transients at the membrane surface of snail neurons were enhanced in HCOT-containing media compared with non-HCOf-buffered solutions (Thomas, 1988). These pH shifts were unaffected by the carbonic anhydrase inhibitor acetazolamide, suggesting that carbonic anhydrase does not play a role in pH buffering in the extracellular space of snail brain. By contrast, in mammalian brain, interstitial pH transients were amplified by acetazolamide (Kraig et aL, 1983; Carlini and Ransom, 1986; Sykova et al., 1988; Sykova, 1989), suggesting that this enzyme could help mitigate rapid acid-base shifts (however, see Section 4.3). The classic equations for CO2/HCO~ may also be inappropriate when pH transients occur within a small interstitial volume. Under these conditions, surrounding tissue would constitute a relatively infinite source of freely exchangeable buffer, pH shifts

T i m REGULATION AND MODULATION OF

would therefore by mitigated by local physicochemical buffering, and by diffusion of fresh buffer (both CO, and HCO~") to the region. The relative contribution of diffusion would depend on the radius of interstitial volume involved, and the kinetics of the buffering reactions. Since diffusion over a few microns would require only milliseconds, for extremely localized perturbations, diffusion would play an important role. Indeed, in the absence of carbonic anhydrase, it is likely that diffusion would be the predominant mitigating process. One consequence of diffusion would be to impart a time and space dependence to the apparent buffering capacity. Thus, at long times, the buffering capacity would seem infinite, since all components of the buffer system would eventually equilibrate with the surround. 1.4. INTRACELLULARBUFFERING Shifts in intracellular pH (pHi) may be mitigated by physicochemical buffers, metabolic reactions and membrane transport systems. In a broad sense, all three processes contribute to intrace[lular "buffering". However, the regulation of intrace[lu[ar pH by transport across the plasma membrane is usually distinguished from strictly intracellular processes. The total intracellular buffering power of cells (~T) is generally considered to consist of two components: the CO.,/HCO3-derived buffered power (]~b), and the non-HCO~- or "intrinsic" buffering power (/~i), where ]~T=~b+~i. The intrinsic component includes physicochemical buffering (e.g. by phosphates, titratable groups on proteins, etc.), as well as the consumption or production of acid by metabolism or organelles. ]~b depends on the intracellular HCO~concentration ([I"[CO~']i) and is given by Eqns (10) and (11). The contribution of/~b to the total intracellular buffering power therefore depends on pHi and the ambient Pco~. Compared with aquatic animals, terrestrial species maintain a higher [I-ICO; ]i, and therefore derive a greater fraction of intracellular buffering power from/~b. In mammalian brain, with a mean [HCO; ]i of approximately 10 raM,/~b averages 23 raM, while ]~i is 16-20 mM (Siesjo and Messeter, 1971). Experimental differentiation among physicochemical buffering and cellular homeostatic responses has never been completely possibly. In early studies of brain pH regulation, intracellular buffering was operationally defined as the final change in intracellular pH following an imposed shift in acid-base status. Typically, animals were made hypo- or hypercapnic (i.e. blood Pep2 lowered or raised respectively) and the average intracellular pH (PHi) of brain determined by weak acid distribution techniques (Ponten, 1964; Roos, 1965; Kjailquist et al., 1969; Roos, 1971). A "buffering capacity" (,~.) was then expressed as ;. -- d log Pco______~.2 dpHi

(16)

Values of ~. for rat brain ranged from 1.5 (Ponten, 1966) to 2,8 (Roos, 1971). In these studies, ,;. undoubtedly included the contribution of plasma membrane transport as well as intracellular buffers. Siesjo and Messeter (1971) attempted to sort out these JPN

~/~-D

pH IN TH~ NERVOUS SYSTEM

405

factors by titrating rat brain homogenates with CO2, in order to estimate the intrinsic intracellular buffering power. The value of ~. for the homogenate ranged from 1.6--1.7. These results can be related to the more familiar expression of buffering capacity (1~ = dB/dpH) as follows: ,~. = d log Pep2 -- l + ~i dpH i 2.3[HCO~]i

(17)

(Karmann and Held, 1972). Note that to convert ~. to /~, one must specify pHi, Pep2 and calculate [HCO; ]i. Assuming a/'co, of 40 mm Hg, a PHi of 7.00, and a [HCOj-]i of 10raM, an intrinsic ~. of 1.7 would correspond to a/3i of 16 mM. In studying the buffering power of brain homogenates, the contribution of neurones vs glial cells could not be addressed. Nonetheless, estimates of cytoplasmic buffering power in single neurons and glial cells have yielded similar values (Table I). In such studies, cells are usually subjected to a defined intracellular acid or base load, while the subsequent shift in pHi is measured. With an acid load, the resulting pH~ transient can be markedly diminished by the stimulation of acid extrusion, causing a rapid recovery of pHi and an overestimation of buffering power. To compensate for such effects, the phi recovery has often been extrapolated back to the time of onset of the pH change (Thomas, 1976b; Aickin and Thomas, 1977a). Alternatively, acid transport systems have been pharmacologically inhibited during an acid load (Boron et al., 1979). A critical discussion of these methods can be found in the review of Roos and Boron (1981). Note that while these approaches compensate for, or eliminate, the contribution of membrane-bound acid transport systems, the results do not distinguish between the intracellular components of buffering. The reversible consumption of acid by metabolic reactions or organelles may therefore contribute an uncertain fraction of the experimentally determined ]~i.

2. INTRACELLULAR pH A critical discussion of the techniques of pHi measurement is beyond the scope of this review and lengthy treatments of this topic may be found elsewhere (Waddel and Bates, 1969; Roos and Boron, 1981). The term "intraceUular pH" is often used synonymously with "cytoplasmic pH", however, in some instances an experimentally determined pHi could include a non-cytosolic contribution. Measurements of average pHi using whole tissue techniques are particularly subject to such uncertainties. These methods have played little role in the study of cellular pH regulation, as they lack sufficient spatial and temporal resolution. Nonetheless, when compared on the same preparation, comparable values of phi have been obtained using weak acids and pH microelectrodes (Boron and Roos, 1976). Moreover, in mammalian brain, direct measurements of glial pH i with microelectrodes (Chesler and Kraig, 1987, 1989) have yielded results nearly identical to whole tissue values obtained with a variety of techniques (Table 5).

NI. CHESLER

-/,06

TABLE i. INTRINSICINTRACELLULARBUFFERINGPOWER(fli) IN NEURALTI$SL~ Whole Tissue Rat brain Invertebrate Neurons Squid Axon Snail H. Aspersa

A. monteryensis (pleural) A. monteryensis (pedal)

Crayfish Barnacle Leech Vertebrate neurons Lamprey Rat synaptosomes Glial Cells Leech

B~

Temp.

Method

t9

37

Homogenate

20 9 11 25 30 11 9-12 9-20 4-9 12-26 25 14-18 16.7 12-33

21 __.~ 22 RT RT RT RT RT 12 12 25 20 RT RT

Homogenate NH: NH: CO: CO: H ~ injection H* injection Weak acid or base H + injection H + injection CO., CO,. NHf CO,. NHZ

16

23

NHZ

30-48 ~ 50

20 30

Homogenate NHf

20-30

22-25

CO,

Reference Siesjo and Messeter. 1971 Spyropoulos. 1960 Boron and DeWeer, 1976 Boron and Russell. 1983 Thomas. 1974 Thomas, 1976b Thomas, 1976b Meech and Thomas, 1980 Szatkowski and Thomas. 1989 Ahmed and Connor, 1980 Ahmed and Connor, 1980 Moody, 1981 Brown and Meech. 1979 Schlue and Thomas, 1985 Deitmer and Schlue. 1987 Chesler. 1986 Jean et al.. 1985 Nachshen and Drapeau, 1988 Deitmer and Schlue, 1987

fl~ in millimoles per liter, T in degrees Celsius. Abbreviation: RT -- room temperature.

2.1. THE DISTRIBUTION OF H + ACROSS CELL MEMBRANES Measurements of intracettular pH have established that H ÷ is non-passively distributed across the plasma membrane of nearly all animal cells (for reviews see Caidwell, 1956; Waddel and Bates, 1969; Roos and Boron, 1981; Thomas, 1984; Frelin et al., 1988). A purely passive H ÷ distribution would predict the following relationship: RT

Em= E.. = - T

[H÷]0

(18)

where Em is the membrane potential, EH. is the equilibrium potential for H +, F is the Faraday, R is the gas constant and T is temperature in Kelvin. Given a membrane potential of - 59 mV and a pH0 of 7.3, a pH~ of 6.3 would be predicted at 25°C. However, pHi is typically 7.0 or greater in animal cells. Thus, E a is more positive than E=, and an inward electrochemical gradient exists for H ÷. Because of this inward gradient, cells must extrude acid by energy-dependent means in order to maintain pH~ near 7.0 (see Section 3). 2.2. AVERAGEINTRACELLULARpH oF BRAIN In mammalian brain, weak acid distribution techniques have yielded an average pH i of approximately 7.0 in vivo (Table 5). Similar values have been obtained using fluorescent (Sundt et al., i978; Csiba et aL, 1983), colorimetric (Kogure et aL, 1980; LaManna and McCracken, 1985; LaManna, 1989) or nuclear magnetic resonance techniques (Ackerman et al., 1980; Petroff et aL, 1985) (see Table 5). Common to whole tissue techniques is the operational treatment of brain as a homogeneous macroscopic system, an approach which must ignore

cellular heterogeneity. Thus, Messeter and Siesjo (1971) referred to the pH~ obtained by weak acid methods as an "equivalent intracellular pH" regarded as "the p H existing in a homogeneous compartment which has the same bicarbonate concentration and the same CO: tension as the corresponding mean values for the intracellular space." While these authors

acknowledged the possibility of differences in pH among intracellular compartments, available techniques could not provide the spatial or temporal resolution required to address this issue. It is now recognized that extremely rapid acid-base shifts occur in brain under normal and pathological conditions (Section 4). Moreover. there can be striking differences in the acid-base behavior of neurons and glial cells. Thus, while whole tissue techniques may provide an accurate measure of average pHi, they are poorly suited for the study of acid-base dynamics. Although numerous in viro studies indicate an average p h i in mammalian brain near 7.0, values of 7.5-7.8 have been reported in brain slices using neutral red as a pH indicator (LaManna et al., 1987: Sick et al., 1989). A somewhat lo~er range (7.2-7.5) was noted using creatine kinase and N M R techniques in brain slices (Kass and Lipton. 1982; Whittingham et al., 1984; Sick and Balestrino. 1988; Kauppinen et al., 1989). By contrast, a D M O study reported an average pH i of approximately 6.9 (Hertz et al., 1970). It is unclear why brain slice pH, seems to vary over such a large range. The relatively high pH~ of hippocampal slices fell to near 7.0 in the presence of the N a - / H ÷ exchange inhibitor amiloride, suggesting that the activity of the Na ÷ H - exchanger may be altered in brain slices (LaManna et al.. 1987). These effects could be related to the transient anoxia associated with slice preparatio n, since a high pH~ has also been reported following reversible brain ischemia (Mabe et al., 1983).

THE R£GULATION AND MODULATION OF

pH IN THE NERVOUS SYSTEM

407

TABLE2. INTRACELLULARpH oF INVERTEBRATENEURONS PHi

Temp.

PH0 /Buffer

Method

Reference

7. I0 7.25-7.45 7.0 7.32 7.35 7.36 7.3

---23 22 22 --

7.04-8.05/? 7.9I SW ?/$W 7.6-7.8/HEPES-TRIS 7.70/HEPES-TRIS 7.70/HEPES-TRIS 7.95-8.05/SW

GE GE SbE GE GE DMO GE

Caldwell, 1958 Spyropoulos, 1960 Bicher and Ohki, 1972 Boron and DeWeer. 1976 Boron and Roos, 1976 Boron and Roos, 1976 Caldwell, 1958

7.26 7.41 ~ 7.2 7.35 7.3

-RT 12 12 20

7.81/? 8.0/TRIS 7.4/MOPS 7.6/MOPS 7.5:TRIS

GE GE GE GE GE

Kostyuk et al., 1969 Thomas, 1974 Ahmed and Connor, 1980 Connor and Hockberger, 1984 Brown and Meech. 1979

7.12 7.23

RT 23

7.4HEPES 7.35HEPES

GE LIX

Moody, 1981 Moser, 1985

7.31 7.28 7.20 7.32 7.27

RT RT RT RT RT

7.4/HEPES 7.4/HEPES 7.4/CO:-Bic 7.4/HEPES 7.4/COrBic

LIX LIX LIX LIX LIX

Squid axon L. forbesi L. pealeii

Crab axon Snail H. pomatia H. asl~rsa A. monteryensis Barnacle Crayfish P. clarkii A. fluriatilis

Leech Retzius cells

Schlue and Thomas. 1985 Deitmer and Schlue. 1987 Deitmer and Schlue, 1987 "Noxious" cells Dcitmer and Schlue, 1987 Deitmer and Schlue, 1987 T in degrees Celsius. Abbreviations: GE -- glass electrode, SbE = antimony electrode, DMO -- 5,5-dimethyloxazolidine2,4-dione, LIX -- liquid ion exchanger, SW = sea water, Bic -- bicarbonate. 2.3. INTRACELLULARpH OF INVERTEBRATENEURONS Microelectrode studies of pH i in neurones were first performed on large invertebrate cells (Table 2). Caldwell (1958) used a 50/am glass pH electrode and a separate reference pipette, inserted longitudinally into squid giant axons. With pH0 ranging from 7-8, the pHi of the axoplasm was about 7.10, a value far too high to be explained by a passive distribution of hydrogen ions. In subsequent studies, neuronal pH~ varied with species, temperature, extraceilular buffer, or measurement technique, however, a non-passive distribution of H + was always noted (Table 2). Using a glass electrode, Spyropoulos (1960) obtained a phi for squid axoplasm of 7.25-7.45. Bicher and Ohki (1972) studied squid axons with an antimony electrode, and reported an axoplasmic pH~ of 7.0. Using a 10-15/~m diameter glass electrode developed by Hinke (1967), Boron and DeWeer (1976) found a pHi of 7.32 in squid axoplasm. The development of pH microelectrodes with tip diameters in the micron range permitted successful radial penetration of neuronal cell bodies. Kostyuk et al. (1969) reported a pH i of 7.26 in Hell.,: neurons using a design in which pH sensitive glass protruded from the tip of a microcapillary. Thomas (1974) developed a recessed tip electrode, and reported a pHi

of 7.41 in H e l i x neurones. Comparable values were obtained in subsequent glass microelectrode studies of snail neurons (Ahmed and Connor, ! 980; Connor and Hockberger, 1984), barnacle photoreceptors (Brown and Meech, 1979), and crayfish neurons (Moody, 1981). More recently, double-barreled pH microelectrodes have been employed, based on an H+-selective liquid exchanger (LIX) (Ammann et al., 1981). Using both LIX and glass pH electrodes in the study of snail neurons, Schlue and Thomas (1985) reported comparable accuracies. 2.4. INTRACELLULARpH OF VERTEBRATENEURONS Due to the relatively small size of most vertebrate neurons, microelectrode studies of intraceUular pH have been limited to a few nerve cells with large cell bodies (Table 3). Double-barreled LIX electrodes were used to study lamprey giant reticulospinal neurons (Chesler and Nicholson, 1985; Chesler. 1986). The pH~ of these neurons (7.44) was generally higher than interstitial pH (6.9-7.4), indicating that an alkaline pH~ could be maintained despite considerable extracellular acidosis. Endres et al. (1986b) used a similar LIX electrode to study frog spinal motoneurons. Although a mean value was not reported, their records suggest that pHi ranged from 6.9-7.2.

TABLE 3. INTRACELLULARpH OF VERTEBRATENEURONS

Lamprey brainstem Frog motoneurons Rat sympathetic Brain synaptosomes Cultured rat brainstem Cultured rat Purkinje

phi

Temp.

7.44 7.43 6.9-7.2 7.03

23 23 18-21 37

6.94 7.18 7.07

30 -37

pH0/Buffer

Method

7.35/CO:-Bic 7.4-7.5/HEPES 7.2-7.5/COrBic 7.4/HEPES

LIX LIX LIX BCECF

Chesler, 1986 Chesler, 1986 Endres et aL, 1986b " Tolkovsky and Richards, 1987

BCECF BCECF BCECF

Nachschen and Drapeau, 1988 Richards and Pocock, 1989 Gaillardet aL, 1989 Gaillard et al., 1989

7.4/HEPES 7.4/CO,.-Bic 7.4/CO.,-Bic

Reference

7.37 37 HEPES BCECF T in degrees Celsius. Abbreviations: LIX = liquid ion exchanger, BCECF = 2'7"-biscarboxyethyl-5,6 carboxy fluorescein.

408

M. CHESLER

TABLE4. INTRACELLULARpH OF GLIALCELLS Rat cortical astrocytes Mudpuppy Leech Cultured cells Rat and mouse astrocytes

Mouse oligodendrocytes Glioma cells

pH~

Temp.

PH0/Buffer

Method

Reference

7.10 7.04 7.39 7.32 6.87

37 37 --

m vivo CO,.-Bic 7.5/HEPES 7.4/HEPES

LIX LIX LIX LIX LIX

Chesler and Kraig, 1987 Chesler and Kraig, 1989 Astion et al., 1989b Astion e t a / . . 1989b Deitmer and Schlue, 1987

7.29/CO2-Bic COz-Bic HEPES CO2-Bic 7.2-7.4/CO:-Bic 7.4/HEPES 7.3/HEPES

DMO DMO BCECF BCECF LIX BCECF BCECF

7.06 7.4-7.5 ~7.1 6.9 7.6 7.0-7.3 7.23

in vivo

RT --

-

37 37 37 37

T in degrees Celsius. Abbreviations: LIX=liquid ion exchanger, D M O =

Kimelberg, 1983 Woodbury et al., 1984 Boyarski et al., 1988 Boyarski et al., 1988 Kettenman and Schlue, 1988 Jean et aL, 1986 Jakubovicz et aL, 1987 5,5-dimethyloxazolidine-2,4-dione,

BCECF = 2'7'-biscarboxyethyl-5.6earboxy fluorescein.

In recent studies of mammalian nerve cells maintained in tissue culture, measurements using the fluorescent pH indicator BCECF have revealed similar pHi values. These include a pHi of 6.94 in rat synaptosomes (Naschen and Drapeau, t988), 7,03 in rat sympathetic neurons (Tolkovsky and Richards, 1987), 7.18 in rat neonatal brainstem neurons (Richards and Pocock, 1989) and 7.07 in rat cerebellar Purkinje cells (Galliard et al., 1989). Notably, in neonatal brainstem neurons, values ranged from 6.8 to 7.8 (Richards and Pncock, 1989), indicating that pHi can vary considerably among individual cells in tissue culture.

2.5. INTRACELLULARpH OF GLIAL CELLS

The earliest reports of PHi in glial cells relied on population measurements of primary cultures or neoplastic cell lines, DMO studies of mammalian astrocytes revealed a range of values (7.0-7.5), which appeared to depend largely on culture conditions (Kimelberg, 1983). In cultured glioma cells, pHi measured by fluorescent techniques ranged from 7.0-7.3 (Jean et al., 1986; Jakubovicz et al., 1987). The pHi of single glial cells has now been studied in a few non-mammalian preparations (Table 4). In the leech, pHi was greater in HCO/'-buff¢red media, averaging 6.85 in HEPES buffer vs 7.18 in HCO/'buffeted solutions (Deitmer and $chlue, 1989), In the mudpuppy optic nerve, Astion et al. (1989b) reported an average pHi of 7.32 in HCO/--free and 7.39 in HCO/'-buffered media. In mammalian brain, Chesler and Kraig (1989) recorded pH, from rat cortical astrocytes/n v/vo, and reported a mean pHi of 7.04. A similar value (--7.1) was recently noted in single cultured rat astrocytes (Boyarsky et al., 1988), but in cultured mouse oligodendrocytes a phi of 7.6 was reported (Kettenman and Schlue, 1988). In comparing such results, it should be borne in mind that the pHi found in tissue culture may have little relation to pHi in vivo. It would therefore be premature to conclude that oligodendrocytes have a phi significantly higher than that of astrocytes or neurons.

2.6. HETEROGENEITYOF INTRACELLULARpH

In the intact nervous system, a few instances of cellular acid-base heterogeneity have been reported. In snails, Ahmed and Connor (1980) noted a marked difference in the intrinsic buffering power of neurons from the pleura1 vs pedal ganglia (Table 1). In the leech, neurons were found to have a pHi considerably higher than that of glial cells (Deitmer and Schlue. 1987). The extent of l~terogeneity in other systems is unclear. It is notable that whole tissue values for the pH~ of mammalian brain (Table 5) do not differ significantly from in vivo pH microelectrode measure. ments in single astrocytes (Chesler and Kraig, 1987, 1989). This indicates that baseline pHi is the same in gila and neurons, when averaged over millions of cells. However, as discussed further below, the pHi of single brain cells may be governed largely by local circuit activity. In this respect, the dynamic acid-base behavior of glial cells appears to differ markedly from that of neurons (Section 5.2). Thus, at the local level, there probably exists marked heterogeneity of pH~ across the tissue. During iscbemia, intracellular acid-base heterogeneity could b~om¢ exaggerated. Based on extracellular pH studies, Kraig et al. (1986) suggested that acid was compartmentalized within ischemic astrocytes. This notion was supported by/n vwo measurements of pH~ from astrocytes w/thin isehemic rat cortex (Kraig and Chesler, 1990). Recent NMR studies also s ~ r t the notion of H ÷ compartrnenration during metabolic disturbances. In cortical slices, replacement of glucose by lactate or pyruvate produced a split of phosphate resonance, suggesting that pH had diverged within two tissue compartments (Kauppinen et al., 1989). In addition to pH differences among cells, it is conceivable that gradients could arise betw¢en regions of a single neuron. Using BCECF fluoreseence imaging, Gillesl~ et al. (1988) found that the growing neurites of PC12 cells had a phi that was 0.10--0.31 pH units more alkaline than the cell body. As modulation of intracellular pH has often been implicated in cellular growth and activation (Busa and Nucciteili, 1984; Moolenaar, 1986) such regional pH variations could play a role in neuronal development and plasticity.

409

THE R~GULATIONAND MODULATIONOF pH L'~TKENERVOUSSYSTEM

Rat brain

Cat brain Dog brain Rat cervicalganglion

Brain slices Cortex

TABLE5. AVF.RAGETBS~ I~C~LLUt.AR pH Temp. pHo/Buffer Method CO, -m vivo DM() m vivo CO2 m vivo NR m vivo -DMO-A m vivo NMR m vivo NMR 7.13 m vivo DMO 7.09 -m vivo UMB 7.12-7.15 m vivo DMO 7.05 -m vivo DMO 7.33 22-27 7.37/CO2-Bic DMO 7.31 18-25 7.4/CO2-Bic

PHi 7.06 7.05 7.06 7.02-7.12 6.87-7.04 7.17

6.83--6.95

37

7.35 Hippocampus

~ 7.2 7.2-7.4 7.64

37 37 37

7.2/CO~-Bic CO2-Bic 7.4/CO2-Bic 7.4/CO2-Bic 7.4/CO2-Bic 7.4/CO2-Bic 7.4/CO2-Bic 7.4/CO2-Bic

DMO NMR CK CK NR CK NR CK

Reference Kjalquist et al., 1969 Roos, 1971 Messeter and Siesjo. 1971 Kogure et al., 1980 Kobatake et al., 1984 Ackerman et al., 1980 Petroff et al., 1985 Roos, 1965 Anderson et al., 1980 Arieff et al., 1976 Brown and Halliwell. 1972 Brown and Oarthwaite. 1979 Hertz et al., 1970 Kauppinen et al., 1989 Kass and Lipton, 1982 Whittingham et al., 1984 LaManna et al., 1987 LaManna et aL, 1987 Sick and Balestrino, 1988 Sick and Balestrino, 1988

7.68 37 7.81 7.53 T in degrees Celsius. Abbreviations: CO2 ffi Van Slyke method, DMO ffi 5,5-dimethyloxazolidine-2,4-dione, NR = neutral red, DMO-A ffi autoradiosraphic DMO technique, NMR ffi nuclear magnetic resonance, UMB ffi umbelliferone fluor* escence, CK ffi creatine kinase. 3. REGULATION OF INTRACELLULAR p H VIA ACID EXTRUSION While it was long recognized that an inward electrochemical gradient for H + was actively mainrained, studies of the regulatory processes were not performed until the 1970s. Indirect evidence for acid extrusion mechanisms was first provided by in v i v o experiments in rat brain (Messeter and Siesjo, 1971). Following hypercapnia (11% COs) of varying duration, average tissue PHi was determined using a weak acid distribution technique. With hypercapnia, the intracellular space was subjected to an acid load as COs diffused across cell membranes and was hydrated to form carbonic acid (Jacobs, 1940). Thus, after 15 rain, average brain pHi fell by approximately 0.10pH. Yet, by 3hr, pHi had recovered, despite sustained hypercapnia. At 48 hr, the apparent buffering power of brain (d[HCO~" ]i/dpHi) was calculated to be 357 raM. Since the ~i of rat brain homogenates was only 18 mM (Siesjo and Messeter, 1971), it was concluded that pHi had to be regulated by mechanisms operating in addition to physicochemical buffers. It was argued that metabolic consumption of acid played the major role in this process since recovery processes occurred over "too short a period to allow appreciable transmembrane fluxes of H + or H C O f ". However, subsequent studies on single cells have established that transmembrane acid extrusion is the principal mechanism of recovery from intracellular acid loads (Thomas, 1984). Note that since the activity of cationic H + equivalents (e.g. NH4+ ) is directly proportional to that of H +, and the activity of anionic equivalents (e.g. O H or H C O f ) is inversely proportional to that of H +, these species all have the same equilibrium potential. Thus, spontaneous "leakage of acid" into cells may occur through the influx of N H 2 or the efflux of O H or HCO~'. Indeed, most of the downhill influx of "acid" is likely to occur via H + equivalents, since the

activity of these species is typically 103-10Lfold greater than that of H +. Similarly, the active "extrusion of acid" may be achieved by the transmembrane flux of a variety of H + equivalents. 3.1. ACID EXTRUSIONMECHANISMSOF INVERTEBRATE NEURONS Studies of acid extrusion on single cells were first performed using cell bodies of snail neurons (Thomas, 1976a, 1977a) and giant axons of squid (Boron and DeWeer, 1976; Russell and Boron, 1976; Boron and Russell, 1983). The typical protocol was to subject the cytosol to an acute acid load, then test the ionic dependence and pharmacologic sensitivity of the subsequent phi recovery. Acid loading was performed by exposure to COs (Thomas, 1974; Boron and DvWeer, 1976; Thomas, 1976b), by the NH4+prepulse technique (Boron and DeWeer, 1976) or by direct intracellular injection of acid (Thomas, 1976b; Boron and Russell, 1983). These protocols have provided the basis for numerous studies of pHi regulation in vertebrate cells (see Thomas, 1984). A critical discussion of these methods can be found in the review of Roos and Boron (1981). In both snail neurons and squid giant axons, the recovery of pHi from acid loading occurred through an electroneutral, energy-dependent process, which required extracellular Na + and HCO~" and intracellular C1- (Russell and Boron, 1976; Thomas, 1977a; Boron and Russell, 1983). This process was inhibited by stilbcne derivatives such as SITS or DIDS, compounds known to block CI-/HCO~" exchange in red blood cells (Cabantchik e t al., 1978). On the basis of these observations, Thomas (1977a) postulated that acid was extruded by a coupled, electroneutral exchange of external Na + for internal H + and external H C O ; for internal C1-. It was suggested that entry of Na + down its electrochemical gradient provided sufficient energy to drive all of

,-tl0

M. CHESLER TABLE 6. EVIDENCEFOR ACID EXTRUSION ~[ECHANISMSIN NEURONSAND GLIA

Na-H Na-H-CI-HCO~ Na-HCO 3 Reference Invertebrates Snail Thomas, 1977 Crayfish Central neurons ÷ Moody, 1981 Stretch receptor '-Moser, 1985 Squid + Boron and Russell, 1983 Leech Neurons + + Schlue and Thomas, 1985 Glial cells + + + Deitmer and Schlue, 1987 Vertebrates Lamprey neurons + + Chesler,1986 Mudpuppy glia + + Astion et al., 1989a, b Cultured ceils Sympathetic neurons + Tolkovsky and Richards, 1987 Oligodendrocytes + + Kettenman and Schtue, 1988 Respective headings refer to Na÷-H + exchange, Na+-H+--Cl--HCO~--coupled acid extrusion and Na +-HCO;-cotransport (see text, Section 3). the coupled ion fluxes. A thermodynamically equivalent stoichiometry was subsequently noted in squid axons. Combined tracer flux and phi studies demonstrated acid extrusion via the net influx of HCO~" and Na ÷ and the net efflux of CI-, in the ratio 2"1:1 (Boron and Russell, 1983). These fluxes were stimulated by an acidic phi, were blocked by SITS, and required external HCO3-, external Na ÷ and internal Cl-. In both squid axon (Russell et al., 1983) and snail neurons (Evans and Thomas, 1984), oppositely directed acid fluxes were obtained by reversing the ionic electrochemical gradients. Several kinetic schemes are consistent with the net exchange of ions in these ratios. These include (Boron, 1985): (1)coupled Na+/H + and CI-/HCO~" exchange (1: 1:1: 1), (2) Na+/HCOi'/C1 - exchange (1:2: i), (3) Na+/COJ-/CI - exchange (1: 1: 1) or (4) NaCOj-/CI- exchange (1 : 1). In squid axons studied in standard extraceUular solutions, the dependence of the acid extrusion rate on external Na +, external HCO~- and internal CI- followed Michaelis--Menten kinetics with apparent Michaelis constants (Kin) of 77, 2.3 and 84 mM respectively (Boron and Russell, 1983). However, Boron (1985) further noted that the apparent K= for either HCO~" or Na +, depended on the external concentration of the other ion. Moreover, when acid extrusion rates were plotted as a function of [NaCO~" ], data from all experiments fell along the same Michaelis--Menten curve, with an apparent K~ for N a C O ; of 80/aM. It was, therefore, postulated that net acid extrusion occurred via the influx of the NaCOi" ion pair in exchange for internal CI- (lk'cker and Duhm, 1978). Experiments u ~ n g the reversible stilbene derivative 4,4'-dinitrostilbene, 2,T-disulfonate (DNDS) provided further support for this model, since DNDS (an anion) was competitive with both HCO~" and Na + (Boron and Knakal, 1989). Although the Na + gradient can provide sufficient energy to power such a mechanism, metabolic inhibitors were originally reported to block acid extrusion in squid axon (Boron and DeWeer, 1976), suggesting a direct dependence on ATP hydrolysis. In fact, the mechanism in squid axon displayed an absolute dependence on internal ATP (Russell and Boron, 1976). Subsequently, this process was found

to be supported by ATP-7-S, an ATP analogue that does not support most ATPases (Boron et al., 1988). These data suggested that the acid extrusion mechanism of squid axon was not a pump directly energized by the hydrolysis of ATP. It was therefore proposed that ATP was required to phosphorylate the transporter or an essential activator of the transporter (Boron et al., 1988). Evidence consistent with Na+/HCOj'/Cl-coupled exchange has been reported in a number of inverte. brate nerve cells (Table 6). These mechanisms have been identified based upon ionic dependence and pharmacological sensitivity. Information about kinetics, in the detail obtained for the squid, is not available. However, it is notable that unlike the q u i d , acid extrusion in snail neurons was not blocked by metabolic inhibitors (Thomas, 1978, 198t) and therefore may not depend upon a phosphorylated component. While snail and squid neurons depend exclusively on Na+/HCO/'/Cl--coupled acid extrusion, other invertebrate nerve cells utilize two independent mechanisms. A dual mechanism was first described in mammalian skeletal muscle, where amiloride,sensitire Na+/H + exchange (reviewed by Aronson, 1985) was found to operate in parallel with a stilbene-sensitire, HCO~'-dependent transporter (Aickin and Thomas, 1977b). A similar dual mechanism was subsequently reported in crayfish (Moody, 1981) and leech neurons (Schlue and Thomas, 1985; Deitmer and Schtue, 1987) and recently in locust neurons (Schwiening and Thomas, 1989). Notably, in a different genus of crayfish, Moser (1985) reported a single Na*/HCO~'/C1 - dependent mechanism in the peripheral stretch receptor. It is unclear whether differ. ences between the crayfish preparations of Moody and Moser represent phylogenetic diversity, or variations among neuronal types. 3.2. ACID EXTRUSIONMFA::HANISM$ OF V~TEeP,ATE NE'u'v.o,'cs

There have been comparatively few investigations of pH regulation in vertebrate neurons. Early tracer flux studies of neuroblastoma cells (Moolenaar et al., 1981) and rat brain synaptosomes (Sauvaigo eta[.,

THE R~GULATIONAND MODULATIONOF pH IN Tim NERVOUSSYS1"EM

1984) demonstrated the presence of an Na+/H + exchanger, however, its role in the regulation of pHi was not established. The only microelectrode study of pH~ regulation in vertebrate neurons was performed on the giant reticulospinal neurons of the lamprey (Chesler, 1986). Lamprey neurons were found to extrude acid using both an amiloride-sensitive Na*/H + exchanger and a separate HCO~'-dependent DIDS-sensitive transporter. Following an acid load, the contribution of each mechanism to the pHi recovery was approximately equal. In HCO~-buffered media, acid extrusion was inhibited after prolonged exposure to Cl--free solutions, and was abolished in Na÷-free solutions, consistent with an Na+/HCO~"/CI-dependent mechanism. Studies of pH i regulation in cultured mammalian neurons have recently been performed using the pH-sensitive fluorescent indicator BCECF. Tolkovsky and Richards (1987) found that the recovery from acid loading in rat sympathetic neurons was inhibited by amiloride, suggesting that acid extrusion was mediated principally by Na +/H + exchange. Although transient exposure to SITS (0.2 m i ) or Cl--free solutions was without effect, addition of HCO~" was found to accelerate the pH~ recovery. In a BCECF study of rat brain synaptosomes (Nachshen and Drapeau, 1988) the rate of pHi recovery was blocked by amiloride and was unaffected by Cl--free solutions or by media containing HCO~-, SITS or DIDS. Since the rate of recovery of pH i was roughly equal in HCO~--containing and HCO~--free media, it was concluded that HCO~- played no role in the acid extrusion process. However, since the total intracellular buffering power is higher in HCO~-containing media (Thomas, 1976b), an equal rate of pHi recovery in this solution implies that HCO~- caused a greater rate of acid extrusion. Thus, the results of Nachsen and Drapeau (1988) appear to ix in agreement with those of Tolkovsky and Richards (1987). pH regulation has recently been studied in rat brainstem neurons and cereixllar Purkinje cells in culture. Acid extrusion mechanisms varied markedly among cultured brainstem neurons (Richards and Pocock, 1989). Most cells required external Na + to recover from an acid load, while some also required external HCO~". Remarkably, in a significant number of neurons, recovery of pH i was observed in the absence of external Na + and HCO~-. In cultured Purkinje cells (Galliard et aL, 1989), the regulation of pH i appeared to be regulated by an amiloride-sensitire Na+/H + exchanger and an Na+-independent CI-/HCO~" exchanger, the latter being responsible for a lower steady state pH i in HCO~--buffered media. While these results are preliminary, they suggest a diversity of acid transport mechanisms in the central nervous system. 3.3. ACID EXTRUSION MECHANISMSOF GLIAL CELLS The presence of acid transport mechanisms in the membrane of glial cells was first suggested by tracer flux studies of primary astrocyte and glioma cell cultures. In rodent astrocyte cultures, fluxes of ~Cl were inhibited by SITS (Kimelberg et al., 1979) and stimulated by HCO~'-containing media (Kimelixrg, 1981), suggesting the presence of a CI-/HCO~"

411

exchanger. Similar results were later obtained in LRM55 glioma cells (Wolpaw and Martin, 1984). Kimelberg et al. (1979) provided evidence for Na ÷dependent acid extrusion in glial cells, by demonstrating that cultured astrocytes could acidify the bathing medium when external Na + was returned. In subsequent studies, amiloride-sensitive '2Na fluxes were demonstrated in cultured astrocytes (Kimelixrg and Ricard, 1982) and glioma cells (Benos and Sapirstein, 1983; Jean et al., 1986). Although this work established the presence of an Na*/H + exchanger in glial cells, the role of this transporter in the regulation of pH i remained unclear. Using BCECF to measure pHi in glioma cell suspensions, Jean et al. (1986) and Jakubovicz et al. (1987), demonstrated that glioma cells utilize amiioride-sensitive Na+/H + exchange to recover from acid loads. Whether HCO~--dependent mechanisms could also participate in pH i recovery was not addressed, as these studies were performed strictly in HCO~'-free media. The first study of pH regulation in single gliai cells was performed in the loach, using double-barreled LIX pH microelectrodes (Dietmer and Schlue, 1987). In nominally HCO~'-free media, 2raM amiloride slowed the pH i recovery from an acid load by approximately 50%. In HCO~'-containing media, recovery was completely blocked by Na+-free, and partly inhibited by SITS, or Cl--free solutions (Deitmer and Schlue, 1989). These data suggested a dual pH i regulatory system: an Na÷/H + exchanger and a separate Na+/HCO~'/Cl--delxndent acid extrusion mechanism. In addition, it was noted that transition from HEPES to HCO~'-buffered media caused a rapid rise in pH i and a membrane hyperpolarization. These HCO~'-stimulated effects were blocked by DIDS and Na+-free solutions, but were unaffected by C1--free media, amiloride or furosemide (Deitmer and Schlue, 1989). These results suggested the presence of an electrogenic Na+/HCO~ cotransporter (Boron and Boulpaep, 1983). It was therefore proposed that three acid transport mechanisms operate in leech glial cells: an Na+/H + exchanger, an Na+/ HCO~"/Cl--dependent electroneutral transporter and an inwardly directed Na+/HCO~" cotransporter with a stoichiometry of 2:1. Preliminary reports suggest that acid may be extruded from cultured mammalian astrocytes by similar mechanisms (Boyarsky et al., 1988, 1989). Evidence for electrogenic Na+/HCO~" cotransport has also been found in glial ceUs of the mudpuppy optic nerve. Astion et al. (1988, 1989a) demonstrated that transition from HCOi'-free to HCOi" containing solutions produced a hyperpolarization that was Na +dependent, SITS-sensitive, and Cl--independent. Further studies with pH-sensitive microelectrodes indicated that recovery of pHi from acid loads was mediated by Na÷/H ÷ exchange and an electrogenic process that was, Na+/HCO~'-dependent, SITSsensitive and Cl--independent (Astion er aL, 1986b). In cultured oligodendrocytes, recovery from acid loads was also found to ix mediated by Na+/H + exchange, and a separate, Na+/HCO~'-dependent, Ci--independent mechanism (Kettenman and Schlue, 1988). However, unlike glial cells of the leech and mudpuppy, Na+/HCO~'-dependent acid extrusion did not appear to be electrogenic.

412

M. CHESLEg

The presence of inward Na+/HCO3- cotransport in glia may have several functional implications. Independence of pH and CI- regulation may be desirable since glial cells are subject to substantial increases ( > 50%) in intracellular CI- during synchronous neuronal activity (Ballanyi et al., 1987). In instances where Na+/HCO~- cotransport is electrogenic, transport could be facilitated by membrane depolarization. This could result in a rise of gtial pHi during neuronal activity (Chesler and Kraig, 1987, 1989). The effect of membrane polarization of glial pH~ is discussed further in Section 5.2. 3.4. THE INFLUENCEOF ACID EXTRUSION ON RESTINGINTERSTITIALpH The steady state interstitial pH (pH0) of the brain is determined largely by regulatory mechanisms of the blood brain barrier and the cerebral spinal fluid. These subjects have been covered extensively in recent reviews (Nattie, 1983; Fencl, 1986; Kazemi and John= son, 1986). However, a number of studies have established that pH0 in mammalian brain is approximately 0.10 pH unit more acid than arterial blood or CSF (Cragg et al., 1977; Javaheri et al., 1983; Kraig et aL, 1983, 1986; Mutch and Hansen, 1984; Siesjo et al., 1985). The independent acid-base variables which may account for this difference are the tissue Pcoz and the SID (i.e. [HCOf]o). Both theoretical considerations and experimental evidence suggest that the /'co: of brain and CSF are nearly equal, and are approximately 1 torr greater than the arithmetic mean of the arterial and venous Poe: (Ponten and Siesjo, 1966; Plum and Price, 1973; Ahmad et al., 1976). Accordingly, [HCOf ] of interstitial space must be lower than that of CSF, suggesting that acid is continuously extruded from brain cells. A pronounced interstitial acidosis has been described in several in vitro preparations. Bath-tissue pH gradients of several tenths of a pH unit were reported in isolated turtle cerebellum (Nicholson et aL, 1985; Chesler and Chan, 1988), lamprey brainstem (Chesler, 1986, 1987) and rat hippocampal slices (Balestrino and Somjen, 1988). Because of diffusion limitations in vitro, oxygen tension falls off steeply towards the center of preparations thicker than 400-500 /am (Fujii et al., 1982). The anaerobic production of lactic acid is therefore likely to play a major role in the generation of these gradients. Classical acid extrusion mechanisms appear partly responsible for the steady state interstitial acidosis seen in vitro. In the isolated lamprey brainstem, pH0 is approximately 7.10 when superfused with an HCOf-free media of pH 7.4. Although Na+/H ÷ exchange is utilized in recovery from intracellular acid loads in this preparation (Chesler, 1986), prolonged application of 1 mM amiloride increased pH0 by less than 0.10 (Chesler, 1987). Thus less than one third of the standing bath-brain pH gradient could be attributed to Na+/H + exchange. Since the medium lacked HCOj-, it was suggested that the non-ionized (Jacobs, 1940; Roos, 1975) or carrier mediated diffusion of lactic acid (deHemptinne et al. 1983; Siebens and Boron, 1987; Mason and Thomas, 1988) might play a role. Recent studies of cultured neurons and astrocytes suggest that glial cells have particularly

effective mechanisms for the extrusion of lactic acid (Walz and Mukerji, 1988a, 1988b). Whether gila contribute predominantly to the steady-state generation of interstitial acid in rive is Jess clear.

4. MODULATION OF BRAIN EXTRACELLULAR pH BY ELECTRICAL ACTIVITY The electrical activity of neurons results in rapid shifts in extracellular ion activity. Activity-related changes in extracelluar Ca -'+ and K + have received particular attention (Nicholson, 1980; Walz and Hertz, 1983; Sykova, 1983), as these ions have been implicated in physiological signalling (Orkand et al., 1973; Llinas, 1979; Pentreath and Kai-Kai, 1982). Although activity-dependent shifts in brain extracellular pH have long been recognized, limits in spatial and temporal resolution of pH recordings have often distorted these events. With technical improvement over the last decade, it is now established that neuronal activity produces stereotyped alkaline and acid shifts in brain interstitial space. 4. !. TECHNICALCONSIDERATIONS A few technical points should be addressed regarding the study of extracellular pH shifts. Attempts to record signals from brain have generally relied on the use of miniaturized pH electrodes. Such electrometric pH recordings are by definition, differential measurements, which require a stable reference potential (see Waddei and Bates, 1969; Roos and Boron, 1981). However, the local DC potential of the brain extracellular space can undergo shifts of several millivotts during synchronous neuronal activity. Thus, to accurately record rapid pH transients in brain, local DC shifts must be recorded identically and coincidently, by the pH and reference elements. If the two electrodes record a different DC transient, the differential voltage will be erroneously interpreted as a pH signal. In instances where electrical events are propagating through cortex, as in seizure or spreading depression, a spatially segregated pH and reference electrode will record DC shifts of different magnitude and time course, resulting in highly distorted pH records. An erroneous alkaline transient will be recorded if the negative DC shift occurs first, or is greatest, at the pH electrode. The opposite circumstance will produce an acid artifact. Similar errors can arise if pH and reference electrodes do not record DC shifts with comparable response times. 4.2, HISTORICALPERSPECTIVE In recent studies, pH0 measurements have been performed using pH microdectrodes with tip diameters in the micron range. Subtraction errors have been avoided by placing reference elements in close proximity to pH electrodes (Urbanics et aL, 1978), or by using double=barreled pH microelectrodes (Kraig et aL, 1983). In much of the older literature, however, pH and reference electrodes were millimeters in diameter, were placed on the pia, and were separated by considerable distances. In the earliest studies, such

THE REGULATIONAND MODULATIONOF pH IN THE NERVOUSSYSTEM

surface measurements were performed during induced seizure activity (Dusser deBarenne et al., 1937, 1938; Jasper and Erickson, 1941; Wang and Sonnenschein, 1955). A seizure-related surface acidosis, up to several tenths of a pH unit, was consistently observed. The associated findings of increased brain lactate suggested that the seizure-induced acidosis was a manifestation of anaerobic respiration (Stone et al., 1945). In primate cortex, Dusser de Barrenne and colleagues (1938) reported that at the onset of seizure, surface acidification was preceded by a large alkalinization (~0.25 pH). However, in subsequent studies of cat cortex, an early alkaline shift was not observed (Jasper and Erickson, 1941; Wang and Sonnenschein, 1955). During spreading depression (SD), Tschirgi et al. (1975) described large ( ~ 0.2 pH) surface alkaline-acid transients in rabbit and cat. However, Rapoport and Marshall (1964) reported no surface alkaline shift during SD in rabbit cortex, and an acid shift of less than 0.03 pH. The development of pH microelectrodes with tip diameters in the micron range permitted electrodes to be inserted into the cortex without causing extensive tissue damage. Urbanics et al. (1978) used pH electrodes with tip diameters of 1--4mM to record interstitial pH changes during direct electrical stimulation of cat cortex. These studies were the first to accurately record local pHo transients, due to the proximate placement of reference elements and the attention paid to subtraction of DC shifts. An early alkaline transient of 0.10 pH units was consistently observed during 10-20sec of repetitive stimulation. Subsequently, pH fell by 0,1-0.2pH units for approximately l-2min (Fig. I B). Similar results were described in a brief note by Heuser et al. (1977). The majority of interstitial pH recordings have since been obtained using a liquid membrane pH sensor based on the proton ligand tridodecylamine (Ammann et al., 1981). The first study using this exchanger was performed in rat cerebellum by Kraig et aL (1983). Repetitive electrical stimulation of the parallel fibers produced a brief alkaline transient in the molecular layer (approximately 0.02pH), which did not persist throughout the stimulus train (Fig. 1), and was superceded by an acid shift exceeding 0.10pH units. This late acid transient peaked during stimulation, and required 1-2 min to recover to baseline. Simultaneous recordings with K+-sensitive microelectrodes demonstrated that the recovery from the acid shift had a time course similar to the post-stimulus [K+]0 undershoot. During spreading depression, a larger alkaline-acid sequence was observed. However, superfusion of the cerebellum with 20mM K + produced a slow acid shift without an initial alkalinization. Evoked pH0 transients have now been studied in a variety of preparations (Fig. 1 and Tables 7 and 8). In the majority of cases, neuronal activation produced an interstitial alkaline--acid transient, although a variety of other sequences have been noted. Available data suggest that the alkaline and acid transients arise through different mechanisms. The origins of these components are therefore considered separately.

413

4.3. EXTRACELLULARALKALINE SHIFTS

Urbanics et al. (1978) noted that local blood flow increased during the time course of cortical stimulation. They reasoned that enhanced blood flow would augment washout of CO,,, and thereby cause an early interstitial alkaline shift. However, in vitro measurements in the isolated turtle cerebellum (Fig. IA) demonstrated that an alkaline shift could occur in the absence of blood flow (Kraig et al., 1983; Nicholson et al., 1985). Similar results were subsequently obtained in a variety of isolated preparations (Fig. 1) (Chesler, 1985; Endres et al., 1985, 1986a, b; Carlini and Ransom, 1986; Chesler and Chan, 1988; Davis et al., 1987; Chvatal et al., 1988; Coles et al., 1988). Recently, in response to light, alkaline shifts were reported in the retina of the drone (Coles et al., 1988) and the frog (Borgula et al. 1989), indicating that physiologic stimuli can give rise to these phenomena. Chesler and Chan (1988) noted that evoked pH0 transients could be amplified by replacing 35mM CO2/HCO~'-buffer with 15ram HEPES (Fig. 2A). Since the field potentials were not similarly enhanced, this result was attributed to the lower buffering power of the HEPES-buffered media. The enhancement of the alkaline response in HEPES-buffered media permitted the resolution of several details. In 10mM HEPES at room temperature, a single stimulus to the parallel fibers produced a pure alkaline shift of less than 0.01 pH unit. This response began 70-100 msec after the stimulus artifact, peaked in 720 msec and required approximately 5 sec to recover to baseline (Fig. 2B). In hippocampal slices studied at room temperature with faster optical techniques, a similar alkaline shift was recorded with a latency of 20 msec (Krishtal et al., 1987). The longer latencies recorded in turtle cerebellum may be attributed to the slower response time of the pH microelectrode. Because the response to a single shock required seconds to peak and recover, these alkalinizations summed at 10 Hz. During the first second of stimulation, the summation was linear, however, thereafter, an acid shift became evident. This indicated that the early alkaline shift was partially masked by the late acidification, particularly during stimulus trains of several seconds duration. Nonetheless, in solutions containing 10ram HEPES, stimulation of the parallel fibers evoked alkaline shifts approaching 0.40 pH units (Fig. 2C). Note that in the in vivo skate cerebellum, where [HCO~']0 is relatively low, Rice and Nicholson (1988) also noted comparatively large alkaline shifts (compare records of Fig. IA). Insights into the origin of interstitial pH transients have been provided by pharmacologic studies, in which the response was inhibited or enhanced. A meaningful interpretation of such experiments requires that an agent does not significantly alter the electroresponsiveness of the preparation. If extracellular field potentials or stimulus-evoked extracellular K + responses are found to be enhanced or diminished by a treatment, concurrent effects on pH transients could be indirect. Without such indices of tissue electroresponsiveness, pharmacologic studies of evoked pH shifts may be di~cult to interpret.

414

M.

C~LER

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Fxo. I. Stimulus-evokedinterstitialpH transients.In allrecords,alkalineshiftsare downward deflections. A. Cerebellum. Electricalstimulationof parallelfibersinrat (20 Hz, from Kraig et al., 1983),turtle(l0 Hz, from Nicholson et eL, 1985)and skate (I0 Hz, from Rice and Nichoison, 1988) produced initialalkaline transientsfollowed by prolonged acid shifts.B. Cortex. Electricalstimulationof corticalsurfaceof rat (20 Hz, from Cheslerand Kraig, 1987)and cat (15 Hz, from Urbanics el eL, 1978).Note the lack of initial alkalineshiftin rat.C. Hippocampus. Electricalstimulationof angular bundle in rat(20 Hr.,from Somjen, 1984), and guinea pig kippocampal slice(dentate granular layer, 5 Hz, and stratum pyramidale, I0 Hz, in 26raM HCO~'/5% CO2, from Carlini and Ransom. 1986). Note regional differencesin the slice preparation. D. Spinal cord, dorsal horn. Electricalstimulationof dorsal root in frog (30 Hz, 20 m M HCO~" buffer, from Chvatal et el., 1988) and rat (t00Hz, from Sykova et eL, 1988). Note the rapid, prominant acid shifts. E. Isolated nerve bundles. Electrical stimulation of rat vagus nerve (30 Hz, from Endres et al. 1986), mature (100 Hz) and immature (15 Hz) rat optic nerve (from Davis et el., 1987). Note occurrence of initial alkaline shifts. F. Retina. Light stimulation of retina in drone (I0 mM MOPS buffer, from Coles et el., 1988) and frog produced pure alkaline transients (27.5 mM HCO~/5% CO2, from Borgula et al., 1989). Duration of time bars under records (thin lines) is given by time notations under regional headings. Thick bars indicate duration of stimulation. Amplitude calibration bar in A represents 0.2 pH units in all records of rows A and C-F. For records in row B, calibration bar represents 0.4 pH units. Asterisk indicates/n c.ivo preparation.

THE REGULATIONAND MODULATIONOF pH IN THE NERVOUSSYSTEM

415

TABLE7. STIMULUS-EvOKEDEx'rRACELLULARH + TRANSmN'rsIn Vi~o

T

Stim

~.T

l'J, T

,LTJ.

Reference

Cortex Cat ES ES

+ 4-

Heuser et al., 1977 Urbanics et al., 1978

+

Chesler and Kraig, 1987, 1989 Siesjo et al., 1985

Rat

ES Seizure

Cerebellum Rat

+ 4,

SD SD SD

+

Lehmenkuhler et al., 1981 Mutch and Hansen, 1984 Chesler and Kraig, 1987, 1989

ES, SD High K +

+

Kraig et al., 1983 Kraig et al., 1983

ES, SD

+

Rice and Nicholson. 1988

ES, SD

+

Somjen, 1984

+ +

Skate Hippocampus Rat Spinal cord Rat

ES

+

Sykova etal., 1988

Arrows indicate sequential direction of H + activity following onset of stimulus (alkaline downward, acid upward). Abbreviations: ES -- electrical stimulation, SD -- spreading depression. In the in vivo rat cerebellum, superfusion with Mn 2+ (which blocks synaptic transmission from parallel fibers to Purkinje cells; Nicholson et aL, 1978) selectively abolished the interstitial alkaline shift (Kraig et al., 1983). Analysis of the extracellular field potentials revealed that the excitability of the parallel fibers was not depressed by Mn +2. These observations indicated that the alkaline and acid components had separate mechanisms and suggested that the alkaline shift was a consequence of "synaptic transmission or dendritic electroresponsivencss" (Kraig et al., 1983). Similar results with divalent Ca 2+ channel blockers were obtained in the isolated turtle cerebellum (Chesler and Chan, 1988) and in slices of olfactory cortex (Endres et al., 1985) and hippocampus (Carlini and Ransom, 1986; Krishtal, 1987).

However, a stimulus-evoked alkaline shift has also been observed in the extraceIlular space of vagus nerve (Endres et al., 1986a), optic nerve (Davis et al., 1987) and skeletal muscle (Dubuisson, 1939; Gcbert and Freidman, 1973), indicating that such phenomena are not dependent on synaptic transmission or dendritic responsiveness per se. In the isolated turtle cerebellum, both the alkaline shift and synaptic transmission were blocked by the excitatory amino acid antagonist kynurenic acid (Chesler and Chan, 1988). Simultaneous observation of evoked field potentials demonstrated that the excitability of the parallel fibers was not depressed by this agent. If it is assumed that kynurenate acted post-synaptically to block synaptic transmission, these results indicate that the alkaline shift is

TxBt~ 8. STIMULUS-EvOKEDEXTILA.CELLULAKH + TP.A~sE~rsIn Vitro T

J,

~T

+

+ + +

Turtle cerebellum ES ES ES

Frog spinalcord Excitatory amino acids ES Lamprey brain High K + Olfactory cortex ES Hippocampus ES ES Isolated nerves Vagus, ES Immature optic, ES Mature optic, ES Retina Drone, LS Frog, LS

T~T

~T~

Kraig et o2.,1983 Nicholson et aL, 1985 Chesler and Chan, 1988 +

+

Endres et al., 1986b +

+ + +

4. 4. +

Chvatal et aL, 1988 Chesler, 1985 Endres et al., 1985

+ 4. +

Reference

Carlini and Ransom, 1986 Krishtal et aL, 1987 Endres et ai., 1986a Davis et al., 1987 Davis et al., 1987 Coles et al., 1988 Borgula et al., 1989

Arrows indicate sequential direction of H + activity following onset of stimulus (alkaline downward, acid upward). Abbreviations: ES = electrical stimulation, LS = light stimulation.

416

M . C I-LESLER

15 mM HEPES

35 mM HCO 3 -

A

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5%

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0.03

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FiG. 2. Evoked pHo transients of turtle cerebellum in bicarbonate-free media. Parallel fibers were stimulated with surface electrodes/n vitro. A. Amplification o f ' ~ by transition from 35 ram HCO~ to 15 mM HEPES-buffered media. B. Alkaline shifts evoked by a single ~hock to the parallel fibers: Note expanded sweep in ri$ht-hand record. C. Maximal ~ ] i n e shift evoked by prolonged parallel fiber stimulation. H + increases before stimulation is terminated. Note the abrupt increase in the rate of acidification at the end of the stimulus train. From Chesler and Chan, 1988.

generated subsequent to transmitter release. Thus, the response in cerebellum does not appear to be generated by the parallel fibers or their presynaptic release sites. Synaptic transrmssion from parallel fibers to Purkinje cells can also be inhibited by superfusion of GABA (Hackett, 1974; Malenka and Kocsis, 1982). Accordingly, application of I mM GABA to the turtle cerebellum blocked both the alkaline shift and the postsynaptic component of the extracellular field potential (Cbesler and Rice, 1989). However, interpretation of this finding is not yet complete, since it remains uncertain whether GABA affects parallel fiber-Purkinje cell transmission by a presynaptic as well as a postsynaptic action. Because the alkaline shift was blocked by Mn 2+, Kraig et aL (1983) su88ested that dendritic calcium electroresponsiveness may be implicated in the mechanism. However, in the drone retina, light induced alkaline shifts were found to be insensitive to Zn 2+, Cd 2÷ and low Ca 2÷ solutions (Coles et al., 1988). In addition, in turtle cerebellum, synaptic transmission was abolished in the presence o f Mn, z + and Cd 2+ yet, alkaline shifts evoked by glutamate iontophoresis were not blocked (Chesler and Rice, 1989). Thus, interstitial alkaline shifts may be generated by Ca 2+independent mechanisms. Kraig et al. (1983) noted that application of 4 mM acetazolamide to the cerebellar superfusate increased the amplitude of evoked pl-Ie transients severalfold. This result suggested that carbonic anhydrase played a role in interstitial buffering. Because the

uncatalyzed hydration of CO2 is 400-times slower than the dehydration reaction (Maren, 1967), fast alkaline pH transients would be expected to be preferentially enhanced by inhibition of carbonic anhydrase. Indeed, Kraig et aL (1983) noted that acetazolamide increased the alkaline shift 2-3.fold compared with the acid shift. Similar concentrations also enhanced alkaline responses in hippocampal slices (Carlini and Ransom, 1986) and frog spinal cord (Chvatal et aL, 1988). The interpretation of these results may not be straightforward, since it is unclear how an intracellular enzyme could rapidly speed extracellular buffering reactions. Moreover, in these studies millimolar concentrations of acetazolamide were employed, whereas the 150 of this drug is approximately 10-gM (Maren, 1967). At these elevated concentrations, nonspecific effects, such as inhibition of anion transport are possible (Cousin et al., 1975, Cousin and Motais, 1976). If this were the case, selective enhancement of alkaline responses could be explained by the inhibition of HCOi'-dependent acid extrusion mechanisms. In support of this notion, Walz (1989) recently found that stimulus-evoked alkaline shifts were selectively enhanced by superfusion with the HCO3- transport inhibitor SITS. However, pre-exposure to amiloride had little effect on the alkaline shift in both HCO~'-free (Chesler and Chan, 1988) and HCO~'-containing media (Walz, 1989). Thus, Na+/H + exchange does not appear to play an important role in the generation or modification of the interstitial alkaline shift.

THE REGULATION AND MODULATION OF pH IN THE NERVOUS SYSTEM

Kraig et al. (1983) noted that in principle, an interstitialalkaline shiftcould arise due to stimulusevoked shrinkage of the extracellularspace (Phillips and Nicholson, 1979; Dietzel et al., 1980). Since COs readily"crosses cell membranes, a reduction in extraceIlular volume fraction would selectively concentrate HCO/" ions causing a risein pH0. However, the enhancement of the alkaline shiftin nominally HCO/'-free solutions argues against this hypothesis. Moreover, in such media, the stimulusevoked alkalineshiftwas blocked by M n 2+, while the simultaneous volume shrinkage was unaffected (Chesler and Chan, 1988). However, as analogous experiments have not been performed in HCO/'buffered media, the possibility of a volume-dependent component of the alkaline shift cannot be fully excluded. Further insights into the mechanism of the extracellular alkaline shift may be gained by considering the magnitude of the underlying acid-base perturbation (Chesler and Chan, 1988). In a hypothetical closed system, 10 mM HEPES-buffered media could provide a maximum buffering power of 5.8 mM. In this setting, the pH response to a single stimulus in turtle cerebellum (~ 0.007 pH, Fig. 2B) would require an increase in the interstitial SID of approximately 50/~M. With repetitive stimulation, alkaline shifts of nearly 0.40 (Fig. 2C) would require the equivalent addition of 2.3 mM strong base to the interstitial compartment (Chesler and Chart, 1988). Since the system is actually open with respect to all acid-base species, an even larger load is required. It therefore seems unlikely that the influx of a weak acid or efflux of a weak base is involved, since compared with a strong acid-base flux, a substantially greater concentration change would be necessary to account for the akaline shift. During electrical stimulation of brain, shifts in extracellular strong ions such as K + occur with a time course similar to alkaline transients (Nicholson, 1980; Sykova, 1983; Walz and Hertz, 1983) and undergo concentration changes comparable to the required increase in strong base. As these ion shifts are mainly due to the opening of transmitter and voltage-gated channels (Nicholson et al., 1978, Hounsgaard and Nicholson, 1983) the alkaline shift might similarly arise due to a channel-mediated flux of H +, or an H + equivalent (e.g. OH-, HCO/', NH4+, etc.). Since the transmembrane electrochemical gradient is inward for H + and positively charged equivalents, and outward for OH- and negatively charged equivalents, the opening of an appropriate channel should cause an extracellular alkaline shift. Channel-mediated fluxes of acid equivalents are not unprecedented in excitable tissue. In snail neurons, a voltage-dependent conductance with the characteristics of an H + channel, has been extensively described (Thomas and Meech, 1982; Byerly et al., 1984; Meech and Thomas, 1987; Thomas, 1988). This conductance can significantly alter intracellular and extracellular pH (Thomas, 1988), however, its activation requires depolarization to approximately 0 mV (Byerly et al., 1984). Thus, while small depolarizations can elicit surface alkaline shifts in snail neurons, the role of the H + conductance has been questioned (Thomas, 1988, 1989).

417

Kaila and Voipio (1987) recently demonstrated that HCO/" fluxes through G A B A - A channels (Bormann et al., 1987) can cause an intracelIularacid shift and an extracellularalkaline shift in crayfish muscle. This observation raised the intriguingpossibility that stimulus-evoked alkaline shifts in brain could be mediated by HCO/" fluxes through G A B A gated CI- channels. However, in the cerebellum, the parallel-fiber-inducedalkaline shiftwas enhanced by superfusion with HCO/'-free media (Chesler and Chan, 1988), suggesting littledependence on external HCO/'. Moreover, the response was not blocked by the G A B A - A antagonist picrotoxin (Chen et al., 1990). Whether GABA-dependent HCO/--fluxes may contribute to extracellularalkalinizationunder other circumstances or in other systems remains to be determined. In view of the availableevidence, a specificmechanism for the generation of the extracellularalkaline shift is not yet apparent. The description of this phenomenon in muscle, nerve and brain, indicates that it is a general property of excitable tissue.Thus, a variety of ligand or voltage-gated channels may permit the passage of H + or an acid equivalent. Although widespread in occurrence, the magnitude of the alkaline shift varies markedly among brain regions, and may therefore be dependent on the distribution of particular ion channels. A striking example of this regional heterogeneity occurs in the hippocampus, where stimulus-evoked alkaline shifts of 0.20 p H units were evoked in the stratum pyramidale (Carlini and Ransom, 1986). In the dentate region, the alkaline shifts were five-fold smaller (Fig. IC). Finally, while it is most parsimonious to propose a single cause for the stimulus-dependent alkalinization, it is plausible that a number of mechanisms contribute to thisp H shift.For example, light-evoked alkaline shiftsin the subretinal space may arise due to the transientinhibitionof a glycolyticacid source (Yamamoto and Steinberg, 1989). In other systems, channel-mediated bicarbonate fluxes (Kaila and Voipio, 1987) or extracellularvolume shifts (Kraig et al., 1983) may play a role.The contribution of such mechanisms should be more fully explored.

4.4. EXTRACELLULARACIDSHIFTS Interstitial acidosis has long been recognized as a consequence of seizure or repetitive electrical stimulation (Dusser de Barenne et al., 1937, 1938; Jasper and Erickson, 1941; Wang and Sonnenschein, 1955; Urbanics et al., 1978; Kraig et al., 1983). In rat spinal cord, it was recently shown that prolonged interstitial acid shifts arise as a result of physiologic stimuli (Sykova et al., 1990). Noxious stimulation of the hind paw was found to cause an acidification as large as 0.05 pH units, which persisted for more than 2 h. While these findings highlight the potential significance of interstitial acid shifts, the mechanisms underlying these events remain poorly understood. Current evidence suggests that three mechanisms may be involved: (1) dflux of lactic acid from giia and neurons (2) classic acid extrusion mechanisms such as Na+/H + exchange and Na+/HCO/'/C1 - coupled

418

M. C~SLER

transport and (3) electrogenic Na+/HCO3--cotrans port into glial cells. Kraig et aL (1983) noted that activity-dependent acid shifts had a time course which paralleled the post-stimulus undershoot of extracellular K +. This suggested that extracellular acidification had its origin in the metabolic events related to the restoration of brain ion homeostasis. In support of this idea, superfusion of the cerebellum with 40 mM K ÷ caused an interstitial acidification of 1-0.2 pH units. Since application of ouabain did not prevent this K+-evoked acid shift, these authors suggested that the acidification was not coupled to N a * / K + transport. In contrast, Endres et aL (1986a) found that the stimulus-evoked acidification of vagus nerve was blocked by ouabain, implicating Na+/K + transport in their preparation. In rat cerebellum, superfusion with fluoride, a glycolytic inhibitor, restored PH0 to normal despite elevated [K+]0(Kraig et al., 1983). It was proposed that lactic acid production accounted for the interstitial acid shift, since lactate is produced distal to the glycolytic step inhibited by fluoride. Further support for this notion was provided by Spuler et al. (1987) who noted that stimulus-evoked acid shifts were inhibited by solutions in which glucose was omitted or replaced by pyruvate. The production of lactic acid during repetitive brain stimulation is not unexpected, since increased production of lactate during seizure (Stone et al., 1945; Sacktor et al., 1966; King et al., 1967; Folbergrova et al., 1969) or spreading depression (Krivanek, 1962; Mutch and Hansen, 1984) has long been recognized. It is notable that glycolysis may be enhanced during seizure, although adequate oxygenation of blood is maintained (Chapman et aL, 1977). Thus, Lipton and Robacker (1983) have proposed that the brain utilizes nonoxidative energy sources to meet the rapid demands of K + homeostasis. Indeed, evidence suggests that lactic acid production may increase as a result of normal neuronal activity. Recent positron emission tomography studies in humans support this view. During physiologic increases in neural activity, glucose uptake and blood flow were found to increase far more than oxygen consumption (Fox et al., 1988). While evidence suggests that extracellular acid shifts can result from lactic acid production, the manner by which acid appears in the extracellular space is not clear. In this regard, the role of classic acid transport mechanisms is not well established. Anion transport inhibitors increased acid transients in rat cortex (Mutch and Hansen, 1984) but decreased the acidification in frog spinal cord (Sykova et aL, 1988), while amiloride inhibited acid shifts in both preparations. A major uncertainty in these studies is whether the transport inhibitors affected the underlying electrophysiologic events which trigger the acid shifts. For instance, in the turtle cerebellum, 2ram amiloride had little immediate effect, however, exposure for more than 10min depressed both the PH0 transients and the associated field potentials (Chesler and Chan, unpublished observation). In addition to classic acid-extrusion mechanisms, nonionic (Roos, 1975), or carrier-mediated efflux

of lactic acid could contribute to stimulus-evoked acid shifts (Spencer and Leninger, 1976; Leeks and Halstrap, 1978; Deuticke et al., 1982, De Hemptinne et al., 1983; Siebens and Boron, 1987: Mason and Thomas, 1988). Since elevated [K" ]0 caused the rapid appearance of lactate in the medium surrounding cultured astrocytes (Walz and Mukerji, 1988a, 1988b), glial cells could be implicated in the generation of extracellular lactic acidosis. Recent studies indicate that glia play an additional role in acidification of the extracellular space (Sections 5.2 and 6). Whereas classical acid extrusion mechanisms are activated by intracellular acidosis (Roos and Boron, 1981; Thomas, 1984) glia appear to rapidly extrude acid in response to membrane depolarization (Chesler and Kraig, 1987, 1989). Data from glia cells of the leech (Deitmer and Schlue, 1989) and mudpuppy (Astion and Orkand 1988; Astion et aL, 1989a) have implicated inward, electrogenic, Na+/HCOj - cotransport in these responses. Since glial cells depolarize during neuronal activity (Orkland et al., 1966), this mechanism may account for the rapid extracellular acid shifts noted in some preparations (Fig. 1, Table 7). In hippocampal slices, Krishtal et al. (1987) described a stimulusevoked acidification with a latency of only 10 msec. While the authors suggested that the exocytosis of acidic synaptic vesicles may be responsible for this rapid acid response, depolarization-mediated acid extrusion from glial cells cannot be excluded. In summary, stimulus-evoked acid shifts are likely to have multiple components with different time courses. Electrogenic Na+/HCO3- cotransport into glial cells could account for early acid shifts, as this transport mechanism would respond instantly to the rapid rise of[K + ]o associated with increased neuronal activity. As lactic acid accumulated in neurons and glial cells, it might br extruded in a nonionic or facilitated manner. If the rate of acid production exceeded the rate of extrusion, intracellular acidosis would ensue, stimulating the classic acid extrusion systems such as Na+/H + exchange and Na+/HCO~-/ Cl--coupled transport.

5. MODULATION OF INTRACELLULAR

pH Activity-dependent pH transients in brain interstitial space are accompanied by rapid acid'base shifts in the intracelluar compartment. In the past, these dynamics were obscured by techniques which provided a spatial-temporal average of intraceliular pH. For instance, in response to seizure, weak acid distribution methods indicated an average intracellular acid shift of 0.20 pH units (Siesjo et aL, 1985). However, measurements on single cells demonstrated that at the onset of activity, glial cells alkalinize, then acidify (Chesler and Kraig, 1987, 1989), while nerve cells tend to acidify in response to depolarizing stimuli (Thomas, 1977b; Ahmed and Connor, 1980; Chesler, 1985; Endres et al., 1986b; Meech and Thomas, 1987). This response heterogeneity underscores the importance of studying activity-dependent pH shifts at the cellular level.

ThE REGULATION AND MODULATION OF pH 5.1. NEURONALpHi T R A N S m N ~ In most cases, electrical activity or depolarization of nerve cells has been found to produce an intracellular acid 'shift.In barnacle photoreceptors, light stimulation caused a slow fall in pHi (Brown and Meech, 1979). In snail neurons, high K + solutions produced an acidification of approximately 0.I0 p H unit (Thomas, 1979). A similar response to elevated K + was noted in lamprey neurons (Chesler, 1985). Superfusion of excitatory amino acids also caused a slow acidification of frog spinal motoneurons (Endres et ai., 1986b). Ahmed and Connor (1980) noted that repetitive electrical activity caused a cytoplasmic acidification of approximately 0.05 pH units. The acid shift was eliminated when Ca' + was removed from the external medium, suggesting that Ca '-+ accumulation was responsible for the fall in intracellular pH. In support of this notion, direct injection of Ca" + into snail neurons has been shown to produce a fall in intracellular pH (Meech and Thomas, 1977). This response was inhibited by ruthenium red (Meech and Thomas, 1980), suggesting that Ca :+ mediated acidification was due to mitochondrial calciumhydrogen exchange (Chance, 1965). However, in cultured cerebellar Purkinje cells, where Ca" + electroresponsiveness may produce substantial localized increases in dendritic Ca-'* (Liinas and Sugimori, 1980; Tank et al., 1988), K+-mediated depolarization produced a Cd'+-sensitive, intracellular alkalinization (Gaillard et al., 1989). The mechanism of this interesting effect is not yet known. Mechanisms other than accumulation of Ca" + may be involved in some of these acid responses. For example, channel-mediated fluxes of H + equivalents would generally be expected to acidify cells, as demonstrated by the HCO~- efflux through GABA-A channels of crayfish muscle (Kaila and Voipio, 1987). However, it should be borne in mind that when studying intracellular pH responses in a nondissociated preparations, interpretation of results can easily be confounded by interstitial pH responses. For instance, if a stimulus paradigm raises [K + ]o, interstitial pH can fall (Kraig et al., 1983), and in turn acidify the cell under study. In this respect, it is interesting that in cultured neurons (where the interstitial space is less significant), elevation of [K+]o did not affect pH i (Tolkovsky and Richards, 1987). Connor and Hockberger (1984) found that the pH i of gastropod neurons could be modulated by intracellular injection of cyclic nucleotides. The predominant response to cyclic AMP or GMP was a slow acidification of approximately 0.10 pH units. However, in half the cells tested, cyclic AMP caused a biphasic alkaline-acid response, while cyclic GMP produced a pure acidification. The mechanism of these pH shifts and their relationship to the normal activity of nerve cells is obscure. Control experiments indicated the hydrolysis of the nucleotides, Ca '-+ accumulation, and Na+/K + transport were not involved. It is notable, however, that in barnacle muscle, cyclic AMP can stimulate the Na+/CI-/ HCO~'-dependent acid extrusion mechanism (Boron et al., 1978). Since a similar carrier is found in gastropod neurons (Thomas, 1977a), the nucleotide-

419

IN THE NERVOUS SYSTEM

evoked alkaline responses may have been related to the enhancement of acid extrusion. 5.2. GLIALpHi TRANSIENTS Recent in vivo studies of cortical astrocytes indicated that glial intraceilular pH is sensitive to membrane potential, and is therefore modulated by the release of K + from electrically active neurons (Chesler and Kraig, 1987, 1989). Cortical stimulation was associated with a rise in glial pHi of several tenths of a pH unit (Fig. 3), which was correlated with the degree of membrane depolarization. During spreading depression, when the membrane potential approached 0 mV, glial pH increased by as much as 0.80 pH units. Since these pH shifts occurred despite an inward H + gradient, and required an increase in the cytoplasmic S I D of as much as 60 raM, it was proposed that the mechanism involved energydependent acid extrusion. To determine whether glial depolarization was required to trigger the alkaline response, Chesler and Kraig (1989) stimulated the rat cortex in the presence of extraccllular Ba: + (Fig. 4A). Under these conditions glial cells hyperpolarize during neuronal activity (Ballanyi et al., 1987). This reversed response has been attributed to the increased resistance of the glia[ membrane (Walz et al., 1984), and the consequent enhanced electrogenic effect of the Na+/K + pump (Ballanyi et aL, 1987). In this setting, either no pH shift, or a small acidification occurred, despite the fact that [K+]o increased in a normal manner (Chesler and Kraig, 1989). This demonstrated that depolarization per se was required to elicit the glial alkaline shift. Furthermore, the lack of a symmetrical acidification during membrane hypcrpolarization indicated that the alkaline shift was not due to acid flux across a simple conductive pathway. In mammalian glia, the nature of the transport mechanism responsible for the depolarizationinduced alkaline shift is not established. Since the process appears to depend upon membrane potential, an electrogenic acid transport system may be responsible. Electrogenic bicarbonate transport was described in the basolateral membrane of renal tubule cells, where the net efflux of one Na + ion was coupled to the efflux of at least two HCO~" ions (Boron and Boulpaep, 1983). In glial cells of mudpuppy optic

PH i 7.3

30sec

-'°

-

Vi

... -90

~ 20Hz

20Hz

FIG. 3. Intracellular pH transients in a rat cortical astrocyte. Cells were impaled with double-barreled pH microelectrodes in vivo. Electrical stimulation of the cortical surface caused a rapid intracellular alkaline shift correlated with the envelope of membrane depolarization. Note the progressive acid shift after delivery of repetitive trains. From Chesler and Kraig, 1989.

420

M. CHESLER

A

control PHi

6.7 7.~ [

8a 2 ÷

"~''-~'~-~ 30see

-60 -90

-10Hz

B

control

10Hz

B a2+

7.2 10Hz

10Hz

7.6

lrnin

FIG. 4. Effect of Ba: + on evoked pH transients in rat cortex. A. pH~ recording from cortical astrocyte in vivo. Superfusion of the cortical surface with 0.5 mM Ba2+ caused glial cell to hyperpolarize during cortical stimulation and blocked the stimulus-evoked rise in pHi. B. Superfusion of the cortical surface with 0.5 mM Ba:÷ produced an initial interstitial alkaline transient during cortical stimulation. nerve (Astion and Orkand, 1988, Astion et al., 1989a, b) and the leech (Deitmer and Schlue, 1989), a similar, but inwardly directed electrogenic Na+/ H C O f cotransporter has recently been reported. Electrogenic Na+/HCO~" cotransport could be responsible for the depolarization-induced alkaline shift of mammalian astrocytes. However, an electrogenie transporter might respond to hyperpolarization. In cortical astrocytes, hyperpolarization did not produce a significant acid shift (Chesler and Kraig, 1989). This may be due to the proximity of the membrane potential to the equilibrium potential of the transporter, as noted in glial cells of the leech (Deitmer and Schlue, 1989). If the stoichiometry of H C O f : N a + cotransport is 2: l, the equilibrium potential is given by RT [Na+ ]o[HCOf ]~ Es,nco, = - - - f - In [Na+ ]i [ H C O ; ]~

(19)

(see Boron and Boulpaep, 1983; Deitmer and Schlue, 1989). For mammalian cortical astrocytes, given an intracellular Na + activity of 25 mM (Ballanyi et al., 1977), an Na + activity coefficient of 0.75, and HCO/activities of 10 and 20 mM for intracellular and extracellular compartments respectively, the equilibrium potential of the transporter would lie between - 7 0 and - 8 0 mV. Chesler and Kraig (1989) recorded an average membrane potential of - 7 3 mV in mammalian astrocytes. The lack of an acid shift during hyperpolarization may therefore be consistent with electrogenic Na+/HCO/- cotransport. However, two additional observations are noteworthy. First, cultured oligodendrocytes appear to have an electroneutral Na+/HCO/- cotransporter, but nonetheless responded to K+-mediated depolarization with a delayed alkaline shift (Kettenman and Schlue, 1988). Second, the depolarization-induced

alkalinization of mammalian astrocytes was not abolished in nominally HCOf-free, HEPES-buffered, media (Boyarsky et al., 1988). However, in this abstract, the respective rates of alkalinization in the two solutions were not reported. Since the HCOf concentration in nominally HCOf-free media is likely to be a few hundred micromolar, a carrier with a millimolar Michaelis constant for HCOf might produce a sizable, albeit slow, alkaline shift. A depolarization-mediated alkalinization, which could only partly be explained by Na+/HCO~ - cotransport, has been described in salamander renal tubule cells (Siebens and Boron, 1989a, b). Based on sensitivity to appropriate transport blockers, and a dependence on external lactate (Siebens and Boron, 1989b), lactate transport was implicated in the mechanism. However, this transporter appeared to be electroneutral (Siebens and Boron, 1987). It was, therefore, unclear how alkalinization was coupled to membrane potential. While astrocytes are capable of rapidly extruding lactic acid (Walz and Mukerji, 1988a, b), it is not known whether lactate transport plays a role in the depolarization-induced alkalinization of glia.

6. RELATIONSHIP OF INTRACELLULAR AND EXTRACELLULAR pH SHIFTS Although it is clear that neuronal activity causes a variety of pH shifts in the neuronal, glial and interstitial space, intercompartmentai relationships are obscure. During neuronal activity, astrocytes alkalinize, suggesting that these cells rapidly extrude acid. However, at the onset of stimulation, pH microelectrodes have recorded alkaline as well as acid shifts in the extracellular space (Fig. l, Tables 7 and 8). Results obtained in rat cortex suggest an explanation for these varied responses (Chesler and Kraig, 1989). Cortical stimulation produced little immediate change in pH0, followed by a gradual acidification. However, in the presence of Ba 2+, stimulation caused an immediate extracellular alkaline shift (Fig. 4B). Ba 2+ was similarly found to enhance extracellular alkaline shifts in olfactory cortex slices (Endres et aL, 1985). In contrast, Ba 2+ blocked the glial alkaline shift. Thus, the alkalinization of glial cells, and the alkalization of the interstitial space, appear to arise through different mechanisms. If it is assumed that the glial alkaline shift occurs due to acid extrusion, then blocking this process with Ba 2+ would subtract an acid load from the interstitial compartment. Consequently, any extracellular alkalinizing process would be unmasked or enhanced. The following hypothesis arises from these observations. At the onset of neuronal activity, extracellular pH is determined by the relative efficacy of two opposing processes: an acidifying action, due to rapid acid effiux from glial cells, and an alkalinizing action associated with neuronal activation. The latter process may be due to channel-mediated acid influx into neurons (Fig. 5A). In regions where these processes were equivalent, little or no extracellular pH change would occur at the onset of neuronal activity. This was noted in rat cortex at the onset of seizure (Siesjo

TIlE REGULATION AND MODULATION OF pH IN THE NERVOUSSYSTEM

A

B

EARLY

°....// /) (

Ned,on

421

LATE

Gila

~'/~Neuron

FiG. 5. Hypothetical net flux of H + equivalents among glia, neurons and interstitial space during neuronal activity. A. At onset of synchronous neuronal activity, a net efflux of acid equivalents from the glial compartment and influx of acid equivalents into neurons is postulated. B. With prolonged synchronous activity, a net efttux of acid equivalents from both glia and neurons is postulated. et al., 1985) or electrical stimulation (Chesler and

Kraig, 1987, 1989). In preparations with a dominant alkalinizing mechanism, an initial rise in extracellular pH would occur, as noted in the extracellular space of cerebellum (Kraig et al., 1983; Chesler and Chan, 1988) and hippocampus (Carlini and Ransom, 1986). Where the glial response predominated, electrical activity would result in a rapid extracellular acidification, as noted in spinal cord (Chvatal et al., 1989). Developmental observations in the rat optic nerve are consistent with these notions (Davis et al., 1987). In immature nerves, prior to glial proliferation, stimulation produced an early extracellular alkaline shift. However, in mature nerves, in which glial proliferation had occurred, stimulation produced a pure acid shift. With sustained, synchronous neuronal activity, the interstitial space has been found to acidify in all preparations. Glial cells have been shown to acidify under ischemic conditions (Chesler and Kraig, 1989), due perhaps, to the production of lactic acid. Neurons may also acidify after prolonged activity, due to the continuous influx of acid equivalents and the production of lactic acid. Etttux of lactic acid from both gila and neurons may therefore be accompanied by classic acid extrusion (e.g. Na+/H + exchange or Na+/HCO/"/Cl--coupled transport) activated by the fall in pHi. The interstitial space would therefore be subject to an acid load from both compartments (Fig. 5B), accounting for the well described, prolonged acid shift.

7. SIGNIFICANCE OF pH MODULATION In addressing the significance of activity-dependent pH modulation, the sensitivity of cellular processes to changes in pH must be weighed against the magnitude of these shifts. Although enzymes and channels are clearly affected by large changes in pH, relatively few processes show marked sensitivity in the physiological range (Busa and Nueciteili, 1984). A frequently cited exception is the rate of the phosphofructokinase step in the glycolytic pathway. With an alkalinization of only 0.10 pH units, the rate of the in vitro reaction increased from close to zero to almost full activity (Trivedi and Danforth, 1966). AccordJPN 34, ~---E

ingly, this enzyme has been cited as a link between shifts of intracellularp H and modulation of cell function (Fidelman et al., 1982). A m o n g channels, the gap-junctional complex is notable for its sensitivityto physiologically-relevantshiftsin intracelIular pH. Gap junctional conductance can display a steep risewith alkalinizationson the order of 0.10 pH units (Spray et aL, 1981). NMDA-mediated synaptic transmission may be particularly sensitive to shifts in extracellular pH. In mammalian central neurons, NMDA-evoked currents were significantly enhanced by extracellular alkaline shifts of a few tenths of a pH unit (Morad et aL, 1988). While artificial stimulation can significantly modulate intracellular and extracellular pH of the brain, it is unclear whether physiologically relevant pH shifts arise in rive. Several considerations suggest that significant pH shifts occur. Glial cells of striate cortex have been shown to depolarize by 5-7 mV in response to appropriate visual stimuli (Kelly and Van Essen, 1974). Astrocytic depolarizations of this magnitude were associated with intracellular akaline shifts as large as 0.10pH unit (Chesler and Kraig, 1989). Thus, an appropriate increase in glycolyticrate and gap junctional coupling may occur at the moment neurons become repetitivelyor synchronously active. In spinal cord (Sykova et aL 1989) and retina (Coles et al., 1988; Borgula et aL, 1989) extracelIularp H has been shown to be modulated by physiologic stimuli. Although these responses were lessthan 0.I0 p H unit, local p H shifts could be larger. For instance, an alkaline source localized near a subsynaptic space, might significantlyenhance NMDA-mediated postsynaptic currents (Morad et aL, 1988) yet not be fully detectable by an electrode tip tens or hundreds of microns distant. The effects of extracellular p H shifts could be particularlyrelevant to the onset and termination of seizure.In mammalian brain slices,extracellularalkaIosiscan induce epileptiforrnactivitywhich is blocked by N M D A antagonists (Aram and Lodge, 1987). Indeed, systemic respiratory alkalosis has long been associated with a predisposition to seizure (Foerster, 1924). Thus, rapid regulation of interstitialp H at the onset of neuronal activity may be critical to the maintenance of normal excitability.Depolarizationinduced effluxof acid from glialceilscould serve such

422

M. C~SLER

a regulatory function. Since this response would be graded with extracellutar K ~ accumulation, glial cells could precisely regulate extracellular pH over a wide range of local neuronal activity.

8. C O N C L U D I N G R E M A R K S Research over the last decade has established that substantial, localized, acid-base fluxes occur during neuronal activity. In light of these findings, the generally-held notion that brain p H is tightly regulated, must yield to a more fluid conceptualization. The idea o f pH homeostasis need not imply pancellular homogeneity or rigid stability. The robust acid-base responses characteristic of neural tissue must be incorporated into the conceptualization o f brain function at the level of local circuits and single cells. F r o m the standpoint of the membrane biophysicist, the mechanisms underlying activity-dependent pH shifts are of interest, and detailed mechanistic studies are needed. However, it must also be stressed that little is known about the magnitude and localization of these events in the intact nervous system. Thus, progress in the understanding of brain pH regulation will ultimately depend on mechanistic approaches, carried out in vitro, as well as technically difficult, but highly relevant, pH recordings performed in vivo.

Acknowledgments--I would like to thank Drs Charles Nicholson, Margaret Rice and Richard Kraig for their critical reading of the manuscript and many helpful suggestions. Technical assistance was provided by Ms Susan Lim. Supported by PHS grant NS27011-01.

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