127

Journal of Phy8iology (1990), 422, pp. 127-144 With 8 figure8 Printed in Great Britain

ACIDOSIS AND BLOCKADE OF ORTHODROMIC RESPONSES CAUSED BY ANOXIA IN RAT HIPPOCAMPAL SLICES AT DIFFERENT TEMPERATURES BY K. KRNJEVIC AND W. WALZ* From the Anaesthesia Research Department, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6

(Received 27 April 1989) SUMMARY

1. Interstitial pH (pH.) and field responses (to stratum radiatum stimulation) were recorded simultaneously with double-barrelled microelectrodes in the CAI region of hippocampal slices from Sprague-Dawley rats. 2. Both the relative acidity and amplitude of field responses increased with depth, reaching a maximum near the centre of the slice. When the temperature was raised from 22 to 37 °C, this pH. gradient was > 2 times steeper, but the field responses were much diminished. 3. Standard anoxic tests (substituting 95 % N2 + 5 % CO2 for 95 % 02 + 5 % CO2, for 2 min) tended to reduce pH0 and population spikes, but these effects were highly temperature sensitive: at 22 °C the blocking rate was only 12 3 + 4 6 % and ApHo -0018+0-0157 units, both per minute; corresponding changes at 34-35 °C were 67-6 + 11 9 % and -0O065 + 0-0046 units per minute. Highly significant linear correlations between rates of block and ApHR gave a mean slope of 904 + 17-6 % per 0-1 unit of acid change. 4. Anoxia caused similar temperature-dependent increases in acidity in stratum pyramidale and radiatum, but in the latter field responses (EPSPs) were much less depressed after 2 min of anoxia. 5. When slices were superfused with acid medium (low [HCO3-j), much greater reductions in pHo were needed to depress responses, giving a mean slope of 17-7 % per 0.1 pH unit. 6. In glucose-free medium, there was a slow alkaline shift in pHo (0-13 +0-036 units); population spikes and the acid transients evoked by anoxia disappeared. 7. It was concluded that acidosis cannot be the immediate cause of the similar depressions of postsynaptic excitability seen during anoxia and hypoglyeaemia. 8. In further tests, DL-p-hydroxyphenyl-lactic acid, a blocker of lactate transport, failed to diminish acid transients evoked by anoxia, indicating that these are not mediated principally by lactate transport. -

* On leave from the Department of Physiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

MS 7661

128

K. KRNJEVIC AND W. WALZ INTRODUCTION

Anoxia causes drastic changes in behavioural, electrophysiological or biochemical manifestation of brain function (Himwich, 1951; Cohen, 1973; Siesj6, 1978; Hansen, 1985). A question that is of great interest is, how are these interrelated? And to what extent do they form a causal chain? The principal electrophysiological effects of anoxia include a loss of both on-going and evoked electrical activity (Sugar & Gerard, 1938; Siesj6, 1978; Hansen, 1985). In the hippocampus, synaptically evoked spike responses disappear early (Yamamoto & Kurokawa, 1970; Hansen, Hounsgaard & Jahnsen, 1982; Lipton & Whittingham, 1984); but they soon return if anoxia does not persist for more than a few minutes. In biochemical terms, one of the first and clearest effects of cerebral anoxia is a rise in lactate production and in tissue acidity (Himwich, 1951; Cohen, 1973; Mutch & Hansen, 1984; Siesj6 & Wieloch, 1985; Tanimoto & Okada, 1987; Schiff & Somjen, 1987). The very early onset of increased lactate formation - which is especially marked in cortex and corresponds to the first appearance of slow waves in the electrocorticogram (as already emphasized by Craig & Beecher, 1943) - suggests that acidosis itself may be responsible for the loss of population spikes during anoxia. Acidosis indeed depresses Ca2+ influx in some excitable cells (e.g. Sperelakis & Schneider, 1976; Umbach, 1982). A block of Ca2+ current would abolish synaptic transmission; but neither anoxia nor exogenous acidosis produces a very early failure of excitatory synaptic potentials (EPSPs), which indeed tend to persist for at least 1 or 2 min after the disappearance of spike responses (Schiff & Somjen, 1985; Fujiwara, Higashi, Shimoji & Yoshimura, 1987; Balestrino & Somjen, 1988; Leblond & Krnjevic, 1989). We therefore compared pHo changes with the simultaneous depression of synaptically evoked population spikes elicited by anoxia, over a wide range of temperatures, because this greatly affects anoxic phenomena as well as underlying disturbances of metabolism (Himwich, 1951; Samson, Balfour & Dahl, 1960; Tanimoto & Okada, 1987, etc.). These findings have been reported briefly (Krnjevi6 & Walz, 1989). METHODS

Slices Conventionl transverse slices (400-500 ,um thick) were cut with a Mcllwain Tissue Chopper after rapidl dissecting out the hippocampus of ether-anaesthetized or decapitated SpragueDawley male rats (- 200 g). The hippocampal dissection was performed in artificial cerebrospinal fluid (ACSF), cooled to 4 °C, with the following composition (in mM): Na+, 151-25; K+, 3 0; Ca , 2-0; Mg2+, 2-0; Cl-, 135; HCO3-, 26; H2P04-, 1 25; and glucose, 10. The ACSF and slices were aerated with 95 % 02 and 5 % CO2 throughout, except during anoxic tests when 02 was replaced by N2 (Pco2 remaining nominally constant). Slices were either kept in a holding vessel (at room temperature) or placed in a Haas-type recording chamber where they lay on nylon fabric, at the interface between aerated ACSF (below, flowing at a rate of 1-15 ml/min) and a rapid stream of warm humidified 02-CO2 mixture (1-5 1/min, above). pH electrodes The method of preparing the electrodes was that described by Borrelli, Carlini, Dewey & Ransom (1985). Micropipettes were pulled from double-theta glass tubing of 3 0 mm outer diameter. The -

ANOXIC pHo CHANGES AND BLOCK OF RESPONSES

129

most satisfactory performance was given by electrodes with tips of 2-4,um. They were silanized by injecting 4% tri-N-butylchlorosilane (in carbon tetrachloride) - prepared 2 days earlier - and then heating the electrodes to 450-500 °C for 5 min. About 1 1 of the proton cocktail (Fluka Chemical Corp., Ronkonkoma, New York, USA) was then introduced by 'back-filling' into the tip of the H+sensitive channel, making a column 3-4 mm long; most of the remainder was filled with 100 mmNaCl and 20 mM-HEPES (N-[hydroxyethyl]piperazine-N'-[2-ethanesulphonic acid]) buffer at pH 6-0. The other, reference channel contained 154 mM-NaCl and 20 mM-HEPES at pH 7 0. A layer of mineral oil at the top of each barrel prevented evaporation and the formation of a lowresistance path between the channels. They were connected to the head stages of a WPI Electrometer (FD 223) by Ag-AgCl leads.

Calibrations The electrodes were calibrated first in HEPES buffer at pH 7 5, 7 0 and 6-5, and then in ACSF at pH 7-35 (the 'normal' solution), 6-8 and 6-45 - prepared by varying the HC03- concentration - flowing through the recording chamber, and equilibrated with 95 % 02 + 5 % CO2. The sensitivity of the electrodes was always confirmed by repeating the calibrations at the end of recording in slices. The pH of the calibrating solutions was verified with a laboratory pH-meter (Acumet, Fisher Instruments) and standard commercial buffers. Useful electrodes gave responses having a slope of 50-70 mV per pH unit. In 5 % C02-equilibrated ACSF at 22-25 °C, the slopes were somewhat greater (62-8 + 3-06 mV, for ten series of tests on seven electrodes) than in HEPES buffers (53-5 + 3X87 mV, for eight electrodes). (The not uncommon supra-Nernstian responses have been noted by previous authors, e.g. Mutch & Hansen (1984).) -

Temperature sensitivity Calibrations were repeated at different temperatures, spanning approximately the range over which recordings were made. The slopes of responses were somewhat steeper at 33-35 °C than at 21-23 °C: by 11 9+2-24% for a 9-56+0426°C increase in temperature (n = 10). The most appropriate calibrations were used throughout. In ACSF, at constant Pco2 and [HCO3-j, the electrode potential was much more sensitive to temperature than expected (taking into account changes in CO2 solubility). Thus in six slices, a rise from 22-5 + 0-12 to 33-8 + 0-42°C was associated with a potential change of - 258 + 197 mV, equivalent to an apparent alkaline shift of 0-426 + 0-657 pH units (which is contrary to clear evidence of increased acidity at higher temperatures, cf. Fig. 1). These anomalies - as well as the marked increase in noise sometimes evident at higher temperatures (Fig. 2) - can be ascribed mainly to the thermoelectric artifacts to which 'long-column' electrodes are especially prone (Vaughan-Jones & Kaila, 1986; Reid, Marrannes & Wauquier, 1988). In further tests, no responses of the electrodes could be detected when [K+] was varied in the range 3-12 mm (in agreement with Borelli et al. 1985) or when changing to glucose-free ACSF. Presumably because of a slightly lower CO2 content, ACSF equilibrated with 95 % N2 + 5 % CO2 was consistently a little more alkaline (by 0 043 + 0-0075 pH units) than ACSF equilibrated with 95 % 02 and CO2. This accounts for comparable small alkaline responses observed during control anoxic tests made in the 'interface' chamber while measuring the pH in a thin layer of flowing ACSF.

Hippocampal recording site and stimulation All the recordings were made in the CAI region, mostly at depths of 100-250 ,sm, in the stratum pyramidale. Population spikes were evoked by 0-1-02 ms constant-current pulses applied through a bipolar electrode in stratum radiatum. In a few cases, the H+-sensitive microelectrode was inserted into the stratum radiatum. Anoxia and temperature changes Anoxia was produced by switching the gas supply from 95 % 02 and 5 % CO2 to 95 % N2 and 5 % CO2. In previous similar experiments (Leblond & Krnjevic, 1989), measurements with Po2 electrodes at the surface of slices showed a relatively constant delay of 9 s before PO begins to drop rapidly, reaching 90% of the minimum by 28 s. On returning to normal oxygenation, PO2 starts to rise after 4 s and reaches 90 % of its plateau level by 12 s. -

5

PHY 422

K. KRNJEVIC AND W. WALZ 130 The temperature of the bath could be accurately set and maintained within < + 0-1 °C by a thermistor-controlled circuit. RESULTS

Interstitial pH in slices As reported previously (Balestrino & Somjen, 1988; Walz, 1989), the interstitial pH (pH.) at the start or end of recording was consistently more acid (by 0-1-0-2 pH 100pm 250pm

^~\/t. r%f

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Fig. 1. Interstitial acidosis and field potentials both increase with depth. A andB, recordings at different depths (from upper surface), at low and high temperature as indicated; examples of population spikes at three different depths are illustrated above. C, recording in stratum radiatum of same slice; D further data from stratum pyramidale of another slice. A, pH; 0, population spike.

units) than ACSF (pH 7-35). Because in the interface chamber pH electrodes could not be quickly calibrated in situ, there was no precise estimate of absolute pH for most of the runs illustrated in the figures. But it can be presumed that basal pH was in the range of 7-1-7-3. In agreement with Walz's (1989) observations, the maximum

of acidity was quite deep in the slice, some 300 ,um from the upper surface. This is the depth where the largest field responses were recorded, as can be seen when the changes in pHo and in amplitude of the population spike are plotted against depth (Fig. 1). Both the acidity and responses tended to increase with depth, roughly in parallel though the acidity consistently had its maximum 50-100 /tm deeper and its distribution was more sharply skewed. Another point of interest is that changes in temperature had marked and opposite effects on the response and on the pHo gradient: as shown by the data in A and B, recorded in the stratum pyramidale of

near

131

ANOXIC pHo CHANGES AND BLOCK OF RESPONSES

the same slice, a rise in temperature from 21 9 to 37-5 °C sharply reduced the population spike amplitude and latency (without changing its depth distribution), whereas the acidity was greatly enhanced. Comparable depth distributions are also plotted for the stratum radiatum of the same slice (C) and stratum pyramidale of another slice (D).

Effects of brief anoxia Recording in stratum pyramidale When recording from the CA1 pyramidal cell layer at 32-35 °C, orthodromic spikes are almost completely abolished after 2 min of anoxia (Cherubini, Ben-Ari & Krnjevi6, 1989; Leblond & Krnjevic, 1989). The degree and rate of block proved to be highly sensitive to the temperature. Plots of the normalized amplitude of the population spikes during and after 2 min anoxic tests, repeated at several different temperatures, are illustrated in Fig. 2; they are superimposed on the original pH,o traces, recorded simultaneously at the same point, to emphasize the striking correspondence between the changes in responses and in pHo. In A, recorded at 22-7 °C, anoxia depressed the responses by a maximum of only 20 %, and there was a small alkaline shift in PHo. By 25-8 °C, anoxia produced a major block of responses (by > 80%) accompanied by a net increase in acidity (after an initial shift in the alkaline direction). Further heating intensified both the block of responses and the acidity evoked by anoxia, until by 37-0 °C there was an almost total blockade within 1 min and the pHo fell by 0-20 units. It can be seen also that responses typically started to diminish as the acidity began to increase. In thirty-eight anoxic tests, at different temperatures, the fall in pHo started 1-55 + 1-53 s (mean + S.E.M.) after the onset of population-spike depression. This time difference is hardly significant. (Only in one slice was there a consistent 15-30 s delay before the pH changes started, which was probably due to an electrode artifact.) The depression and acidity reached their maxima about 30 s after the return of 02. The recovery of responses and pH became progressively slower and less complete at the higher temperatures. These temperature-related changes were largely reversible when slices were allowed to return to 33 °C (filled circles). From the time taken to reach a maximum depression of responses, a blocking rate could be calculated for each temperature. The marked effect of temperature is indicated by a simple comparison of the mean changes (+ S.E.M.) observed in different experiments at 22-23 0C, a blocking rate of only 12-3 + 4-6 % (n = 11) per minute and no regular shift in pH (- 0-018 + 0-0157 unit per minute); and at 34-35 °C, a 5*5-fold higher blocking rate of 67-6 + 119 % per minute (n = 9) and a clear acid shift (- 0-065 + 0-0046 (n = 11) pH units per minute). A closer examination of data from individual experiments revealed a monotonic increase in blocking rate with temperature (open circles in Fig. 3 C), which paralleled the increases in acidity (open triangles). Hence, as shown by the open circles in Fig. 3D, there was a virtually linear relation between the rates of blocking and of rise in acidity (r = 0-956). The regression lines for the data obtained in eleven different slices gave a mean slope of 90-4 + 17-64 % block per 0-1 unit of acid pH change. These results are summarized in Table 1. 5-2

132

K. KRNJEVIC AND W. WALZ 120 100 80 60

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Fig. 2. Identical anoxic tests (for 2 min) evoke progressively greater interstitial acidosis and block of population spikes as temperature is raised. At each temperature, the plot of normalized amplitude of population spikes (0) is superimposed on the actual holding potential (VH) trace (note increase in noise at higher temperatures in D and E). Data were all obtained from the pyramidal layer of one slice, in the sequence A to E; the temperature was then returned to 33 °C, at which the observations were repeated (@). The pH calibration in E is equivalent to 0-14 pH units in A.

133

ANOXIC pHo CHANGES AND BLOCK OF RESPONSES

Stratum radiatum N2 for

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Fig. 3 In stratum radiatum, anoxia for 2 min produces a similar acidosis, but the field

(EPSP) is much less depressed than in stratum pyramidale; note minimal depression of field, even at 37-5 °C (B). The graph in C shows a parallel marked increase in anoxic blocking rate (0) and change in pHo (A) with increase in temperature, in stratum pyramidale of same slice; but in the corresponding plots for stratum radiatum (@ and A), there is a clear dissociation between these variables (dashed lines): hence, plots (D) of blocking rates vs. pHo have a much steeper slope for stratum pyramidale (0) than for stratum radiatum (@). The pH calibration applies to both A and B.

K. KRNJEVIC AND W. WALZ

134 6-6 6.8 Q a.

a)

-~ > .a E _ en 0 -0

CL

Q 0cL E

30

50 40 Time (min)

Fig. 4. Superfusion with acid ACSF (low HCO3-, at 34-5 °C) is much less effective than anoxia in producing block of population spikes (0). A, simultaneously recorded pH0. Examples of population spikes seen at various times are illustrated. TABLE 1. Changes in pH, and depression of population spikes caused by 2 mim of anoxia. acid ACSF (pH 6 4) and glucose deprivation: mean values (± standard error of the mean, number of observations) Rate of Corresponding A pHo Slope of block vs. pH block Blocking Temperature agent (OC) (/min) (%/0/-1 Ullit) (%/min) 12-2 -0-018 Anoxia 22-23 -904 (±0+0157) (±4-6, 11) Anoxia 676 -0(065 34-35 (+17-6. 11) (±11 9, 9) (±0 0046) - 17 7 - 0-0203 Acid AC'SF 3-7 33-35 (±2-28, 3) (±0-0015) (±0-58, 3) Glucose-free 50 33-34 + 0-0087 +702 (+22-3. 5) AC'SF (±0-00173) (±0-88, 5) Anoxia in 33-34 + 0-0032 absence of glucose (±0+0070, 5)

In one slice, there was no acidosis during anoxia (for 2 min as usual) at any temperature between 225 and 370 °C: the population spike, however, was also exceptionally resistant to anoxia, being reduced by only 2-5 and 6% at 225 and 33.5 0C respectively. Though unusual, this behaviour was in keeping with a strong correlation between acidosis and block of responses.

ANVOXIC pHo CHANGES AND BLOCK OF RESPONSES

135

Recording in stratum radiatum A direct comparison between temperature-dependent changes in the different strata was made in only four experiments. As previously reported (e.g. Fujiwara et al. 1987), the extracellular field in stratum radiatum - which reflects mainly the EPSPs generated in apical dendrites - was much more resistant to anoxia (Fig. 3A and B). Although the pH changes were almost identical in amplitude and time course at the somatic and dendritic levels (cf. open and filled triangles in Fig. 3C), the depression of the EPSP fields was much less severe. Hence, the corresponding plot (filled circles) in Fig. 3D indicates a very weak correlation between EPSP amplitude and acidity (in agreement with Balestrino & Somjen, 1988).

Blocking effects of acid solutions In view of the impressive correlation between N2-evoked acidosis and blockade of responses, it seemed of interest to examine the effects of acid ACSF on responses recorded under normal conditions of oxygenation. The results of such an experiment are plotted in Fig. 4. It can be seen that perfusion with a low-HC03- solution (pH 6 4) led to a progressive acidosis, which indeed depressed the population spikes. The effect, however, was not only very slow (4-8 % per minute) - which is not surprising in view of the slow time course of pHo change (0-0214 unit/min) - but very much larger changes of pHo were required than those seen during anoxia. Thus, during the preceding 2 min anoxic test (at left in Fig. 4), the population spike was reduced by a maximum of 87 %, though pH,, fell by only 0-1 unit. During acid perfusion, by contrast, the blockade reached the same 87 % level only when pH. fell to 6-7 (a sixfold greater drop). This strong, if slow, blocking action was fully reversible. Similar results were obtained in two other experiments. Overall, the apparent sensitivity to exogenous pH0 change was 17-7 + 2-28 % depression per 0 I pH unit, as against 90 4 + 17 6 % per 0 I unit during anoxic tests (see above). Anoxia in the absence of glucose Because the mammalian brain has little in the way of glycogen reserves, and there is no significant alternative to the glycolytic pathway for the production of ATP (Himwich, 1951; Samson et al. 1960; Cohen, 1973; Siesj6, 1978), anoxic acidosis should greatly diminish or even disappear when glucose is removed (Hochachka & Mommsen, 1983; Siesj6 & Wieloch, 1985). In six experiments, the normal 10 mM-glucose-containing ACSF was temporarily replaced by glucose-free ACSF. In five cases, the predominant result was a progressive alkalosis, leading to a maximum pH increase of 0-13+0036 units, after 35-7 + 6 7 min of glucose-free superfusion. This alkaline shift is shown by the pH electrode traces in Fig. 5 and its relatively rapid reversal is evident in Fig. 5C. The variable character of such alkalosis is indicated by the upper plots (triangles) of Figs 6 and 7 (from two other slices). All three figures also illustrate the disappearance of anoxic acidosis during glucosefree perfusion. In Fig. 5A, when glucose was present the standard 2 min anoxic test elicited a typical delayed acidotic response (ApH, -0 065). In B, in the absence of glucose, only a further alkaline shift is seen during a similar period of anoxia; but in

136

K. KRNJEVIC A_ND W. WALZ

C, after the return of glucose, anoxia again evoked acidosis (-0060 pH units). A virtual elimination of anoxic acidosis is also evident in Figs 6 and 7. In the five slices, the maximum pH shifts evoked by 2 min anoxic tests changed from -011 + 0021 in the presence of glucose to +00064+0014 in its absence, with a return to -0052 + 0-0116 subsequently. N2

A

33-3 OC

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V~~~~~~~~~~~~~~~~~~~~~ N2

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Glucose-free s t+ " tfflX

+

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Fig. 5. In the absence of glucose, the slice becomes more alkaline and anoxia no longer evokes acid shift. After the initial control run in the presence of 10 mM-glucose (normal ACSF; A, superfusion with glucose-free ACSF began at the arrow in B; note lower paper speed during first part of B. and subsequent return to higher speed (indicated by increased spacing of stimulus artifacts, which were at a constant rate throughout A-D). Return of normal ACSF was followed by relative acidosis and reappearance of anoxic acid shifts. D, large acidosis, elicited by prolonged anoxia, could be fully reversed by switching to glucose-free ACSF. All recordings from same slice, at 123 ,um.

The crucial role of glucose could also be demonstrated by switching to glucose-free ACSF during a prolonged anoxia. Thus, in Fig. 5 the large acidotic response was completely reversed by the withdrawal of glucose. A combination of prolonged anoxia and glucose lack may, however, be irreversibly damaging for cellular metabolism, judging by the diminished tendency to acidosis evident after the return of glucose, which presumably indicated a reduced capacity either for ATP formation (by glycolysis) or utilization. In two experiments, the glucose-free perfusate initially caused a gradual acidosis and increase in excitability (Fig. 7) leading to spreading depression-like events (either 'spontaneous' or N2-evoked), manifested by striking fluctuations in response

ANOXIC pHo CHANGES AND BLOCK OF RESPONSES

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amplitude and the characteristic sequence of alkaline-acid shifts (Kraig, FerreiraFilho & Nicholson, 1983; Somjen, 1984; Mutch & Hansen, 1984). In the experiment illustrated in Fig. 7, this was followed by the usual slow and progressive alkalosis; but in the other experiment, the interstitial pH remained at a slightly acid level during 38 min of glucose-free perfusion, and the return of glucose was followed by a sharp, irreversible further increase in acidity. 71

7.3

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Fig. 6. Another example (from another slice) of alkaline shift and loss of anoxic acidosis produced by deprivation of glucose (A); inset traces and plot below (0) illustrate corresponding depression of population spikes. Recording was at 250,utm depth and 329 0C.

Changes in responses during glucose-free perfusion In agreement with previous observations (Bachelard, Cox & Drower, 1984), evoked responses gradually disappeared in the absence of glucose. Thus in five slices population spikes were totally blocked after 23tO+ 4*3 min (in all cases the temperature was 33-34 5 °C). The depression tended to increase in parallel with the alkalosis, but it seemed to be much enhanced by anoxic tests (as in Fig. 6). A curious observation (Fig. 7) was an unexpected temporary (though only partial) recovery in two slices of the fully blocked response during anoxia (previously reported by Roberts, Sick & Solow, 1987). Because of the deleterious effect of prolonged lack of glucose - perhaps significantly reinforced by even brief anoxic tests - in most experiments there was incomplete recovery of responses after the return of glucose: Figs 6 and 7 show two extreme cases. Overall, the responses recovered only 37~4+ 16*7 % of their initial size after 26±4*7 min (n = 5).

K. KRNJEVIC AND W. WALZ

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Effects of block of lactate transport The traditional explanation (Siesjo, 1978; Hansen, 1985) for the acid change in pHo observed during anoxia is a rapid increase in intracellular lactic acid, produced by glycolysis, and its rapid efflux, presumably via the well-known lactate transport N2 Glucose-free -0.2

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Fig. 7. In some cases, the alkalosis evoked by glucose deprivation was preceded by a short phase of increased acidosis, associated with spreading depression (SD). Two such episodes of SD (marked by arrows) are characterized by a biphasic (alkaline-acid) shift of pH and transient large enhancement of fields. After the second SD, population spikes failed to recover (except briefly during the second anoxic test). Recording was at 120 ,um and 33-1 'C.

system (Spencer & Lehninger, 1976; Mason & Thomas, 1988). Lactate release from cultured cerebral neurones and astrocytes is very much depressed by DL-phydroxyphenyl-lactic acid (HPL; Walz & Mukerji, 1988), which might therefore be expected to reduce the magnitude and time course of the pHo change evoked by anoxia.

As an acid, 8 mM-HPL lowered the pH of ACSF by 0-19 +0-018 units. Hence, superfusion of slices with such ACSF produced a slow fall in pHo (Fig. 8). By 30-50 min this neared a plateau, equivalent to a mean reduction in pH0 by -0-144 + 0023 (very similar to the change in pHo produced by 10 mM-lactic acid; Walz & Harold (1988)). The acid transients evoked by anoxia, however, were not diminished: even when pHo was at the plateau, its peak rate of fall during anoxia was 8-6+3-28%

ANOXIC pHO CHANGES AND BLOCK OF RESPONSES A 33-1 0C

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Fig. 8. A blocker of lactate transport (DL-p-hydroxyphenyl-lactic acid, HPL, 8 mM) did not reduce acid shifts evoked by 2 min anoxic tests. A, continuous VH traces show, on the first line, usual acidosis evoked by 2 min of anoxia; on the second line (at lower speed), slow drop in pH directly caused by acid HPL; and then after resetting baseline (at asterisk) and return to faster trace, marked acidosis evoked again by anoxia; in the third line, there is a final anoxic test, when pH0 reached a plateau, and then the slow rise in pH. that followed the end of HPL application. B, plot of similar data from another experiment, including amplitude of population spike (0, below) and pH. (A, above). Anoxic tests (N2) and HPL (8 mM) application are indicated.

139

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K. KRN-hJEVIC AN..VD W' WVALZ

greater than during the pre-HPL control tests. The decay phase was consistently slower during HPL superfusion, showing a persistent tail (Fig. 8A), which can also be ascribed to some underlying slow reduction in pHo, directly due to the acid superfusate; if time to half-decay was estimated with reference to the post-anoxic baseline, there was no evidence of any significant change. A further point of interest was a marked depression of population spikes (by 40-k50%); as can be seen in Fig. 8B, this was poorly correlated with the acid shift, in particular being little or only very slowly reversible. DISCUSSION

Higher acidity in resting slices A more acid slice interior - already noted by Schiff & Somjen (1987), Balestrino & Somjen (1988) and Walz (1989) - is to be expected in view of the significant lactate formation observed in brain slices even under fully aerobic conditions (Lipton & Whittingham, 1984). In terms of excess acid, the depth distribution gives a good fit to a parabola, such as would be expected for a slab with steady acid production, at an equal rate throughout its volume (Hill, 1928). Taking into account the slice thickness, probable tortuosity factor and extracellular volume fraction, a 10-15 mm excess of acid near the centre would be consistent with lactate production at a rate of 1-2 ,umol cm-3 min-' (in fair agreement with a previous estimate of 099,umol g-' min-'; Benjamin & Verjee, 1980). Although the increase in acidity at the higher temperature may contribute to the depression of the population spike (Balestrino & Somjen, 1988), the main factor is likely to be poorer synchronization of the briefer unitary spikes (Thompson, Masukawa & Prince, 1985). pH changes during anoxia The initial small alkaline shift (< 0 05 units) - at low temperature often the only change recorded - can be ascribed mainly to consistently lower CO2 content of the N2 + CO2 mixtures used in these experiments (see Methods). In addition, as suggested by Kraig et al. (1983), some extracellular alkalosis may follow a net flux of isotonic NaCl from the extracellular space as a result of reduced Na+ outward pumping in the absence of oxygen, and perhaps Na+ influx mediated by Na+-H+ exchange or by transmitter release (Siesj6 & Wieloch, 1985; Grinstein, Rotin & Mason, 1989). A further possibility is that it reflects a rise in pHi, as observed in cardiac and other muscle (de Hemptinne, Marrannes & VanHeel, 1982; Allen, Morris, Orchard & Pirolo, 1985; Mainwood & Renaud, 1985; Ellis & Noireaud, 1987), which can be ascribed to proton absorption by phosphocreatine (PCr) break-down (Allen et al. 1985; Mainwood & Renaud, 1985). With increasing temperature and the rapid acceleration of glycolysis (cf. Samson et al. 1960; Higerdahl, Harp, Nillson & Siesj6, 1975) lactic acid formation is no longer matched by PCr break-down, so there is a net increase in proton liberation, SOOI reflected in a net fall in pH,. This might be expected to be mediated by an accelerated outward transport of lactic acid. But the lack of effect of the lactate transport antagonist HPL (Spencer & Lehninger, 1976; Walz & Mukerji, 1988) indicates another predominant mechanism of H+ efflux, most likely by transmembrane

ANOXIC pHO CHANGES AND BLOCK OF RESPONSES 141 diffusion of C02, as well as perhaps Na+-H+ exchange (Mainwood & Renaud, 1985; Chesler, 1987; Schlue & Deitmer, 1987; Tolkovsky & Richards, 1987). The progressively earlier and larger ApH0 generated at higher temperatures thus probably reflects a more rapid increase in intracellular acidity (as in cardiac Purkinje fibres; Bright & Ellis, 1988). The continued fall in pH. for some 30 s after the return of 02 may be due to the early resynthesis of PCr and the consequent liberation of protons (Ellis & Noireaud, 1987).

Correlated changes in responses during anoxia The present finding that anoxia causes a much greater and more rapid block of responses at temperatures above 30 °C is fully in agreement with previous intracellular observations. Thus, in the experiments of Morris, Krnjevic & Leblond (1988) anoxia evoked much smaller changes in membrane properties at room temperature (notably a 3-fold smaller conductance increase) than at 33 'C. The early block of population spikes is probably due mainly to these postsynaptic changes, rather than the depression of EPSPs which was consistently slower and often much less pronounced (as emphasized by Fujiwara et al. 1987). The striking correlation between increasing acidity and the rate at which population spikes diminished during anoxia at various temperatures inevitably suggests that the acidosis itself may be directly responsible for the underlying loss of excitability, owing to increase in K+ conductance (GK) (Hansen et al. 1982; Fujiwara et al. 1987; Leblond & Krnjevic, 1989). The extracellular acidosis, however, cannot be the immediate cause of anoxic block, in view of the rather weak effects of exogenous acidosis (perfusion with reduced HCO3-): a depression of responses by only 18 % per 0-1 pH. unit, compared to 100 % per 041 pH0 unit during anoxia. Does this mean that a fall in pHi is a crucial event, and that external acidification is very ineffective because it has little effect on pHi (Lipton & Korol, 1981; Balestrino & Somjen, 1988)? This may well be the case under normoglyeaemic conditions. On the other hand, glucose deprivation also causes a blockade of transmission, associated with virtually identical changes in membrane potential and conductance (Spuler, Endres & Grafe, 1988), but there is a consistent tissue alkalosis. There are two possible explanations: either GK is triggered by the same agent, released during both anoxia and hypoglycaemia, or different mechanisms operate in the two conditions. One suggested mechanism, activation of GK by a rise in free [Ca2+]i (Krnjevic, 1975), has been observed in some cells during normoglycaemic anoxia (Biscoe, Duchen, Eisner, O'Neill & Valdeolmillos, 1988), though evidence for its operation in hippocampal neurons remains inconclusive (Leblond & Krnjevic, 1989). Nevertheless, in view of the well-known interactions between pHi and [Ca2+], (Meech & Thomas, 1977; Bers & Ellis, 1982; Vaughan-Jones, Lederer & Eisner, 1983; Kaila, Vaughan-Jones & Bountra, 1987) a very early fall in pHi may be the initial stimulus for a rise in free [Ca2+], which then activates GK. But whether (and how) glucose lack would lead to an early rise in [Ca2+]i, or the liberation of some other messenger, is a matter of conjecture. In conclusion, although interstitial acidosis is a strikingly early and reproducible consequence of anoxia, that is both highly sensitive to temperature and predictive of

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the intensity of anoxic blockade of postsynaptic responses - and no doubt must have important short- and perhaps long-term consequences for cellular function nevertheless it is unlikely to be immediately responsible for the characteristically rapid, but reversible, loss of hippocampal excitability. We thank Dr G. Mainwood for reading the manuscript and making very helpful cornments. N. Schestakowich and G. Bowen for technical assistance, and J. Watson for typing the manuscript. K. Krnjevi6 is grateful to the Medical Research Council of Canada for financial support. REFERENCES

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