Effects of C02 on excitatory transmission apparently caused by changes in intracellular pH in the rat hippocampal slice

BRAIN RESEARCH ELSEVIER

Brain Research 706 (1996) 210-216

Research report

Effects of CO 2 on excitatory transmission apparently caused by changes in intracellular pH in the rat hippocampal slice Junhee Lee c, Tomi Taira a,b, Pekka Pihlaja a Bruce R. Ransom c, Kai Kaila a,* Department of Biosciences, Division of Animal Physiology, P.O. Box 17, FIN-O0014 University of Helsinki, Helsinki, Finland b Institute of Biomedicine, Department of Physiology, P.O. Box 9, FIN-O0014 University ofHelsinki, Helsinki, Finland c Department of Neurolog)~, Yale Medical School, New Haven, CT 06510, USA

Accepted 13 September 1995

Abstract It is generally known that hyperventilation produces an increase in neuronal excitability. However, the mechanism whereby a change in CO 2 partial pressure (Pco 2) leads to changes in neural excitability is not known. We have studied this phenomenon in rat hippocampal slices using double-barrelled microelectrodes for simultaneous recording of field excitatory postsynaptic potentials (EPSPs) and extracellular pH in stratum radiatum of area CA1. A drop in Pco 2 from the control level, 36 mmHg to 7 mmHg, produced an increase in extracellular pH of 0.4-0.6 pH units and a transient increase in EPSP slope by about 20-30%. Despite the stable extracellular alkalosis, the EPSP reverted back to its original level within 10 min. Switching back to 36 mmHg Pco 2 restored the original extracellular pH and caused a transient decrease in the EPSP slope. Pharmacological blockade of N M D A receptor a n d / o r GABA A receptor had no influence on the effects of CO 2. An increase in Pco 2 to 145 mmHg led to a stable fall in extracellular pH by 0.6 units and to a transient 30-50% decrease in EPSP slope. The above results indicate that the CO2-induced changes in neuronal excitability were not caused by changes in extracellular pH but they might have been mediated by changes in intracellular pH. Indeed, exposing the slices to the permeant weak base, trimethylamine (20 mM), which is known to produce a rise in intracellular pH, increased the EPSP slope by 50-70%. Application of 20 mM propionate (a permeant weak acid) decreased the EPSP slope by 40-60%. We conclude that the transient changes in the EPSP seen in response to changes in P¢o 2 are mediated by in intracellular pH. Keywords: CO 2 partial pressure; Extracellular pH; Intracellular pH; Neural excitability; Respiratory alkalosis

1. Introduction

Changes in the arterial C O 2 partial pressure (Pco 2) have powerful effects on brain function [2,4,15]. As demonstrated already in 1924 by Foerster [11], hyperventilation (which, by definition, is associated with a fall in arterial Pco 2) can precipitate an epileptic seizure in susceptible persons [16], a finding that is still in routine clinical use for the detection of petit mal epilepsy. In individuals with no epileptic tendencies, hyperventilation leads to unpleasant sensations of drowsiness and numbness, which result from increases in neuronal excitability as evidenced by a pronounced enhancement of lowfrequency activity and a negative DC shift in the electroen-

* Corresponding author. Fax: (358) (0) 191-7301; E-maih [email protected] 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01214-1

cephalogram [4,22,33]. In contrast to this, an increase in Pco 2 is known to lead to a decrease in nervous excitability and to have an anticonvulsant action in vivo [3,4]. The enhanced excitability produced by reduction in Pco2 is not due to the associated fall in cerebral blood flow because it occurs in vitro where the confounding variable of blood flow is eliminated [2]. Despite the wealth of experimental data on the influence of Pco 2 on neural excitability, it is still unclear whether the changes in neuronal function are mediated by a direct action of molecular CO 2 on the neuronal plasma membrane, or whether the changes in excitability are secondary to changes in extra- or intraneuronal pH [2,4]. In the present work on rat hippocampal slices, we have examined the effects of changes of Pco 2 on the field excitatory postsynaptic potential (EPSP) evoked monosynaptically in the CA1 region by low-frequency stimulation of Schaffer collaterals. Some preliminary findings of this study have appeared in abstract form [26].

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2. M a t e r i a l s a n d m e t h o d s

The experiments were done on slices (400 /zm thickness), cut transversally by means of a vibratome from hippocampi of Wistar rats (100-150 g) which were decapitated under pentobarbital anaesthesia (30-40 mg kg - ] , i.p.). The slices were maintained at room temperature and thereafter transferred one at a time to an interface-type recording chamber (volume, 0.6 ml; flow rate, 1.5 ml/min). Slices were allowed to recover for about an hour before recording began. The experimental temperature was 32°C. The physiological solution contained (in mM) NaC1 124, KC1 3.0, CaC12 2.0, NaHCO 3 25, NaH2PO 4 1.1, MgSO 4 2.0 and D-glucose 10. The solution was gassed with 5%, 1% and 20% CO 2 in oxygen. The corresponding Pco 2 values are (in mmHg) 36, 7.3 and 145. This yielded a solution pH of 7.4, 8.03 and 6.72, respectively. For simplicity, these will be referred to below as the 5%, 1% and 20% CO 2 solutions. A stream of the same gas mixture was (following warming and humidifying) continuously passed over the preparation. The change of the interstitial pH following a change in Pco 2 had a halGtime of about 20 to 40 s. Extracellular pH (pH o) was measured using double-barrelied H +- selective microelectrodes. The pipettes were pulled from borosilicate glass (2GC150FS, Clark Electromedical, Pangbourne, Reading, UK), and the non-filamented barrel was exposed to vapor of dimethyltrimethyl-silylamine (TMSDMA; Fluka, Buchs, Switzerland) at 200°C. The pipettes were bevelled dry, and the non-filamented barrel was back-filled (using pressure) with a solution containing 100 mM NaC1, 200 mM HEPES and 100 mM NaOH (pH 7.6). The filamented reference barrel was filled with 150 mM NaC1. A short column of the H ÷ sensor (Fluka 95291) was taken into the tip of the silanized barrel by using slight suction. The resistance of the H ÷sensitive barrel was 15-20 G O and that of the reference barrel, 20-50 M/2. The electrodes had a slope of 52-59 mV for a unit change in pH. Recordings were made in stratum radiatum of area CA1. The tip of the bipolar stimulus electrode was positioned at some distance (about 1.5 mm) from the recording electrode to provide orthodromic stimulation (0.1 ms, 0.05 Hz) of Schaffer collaterals. Stimulus intensity was set to evoke 50-60% of the maximal EPSP amplitude. The NMDA (N-methyl-D-aspartate) receptor antagonist AP5 (o-2-amino-5-phosphonopentoate, 40-80 /xM), the AMPA (a-amino-3-hydroxy-5-methylisoxazolate-4-propionic acid) receptor antagonist CNQX (6-cyano-2,3-dihydroxy-7-nitroquinoxaline, 20-40 /xM) and the GABA A receptor antagonist PiTX (picrotoxin, 100 /zM) were applied in the perfusion solution. In solutions containing trimethylamine (TriMA), NaC1 was replaced by an equivalent amount of TriMA-C1. In order to maintain a constant extracellular CI- concentra-

tion (104 mM) in experiments involving propionate, we used a control solution which was obtained from the standard physiological solution by substituting 20 mM Na-methanesulfonate (NaMS) for 20 mM NaCI. Na-propionate was then substituted for an equivalent amount of NaMS. In some experiments, 20 mM NaC1 was replaced by 20 mM N-methyl glucamine chloride. The chemicals were purchased from Tocris Neuramin (Bristol, UK) and from Sigma (St Louis, MO, USA). The present data are based on experiments done on a total of 61 slices. Each result described below was confirmed in at least five preparations.

3. Results 3.1. E f f e c t o f c h a n g e s in P c o 2 o n p H o

As shown in previous studies [27,35], the interstitial pH of a hippocampal slice under the present experimental conditions (with 5% CO 2) is somewhat more acid (by 0.05-0.25 pH units) than the bulk solution (pH 7.4). A decrease in Pco 2 following superfusion with the 1% CO 2 solution produced a mean increase in pH o to 7.5, and an increase in Pco 2 (20% CO 2 solution) resulted in a pH o of about 6.6. The changes in the pH o were somewhat smaller than those in the bulk solution, as is evident from Fig. 1 which illustrates the relationship between the changes in pH o and solution pH for the three levels of Pco 2. A possible explanation for this may be that lactate production, which would be expected to generate an acid load in the tissue, is enhanced by hypocapnia and inhibited by hypercapnia [18]. From a technical point of view, these results emphasize that quantitative studies on the physiological effects of external pH in brain slices are meaningless without direct measurements of the interstitial pH.

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3.2. Effect o f a decrease in P c o 2 on EPSPs In the experiment shown in Fig. 2, pH o was 7.2 under control conditions (i.e., 5% CO 2 and pH 7.4.in the bathing solution). Upon a decrease in Pco 2 (1% CO 2, solution pH 8.0), a rapid increase in pH o to about 7.5 took place. This increase in pHo was coupled to a prompt increase in both the amplitude and slope of the field EPSP. A striking feature of the enhancement of the EPSP was its transient character: during a maintained (10-15 min) exposure to the low-Pco 2 solution, both the EPSP amplitude and slope started to decline, levelling off close to their control values. Switching back from the low-Pco z solution to the control solution brought about a fast fall in pHo to the original control level, which was now linked to a transient decrease in EPSP amplitude and slope. Effects of the above kind were seen in 17 out of 22 slices studied (increase in the EPSP slope and amplitude 32.0 _+ 5.5% and 30.0 _+ 3.8%, respectively; mean + S.E.M. are given), but in the rest of the preparations examined, little change in EPSP slope or amplitude could be observed. The transient nature of the on- and off-effects on EPSP induced by a step fall in PCO 2 clearly indicated that the changes in excitatory transmission were not directly related to either PCO 2 o r pH o, because these variables assumed stable new values with monophasic time courses. Below, we will present evidence to suggest that the effects

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A rise in external pH enhances synaptic responses mediated by the NMDA subtype of glutamate-gated receptor channels [28,29,32], while an opposite dependence on pH is characteristic for GABA A receptors in several kinds of preparations [12-14]. Hence, it could be assumed that the enhancement of the EPSPs under conditions of high pH o might be due to a potentiation of NMDA receptormediated excitation, or to a suppression of GABA A receptor-mediated inhibition (cf. [19] and [20]), or both. As already pointed out above, the results described so far do not suggest a major role for pH o in the influence of CO 2 on EPSP slope and amplitude. Nevertheless, to gain further information on this question, we examined the effects of AP5 (an NMDA antagonist) and PiTX (a GABA A antagonist). However, neither of the two antagonists was capable of abolishing the transient changes in EPSP slope and amplitude induced by the wash-in and wash-off of the low-Pco 2 solution. Furthermore, as shown in Fig. 3, this absence of an influence of AP5 and PiTX was evident also in experiments where the two drugs were simultaneously applied. As expected (cf. [36]), application of PiTX (both in the absence and presence of AP5) gave rise to epileptic discharges (see Fig. 3). A detailed analysis of the influence of Pco 2 on these late responses was not carried out, but a

J. Lee et al. / B r a i n Research 706 (1996) 210-216

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In view of the qualitatively opposite effects of hypoand hypercapnia on nervous excitability (see section 1), we examined the effects of an increase in Pco 2 on the slope and amplitude of the field EPSP. Fig. 4 shows an experiment where a slice was exposed for 10 min to the 20% CO 2 solution (pH 6.7). This produced a steplike fall in pH o to 6.75. Again, transient changes in the EPSP were associated with the onset and end of the P c o 2 / p H o step. There was first an initial decrease in EPSP slope and amplitude both of which recovered close to their original values despite the maintained high Pco 2 and low pH o. Immediately upon re-establishment of the original PEO2, a pronounced transient enhancement of the EPSP took place which was, again, followed by a recovery towards the original base-line level. In contrast to the slice-to-slice variability of the EPSP change linked to a decrease in Pco 2 from 5% to 1%, a clear decrease in EPSP slope ( - 5 4 . 7 _ 2.3%) and amplitude ( - 4 2 . 8 ___5.7%),was seen in all slices (n = 10) exposed to 20% CO 2. It might be noted that, in the present experiments, the increase in Pco 2 was achieved at the expense of the oxygen partial pressure (see section 2). However, hypoxia

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as a cause of the effects described above (see, e.g., [38]) is unlikely in view of the rather modest decrease in the oxygen level. It is also difficult to see how a reduction in the oxygen concentration could result in transient changes in excitability of the kind described above.

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Exposure of cells to weak acids or bases which have a high membrane permeability in their neutral form has been frequently used for experimental displacement of pH i [23,31]. TriMA is a weak base that is widely employed to induce an intracellular alkalosis (e.g. [10]) and, hence, it was of interest to observe that application of 20 mM TriMA induced a rapid, transient enhancement of the EPSP (slope: 92.0 + 26.2%; amplitude: 68.3 + 14.8%), which was followed by an undershoot following withdrawal of the weak base (Fig. 5). This kind of temporal behaviour is in full agreement with a pHi-mediated action of TriMA (see section 4). In the experiments with TriMA, there was a slight fall in the Na ÷ concentration of the medium, from 150 mM to 130 mM. That the reduction in sodium played no role in the above results was checked in control experiments, which showed that replacing 20 mM Na ÷ with N-methyl glucamine (which is not expected to affect pH i) had no effect on the EPSP. Exposure of the slice to 20 mM propionate, a weak acid known to produce a fall in pH i (e.g. [31]) brought about a decrease in the EPSP slope ( - 31.7 + 5.7%) and amplitude ( - 14.3 _ 4.9%) (Fig. 6). In several slices, the effect of a prolonged application of propionate was not reversible, and hence, the duration of exposure was limited to 5 min.

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With the notable exception of certain neurones involved in the regulation of breathing, neural excitability is enhanced following respiratory alkalosis and suppressed with acidosis (see section 1). However, the critical factor that couples excitability to changes in P c o 2 / p H has not been identified, and there are several alternative explanations that have to be considered. It is a well-established fact that a change in the ambient Pco 2 level has an effect on both pH o and pH i in nervous tissue [25]. In particular, a decrease in Pco 2 produces a tissue alkalosis (respiratory alkalosis) and an increase has the opposite effect (respiratory acidosis) [2,4,33]. In addition to effects mediated by changes in pHi, or by changes in pHo, molecular CO 2 is lipophilic and can itself act as an anaesthetic gas.

25

4.1. Transient effect o f CO 2 on EPSPs

Time (rata) Fig. 6. Effect of 20 mM propionate on field EPSPs. The graph shows the time course of the changes in EPSP slope, and the letters A - C refer to the specimen recordings above where the stippled traces reproduce the control response seen in A.

This may explain the observation that the wash-off of propionate was not followed by a clear overshoot of the kind that might be expected to take place in the light of the observations with an increased Pco z. Net fluxes of acid equivalents mediated by transmembrane diffusion of weak acids and bases will also affect pH o [8]. However, as shown in Fig. 7, the pH o changes caused by application of 20 mM TriMA or propionate were rather small (0.1 pH units or less), and control experiments with changes in the bicarbonate concentration of the perfusion solution showed that changes in pH o of this magnitude had no detectable effect on EPSP slope or amplitude (not illustrated). This result is not surprising when considering that the large changes in pH o that took place in experiments with alterations in Pco 2 did not seem to exert any major action on the EPSP (cf. Figs. 1-4).

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In the present study, we have examined the influence of changes in Pco 2 on excitatory transmission in an extensively studied in vitro model, the hippocampal slice, where stimulation of the Schaffer collaterals evokes monosynaptic excitatory potentials in the pyramidal neurones in the CA1 region [1]. A key observation made in the present work is that a stepwise change (fall or increase) in Pco 2 produces a transient effect (potentiation or suppression, respectively) of excitatory transmission mediated by the A M P A subtype of glutamate receptors. In addition to these 'on-effects', a transient effect of the opposite kind (an 'off-effect') was consistently associated with the re-establishment of the normal CO 2 partial pressure. The transient nature of the effects of CO 2 on EPSP amplitude and slope excludes the possibility that the influence of CO 2 was directly caused by the concomitant change in pH o or by molecular CO 2 as the changes in pH o and Pco 2 were monophasic and stable. In fact, the present observations suggest a surprisingly high insensitivity of monosynaptic A M P A receptor-mediated transmission to extracellular H ÷ ions: within the pH o range examined (about 6.6-7.5; cf. Fig. 1), the steady-state values of EPSP slope and amplitude remained relatively constant (Figs. 2-4). The transient character of the CO2-induced changes in EPSP slope and amplitude can be readily explained on the basis of the general properties of intracellular pH regulation. In practically all types of nucleated cells, including neurones, intracellular alkaline and acid loads activate regulatory mechanisms that serve to maintain pH i within a narrow range [5,6,31,37]. The typical response to an intracellular acid or alkaline load is, initially, a respective transient fall or rise in pHi. In particular, a step decrease in PCO 2 produces an alkaline load while a rise in Pco 2 produces an acid load [30]. However, these changes in pH i are not persistent because regulatory mechanisms bring pH i back toward the original steady state level. A subse-

J. Lee et al. /Brain Research 706 (1996) 210-216

quent removal of the acid or alkaline load brings about a respective overshoot or undershoot in pH i . It is evident, therefore, that the transient changes in EPSP slope and amplitude observed in response to changes in Pco 2 are fully compatible with the idea that pH i acts as the critical link mediating the actions of CO 2 on excitatory transmission. In the present experiments, a step decrease from 5% to 1% CO 2 (36 to 7 mmHg) gave a peak enhancement in the EPSP slope of about 30%, while a rise to 20% CO 2 (145 mmHg) produced a 50% decrease. When considering the quantitative aspects of the influence of CO 2 on the monosynaptic EPSP, it should be noted that the maximum EPSP change is probably related to the maximum change of pH i which, in turn, is governed by the rate of change of Pco 2 within the tissue. In the present experiments, the Pco 2 change within the slice had a half-time of about 20-40 s, as judged on the basis of the rate of change of pH o (cf. Figs. 2 and 4). Therefore, effects on EPSP of the present kind are not likely to be detected under experimental conditions where the change in tissue Pco 2 is not achieved at a sufficient rate. This may explain the fact that Balestrino and Somjen [2] observed only rather small, inconsistent effects of Pco 2 on hippocampal EPSPs. On the other hand, it is likely that the effects of CO 2 on EPSP slope and amplitude would be more pronounced under experimental conditions allowing even faster change in tissue Pco 2 than what was achieved in the present study.

215

two receptors were involved in the influence of Pco 2 on monosynaptic EPSPS examined here. In fact, the absence of a role for NMDA receptors in the EPSP enhancement seen at a low Pco 2 is not surprising, since little activation of NMDA receptors takes place at low stimulation rates [7], such as those (0.05 Hz) employed in the present study. With regard to PiTX, it is noteworthy that a fast component of GABA A receptor-mediated inhibition "might have an influence on the early phase of the EPSP under experimental conditions of the present kind [21]. However, PiTX did not abolish the influence of a low Pco 2 on the EPSP, which excludes a major contribution by GABA A receptors to the effects examined. In fact, it would be rather difficult to explain how a step increase in pH o acting on GABA A receptors would lead to a transient potentiation of excitatory transmission. 4.4. Conclusions

To summarize, the present findings suggest that changes in the slope and amplitude of monosynaptic AMPA receptor-mediated EPSPs in response to changes in Pco 2 are caused by changes in pH i . Since neuronal pH i is tightly regulated and perturbations are rapidly corrected, the EPSP changes are transient and they are likely to show a strong dependence not only on the extent, but also on the rate, of Pco 2 changes. An important task in future work is to identify the intracellular site(s) that convey the effect of pH i to EPSP generation.

4.2. Effects of TriMA and propionate

The above idea gained further support by the findings that application of a membrane-permeant weak base (TriMA) or a weak acid (propionate) also induced large changes in EPSP slope and amplitude. Taken together, the present findings on the actions of CO 2, TriMA and propionate are all compatible with the assumption that a rise in pH i leads to a potentiation, and a fall in pH i leads to a suppression, of excitatory transmission. We have recently obtained direct experimental support for this hypothesis in preliminary experiments involving simultaneous measurement of field EPSPs and pH i in rat hippocampal slices loaded with a fluorescent intracellular pH probe (Taira, T., BjSrkholm, S. and Kaila, K., unpublished observations). The target site for this kind of intracellular H + modulation cannot be identified on the basis of the present results but there are several possibilities, including presynaptic voltage-gated Ca 2÷ or K + channels that might be modulated by intracellular H ÷ ions [9,24]. 4.3. Absence of a role for NMDA and GABA A receptors

While it is highly likely that, due to their sensitivity to external H ÷ ions (see above), NMDA and GABA A receptors play a role in pH modulation of nervous function under certain conditions (e.g. [17,18,28,34]), neither of the

Acknowledgements This work was supported by grants from the Academy of Finland and from the Sigrid Juselius Foundation. J.L. was a Howard Hughes Medical Institute Medical Student Research Training Fellow.

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[26]

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