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Upregulation of the hyperpolarization-activated cation current in rat thalamic relay neurones by acetazolamide

Thomas Munsch and Hans-Christian Pape

MS 8773 Received 28 September 1998; accepted after revision 8 June 1999.

	ABSTRACT

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1. The effect of inhibition of brain carbonic anhydrase (CA) on the hyperpolarization-activated cation current (I_h) of thalamocortical (TC) neurones of the rat ventrobasal thalamic complex (VB) was investigated in an in vitro slice preparation using the whole-cell patch-clamp technique and fluorescence ratio imaging of the pH indicator 2',7'-bis(carboxyethyl)-5(and -6)-carboxyfluorescein (BCECF).

2. Recording of I_h before and after addition of 0·4-0·8 mM acetazolamide to the bathing fluid revealed a significant shift in the voltage dependence of activation (V_½) of 5-7 mV to more positive potentials.

3. Simultaneous recording of I_h and BCECF fluorescence ratio (F₄₂₀/F₄₉₅) revealed an increase in I_h amplitude accompanied by an intracellular alkalinization upon application of acetazolamide. The CA inhibitor ethoxyzolamide (EZA, 50 µM) also led to an intracellular alkalinization and a subsequent 4-5 mV positive shift of V_½ of I_h.

4. Acetazolamide and EZA both profoundly slowed the rapid fall of pH_i upon switching from Hepes- to CO₂/HCO₃^--buffered superfusate, indicating intracellular CA isoforms in TC neurones.

5. In slices bathed in Hepes-buffered saline, addition of acetazolamide had no effect on the amplitude and time course of activation of I_h, indicating that the action of acetazolamide on I_h was dependent on the presence of HCO₃^-.

6. Under current-clamp conditions, the neuronal response to hyperpolarizing current pulses in the presence of acetazolamide was decreased as compared to control. This resulted in a strongly reduced ability of TC neurones to produce rebound Ca²⁺-mediated spikes.

7. The present results implied that in TC neurones acetazolamide led to an intracellular alkalinization which causes, due to its pH sensitivity, an increase in I_h.

	INTRODUCTION

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Acetazolamide, a selective sulfonamide inhibitor of carbonic anhydrase, is widely used as an antiepileptic agent for the treatment of, among others, absence seizures (Reiss & Oles, 1996), yet the mechanistic basis of its therapeutic action remains unclear (Wilder & Bruni, 1982). Its anticonvulsant action is related to the selective non-competitive inhibition of brain carbonic anhydrase, which catalyses the reversible hydration of carbon dioxide (Millchap et al. 1955; Maren, 1967). In the brain, carbonic anhydrase is found predominantly in glial cells (Giacobini, 1962; Cammer, 1984; for review see also Ridderstrale & Wistrand, 1998), but it is also present in neurones (Nógrádi & Milhály, 1991; Pasternack et al. 1993), or membrane bound with its catalytic side facing the extracellular space (Diaz et al. 1982). Inhibition of carbonic anhydrase, as, for example, with acetazolamide or ethoxyzolamide, has been shown to affect steady-state extracellular pH as well as transient extracellular pH shifts related to neuronal activity (Kraig et al. 1983; Walz, 1989; Kaila et al. 1992; Chen & Chesler, 1992; Rose & Deitmer, 1995a,b). As pH_o shifts are accompanied by corresponding pH_i shifts and vice versa, it is expected that such pH shifts will affect a variety of ligand- and voltage-gated ion channels.

In the preceding paper we showed that the hyperpolarization-activated cation currrent (I_h) of TC neurones is greatly affected by changes in intracellular pH (Munsch & Pape, 1999). TC neurones are capable of generating intrinsic oscillations which are under control of the I_h acting as a pacemaker current (McCormick & Pape, 1990). Therefore, changes in pH_i, whether activity related or experimentally induced, for instance by therapeutically active substances, can be assumed to affect the timing of membrane oscillations in TC neurones. TC neurones form part of the thalamocortical network, which can generate highly synchronized oscillations, most probably occurring during electroencephalographic synchronization during sleep (Steriade et al. 1994). The regulation of the oscillatory mechanisms is particularly important for the control of the sleep-waking cycle (McCormick & Bal, 1997). In addition, there is ample evidence that the circuits and mechanisms that normally sustain synchronized oscillatory activity during sleep are those that are critically involved in the generation of synchronous spike and wave discharges during generalized absence seizures (Steriade et al. 1994).

In the present study we therefore investigated a possible effect of the carbonic anhydrase inhibitor and antiepileptic drug acetazolamide on the I_h of TC neurones. We found that acetazolamide caused an intracellular alkalinization in TC neurones which resulted in a persistent upregulation of I_h. It is hypothesized that this mechanism of action of acetazolamide may contribute to the anticonvulsant action of the drug in the treatment of seizures.

	METHODS

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Recordings were performed on TC neurones in an in vitro slice preparation of the thalamus from juvenile Long-Evans rats. The methods for preparation, electrophysiological recording of I_h and pH_i imaging are described in the preceding paper (Munsch & Pape, 1999).

Slices were superfused using a roller pump driven system. To achieve rapid solution changes, bath perfusion was increased from 2·5 to 10 ml min^-1 (bath volume 1 ml).

The pipette solution for recording low voltage-activated (LVA) Ca²⁺ currents was composed of (mM): caesium gluconate, 55; caesium citrate, 10; NaCl, 10; tetraethylammonium (TEA)-Cl, 20; Hepes, 20; MgCl₂, 1; KCl, 1; EGTA, 0·2; Mg-ATP, 3; Na₂-GTP, 0·5; phosphocreatine, 15; lidocaine N-ethyl bromide (QX-314), 5; pH adjusted to 7·25 with KOH. LVA Ca²⁺ currents were selectively activated by step depolarizations to -40 mV from hyperpolarizing prepulses of 1 s duration to -90 mV to de-inactivate all LVA Ca²⁺ channels.

All substances were obtained from Sigma (St Louis, MO, USA), except for Hepes (SERVA, Heidelberg, Germany). Acetazolamide and ethoxyzolamide were dissolved directly in ACSF immediately before use. Ultrasonication for 1 min greatly enhanced their solubility in ACSF. Drugs were applied with the bathing solution.

The data are presented as means ± S.D. or ± S.E.M., as indicated. Statistical significance was tested with an unpaired t test for small samples (Dixon & Massey, 1969) or Student's paired t test using the statistics package provided by Origin, v. 4.0 (Microcal, Northampton, MA, USA), as indicated.

	RESULTS

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Whole-cell recordings were obtained from neurones of the ventrobasal thalamic nucleus that exhibited basic electrophysiological properties characteristic of thalamic relay neurones (Jahnsen & Llinás, 1984a,b). A representative sample of TC neurones from VB possessed an average membrane resting potential of -65 ± 3·5 mV (n = 15), as measured in current-clamp mode at zero current. Mean input resistance was 298 ± 75 M (n = 15) at -48 mV holding potential in voltage clamp mode. Mean cell capacitance, as compensated and read from the C_slow compensation circuit of the amplifier, amounted to 147 ± 23 pF (n = 15). All recorded neurones produced a low-threshold Ca²⁺ spike crowned by a burst of fast Na⁺/K⁺ spikes upon depolarization from hyperpolarized values of the membrane potential (see e.g. Fig. 8).

Effect of carbonic anhydrase (CA) inhibitors on I_h

I_h currents evoked under control conditions in TC neurones by hyperpolarizing voltage steps from -48 to -103 mV are shown in Fig. 1A. Upon bath application of acetazolamide (0·4 mM), step hyperpolarizations elicited h-currents with increased amplitudes during the 2 s hyperpolarization and faster activation time course as compared to currents recorded under control conditions. This effect was only partially reversible as revealed by the current responses after 30 min washout of acetazolamide. The effect of acetazolamide was due to a significant shift of the voltage dependence of activation of I_h (Fig. 1B). Mean half-activation potential shifted significantly (P 0·01) from -91·1 ± 1·9 mV (k = 8·5 ± 0·6, n = 6) under control conditions to -84·4 ± 2·1 mV (k = 8·8 ± 0·7, n = 6) in the presence of 0·4 mM acetazolamide. After 30 min of washout of acetazolamide I_h activation remained positively shifted (V_½ = -85·4 ± 3·9 mV, k = 9·1 ± 0·9, n = 6) as compared to control. The fully activated conductance underlying I_h or the extrapolated reversal potential were not significantly (P > 0·1) different between control and acetazolamide-treated cells (G_h = 11·2 ± 3·3 vs. 12·3 ± 3·7 pA mV^-1 and V_rev = -19·6 ± 4·4 vs. -20·7 ± 4·7 mV) (Fig. 1C).

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Figure 1. Effect of acetazolamide on Ih of thalamic relay neurones A, examples of current families recorded prior to, during and after 30 min washout of 0·4 mM acetazolamide (AZA). Mean steady-state activation curve (B) and fully activated I-V relationship (C) of Ih recorded under different conditions as indicated. Lines in B and C represent best fits of a Boltzmann distribution and linear regression analysis to the data points, respectively. Data represent mean values and S.E.M. (n = 6). Recordings were obtained in the presence of 1 mM Ba2+ to isolate Ih. Voltage protocol is shown in inset.

The positive shift of the I_h activation curve by acetazolamide indicated that this carbonic anhydrase inhibitor was acting through a change in the intracellular pH of TC neurones. To investigate this possibility, we simultaneously recorded I_h and BCECF fluorescence ratio in neurones filled with the indicator via the patch pipette. Application of acetazolamide (0·4 mM) indeed caused a rapid intracellular alkalinization which only partially recovered following washout of the substance (Fig. 2A). At this concentration, acetazolamide decreased F₄₂₀/F₄₉₅ by 19·7 ± 5·0 % from baseline (n = 12). By estimation of the magnitude of shifts in pH_i from TMA- and lactate-induced changes in BCECF fluorescence (see preceding paper) this would correspond to an intracellular alkalinization of 0·19 pH units. Concomitant with the acetazolamide-induced increase in pH_i, an increase in I_h amplitude and time course of activation occurred (Fig. 2A and B). Since the speed of the alkaline shift may point to an extracellular mechanism of action of acetazolamide, we also used ethoxyzolamide, a CA inhibitor with established membrane permeability (Maren, 1967). Application of ethoxyzolamide (0·05 mM) caused a rapid decrease in F₄₂₀/F₄₉₅ of 9·0 ± 1·8 % (n = 5), indicating an intracellular alkalinization by about 0·08 pH units (Fig. 3A). The ethoxyzolamide-induced rise in pH_i was accompanied by a significant (P 0·05, paired t test) positive shift of the mean half-activation potential of I_h from -91·6 ± 1·6 mV (k = 9·7 ± 1·0, n = 5) under control conditions to -87·3 ± 2·4 mV (k = 9·6 ± 0·7, n = 5) in the presence of ethoxyzolamide (Fig. 3B).

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Figure 2. Increase in Ih by acetazolamide-induced intracellular alkalinization A, plot of normalized BCECF fluorescence ratio (F420/F495 expressed as a percentage, left abscissa) and Ih, measured as steady-state current minus instantaneous current (right abscissa), evoked by hyperpolarizing voltage steps of 2 s duration to -98 mV. Downward deflection of F420/F495 indicates an alkalinization. At times marked by a and b the corresponding current responses shown in B were recorded. B, current responses to hyperpolarizing voltage steps to -98 mV recorded from a VB neurone before (a) and during (b) application of 0·4 mM acetazolamide.

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Figure 3. Shift in half-maximal activation voltage of Ih by ethoxyzolamide-induced intracellular alkalinization A, plot of BCECF fluorescence ratio (F440/F495, expressed as a percentage) versus time measured in a TC neurone before, during and after application of 0·05 mM ethoxyzolamide (EZA) with the bath solution. Downward deflection of F440/F495 indicates an alkalinization. B, steady-state activation curves of Ih prior to and during application of 0·05 mM EZA. Lines in B represent best fits of a Boltzmann distribution to the data points. Data represent mean values and S.E.M. (n = 5).

Dependence on bicarbonate of acetazolamide action

Carbonic anhydrase catalyses the reversible hydration of CO₂ (Maren, 1967), implying that an acetazolamide-induced alkalinization and a resulting shift in the voltage dependence of I_h requires the presence of bicarbonate (HCO₃^-). Indeed, when the thalamic slices were superfused with Hepes-buffered saline, substituting HCO₃^--buffered solution for at least 30 min prior to recording of I_h, addition of acetazolamide (0·4 mM) had no measurable effect on I_h properties (Fig. 4, n = 7). This suggested that the action of acetazolamide on I_h is related to inhibition of carbonic anhydrase. Moreover, the results strongly argue against a direct effect of acetazolamide on the channels carrying I_h.

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Figure 4. Lack of acetazolamide effect in Hepes-buffered saline Plot of Ih, measured as steady-state current minus instantaneous current, evoked by hyperpolarizing voltage steps to -98 mV (duration 2 s), versus time. Application of AZA is indicated by the bar above data points. The inset shows current responses to -98 mV hyperpolarizing test steps recorded before (a) and during (b) application of AZA. Recordings were obtained 30 min following substitution of HCO3-- by Hepes-buffered saline.

To address the question of whether there are intracellular CA isoforms in TC neurones, we monitored BCECF fluorescence in single cells filled via a patch pipette and rapidly changed from Hepes- to CO₂/HCO₃^--buffered superfusate and back again. The rapid intracellular acidification seen under these conditions (Fig. 5A and B) is suggested to reflect the diffusion of CO₂ into TC neurones and its hydration to H⁺ and HCO₃^-. In the presence of the CA inhibitors acetazolamide (0·4 mM, n = 4) and ethoxyzolamide (0·05 mM, n = 4) the rapid acidification upon CO₂/HCO₃^- exposure was profoundly slowed (Fig. 5A and B). Sometimes (2/4 cells exposed to acetazolamide and 2/4 cells exposed to ethoxyzolamide) a slow alkalinization developed upon CO₂/HCO₃^- exposure in the presence of CA inhibitors (data not shown). These alkaline shifts were not further investigated in this study.

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Figure 5. Effect of CA-inhibitors on CO2-induced intracellular acid shifts Switching from Hepes- to CO2/HCO3--buffered solution and back again induced a rapid intracellular acidification to a plateau level followed by a slower return to the initial values. Upward deflection of F440/F495 indicates an acidosis. Addition of 0·4 mM AZA (A) or 0·05 mM EZA (B) profoundly slowed the intracellular acidification. Within the duration of recording of F440/F420 there was no recovery following washout of both CA inhibitors.

Additivity of acetazolamide- and cAMP-mediated effects on I_h

The I_h current in TC neurones is under control of the intracellular adenylate cyclase-cAMP system (reviewed by Pape, 1996). To test whether the acetazolamide-induced shift of I_h activation is mediated by pH-dependent modulation of adenylate cyclase activity, we used forskolin (10 µM) and cAMP (100 µM) to near-maximally shift the voltage dependence of I_h activation to more positive potentials. In the presence of the adenylate cyclase activator forskolin, application of acetazolamide (0·4 mM) further shifted the half-activation potential from -85·2 ± 1·8 mV (k = 8·5 ± 1·0, n = 6) to -79·3 ± 2·0 mV (k = 8·5 ± 1·2, n = 6, P 0·01) (Fig. 6A and B). Similarly, with 100 µM cAMP in the pipette solution, which shifts the voltage dependence of activation most probably by binding to the channels underlying I_h (reviewed by Pape, 1996), acetazolamide significantly (P 0·05, paired t test) shifted V_½ values from -88·6 ± 2·6 mV (k = 8·4 ± 0·7, n = 4) to -85·9 ± 3·3 mV (k = 8·6 ± 0·9, n = 4) (Fig. 6C and D). These findings of additive effects of activation of the adenylate cyclase-cAMP system and acetazolamide suggested that the acetazolamide-induced shift in the voltage dependence of I_h activation is mediated through a pathway independent of that involving adenylate cyclase.

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Figure 6. Additivity of the effects of acetazolamide and adenylate cyclase on Ih A, examples of current responses to hyperpolarizing voltage steps from -48 to -98 mV recorded under control conditions, in the presence of forskolin (10 µM), and during addition of AZA (0·4 mM) to the forskolin-containing solution. B, steady-state activation curves of Ih recorded under control conditions, after bath application of 10 µM forskolin and after addition of 0·4 mM acetazolamide (AZA) in the presence of forskolin (n = 6). C, family of current responses to voltage steps to -98 mV with cAMP (100 µM) in the pipette solution (control) and after addition of AZA (0·4 mM) with the bathing solution. D, steady-state activation curves recorded with 100 µM cAMP in the patch pipette before and after addition of 0·4 mM AZA to the saline. Continuous lines in B and D are best fits of a Boltzmann distribution. Data represent means ± S.E.M. of 6 and 4 experiments, as indicated.

Lack of acetazolamide effect on LVA Ca²⁺ currents

Benzolamide, another highly potent inhibitor of carbonic anhydrase (Maren, 1977), was found to directly inhibit low-threshold calcium currents of rat hippocampal pyramidal neurones (Gottfried & Chesler, 1995). To test for a possible effect of acetazolamide on LVA Ca²⁺ (I_LVA) currents in TC neurones, the current responses of TC neurones evoked by step depolarizations to -40 mV following a hyperpolarizing prepulse to -90 mV for 1 s were recorded before and during the presence of acetazolamide (0·4 mM). A typical experiment is shown in Fig. 7. LVA Ca²⁺ currents typically displayed run-down of about 12 % in 10 min and addition of acetazolamide caused no further reduction in I_LVA amplitude (n = 10), largely ruling out a direct effect of acetazolamide on I_LVA.

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Figure 7. Lack of acetazolamide effect on LVA Ca2+ current Plot of LVA Ca2+ current (ILVA), evoked by step depolarizations to -40 mV from 1 s hyperpolarizing prepulses to -90 mV, versus time. The black bar above data points indicates time of acetazolamide (AZA) application. The inset shows two representative current traces obtained before (a) and during (b) application of AZA.

Possible functional consequences of acetazolamide-induced modulation of I_h

The enhancement of I_h by various types of neurotransmitters has been shown to result in a decreased voltage response to hyperpolarizing stimuli and a resulting dampening of Ca²⁺-mediated burst activity (Pape & McCormick, 1989; reviewed by McCormick & Bal, 1997). We tested the hypothesis that enhancement of I_h by acetazolamide may have similar consequences on the neuronal response to hyperpolarizing current pulses, in particular the influence of modulation of I_h on the generation of rebound burst discharges. Under current-clamp conditions, the voltage responses to hyperpolarizing current steps (duration 2 s, amplitude -20 to -50 pA) were recorded before and during the presence of acetazolamide (0·4 mM) (Fig. 8). Fast inward rectification was blocked by 100 µM extracellular Ba²⁺ (Williams et al. 1997). Application of acetazolamide resulted in a small depolarization of 1-2 mV and a decrease in apparent input resistance (n = 6). The decrease in hyperpolarizing voltage deflection resulted in a distinct reduction in the neurones' ability to generate rebound Ca²⁺ spikes (Fig. 8). These results indicate that acetazolamide decreases rebound burst firing in TC neurones by depolarization of the membrane and reduced voltage responses to hyperpolarizing stimuli, presumably due to a decrease in de-inactivation of I_LVA (cf. McCormick & Bal, 1997).

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Figure 8. Effects of acetazolamide on neuronal responses to hyperpolarizing current pulses Voltage traces recorded under current-clamp conditions from a VB neurone before (control) and during bath application of 0·4 mM acetazolamide in the presence of 100 µM extracellular Ba2+. Voltage deflections were evoked by 2 s injections of -20 and -50 pA hyperpolarizing current, respectively. Note absence of low-threshold Ca2+ spikes in the presence of acetazolamide. The large low-threshold spike after -50 pA current injection under control conditions was truncated.

	DISCUSSION

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The present results demonstrate that application of the CA inhibitor acetazolamide induces an intracellular alkalinization and resulting shift in the activation curve of I_h in TC neurones, which led to a persistent upregulation of I_h. In the following sections, the hypothesis is discussed that these mechanisms exert a dampening influence on rhythmic electrical activity in the thalamocortical system and thereby contribute to the antiepileptic properties of acetazolamide in the treatment of epileptic seizures.

Effect of acetazolamide on pH_i

In brain tissue, CA is mainly localized within glia (Sapirstein et al. 1984; Cammer, 1984; Ridderstrale & Wistrand, 1998). In glial cells from the leech central nervous system, inhibition of intracellular CA by ethoxyzolamide had only scarcely detectable effects on steady-state pH_i and stimulus-evoked pH_i transients (Rose & Deitmer, 1995b). But evidence is accumulating that CA might also be present in neurones (Nógrádi & Milhály, 1991; Pasternack et al. 1993). In the present study it is demonstrated that inhibition of CA by acetazolamide and ethoxyzolamide caused an increase in steady-state pH_i of TC neurones, presumably due to the intracellular accumulation of HCO₃^-. The accumulation of intracellular HCO₃^- has been suggested to contribute to the anticonvulsant effects of CA inhibition (Millchap et al. 1955; Tower, 1960). The present study provides a physiological demonstration of intracellular CA in TC neurones. Intracellular acidification upon rapid changes in P_CO2 has been observed in a variety of cells, including crayfish muscle (Kaila et al. 1990), rat cortical and hippocampal neurones (Ou-yang et al. 1993; Pasternack et al. 1993) and retinal Müller glial cells (Newman, 1994). The demonstation of intracellular CA activity in TC neurones does not rule out possible extracellular mechanisms of action of CA inhibitors, which might contribute to the effects on intracellular pH shifts. However, the fast intracellular alkalinization is more indicative of a intracellular site of action, because changes in extracellular pH caused only small changes in pH_i, which were insufficient to modulate I_h (Munsch & Pape, 1999). Also, it seems unlikely that changes in the buffering status within the cell contribute considerably to the rapid alkalinization upon exposure to CA inhibitors.

Effect of acetazolamide on I_h

The CA inhibitor benzolamide, which is barely membrane permeable, was shown to inhibit LVA Ca²⁺ channels of hippocampal pyramidal neurons directly and in a bicarbonate-independent way (Gottfried & Chesler, 1995). Based upon this observation it was suggested that inhibition of CA or changes in extracellular buffering were not involved in the action of benzolamide on LVA Ca²⁺ channels. Recording of I_LVA in TC neurones revealed no direct effect of acetazolamide on LVA Ca²⁺ channels. However, at present one cannot rule out that pH_i-mediated effects of acetazolamide on I_LVA might contribute to the reduced ability to generate LTSs. A fuller understanding of the mechanisms of acetazolamide on thalamic relay cell activity therefore awaits direct testing of the effects of this drug on thalamic network oscillations.

Augmentation of I_h by acetazolamide and EZA was strongly dependent on the presence of HCO₃^-, supporting the role of inhibition of intracellular CA in the chain of mechanisms leading to upregulation of I_h. Due to the pH sensitivity of I_h activation, any transient or steady-state change in pH_i, including changes in metabolic production of CO₂ and lactate, exchange of H⁺ for Ca²⁺ from intracellular binding sites after rises of intracellular free Ca²⁺ and changes in the transport or flux of acid or base equivalents across the membrane can be assumed to participate in the control of I_h. In the preceding paper we have shown that activity-related transient intracellular acidification led to a downregulation of I_h. Here we demonstrate that intracellular alkalinization due to inhibition of CA causes an upregulation of I_h. This will make I_h a preferential target of pH_i-affecting changes during electrical and metabolic activity in neurones.

Model of acetazolamide action on I_h and possible functional consequences

On the basis of the results obtained in the preceding (Munsch & Pape, 1999) and present studies, we suggest a model of the action of acetazolamide on I_h of TC neurones. Acetazolamide rapidly enters neurones due to its membrane permeability. Inside the cells it blocks intracellular CA. Since this enzyme plays a pivotal role in the efficacy of the CO₂/HCO₃^--buffer system (Maren, 1963), inhibition of intracellular CA is expected to slow conversion of HCO₃^- into CO₂. Different mechanisms for pH regulation, as for instance, the Na⁺-dependent Cl^--HCO₃^- exchange (Schwiening & Boron, 1994; Baxter & Church, 1996), may contribute to HCO₃^- accumulation. Together with the removal of acid equivalents from the cell this will result in an increase of the intracellular pH. Due to its direct pH sensitivity, the intracellular alkalinization will lead to a positive shift in the activation curve and thence an upregulation of I_h. The resulting membrane depolarization from resting levels and decrease in amplitude of hyperpolarizing responses can be assumed to reduce the probability of rebound Ca²⁺ burst firing in TC neurones.

These effects, in turn, may have important functional consequences for the temporal shaping of rhythmic electrical discharges in the thalamocortical network. In fact, a positive shift in the I_h activation curve upon an increase in intracellular Ca²⁺ concentration during spindling oscillations in TC neurones in vitro has been shown to contribute to termination of oscillatory activity (Lüthi & McCormick, 1998). Based upon these observations it was suggested that a similar mechanism contributes to the waning phase of sleep spindles in the thalamocortical system in vivo. It is thus tempting to speculate that the pH-dependent upregulation of I_h by acetazolamide may similarly act to control rhythmic electrical discharges in these cells, particularly those that are associated with spike-and-wave discharges during absence seizures. The following line of evidence is supportive of this conclusion. First, the mechanisms and circuits that normally sustain spindle waves have been found to be critically involved in the production of spike-and-wave discharges during absence seizures (Steriade et al. 1997; but see also Steriade & Contreras, 1995; Pinault et al. 1998). Second, the highly synchronized neuronal discharges associated with epileptiform activity result in a significant shift in pH_o (Caspers & Speckmann, 1972; Somjen, 1984; de Curtis et al. 1998), presumably caused by a rapid intracellular acidification due to transmembrane movement of protons from the extracellular space into neurones (Kraig et al. 1983). Third, the I_h current is under control of the intracellular H⁺ concentration (Munsch & Pape, 1999); intracellular acidification during epileptiform discharges will result in a negative shift in the I_h activation curve, thereby counteracting the Ca²⁺-mediated positive shift in I_h activation and thence prolonging the duration of synchronized rhythmic activity. Fourth, application of acetazolamide and resulting intracellular alkalinization positively shifts I_h activation, thereby helping to restore the normal balance between mechanisms of up- and downregulation of I_h and thence to support the termination of synchronized rhythmic activity. Fifth, these effects, in turn, may contribute to the antiepileptic properties of acetazolamide. Indeed acetazolamide is still in use in adjunctive therapy or monotherapy for patients responding poorly to other antiepileptics, including the treatment of absence epilepsies (Reiss & Oles, 1996). Unfortunately, therapeutic cerebrospinal fluid concentrations of acetazolamide have not yet been determined (Reiss & Oles, 1996). The recommended dosage for the treatment of adult humans (500-1000 mg day^-1; Oles et al. 1989) seems low compared with the concentration of acetazolamide (0·4 mM) used in the present in vitro study. However, acetazolamide is long-term administered during the treatment of epilepsies, and cells containing CA have been found to accumulate acetazolamide (Inui et al. 1982).

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Acknowledgements

Thanks are due to Mrs R. Ziegler for excellent technical assistance and to Dr T. Budde for comments on this manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Pa 336/13-1) and the Human Capital and Mobility Program of the European Science Foundation (CHRX-CT9-40543).

Corresponding author

T. Munsch: Otto-von-Guericke Universität, Medizinische Fakultät, Institut für Physiologie, Leipzigerstrasse 44, D-39120 Magdeburg, Germany.

Email: thomas.munsch{at}medizin.uni-magdeburg.de

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