The mechanisms that control the periodicity of spontaneous epileptiform cortical potentials were investigated in the in vitro isolated guinea pig brain preparation. A brief intracortical application of bicuculline in the piriform cortex induced spontaneous interictal spikes (sISs) that recurred with high periodicity (8.5 ± 3.1 sec, mean ± SD). Intracellular recordings from principal neurons showed that the early phase of the inter-sIS period is caused by a GABAb receptor-mediated inhibitory potential. The late component of the interspike period correlated to a slowly decaying depolarization abolished at membrane potentials positive to −32.1 ± 5.3 mV and was not associated with membrane conductance changes. Specific pharmacological tests excluded the contribution of synaptic and intrinsic conductances to the late inter-sIS interval. Recordings with ion-sensitive electrodes demonstrated that sISs determined both a rapid increase in extracellular K+ concentration (0.5–1 mm) and an extracellular alkalinization (0.05–0.08 pH units) that slowly decayed during the inter-sISperiod and returned to control values just before a subsequentsIS was generated. These observations were not congruous with the presence of a silent period, because both extracellular increase in K+ and alkalinization are commonly associated with an increase in neuronal excitability. Extracellular alkalinization could be correlated to an sIS-induced intracellular acidification, a phenomenon that reduces cell coupling by impairing gap junction function. When intracellular acidification was transiently prevented by arterial perfusion with NH4Cl (10–20 mm), spontaneous ictal-like epileptiform discharges were induced. In addition, the gap junction blockers octanol (0.2–2 mm) and 18-α-glycyrrethinic acid (20 μm) applied either via the arterial system or locally in the cortex completely and reversibly abolished the sIS. The results reported here suggest that the massive cell discharge associated with an sIS induce a strong inhibition, possibly secondary to a pH-dependent uncoupling of gap junctions, that regulatessIS periodicity.
When a condition of hyperexcitability occurs in the cortex, epileptiform events such as interictal spikes or ictal discharges arise spontaneously. In vivo and in vitro studies that used different acute and chronic models of epileptogenesis showed that spontaneous interictal spikes (sISs) can recur with a period variable between 1 and 10 sec depending on the experimental condition (Prince 1971; Lebovitz, 1979; Traub and Wong, 1982; Rutecki et al., 1985; Schneiderman and Mac Donald 1989; Chamberlin et al., 1990; Leung, 1990; Perez-Velasquez et al., 1994; Pelletier and Carlen, 1997). Clinical observations demonstrated that periodic spiking activity is a common phenomenon in lesional human epilepsy (Chatrian et al., 1964) and in idiopathic benign partial epilepsies of childhood (Beaussat et al., 1972). According to the work of Lebovitz (1979), autorhythmicity of sISs derives from the functional suppression of the propagation to the soma of the spontaneous synaptic events generated distally in the dendrites. This observation introduced the idea thatsIS periodicity is not simply attributable to progressive buildup of excitation but might be caused by a prolonged and powerful phasic inhibition that follows the synchronous paroxysmal discharge associated with the sIS itself. This assumption will be tested here in an acute model of focal epileptogenesis induced in the piriform cortex of the in vitro isolated guinea pig brain preparation. Previous studies on this model demonstrated that a transient ejection of bicuculline in the piriform cortex inducesISs that recur periodically and persist for hours, even when the drug is washed out (de Curtis et al., 1994, 1998; Forti et al., 1997). The sISs are sustained by a primary burst associated with the activation of an intrinsic calcium spike, followed by a large recurrent glutamatergic synaptic potential propagated along the diffuse intrinsic associative fiber system of the piriform cortex (Haberly and Bower, 1989; Biella and de Curtis, 1995). The intracellular and extracellular events associated with the interspike silent period will be analyzed in the present study.
MATERIALS AND METHODS
Experiments were performed on young adult guinea pigs (200–250 gm; Charles River, Calco, Italy). The procedures for the isolation of the brain have been previously described in detail (Llinas et al., 1981; de Curtis et al., 1991; Muhlethaler et al., 1993). Briefly, after barbiturate anesthesia (Pentotal, 20 mg/kg, i.p.) and after cardiac perfusion with cold saline solution, the brain was extracted and transferred to an incubation chamber. The in vitro brain was perfused with an oxygenated solution (5% CO2-95% O2) via a cannula inserted into the basilar artery. The composition of the perfusate was: NaCl 126 mm, KCl 2.3 mm, NaHCO3 26 mm, MgSO41.3 mm, CaCl2 2.4 mm, KH2PO4 1.2 mm, glucose 15 mm, HEPES 5 mm, thiourea 0.4 mm, and 3% dextran 70.000, pH 7.3. The perfusion rate was 5.5–6 ml/min. The experiments were performed at 32°C. Bicuculline methiodide (2 mm; Research Biochemicals, Natick, MA) dissolved in the perfusate was injected for 10 sec at 800 μm depth in the anterior piriform cortex (APC) through a 8- to 10-μm-tip diameter glass pipette connected to a graduated syringe. One hundred fifty to 200 nmol of bicuculline were delivered in one single ejection; the pipette was removed after bicuculline application. Bicuculline was washed out within 90 min (de Curtis et al., 1994; Forti et al., 1997). The experimental protocol has been reviewed and approved by the Commitee on Animal Care and Use of the Istituto Nazionale Neurologico.
Although the study is centered on spontaneous events, a bipolar silver wire electrode was positioned on the lateral olfactory tract (LOT) to evoke responses in the APC and to locate the depth of the extracellular recording electrodes (3–10 MΩ resistance micropipettes filled with 0.9% NaCl). Intracellular recordings were performed from principal neurons in layers II and III with sharp electrodes filled either with 3m K-acetate or with 2 m K-acetate and 1% biocytin (50–120 MΩ resistance). Extracellular and intracellular activity was recorded with a Neurodata (New York, NY) amplifier. Extracellular K+ concentration and pH were measured with ion-sensitive microelectrodes (ISMs) pulled from borosilcate glass capillaries with filament (WPI; electrode tip diameter, 2–4 μm) acid-cleaned and dryed at 100°C. The pipettes were then exposed for 1 min to dimethyldichlorosylane vapors (14896; Fluka, Neu-Ulm, Germany) and baked at 120°C for 2 hr. The K+-ISMs were filled at the tip with potassium ionophore I-cocktail A (Fluka 60031) and were back-filled with 200 mm KCl. The pH-ISM tips were filled with hydrogen ionophore II-cocktail A (Fluka 95297) and back-filled with a buffer solution (in mm: NaCl 100, HEPES, and NaOH 10, pH 7.5). K+ and pH calibration solutions were similar to the perfusate used during the experiment, with either KCl or NaHCO3 substituted for the corresponding moles of NaCl; for the K+-ISM the perfusate solution was modified to contain 1–20 mm K+. For the pH-ISM the perfusate solution was modified from pH 6.0 to 8.0 (pH range measured with the conventional macro-pH electrode calibrated with commercial buffers). The signals were amplified with a high input impedance head stage and an Axoclamp 2B amplifier (Axon Instruments). The pH-ISMs had a response of 50–55 mV for unit change in pH. The data were accepted only if the calibration curves obtained before and after the experiments did not differ by >5%. Signals were stored on a Biological 2602 digital tape recorder for off-line analysis with a Digital Microvax 3400 computer system.
Octanol (0.2 mm; Sigma, St. Louis, MO)(S)-34-methyl-4-carboxyphenylglycine (MCPG, 2 mm; Tocris), 18-α-glycyrrethinic acid (Sigma, 20 μm), and NH4Cl (10–20 mm, substituted for NaCl; Sigma) were delivered by arterial perfusion. When octanol and MCPG were applied locally in the extracellular space, a 10-fold concentration was used. QX-314 (80 mm; Research Biochemicals) dissolved in 1 m K-acetate, pH 7.3, was applied by intracellular diffusion through the recording pipette. When micropipettes filled with biocytin were used for intracellular staining, the isolated brains were fixed overnight with 4% paraformaldehyde. Coronal sections (100 μm thick) were then processed for avidin–HRP visualization and were counterstained with neutral red to locate the cells within the cortical layers (Forti et al., 1997).
The silent inter-sIS period was analyzed by performing simultaneous extracellular field recordings and intracellular recordings from principal neurons in layers II and III of the APC at the bicuculline focus. Average resting membrane potential was −75.20 ± 6.47 mV (mean ± SD). No differences in the electrophysiological behavior during the inter-sIS period were observed between the layer II (n = 29) and layer III (n = 9) cells.
As previously reported (de Curtis et al., 1998), a single injection of bicuculline in the deep layers of the APC induces sISs that recur periodically every 8.5 ± 3.1 sec (Fig.1 A) and persist when bicuculline is washed out. The frequency of the sISs became regular in an individual brain 10 min after the application of bicuculline. Previous studies demonstrated that the extracellularly recorded sIS correlates to a primary burst of action potentials subtended by a calcium spike, followed by a secondary depolarization mediated by a recurrent glutamatergic excitatory potential (Forti et al., 1997; de Curtis et al., 1998; also see Traub and Wong, 1982; Traub et al., 1993). During the silent inter-sISs period a slowly decaying depolarization was recorded intracellularly. The activation of the next sISoccurred exclusively when the membrane potential returned to resting values (Fig. 1 A). As previously demonstrated, the early portion of the inter-sIS period is attributable to a K+-dependent GABAb receptor-mediated inhibitory potential (de Curtis et al., 1998). The GABAb inhibitory potential was identified as a hyperpolarizing afterpotential when the membrane was depolarized to values more positive then −75 mV by injecting intracellularly a steady current (Fig. 1 B, right trace, arrow). The modifications of membrane potential induced by current injection did not change the frequency of sIS repetition. The late, slowly decaying component of the inter-sISpotential showed no voltage reversal but was abolished at membrane potentials positive to −32.1 ± 5.3 mV (n = 13). The late inter-sISs period can be imputed either to the activation of an inhibitory membrane conductance or to a passive decrease in excitability associated with microenvironmental changes in the extracellular space.
To verify whether the late inter-sIS potential correlated to the activation of a membrane conductance, cellular membrane resistance was tested continuously during several consecutive sISs by evaluating the voltage response to a 50–100 msec hyperpolarizing current pulse at 2–3 Hz. As illustrated in Figure2, membrane resistance decreased just after the sIS, returned to pre-sIS values within 2 sec, and was not altered during the late part of the inter-sIS potential (n = 8). In Figure2 B the voltage responses to the current pulse just before an sIS (a) and 600 msec (b) and 3 sec (c) after the sIS are superimposed. The possible activation of a membrane conductance during the late inter-sIS period was further evaluated by analyzing the tonic firing induced by membrane potential depolarization (Fig.3). The activation of the conductance associated with the post-sIS GABAb inhibitory potential shunted the tonic firing evoked by membrane depolarization to −57 mV (Fig. 3 A). The pre-sIS firing frequency was completely reestablished within 2 sec from the sIS onset (n = 4). The graph in Figure 3 B shows the same effect in a different neuron. The return of firing rate to control values after the sIS is illustrated for two different levels of depolarization (−40 and −53 mV). These results suggest that no significative changes in membrane conductance correlate to the late part of the inter-sIS potential.
The possibility that the slow inter-sIS potential could be attributable to distal voltage-dependent dendritic conductances activated by the sustained sIS-related bursting was evaluated by hindering bursting activity either by passively diffusing the sodium channel blocker QX-314 (80 mm) through the intracellular electrode (Fig.4 A; n = 8) or by hyperpolarizing membrane potential via steady current injection (Fig. 4 B; n = 3). In both types of experiments the slow depolarizing potential was not abolished.
Finally, because slow depolarizing potentials dependent on nonionotropic glutamate receptors have been demonstrated in cortical neurons (Bianchi and Wong, 1995), the effect of the metabotropic receptor antagonist MCPG applied either by arterial perfusion (2 mm; n = 2) or locally in the tissue (20 mm; n = 2) was tested. The drug did not affect the sIS periodicity (data not shown).
These results suggest that the inter-sIS period is not associated with the activation of an intrinsic or synaptic membrane conductance. As a possible alternative, modifications of ion concentrations in the extracellular space could lead to inhibition during the silent period. Because transmembrane movement of K+ and protons is known to follow massive and synchronous bursting activity (Moody et al., 1974; Heinemann et al., 1977; Somjen, 1984; Jarolimek et al., 1989) (for review, see Jefferys, 1995), the modifications of K+ concentration and pH in the extracellular space were measured during periodicsISs with ion-selective electrodes. The sISrecorded extracellularly consistently correlated to a rapid increase in extracellular K+ concentration (peak concentration changes of 0.5–1 mm from a baseline of 3.5 mm;n = 4; Fig. 5). The recovery to control values coincided with the activation of the nextsISs.
Similarly, a fast-rising extracellular alkaline shift (ranging between 0.05 and 0.08 pH units from a baseline of 7.3; n = 5) that completely decayed before the activation of the nextsIS was observed during the inter-sIS period (Fig. 6 A). High-frequency afferent stimulation that mimics bursting activity has been shown to induce fast extracellular alkaline transients with rise times in the order of the tens of milliseconds (Gottfried and Chesler, 1996), compatible with the rapid changes we observed. The periodicity of the sISs (Fig. 6 B, top two traces) was disrupted when a tetanic stimulation was applied to the lateral olfactory tract (Fig. 6 B, bottom two traces). High-frequency stimulation also induced a large extracellular K+ shift that returned to control values with a time course similar to that of pH changes (data not shown). The discontinuation of the periodicity correlated to a very large alkaline shift, and the reappearance of the sISs coincided with the return of pH to control values (Fig.6 B). Both increase in K+ and alkalinization of the extracellular environment are known to increase neuronal excitability by several means (Rutecki et al., 1985; Church and McLennan, 1989; Chamberlin et al., 1990; Gottfried and Chesler, 1994; Tombaugh and Somjen 1996; Deitmer and Rose, 1996). Because increases in K+ and alkalinization occur after a single sIS, a gradual transition toward the development of an ictal event would be expected if excitability were enhanced. On the contrary, in our experimental conditions the probability of occurrence of an sIS is reduced during the inter-sIS period, as demonstrated by the inability to generate sISs from subthreshold potentials in a condition of increased K+ illustrated in Figure 5 (top trace, arrowheads).
The observed extracellular alkalinization could be attributable to a rapid transmembrane movement of protons into the neurons associated with the massive bursting and the secondary recurrent synaptic excitation during the sIS (Kraig et al., 1983; Chen and Chesler, 1992; Hartley and Dubinsky, 1993; Deitmer and Rose, 1996). Intracellular acidification is known to decrease gap junctional conductance (Spray et al., 1981; Perez-Velasquez et al., 1994). If the intracellular acidification determined inter-sIS inhibition via a blockade of the nonsynaptic transfer of excitation among PC neurons, then reducing acidification should cause an increase in the frequency of sISs and should promote a transition toward an ictal discharge. Ammonium chloride (NH4Cl), a compound known to induce transient intracellular alkalinization followed by acidification (Giaume and Korn, 1982; Thomas, 1984; Perez-Velasquez et al., 1994), was used to test this hypothesis. NH4Cl applied via the arterial system (10–20 mm; n = 6) induced a gradual increase in excitability followed by a prolonged inhibition (up to 1 hr) on NH4Cl washout. The extracellular recordings illustrated in Figure7 A show that the transient increase in excitability is characterized by the activation of spontaneous, long-lasting (1–2 sec) afterdischarges (after 2–5 min of perfusion) followed by a disappearance of the sIS (after 5–10 min of perfusion). The polysynaptic component of the field responses evoked by low-intensity LOT stimulation, but not the monosynaptic response, was reduced by NH4Cl, suggesting that the intrinsic excitability of PC neurons was not increased by NH4Cl (data not shown). When intracellular recordings were performed, no changes in input membrane resistance were observed during NH4Cl perfusion (n = 2; data not shown). The effects of NH4Cl were reverted within 40–60 min after washout. In two of six tests prolonged ictal discharges were observed during NH4Cl perfusion (Fig. 7 B, middle traces). The LOT stimulation-evoked activity was preserved during the silent period (Fig. 7 B, middle trace, arrowhead), suggesting that the NH4Cl-induced inhibition was not coupled to a spreading depression-like phenomenon.
Because a decrease in electrotonic coupling between neurons could substantially reduce excitability, we tested the possibility that a reduction of gap junction function could prevent further sISto be generated. The gap junctional blocker octanol (Peinado et al., 1993) applied either by arterial perfusion (0.2 mm;n = 5) or locally in the cortex (2 mm;n = 2) induced a complete and reversible abolition of the sIS (Fig.8 A). Because octanol has been shown to decrease T-type calcium conductances (Scott et al., 1990), we tested the effect of the T-type calcium condutance blocker nickel to exclude the involvement of high-threshold calcium spike modulation in the abolition of sISs. Local application of nickel (50 μm; n = 2) did not modifysIS periodicity (data not shown). Arterial perfusion with another gap junction blocker, 18α-glycyrrhetinic acid (20 μm; Davidson et al., 1986; Blanc et al., 1998), reduced the population spike component of the sIS and progressively decreased their periodicity until a quasi-complete abolition was obtained after 5 min (Fig. 8 B; n = 3). In two experiments the effect was reverted after 5–10 min of washout.
The study demonstrates that spontaneous interictal potentials induced by transient application of bicuculline in the piriform cortex of the isolated guinea pig brain recur periodically. Two possible conditions may account for the inter-sIS silent period: (1) the hyperexcitable cortical network requires some time to build up and to reach threshold for the activation of the next spontaneous synchronous event after the activation of a sIS; or (2) the cortex is transiently inhibited after an sIS and cannot be reexcited until such inhibition is removed.
Our experiments demonstrate that a single sIS induces extracellular ionic shifts that would be expected to produce excitation but paradoxically are associated with a predominant inhibition. Moody et al. (1974) demonstrated that K+ elevation is maximal 150–400 msec after an interictal population burst and slowly decays with a time course of several seconds. Similarly, in our experiments the extracellular K+ concentration was maximally increased just after an sIS and remained elevated for 5–10 sec. High extracellular K+ is known to slow down repolarization of presynaptic terminals and to increase the presynaptic release of neurotransmitter, therefore augmenting the duration and frequency of spontaneous EPSPs (Chamberlin et al., 1990;Traub and Dingledine, 1990). The probability of activating spontaneous EPSPs in our model should be maximal ∼2 sec after the precedingsIS discharge, when recurrent GABAb synaptic inhibition ceases and extracellular K+ is still elevated (Traub and Dingledine, 1990). As a consequence, if the buildup of synaptic excitation would be the only mechanism for sIS generation,sISs should be facilitated at the end of post-sISGABAb-mediated inhibitory potential. This is not the case, becausesISs are reactivated after a much longer period (5–10 sec) in our experiments.
A condition of enhanced excitability would also be expected to result from the extracellular alkalinization induced by the synchronous neuronal firing associated with the sIS (for review, seeChesler and Kaila, 1992; Deitmer and Rose, 1996). Several studies demonstrated that extracellular alkalinization enhances Na+ and Ca+2 voltage-activated conductances in hippocampal neurons (Tombaugh and Somjen, 1996), increases NMDA receptor-dependent activity (Tang et al., 1990;Traynelis and Cull-Candy, 1990; Gottfried and Chesler, 1994), and promotes seizure activity (Jarolimek et al., 1989). Thus, like the increases in extracellular K+, the post-sIS alkalinization produces a condition in the extracellular microenvironment that would favor excitation, in striking contrast with the observation of a silent inter-sIS period. The absence of such an increase in excitability is demonstrated by the observation that spontaneous potentials below the threshold for an sIS (Fig. 5, arrows) do not evoke a population spike during the silent period, despite the concurrent elevations in extracellular K+ and pH. Indeed, a transient decrease in excitability occurs during these ionic shifts induced by the activation of sIS.
A transient inhibition during the silent period could be attributable either to activation of a shunting conductance or to reduced coupling between neurons. As mentioned before, the early phase of the inter-sIS potential correlates to a GABAb receptor-mediated inhibitory synaptic potential (de Curtis et al., 1998). The late component of the inter-sIS period is paralleled by a slowly decaying depolarization that is (1) abolished at membrane potentials positive to −35 mV, (2) not coupled to a reduction in membrane resistance (Fig. 2), and (3) not dependent on the intracellular activation of the sIS burst. These observations suggest that the slow inter-sIS depolarization is not mediated by the activation of an established synaptic or intrinsic membrane conductance. Bal and McCormick (1996) showed that the slow depolarizing potentials responsible for the interspindle silent period are mediated by the persistent activation of a hyperpolarization-activated cation current in thalamic neurons. The possible role of such a current can be excluded in our experimental conditions, because the selective intracellular blocker of the hyperpolarization activated conductance, QX-314 (Perkins and Wong, 1995), does not abolish the inter-sIS slow depolarization (Fig. 4 A). In cortical neurons metabotropic glutamate receptors have been shown to sustain slow depolarizing events (Bianchi and Wong, 1995) that might presynaptically depress synaptic transmission (Burke and Hablitz, 1994) and could be responsible for the inter-sIS silent period. This inhibitory mechanism is ruled out in our model by the demonstration that the metabotropic glutamate receptor MCPG does not alter sIS periodicity. We concluded that none of the membrane conductances known to reduce excitability in cortical neurons is activated during the late depolarizing potential associated with the inter-sIS period.
We noticed that the return to resting membrane potential values of the slow post-sIS depolarization paralleled the slow, monotonic decay of K+ concentration measured with ion-selective electrodes during the silent period. This observation suggest that the post-sIS membrane depolarization could be attributable to a modification of the K+ equilibrium potential consequent to the transient elevation of the extracellular K+ concentration associated with the populationsIS discharge (Whisler and Johnston, 1978; Rutecki et al., 1985; Haglund and Schwartzkroin, 1990; Otis et al., 1993). The post-sIS depolarization had an amplitude of 4–6 mV just after the spike component of the sIS recorded intracellularly with QX-314, a condition that blocks the GABAb-mediated IPSP and reveals the slow depolarization. Assuming that (1) at resting membrane potential values (approximately −80 mV) the membrane of piriform cortex neurons is permeable mainly to K+, (2) the [K+]i measured at 32°C for a −87 mV K+ equilibrium potential (Otis et al., 1993) is 100 mm, and (3) the experimental [K+]o is 3.5 mm, the 0.5–1 mm increase in [K+]o measured in our experiments just after the sIS determined according to the Nernst–Planck equation a 3.5–6.5 mV shift in the K+equilibrium potential, compatible with the amplitude of the observed depolarization.
The extracellular alkalinization associated with neuronal activity is supposed to be caused by a rapid intracellular acidification mediated by transmembrane flow of protons from the extracellular space into neurons (Kraig et al., 1983) either through ion channels activated by excitatory and inhibitory neurotransmitters or by calcium–proton exchange (Chen and Chesler, 1992; Smith et al., 1994) (for review, see Chesler and Kaila, 1992; Deitmer and Rose, 1996). NH4Cl has been applied in our experiments to test whether by preventing intracellular acidification we could increase excitability and interfere with the mechanisms that generate the silent inter-sIS period. NH4Cl is known to enter cells via K+ channels (Moser, 1987) and to induce a transient intracellular alkalinization followed by intracellular acidification on washout (Giaume and Korn, 1982; Thomas, 1984;Perez-Velasques et al., 1994). The results show that the intracellular alkalinization presumably induced during the early phases of NH4Cl perfusion promotes a fast transition toward ictal-like discharges (Fig. 7 A,B). Moreover, during the NH4Cl washout, when intracellular acidification is expected to occur, sISs are completely abolished. These observations strongly suggest that (1) a marked enhancement in neuronal excitability is produced by contrasting sIS-induced intracellular acidification, and (2) intracellular acidification promotes a condition of low excitability during which no sIS can be generated. The possibility that the transitory enhancement in excitability we observed could be attributable to NH4Cl-induced increase of calcium conductances, as demonstrated in chick sensory neurons (Mironov and Lux, 1993), can be excluded because (1) membrane input resistance did not change during NH4Cl perfusion, and (2) the polysynaptic component of the field responses evoked by low-intensity LOT stimulation below the threshold for the activation of interictal epileptiform discharge, but not the monosynaptic response, was reduced by NH4Cl.
Both intracellular acidification or extracellular alkalinization have been shown to influence dye coupling (Church and Baimbridge, 1991) and bursting behavior of neurons (Church and McLennan, 1989; Valiante et al., 1996) by reducing gap junction function (Spray et al., 1981) and consequently by decreasing synchronization of neuronal firing (Perez-Velazquez, 1994). This mechanism could hypothetically account for the transient decrease in intrinsic neuronal excitability during the inter-sIS period. Nonsynaptic intercellular diffusion of action potentials generated during the sIS might be reduced by transient gap junction impairment so that population excitability decreases and repetitive activation of the neurons that just generated a sIS is prevented. The abolition of sISs induced by application of gap junction blockers octanol and 18α-glycyrrhetinic acid supports this hypothesis.
The results shown here suggest that changes in pH should not be considered an inconsequential epiphenomenon generated during interictal spiking but, rather, may be a primary factor that regulates neuronal excitability and controls epileptiform discharges. Patterns of recurrent interictal activity such as periodic sharp waves or spikes similar to those described in this study have been described in experimental models of epileptogenesis and are commonly observed as interictal electroencephalographic abnormalities in epileptic patients suffering of idiopathic or postlesional partial epilepsy (Chatrian et al., 1964; Beaussat et al., 1972). Even if the basic alterations expressed in different epileptic conditions are highly heterogeneous, common mechanisms similar to those described in the present paper could be postulated for the generation of periodic epileptiform events, and their functional implications can be exploited to understand the process of epileptogenesis.
Partial support was provided by the Italian Health Ministry through Pharmacia-Upjohn, Italy (PNR-MURST Grant 188.8.131.52). G.B. is supported by a Human Frontier Science Program fellowship (Grant RG 19/96). We thank Dario Brambilla and Carlo Rossetti for the technical help with the preparation of the ion-selective recordings.
Correspondence should be addressed to Marco de Curtis, Department of Experimental Neurophysiology, Istituto Nazionale Neurologico, via Celoria 11, 20133 Milan, Italy.