INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Epileptiform activity is abnormally enhanced electrical activity associated with an imbalance between excitatory and inhibitory synaptic mechanisms and/or alterations of neuronal intrinsic properties. Evidence obtained in vitro has shown that several cellular mechanisms may lead to epileptiform activity. Indeed, reduced GABAergic inhibitory synaptic activity (e.g., Federico and MacVicar 1996; McBain 1994; Rodriguez-Moreno et al. 1997; Wong and Miles 1994) or enhanced glutamatergic excitatory synaptic activity (e.g., Federico and MacVicar 1996; Johnston and Brown 1981; Traub et al. 1993) may lead to epileptic-like phenomena. Alterations of neuronal intrinsic properties may also participate in in-vitro models of epileptogenesis (e.g., Biervert et al. 1998; Johnston and Brown 1981; Prince 1993; Traub and Jefferys 1994). However, the specific extrasynaptic conductances involved and the mechanisms mediating their regulation are poorly understood.

Potassium conductances regulate neuronal excitability (e.g., Hille 1992; Storm 1990) and are susceptible to modulation. Therefore they are good candidates for the extrasynaptic elements involved in the regulation of epileptiform activity (Beck et al. 1996; Biervert et al. 1998; Rutecki et al. 1987). Specifically, Ca²⁺-dependent K⁺ conductances may be extremely relevant because they control cellular excitability (Araque and Buño 1995; Araque et al. 1998; Borde et al. 1995, 2000; Hille 1992; Madison and Nicoll 1984; Storm 1990). Indeed, it has been suggested that post-spike after-hyperpolarization (AHP), which regulates neuronal excitability, controls epileptogenesis (Behr et al. 2000; Empson and Jefferys 2001; Matsumoto and Ajmone-Marsan 1964; Verma-Ahuja et al. 1995).

In CA1 pyramidal neurons, an apamin-insensitive Ca²⁺-activated K⁺ current with slow kinetics (sI_AHP) mediates the slow AHP (Sah 1996; Sah and Bekkers 1996). This sI_AHP provides a negative feedback control of neuronal excitability and is susceptible to modulation by several neurotransmitters (e.g., Charpak et al. 1990; Madison et al. 1987; Storm 1990). Although the role of the sI_AHP in regulating normal neuronal excitability is well established, the contribution of the sI_AHP in controlling abnormal epileptiform activity is not fully studied.

Hippocampal epileptiform activity most likely initiates in the CA3 and spreads to the CA1 region. Understanding the cellular mechanisms that mediate and control the spread of epileptiform activity to CA1 is a relevant issue. Therefore we used electrophysiological and photometric Ca²⁺ measurements in hippocampal slices to investigate the possible participation of the sI_AHP of the CA1 pyramidal neurons in epileptiform activity. We show that the increased synaptic activity evoked by 4AP or 4AP+Mg²⁺-free treatment reduced the sI_AHP via the activation of postsynaptic group I/II mGluRs; we also show that the increased excitability caused by inhibition of the negative feedback provided by the sI_AHP contributes to epileptiform activity. Therefore the cooperative action both of abnormal network activity and intrinsic cellular mechanisms is required for 4AP or 4AP+Mg²⁺-free-induced epileptiform activity in the CA1 region.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Wistar rats (13-17 days old) were decapitated and their brains were rapidly removed and placed in ice-cold artificial cerebrospinal fluid (ACSF) gassed with a 95% O₂-5% CO₂ mixture to attain a pH of 7.2-7.4. Transverse brain slices (400-450 µm thick) were cut with a Vibratome (Pelco 101, Series 1000, St. Louis, MO) and incubated >1 h at room temperature (21-24°C) in ACSF that contained (in mM) 124 NaCl, 2.69 KCl, 1.25 KH₂PO₄, 2 MgSO₄, 26 NaHCO₃, 2 CaCl₂, and 10 glucose and was gassed with 95% O₂-5% CO₂. Slices were then transferred to an immersion recording chamber placed on the stage of an inverted microscope (Nikon DIAPHOT-TMD, Tokyo, Japan) and superfused (1.5 ml/min) at room temperature with gassed ACSF. The exchange of solution in the recording chamber was usually complete within 4 min. To reduce the effects of GABA_A-mediated inhibitory synaptic transmission, some experiments were performed in the presence of 50 µM picrotoxin or 50 µM bicuculline.

To induce epileptiform activity, the ACSF contained 100 µM 4-aminopyridine (4AP) either with or without MgSO₄ (4AP+Mg-free ACSF).

Electrophysiology

Recordings were made using the whole-cell configuration of the "blind" patch-clamp technique. Recorded cells were identified as CA1 hippocampal pyramidal neurons according to their placement in the pyramidal layer and their electrophysiological properties (see, e.g., Borde et al. 1995, 2000). In some cases, cells were visualized under a microscope equipped with infrared and differential interference contrast imaging devices and a 40× water immersion objective (see following text). In these cases, morphological criteria confirmed the electrophysiological identification of cells. Patch electrodes were fabricated from borosilicate glass capillaries (R-Series 1B150F-4, WPI, Sarasota, FL) with a Brown-Flamming model P-80 micropipette puller (Sutter Instruments, Novato, CA). Pipettes had resistances of 6-10 M when back-filled with an "internal" solution that contained (in mM) 150 KMeSO₄, 10 HEPES, and 4 ATP-Na₂ (added immediately before filling) and that was buffered to pH 7.2-7.3 with KOH (280-290 mOsm).

Recordings were obtained with an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) either in the current-clamp bridge mode or the continuous single electrode voltage-clamp mode. Fast and slow whole-cell capacitances were neutralized and series resistance was always compensated (~70%). Cells were considered only when the series resistance did not change >12% throughout the experiment. In voltage-clamp experiments, the membrane potential (Vm) was either held at 60 mV when excitatory postsynaptic currents (EPSCs) were recorded or at 50 or 60 mV when the Ca²⁺ activated K⁺ currents that mediate after-hyperpolarization (I_AHP) were evoked by voltage command pulses (200 ms) to 20 mV. The sI_AHP magnitude was quantified from the area under the current trace, measured 100-200 ms after the end of the pulse (after the medium I_AHP was negligible; see RESULTS).

Signals were low-pass filtered at 3 kHz and fed to a Pentium-based PC through a DigiData 1200 interface board (Axon Instruments). The pCLAMP 7 software (Axon Instruments) was used for stimulus generations, data display, acquisition, and storage.

Stimulation

Bipolar nichrome wire electrodes (80 µm diameter) were connected to a stimulator and isolation unit (Grass S88, Quincy, MA) and placed under visual guidance in the stratum radiatum near the border of the CA1 pyramidal neurons to stimulate Schaffer collateral-commissural (SC) afferents. Stimulation was with single pulses (50 µs, 0.3 Hz) adjusted in intensity to evoke EPSCs below the threshold for evoking "unclamped" action currents (Fig. 1D).

View larger version (20K): [in this window] [in a new window]   Fig. 1. 4-aminopyridine (4AP)+Mg-free artificial cerebrospinal fluid (ACSF) increases spontaneous synaptic activity, reduces the slow Ca2+-activated K+ current (sIAHP), and induces epileptiform activity. A and B: representative current- and voltage-clamp recordings, respectively, in control, 4AP+Mg-free ACSF, and after washout. Note the reversible increase in the frequency and amplitude of spontaneous excitatory postsynaptic potentials and spontaneous excitatory postsynaptic currents (sEPSCs) induced by 4AP+Mg-free ACSF. Characteristic slow depolarizations (or slow inward currents) and action potential bursts (or "unclamped" action currents) were observed under current- and voltage-clamp conditions. C: representative traces showing the sIAHP evoked by 200 ms depolarizing pulses (from 50 mV to 20 mV) (left, bottom trace) in control, 4AP+Mg-free ACSF, and after washout. D: superimposed EPSCs (n = 10) evoked by Schaffer collateral-commissural stimulation in control, 4AP+Mg-free ACSF, and after washout. Holding potential was 60 mV. B and C: same cell.

Measurement of intracellular Ca²+ variations

Cells were visualized under an Olympus BX50WI microscope (Olympus Optical, Tokyo, Japan) equipped with infrared and differential interference contrast imaging devices and a 40× water immersion objective. Patch pipettes were filled with standard internal solution containing 10-20 µM fluo-3 (Molecular Probes, Eugene, OR). Cells were illuminated with a xenon lamp at 490 nm using a monochromator Polychrome II (T.I.L.L. Photonics, Planegg, Germany). Fluorescence intensity was collected by a photomultiplier tube (model R928, Hamamatsu Photonic System, Bridgewater, NJ) from a variable rectangular window (side 25-50 µm) that included part of the neuronal soma and the proximal apical dendrite. Cells were illuminated for 20-100 ms every 100-150 ms and the fluorescence signal collected was integrated by using the T.I.L.L. Photonics photometry system.

Chemicals

(S)--methyl-4-carboxyphenylglycine (MCPG), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and D()-2-amino-5-phosphonopentanoic acid (AP5) were obtained from Tocris Cookson (Bristol, UK). MCPG and t-ACPD were prepared from concentrated stock solutions (100 mM) and added to the ACSF at the desired concentration immediately before use. All other drugs were from Sigma (St. Louis, MO).

All experiments were performed at room temperature (20-23°C). Statistical differences were established using the two-tailed Student's t-test unless stated otherwise. Data are expressed as means ± SE. Analysis of spontaneous EPSCs (sEPSCs) was performed with ACSPLOUF software (obtained from Dr. Pierre Vincent, University of California, San Diego). The cumulative probabilities of the amplitude and frequency of the sEPSCs recorded during 1-3 min in the different conditions were estimated. The mean frequency of the sEPSCs was calculated in 1-s bins. Statistically significant differences were established at P < 0.05 using the Kolmogorov-Smirnov test. After the significant differences between the different conditions in each cell were assessed, Student's t-test was used to compare the averaged sEPSC mean frequency with established statistical differences of the pooled data.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Results are based on recordings obtained from 69 CA1 pyramidal neurons that exhibited a stable resting Vm between 55 and 66 mV. Control recordings of spontaneous activity showed that neurons were silent, had a resting Vm of 59.0 ± 0.7 mV (n = 22), and displayed very few spontaneous excitatory postsynaptic potentials (sEPSPs) or sEPSCs (mean frequency 0.27 ± 0.02 s¹; n = 12) (Fig. 1, A and B).

4AP+Mg-free increased spontaneous synaptic activity and evoked epileptiform bursts

After control recordings, slices were superfused with 4AP+Mg-free ACSF while the activity was continuously monitored under current- or voltage-clamp conditions. Epileptiform activity was characterized in current-clamp recordings by spontaneous depolarizations and bursts with multiple action potentials (Fig. 1A, 4AP+Mg-free, right trace). Slow, long-lasting inward currents and bursts of action currents caused by "unclamped" action potentials typified the epileptiform activity recorded under voltage-clamp conditions (Fig. 1B, 4AP+Mg-free, right trace). Epileptiform activity appeared 681 ± 55 s (n = 20) after the onset of superfusion. Spontaneous bursts had durations of 18.0 ± 2.1 s with 26 ± 4 spikes/burst (mean spike amplitude during the burst was 47.1 ± 0.7 mV) and burst frequencies were 0.3-3.0 bursts/min (n = 28). These characteristics are in agreement with the properties of the in vitro epileptiform discharges in CA1 pyramidal neurons in rat hippocampal slices described by several authors (e.g., Avoli et al. 1996; Jones and Heinemann 1988; Perreault and Avoli 1991).

Spontaneous synaptic activity was continuously monitored under current- or voltage-clamp conditions (Fig. 1, A and B, respectively) and the frequency and amplitude of sEPSPs or sEPSCs were quantified. Epileptiform activity was always preceded (230 ± 29 s before the first depolarization burst; n = 20) by a marked increase in the frequency and amplitude of sEPSCs from 0.27 ± 0.02 s¹ and 11.2 ± 0.6 pA in control (Fig. 1B, control) to 15.04 ± 0.67 s¹ and 37.5 ± 0.9 pA in 4AP+Mg-free ACSF (Fig. 1B, 4AP+Mg-free, left trace; n = 13; P < 0.001). This increase occurred in all cells tested (n = 54) and was clearly revealed by comparing the corresponding cumulative probability plots (e.g., Fig. 2, A and B; P < 0.001). The amplitude of EPSCs evoked by SC stimulation was also dramatically increased (from 39.1 ± 3.7 pA in control to 186.4 ± 21.3 pA; n = 9), which usually triggered a single unclamped action current and occasionally a brief burst (Fig. 1D). This enhancement of spontaneous and evoked synaptic transmission and epileptiform activity could be fully reversed after a 10 min washout in control ACSF (e.g., Figs. 1 and 2).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Effects of 4AP+Mg-free ACSF on spontaneous synaptic activity and sIAHP. A and B: cumulative probability plots of sEPSC frequency and amplitude, respectively, recorded during 1-3 min in control (squares), 4AP+Mg-free (circles), and after washout (triangles). C: mean sEPSC frequency in control and in 4AP+Mg-free ACSF (n = 17). D: mean relative sIAHP area in control and in 4AP+Mg-free ACSF (n = 17). Significant differences with respect to control were estimated with Student's t-test at P < 0.001 (***).

sI_AHP was inhibited during epileptiform activity

We further analyzed the changes in the sI_AHP induced by superfusion with 4AP+Mg-free ACSF. The sI_AHP evoked by 200 ms pulses to 20 mV from the 60 or 50 mV holding potential consisted of a large, slowly decaying outward tail current that followed the depolarizing pulse (Fig. 1C, control) and decayed with a single exponential rate ( = 2343 ± 166 ms; n = 25). The amplitude of the sI_AHP usually increased with time after establishing the whole-cell configuration, stabilized in ~10 min, and attained a mean peak amplitude of 97 ± 17 pA at 60 mV (n = 33). In addition to the enhancement of spontaneous synaptic activity induced by 4AP+Mg-free ACSF, the sI_AHP area was reduced to 45.0 ± 6.3% from control values (P < 0.001; n = 16; Figs. 1C and 2D). Recovery of the sI_AHP following a washout was always associated with the recovery to control values of the normal spontaneous and evoked synaptic transmissions and of the normal electrical activity (Figs. 1 and 2).

Mechanisms regulating the sI_AHP

Because the sI_AHP of CA1 pyramidal neurons is not affected by micromolar concentrations of 4AP per se (see below; cf. Storm 1990), and because the induction of epileptiform activity was identical in the presence or absence of magnesium (n = 7; see below), the reduction of the sI_AHP was not caused by a direct effect of the 4AP+Mg-free treatment. Furthermore, the sI_AHP was not significantly modified by 4AP+Mg-free ACSF (116 ± 13% from control values; n = 4) after blocking excitatory synaptic transmission with 20 µM CNQX and 50 µM AP5, again indicating that the sI_AHP was not directly modulated by 4AP+Mg-free ACSF. The properties of epileptiform activity induced by the 4AP+Mg-free treatment, as well as the sI_AHP reduction, were similar in the presence of 50 µM picrotoxin (n = 5, not shown) or 50 µM bicuculline (n = 3, not shown), indicating that GABA_A receptors are not involved.

As described in the preceding text, increased spontaneous synaptic activity was accompanied by a reduction of the sI_AHP (Figs. 1, A-C, and 2). The inhibition of the sI_AHP and the enhancement of spontaneous synaptic activity always preceded (n = 17) the epileptiform bursts (Fig. 3A). These results suggest that the inhibition of the sI_AHP was associated with increased synaptic activity. Indeed, Fig. 3A shows that the temporal profile of the sI_AHP area reduction following 4AP+Mg-free treatment was the mirror image of the increased sEPSC frequency. Both variables were clearly correlated, as deduced from the good linear regression fit of their relationship (r = 0.94; Fig. 3B), which again supports the idea that increased synaptic activity is associated with sI_AHP inhibition.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Temporal profile of 4AP+Mg-free ACSF effects on synaptic activity and sIAHP. A: mean sEPSC frequency (filled symbols) and mean relative changes in sIAHP area (open symbols). Bottom bar: occurrence times of the first epileptiform bursts after the onset of superfusion with 4AP+Mg-free ACSF (n = 5). B: mean relative sIAHP area vs. mean sEPSC frequency. Values are from the data in A. Data were fitted to a linear regression (continuous line; r = 0.94).

It can be argued that the sI_AHP would appear to be reduced if 4AP+Mg-free-induced synaptic activity decreased the extent of the space-clamped cell membrane. However, this interpretation seems to be unlikely because membrane input resistance (estimated from the linear regression of the voltage-current relationship evoked by 200 ms hyperpolarizing current pulses) was not significantly affected by 4AP+Mg-free ACSF (87 ± 8 M in control and 70 ± 5 M in 4AP+Mg-free ACSF; P = 0.1; n = 6).

In addition, synaptic enhancement caused by 4AP+Mg-free ACSF did not modify the sI_AHP when MCPG was present (see Fig. 4), indicating that the sI_AHP was not reduced per se by a potential conductance increase induced by the enhanced synaptic activity. A possible MCPG-evoked rise in Ca²⁺ influx, which would increase the activation of the sI_AHP, thus counteracting possible sI_AHP reduction caused by space-clamp error, can also be ruled out because superfusion with MCPG alone did not significantly modify the intracellular Ca²⁺ signal (112.1 ± 11.5% from control values; n = 5) or the sI_AHP amplitude (see Fig. 4). Therefore these results imply that synaptic activity-induced sI_AHP reduction is not caused by significant space-clamp errors.

Taken together, these results suggest that enhancement of synaptic activity induced by 4AP+Mg-free ACSF is associated with sI_AHP reduction.

Inhibition of the sI_AHP is mediated via activation of mGluRs

The sI_AHP in hippocampal pyramidal neurons may be regulated via network activity involving the release of several transmitters, including glutamate and acetylcholine (ACh), by activation of metabotropic glutamate receptors (mGluRs) and muscarinic ACh receptors (mAChR) (e.g., Charpak et al. 1990; Madison et al. 1987; Storm 1990). Therefore reduction of the sI_AHP may be caused by activation of mGluR and/or mAChR caused by the augmented glutamate and ACh released by the increased synaptic activity stimulated by the 4AP+Mg-free treatment. We therefore investigated the effects of mGluR and mAChR antagonists to determine whether sI_AHP inhibition was associated with the activation of those receptors.

We first tested the effects of MCPG, an antagonist of the group I and II mGluRs (Conn and Pin 1997). We superfused 0.5-1.0 mM MCPG, which was added to the control ACSF, for 10 min. A slice was then superfused with 4AP+Mg-free ACSF containing 0.5-1.0 mM MCPG for at least 30 min. Figure 4A shows representative traces of the spontaneous synaptic activity recorded in the presence and absence of MCPG and the corresponding sI_AHP (Fig. 4B). MCPG did not change spontaneous synaptic activity or sI_AHP magnitude. Furthermore, although enhancement of spontaneous synaptic transmission by the 4AP Mg-free treatment was still present under MCPG (Fig. 4, A and C), the sI_AHP was not significantly affected (Fig. 4, B and E) and epileptiform activity was not observed. After a washout in control ACSF (>50 min), superfusion of 4AP+Mg-free ACSF without MCPG markedly reduced the sI_AHP and induced epileptiform activity (Fig. 4, A, B, and E). These effects of MCPG on 4AP+Mg-free-induced spontaneous synaptic activity and sI_AHP regulation are quantitatively displayed in Fig. 4, D and E. In addition, the absence of sI_AHP inhibition in the presence of MCPG confirms that the sI_AHP is not directly affected by micromolar concentrations of 4AP (cf. Storm 1990).

View larger version (22K): [in this window] [in a new window]   Fig. 4. (S)--methyl-4-carboxyphenylglycine (MCPG) prevents sIAHP regulation and the induction of epileptiform activity but does not block the increase in synaptic activity induced by 4AP+Mg-free ACSF. A: representative voltage-clamp recordings in control, during superfusion with 800 µM MCPG, 4AP+Mg-free plus MCPG, after washout, and 4AP+Mg-free ACSF. B: sIAHP evoked by 200 ms depolarizing pulses from 60 to 20 mV in the same solutions as in A. Note the increased synaptic activity under 4AP+Mg-free ACSF plus MCPG and the absence of sIAHP reduction and epileptiform activity. However, 4AP+Mg-free ACSF without MCPG induced synaptic activity enhancement, sIAHP inhibition, and epileptiform activity. C: cumulative probability plots of sEPSC frequency (left) and amplitude (right) recorded for 1-3 min in control (squares), 4AP+Mg-free ACSF plus MCPG (circles), and after washout (triangles). A-C: same neuron. D and E: mean sEPSC frequency and mean relative sIAHP area, respectively, under different experimental conditions as indicated by - (absence) and + (presence) (n = 10). Significant differences with respect to controls were established by Student's t-test at P < 0.001 (***).

To control whether MCPG changed the initial control conditions, we analyzed the effects of MCPG superfused in isolation for up to 30 min. We found that 1) neither the frequency or amplitude of sEPSCs was significantly affected (n = 10) (Fig. 4, A and D), 2) synaptic enhancement evoked by 4AP+Mg-free ACSF was not significantly modified (n = 10) (Fig. 4D), and 3) the sI_AHP was unaffected by MCPG (in MCPG, sI_AHP area was 116.3 ± 10.2% from control; n = 10) (Fig. 4E). These results indicate that synaptic activity and the sI_AHP were unaffected by superfusion with MCPG. Furthermore, these data also imply that the effect of MCPG on regulation of the sI_AHP is not mediated presynaptically.

After the sI_AHP had been reduced and epileptiform activity was induced by 4AP+Mg-free ACSF, further addition of MCPG did not reverse sI_AHP inhibition and failed to block epileptiform bursts (n = 5; not shown) (cf. Arvanov et al. 1995). Therefore MCPG prevented the induction but not the maintenance of epileptiform bursts, implying that after the sI_AHP was inhibited and epileptiform activity was produced, sustained activation of mGluRs was not required.

Taken together, these data indicate that the enhanced spontaneous synaptic activity evoked by 4AP+Mg-free ACSF inhibits the sI_AHP through the activation of postsynaptic group I/II mGluRs, leading to epileptic discharges.

mAChRs are not involved in sI_AHP inhibition

In addition to mGluRs, mAChRs can also modulate the sI_AHP in CA1 hippocampal pyramidal neurons (e.g., Charpak et al. 1990; Madison et al. 1987; Storm 1990). The enhanced synaptic transmission evoked by 4AP+Mg-free ACSF could lead to increased release of ACh, which, acting on mAChRs, could also inhibit the sI_AHP, ending in epileptiform activity.

We analyzed the effects of 10 µM atropine, a broad-spectrum mAChR antagonist, superfused 10 min before switching to 4AP+Mg-free ACSF plus atropine. In the presence of atropine, increased spontaneous synaptic activity was not prevented, the sI_AHP was still reduced (50.9 ± 4.2% from control, which is not significantly different from the reduction observed in the absence of atropine; n = 5), and epileptiform activity was generated (not shown). These data indicate that under the conditions of our study, the sI_AHP is not modulated by mAChRs to induce epileptiform activity.

sI_AHP inhibition is not paralleled by a reduction of the intracellular Ca²+ signal

It has been reported that mGluRs can modulate voltage-gated Ca²⁺ channels in several systems (e.g., Choi and Lovinger 1996; Takahashi et al. 1996). Because the channels underlying the sI_AHP are Ca²⁺-dependent (Borde et al. 1995, 2000; Sah 1996; Sah and Bekkers 1996), the mGluR-dependent inhibition of the sI_AHP may be an indirect effect mediated through a reduction of the intracellular Ca²⁺ signal. Alternatively, mGluRs could activate a path directly leading to the inhibition of the sI_AHP channels (Charpak et al. 1990).

To test these possibilities, we simultaneously recorded the intracellular Ca²⁺ variations and the corresponding sI_AHP evoked by depolarizing pulses before and during the generation of 4AP+Mg-free-induced epileptiform activity (Fig. 5). Figure 5, A and B, shows representative traces of the intracellular Ca²⁺ variations and of the sI_AHP, respectively, under control conditions and during epileptiform activity induced by 4AP+Mg-free ACSF. The intracellular Ca²⁺ signal increased in the presence of 4AP+Mg-free ACSF (134.3 ± 11.2 from control values; P < 0.05; n = 7), as expected from the absence of a magnesium block of voltage-gated Ca²⁺ currents (Carbone et al. 1997). However, the sI_AHP was reduced (to 46.0 ± 4.7% from control values; P < 0.001; n = 7) (Fig. 5C).

View larger version (13K): [in this window] [in a new window]   Fig. 5. The sIAHP but not the intracellular Ca2+ signal is inhibited during epileptiform activity. A and B: representative traces of the intracellular Ca2+ variation and the corresponding sIAHP, respectively, evoked by 200 ms depolarizing pulses from 50 to 20 mV in control and in 4AP+Mg-free ACSF. C: mean relative changes of the intracellular Ca2+ signal variation and the sIAHP area in control (black bars) and in 4AP+Mg-free ACSF (gray bars) (n = 7). Significant differences with respect to controls were established by Student's t-test at P < 0.05 (*) and P < 0.001 (***).

Because voltage-gated Ca²⁺ currents can be modified by changes in the extracellular magnesium concentration (e.g., Carbone et al. 1997), we performed two sets of additional experiments, under more controlled conditions, to further investigate whether changes in intracellular Ca²⁺ variation could account for mGluR-mediated sI_AHP reduction. First, experiments were performed in the presence of Mg²⁺ to maintain the extracellular divalent cation concentration constant. Under this condition, superfusion with 4AP increased spontaneous synaptic activity, reduced the sI_AHP (to 37.0 ± 8.6% from control values; P < 0.001; n = 5), and evoked epileptiform activity. However, no significant changes were observed in the intracellular Ca²⁺ signal (116.2 ± 14.7% from control values; n = 5). Second, slices were superfused with Mg-free ACSF and then exposed to 4AP+Mg-free ACSF. Again, synaptic activity was enhanced, the sI_AHP was reduced (to 41.9 ± 10.4% from values in Mg-free ACSF; P < 0.01; n = 7), and epileptiform activity was generated. However, the intracellular Ca²⁺ signal was not significantly modified (108.1 ± 17.6% from Mg-free ACSF values; n = 7).

Taken together, these results imply that mGluR-mediated inhibition of the sI_AHP is not caused by a block of voltage-gated Ca²⁺ channels or of Ca²⁺ released from intracellular stores, suggesting a direct regulation somewhere along the intracellular activation pathway of the sI_AHP.

sI_AHP reduction per se does not generate epileptiform activity

The preceding experiments show that increased synaptic activity is not sufficient to induce epileptiform activity but that mGluR-dependent modulation of the sI_AHP is also required. To further investigate the contribution of the sI_AHP, we attempted to determine whether sI_AHP inhibition was sufficient to induce epileptiform activity in the absence of enhanced spontaneous synaptic activity. We therefore used different pharmacological agents that are known to inhibit the sI_AHP.

Because our results support mGluR-dependent modulation of the sI_AHP, we analyzed the effects of the nonselective mGluR agonist t-ACPD on the sI_AHP and epileptiform activity. Initially, the slice was superfused with control ACSF plus 20 µM t-ACPD for 20 min. The slice was then superfused with 4AP+Mg-free ACSF containing 20 µM t-ACPD for an additional 20 min. Figure 6, A and B, shows representative traces of spontaneous synaptic activity and the corresponding sI_AHP, respectively, recorded in the presence and absence of t-ACPD and after washout. Spontaneous synaptic activity was unchanged (Fig. 6D) whereas the sI_AHP was strongly reduced by t-ACPD (Fig. 6E). However, no epileptiform activity was observed (n = 6). Subsequent treatment with 4AP+Mg-free in the presence of t-ACPD enhanced synaptic activity, reduced the sI_AHP, and evoked epileptiform activity (Fig. 6, A-E), reproducing the effects described in the preceding text (e.g., Fig. 1). Low-level synaptic activity and the large sI_AHP were recovered after washout (Fig. 6, A and B). This action of t-ACPD indicates that the sI_AHP reduction was not sufficient to evoke epileptiform activity.

View larger version (22K): [in this window] [in a new window]   Fig. 6. The sIAHP reduction by (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) was not sufficient to induce epileptiform activity. A: representative voltage-clamp recordings in control, during superfusion with 20 µM t-ACPD, 4AP+Mg-free plus t-ACPD, and after washout. B: sIAHP evoked by 200 ms depolarizing pulses from 50 to 20 mV in the same solutions as in A. Note that t-ACPD reduced the sIAHP but did not increase spontaneous synaptic activity and did not induce epileptiform activity. However, later addition of 4AP+Mg-free ACSF induced synaptic activity enhancement, sIAHP inhibition, and epileptiform activity. C: cumulative probability plots of sEPSC frequency (left) and amplitude (right) recorded for 1-3 min in control (squares), t-ACPD (triangles), and t-ACPD plus 4AP+Mg-free (circles). A-C: same neuron. D and E: mean sEPSC frequency and mean relative sIAHP area, respectively, under the different experimental conditions as indicated by - (absence) and + (presence) (n = 6). Significant differences with respect to controls were established by Student's t-test at P < 0.001 (***).

We also tested the effects of other agents that inhibit the sI_AHP, such as histamine and isoproterenol (Haas and Konnerth 1983; Madison and Nicoll 1982). As with t-ACPD, histamine and isoproterenol did not significantly increase spontaneous synaptic activity but did reduce the sI_AHP without inducing epileptiform activity (Fig. 7), indicating that the sI_AHP reduction was not sufficient to evoke epileptiform activity (cf. Williams et al. 1993).

View larger version (18K): [in this window] [in a new window]   Fig. 7. The sIAHP is necessary but not sufficient to induce epileptiform activity. Effects of 10 µM histamine (n = 4), 10 µM isoproterenol (n = 4), 20 µM t-ACPD (n = 6), 4AP+Mg-free ACSF plus 0.8 mM MCPG (n = 10), and 4AP+Mg-free ACSF (n = 14) on the sIAHP and spontaneous synaptic activity. Histamine, isoproterenol, and t-ACPD reduced the sIAHP but did not increase synaptic activity and failed to induce epileptiform activity. 4AP+Mg-free ACSF plus MCPG increased synaptic activity but failed to induce epileptiform activity. Epileptiform activity was induced only when the sIAHP was inhibited and spontaneous synaptic activity was increased by the 4AP+Mg-free treatment. Statistically significant differences relative to controls were established by Student's t-test at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Figure 7 summarizes the effects of the pharmacological agents applied to spontaneous synaptic activity and the sI_AHP. Histamine, isoproterenol, and t-ACPD reduced the sI_AHP but did not increase spontaneous synaptic activity and failed to induce epileptiform activity, indicating that the sI_AHP reduction was not sufficient to evoke epileptiform activity. Increased synaptic activity induced by 4AP+Mg-free ACSF was also unable to produce epileptiform activity when mGluR-mediated inhibition of the sI_AHP was prevented by MCPG, indicating that sI_AHP reduction is required to induce epileptiform activity. Finally, epileptiform activity was only induced when both enhanced spontaneous synaptic activity and sI_AHP inhibition were present.

Taken together, these results indicate that the cooperative action of synaptic enhancement and the resulting inhibition of the sI_AHP is required to induce epileptiform activity in the CA1 region.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Several studies established the important role of the sI_AHP as negative feedback controlling the normal excitability of several types of neurons (e.g., Borde et al. 1995, 2000; Empson and Jefferys 2001; Storm 1990). However, the participation of this current in the generation of abnormal hyperexcitable states, such as epileptiform activity, has not been sufficiently studied. Because the sI_AHP is crucial in maintaining normal neuronal excitability, we hypothesize that a reduction of this current could be involved in the appearence of epileptiform activity.

To test this hypothesis, we used the 4AP model of in-vitro epileptiform activity (Perreault and Avoli 1991; Rutecki et al. 1987) and electrophysiological and photometric Ca²⁺ techniques on rat hippocampal slices to investigate the regulation of the sI_AHP of CA1 pyramidal neurons during epileptiform activity. Our results show that superfusion with 4AP or 4AP+Mg-free ACSF induced a dramatic increase in spontaneous synaptic activity that was paralleled by a sustained reduction of the sI_AHP and, finally, by the generation of characteristic epileptiform activity with repetitive spontaneous depolarizations and action potential bursts.

We present evidence that sI_AHP inhibition is mediated via the postsynaptic activation of group I/II mGluRs by augmented glutamate release induced by increased synaptic activity. We show that the sI_AHP channels are inhibited without changes in intracellular Ca²⁺ signaling. Because the sI_AHP is a key element in the regulation of neuronal excitability, we propose that its inhibition contributes to the increased neuronal excitability that results in epileptiform activity. We also show that inhibition of the sI_AHP per se is not sufficient to induce epileptiform activity because when it is blocked by agents that do not increase synaptic activity it does not lead to epileptic-like discharges. Therefore cooperative action between enhanced synaptic activity and sI_AHP inhibition is required. We conclude that in the 4AP in-vitro model of epileptogenesis, both the enhancement of synaptic transmission and the resulting synaptically induced sI_AHP inhibition are complementary elements in the appearance of abnormal activity in the CA1 hippocampal region.

Our data also confirm previous observations suggesting that the epileptic ictal state observed in vivo is accompanied by reduced AHP (Matsumoto and Ajmone-Marsan 1964).

The present results indicate that mGluRs modulate the sI_AHP without affecting the intracellular Ca²⁺ signal. Calcium measurements correspond to global changes occurring in the soma and apical dendrites. Further studies outside the scope of the present work are required to determine whether local Ca²⁺ changes close to the sI_AHP channels are different from those recorded with our methodology. Nevertheless, our results closely agree with previous reports suggesting that reduction of the sI_AHP evoked by quisqualate or muscarine is not mediated by sustained Ca²⁺ variations or blockade of voltage-dependent Ca²⁺ channels (Charpak et al. 1990; Knöpfel et al. 1990).

Epileptiform discharges are initiated by excitatory postsynaptic potentials (Prince 1978). In close agreement, our results, which show that 4AP or 4AP+Mg-free ACSF incremented spontaneous EPSC and inhibitory postsynaptic current activity, underscore the importance of enhanced glutamatergic excitatory synaptic transmission. We show that blocking GABA_A inhibition does not significantly modify the emergence of epileptiform activity in our model. However, the participation of synaptic inhibition in the genesis of epileptic activity should not be diminished, especially when considering other models of experimentally induced epileptiform activity (e.g., Wong and Miles 1994) (see INTRODUCTION).

The participation of metabotropic glutamate receptors in epileptiform activity has been examined in several experimental animal models (Aronica et al. 1997; Arvanov et al. 1995; McBain 1994; McDonald et al. 1993; Merlin and Wong 1997; Merlin et al. 1995; for review, see Wong et al. 1999) and in human patients (Blümcke et al. 2000), but the cellular mechanisms involved remain unclear. The present demonstration, that the sI_AHP is inhibited by mGluR activation induced by enhanced spontaneous synaptic transmission, provides a cellular mechanism for the involvement of mGluR in epileptic activity.

MCPG has been described as having no effect on membrane potential, input resistance, or spike frequency adaptation per se, but inhibiting the postsynaptic effects induced by t-ACPD in CA1 hippocampal pyramidal neurons (Davies et al. 1995). We report that neither the frequency or the amplitude of the spontaneous EPSCs were significantly affected by MCPG and that the synaptic enhancement evoked by 4AP+Mg-free ACSF was unaffected by MCPG. Similarly, in CA1 stratum oriens inhibitory interneurons, MCPG abolished the abnormal periodic activity induced by elevated potassium without modifying sEPSC activity (McBain 1994). Therefore the effects of MCPG are not mediated presynaptically, indicating a postsynaptic regulation of the sI_AHP.

MCPG antagonizes group I (mGluR1 and mGluR5) and group II (mGluR2 and mGluR3) metabotropic glutamate receptors (Conn and Pin 1997). Further pharmacological experiments are required to define the I/II mGluR subtype involved.

In conclusion, we provide original evidence demonstrating that abnormal enhancement of spontaneous excitatory glutamatergic synaptic activity inhibits the sI_AHP via activation of postsynaptic group I/II mGluRs. This inhibition reduces the negative feedback provided by the sI_AHP, which leads to increased excitability that contributes to epileptiform activity. Therefore we propose that epileptiform activity is generated by a synaptically mediated cellular mechanism, caused by the enhancement of glutamatergic excitatory synaptic activity, that modifies postsynaptic excitability via changes in membrane intrinsic properties.