HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 96/6/3028    most recent 00434.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by de Sevilla, D. F. Articles by Buño, W. Search for Related Content PubMed PubMed Citation Articles by de Sevilla, D. F. Articles by Buño, W.

Calcium-Activated Afterhyperpolarizations Regulate Synchronization and Timing of Epileptiform Bursts in Hippocampal CA3 Pyramidal Neurons

Instituto Cajal, Consejo Superior de Investigaciones Científicas, Madrid, Spain

Submitted 25 April 2006; accepted in final form 30 August 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Calcium-activated potassium conductances regulate neuronal excitability, but their role in epileptogenesis remains elusive. We investigated in rat CA3 pyramidal neurons the contribution of the Ca²⁺-activated K⁺-mediated afterhyperpolarizations (AHPs) in the genesis and regulation of epileptiform activity induced in vitro by 4-aminopyridine (4-AP) in Mg²⁺-free Ringer. Recurring spike bursts terminated by prolonged AHPs were generated. Burst synchronization between CA3 pyramidal neurons in paired recordings typified this interictal-like activity. A downregulation of the medium afterhyperpolarization (mAHP) paralleled the emergence of the interictal-like activity. When the mAHP was reduced or enhanced by apamin and EBIO bursts induced by 4-AP were increased or blocked, respectively. Inhibition of the slow afterhyperpolarization (sAHP) with carbachol, t-ACPD, or isoproterenol increased bursting frequency and disrupted burst regularity and synchronization between pyramidal neuron pairs. In contrast, enhancing the sAHP by intracellular dialysis with KMeSO₄ reduced burst frequency. Block of GABA_A–B inhibitions did not modify the abnormal activity. We describe novel cellular mechanisms where 1) the inhibition of the mAHP plays an essential role in the genesis and regulation of the bursting activity by reducing negative feedback, 2) the sAHP sets the interburst interval by decreasing excitability, and 3) bursting was synchronized by excitatory synaptic interactions that increased in advance and during bursts and decreased throughout the subsequent sAHP. These cellular mechanisms are active in the CA3 region, where epileptiform activity is initiated, and cooperatively regulate the timing of the synchronized rhythmic interictal-like network activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Changes in neuronal excitability and synaptic efficacy contribute to induce the coordinated network activity needed to generate abnormal epileptiform bursting. In hippocampal pyramidal neurons a key role in the control of excitability is carried out by Ca²⁺-activated K⁺ currents with medium (mI_AHP) and slow (sI_AHP) deactivation kinetics that mediate the medium AHP (mAHP) and the slow AHP (sAHP), respectively (Borde et al. 1995, 2000; Carrer et al. 2003; Sah and Bekkers 1996; reviewed in Sah and Faber 2002; Stocker 2004; Storm 1987; Vogalis et al. 2003). It has been recognized that an abnormal regulation of the sI_AHP/sAHP may contribute to epileptogenesis (Alger and Nicoll 1980; Alger and Williamson 1988; Martín et al. 2001; Matsumoto and Ajmone-Marsan 1964; Traub et al. 1993; reviewed in de Curtis and Avanzini 2001; McCormick and Contreras 2001). However, important aspects of the contribution of the sAHP to abnormal hyperexcitable states remain under debate. In addition, little attention has been paid to the contribution of the mI_AHP/mAHP to epileptogenesis (Alger and Williamson 1988; Empson and Jefferys 2001; Garduño et al. 2005; McCown and Breese 1990; Verma-Ahuja et al. 1998).

We centered our analysis on the contribution of both I_AHPs/AHPs to epileptogenesis in CA3 pyramidal neurons, the region where hippocampal epileptiform activity is initiated (Colom and Saggau 1994; Luhmann et al. 2000; MacVicar and Dudek 1982; Miles and Wong 1983; Schwartzkroin and Prince 1978). We show that 1) a downregulation of the mI_AHP/mAHP paralleled the emergence of epileptiform bursting; 2) when the mI_AHP/mAHP was reduced or enhanced by pharmacological manipulations, bursts were increased or blocked, respectively; 3) manipulations that decreased the sI_AHP/sAHP increased bursting frequency and decreased network synchronization; 4) in contrast, increasing the sI_AHP/sAHP reduced bursting frequency; and 5) bursting in pyramidal neuron pairs was synchronized by excitatory synaptic interaction that increased shortly in advance and during bursts and decreased throughout the subsequent sAHP. The rhythmic bursting network activity that characterizes CA3 epileptogenesis is regulated by intrinsic cellular mechanisms where the mAHP and the sAHP play different roles, but nevertheless act cooperatively to regulate the synchronized bursting that characterizes the interictal-like network activity in the CA3 region. These cellular mechanisms may also be an integral part in the normal function of hippocampal region by regulating networks dynamics, such as the theta rhythm, where bursts of synchronous population activity occur (e.g., Buño et al. 1978) and are reset by interictal spikes in vivo (Lerma et al. 1984).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Slice preparation

Procedures of animal care, surgery, and slice preparation were in accordance with the guidelines laid down by the European Communities Council. Juvenile Wistar rats (12–15 days) were decapitated and their brains were quickly removed and placed in ice-cold control artificial cerebrospinal fluid (ACSF). The composition of the ACSF was (in mM): 124 NaCl, 2.69 KCl, 1.25 KH₂PO₄, 2 MgSO₄, 26 NaHCO₃, 2 CaCl₂, and 10 glucose. The ACSF was continuously gassed with a 95% O₂-5% CO₂ mixture to attain a pH of 7.3–7.4. Transverse hippocampal slices (400 µm thick) were prepared using a Vibratome (Pelco 101, Series 1000, St. Louis, MO), incubated >1 h at room temperature (20–22°C), and were transferred to a recording chamber (about 1 ml) placed on an inverted (Nikon TMS, Tokyo, Japan) or an upright microscope (Olympus BX51WI, Tokyo, Japan) equipped with infrared differential interference contrast video microscopy and a x40 water-immersion objective. Slices were superfused with gassed ACSF at a rate that completely exchanged the solution in the chamber within about 3 min and maintained at room temperature and in some cases at 32–34°C.

Electrophysiology

Whole cell recordings from pyramidal cells placed in the ventral branch of the CA3 region (Fig. 1A) were both in the current- and voltage-clamp modes with (4–8 M) patch-pipettes connected to an Axoclamp-2A amplifier (Axon Instruments, Foster City, CA). Pipettes were filled with a solution that contained (in mM): 135 K-gluconate, 1 EGTA, 10 KCl, 10 HEPES, 2 ATP, 0.4 GTP, and 1 MgCl₂, buffered to pH 7.2–7.3 with KOH.

View larger version (27K): [in this window] [in a new window]   FIG. 1. A slow afterhyperpolarization (sAHP) and outward current follow epileptiform bursts. A: schematic diagram showing the experimental recording and stimulation setup; pyramidal neurons in gray indicate the recording site. B: representative current-clamp recordings showing examples of the 2 types of responses evoked by de- and hyperpolarizing pulses in 2 CA3 pyramidal cells. C, left: current-clamp recording in control solution. D, left: autocorrelation functions calculated with data (2 min long) from same cell. Cell was "silent" and the autocorrelation function tended to be flat (except for the expected narrow peak at brief delays) in control artificial cerebrospinal fluid (ACSF). E, left and F, left: same as C and D, but >20 min after the onset of superfusion with 4-aminopyridine (4-AP) + Mg2+-free ACSF. Repetitive spike bursts (truncated, as in most other cases) followed by sAHP (interrupted arrows) were induced during epileptiform activity [continuous arrows indicate the absence of the medium afterhyperpolarization (mAHP)]. Autocorrelation function shows periodic peaks separated by slow waves that reach negative estimate values. C–F, right: same as C–F, left, but in voltage-clamp conditions. Note the increased synaptic activity indicated between open arrows and its absence during the slow Ca2+-activated K+ current (sIAHP). Bursts were truncated, as in most other figures.

Stimulation

Synaptic responses were evoked by mossy fiber (MF) stimulation through a pair of nichrome wires ( 60 µm) separated about 100 µm, insulated except at the tips, and placed in the stratum lucidum about 500 µm away from the recorded neuron (Fig. 1A). Electrodes were connected to a stimulator unit (Cibertec, Madrid, Spain) driven by the Clampex program (Axon Instruments). Stimulation was with brief barrages of three current pulses (barrage 110 ms; pulse 0.25 ms; repetition rate 0.1 s^–1).

Induction of epileptiform activity

Epileptiform activity was induced with 50–100 µM 4-AP added to a modified Mg²⁺-free ACSF that contained (in mM): 124 NaCl, 2.69 KCl, 1.25 KH₂PO₄, 26 NaHCO₃, 2 CaCl₂, and 10 glucose. 4-AP increases excitability and presynaptic glutamate release by block of the transient A-type K⁺-mediated current (I_A). Responses mediated by released glutamate by N-methyl-D-aspartate (NMDA)–receptor activation are enhanced in Mg²⁺-free solutions by relieving the voltage-dependent block by extracellular Mg²⁺. We previously showed that 4-AP + Mg²⁺-free and 4-AP in control ACSF had identical epileptogenic effects in the CA1 region (Martín et al. 2001). We used the Mg²⁺-free ACSF because the induction of epileptiform activity was faster and stable for a longer period of time than with 4-AP per se (as tested in six cells not included in this study).

Pharmacology

All the following drugs were added to the solutions and superfused in some experiments: Picrotoxin (PTX; 40 µM), to block -aminobutyric acid (GABA_A)–mediated synaptic inhibition, and saclofen (100 µM), to block GABA_B inhibition. Bicuculline (50 µM), to block GABA_A inhibition; the drug also inhibits the mAHP/mI_AHP (Debarbieux et al. 1998; Stocker et al. 1999). Apamin (100 nM), which specifically blocks small conductance (SK) Ca²⁺-activated K⁺-mediated channels and the mI_AHP/mAHP (see DISCUSSION). EBIO (1-ethyl-2-benzimidazolinone), which enhances channel activity and the Ca²⁺-dependent AHPs in neurons (Pedarzani et al. 2001; reviewed in Stocker 2004). EBIO was the first benzimidazolinone described as an activator of both SK channels and Cl^– secretion (Devor et al. 1996). EBIO was prepared as a stock solution in DMSO, stored at –18°C, diluted before use, and added at concentrations between 200 µM and 1 mM. The DMSO at the concentrations used had no effect on membrane properties or synaptic potentials (n = 4; Garduño et al. 2005). t-ACPD [(±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid, 20 µM], a nonselective metabotropic glutamate receptor (mGluR) agonist; carbachol (CCh, 10 µM), a wide-spectrum nonhydrolysable cholinergic agonist; or isoproterenol (5–10 µM), a -adrenergic agonist—all three are unspecific blockers of the sI_AHP/sAHP. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 20 µM), which specifically blocks non-NMDA glutamate receptors. In addition, atropine (10 µM) that inhibits muscarinic receptors, MCPG [(S)--methyl-4-carboxyphenylglycine, 0.5–1.0 mM], or LY341495 (20 µM), group I and group II mGluR antagonists, were superfused throughout the experiment and starting 10 min before switching to the 4-AP solution. Chemicals were purchased from Sigma (St. Louis, MO), Tocris Cookson (Bristol, UK), and Alomone Labs (Jerusalem, Israel).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Results are based on 137 pyramidal neurons from the ventral branch of the CA3 region (Fig. 1A) that were recorded at room temperature and exhibited a stable mean V_m of –69.3 ± 1.9 mV and an input resistance (R_in) of 195.5 ± 33.3 M. In addition, experiments (n = 18) were also performed at 32–34°C and the mean V_m was –62.3 ± 2.1 mV and the R_in 175.5 ± 45.8 M, respectively. Neurons were silent in control conditions and either an initial burst followed by a silence (n = 48 or roughly 35%) or a sustained response with little frequency adaptation (n = 89 or roughly 65%) were evoked by suprathreshold depolarizing pulses. The V_m and R_in were not statistically different in both neuron types and responses evoked by hyperpolarizing pulses always displayed repolarizing sag that was essentially identical in bursting and slowly adapting neurons (Fig. 1A).

Evolution of epileptiform activity

The AHP terminates epileptiform bursts. In current-clamp conditions pyramidal neurons did not show spontaneous spike activity in control ACSF (i.e., were "silent"), did not reveal V_m oscillation, and spontaneous synaptic activity was scarce (Current-clamp, Fig. 1C). Superfusion with 4-AP (50–100 µM) induced epileptiform activity in all the CA3 pyramidal neurons analyzed (n = 104). The abnormal activity started after nearly 10–15 min of superfusion with the 4-AP solution and was initially characterized by repetitive single spikes and spike bursts. This mixed activity rapidly changed to continuous spike bursts (3.3 ± 0.1 spikes, 258.2 ± 30.9 ms duration; at 0.23 ± 0.03 s^–1; n = 35, selected at random from the sample) and stabilized in about 10–20 min (Luhmann et al. 2000; Perreault and Avoli 1992). Each burst rode on a large and prolonged depolarizing wave termed paroxysmal depolarization shift (PDS) that was always terminated by an AHP (Fig. 1E) (Garduño et al. 2005; Goldensohn and Purpura 1963; Martín et al. 2001; Matsumoto and Ajmone-Marsan 1964; reviewed in Avoli et al. 2002; de Curtis and Avanzini 2001; McCormick and Contreras 2001). Brief volleys of synaptic activity could precede or follow spike bursts but were absent or markedly reduced during the sAHP (Voltage-clamp, Fig. 1E). The postburst AHP could display an initial faster and a subsequent slower component (Current-clamp, Fig. 1E). The AHP component with faster decay kinetics (30–90 ms; 5.1 ± 1.8 mV; n = 35; in control conditions, measured at –50 mV) could be reduced in amplitude (by 72. 2 ± 9.3% from control values; 10/35 cells or roughly 29% measured at –50 mV) or disappear altogether (25/35 neurons or roughly 71%) during the evolution of the epileptiform activity (continuous arrows, Current-clamp, Fig. 1E). In contrast, the AHP with slower decay kinetics was always present, it decayed to baseline in 3.7 ± 0.2 s, had a peak amplitude of 10.1 ± 0.3 mV (n = 35), and did not change (97 ± 5.7% of control; P > 0.05; same cells, measured at –50 mV) with time during the epileptiform activity (interrupted arrows, Current-clamp, Fig. 1E). The epileptogenic effects of the 4-AP reverted after a prolonged washout (>20 min; n = 12).

We centered our analysis on this type of bursting that has been termed interictal-like activity (Avoli et al. 1993; reviewed in Avoli et al. 2002; de Curtis and Avanzini 2001; McCormick and Contreras 2001). In some cases (10/35 cells or roughly 29%), ictal-like activity was also generated, characterized by bursts at higher frequency, riding on a sustained depolarization (Schiller 2004; Traub et al. 1993; reviewed in Avoli et al. 2002; de Curtis and Avanzini 2001; McCormick and Contreras 2001), and not followed by AHPs (see following text).

It is noteworthy that both during the mixed initial bursting–single-spike activity and the subsequent interictal-like activity bursts were essentially identical and always synchronized in paired recordings, indicating that similar population activity was occurring in the network during both periods (see following text).

We also calculated autocorrelation functions that provide an estimation of the membrane potential oscillations during the control and abnormal interictal-like activity (n = 10). In control conditions autocorrelations were flat (Current-clamp, Fig. 1D), consistent with the absence of oscillations. During the interictal-like activity autocorrelations revealed periodic peaks separated by slow waves (Current-clamp, Fig. 1F), in harmony with the rhythmic repetitive PDS topped by bursts followed by AHPs.

There were no significant differences between the above-described activity recorded at room temperature and the one induced by 4-AP at 32–34°C (3.8 ± 0.6 spikes, 300.4 ± 42.8-ms duration; at 0.33 ± 0.06 s^–1; n = 10) and the faster (30–90 ms; 4.5 ± 1.5 mV; n = 10) and slower (2.8 ± 0.4 s; 5.6 ± 0.9 mV) AHPs. In addition, both the mAHP and sAHP showed similar behaviors, with the former decreasing or disappearing with the evolution of the abnormal activity and the latter remaining unchanged, respectively.

Under voltage-clamp mode cells were silent in control conditions (Voltage-clamp, Fig. 1D) and the abnormal activity evoked in all cells (n = 33) by the 4-AP challenge (100 µM) was initially (about 10–15 min) typified by single or bursts of "unclamped" action currents riding on an inward current wave followed by a long-lasting outward "tail" current. The inward and outward currents correspond to the PDS and the sAHP recorded under current-clamp mode, respectively. The activity then stabilized at a frequency of 0.18 ± 0.02 s^–1 after about 20 min of superfusion with the 4-AP solution. Burst duration was 170.7 ± 23.9 ms and the number of action currents per burst was 2.7 ± 0.2, respectively (n = 33). Differences in the characteristics of the abnormal activity under current- and voltage-clamp modes may be explained by the distinct recording methods.

The large prolonged outward current that followed bursts could show (12/33 cells or roughly 36%) an early, briefer higher-amplitude (50–150 ms, 39.8 ± 9.8 pA, measured at –50 mV in control conditions) component that usually disappeared with the evolution of the epileptiform activity (9/12 or 75%) or was substantially reduced in amplitude (by 65 ± 9.3% from control values; 3/12 cells or 25%, measured at –50 mV) (filled arrows in Voltage-clamp, Fig. 1E). A slower current (25.7 ± 1.3 pA, decay 5.6 ± 2.6 s; n = 33, measured at –50 mV) was always present and did not change (102 ± 9.1%; P > 0.05; same ells) during epileptiform activity (Voltage-clamp, Fig. 1E). Accordingly, the corresponding autocorrelation functions were flat in control conditions (Voltage-clamp, Fig. 1D) and showed periodic peaks at the bursting frequency during epileptiform activity (Voltage-clamp, Fig. 1F). The spontaneous synaptic activity was clearly reduced during the slow outward currents (open arrows, Voltage-clamp, Fig. 1E).

Synaptic inhibition does not contribute to the AHPs

Voltage- and Ca²⁺-activated K⁺ conductances or synaptic inhibition could contribute to the afterhyperpolarization. Block of GABA_A inhibition with PTX (40 µM) did not change the frequency of epileptiform burst (0.16 ± 0.01 s^–1; P > 0.05; n = 8), burst duration (179.4 ± 22.1 ms; P > 0.05; same cells), number of action currents per burst (3.0 ± 0.1 P > 0.05; same cells), nor the amplitude and duration (26.5 ± 0.3 pA; 5.2 ± 0.9 s; P > 0.05, same neurons) of the outward current that follows epileptiform bursts (Fig. 2, A–C). Moreover, block of GABA_B inhibition with saclofen (100 µM) did not modify the epileptiform activity, the peak amplitude (89.6 ± 10.7% of control; P > 0.05; n = 5) of the outward current, or the area under the outward current (91.3 ± 13% of control; P > 0.05; n = 5; Fig. 2D). The values shown correspond to measurements performed when the interictal-like activity had stabilized >20 min after the first spike bursts. These results challenge the notion of an important contribution of postburst GABAergic inhibition to the postburst AHPs in our experimental conditions. Indeed, the duration of the postburst GABA-mediated inhibition, when present (Fig. 2E and F), is much shorter than the postburst AHPs (reviewed in de Curtis and Avanzini 2001), suggesting that additional mechanisms, such as the sI_AHP/sAHP, must be active in our experimental conditions even if GABAergic inhibition is present.

View larger version (26K): [in this window] [in a new window]   FIG. 2. -Aminobutyric acid types A and B (GABAA and GABAB, respectively)–mediated currents do not contribute to the slow outward currents. A: epileptiform action current burst and subsequent slow outward current were induced after (20 min) superfusion with 4-AP + Mg2+-free ACSF under voltage clamp. B: adding picrotoxin (PTX, 40 µM) did not modify the slow outward current. Note the absence of medium Ca2+-activated K+ current (mIAHP, arrows). C: superimposed records A and B. D: summary data showing the mean peak amplitude of the slow outward current in control conditions and 20 min after the onset of superfusion with 4-AP + Mg2+-free ACSF and when PTX (40 µM; n = 8), bicuculline (50 µM; n = 5), and saclofen (100 µM; n = 6) were added. E: repetitive outward currents corresponding to inhibitory postsynaptic potentials (IPSPs) evoked at the onset (about 5 min) of 4-AP superfusion. F: brief inward repetitive action current burst followed by the sIAHPs, characteristic of the interictal-like activity recorded later (>10 min) in the same cell. All recordings were at a –50 mV holding potential (Vh).

We first tested the effects of an intracellular pipette solution containing Cs-gluconate, which blocks all K⁺-mediated currents in the recorded cell (n = 11) without affecting other neurons in the bursting network. Intracellular Cs⁺ blocked the outward currents and the hyperpolarizations that followed epileptiform bursts in all cells (Fig. 3A), consistent with the AHPs being K⁺-mediated conductances without contribution of Cl^–-mediated GABA_A inhibition. An inward current (IC) (19.8 ± 7.9 pA, measured 50 ms after burst termination; IC, Fig. 3A) or an afterdepolarization (ADP) that followed bursts (15.9 ± 8.3 mV, measured 50 ms after burst termination) and that had decay kinetics similar to that of the mI_AHP/mAHP was usually unmasked (7/11 or roughly 64%) with intracellular Cs⁺ (see following text).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Two components of the AHP that follow epileptiform bursts are mediated by K+ conductances. A: representative voltage-clamp trace obtained with a Cs-gluconate–filled electrode 15 min after the onset of 4-AP superfusion. Note epileptiform burst of inward unclamped action currents (truncated) followed by prolonged inward current (IC) and total absence of outward currents. B: representative voltage-clamp trace showing the mIAHP and sIAHP evoked by depolarizing command pulses (shown above) in control ACSF (top) and after block of the mIAHP with apamin (bottom). C: representative voltage-clamp traces of outward "tail currents" evoked by brief depolarizing command pulses (shown above). C, left: in control ACSF the currents are the early mIAHP and late sIAHP components, respectively. C, middle: mIAHP is isolated after blockade of the sIAHP with (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD, 20 µM). C, right: with t-ACPD, during epileptiform activity induced by 4-AP, the isolated mIAHP is also inhibited. D: summary data showing amplitude modifications of the mIAHP and sIAHP (% of control values) when apamin (100 nM; n = 9) and carbachol (CCh, 10 µM; n = 6) were added to the control solution and 20 min after the onset of the epileptiform activity when the 4-AP + Mg2+-free (n = 10) and the 4-AP + Mg2+-free + CCh solutions (n = 6) were superfused. All recordings were at a –50 mV Vh.

The molecular identity of the Ca²⁺-activated K⁺ channels mediating the mI_AHP is known (Bond et al. 2004; Sailer et al. 2002; Stocker 2004; Stocker et al. 1999; Villalobos et al. 2004; however, see DISCUSSION). In addition, the Kv7/KCNQ M-current and the hyperpolarization-activated I_h were previosuly shown to contribute to the mI_AHP/mAHP (Gu et al. 2005; Storm 1987; Young et al. 2004). In contrast, the channels mediating the sI_AHP are different and their nature has not been clarified (Bond et al. 2004; Sah and Faber 2002; Villalobos et al. 2004). There is no known specific blocking agent for the sI_AHP that is insensitive to tetraethylammonium (TEA) and micromolar concentrations of 4-AP (Alger and Williamson 1988; Martín et al. 2001; reviewed in Sah and Faber 2002; Stocker 2004). The clotrimazole analogue UCL2027-2 (PZ323) has been shown to induce a relatively selective inhibition of the sAHP in cultured hippocampal neurons (Shah et al. 2001), but bath application of PZ323 (5–10 µM) had no significant effects on the sAHP in CA3 pyramidal neurons in our experimental conditions (data not shown, n = 2), even when GABAergic inhibition was blocked with PTX (40 µm) and saclofen (100 µM; n = 3). However, the sI_AHP is strongly inhibited in a nonspecific manner by muscarinic, adrenergic, and mGluR agonists (e.g., Borde et al. 2000; Madison and Nicoll 1986; Martín et al. 2001; Melyan et al. 2002; Pedarzani and Storm 1993; reviewed in Sah and Faber 2002; Stocker 2004). We investigated the action of t-ACPD (20 µM; n = 6) added to the 4-AP solution (t-ACPD + 4-AP, Fig. 3C) and in a few cases (n = 5) to the control ACSF (+t-ACPD, Fig. 3C). The t-ACPD challenge (Fig. 3C), CCh (10 µM; n = 6; Fig. 3D), and isoproterenol (10 µM; n = 5; see following text) inhibited the slow outward component, consistent with this current being the sI_AHP. Therefore the I_AHPs/AHPs that follow epileptiform burst are most likely the faster initial mI_AHP that mediates the mAHP and the late slower sI_AHP that underlies the sAHP.

A reduction of the mI_AHP/mAHP parallels the induction of epileptiform bursts

When the mI_AHP was isolated after blocking the sI_AHP with t-ACPD the mI_AHP was not modified, but after the induction of epileptiform activity there was a gradual and marked reduction of the mI_AHP (to 40.8 ± 4.3% of control values; P < 0.001; n = 6) (t-ACPD + 4-AP, Fig. 3C).

To further analyze the possible modifications of the I_AHPs/AHPs that follow epileptiform bursts we compared them with the I_AHPs/AHPs induced by identical depolarizing current pulses both in control conditions and during interictal-like activity (n = 11). With this methodology the changes of the I_AHPs/AHPs during the epileptiform activity could be compared with the I_AHPs/AHPs in control conditions in the same cells. In addition, the contributions of variations in the burst characteristics to the I_AHPs/AHPs during the interictal-like activity were minimized. Moreover, manipulations that modified the I_AHPs/AHPs that followed interictal-like bursts induced parallel changes of the pulse-evoked I_AHPs/AHPs, suggesting that they were mediated buy the same conductances.

In current-clamp conditions the pulse-evoked mAHP was reduced in amplitude (^bx40.1 ± 6.2% of control values; P < 0.001; n = 6) or even disappeared altogether (n = 5) when measured 10–20 min after the establishment of the abnormal activity (Fig. 4, A–D). The mAHP did not recover after a prolonged nearly 45-min washout. In contrast the pulse-evoked sAHP did not change during the abnormal bursting activity (96.9 ± 4.3% of control; P > 0.05; n = 11) (Fig. 4, A–D). Under voltage-clamp conditions the control pulse-evoked mI_AHP was reduced in amplitude (by 45.9 ± 5.7% from control values; P < 0.001; 8/11 cells or roughly 73%) and could even disappear (3/11 cells or roughly 27%) during the epileptiform activity (Fig. 4, E–G), whereas the sI_AHP did not change (98.2 ± 5.9% of control; P > 0.05; n = 9).

View larger version (31K): [in this window] [in a new window]   FIG. 4. A reduction of the mIAHP/mAHP parallels the induction of epileptiform activity, whereas the sIAHP/sAHP is not modified. A: superimposed current-clamp records of pulse-evoked AHPs in: control ACSF (black continuous record), showing the mAHP (arrow) and the sAHP; during epileptiform activity induced by 4-AP (dashed record), with bursts before and after the AHPs (open arrows); and during a washout 20 min after the withdrawal of the 4-AP (gray record). B: superimposed representative current-clamp traces (n = 3) obtained in control ACSF showing the spike burst and the sAHP evoked by depolarizing current pulses (shown below). C: same as B, but during epileptiform activity after 30 min of superfusion with 4-AP + Mg2+-free ACSF. sAHP that follows epileptiform bursts is similar to the one evoked by depolarizing pulses. Note the lack of abnormal bursting during the sAHPs (double-headed arrows). B and C: same neuron. D: superimposed traces B and C showing the reduction of the mAHP (arrow) but not of the sAHP during epileptiform activity. E and F: same as B and C, but under voltage-clamp, showing the epileptiform bursts of inward unclamped action currents followed by small IAHPs and the larger pulse-evoked IAHPs. Note the absence of bursts (double-headed arrows) during the pulse-evoked IAHPs with epileptiform activity. G: same as D, but showing superimposed IAHPs; note reduction of the mIAHP (arrow) and unchanged sIAHP during epileptiform activity.

View larger version (59K): [in this window] [in a new window]   FIG. 5. Manipulations that inhibit and enhance the mIAHP strengthen and weaken bursting, respectively. A: current-clamp record showing epileptiform bursting under 4-AP. Note the increased paroxysmal depolarization shift (PDS) amplitude and burst duration (arrows) when apamin (100 nM) was added, and the recovery of the abnormal bursting 20 min after the removal of apamin; without important changes in the interburst intervals. B: expanded versions of the recordings enclosed by the dotted rectangles in A. Note the prolonged PDSs and bursts followed by afterdepolarization (ADP, arrows). C: current-clamp record showing epileptiform bursting under 4-AP. Note the increased mAHP (arrows) and the gradual reduction of burst and prolonged silences (i.e., block of epileptiform activity) when 1-ethyl-2-benzimidazolinone (EBIO, 400 µM) was added. Abnormal bursting recovered 15 min after the removal of EBIO.

In some cases apamin was effective in enhancing bursts even in cells where the mAHP was not evident (4/14 or roughly 29%), probably indicating that the mAHP was nevertheless present but masked by the ADP. Moreover, in those cases the ADP could be revealed under apamin (Fig. 5B). The ADP is also exposed in CA3 pyramidal cells when the sAHP is blocked under muscarinic receptor activation (e.g., McQuiston and Madison 1999). Therefore the increased ADP could result from an increased acetylcholine (ACh) release arising from the activation of cholinergic afferents during the abnormal activity. However, superfusion of atropine (10 µM) starting 10 min before applying the 4-AP challenge did not modify bursting and did not change the ADP nor the subsequent sAHP (n = 4; data not shown), suggesting that an increased release of ACh was not functional in our conditions and that other mechanisms were active (Schiller 2004). It is noteworthy that apamin added to the control ACSF at similar concentration did not generate abnormal activity (n = 4; data not shown), suggesting that other processes besides the block of the mI_AHP/mAHP were needed to induce the abnormal activity. The added process could be the increased ADP induced by the epileptiform activity, a possibility that waits to be investigated.

Agonists of mGluRs may reduce both the sAHP and mAHP in CA3 pyramidal neurons (Young et al. 2004), an action that could be caused by activation of mGluR induced by the increased glutamate release that parallels the epileptiform activity as occurs in CA1 pyramidal cells (Martín et al. 2001). In addition, activation of mGluRs may also induce a reduction of the background conductance and the activation of a voltage-gated inward current that contributes to the ADP (Chuang et al. 2002; Young et al. 2004). However, neither MCPG (0.5–1.0 mM) nor LY341495 (20 µM), which block type I and type II mGluRs, prevented the mI_AHP/mAHP reduction that paralleled the epileptiform activity (n = 4; data not shown), suggesting that other mechanisms were active to depress the conductance. This view is consistent with the action of the mGluR agonist t-ACPD that inhibited the sI_AHP but not the mI_AHP (Fig. 3C).

The sI_AHP/sAHP regulates the interburst interval and rhythmicity

Another important issue that remains to be clarified is what factors determine the timing of the periodic network interictal-like activity. Therefore we tested the possible contribution of the sI_AHP/sAHP to the timing of the bursting activity. The constancy of epileptiform bursts characteristics in different cells suggests that a uniform influx of Ca²⁺ was induced in all cells in the bursting network. Therefore we hypothesized that: 1) bursts were supported by the increased excitability caused by the reduction of the mI_AHP/mAHP and 2) the synchronized bursting and the resulting massive influx of Ca²⁺ activated the sI_AHP/sAHP that had similar characteristics and tended to terminate at fixed intervals after a network burst. This view is in accordance with the absence of epileptiform bursts and the reduction of the synaptic activity during the sI_AHP/sAHP. It also agrees with the occurrence of the subsequent synchronized bursts in the network when sI_AHP/sAHP had terminated, as observed with paired recordings (see following text). This notion is consistent with the synchronized bursting being caused by a recovery of the V_m and excitability that brought cells in the network to fire in close synchrony to a level of population firing that depolarized neurons and triggered the burst (Menéndez de la Prida et al. 2006).

We further tested the above assumptions in two ways. First, by estimating the probability of occurrence of epileptiform bursts before and after pulse-evoked sI_AHP/sAHPs, we found that there was a dramatic reduction of bursting both in current-clamp (by 77.9 ± 0.8% from control values; P < 0.01; n = 10) and voltage-clamp conditions (by 60.2 ± 0.3% from control values; P < 0.01; n = 10) during the pulse-evoked sI_AHP/sAHP (Fig. 6, A–C). In addition, under current-clamp mode the pulse-evoked burst–sAHP sequence induced a reset of the abnormal bursting interictal-like activity that was characterized by repeated bursts that tended to occur at specified times after the pulse. The successive bursts were synchronized by the consecutive pulse-evoked burst–sAHP sequences (Fig. 6A). Second, we tested the effects of blocking the sI_AHP/sAHP during the epileptiform activity with t-ACPD (20 µM) that disrupted rhythmicity, reduced the silent interval that followed pulse-evoked bursts, and increased the frequency of epileptiform bursts by 175.5 ± 22.3% (P < 0.005, n = 6). Therefore under block of the sI_AHP with t-ACPD epileptiform bursts occurred at irregular intervals and with similar probability through the record (Fig. 6D), suggesting that synchronization was not a direct consequence of the bursts but resulted from the combined pulse-evoked burst–sAHP sequence.

View larger version (60K): [in this window] [in a new window]   FIG. 6. Epileptiform bursts are inhibited during the sIAHP/sAHP and preceded by increased synaptic activity. A: superimposed representative current-clamp traces showing pulse-evoked sAHPs (n = 6, as in the rest of the traces) after 20 min of superfusion with 4-AP. Note the synchronization of bursts after the pulse-evoked sAHP; i.e., the sAHP "resets" the ongoing interictal-like activity. B, top: superimposed current-clamp traces showing pulse-evoked sAHPs 20 min after superfusion with 4-AP. Note the absence of bursts during the sAHP (double-headed arrow). B, bottom: histogram showing epileptiform burst occurrences (%) before and after spike bursts and sAHPs obtained from the same cell as in B, top. C, top and bottom: same as B, top and bottom, but in voltage-clamp conditions in another cell. D, top and bottom: same as C, top and bottom, but 30 min after onset of superfusion with 4-AP and added t-ACPD (20 µM) in another neuron. Note the absence of sIAHP and of burst synchronization. Histograms in each experimental group (n = 10 cells) were constructed with responses evoked by 20 successive stimuli in each case. E: current-clamp recordings, showing examples of the increased synaptic activity (dotted squares) immediately preceding (about 1.5 s) epileptiform bursts (i.e., "follower cell"). F: same as E, but the increased synaptic activity (dotted squares) occurred after (about 1.0 s) bursts in another neuron (i.e., "leader cell"). G: same as E, showing complete bursting cycle and interburst interval (IBI). E and G: same cell. H: histogram showing postsynaptic potential occurrences % after (n = 6 cells) and before (n = 5 neurons) bursts. Data were averaged from synaptic potentials paralleling 10 successive bursts in each cell. IBI was normalized by scaling to the briefest one.

These results are consistent with the regulation of network interactions by the sAHP that probably acts by decreasing population activity by reducing excitability in the cells that compose the bursting network. Many neurons showed synaptic potentials during bursts (21/35 or 60.0%), suggesting that they fired in close synchrony with other cells in the network (see following text). The above determinations were made in the first 20 min after the onset of the interictal-like activity. However, synaptic activity preceding and following burst was scarce (4/35 or 11.4%) later during the evolution of epileptiform activity, suggesting that burst synchronization improved in the network with the evolution of the abnormal activity (see following text). The above results are consistent with the burst–sAHP sequence being a key factor in the regulation of the frequency and synchronization of the bursting rhythm, whereas the burst per se was not because desynchronization was observed in conditions where the burst persisted and the sAHP was absent. The abnormal activity was inhibited by 20 µM CNQX (n = 3; data not shown), indicating that excitatory synaptic interactions were of major importance in the genesis of the interictal-like activity.

We verified the above assumptions by recording pairs of CA3 pyramidal neurons (about 50–100 µm apart; n = 22 pairs). We could not detect synaptic interactions between pairs, but their absence is not surprising in view of the low probability of functional excitatory interconnections in acute slices (Miles and Wong 1986).

First, during epileptiform activity we blocked the sAHP with 10 µm CCh (n = 5 pairs) that desynchronized bursting between neurons and increased the bursting rate (by 148.3 ± 18.3%, 3/10 cells or 30%). The ACh challenge also usually increased synaptic activity (4/10 cells or 40%). Both effects were probably caused by the depolarization and the increased excitability induced by CCh in the cells composing the network. Indeed, CCh inhibits the sI_AHP/sAHP and also increases excitability by blocking several K⁺-mediated conductances (reviewed in Storm 1987). However, it was previously reported that at higher temperature (about 32°C) and under GABA_A blockade with bicuculine, instead of desynchronization, CCh per se induces synchronized population activity in the CA3 region in vitro (Psarropoulou and Dallaire 1998). The synchronized activity is blocked by muscarinic antagonists and is thought to be mediated by local excitatory circuits enhanced by muscarinic activity in the absence of inhibition. We tested the effects of CCh applied at 32–34°C on the behavior of pyramidal neuron pairs (separated by about 100 µM; n = 5 pairs) in experiments in which GABA_A inhibition was blocked with PTX (40 µM) in replacement of the bicuculline used by Psarropoulou and Dallaire (1998) because this drug also blocks the mAHP (Debarbieux et al. 1998; Stocker et al. 1999), thus favoring epileptogenesis. Synchronized bursts (5.1 ± 1.1 spikes, 340 ± 20.8 ms duration, at 0.38 ± 0.1 s^–1, n = 5) were induced by CCh (10 µM) (CCh, Fig. 7A), thus confirming the results of Psarropoulou and Dallaire (1998). In addition, synchronized bursts at a higher more irregular rate (8.3 ± 1.8 spikes, 450 ± 30.2 ms duration, at 0. 75 ± 0.1 s^–1, n = 5 pairs) continued when 4-AP (100 µM) was added to the CCh solution (CCh + 4-AP, Fig. 7A). In contrast, the synchronized interictal-like bursting activity induced by 100 µM 4-AP (4-AP, Fig. 7B) was totally desynchronized by adding CCh (10 µM), confirming our results obtained at room temperature (n = 4 pairs). The above differences between the effects of superfusion with CCh before and after the induction of the interictal-like activity by 4-AP at high temperatures might be caused by divergence in the sequences of the blockade of different potassium conductances by both agents, especially because the muscarinic metabotropic activity may be long lived, may induce potentiation (Fernández de Sevilla et al. 2005), and may disclose an ADP (McQuiston and Madison 1999). In any case the differences are highly interesting and should be analyzed in detail in future studies.

View larger version (60K): [in this window] [in a new window]   FIG. 7. Paired recordings: effects of blocking or enhancing the sAHP in one of the cells. A, left: representative current-clamp recordings in a pair of pyramidal neurons (1 and 2; about 50-µm separation) showing rhythmic bursting activity induced by CCh at 32–24°C (10 µM). A, right: after adding 4-AP (50 µM) the bursting frequency increased and bursts remained synchronized in both neurons. B, left: records showing synchronized bursting interictal-like activity induced by 4-AP (50 µM). B, right: when 50 µm CCh was added bursts were at a higher frequency in both cells and totally desynchronized. C1: representative traces obtained with an electrode filled with the KMeSO4 solution that enhances the sAHP. D2: simultaneous recording with the normal K-gluconate electrode solution. Note the absence of burst synchronization and the more prolonged and larger sAHP in D1 during the interictal-like activity. Cells synchronized at the onset of the ictal episode. E: digitized differential interference contrast (DIC) image of the pyramidal neuron pair corresponding to the recordings shown in D.

Second, we tested the contribution of the sAHP to the bursting rhythm by enhancing the sAHP in one cell with an intracellular KMeSO₄ solution (Zhang et al. 1994), whereas the other cell was recorded with normal K-gluconate pipette solution (n = 4 pairs). The cells initially (<20 min after the start of the abnormal bursts) did not burst in synchrony. The neuron dialyzed with KMeSO₄ showed larger sAHPs of longer duration and tended to burst at a lower frequency than the other neuron (by 30.9 ± 5.2%, P < 0.005, n = 4 pairs) (Interictal, Fig. 7C). However, both cells fired in synchrony during the ictal-like episodes when the sAHPs were substantially reduced in both cells and a prolonged depolarization was evoked (Ictal, Fig. 7C). In addition, both cells also tended to burst in synchrony later (>20 min) with the evolution of the epileptiform activity (data not shown).

Third, we recorded pairs with one cell loaded with Cs-gluconate to test the effects blocking all K⁺ conductances in the recorded cell (n = 5 pairs) without affecting other neurons in the bursting network or the cell recorded with the normal intracellular solution. With intracellular Cs⁺ the AHPs were completely blocked and the Cs⁺-loaded cells initially (<20 min after the onset of bursts) tended to burst irregularly at a higher rate (by 192.3 ± 7.2%, P < 0.001, n = 5 pairs) and were not synchronized. However, later (>20 min) both cells tended to burst in synchrony, although bursts were much longer and were followed by ADPs in the neuron dialyzed with Cs⁺ (n = 6) (Fig. 8A). Pulse-evoked bursts were followed by AHPs in control conditions but ADPs that could generate plateaulike depolarization topped by prolonged spike bursts were evoked under Cs⁺ (ACSF, Fig. 8A).

View larger version (57K): [in this window] [in a new window]   FIG. 8. Paired recordings: abnormal bursting in cells dialyzed with Cs+ and 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) and with control intracellular solution. A, left: representative current-clamp records obtained in control conditions showing pulse evoked responses (the 200-ms pulse shown below) of a neuron with control intracellular solution (1) and another dialyzed with Cs+ (2). Note sAHP in (1) is absent in (2) and a plateau-like ADP is evoked in (2). A, center: abnormal bursting activity induced by 4-AP (during the first 20 min) showing the irregular high-frequency bursting of the neuron dialyzed with Cs+ (2) and the much lower rate bursting of the cell with control intracellular solution (1). A, right: records obtained 30 min after, showing partial synchronization with occasional out-of-phase bursts. Note that more and much longer bursts followed by ADPs are evoked under Cs+ (2). B, left and middle: same as A, left, but one neuron loaded with control solution (1) and the other with BAPTA (2). Effects were comparable to those of Cs+, but the bursting rate in the BAPTA-loaded cell was lower. B, right: superfusion with isoprotereonol (10 µM) completely desynchronized bursting that was at a higher rate in the BAPTA-loaded cell (2). Note also the increased spontaneous synaptic activity.

The above results offer additional support to the notion that the sI_AHP/sAHP plays a key role in determining the interburst cycle and regulating the frequency and synchronization of the bursting network. The results also explain why, when the sAHP has different kinetics in different cells, bursts may not be entirely synchronized. However, bursts became synchronized later even if the AHPs and other K⁺-mediated currents were inhibited in one of the recorded cells, indicating that other neurons in the network with active AHPs and K⁺-mediated conductances were driving the recorded neurons.

Synaptic excitation synchronizes CA3 pyramidal neuron ensembles

As described above, in many cells there was an increase in synaptic activity preceding or following the interictal burst in the recorded cell (Fig. 10A). The synaptic activity increased in amplitude with imposed hyperpolarization and decreased with depolarization, consistent with excitatory postsynaptic potentials (EPSPs, not shown). Therefore the bursting network was driven by mutual excitatory interconnections that depolarized and drove the recorded cell to the bursting threshold (Menédez de la Prida et al. 2006).

View larger version (39K): [in this window] [in a new window]   FIG. 9. Paired recordings: burst synchronization increases with the evolution of epileptiform activity. A, left: current-clamp records showing irregular and partially synchronized bursting early (10 min) during superfusion with 4-AP. B, left: time-expanded version of framed record in A, left. A, right: cross-correlation of data in A, obtained from a 2-min-long recording. Gray graph is a time-expanded version of the black record. C and D, left and right: same as A and B, but later (20 min) when the abnormal activity had stabilized. Note higher bursting rate, the increased regularity and synchronization of the rhythmic bursting, and the reduction of delay between bursts in both cells from about 20 to about 2 ms with the evolution of the abnormal activity.

View larger version (53K): [in this window] [in a new window]   FIG. 10. Paired recordings: firing synchronization during epileptiform activity; activation of a single neuron can drive the interictal-like activity. A, left: change from synchronous excitatory postsynaptic potentials (EPSPs, dotted arrows), to simultaneous bursts and bursts and EPSPs (open arrow) in 2 neurons (1 and 2). A, right: EPSPs could precede bursts and occur simultaneously with burst in the other neuron (dotted arrows) later during the establishment of the abnormal activity in two neurons (1 and 2). B: superimposed traces (n = 10) showing that the activation of one neuron (1) with a brief depolarizing pulse could "drive" a simultaneously recorded cell (2). Dotted arrows indicate the silent period during the sAHPs.

Additional support to the increased excitatory synaptic interactions was provided by paired recordings, which revealed that initially during the interictal-like activity (5–10 min) EPSPs could occur simultaneously in both cells (18/22 pairs or 86%), consistent with a common excitatory input from other neurons in the network (arrows, left records, Fig. 10A). Later with the stabilization of the abnormal activity (20–30 min) simultaneous EPSPs disappeared and coincident spikes and EPSPs could occur during bursts (20/22 or 90%) (arrows, right records, Fig. 10A). These changes are also consistent with an increase in excitability and excitatory synaptic interactions within the network.

The above results suggest that the activation of a single cell could eventually, as a consequence of the increase synaptic interactions, control the interictal-like network activity. Therefore we used paired recordings and direct stimulation of one of the recorded cells with a brief high-intensity current pulse that evoked the burst–sAHP sequence to test the possible evolution of network interactions. Activation of either of the neurons of the pair never reveled direct excitatory connections in control conditions in our sample (n = 22). However, when the interictal-like activity had stabilized (>20 min) a brief current pulse in one cell, which induced the usual spike burst followed by a silent period, was paralleled by a brief period of increased bursting activity followed by a silent interval in the other neuron (Fig. 10B). This synchronization of activity suggests that the pulse-evoked burst and subsequent sAHP in the recorded cell induced changes in the excitability of the other neuron (4/22 pairs or roughly 18%), implying that activation of a single neuron could control bursting activity in the network (e.g., Menéndez de la Prida et al. 2006; Miles and Wong 1983), probably by indirect excitatory interconnections that increased or synapses that were silent and became active with the evolution of the epileptiform activity.

We also analyzed the effects of stimulating an excitatory synaptic input on burst synchronization and timing between simultaneously recorded CA3 pyramidal neurons (n = 7 pairs) under block of GABA_A inhibition with 40 µM PTX. At the onset of the abnormal activity the burst–sAHP sequence evoked by MF stimulation inhibited the epileptiform bursts during a somewhat variable interval in both cells that terminated by bursts that were not well synchronized (15 min + Stimulation, Fig. 11A). Later, when the epileptiform activity had stabilized, the burst–sAHP sequences evoked by MF stimulation were of similar duration and inhibited epileptiform bursts during a similar interval (4.5 ± 0.5 s, n = 6) that terminated by well-synchronized bursts when the sAHP ended in both neurons (30 min + Stimulation, Fig. 11A). The synchronization, estimated by measuring the reduction of the temporal dispersion between bursts in successive responses, increased by 208 ± 18.3% (P < 0.001, n = 6). Therefore the sAHP evoked by synaptic stimulation silenced the cells during a relatively fixed interval that synchronized and timed the subsequent rhythmic bursting activity. In addition, the response evoked by the MF stimulation was modified during the evolution of the epileptiform activity because in control conditions EPSPs were smaller, evoked bursts with fewer spikes, and were followed by a mAHP (Control, Fig. 11B), whereas later when the interictal-like activity had stabilized EPSPs were larger and evoked longer bursts with more spikes (the number of spikes increased by 167.3 ± 17.3%, P < 0.005, n = 7) and were followed by an ADP in all seven cells (30 min, Fig. 11B). Therefore both increases in synaptic efficacy and postsynaptic excitability contributed to the augmented network synchronization that developed with the evolution of the abnormal activity.

View larger version (26K): [in this window] [in a new window]   FIG. 11. Paired recordings: effects of MF stimulation on burst synchronization increase during epileptiform activity. A, left: superimposed records (n = 8) of bursting activity in two neurons (1 and 2) early after superfusion with 4-AP during mossy fiber (MF) stimulation. Stimulation (MF stim) generated a burst followed by sAHPs of slightly different durations in both neurons that inhibited bursting (dotted arrows); the subsequent bursts occurred at the end of the sAHPs (open arrow). A, right: later during the establishment of the abnormal activity, the sAHPs (dotted arrows) had similar durations in both cells and bursts occurred simultaneously at the termination of the sAHPs (i.e., the post sAHP activity was synchronized in both neurons, open arrows). B: superimposed averages (n = 10) showing the response evoked by the MF stimulation in control conditions (black trace) and 30 min after the establishment of the interictal-like activity in neuron 1 (gray trace). Note that mAHP recorded in the control (black trace) was substituted by an ADP (arrows) during the epileptiform activity (gray trace).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
We present evidence demonstrating that in the CA3 region, where epileptiform activity is initiated and from there spreads to other hippocampal regions (Colom and Saggau 1994; Luhmann et al. 2000; MacVicar and Dudek 1982; Miles and Wong 1983; Schwartzkroin and Prince 1978), the interictal-like activity in pyramidal neurons is precisely regulated by two Ca²⁺-activated K⁺-mediated currents with different kinetics. A downregulation of the mI_AHP/mAHP and the resulting increased excitability are central mechanisms contributing to the generation and regulation of the network bursts that characterize CA3 interictal-like activity. The results also point to a key role of the decreased excitability induced by the activation of the postburst sI_AHP/sAHP in controlling both the frequency and synchronization of the interictal-like network activity.

We show that there is a close relationship between the decline of the mAHP amplitude, the increased excitability, and the induction of interictal-like activity. Pharmacological manipulations that reduce the mAHP, as bath-applied apamin, that specifically inhibits SK Ca²⁺-activated K⁺-mediated channels, caused a marked increase of the bursting activity (McCown and Breese 1990). In contrast, EBIO that enhances Ca²⁺-activated K⁺ currents, and in CA3 pyramidal cells specifically augments the mI_AHP/mAHP, reduced bursting or even blocked the epileptiform activity (Garduño et al. 2005). The above-discussed results are consistent with a key involvement of the mI_AHP/mAHP acting as a negative feedback in the regulation of excitability and in the genesis of CA3 epileptogenesis.

Participation of SK channels in the genesis of the mI_AHP/mAHP has recently been questioned (Gu et al. 2005) because, although a clear SK-mediated apamin-sensitive component was evoked by brief depolarization under voltage-clamp mode, these authors could not evoke the Ca²⁺-activated K⁺-mediated current with similar depolarizations under current-clamp mode. Several ionic conductances may contribute to the mI_AHP/mAHP besides the apamin–EBIO-sensitive Ca²⁺-activated K⁺-mediated SK component. The Kv7/KCNQ M-current at depolarized (> –60 mV) and the H-current at hyperpolarized V_ms (< –70 mV) may add to the mI_AHP/mAHP (Bond et al. 2004; Sah and Faber 2002; Sailer et al. 2002; Stocker 2004; Stocker et al. 1999; Villalobos et al. 2004). Nevertheless, an apamin–EBIO-sensitive component without an important contribution of the M-current was found in our experimental conditions because both t-ACPD and CCh, which are potent inhibitors of the M-current, did not induce important reductions of the mI_AHP/mAHP. Activation of the H-current was not expected in our experiments because V_m values of > –75 mV at which the H-current is activated were never reached. The divergence between our results and those mentioned above may either reside in differences in the channels expressed in CA1 and CA3 pyramidal cells (Chuang et al. 2002; Sailer et al. 2002; Schiller 2004; Schwartzkroin and Prince 1980; Young et al. 2004) or as a result of the masking of the muscarinic M-type component by the ADP (McQuiston and Madison 1999).

Present results could indicate the mAHP may be masked by the enhancement of an inward current that underlies the ADP that effectively contributes to increase excitability (Chuang et al. 2002; Sailer et al. 2002; Schiller 2004; Schwartzkroin and Prince 1980; Young et al. 2004). Consistent with this view—when the mI_AHP/mAHP was not apparent during epileptiform activity—apamin always enhanced bursts and could disclose an ADP, suggesting that the apamin-sensitive SK Ca²⁺-activated K⁺-mediated component of the mAHP was nevertheless present and masked the ADP (McCown and Breese 1990). The mechanisms that induce the downregulation of the mI_AHP/mAHP and the enhancement of the ADP during epileptiform activity could be of key importance but remain to be investigated.

Several studies established the crucial role of the sI_AHP/sAHP as negative feedback controlling excitability in normal conditions (reviewed in Sah and Faber 2002; Stocker 2004; Vogalis et al. 2003) and during epileptiform activity (reviewed in de Curtis and Avanzini 2001; McCormick and Contreras 2001). Present results suggest that after each synchronized epileptiform burst the resulting massive Ca²⁺ influx activates the sI_AHP/sAHP that sets the interburst interval of the interictal-like activity by reducing the excitability of the network, thus decreasing synaptic interactions and burst generation during a relatively fixed time interval after each burst. The intracellular Ca²⁺ concentration is then gradually reduced, inducing the deactivation of the sI_AHP/sAHP that leads to the return of the V_m, the membrane conductance, and the excitability to previous values, favoring the generation of a subsequent burst. The increased excitability would bring cells in the network to firing threshold, thus increasing synaptic interactions to a level of population firing that depolarizes neurons and triggers the burst. Paired recordings show that when the epileptiform activity has stabilized, the activation–deactivation of the sI_AHP/sAHP tend to occur in parallel with practically identical duration in the pyramidal cells that form part of the bursting network. Therefore the burst–sAHP sequence is unceasingly repeated at relatively fixed intervals, determined by the duration of the sAHP, thus setting the bursting cycle and timing the rhythmic interictal-like network activity.

The above conclusions were sustained by the effects of inhibiting the sI_AHP/sAHP with CCh, isoproterenol, or t-ACPD that increase the bursting frequency and disrupt burst regularity and synchronization between pairs of pyramidal neurons. Although those drugs have other actions besides a potent inhibition of the sI_AHP/sAHP they share essentially identical effects on the abnormal interictal-like activity, suggesting an action most likely exclusively caused by the inhibition of the sI_AHP/sAHP (Borde et al. 2000; Buño et al. 2004; Martín et al. 2001). In addition, enhancing the sI_AHP/sAHP in one of the recorded neurons by filling the electrode with a solution containing KMeSO₄ (Zhang et al. 1994) induced burst desynchronization because it increased the interburst interval, whereas the other cell (recorded with the normal K-gluconate intracellular solution) showed a briefer sAHP and a higher bursting rate (present results). Burst desynchronization was also observed when, in one of the neurons of the pair, K⁺-mediated currents were blocked with intracellular Cs⁺ or when the AHPs were inhibited by chelating intracellular Ca²⁺ with BAPTA. All the above effects are consistent with the sI_AHP/sAHP being central in determining the interburst interval, the frequency, and the synchronization of the bursting network.

Changes of the sAHP induced by extracellular drug applications caused stable modifications of the interictal-like activity because they act on all the cells of the network. In contrast, the changes occurred only in the dialyzed cell and exclusively early on during the evolution of epileptiform activity (<20 min) when the sAHP was modified by intracellular drug applications (i.e., KMeSO₄, Cs⁺, or BAPTA). However, when epileptiform activity stabilized the dialyzed neuron showed partial burst synchronization in the three different experimental conditions (i.e., intracellular KMeSO₄, Cs⁺, or BAPTA), suggesting that the dialyzed cell was "driven" by the convergent synaptic activity generated by bursting cells in the network. The switch from desynchronization to partial synchronization is consistent with an improvement in excitatory interconnections with the progression of the interictal-like activity, although the mechanisms that mediate these effects remain to be investigated. A factor that could contribute is the necessary recovery of synaptic function after a burst before another burst can be initiated (Staley et al. 1998).

In harmony with a key influence of the sI_AHP/sAHP in controlling the bursting frequency, we show that the ongoing rhythm can be effectively "reset" by introducing a pulse-evoked or a synaptically evoked burst followed by a sI_AHP/sAHP. Interestingly, a similar reset of the hippocampal theta rhythm was induced by synaptic inputs in vivo (Buño et al. 1978), suggesting that similar mechanisms may be functional in normal conditions.

The unlike behaviors of the sAHP and mAHP suggest that both conductances may be regulated by different mechanisms. It was recently shown that activation of the kinate (KA) receptor subunit GluR6 mediates a metabotropic inhibition of the mI_AHP/mAHP that increases excitability and firing frequency in CA3 pyramidal cells (Fisahn et al. 2005). Therefore a likely explanation for the decreased mI_AHP/mAHP is that it is mediated by the activation of KA receptors caused by the abnormally enhanced synaptic activity and glutamate release that characterize epileptiform activity—an interesting possibility that remains to be investigated. We previously showed that glutamate released during epileptogenesis inhibited the sI_AHP/sAHP in CA1 pyramidal neurons and that this effect was mediated by group I and group II mGluRs because it was prevented by superfusion with MCPG, an action that is mediated downstream of the increased intracellular Ca²⁺ signal (Martín et al. 2001). However, the sAHP was not decreased and the reduction of the mAHP was not prevented by MCPG or LY341495 (present results), suggesting that the mGluR-mediated inhibition was not functional. In addition, the consistency of the sAHP during epileptiform activity also suggests that the KA-mediated inhibition of the sAHP (Fisahn et al. 2005) was absent in our experiments.

With paired recordings activation of a single cell never revealed direct excitatory connections in control conditions but when interictal-like activity stabilized a similar brief current pulse in one cell it could induce a burst followed by a sAHP that was occasionally paralleled by a brief period of increased activity followed by a silent interval and bursting in the other neuron. Therefore activation of a single neuron might control network activity probably by indirect excitatory interconnections that eventually increase or become functional with the evolution of the epileptiform activity, suggesting dynamic modifications in excitability and synaptic efficacy during the abnormal activity.

Other differences between the activation of the mAHP and sAHP may also play a role in the dissimilar behaviors of both conductances during epileptiform activity. Indeed, the sAHP is inhibited by nifedipine but unaffected by omega-conotoxin GVIA. In contrast, the mAHP is insensitive to nifedipine and inhibited by omega-conotoxin GVIA, suggesting that different voltage-gated Ca²⁺ channels mediate the major calcium influx pathway for activation of both conductances (Borde et al. 2000; Empson and Jefferys 2001; Viana et al. 1993). These Ca²⁺ channels may be regulated in different ways by epileptiform activity, thus determining the different behaviors of both conductances (Empson and Jefferys 2001). In addition, sAHP channels require an elevation of Ca²⁺ in the cytoplasm, rather than at the membrane, consistent with a role for a cytoplasmic intermediate between Ca²⁺ and the sAHP channels (Abel et al. 2004), where Ca²⁺-induced Ca²⁺ release may play a role (Borde et al. 2000), suggesting that the sAHP and mAHP are regulated by different mechanisms during epileptiform activity and that those that inhibit the mAHP are active, whereas those that influence the sAHP are inoperative in CA3 pyramidal cells.

GABAergic inhibition does not contribute to the AHPs that follow bursts in our experimental conditions. Controversy exists regarding the contribution of GABA receptors to the hyperpolarization that follows epileptiform bursts because both a significant involvement (Perez-Velazquez and Carlen 1999; reviewed in Delgado-Escueta et al. 1986; Wong and Miles 1994) and an irrelevant contribution have been reported. The latter was attributed to a GABA_A-mediated excitation (Perrault and Avoli 1992) or a decreased inhibition, either attributable to a postsynaptic depolarizing shift in the reversal potential of the GABA-mediated currents (Lamsa and Taira 2003) or to an NMDA-dependent block of GABA inhibition (Stelzer et al. 1987). Presynaptic mechanisms may also contribute by a decrease in GABA release sharing properties with the depolarization-induced suppression of inhibition (Le Beau and Alger 1998). A presynaptic downregulation of GABA release from CA3 interneurons mediated by a metabotropic action on the GluR6 subunit of KA receptors was previously demonstrated (Min et al. 1999; Rodriguez-Moreno and Lerma 1998). The reduced GABA release would diminish both GABA_A and GABA_B inhibition, thus explaining our present results. In addition, the GABA_B-mediated presynaptic inhibition of glutamate release may be substantially reduced after seizures in the hippocampus (Poon et al. 2006), an action that would increase glutamate release and seizure susceptibility. Finally, prolonged epileptiform activity may reduce GABAergic inhibition in vitro by rapid internalization of GABA receptors (Goodkin et al. 2005).

The existence of different mechanisms for the decline of inhibitory feedback during epileptiform activity strongly suggests a major contribution of the reduction of inhibition to the abnormal activity. Therefore the above effects could increase network excitability and induce the abnormal activity by the cooperative action of downregulating both synaptic inhibition and the mI_AHP/mAHP. The reduction of inhibition in some types of epilepsies but not in others may explain why barbiturates and benzodiazepines that act on GABA receptors to inhibit some types of epilepsies are totally ineffective in controlling other forms (Treiman et al. 1998).

Interesting are the differences between the epileptiform activities induced by the identical procedures in the CA1 (Martín et al. 2001) and the CA3 pyramidal cells (present results) because they indicate different network dynamics that could determine the higher susceptibility of the CA3 region to develop and command the induction of epileptic activity. In both regions there is an increased synaptic activity during epileptogenesis (Martín et al. 2001; Rutecki et al. 1987; present results) that activate mGluRs that inhibit the sI_AHP/sAHP in CA1 pyramidal neurons (Martín et al. 2001). In contrast, reduction of the sI_AHP/sAHP is absent and instead the mI_AHP/mAHP is reduced by yet unknown mechanisms during epileptiform activity in CA3 pyramidal cells (present results). The CA1–CA3 differences could also depend on the distinct characteristics of the Ca²⁺-dependent K⁺ currents in CA1 and CA3 pyramidal neurons, especially because inhibition of the mI_AHP/mAHP in CA3 pyramidal neurons may disclose an inward current that could greatly enhance the excitability and contribute to bursting discharges by mediating a postburst ADP (Chuang et al. 2002; Sailer et al. 2002; Schiller 2004; Schwartzkroin and Prince 1980; Young et al. 2004), whereas this inward current and ADP are not important in CA1 pyramidal neurons (Sailer et al. 2002; Schwartzkroin and Prince 1980). Network properties may also contribute to the differences because the CA3 region is characterized by recurrent excitatory connections made by axon collaterals of pyramidal cells, whereas recurrent connections are minor in the CA1 region (reviewed in Knowles 1992).

In conclusion present results suggest that the interictal-like bursting is sustained by active processes where the mI_AHP/mAHP and the sI_AHP/sAHP play crucial roles that are initiated by the massive Ca²⁺ influx during a burst. After the burst network excitability first decreases and then increases as the intracellular Ca²⁺ concentration rises by the calcium influx and then declines with the intracellular Ca²⁺ buffering and extrusion mechanisms. During this period the Ca²⁺-activated mI_AHP/mAHP and sI_AHP/sAHP act cooperatively and in sequence. First, the reduction of the mI_AHP/mAHP increases neuronal excitability and facilitates bursting, resulting in a massive Ca²⁺ influx. Finally, the sI_AHP/sAHP is strongly activated during the interburst interval because the Ca²⁺ concentration is high as a result of the substantial Ca²⁺ influx. The increased Ca²⁺ concentration activates the sAHP that reduces excitability, network interactions, and bursting until the Ca²⁺ concentration is lowered by Ca²⁺-buffering extrusion, ultimately reducing the sI_AHP/sAHP and thus recovering the excitability that increases population activity to generate a subsequent burst.

Finally, we emphasize that acute animal models may not provide unambiguous information on the pathophysiology of chronic and human epileptogenesis. Even with these intrinsic limitations acute animal models are particularly fruitful in revealing the cellular pathophysiology of epileptic disorders, including synaptic and ionic channel alterations that cannot be completely answered with human investigation. Moreover, the development of novel models and of innovative analysis techniques may broaden our knowledge of the processes underlying epilepsy, may be useful in the understanding of human epilepsy, and inspire the development of new treatment strategies.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by Ministerio de Ciencia y Tecnología Grants BFI2002-01107 and BFU2005-07486) and Comunidad Autónoma de Madrid (Spain) Grant GR/SAL/0877/2004) to W. Buño. D. Fernandez de Sevilla was previously awarded a postdoctoral contract funded by Ministerio de Ciencia y Tecnología Grant BFI2002-01107 and Comunidad Autónoma de Madrid Grant GR/SAL/0877/2004, and at present by a Ministerio de Ciencia y Tecnología Grant BFU2005-07486. J. Garduño was a postdoctoral fellow supported by a Fundación Carolina (Spain) fellowship. E. Galván was funded by a Dirección General de Estudios de Postgrado–Universidad Nacional Autónoma de México (Mexico) fellowship.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Many thanks to Dr. A. Araque for valuable suggestions.

Present addresses: J. Garduño, Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autónoma de México, México Distrito Federal, Mexico; E. Galván, Departamento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México Distrito Federal, Mexico.

	FOOTNOTES

 
^* D. Fernández de Sevilla and J. Garduño contributed equally to this work.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: W. Buño, Instituto Cajal, CSIC, Av. Dr. Arce 37, 28002, Madrid, Spain

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
Abel HJ, Lee JC, Callaway JC, and Foehring RC. Relationships between intracellular calcium and afterhyperpolarizations in neocortical pyramidal neurons. J Neurophysiol 91: 324–335, 2004.[Abstract/Free Full Text]

Alger BE and Nicoll RA. Epileptiform burst after hyperpolarization: calcium-activated potassium potential in hippocampal CA1 pyramidal cells. Science 210: 1122–1124, 1980.[Abstract/Free Full Text]

Alger BE and Williamson A. A transient calcium-activated potassium component of the epileptiform burst afterhyperpolarization in rat hippocampus. J Physiol 399: 191–205, 1988.[Abstract/Free Full Text]

Avoli M, D’Antuono M, Louvel J, Kohling R, Biagini G, Pumain R, D’Arcangelo G, and Tancredi V. Network and pharmacological mechanisms leading to epileptiform synchronization in the limbic system in vitro. Prog Neurobiol 68: 167–207, 2002.[CrossRef][Web of Science][Medline]

Avoli M, Psarropoulou C, Tancredi V, and Fueta Y. On the synchronous activity induced by 4-aminopyridine in the CA3 subfield of juvenile rat hippocampus. J Neurophysiol 70: 1018–1029, 1993.[Abstract/Free Full Text]

Bond CT, Herson PS, Strassmaier T, Hammond R, Stackman R, Maylie J, and Adelman JP. Small conductance Ca²⁺-activated K⁺ channel knock-out mice reveal the identity of calcium-dependent afterhyperpolarization currents. J Neurosci 24: 5301–5306, 2004.[Abstract/Free Full Text]

Borde M, Bonansco C, de Sevilla F, Le Ray D, and Buño W. Voltage-clamp analysis of the potentiation of the slow Ca²⁺-activated K⁺ current in hippocampal pyramidal neurons. Hippocampus 10: 198–206, 2000.[CrossRef][Web of Science][Medline]

Borde M, Cazalets JR, and Buño W. Activity-dependent response depression in rat hippocampal CA1 pyramidal neurons in vitro. J Neurophysiol 74: 1714–1729, 1995.[Abstract/Free Full Text]

Buño W, Fernández de Sevilla D, and Fuenzalida M. The slow after hyperpolarization selectively shunts the NMDA component of glutamatergic EPSPs in CA1 pyramidal neurons in rat hippocampal slices. Program Number 169.5. 2004 Abstract Viewer and Itinerary Planner. Washington, DC: Society for Neuroscience, 2004, Online.

Buño W, García-Sánchez JL, and García-Austt E. Reset of hippocampal rhythmical activities by afferent stimulation. Brain Res Bull 3: 21–28, 1978.[Medline]

Carrer HF, Araque A, and Buño W. Estradiol regulates the slow Ca²⁺-activated K⁺ current in hippocampal pyramidal neurons. J Neurosci 23: 6338–6344, 2003.[Abstract/Free Full Text]

Chuang S-C, Zhao W, Young SR, Conquet F, Bianchi R, and Wong RKS. Activation of group I mGluRs elicits different responses in murine CA1 and CA3 pyramidal cells. J Physiol 541: 113–121, 2002.[Abstract/Free Full Text]

Colom LV and Saggau P. Spontaneous interictal-like activity originates in multiple areas of the CA2–CA3 region of hippocampal slices. J Neurophysiol 71: 1574–1585, 1994.[Abstract/Free Full Text]

Debarbieux F, Brunton J, and Charpak S. Effect of bicuculline on thalamic activity: a direct blockade of IAHP in reticularis neurons. J Neurophysiol 79: 2911–2918, 1998.[Abstract/Free Full Text]

de Curtis M and Avanzini G. Interictal spikes in focal epileptogenesis. Prog Neurobiol 63: 541–567, 2001.[CrossRef][Web of Science][Medline]

Delgado-Escueta AV, Ward AA, Woodbury DM, and Porter RJ. New wave of research in the epilepsies. In: Advances in Neurology, edited by Delgado-Escueta AV, Ward AA Jr, Woodbury DM, and Porter RJ. New York: Raven Press, 1986, vol. 44, p. 3–55.[Medline]

Devor DC, Singh AK, and Frizzell RA. Modulation of Cl^– secretion by benzimidazolones. I. Direct activation of a Ca(2+)-dependent K⁺ channel. Am J Physiol Lung Cell Mol Physiol 271: L775–L784, 1996.[Abstract/Free Full Text]

Empson RM and Jefferys JGR. Ca²⁺ entry through L-type Ca²⁺ channels helps terminate epileptiform activity by activation of a Ca²⁺ activated after hyperpolarization in hippocampal CA3. Neuroscience 102: 297–306, 2001.[CrossRef][Web of Science][Medline]

Fernández de Sevilla D, Fudenzalida M, and Buño W. Cholinergic induction of long-term potentiation in CA1 pyramidal neurons. Program Number 498.11. 2005 Abstract Viewer and Itinerary Planner. Washington DC: Society for Neuroscience, 2005, Online.

Fisahn A, Heinemann SF, and McBain CJ. The kainate receptor subunit GluR6 mediates metabotropic regulation of the slow and medium AHP currents in mouse hippocampal neurones. J Physiol 562: 199–203, 2005.[Abstract/Free Full Text]

Garduño J, Galván E, Ferández de Sevilla D, and Buño W. 1-Ethyl-2-benzimidazolinone (EBIO) suppresses epileptiform activity in in vitro hippocampus. Neuropharmacology 49: 376–388, 2005.[CrossRef][Web of Science][Medline]

Goldensohn ES and Purpura DP. Intracellular potentials of cortical neurons during focal epileptogenic discharges. Science 139: 840–842, 1963.[Abstract/Free Full Text]

Goodkin HP, Yeh J-L, and Kapur J. Status epilepticus increases the intracellular accumulation of GABAA receptors. J Neurosci 25: 5511–5520, 2005.[Abstract/Free Full Text]

Gu N, Vervaeke K, Hu H, and Storm JF. Kv7/KCNQ/M and HCN/h, but not KCa2/SK channels, contribute to the somatic medium after-hyperpolarization and excitability control in CA1 hippocampal pyramidal cells. J Physiol 566: 689–715, 2005.[Abstract/Free Full Text]

Knowles WD. Normal anatomy and neurophysiology of the hippocampal formation. J Clin Neurophysiol 9: 252–263, 1992.[Web of Science][Medline]

Lamsa K and Taira T. Use-dependent shift from inhibitory to excitatory GABAA receptor action in SP-O interneurons in the rat hippocampal CA3 area. J Neurophysiol 90: 1983–1995, 2003.[Abstract/Free Full Text]

Le Beau FE and Alger BE. Transient suppression of GABA_A-receptor-mediated IPSPs after epileptiform burst discharges in CA1 pyramidal cells. J Neurophysiol 79: 659–669, 1998.[Abstract/Free Full Text]

Lerma J, Herreras O, Muñoz D, and Solis JM. Interactions between hippocampal penicillin spikes and theta rhythm. Electroencephalogr Clin Neurophysiol 57: 532–540, 1984.[CrossRef][Web of Science][Medline]

Luhmann HJ, Dzhala VI, and Ben-Ari Y. Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro. Eur J Neurosci 12: 2757–2768, 2000.[CrossRef][Web of Science][Medline]

MacVicar BA and Dudek FE. Local circuit interactions in rat hippocampus: interactions between pyramidal cells. Brain Res 242: 341–344, 1982.[CrossRef][Web of Science][Medline]

Madison DV and Nicoll RA. Cyclic adenosine 3'5'-monophosphate mediates beta-receptor actions of noradrenaline in rat hippocampal pyramidal cells. J Physiol 372: 245–259, 1986.[Abstract/Free Full Text]

Martín ED, Araque A, and Buño W. Synaptic regulation of the slow Ca²⁺-activated K⁺ current in hippocampal CA1 pyramidal neurons: possible implication in epileptogenesis. J Neurophysiol 86: 2878–2886, 2001.[Abstract/Free Full Text]

Matsumoto H and Ajmone-Marsan C. Cortical cellular phenomena in experimental epilepsy: ictal manifestations. Exp Neurol 9: 305–326, 1964.[CrossRef][Web of Science][Medline]

McCormick DA and Contreras D. On the cellular and network bases of epileptic seizures. Annu Rev Physiol 63: 815–846, 2001.[CrossRef][Web of Science][Medline]

McCown TJ and Breese GR. Effects of apamin and nicotinic acetylcholine receptor antagonists on inferior collicular seizures. Eur J Pharmacol 187: 49–58, 1990.[CrossRef][Web of Science][Medline]

McQuiston AR and Madison DV. Muscarinic receptor activity induces an afterdepolarization in a subpopulation of hippocampal CA1 interneurons. J Neurosci 19: 5703–5710, 1999.[Abstract/Free Full Text]

Melyan Z, Wheal HV, and Lancaster B. Metabotropic-mediated kainate receptor regulation of IsAHP and excitability in pyramidal cells. Neuron 34: 107–114, 2002.[CrossRef][Web of Science][Medline]

Menéndez de la Prida L, Huberfeld G, Cohen I, and Miles R. Threshold behavior in the initiation of hippocampal population bursts. Neuron 49: 131–142, 2006.[CrossRef][Web of Science][Medline]

Miles R and Wong RKS. Single neurones can initiate synchronized population discharge in the hippocampus. Nature 306: 371–373, 1983.[CrossRef][Medline]

Miles R and Wong RKS. Excitatory synaptic interactions between CA3 neurones in the guinea-pig hippocampus. J Physiol 373: 397–418, 1986.[Abstract/Free Full Text]

Min MY, Melyan Z, and Kullmann DM. Synaptically released glutamate reduces gamma-aminobutyric acid (GABA)ergic inhibition in the hippocampus via kainate receptors. Proc Natl Acad Sci USA 96: 9932–9937, 1999.[Abstract/Free Full Text]

Pedarzani P, Mosbacher J, Rivard A, Cingolani LA, Oliver D, Stocker M, Adelman JP, and Fakler B. Control of electrical activity in central neurons by modulating the gating of small conductance Ca²⁺-activated K⁺ channels. J Biol Chem 276: 9762–9769, 2001.[Abstract/Free Full Text]

Pedarzani P and Storm JF. PKA mediates the effects of monoamine transmitters on the K⁺ current underlying the slow spike frequency adaptation in hippocampal neurons. Neuron 11: 1023–1035, 1993.[CrossRef][Web of Science][Medline]

Perez-Velazquez JL and Carlen PL. Synchronization of GABAergic interneuronal networks in seizure-like activity in the rat hippocampal slice. Eur J Neurosci 11: 4110–4118, 1999.[CrossRef][Web of Science][Medline]

Perreault P and Avoli M. 4-Aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus. J Neurosci 12: 104–115, 1992.[Abstract]

Poon N, Kloosterman F, Wu C, and Leung LS. Presynaptic GABAB receptors on glutamatergic terminals of CA1 pyramidal cells decrease in efficacy after partial hippocampal kindling. Synapse 59: 125–134, 2006.[CrossRef][Web of Science][Medline]

Psarropoulou C and Dallaire F. Activation of muscarinic receptors during blockade of GABA(A)-mediated inhibition induces synchronous epileptiform activity in immature rat hippocampus. Neuroscience 82: 1067–1077, 1998.[CrossRef][Web of Science][Medline]

Rodriguez-Moreno A and Lerma J. Kainate receptor modulation of GABA release involves a metabotropic function. Neuron 20: 1211–1218, 1998.[CrossRef][Web of Science][Medline]

Rutecki PA, Lebeda FJ, and Johnston D. 4-Aminopyridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition. J Neurophysiol 57: 1911–1924, 1987.[Abstract/Free Full Text]

Sah P and Bekkers JM. Apical dendritic location of slow after hyperpolarization current in hippocampal pyramidal neurons: implications for the integration of long-term potentiation. J Neurosci 16: 4537–4542, 1996.[Abstract/Free Full Text]

Sah P and Faber ES. Channels underlying neuronal calcium-activated potassium currents. Prog Neurobiol 66: 345–353, 2002.[CrossRef][Web of Science][Medline]

Sailer CA, Hu H, Kaufmann WA, Trieb M, Schwarzer C, Storm JF, and Knaus H-G. Regional differences in distribution and functional expression of small-conductance Ca²⁺-activated K⁺ channels in rat brain. J Neurosci 22: 9698–9707, 2002.[Abstract/Free Full Text]

Schiller Y. Activation of a calcium-activated cation current during epileptiform discharges and its possible role in sustaining seizure-like events in neocortical slices. J Neurophysiol 92: 862–872, 2004.[Abstract/Free Full Text]

Schwartzkroin PA and Prince DA. Cellular and field potential properties of epileptogenic hippocampal slices. Brain Res 147: 117–130, 1978.[CrossRef][Web of Science][Medline]

Schwartzkroin PA and Prince DA. Effects of TEA on hippocampal neurons. Brain Res 185: 169–181, 1980.[CrossRef][Web of Science][Medline]

Shah MM, Miscony Z, Javadzadeh-Tabatabaie M, Ganellin CR, and Haylett DG. Clotrimazole analogues: effective blockers of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurones. Br J Pharmacol 132: 889–898, 2001.[CrossRef][Web of Science][Medline]

Staley KJ, Longacher M, Bains JS, and Yee A. Presynaptic modulation of CA3 network activity, Nat Neurosci 1: 201–209, 1998.[CrossRef][Web of Science][Medline]

Stelzer A, Slater NT, and ten Bruggencate G. Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy. Nature 326: 698–701, 1987.[CrossRef][Medline]

Stocker M. Ca(2+)-activated K⁺ channels: molecular determinants and function of the SK family. Nat Rev Neurosci 5: 758–770, 2004.[CrossRef][Web of Science][Medline]

Stocker M, Krause M, and Pedarzani P. An apamin-sensitive Ca²⁺-activated K⁺ current in hippocampal pyramidal neurons. Proc Natl Acad Sci USA 96: 4662–4667, 1999.[Abstract/Free Full Text]

Storm JF. Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. J Physiol 385: 733–759, 1987.[Abstract/Free Full Text]

Traub RD, Miles R, and Jefferys JG. Synaptic and intrinsic conductances shape picrotoxin-induced synchronized afterdischarges in the guinea-pig hippocampal slice. J Physiol 461: 525–547, 1993.[Abstract/Free Full Text]

Treiman DM, Meyers PD, Walton NY, Collins JF, Colling C, Rowan AJ, Handforth A, Faught E, Calabrese VP, Uthman BM, Ramsay RE, and Mamdani MB. A comparison of four treatments for generalized convulsive status epilepticus. Veterans Affairs Status Epilepticus Cooperative Study Group. N Engl J Med 339: 792–798, 1998.[Abstract/Free Full Text]

Verma-Ahuja S, Evans MS, and Espinosa JA. Evidence of increased excitability in GEPR hippocampus preceding development of seizure susceptibility. Epilepsy Res 31: 161–173, 1998.[CrossRef][Web of Science][Medline]

Viana F, Bayliss DA, and Berger AJ. Multiple potassium conductances and their role in action potential repolarization and repetitive firing behavior of neonatal rat hypoglossal motoneurons. J Neurophysiol 69: 2150–2163, 1993.[Abstract/Free Full Text]

Villalobos C, Shakkottai VG, Chandy KG, Michelhaugh SK, and Andrade R. SKCa channels mediate the medium but not the slow calcium-activated afterhyperpolarization in cortical neurons. J Neurosci 24: 3537–3542, 2004.[Abstract/Free Full Text]

Vogalis F, Storm JF, and Lancaster B. SK channels and the varieties of slow after-hyperpolarizations in neurons. Eur J Neurosci 8: 3155–3166, 2003.

Wong RKS and Miles R. Study of GABAergic inhibition and GABA_A receptors in experimental epilepsy. In: Epilepsy: Models, Mechanisms and Concepts, edited by Schwartzkroin PA. Cambridge, UK: Cambridge Univ. Press, 1994, p. 424–436.

Young SR, Chuang SC, and Wong RK. Modulation of afterpotentials and firing pattern in guinea pig CA3 neurones by group I metabotropic glutamate receptors. J Physiol 554: 371–385, 2004.[Abstract/Free Full Text]

Zhang L, Weiner JL, Valiante TA, Velumian AA, Watson PL, Jahromi AA, Schertzer S, Pennefather P, and Carlen PL. Whole-cell recordings of the Ca²⁺-dependent slow after hyperpolarization in hippocampal neurons: effects of internally applied anions. Pfluegers Arch 426: 247–253, 1994.[CrossRef][Web of Science][Medline]

This article has been cited by other articles:

				S. R. Young, R. Bianchi, and R. K. S. Wong Signaling Mechanisms Underlying Group I mGluR-Induced Persistent AHP Suppression in CA3 Hippocampal Neurons J Neurophysiol, March 1, 2008; 99(3): 1105 - 1118. [Abstract] [Full Text] [PDF]

				D. Fabo, Z. Magloczky, L. Wittner, A. Pek, L. Eross, S. Czirjak, J. Vajda, A. Solyom, G. Rasonyi, A. Szucs, et al. Properties of in vivo interictal spike generation in the human subiculum Brain, February 1, 2008; 131(2): 485 - 499. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 96/6/3028    most recent 00434.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by de Sevilla, D. F. Articles by Buño, W. Search for Related Content PubMed PubMed Citation Articles by de Sevilla, D. F. Articles by Buño, W.