Excitatory gaba input directly drives seizure-like rhythmic synchronization in mature hippocampal CA1 pyramidal cells

Neuroscience 119 (2003) 265–275

EXCITATORY GABA INPUT DIRECTLY DRIVES SEIZURE-LIKE RHYTHMIC SYNCHRONIZATION IN MATURE HIPPOCAMPAL CA1 PYRAMIDAL CELLS Y. FUJIWARA-TSUKAMOTO,a,b1 Y. A. NAMBUa,b AND M. TAKADAa,b

ISOMURA,a,b,c*1

Key words: afterdischarge, chloride influx, GABAA receptor, temporal lobe epilepsy.

a Department of System Neuroscience, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan

GABA is generally known as the major inhibitory transmitter in the CNS of adult animals. However, intense activation of GABAA receptors by repetitive synaptic stimulation or exogenous GABA application often produces a large depolarizing response in mature hippocampal pyramidal cells (Alger and Nicoll, 1979; Andersen, et al., 1980). Such intense GABAA receptor activation may cause a drastic change in ionic gradients of GABAA-permeable anions, i.e. the intracellular accumulation of chloride (Cl⫺) ions and the CO2-mediated redistribution of bicarbonate (HCO3⫺) ions, leading to the prominent depolarizing GABAergic response (Staley et al., 1995; Perkins and Wong, 1996; Staley and Proctor, 1999). In addition, the depolarizing response may be enhanced non-synaptically by the extracellular accumulation of potassium (K⫹) ions (Kaila et al., 1997; Smirnov et al., 1999). Thus, hippocampal GABAergic transmission seems dynamically changeable from inhibitory to excitatory by intense repetitive stimulation of GABAergic synapses. However, little is known about the functional significance of such an excitatory GABAergic response in mature hippocampal pyramidal cells. On the other hand, it is well known that similar repetitive electrical stimulation in vivo can evoke a synchronous, rhythmic neuronal activity (‘seizure-like’ or ‘ictal’ afterdischarge) in experimental animals (Goddard et al., 1969; Bragin et al., 1997) and even in human epilepsy patients (Gloor et al., 1982). Particularly, afterdischarge in the hippocampus has been considered to be an experimental model for temporal lobe epilepsy, and, therefore, numerous pathophysiological studies have been directed at elucidating the cellular mechanism of hippocampal afterdischarge in vivo and in vitro (McNamara, 1994). Afterdischarges in normal hippocampal slices have been induced under a variety of conditions that increase neuronal activity, including low Mg2⫹ (Mody et al., 1987; Tancredi et al., 1990; Traub et al., 1994; Ko¨hling et al., 2000), 4-aminopyridine (4-AP) (Perreault and Avoli, 1989, 1991, 1992; Avoli et al., 1996; Lamsa and Kaila, 1997), bicuculline (Borck and Jefferys, 1999), and penicillin (Swann et al., 1993), as well as repeated electrical stimulation (Stasheff, 1989, 1993a,b; Rafiq et al., 1993; Velazquez and Carlen, 1999; Higashima et al., 2000). However, a functional significance of GABAergic transmission in rhythmic synchronization of the hippocampal afterdischarges remains unclear even in these experimental models, probably be-

b

CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan c

The Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8471, Japan

Abstract—GABA, which generally mediates inhibitory synaptic transmissions, occasionally acts as an excitatory transmitter through intense GABAA receptor activation even in adult animals. The excitatory effect results from alterations in the gradients of chloride, bicarbonate, and potassium ions, but its functional role still remains a mystery. Here we show that such GABAergic excitation participates in the expression of seizure-like rhythmic synchronization (afterdischarge) in the mature hippocampal CA1 region. Seizure-like afterdischarge was induced by high-frequency synaptic stimulation in the rat hippocampal CA1-isolated slice preparations. The hippocampal afterdischarge was completely blocked by selective antagonists of ionotropic glutamate receptors or of GABAA receptor, and also by gap-junction inhibitors. In the CA1 pyramidal cells, oscillatory depolarizing responses during the afterdischarge were largely dependent on chloride conductance, and their reversal potentials (average ⴚ38 mV) were very close to those of exogenously applied GABAergic responses. Moreover, intracellular loading of the GABAA receptor blocker fluoride abolished the oscillatory responses in the pyramidal cells. Finally, the GABAergic excitation-driven afterdischarge has not been inducible until the second postnatal week. Thus, excitatory GABAergic transmission seems to play an active functional role in the generation of adult hippocampal afterdischarge, in cooperation with glutamatergic transmissions and possible gap junctional communications. Our findings may elucidate the cellular mechanism of neuronal synchronization during seizure activity in temporal lobe epilepsy. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. 1

Y. Fujiwara-Tsukamoto and Y. Isomura contributed equally to the research described. *Correspondence to: Y. Isomura, Department of System Neuroscience, Tokyo Metropolitan Institute for Neuroscience, 2– 6 Musashidai, Fuchu, Tokyo 183-8526, Japan. Tel: ⫹81-42-325-3881; fax: ⫹81-42-321-8678. E-mail address: [email protected] (Y. Isomura). Abbreviations: ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; 4-AP, 4-aminopyridine; DL-AP-5, DL-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; EGTA, ethylene glycol bis(␤-aminoethyl ether)-N,N,N',N'-tetraacetic acid; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid; LLD, long-lasting depolarization; MCPG, (⫾)-␣-methyl-4carboxyphenylglycine; NMDA, N-methyl-D-aspartic acid; r.m.p., resting membrane potential; 5␣-THDOC, 5␣-pregnane-3␣,21-diol-20-1; TPMPA, (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid; VLJ, liquid junction potential.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00102-7

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cause such pharmacological treatments might have serious non-specific influences on many other unrelated neurons, or because complex inter-regional propagation of neural activity throughout hippocampal or limbic slices might obscure local neuronal processing. To investigate the possible contribution of excitatory GABAergic transmission to the generation of seizure-like synchronous oscillations at a cellular level, we have adopted a simpler hippocampal afterdischarge model. In our model, the CA1 region of normal rat hippocampal slices was isolated from other regions, thus preventing any external input from intruding through the Schaffer collaterals, the perforant path, or other pathways. Also, afterdischarge was consistently induced by simple electrical stimulation in normal extracellular fluid to exclude non-specific pharmacological influences. Using this improved experimental model, we provide the first evidence that dynamically excitable GABAergic transmission plays an essential role in the expression of seizure-like rhythmic synchronization among the CA1 pyramidal cells of the mature hippocampus.

EXPERIMENTAL PROCEDURES Slice preparations Hippocampal slices (400 ␮m thick) were prepared from etheranesthetized, mature (P21–142) or immature (P6 –20) Wistar rats with a microslicer (DTK-1500, Dosaka EM, Kyoto, Japan), and the CA1 regions were routinely isolated from the CA3 and subicular regions. After the recovery for at least 1 h, each slice was transferred to a submerged-type recording chamber continuously circulated with a normal artificial cerebrospinal fluid (ACSF; 30 – 32 °C), which consisted of 124 NaCl, 2.5 KCl, 1.2 KH2PO4, 26 NaHCO3, 1.2 MgSO4, 2.5 CaCl2 and 25 D-glucose (in mM) and was saturated with 95% O2/5% CO2 gas (Isomura et al., 2002).

Electrophysiological recordings To induce the afterdischarge, tetanic stimulation (100 Hz for 0.5 s; intensity 300 – 600, usually 400 ␮A; duration 400 ␮s) was delivered by a monopolar glass stimulating electrode (0.5–1 M⍀, filled with 2.5 M NaCl) placed in the stratum radiatum. Whole-cell patch-clamp recordings were obtained from the pyramidal cells in hippocampal CA1 slices under visual guidance (Isomura and Kato, 1999). In current-clamp mode (I⫽0), membrane potentials (resting membrane potential (r.m.p.), ⫺61.9⫾6.1 mV) were recorded with a patch-clamp amplifier (Axopatch 1D or Axopatch 200B; Axon Instruments, Union City, CA, USA), through glass patch electrodes usually filled with a low-Cl⫺ internal solution containing 140 K-gluconate, 2 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 2 5'-ATP Na2 and 0.5 GTP Na2 (in mM, pH 7.4; liquid junction potential (VLJ)⫹13.7 mV; 5–10 M⍀). KCl was substituted for K-gluconate in the internal solution for experiments in high intracellular Cl⫺ condition (VLJ⫹3.8 mV). In voltage-clamp mode, K-gluconate was replaced to 132 mM Cs-gluconate, the concentration of EGTA was increased to 2 mM, and 5 mM QX-314 (Alomone laboratories, Jerusalem, Israel) was added to it (VLJ⫹12.8 mV). The patch electrode solution used for intracellular blockade of GABAA receptors consisted of 140 KF, 10 HEPES and 0.2 EGTA (in mM, pH 7.4; VLJ⫹6.8 mV). VLJ, pipette and whole-cell capacitances, and series resistance (5– 40 M⍀) were compensated adequately. If necessary, 10 –15 mM biocytin was added to internal solutions and visualized by the avidin– biotin– HRP complex (ABC) method to identify the recorded neurons morphologically or to confirm ‘re-patch-clamp’ recordings from the

same single pyramidal cells. For simultaneous whole-cell and extracellular recordings, field potentials were recorded with another amplifier (Axoclamp 2B, Axon Instruments) through glass electrodes (2–5 M⍀, filled with 2.5 M NaCl) placed in the stratum pyramidale. Intracellular recordings were made with sharp glass microelectrodes filled with 1 M K-acetate (80 –120 M⍀) and amplified with the Axoclamp 2B. Recorded signals were low-pass filtered at 3–5 kHz and digitized at 5 kHz with an A/D interface (Digidata 1200, Axon Instruments).

Pharmacological treatments Bicuculline methiodide, carbenoxolone, octanol, muscimol, NO711, strychnine, 5␣-pregnane-3␣,21-diol-20-1 (5␣-THDOC) and (1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA) were purchased from Sigma, St. Louis, MO, USA; DL-2-amino-5phosphonopentanoic acid (DL-AP-5), 6-cyano-7-nitroquinoxaline2,3-dione (CNQX), (⫾)-␣-methyl-4-carboxyphenylglycine (MCPG), and CGP55845 from Tocris Cookson, Ballwin, MO, USA; and atropine sulfate, carbachol, and picrotoxin from Nacalai Tesque, Kyoto, Japan. Each drug was added to normal ACSF and bath-applied to the recorded slices. Acetazolamide (Nacalai) was added to lowbicarbonate ACSF, in which HCO3⫺ was depleted by replacing NaHCO3 buffer with HEPES buffer (pH 7.4) and bubbled with pure O2. dimethyl sulfoxide (final concentration, 0.05%) was used to dissolve CGP55845 and acetazolamide, as well as for their controls. GABA or muscimol (0.1 mM in saline, Sigma) was applied to the soma of recorded pyramidal cells briefly and repeatedly by pressure (5–10 psi, 10 –150 ms; Picospritzer II, General Valve, Fairfield, NJ, USA) through a glass capillary (tip diameter, 1–2 ␮m). Similarly, 1 mM glutamate (Sigma) was applied to their apical dendrites (10 psi, 10 –20 ms). Bicuculline (5 mM in saline, pH approximately 7.4) was injected locally into the stratum pyramidale for 1–5 s starting at 2– 6 s after the tetanization. As a control, we confirmed that saline injection alone had no obvious effects.

Data analysis and animal experiments All data in the text are expressed as means⫾S.D., and Student’s t-test or analysis of variance (ANOVA) was applied for statistical comparisons. In all experiments, we minimized the number of animals used and their suffering, in accordance with the Guideline for Care and Use of Animals (Tokyo Metropolitan Institute for Neuroscience, 2000).

RESULTS Synaptically induced afterdischarge in hippocampal CA1-isolated slices Employing whole-cell current-clamp recordings, we found that a prolonged train of oscillatory depolarization at 2–7 Hz frequency (seizure-like afterdischarge) can be induced in the pyramidal cells of rat hippocampal ‘CA1isolated’ slices, by a simple, but relatively strong tetanic stimulation at the stratum radiatum in ACSF (Fig. 1A). As previously reported, a large, slow depolarization (Staley et al., 1995; Kaila et al., 1997) and, occasionally, ␤ (10 – 30 Hz) or ␥ (30 –100 Hz) oscillations (Bracci et al., 1999) were also observed for only several seconds after the tetanization. In all neurons examined, the afterdischarge was consistently evoked with or without spiking (Fig. 1B; duration 19.3⫾3.4 s, frequency 4.4⫾1.6 Hz, n⫽52). In most of the pyramidal cells, no or only a single action potential occurred in each cycle of oscillatory depolariza-

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Fig. 1. Seizure-like synchronous membrane-potential oscillations in hippocampal CA1 pyramidal cells. (A) Membrane-potential recording from single pyramidal cells in hippocampal ‘CA1-isolated’ slices (upper right). DG, dentate gyrus; S, subiculum. Rhythmic depolarization at 2–7 Hz frequency (seizure-like afterdischarge) following a slow post-tetanic depolarization was induced by a strong tetanus (100 Hz, 0.5 s; lower trace), but not by a weak (upper trace) or a moderate (middle trace) tetanus. Thick underlines indicate the duration of tetanus, and downward artifacts during the tetani are truncated in all figures. Scale bars⫽0.5 s, 10 mV. Boxes: Synaptic responses evoked by a single shock at the same intensity as each tetanus. Scale bars⫽10 ms, 20 mV. (B) Colored isogram showing the averaged time-frequency distribution of oscillatory depolarizing events during the afterdischarge that was induced by tetanus (small bar). Activity was defined as the number of oscillatory pulses in each of 0.5 s⫻2 Hz bins for individual neurons, and averaged for all neurons (n⫽52). (C) Averaged discharge probability per oscillatory depolarizing event in the 52 neurons analyzed in (B). Error bars⫽S.E.M. (D) Synchronous activity among hippocampal CA1 pyramidal cells during the afterdischarge. Population spikes (field potential, FP) were precisely time-locked to oscillatory depolarizing responses (membrane potential, MP). Thick underline indicates the duration of tetanus. Scale bars⫽1 s, 0.2 mV (FP) or 10 mV (MP). (E) Gradual increase in amplitude of population spikes during the initial 10 s of the afterdischarge. Error bars⫽S.E.M. (n⫽8).

tion, and the probability of their spiking decreased gradually until the end of afterdischarge (Fig. 1C). The afterdischarge was evocable in various different conditions; in

thinner (300 ␮m thick) or non-isolated (the whole hippocampal) slices, in the presence of 2 mM extracellular Mg2⫹, or by tetanizing either the stratum oriens or lacuno-

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Table 1. Pharmacological effects of receptor/enzyme blockers on duration of afterdischarge and total number of oscillatory pulses during afterdischarge (% of control) Treatment

n

Atropine AP-5 CNQX AP-5⫹CNQX Bicuculline Acetazolamide/ low HCO3⫺ CGP55845 Carbenoxolone

(7) (6) (5) (6) (5) (6) (5) (6)

Duration (%) 86.6⫾28.8 62.8⫾12.1* 17.5⫾12.2** 0*** 0*** 23.9⫾10.3*** 129.9⫾22.9 16.1⫾10.4**

Total pulse number (%) 66.3⫾44.9 48.7⫾15.9* 3.7⫾8.3* 0** 0** 3.6⫾5.7*** 181.1⫾53.1* 2.1⫾4.5*

* p⬍0.01; ** p⬍0.001; *** p⬍0.0001 (paired t-test).

sum-moleculare (data not shown). We could also record similar afterdischarge intracellularly with K-acetate-filled sharp microelectrodes, which left intracellular ionic concentrations unaffected (data not shown). The afterdischarge was synchronous among the CA1 pyramidal cells, because their population spikes in field potential were precisely time-locked to membrane potential oscillations in individual pyramidal cells (Fig. 1D; time lag 5.9⫾6.0 ms, n⫽8). The synchronization did not begin immediately after the tetanus, but rather individual neurons seemed to be gradually recruited into a global network oscillation (Fig. 1E; n⫽8, ANOVA P⬍0.001; see also Fig. 1C). Taken together, these findings establish a simple and reliably reproducible model for seizure-like afterdischarge within the hippocampal CA1 local network of mature rats in normal extracellular fluid. Synaptic transmissions involved in hippocampal afterdischarge generation We pharmacologically surveyed the synaptic transmissions that might contribute to the generation of afterdischarge in hippocampal CA1-isolated slices. The muscarinic receptor antagonist atropine did not influence afterdischarge generation (Fig. 2A and Table 1; 10 ␮M, n⫽7), while the muscarinic receptor agonist carbachol greatly depressed it (50 ␮M, data not shown). The afterdischarge was still inducible but was slightly briefer in the presence of the N-methyl-D-aspartic acid (NMDA)-type glutamate receptor antagonist DL-AP-5 (Fig. 2B and Table 1; 50 ␮M, n⫽6). In contrast, the afterdischarge was largely blocked by the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate-type (non-NMDA-type) glutamate receptor antagonist CNQX (Fig. 2C and Table 1; 10 ␮M, n⫽5) and was completely blocked by a mixture of DL-AP-5 and CNQX (Fig. 2D and Table 1; n⫽6). The metabotropic glutamate receptor antagonist MCPG had no apparent effects on afterdischarge generation (500 ␮M, data not shown). These results suggest that non-NMDA-type glutamate receptors, in cooperation with NMDA-type receptors, may participate in the generation of synchronous neuronal oscillations in the CA1 region. The afterdischarge was also abolished by the GABAA receptor antagonists bicuculline (Fig. 2E and Table 1;

25 ␮M, n⫽5) and picrotoxin (50 ␮M, data not shown), and by the carbonic anhydrase inhibitor acetazolamide in a low-bicarbonate ACSF (Fig. 2F and Table 1; 25 ␮M, n⫽6), while the potent GABAB receptor antagonist CGP55845 significantly prolonged the afterdischarge (Fig. 2G and Table 1; 1 ␮M, n⫽5, P⬍0.05). But neither the GABAC receptor antagonist TPMPA (50 ␮M) nor the glycine receptor antagonist strychnine (1 ␮M) showed any remarkable effect on its generation (data not shown). The positive GABAA modulator 5␣-THDOC (20 ␮M) extremely prolonged the slow post-tetanic depolarization (⬎20 s), to mask the subsequent afterdischarge completely (data not shown). As the blockade of CO2–HCO3⫺ exchange by carbonic anhydrase inhibitors or the extracellular depletion of bicarbonate dramatically reduces depolarizing GABAA responses in the adult hippocampus (Grover et al., 1993; Staley et al., 1995; Kaila et al., 1997; Burg et al., 1998), it is possible that the GABAA receptor may mediate excitatory, rather than inhibitory, synaptic responses at a certain site in the oscillating neural network of the hippocampus. Interestingly, acetazolamide is often used clinically as an antiepileptic drug (Diamox) for epilepsy patients, although its exact action on epileptic seizures has not yet been clarified. In addition to these receptor inhibitors, the gap-junction blockers carbenoxolone (Fig. 2H and Table 1; 100 ␮M, n⫽6) and octanol (1 mM, duration 7.8⫾9.0% of control, n⫽6, P⬍0.001) suppressed the afterdischarge reversibly, thereby indicating the possible involvement of electrical coupling in the rhythmic synchronization of the CA1 pyramidal cells (Draguhn et al., 1998; Fukuda and Kosaka, 2000; Schmitz et al., 2001). Thus, excitatory GABAergic and glutamatergic synaptic transmissions and, possibly, gap-junctional interactions are required for the generation of hippocampal afterdischarge. Oscillatory depolarization mediated by excitatory GABAergic input in pyramidal cells The pyramidal cells are expected to receive massive GABAergic inputs directly from local interneurons during the afterdischarge. In fact, the oscillatory depolarizing responses during the afterdischarge were greatly dependent on chloride conductance; the oscillatory responses recorded in high intracellular Cl⫺ condition (144 mM) were much larger than those in low Cl⫺ condition (4 mM) in the same single pyramidal cells (Fig. 3A; discharge probability, control 11.0⫾19.9%, high Cl⫺ 61.0⫾9.5%, n⫽7, paired t-test P⬍0.025; r.m.p., control ⫺57.1⫾1.6 mV, high Cl⫺ ⫺58.4⫾3.6 mV, P⬎0.3). There was no obvious effect on the oscillatory responses by repeating patch-clamp recordings with the same pipette solution (data not shown). Thus, the oscillatory depolarizing responses may contain an anion-dependent component mediated probably by GABAA receptor in the pyramidal cells. It is, therefore, possible that oscillatory responses in the pyramidal cells might be due to depolarizing inputs via the GABAergic interneurons, given that intense GABAA activation during tetanization transiently keeps the reversal potential of GABAA response

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Fig. 2. Pharmacological characterization of afterdischarge. Afterdischarge was inducible in the presence of the muscarinic receptor antagonist 10 ␮M atropine (A), the NMDA receptor antagonist 50 ␮M DL-AP-5 (B), or the GABAB receptor antagonist 1 ␮M CGP55845 (G), whereas it was blocked largely by the AMPA/kainate receptor antagonist 10 ␮M CNQX (C) and completely by both DL-AP-5 and CNQX (D). Application of the GABAA receptor antagonist 25 ␮M bicuculline (E), the carbonic anhydrase inhibitor 25 ␮M acetazolamide in low-bicarbonate ACSF (F), or the gap-junction blocker 100 ␮M carbenoxolone (H) also abolished the afterdischarge. Note that the slow post-tetanic depolarization was depressed by bicuculline application (arrow in E). Spikes are truncated. Scale bars⫽0.5 s, 10 mV.

much higher than the r.m.p. of the pyramidal cells (Staley et al., 1995).

To test this possibility, we applied GABA locally while recording from the pyramidal cells before and after tetani-

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zation. Repeated local GABA applications, which normally hyperpolarized the pyramidal cells, elicited large depolarizing responses for more than 20 s after tetanization (29.2⫾10.0 s, n⫽19), and such responses gradually recovered to the level of normal hyperpolarizing responses (Fig. 3B). Similar results were obtained with using the specific GABAA receptor agonist muscimol instead of GABA (data not shown). In addition, our voltage-clamp recordings at various holding potentials revealed that oscillatory currents were clearly reversed between ⫺25 and ⫺50 mV (⫺37.7⫾8.5 mV, n⫽11), and that the reversal potentials of oscillatory currents were very close (within approximately 5 mV) to those of exogenous GABAergic responses during the afterdischarge (Fig. 3C; n⫽5). Next, we examined whether the oscillatory depolarizing potentials are achieved by a combination of excitatory glutamatergic inputs and inhibitory GABAergic inputs, or by excitatory GABAergic inputs. As shown in Fig. 3D, the oscillatory depolarizing responses were slightly but significantly inhibited, rather than disinhibited (enlarged), by local application of a puff of bicuculline to the stratum pyramidale for several seconds (1–5 s) during the afterdischarge generation (amplitude, control 6.4⫾3.4 mV, bicuculline 4.6⫾2.8 mV, n⫽7, paired t-test P⬍0.025), suggesting that GABAA-mediated component of the oscillatory responses may indeed become excitatory during afterdischarge. Owing to some technical reasons, it might be very difficult to block the GABAA responses in recorded pyra-

Fig. 3. Synchronous oscillatory depolarization driven directly by excitatory GABAergic input in CA1 pyramidal cells. (A) Chloride (Cl⫺) conductance-dependent oscillatory responses during the afterdischarge. Whole-cell recordings were performed in the same pyramidal cells sequentially, at first through the patch-clamp electrode filled with 4 mM Cl⫺ (first, control; r.m.p., ⫺57 mV), and after careful withdrawal of the first electrode, through another electrode with 144 mM Cl⫺

(second, high Cl⫺; r.m.p., ⫺58 mV) (‘re-patch-clamp’ recording). Scale bars⫽1 s, 10 mV. Right: Representative traces of single oscillatory responses indicated by asterisks in the left traces. Scale bars⫽25 ms, 5 mV. (B) Membrane-potential changes in response to repetitive local GABA applications to the stratum pyramidale (filled triangles) before and every 5 s after tetanization (r.m.p., ⫺57 mV). Scale bars⫽5 s, 10 mV. (C) Tetanus-induced oscillatory currents at various holding potentials (VH) in voltage-clamp recordings. The reversal potential of oscillatory currents was almost identical to that of exogenous GABAergic responses (filled triangles) during the afterdischarge. Scale bars⫽ 0.5 s, 200 pA. Inset, The oscillatory currents (circles) and the GABA responses (triangles) plotted against the holding potentials; note the similar reversal potentials. (D) Suppression of oscillatory depolarizing responses by local application of bicuculline during the afterdischarge generation. A puff of bicuculline was applied locally to the stratum pyramidale, for approximately 5 s starting at ⬎2 s after the tetanization. The amplitude of oscillatory responses was indeed suppressed, rather than enhanced, by the bicuculline application (middle, compared with control [left] and washout [right] trials; these traces are sampled at 10 s after the tetanization), suggesting that GABAA response may be depolarizing during afterdischarge. Scale bars⫽100 ms, 5 mV. (E) Complete blockade of synchronous oscillatory depolarization by intracellular loading of the GABAA blocker fluoride ions (F⫺) in a single pyramidal cell (MP) during the network oscillations (FP) (upper left, control, r.m.p. ⫺65 mV; upper right, F⫺-loaded, ⫺66 mV). Slight membrane depolarization due to intracellular F⫺-loading was compensated by constant current injection (approximately ⫺0.2 nA). Scale bars⫽0.1 s, 0.1 mV (FP) or 5 mV (MP). Insets, Successfully evoked EPSP and action potential in the same control or F⫺-loaded neuron. Scale bars⫽25 ms, 5 mV. Lower, Depolarizing responses evoked by local glutamate applied to the stratum radiatum (open triangles) during the blockade of synchronous oscillatory depolarization in an F⫺-loaded single pyramidal cells (MP; r.m.p., ⫺54 mV; injected current, ⫺0.12 nA). Scale bars⫽1 s, 0.1 mV (FP) or 5 mV (MP).

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Fig. 4. Absence of excitatory GABAergic afterdischarge in early developmental stages. (A) Postnatal development of tetanically induced afterdischarge in hippocampal CA1 slices. Left, Field potential traces after tetanus delivery. Note that no oscillatory population spikes appear until the second postnatal week. Scale bars⫽1 s, 0.2 mV. Right, Summary diagram showing developmental appearance of oscillatory events during the first 4 postnatal weeks. Error bars⫽S.E.M. (first postnatal week, n⫽8; 2nd, n⫽9; third, n⫽12; fourth, n⫽13). (B) Representative (top) and averaged (bottom) traces of membrane potentials after tetanization in immature (left; P12–14, n⫽6) and mature (right; P21–27, n⫽8) pyramidal cells. Membrane potentials were averaged after all spikes were deleted. Black and gray lines indicate the mean and S.E.M. Note that the GABAA-dependent, slow post-tetanic depolarization was smaller and briefer in immature than in mature pyramidal cells. Scale bars⫽0.5 s, 10 mV.

midal cells completely by such a local application of bicuculline, because their GABAergic synapses are distributed broadly not only in the stratum pyramidale, but also in the strata oriens, radiatum, and even lacunosum-moleculare (Freund and Buzsa´ki, 1996). We obtained further evidence for the GABAergic nature of the oscillatory responses by using patch-clamp electrodes to load single pyramidal cells with fluoride (F⫺) ions, which inactivate GABAA receptors internally (Bormann et al., 1987; Smirnov et al., 1999). In contrast with control recordings, F⫺-loaded pyramidal cells exhibited no oscillatory responses after tetanization, even though oscillatory population spikes were still expressed in the same slices (Fig. 3E, upper; amplitude, control 8.2⫾3.9 mV, n⫽8; F⫺-loaded 1.0⫾0.4, n⫽6; P⬍0.002). GABAA receptor function seemed to be selectively blocked in these F⫺-loaded neurons, because large EPSPs and action potentials, but not IPSPs, were normally elicited by a single stimulating pulse (Fig. 3E, insets), and because exogenous glutamatergic responses were evocable even in the disappearance of the oscillatory depolarizing responses (Fig. 3E, lower). On the basis of these results, we conclude that the CA1 pyramidal cells are depolarized almost purely by direct excitatory GABAergic input in each cycle of the seizure-like afterdischarge in our experimental condition.

Lack of excitatory GABAergic afterdischarge in early developmental stages We finally looked for a developmental change in the excitatory GABAergic network oscillations, because neonatal GABAA responses are often depolarizing owing to a positive shift in their reversal potential (Ben-Ari et al., 1989; Ben-Ari, 2001; Leinekugel et al., 2002). In our experiments, no synchronous activity was induced until the second postnatal week (Fig. 4A; first week, n⫽8; second, n⫽9; third, n⫽12; fourth, n⫽13; ANOVA P⬍0.001). We failed to induce any afterdischarge in immature slices even by much stronger and longer tetanization or in the presence of 2 ␮M NO-711, a GABA uptake inhibitor (data not shown). Thus, the afterdischarge in mature hippocampus is clearly distinct from the giant depolarizing potentials in neonatal hippocampus (Ben-Ari et al., 1989). In addition, the GABAA-dependent, slow post-tetanic depolarization was smaller and briefer in immature pyramidal cells than in mature ones (Fig. 4B; peak amplitude, immature, 14.6⫾10.0 mV, n⫽6; mature, 27.5⫾5.6 mV, n⫽8; P⬍0.01), indicating that the mature pyramidal cells seem to have more dynamic excitability in response to intense GABAA receptor activation.

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Fig. 5. A hypothetical model for seizure-like rhythmic synchronization (afterdischarge) within hippocampal CA1 region. GABAergic interneurons are stimulated, directly or indirectly via the glutamatergic Schaffer collaterals, during tetanization. Intense GABAA receptor activation leads to massive Cl⫺ influx into the postsynaptic pyramidal cells. As a result, intracellular Cl⫺ accumulation raises the reversal potential of GABAA response more positive than the r.m.p., and occasionally even above the firing threshold, for more than 20 s. In cooperation with possible electrical communication via gap junctions among the presynaptic interneurons, excitatory GABAergic transmission allows the expression of seizure-like synchronization in the postsynaptic pyramidal cells. It remains to be elucidated what neuronal mechanism actually underlies the rhythmicity in this neuronal model.

DISCUSSION Ionic and cellular mechanisms of hippocampal afterdischarge Based on the present results, we propose a hypothetical model for the seizure-like afterdischarge within the hippocampal CA1 network (Fig. 5). According to our model, intense GABAA receptor activation causes a transient Cl⫺ accumulation inside the postsynaptic pyramidal cells, which changes the GABAergic effect from inhibition to excitation. The extent of GABAergic excitation would be determined primarily by the balance between intracellular Cl⫺ accumulation via GABAA receptors (Staley et al., 1995) and subsequent Cl⫺ extrusion by cation-dependent chloride transporters such as KCC2 (Rivera et al., 1999) and by inwardly rectifying chloride channels such as CLC-2 (Smith et al., 1995). Different Cl⫺ balances among individual neurons might result in the diversity in depolar-

izing GABAergic responses (see Fig. 2, control experiments). Moreover, strong tetanization has been reported to change readily extracellular K⫹ and H⫹ concentrations (Kaila et al., 1997; Smirnov et al., 1999). However, it is quite unlikely that extracellular potassium accumulation might directly mediate fast oscillatory depolarization in a rhythmic manner, even if the accumulation can diminish the KCC2-driven Cl⫺ extrusion indirectly. As low KCC2-driven Cl⫺ extrusion results in persistent ⫺ Cl accumulation during development (Rivera et al., 1999), GABAA response is depolarizing in the immature pyramidal cells, such that its reversal potential is kept above the r.m.p. (Ben-Ari et al., 1989). But the afterdischarge we have defined, depending on excitatory GABAergic transmission, is not evoked within the first two postnatal weeks (see Fig. 4). This developmental pattern is very similar to that of low Mg2⫹-induced ictal epileptiform activity (Ko¨hling et al., 2000). The amount of GABA release or the number of GABAA receptors may be insufficient to produce a depolarizing GABAA response to tetanization in the immature hippocampus. Alternatively, the maturation of glutamatergic synapses might be necessary for the developmental appearance of afterdischarge. It should be noted here that the postnatal development of this seizurelike afterdischarge seems in parallel with that of long-term potentiation at glutamatergic synapses in the CA1 region (Isomura and Kato, 1999). Our recorded pyramidal cells might receive only a few glutamatergic inputs during the seizure-like rhythmic synchronization, because both CA3–CA1 connection (Schaffer collaterals) and entorhinal–CA1 connection (perforant path) were always cut in hippocampal CA1-isolated slices, and because the recurrent collaterals of the CA1 pyramidal cells, the only glutamatergic neurons in this region, rarely innervate other pyramidal cells directly (Amaral and Witter, 1995). Certainly, the direct connections among the pyramidal cells can participate in generation of a bicucullinedependent epileptic polysynaptic response (Cre´pel et al., 1997), but these connections did not seem to make a dominant contribution to the afterdischarge generation at least in our experimental model (see Fig. 3E). Instead, the pyramidal cells would receive substantial GABAergic inputs from some types of local interneurons within the CA1 region (Freund and Buzsa´ki, 1996). The excitatory GABAergic inputs synchronizing the pyramidal cells may originate from fast-spiking interneurons such as basket cells and chandelier cells (Buhl et al., 1994; Cobb et al., 1995; Sik et al., 1995; Ylinen et al., 1995), which may, in turn, receive glutamatergic recurrent inputs from the pyramidal cells (Freund and Buzsa´ki, 1996). Therefore, interconnected sets of pyramidal cells and these interneurons might form a positive-feedback circuit that produces rhythmic synchronization during the afterdischarge. This idea is well supported by our observation that a gradual cessation of pyramidal cell firing eventually attenuates afterdischarge expression (see Fig. 1C). Although such fast-spiking interneurons seem critical for the ‘synchronization,’ an additional neuronal mechanism must underlie the ‘rhythmicity’ of the afterdischarge in our network model. The rhythm

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generation might be achieved by intrinsic membrane potential oscillations in the pyramidal cells themselves (Leung and Yim, 1991) or the stratum lacunosum-moleculare interneurons (Chapman and Lacaille, 1999), or by slow, hyperpolarizing, GABAA-mediated inputs from these interneurons (Banks et al., 2000). Such possible rhythm generators of seizure-like afterdischarge could be shared, at least in part, with the generators of hippocampal ␪ rhythms (Bland and Colom, 1993; Freund and Buzsa´ki, 1996). Physiological and pathophysiological significances of excitatory GABAergic transmission There is growing evidence that GABAergic depolarization occurs naturally and functionally in the CNS of adult animals. In suprachiasmatic nucleus neurons, for instance, GABA acts as an excitatory transmitter during daytime, but an inhibitory transmitter at night (Wagner et al., 1997). Depolarizing GABAergic transmission can directly trigger low-threshold spikes that are related to rhythmic activity in reticular thalamic neurons (Bazhenov et al., 1999). GABAA-mediated excitatory responses are elicited in axotomized vagal motor neurons, which have a reduced expression of KCC2 (Nabekura et al., 2002). In the hippocampus, a positive shift in the GABAA reversal potential could function to gate each cycle of a cholinergic ␪-like oscillation, although the shift itself seems too small to drive the next cycle of the ␪ rhythm (Sun et al., 2001). Moreover, several endogenous steroids are capable of converting an inhibitory GABAA response to a large depolarizing response in the mature hippocampus (Burg et al., 1998). Thus, it can be considered that dynamic excitatory GABA transmissions play a critical role in controlling some normal brain functions. On the other hand, a series of pharmacological studies have shown that 4-AP application to hippocampal slices induces an epileptic, long-lasting depolarization (LLD) in the pyramidal cells, which occurs irregularly at less than 0.1 Hz and lasts for 600 –1500 ms (Perreault and Avoli, 1989, 1991). This 4-AP-induced LLD is blocked by GABAA receptor antagonists and by bicarbonate depletion, but not by NMDA or non-NMDA receptor antagonists (Perreault and Avoli, 1992; Lamsa and Kaila, 1997), although the LLD is apparently different from both seizure-like (ictal) and interictal epileptiform activities (Perreault and Avoli, 1992; Avoli et al., 1996). Recently, it was also shown that GABAergic transmission is essential for the generation of hippocampal ictal afterdischarge (Velazquez and Carlen, 1999; Higashima et al., 2000). Furthermore, Ko¨hling et al. (2000) have suggested that low-Mg2⫹-induced gamma oscillations and subsequent ictal activities might be facilitated partly by putative GABAergic depolarization. However, the functional role of excitatory GABAergic transmission in seizure-like (ictal) epileptiform activity has so far been unappreciated despite of numerous pathophysiological studies on temporal lobe epilepsy. Our findings clearly demonstrate that each cycle of the seizure-like afterdischarge is driven by powerful GABAA-mediated excitation of a number of pyramidal cells within the hippocampal CA1

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region. This novel cellular mechanism of rhythmic synchronization may apply as well to the seizure-like activity in other in vivo and in vitro experimental epilepsy models. GABAergic transmission in human temporal lobe epilepsy A loss of hippocampal GABAergic inhibition is believed to underlie the neuronal hyperexcitability in human temporal lobe epilepsy. In fact, it is well known that complete blockade of GABAA receptor can readily induce epileptiform activity in hippocampal slices (Borck and Jefferys, 1999). However, GABAergic neurons and their terminals containing glutamate decarboxylase are preserved in the human epileptic hippocampus (Babb et al., 1989), and the amount of GABA released in the hippocampus is increased during spontaneous seizures in human epilepsy patients (During and Spencer, 1993). And now, a loss of GABA transporters is thought to reduce non-synaptic GABA release by GABA transporter reversal in the hippocampus, leading to temporal lobe epilepsy (During et al., 1995). As a whole this hypothesis looks very attractive, but hippocampal GABA transporters are almost preserved in temporal lobe epilepsy patients (Mathern et al., 1999). In addition, the hypothesis is not sufficient to explain the neuronal mechanism of rhythmic synchronization in hippocampal seizure activity. As we have demonstrated here, excitatory GABAergic transmission may play rather an active role in generating rhythmic synchronization in the focus of an epileptic seizure, even though glutamatergic transmission also mediates synchronized fast propagation into surrounding or distant cortical regions. Interestingly, electrically evoked perceptual hallucinations and illusions, as seen in classical ‘Penfield’s experiments,’ are tightly associated with the occurrence of hippocampal afterdischarge in temporal lobe epilepsy patients (Gloor et al., 1982). The afterdischarge, like hippocampal ␪ rhythms (O⬘Keefe, 1993; Buzsa´ki, 2002), might also activate a neural network system underlying cognitive brain functions. Thus, our findings may shed light on the functional significance of dynamic GABAergic excitation in the normal and abnormal generation of synchronous network oscillations. Acknowledgements—This study was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Japan Science and Technology Corporation, and by Japan Society for Promotion of Science.

REFERENCES Alger BE, Nicoll RA (1979) GABA-mediated biphasic inhibitory responses in hippocampus. Nature 281:315–317. Amaral DG, Witter MP (1995) Hippocampal formation. In: The rat nervous system, 2nd edition (Paxinos G, ed), pp 443–493. San Diego: Academic Press. Andersen P, Dingledine R, Gjerstad L, Langmoen IA, Laursen AM (1980) Two different responses of hippocampal pyramidal cells to application of gamma-amino butyric acid. J Physiol (Lond) 305: 279 –296. Avoli M, Barbarosie M, Lu¨cke A, Nagao T, Lopantsev V, Ko¨hling R (1996) Synchronous GABA-mediated potentials and epileptiform

274

Y. Fujiwara-Tsukamoto et al. / Neuroscience 119 (2003) 265–275

discharges in the rat limbic system in vitro. J Neurosci 16:3912– 3924. Babb TL, Pretorius JK, Kupfer WR, Crandall PH (1989) Glutamate decarboxylase-immunoreactive neurons are preserved in human epileptic hippocampus. J Neurosci 9:2562–2574. Banks MI, White JA, Pearce RA (2000) Interactions between distinct GABAA circuits in hippocampus. Neuron 25:449 –457. Bazhenov M, Timofeev I, Steriade M, Sejnowski TJ (1999) Self-sustained rhythmic activity in the thalamic reticular nucleus mediated by depolarizing GABAA receptor potentials. Nat Neurosci 2:168 – 174. Ben-Ari Y, Cherubini E, Corradetti R, Gaiarsa J-L (1989) Giant synaptic potentials in immature rat CA3 hippocampal neurons. J Physiol (Lond) 416:303–325. Ben-Ari Y (2001) Developing networks play a similar melody. Trends Neurosci 24:353–360. Bland BH, Colom LV (1993) Extrinsic and intrinsic properties underlying oscillation and synchrony in limbic cortex. Prog Neurobiol 41: 157–208. Borck C, Jefferys JGR (1999) Seizure-like events in disinhibited ventral slices of adult rat hippocampus. J Neurophysiol 82:2130 –2142. Bormann J, Hamill OP, Sakmann B (1987) Mechanism of anion permeation through channels gated by glycine and ␥-aminobutyric acid in mouse cultured spinal neurones. J Physiol (Lond) 385:243–286. Bracci E, Vreugdenhil M, Hack SP, Jefferys JGR (1999) On the synchronizing mechanisms of tetanically induced hippocampal oscillations. J Neurosci 19:8104 –8113. Bragin A, Csicsva´ri J, Penttonen M, Buzsa´ki G (1997) Epileptic afterdischarge in the hippocampal-entorhinal system: current source density and unit studies. Neuroscience 76:1187–1203. Buhl EH, Halasy K, Somogyi P (1994) Diverse source of hippocampal unitary inhibitory postsynaptic potentials and the number of synaptic release sites. Nature 368:823–828. Burg M, Heinemann U, Schmitz D (1998) Neuroactive steroids induce GABAA receptor-mediated depolarizing postsynaptic potentials in hippocampal CA1 pyramidal cells of the rat. Eur J Neurosci 10: 2880 –2886. Buzsa´ki G (2002) Theta oscillations in the hippocampus. Neuron 33:325–340. Chapman CA, Lacaille J-C (1999) Intrinsic theta-frequency membrane potential oscillations in hippocampal CA1 interneurons of stratum lacunosum-moleculare. J Neurophysiol 81:1296 –1307. Cobb SR, Buhl EH, Halasy K, Paulsen O, Somogyi P (1995) Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons. Nature 378:75–78. Cre´pel V, Khazipov R, Ben-Ari Y (1997) Blocking GABAA inhibition reveals AMPA- and NMDA-receptor-mediated polysynaptic responses in the CA1 region of the rat hippocampus. J Neurophysiol 77:2071–2082. Draguhn A, Traub RD, Schmitz D, Jefferys JGR (1998) Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro. Nature 394:189 –192. During MJ, Ryder KM, Spencer DD (1995) Hippocampal GABA transporter function in temporal-lobe epilepsy. Nature 376:174 –177. During MJ, Spencer DD (1993) Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet 341:1607–1610. Freund TF, Buzsa´ki G (1996) Interneurons of the hippocampus. Hippocampus 6:347–470. Fukuda T, Kosaka T (2000) Gap junctions linking the dendritic network of GABAergic interneurons in the hippocampus. J Neurosci 20: 1519 –1528. Gloor P, Olivier A, Quesney LF, Andermann F, Horowitz S (1982) The role of the limbic system in experimental phenomena of temporal lobe epilepsy. Ann Neurol 12:129 –144. Grover LM, Lambert NA, Schwartzkroin PA, Teyler TJ (1993) Role of

HCO3⫺ ions in depolarizing GABAA receptor-mediated responses in pyramidal cells of rat hippocampus. J Neurophysiol 69:1541– 1555. Goddard G, McIntyre D, Leech C (1969) A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 25: 295–330. Higashima M, Ohno K, Kinoshita H, Koshino Y (2000) Involvement of GABAA and GABAB receptors in afterdischarge generation in rat hippocampal slices. Brain Res 865:186 –193. Isomura Y, Fujiwara-Tsukamoto Y, Imanishi M, Nambu A, Takada M (2002) Distance-dependent Ni2⫹-sensitivity of synaptic plasticity in apical dendrites of hippocampal CA1 pyramidal cells. J Neurophysiol 87:1169 –1174. Isomura Y, Kato N (1999) Action potential-induced calcium dynamics correlated with synaptic plasticity in developing hippocampal pyramidal cells. J Neurophysiol 82:1993–1999. Kaila K, Lamsa K, Smirnov S, Taira T, Voipio J (1997) Long-lasting GABA-mediated depolarization evoked by high-frequency stimulation in pyramidal neurons of rat hippocampal slice is attributable to a network-driven, bicarbonate-dependent K⫹ transient. J Neurosci 17:7662–7672. Ko¨hling R, Vreugdenhil M, Bracci E, Jefferys JGR (2000) Ictal epileptiform activity is facilitated by hippocampal GABAA receptor-mediated oscillations. J Neurosci 20:6820 –6829. Lamsa K, Kaila K (1997) Ionic mechanisms of spontaneous GABAergic events in rat hippocampal slices exposed to 4-aminopyridine. J Neurophysiol 78:2582–2591. Leinekugel X, Khazipov R, Cannon R, Hirase H, Ben-Ari Y, Buzsa´ki G (2002) Correlated bursts of activity in the neonatal hippocampus in vivo. Science 296:2049 –2052. Leung L-WS, Yim C-YC (1991) Intrinsic membrane potential oscillations in hippocampal neurons in vitro. Brain Res 553:261–274. Mathern GW, Mendoza D, Lozada A, Pretorius JK, Dehnes Y, Danbolt NC, Nelson N, Leite JP, Chimelli L, Born DE, Sakamoto AC, Assirati JA, Fried I, Peacock WJ, Ojemann GA, Adelson PD (1999) Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy. Neurology 52:453–472. McNamara JO (1994) Cellular and molecular basis of epilepsy. J Neurosci 14:3413–3425. Mody I, Lambert JDC, Heinemann U (1987) Low extracellular magnesium induces epileptiform activity and spreading depression in rat hippocampal slices. J Neurophysiol 57:869 –888. Nabekura J, Ueno T, Okabe A, Furuta A, Iwaki T, Shimizu-Okabe C, Fukuda A, Akaike N (2002) Reduction of KCC2 expression and GABAA receptor-mediated excitation after in vivo axonal injury. J Neurosci 22:4412–4417. O'Keefe J (1993) Hippocampus, theta, and spatial memory. Curr Opin Neurobiol 3:917–924. Perkins KL, Wong RKS (1996) Ionic basis of the postsynaptic depolarizing GABA response in hippocampal pyramidal cells. J Neurophysiol 76:3886 –3894. Perreault P, Avoli M (1989) Effects of low concentrations of 4-amminopyridine on CA1 pyramidal cells of the hippocampus. J Neurophysiol 61:953–970. Perreault P, Avoli M (1991) Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices. J Neurophysiol 65:771–785. Perreault P, Avoli M (1992) 4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus. J Neurosci 12:104 –115. Rafiq A, DeLorenzo RJ, Coulter DA (1993) Generation and propagation of epileptiform discharges in a combined entorhinal cortex/ hippocampal slice. J Neurophysiol 70:1962–1974. Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa K, Pirvola U, Saarma M, Kaila K (1999) The K⫹/Cl⫺ co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation. Nature 397:251–255. Schmitz D, Schuchmann S, Fisahn A, Draguhn A, Buhl EH, Petrasch-

Y. Fujiwara-Tsukamoto et al. / Neuroscience 119 (2003) 265–275 Parwez E, Dermietzel R, Heinemann U, Traub RD (2001) Axoaxonal coupling: a novel mechanism for ultrafast neuronal communication. Neuron 31:831–840. Sik A, Penttonen M, Ylinen A, Buzsa´ki G (1995) Hippocampal CA1 interneurons: an in vivo intracellular labeling study. J Neurosci 15:6651–6665. Smirnov S, Paalasmaa P, Uusisaari M, Voipio J, Kaila K (1999) Pharmacological isolation of the synaptic and nonsynaptic components of the GABA-mediated biphasic response in rat CA1 hippocampal pyramidal cells. J Neurosci 19:9252–9260. Smith RL, Clayton GH, Wilcox CL, Escudero KW, Staley KJ (1995) Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific modulation of postsynaptic inhibition. J Neurosci 15:4057–4067. Staley KJ, Soldo BL, Proctor WR (1995) Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors. Science 269:977–981. Staley KJ, Proctor WR (1999) Modulation of mammalian dendritic GABAA receptor function by the kinetics of Cl⫺ and HCO3⫺ transport. J Physiol (Lond) 519:693–712. Stasheff SF, Anderson WW, Clark S, Wilson WA (1989) NMDA antagonists differentiate epileptogenesis from seizure expression in an in vitro model. Science 245:648 –651. Stasheff SF, Hines M, Wilson WA (1993a) Axon terminal hyperexcitability associated with epileptogenesis in vitro: I. Origin of ectopic spikes. J Neurophysiol 70:961–975. Stasheff SF, Mott DD, Wilson WA (1993b) Axon terminal hyperexcit-

275

ability associated with epileptogenesis in vitro: II. Pharmacological regulation by NMDA and GABAA receptors. J Neurophysiol 70: 976 –984. Swann JW, Smith KL, Brady RJ (1993) Localized excitatory synaptic interactions mediate the sustained depolarization of electrographic seizures in developing hippocampus. J Neurosci 13:4680 –4689. Sun M-K, Zhao W-Q, Nelson TJ, Alkon DL (2001) Theta rhythm of hippocampal CA1 neuron activity: gating by GABAergic synaptic depolarization. J Neurophysiol 85:269 –279. Tancredi V, Hwa GGC, Zona C, Brancati A, Avoli M (1990) Low magnesium epileptogenesis in the rat hippocampal slice: electrophysiological and pharmacological features. Brain Res 511:280 – 290. Traub RD, Jefferys JGR, Whittington MA (1994) Enhanced NMDA conductance can account for epileptiform activity induced by low Mg2⫹ in the rat hippocampal slice. J Physiol (Lond) 478:379 –393. Velazquez JLP, Carlen PL (1999) Synchronization of GABAergic interneuronal networks during seizure-like activity in the rat horizontal hippocampal slice. Eur J Neurosci 11:4110 –4118. Wagner S, Castel M, Gainer H, Yarom Y (1997) GABA in the mammalian suprachiasmatic nucleus and its role in diurnal rhythmicity. Nature 387:598 –603. Ylinen A, Solte´sz I, Bragin A, Penttonen M, Sik A, Buzsa´ki G (1995) Intracellular correlates of hippocampal theta rhythm in identified pyramidal cells, granule cells, and basket cells. Hippocampus 5:78 –90.

(Accepted 13 January 2003)