Synaptic interactions between pyramidal cells and interneurone subtypes during seizure-like activity in the rat hippocampus

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1 J Physiol (2004) pp Synaptic interactions between pyramidal cells and interneurone subtypes during seizure-like activity in the rat hippocampus Yoko Fujiwara-Tsukamoto 1,2, Yoshikazu Isomura 1,2,3, Katsuyuki Kaneda 1,2,3 and Masahiko Takada 1,2 1 Department of System Neuroscience, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo , Japan 2 CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama , Japan 3 The Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo , Japan We have recently reported that excitatory GABAergic and glutamatergic mechanisms may be involved in the generation of seizure-like (ictal) rhythmic synchronization (afterdischarge), induced by a strong synaptic stimulation of the CA1 pyramidal cells in the mature rat hippocampus in vitro. To clarify the network mechanism of this neuronal synchronization, dual whole-cell patch-clamp recordings of the afterdischarge responses were performed simultaneously from a variety of interneurones and their neighbouring pyramidal cells in hippocampal CA1-isolated slice preparations. According to morphological and electrophysiological criteria, the recorded interneurones were then classified into distinct subtypes. The non-fast-spiking interneurones located in the strata lacunosum-moleculare and radiatum hardly discharged during the afterdischarge, whereas most of the fast-spiking and non-fastspiking interneurones in the strata oriens and pyramidale, including the basket, chandelier and bistratified cells, exhibited prominent firings that were precisely synchronous with oscillatory responses in the pyramidal cells. Field potential recordings showed that excitatory synaptic transmissions might take place primarily in the strata oriens and pyramidale during the afterdischarge. Restricted lesions in the strata oriens and pyramidale, but not in the other layers, resultedinthecompletedesynchronizationofafterdischargeactivity,andalso,localapplication of glutamate receptor antagonists to these layers blocked the expression of afterdischarge. The present findings indicate that the neuronal synchronization of epileptic afterdischarge may be accomplished in a positive feedback circuit formed by the excitatory GABAergic interneurones and the glutamatergic pyramidal cells within the strata oriens and/or pyramidale of the hippocampal CA1 region. (Received 16 December 2003; accepted after revision 22 April 2004; first published online 23 April 2004) Corresponding author Y. Isomura: Department of System Neuroscience, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo , Japan. isomura@tmin.ac.jp Numerous investigations have been focused on the mechanisms or functions of synchronous and oscillatory phenomena in mammalian brains in physiological or pathological states, including theta and gamma oscillations and epileptic seizure (ictal) activity. In the hippocampus, an intense electrical stimulation (tetanization) readily induces a long-lasting rhythmic synchronization (seizure-like afterdischarge) in the rat in vitro (Stasheff et al. 1989, 1993a,b; Rafiq et al. 1993) and in vivo (Bragin et al. 1997a,b), and in human epilepsy patients (Gloor et al. 1982). The hippocampal seizurelike afterdischarge displays all-or-none triggering, 3 5 Hz Y. Fujiwara-Tsukamoto and Y. Isomura contributed equally to this work. frequency, and tonic- and clonic-like phases. It is therefore considered an excellent experimental model for seizure activity in human temporal lobe epilepsy (Stasheff et al. 1989). Several pharmacological studies suggested that GABAergic transmission might essentially contribute to the generation of this afterdischarge (Higashima et al. 1996; Klapstein & Colmers, 1997; Perez Velazquez & Carlen, 1999; Higashima et al. 2000). As an intense GABA A receptor activation during the tetanization occasionally makes the GABA A response depolarizing in mature hippocampal pyramidal cells (Alger & Nicoll, 1979; Andersen et al. 1980; Grover et al. 1993; Staley et al. 1995; Perkins & Wong, 1996; Kaila et al. 1997; Smirnov et al. DOI: /jphysiol

2 962 Y. Fujiwara-Tsukamoto and others J Physiol ; Staley & Proctor, 1999; Isomura et al. 2003b) and interneurones (Lamsa & Taira, 2003), it is quite possible that GABA A -mediated excitation, rather than inhibition, may be involved in neuronal synchronization of the afterdischarge. In fact, our previous study has shown that each cycle of the afterdischarge is directly driven by excitatory GABAergic transmission per se in mature hippocampal pyramidal cells (Fujiwara-Tsukamoto et al. 2003). On the other hand, it has been assumed that glutamatergic transmission mediated through the AMPAtype and NMDA-type receptors is also required for afterdischarge generation within a local network of the hippocampal CA1 region (Fujiwara-Tsukamoto et al. 2003), as well as for its propagation into other hippocampal regions (Isomura et al. 2003a). The pyramidal cells are the only (or major) glutamatergic neurones in the hippocampal CA1 region, and a number of GABAergic interneurones are innervated by the recurrent collaterals of the pyramidal cells in this region (Freund & Buzsáki, 1996). Therefore, synaptic interactions between the glutamatergic pyramidal cells and the GABAergic interneurones would play a critical role in the generation of afterdischarge in the local CA1 network (Perez Velazquez & Carlen, 1999). However, it remains unknown which subtype(s) of interneurones may be responsible for such interactions and how they may actually communicate with the pyramidal cells to generate the rhythmic synchronization in the hippocampal seizure-like afterdischarge. To elucidate the cooperative GABAergic and glutamatergic mechanism of afterdischarge generation at a network level, we analysed the interneurone subtype and functional circuit that participate in the rhythmic synchronization by recording afterdischarge responses evoked in electrophysiologically or morphologically different interneurones, as well as the neighbouring pyramidal cells, in rat hippocampal CA1-isolated slice preparations. Methods All experiments were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute for Neuroscience, and carried out in accordance with the Guidelines for Care and Use of Animals (Tokyo Metropolitan Institute for Neuroscience 2000). Slice preparation Hippocampal slices (400 µm thick) were prepared with a microslicer (DTK-1500; Dosaka EM, Kyoto, Japan) from Wistar rats (postnatal days) which were deeply anaesthetized with diethyl ether gas and then decapitated. The CA1 region was routinely isolated from the CA3 and subiculum regions (the hippocampal CA1-isolated slice; see Fig.1Aa), to prevent any external input from intruding through the Schaffer collateral, the perforant path, or other pathways. The stratum (s.) oriens and s. pyramidale or the s. lacunosum-moleculare (and a part of s. radiatum) of the CA1 region were lesioned with a surgical knife in some experiments. After at least 1 h of recovery, each slice was transferred to a submergedtype recording chamber continuously circulated with normal artificial cerebrospinal fluid (ACSF; C), which consisted of (mm): 124 NaCl, 2.5 KCl, 1.2 KH 2 PO 4, 26 NaHCO 3, 1.2 MgSO 4, 2.5 CaCl 2 and 25 d-glucose, and was saturated with 95% O 2 :5% CO 2 gas (Fujiwara- Tsukamoto et al. 2003; Isomura et al. 2003a,b). Electrophysiological recordings To induce seizure-like afterdischarge, tetanic stimulation (100 Hz for 0.5 s; intensity 400 µa, duration 400 µs) was delivered through a monopolar glass stimulating electrode (0.5 1 M, filled with 2.5 m NaCl) placed in the s. radiatum, unless otherwise stated. Whole-cell patch-clamp recordings were obtained simultaneously from two hippocampal neurones (mostly pairs of a pyramidal cell and an interneurone) in the hippocampal CA1-isolated slices under visual guidance. The membrane potentials of each neurone pair were recorded with patchclamp amplifiers (Axopatch 1D and Axopatch 200B; Axon Instruments, Union City, CA, USA) through glass patch electrodes filled with an internal solution containing (mm): 140 potassium gluconate, 2 NaCl, 1 MgCl 2, 10 Hepes, 0.2 EGTA, 2 5 -ATP Na 2, 0.5 GTP Na 2 and biocytin (ph 7.4; 5 10 M ) (Fujiwara-Tsukamoto et al. 2003). Electrophysiological parameters, such as firing activity and input resistance, were measured by depolarizing or hyperpolarizing current injections (± pa, 500 ms duration; see Table 1 for details). At the end of the recordings, biocytin was loaded into the recorded neurones by repetitive current injection (+300 pa, 500 ms at 1 Hz for 15 min), followed by further incubation for min in the ACSF. In some experiments, field potentials were recorded with the amplifier(s) through glass electrode(s) (2 5 M, filled with 2.5 m NaCl) (Isomura et al. 2002). 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 application A mixture of dl-2-amino-5-phosphonopentanoic acid (dl-ap-5; 5 mm) and 6-cyano-7-nitroquinoxaline-2,3- dione (CNQX; 1 mm) in saline (ph 7.4) was injected

3 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 963 Figure 1. Seizure-like afterdischarge in hippocampal pyramidal cells and lacunosum-moleculare (LM) interneurones A, simultaneous membrane potential recordings from two pyramidal cells. Aa, schematic diagram of dual whole-cell recordings in hippocampal CA1-isolated slice. Rec., recording electrodes; Stim., stimulating electrode. DG, dentate gyrus; S, subiculum. Ab, morphology of recorded pyramidal cells (PC1, black; PC2, grey). s.l.m., s. lacunosummoleculare; s.r., s. radiatum; s.p., s. pyramidale; s.o., s. oriens; alv., alveus. Ac, membrane potential responses (V m ) to positive and negative current injections (I). Note the prominent spike accommodation. Ad, post-tetanic depolarization (arrows) and seizure-like afterdischarge (dots) in the pyramidal cells. Downward artifacts during tetanization (Tetanus; 100 Hz, 0.5 s) are truncated. Inset, single oscillatory responses of the afterdischarge, specified by the filled triangle in the left traces. B, simultaneous recordings from an LM cell and a pyramidal cell. Ba, recorded LM cell (IN) and pyramidal cell (PC). Bb, firing patterns (upper traces, V m ) and mean firing rates (lower plot) in response to positive current injections (I) in the LM cell. Note the prominent after-hyperpolarization (AHP) following each action potential. Bc, hyperpolarizing responses to negative current injections (left) and I V relation (right; dots, peak values; bars, stable values) in the LM cell. Bd, no, or only a small, afterdischarge response in the LM cell. Inset, single oscillatory responses specified by the filled triangle in the left traces.

4 964 Y. Fujiwara-Tsukamoto and others J Physiol Table 1. Electrophysiological characterization of pyramidal cells and FS/non-FS interneurones Pyramidal Non-FS Non-FS Non-FS FS Non-FS FS (s.p.) (s.l.m.) (s.r.) (s.p.) (s.p.) (s.o.) (s.o.) n 20 21(12) 25(20) 12(5) 5(1) 30(13) 15(13) RMP 1 (mv) 59.9 ± ± ± ± ± ± ± 6.3 R 2 input (M ) 45.3 ± ± ± ± ± ± ± 39.1 Discharge rate 3 (Hz) 37.2 ± ± ± ± ± ± ± 18.1 Adaptation 4 (%) 33.1 ± ± ± ± ± ± ± 7.9 AP width 5 (ms) 1.33 ± ± ± ± ± ± ± 0.13 AHP 6 (mv) 2.3 ± ± ± ± ± ± ± 2.9 AHP duration 7 (ms) 3.7 ± ± ± ± ± ± ± 10.9 A total of 108 recorded interneurones (including 64 morphologically identified interneurones, numbers in parentheses) are classified by discharge rate 3 and somatic location (s.l.m., s. lacunosum-moleculare; s.r., s. radiatum; s.p., s. pyramidale; s.o., s. oriens). FS, fast-spiking interneurone (> 100 Hz per 0.5 na injection); non-fs, non-fast-spiking interneurone (< 100 Hz). 1 RMP, resting membrane potential; 2 R input, input resistance calculated by linear regression from peak voltage changes evoked by negative current injections ( pa). 3 Mean discharge rate per 0.5 na, calculated by linear regression from the numbers of action potentials evoked by positive current injections ( pa for 500 ms). 4 Adaptation (accommodation), ratio of mean discharge frequency of the 11th 15th action potentials to that of the 1st 5th action potentials. 5 Duration of action potential (AP), measured at half-amplitude. 6 Amplitude of after-hyperpolarization (AHP) following single action potential generation. 7 Duration of the AHP, measured at half-amplitude. Each value is expressed as the mean ± S.D. In each row, the values in italic style are significantly different from those of pyramidal cells (P < 0.01). For FS interneurones in s.p. and s.o., the values are significantly different from those of non-fs interneurones in the same layers (P < 0.01). locally to the s. radiatum or the s. oriens/pyramidale by pressure (Picospritzer II; General Valve, Fairfield, NJ, USA; 5 10 p.s.i., 1 2 s) through a glass capillary (tip diameter, 1 2 µm). Both dl-ap-5 and CNQX were purchased from Tocris Cookson, Ballwin, MO, USA. In our preliminary experiment using a dye, the drug injection was expected toinvolveanareaof µm in diameter. As a control, we also confirmed that vehicle injection had no obvious effects on afterdischarge generation. Morphological analysis. For visualizing biocytin-loaded recorded neurones, slices were fixed in 10% formalin in phosphate-buffered saline (PBS) for at least 24 h. The whole slices were immersed, without resectioning, in 0.05 m Tris-HCl buffer (ph 8.0) containing 30% sucrose for 2 3 h, and slowly frozen and thawed twice. The slices were pre-treated with 0.3% H 2 O 2 in Tris-HCl buffer for 20 min at room temperature to diminish endogenous peroxidase activity, rinsed three times in Tris-buffered saline (TBS; ph 8.0), and incubated overnight with avidin biotin horseradish peroxidase (HRP) complex (Vectastain Elite ABC kit; Vector, Burlingame, CA, USA; 200-fold dilution) in TBS containing 0.5% Triton X-100 at 4 C. After washes in 0.05 m Tris-HCl buffer, the slices were pre-incubated with 0.04% NiCl 2 and 0.04% diaminobenzidine (DAB; Sigma, St Louis, MO, USA) in Tris-HCl buffer for 20 min, and H 2 O 2 (final concentration, 0.003%) was added to permit peroxidase reaction for 5 15 min at room temperature. The reaction was ceased by washing the slices with Tris-HCl buffer. The slices were then embedded in Crystal/Mount (Biomeda, Foster City, CA, USA) on glass slides and coverslipped, without dehydration, for digital photography and camera lucida reconstruction under a light microscope. Data analyses All data in the text are expressed as the mean ± s.d., and Student s t test or ANOVA was applied for statistical comparisons. Results Synaptic induction of hippocampal seizure-like afterdischarge As shown in our previous studies (Fujiwara-Tsukamoto et al. 2003; Isomura et al. 2003a), a strong synaptic stimulation (tetanization) induced long-lasting, GABAergic/glutamatergic rhythmic synchronization among the pyramidal cells (seizure-like afterdischarge) in mature hippocampal CA1-isolated slice preparations in normal ACSF. Field potential recordings revealed that the afterdischarge induced by the strong tetanization was synchronized broadly in the whole CA1 region, and

5 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 965 Figure 2. Seizure-like afterdischarge in bistratified and putative chandelier interneurones A, simultaneous recordings from a bistratified cell and a pyramidal cell. Aa, recorded bistratified cell (IN, black) and pyramidal cell (PC, grey). Ab, firing patterns (upper traces, V m ) and mean firing rates (lower plot) in response to positive current injections (I) in the bistratified cell. Note the prominent AHP and fast-spiking properties of this interneurone. Ac, hyperpolarizing responses to negative current injections (left) and I V relation (right; dots, peak values; bars, stable values) in the bistratified cell. Ad, robust seizure-like afterdischarge in the bistratified cell. Inset, single oscillatory responses of the afterdischarge, specified by the filled triangle in the left traces. B, simultaneous recordings from a putative chandelier cell and a pyramidal cell. Ba, recorded putative chandelier cell (IN) and pyramidal cell (PC). Bb, firing patterns (upper traces) and mean firing rates (lower plot) in response to positive current injections in the putative chandelier cell. Note the prominent AHP and fast-spiking properties of this interneurone. Bc, hyperpolarizing responses to negative current injections (left) and I V relation (right) in the putative chandelier cell. Bd, robust seizure-like afterdischarge in the putative chandelier cell. Inset, single oscillatory responses of the afterdischarge, specified by the filled triangle in the left traces.

6 966 Y. Fujiwara-Tsukamoto and others J Physiol Figure 3. Seizure-like afterdischarge in oriens-lacunosum-moleculare (O-LM) and trilaminar interneurones A, simultaneous recordings from an O-LM cell and a pyramidal cell. Aa, recorded O-LM cell (IN, black) and pyramidal cell (PC, grey). Ab, firing patterns (upper traces, V m ) and mean firing rates (lower plot) in response to positive current injections (I) in the O-LM cell. Ac, hyperpolarizing responses to negative current injections (left) and I V relation (right; dots, peak values; bars, stable values) in the O-LM cell. Note the prominent sag of the responses, reflecting I h current. Ad, seizure-like afterdischarge in the O-LM cell. Inset, single oscillatory responses of the afterdischarge, specified by the filled triangle in the left traces. B, simultaneous recordings from a trilaminar cell and a pyramidal cell. Ba, recorded trilaminar cell (IN) and pyramidal cell (PC). Bb, firing patterns (upper traces) and mean firing rates (lower plot) in response to positive current injections in the trilaminar cell. Bc, hyperpolarizing responses to negative current injections (left) and I V relation (right) in the trilaminar cell. Bd, seizure-like afterdischarge in the trilaminar cell. Inset, single oscillatory response of the afterdischarge, specified by the filled triangle in the left traces.

7 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 967 that the largest negative peak of oscillatory afterdischarge responses, which were much slower than population spikes, was observed in s. oriens or s. pyramidale in all of the slices examined (n = 6; data not shown), suggesting that most excitatory synaptic transmissions (and/or nonsynaptic local excitations) may occur in these laminar structures during the afterdischarge. Such spatial profiles of the extracellular afterdischarge responses were always constant irrespective of the stimulating sites for induction (s. oriens or s. pyramidale; n = 4). Hereafter, the seizurelike afterdischarge was induced by tetanizing the middle portion of the s. radiatum in the CA1 region. Seizure-like afterdischarge in different identified interneurones To examine the network mechanism of the seizurelike afterdischarge, we recorded the changes in the membrane potential during the afterdischarge in a variety of hippocampal neurones (20 pyramidal cells and 108 interneurones) in the strata lacunosum-moleculare, radiatum, pyramidale and oriens of the CA1 region, in combination with the neighbouring pyramidal cells in s. pyramidale (Fig. 1Aa). Of the 108 interneurones recorded, 64 well-visualized ones were morphologically identified as lacunosum-moleculare (LM) cells (n = 12), Figure 4. Discharge activities during seizure-like afterdischarge in pyramidal cells and interneurones A, temporal changes in the mean number of action potentials per oscillatory response (cycle) during the afterdischarge in the pyramidal cell and interneurone groups. The interneurones were classified into six distinct subtypes according to their firing properties (fast-spiking, FS, or non-fast-spiking, non-fs) and somatic locations (s.l.m., s.r., s.p., or s.o.) (see Table 1). Dots and error bars indicate the mean and S.D., respectively. B, temporal changes in the mean discharge probability per oscillatory response (cycle) during the afterdischarge in these neurone groups. Dots and error bars indicate the mean and S.E.M., respectively. Only a small number of action potentials were generated during the afterdischarge in the non-fs interneurones in s. lacunosum-moleculare and s. radiatum, as compared to both the FS and the non-fs interneurones in s. pyramidale and s. oriens.

8 968 Y. Fujiwara-Tsukamoto and others J Physiol bistratified cells (n = 4), basket/chandelier (axo-axonic) cells (n = 11), oriens-lacunosum-moleculare (O-LM) cells (n = 2), and other subtypes of cells (n = 35) (Sik et al. 1995; Freund & Buzsáki, 1996). The last group (other sub- types) of cells includes trilaminar cells as well as various monolaminar and bilaminar cells, which have not been classified further by the conventional morphological definition (Parra et al. 1998). Figure 5. Relative discharge timings between pyramidal cells and interneurones during seizure-like afterdischarge A, histograms show averaged discharge probability (P, in 1 ms bins) in the pyramidal cell and interneurone groups, aligned with the onset of oscillatory depolarizing responses (time lag T = 0 ms) in a simultaneously recorded pyramidal cell as a standard neurone during the afterdischarge (8 20 s). Upward columns indicate the probabilities of the first (black) and total (grey) action potentials in relation to the oscillatory responses in test neurones (pyramidal cells or interneurones), and downward black columns indicate those of the first action potentials in relation to the oscillatory responses in the standard pyramidal cells. The discharges in most of the interneurones examined were time-locked to those in the simultaneously recorded pyramidal cells; the interneurones in the s. pyramidale and s. oriens particularly exhibited robust spiking during the afterdischarge. B, upper panels, superimposed traces of the first action potentials in a non-fs (s.r.; left) and an FS (s.p.; right) interneurone, aligned with the onset of the first action potentials in standard pyramidal cells (PC) (dotted lines) during the afterdischarge. Lower panel, temporal changes in relative discharge timing of the non-fs ( ) and FS ( ) interneurones to the pyramidal cells in the time course of afterdischarge.

9 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 969 In a typical pyramidal cell, zero, one, or two action potentials synchronous with those in another pyramidal cell were elicited in each oscillatory depolarizing response during the seizure-like afterdischarge (Fig. 1Ab d), as previously reported (Fujiwara-Tsukamoto et al. 2003). The generation of action potentials was greatly depressed during large post-tetanic depolarization prior to the appearance of afterdischarge in the pyramidal cells, probably owing to a strong GABA A -mediated depolarizing inhibition (i.e. shunting effect; see Alger & Nicoll, 1979) which lasted for several seconds after the tetanization (Fig. 1Ad). The interneurone illustrated in Fig. 1B is an LM cell which was located horizontally around the border between s. lacunosum-moleculare and s. radiatum (Fig. 1Ba; Chapman & Lacaille, 1999). This LM neurone, which had the properties of a non-fast-spiking (non-fs) neurone (Fig. 1Bb and c), exhibited no, or only small, oscillatory responses without spiking during the afterdischarge (Fig. 1Bd). Six of 12 LM cells never discharged and the others displayed only a small number of action potentials while the afterdischarge was expressed in the pyramidal cells. The two interneurones illustrated in Fig. 2 are a bistratified cell and a putative chandelier cell with fastspiking (FS) properties (Fig. 2Ab and c, and Bb and c), both of which were located around the border between s. oriens and s. pyramidale (Fig. 2Aa and Ba). The bistratified cell spread its axon broadly into s. radiatum and s. oriens, but not into s. pyramidale (see Buhl et al. 1996), while the extension of the chandelier cell axon was restricted to the border between s. pyramidale and s. oriens (Buhl et al. 1994b). These interneurones exhibited robust bursting responses (3 8action potentials) during the afterdischarge, which were synchronous with oscillatory responses in the simultaneously recorded pyramidal cells (Fig. 2Ad and Bd). The first discharge of their burst responses frequently preceded the onset of the oscillatory response in the pyramidal cells. These interneurones also discharged at a very high frequency during the preceding post-tetanic depolarization, unlike the pyramidal cells. Three of four bistratified cells and 7 of 11 basket/chandelier cells displayed an outstanding generation of burst spikes during the afterdischarge. Figure 3 shows two representative non-fs interneurones, an O-LM cell in the s. oriens and a trilaminar cell in the s. radiatum. The O-LM cell extended its axon directly towards the s. lacunosum-moleculare, where the axonal arborization developed horizontally (Fig. 3Aa). The O-LM cell discharged at a relatively low frequency, and characteristically displayed a hyperpolarizationactivated cation current (I h )-dependent response to hyperpolarizing current injections (Fig. 3Ab and c; Maccaferri & McBain, 1996). This O-LM cell exhibited one or two action potentials per oscillatory response during the afterdischarge, which were time-locked to oscillatory responses in the nearby pyramidal cell (Fig. 3Ad). Similar results were obtained in the other recorded O-LM cell. The trilaminar cell spread its axon into the three laminar structures of s. radiatum, s. pyramidale and s. oriens (Fig. 3Ba; Pawelzik et al. 2002). This trilaminar cell also displayed steady oscillatory discharges synchronous with those in the pyramidal cell during the afterdischarge (Fig. 3Bb d). Thus, the afterdischarge responses were prominent in FS-type interneurones and also in non-fs-type interneurones within s. pyramidale and s. oriens, whereas they were very weak in non-fs-type interneurones in s. lacunosum-moleculare. Figure 6. Seizure-like afterdischarge in morphologically identified interneurones Left, somatic location plotted against the mean number of action potentials per oscillatory response (cycle) during the afterdischarge (8 20 s; see Fig. 4) in morphologically identified interneurones (blue dots, LM cells; red, bistratified cells; orange, basket/chandelier cells; light blue, O-LM cells; green, trilaminar or other cells). Right, somatic location plotted against the relative discharge timing during the afterdischarge (as defined in Fig. 5) in these identified interneurones. Dots and error bars indicate the mean and S.D., respectively.

10 970 Y. Fujiwara-Tsukamoto and others J Physiol 557.3

11 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 971 Layer-dependent interneurone activity during seizure-like afterdischarge The results obtained from our dual whole-cell patch-clamp recordings suggest that afterdischarge responses in the hippocampal interneurones may be dependent on their intrinsic firingability aswellasontheirsomaticlocationin the CA1 region. Therefore, the 108 recorded interneurones were classified into six distinct groups according to their electrophysiological properties and somatic locations (Table 1; FS interneurones in s. pyramidale (FS (s.p.), n = 5) and s. oriens (FS (s.o.), n = 15); non-fs interneurones in s. lacunosum-moleculare (non-fs (s.l.m.), n = 21), s. radiatum (non-fs (s.r.), n = 25), s. pyramidale (non-fs (s.p.), n = 12), and s. oriens (non-fs (s.o.), n = 30)). Besides these classified cells, only one FS interneurone was found in the s. radiatum in the present experiments (data not shown). We defined the FS and non-fs cells using the criterion of whether or not the mean discharge rate was higher than 100 Hz per 0.5 na current (see Table 1). The amplitudes of after-hyperpolarization (AHP) in all the interneurone groups were significantly larger than those in the pyramidal cells, and also, the FS interneurone groups displayed less spike adaptation (accommodation) and shorter spike width than the non-fs interneurone groups and the pyramidal cells (Table 1), suggesting that the criterion used here was valid. Given that oscillatory afterdischarge responses in the pyramidal cells are driven by excitatory GABAergic inputs, their presynaptic GABAergic interneurones should generate action potentials synchronously. In fact, action potentials in most of the recorded interneurones were synchronous with oscillatory responses in the pyramidal cells during the afterdischarge. However, the mean discharge number per oscillatory response (or the oscillation cycle in the afterdischarge) was very different among the six interneurone groups (Fig. 4A; (in spikes cycle 1 ) non-fs (s.l.m.) interneurones, 0.21 ± 0.38; non-fs (s.r.), 0.58 ± 0.63; non-fs (s.p.), 2.25 ± 2.13; FS (s.p.), 3.70 ± 2.26; non-fs (s.o.), 1.68 ± 1.26; FS (s.o.), 4.20 ± 2.71; one-way ANOVA, P < 0.001). The non-fs (s.l.m.) interneurone group generated much fewer action potentials during the afterdischarge than all of the non-fs (s.p.), non-fs (s.o.), FS (s.p.), and FS (s.o.) interneurone groups (Duncan s multiple range test, at most P < 0.01). The non-fs (s.r.) interneurone group also generated fewer action potentials than all of these groups (P < 0.01). Similarly, the discharge probabilities in oscillatory response were different among the interneurone groups (Fig. 4B; non-fs (s.l.m.) interneurones, 17.1 ± 31.4%; non-fs (s.r), 42.8 ± 42.2%; non-fs (s.p.), 79.2 ± 27.6%; FS (s.p.), 88.9 ± 21.3%; non-fs (s.o.), 71.2 ± 37.1%; FS (s.o.), 86.9 ± 27.0%; one-way ANOVA, P < 0.001). Both of the non-fs (s.l.m.) and the non-fs (s.r.) interneurone groups fired during the afterdischarge at significantly lower probabilities than all of the non-fs (s.p.), non-fs (s.o.), FS (s.p.), and FS (s.o.) groups (Duncan s multiple range test, P < 0.01 each, except P < 0.05 for comparison of non-fs (s.r.) versus FS (s.p.)). Thus, along with the FS interneurones, the non-fs interneurones in s. pyramidale and s. oriens were apparently much more active than those in s. lacunosum-moleculare and s. radiatum during the afterdischarge, although the basic firing properties were very similar among the four non-fs interneurone groups (Table 1). Furthermore, we analysed the temporal firing patterns during the afterdischarge in these interneurone groups. As shown in Fig. 5A, the discharge timings in most of the active interneurones were precisely time-locked to Figure 7. Direct synaptic connections between pyramidal cells and interneurones A, convertible interneurone-to-pyramidal cell connection. Aa, a presynaptic basket cell in s. oriens (IN) and a postsynaptic pyramidal cell (PC). Ab, unitary inhibitory postsynaptic potentials (uipsps) in the pyramidal cell evoked by the basket cell. Ac, superimposed uipsp traces in the paired-pulse protocol (at 50, 100, 150 and 200 ms intervals; two trials each). Ad, transient conversion of evoked uipsps into depolarizing responses after tetanic stimulation. Weak tetanus (pulse duration, 200 µs) inducing a short afterdischarge ( 7 s) was delivered to make the evoked responses clearly visible (upper traces). Relative membrane potential changes in the pyramidal cells are shown below (lower panel, magnified traces 1 5). The arrowhead indicates its resting membrane potential ( 61 mv). B, inhibitory interneurone-to-pyramidal cell connection. Ba, a presynaptic interneurone in the boundary between s. radiatum and s. lacunosum-moleculare (IN) and a postsynaptic pyramidal cell (PC). Bb, uipsps in the pyramidal cell evoked by the interneurone. Bc, superimposed uipsp traces in the paired-pulse protocol. Bd, uipsp evoked during the afterdischarge expression (arrow). Asterisks indicate three cycles of subthreshold oscillatory responses in the pyramidal cell and weak counterparts in the interneurone. C, excitatory pyramidal cell-to-interneurone connection. Ca, unitary excitatory postsynaptic potentials (uepsps) in a postsynaptic interneurone in s. oriens (IN), evoked by a presynaptic pyramidal cell (PC). Cb, superimposed uepsp traces in the paired-pulse protocol. Cc, synchronous spiking of the two neurones during the afterdischarge.

12 972 Y. Fujiwara-Tsukamoto and others J Physiol those in the simultaneously recorded pyramidal cells. This analysis again indicates that the FS and non-fs interneurones in s. pyramidale and oriens discharged more frequently than the non-fs interneurones in s. lacunosummoleculare and s. radiatum. In particular, the first action potentials of oscillatory bursting responses in many of the FS interneurones (10 of 14 cells) tended to precede the first discharges in the simultaneously recorded pyramidal cells (Fig. 5B; paired t test, n = 14, P < 0.001; see also Fig. 2). This suggests that these interneurones are unlikely to inhibit the activity of their postsynaptic pyramidal cells during the afterdischarge. It should be emphasized here that we didn t find any anti-phasic interneurones, which would discharge only in the resting periods of afterdischarge responses evoked in the pyramidal cells. In addition, we found no pace-maker -like interneurones with an intrinsically rhythmic bursting property in the CA1 region. With respect to the temporal firing properties of the afterdischarge, essentially the same results were obtained in the 64 morphologically identified interneurones (Fig. 6). The discharge activity in oscillatory afterdischarge responses depended greatly on the somatic location, rather than the interneurone subtype (Fig. 6, left; identified interneurones in s. lacunosum-moleculare and s. radiatum, 0.38 ± 0.52 spikes cycle 1, n = 32; those in s. pyramidale and s. oriens, 3.17 ± 2.60 spikes cycle 1, n = 32; t test, P < 0.001). Their discharge timings were time-locked to those of the pyramidal cells, and the rate of interneurones with no spiking during the afterdischarge was much larger in s. lacunosum-moleculare and s. radiatum than in s. pyramidale and s. oriens (Fig. 6, right). Synaptic interactions in s. oriens/pyramidale during afterdischarge Next, we confirmed the synaptic communication between the pyramidal cells and the interneurones in s. oriens/ pyramidale during the seizure-like afterdischarge, since both GABAergic and glutamatergic synaptic transmissions are necessary for afterdischarge generation in hippocampal CA1-isolated slice preparations (Fujiwara-Tsukamoto et al. 2003). Thirteen of the 108 interneurones recorded had direct synaptic connections with the simultaneously recorded pyramidal cells (11 interneurone-to-pyramidal cell and 2 pyramidal cell-to-interneurone pairs, consisting of 2 interneurones in s. lacunosum-moleculare, 6 in s. radiatum, 3 in s. pyramidale, and 2 in s. oriens). Consistent with a previous study (Knowles & Schwartzkroin, 1981a), we failed to record any direct excitatory synaptic responses between the CA1 pyramidal cells. Figure 7A illustrates a presynaptic putative basket cell and postsynaptic pyramidal cell pair. This basket cell densely innervated the pyramidal cell at its somatic site in the s. pyramidale in a restricted manner (Fig. 7Aa), and the unitary inhibitory postsynaptic potentials (uipsps) were evoked in the postsynaptic pyramidal cell by the spiking of the basket cell in a normal resting condition (Fig. 7Ab and c; see Buhl et al. 1996). These two neurones discharged synchronously during the afterdischarge (Fig. 7Ad), and once the tetanic stimulation was applied, the evoked unitary responses in the pyramidal cell were transiently converted from hyperpolarizing to depolarizing ones (Fig. 7Ad, lower panel; see also Fujiwara-Tsukamoto et al. 2003). Thus, it is most likely that the interneurones might activate the pyramidal cells, probably by means of excitatory GABAergic synaptic transmissions, within s. oriens/pyramidale during the afterdischarge. In contrast, a presynaptic interneurone located in the boundary between s. radiatum and lacunosum-moleculare always evoked clear uipsps in a postsynaptic pyramidal cell even during the constant expression of afterdischarge (Fig. 7B). On the other hand, Fig. 7C illustrates unitary excitatory postsynaptic potentials (uepsps) in a postsynaptic interneurone in s. oriens, which were evoked by a presynaptic pyramidal cell (Fig. 7Ca and b). These two neurones also discharged synchronously during the afterdischarge, although the spiking activity in the interneurone was more prominent than that in the pyramidal cell (Fig. 7Cc). The recurrent collaterals of the pyramidal cells, major glutamatergic neurones in this area (Freund & Buzsáki, 1996), were distributed exclusively within s. oriens and s. pyramidale (Fig. 8A, n = 12; see Knowles & Schwartzkroin, 1981b). In addition, the distribution of dendrites in s. oriens/pyramidale which originated from the non-fs interneurones in s. lacunosummoleculare and s. radiatum seemed to allow them to discharge more actively during the afterdischarge (Fig. 8B; dendrites (+), 0.61 ± 0.63 spikes cycle 1, n = 14; ( ), 0.12 ± 0.2 spikes cycle 1, n = 13; t test, P < 0.02). Therefore, glutamatergic activation of GABAergic interneurones by the pyramidal cell collaterals may contribute to the synchronization of afterdischarge activity in the s. oriens/pyramidale of the CA1 region. To test this possibility, glutamate receptor antagonists were applied to specific layers of the CA1 region during the afterdischarge. Local application of a mixture of dl-ap-5 and CNQX to s. oriens/pyramidale during the constant expression of afterdischarge significantly depressed the oscillatory responses in the probed pyramidal cells (Fig. 8D; amplitude 14.1 ± 9.5% of control at 5 s after the application, n = 5, P < 0.02). In contrast, the

13 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 973 application of the same mixture to s. radiatum (Fig. 8C; ± 18.2%, n = 5, P > 0.4) or to s. lacunosummoleculare (data not shown) had no effects on afterdischarge expression. The application of a GABA A antagonist to s. pyramidale also depressed the afterdischarge expression, as also shown elsewhere (Fujiwara- Tsukamoto et al. 2003). These results suggest that the recurrent collaterals of the glutamatergic pyramidal cells, which probably connect to the GABAergic interneurones, may play a critical role in the generation of afterdischarge within a local CA1 network. Synchronization of afterdischarge activity in s. oriens/pyramidale The afterdischarge-related synaptic interactions in s. oriens and s. pyramidale imply that a neural circuit for synchronization of afterdischarge activity may exist within these laminar structures. Therefore, we examined whether microsurgical lesions in s. oriens/pyramidale may result in some perturbation in neuronal synchronization. As shown in Fig. 9, synchronized field responses of the afterdischarge were normally recorded in two different sites of intact CA1 slices (Fig. 9A and E; n = 7) and CA1 slices with the s. lacunosum-moleculare lesioned (Fig. 9B and E; n = 6). In contrast, the field responses of the afterdischarge were completely desynchronized between two recording sites in CA1 slices with the s. oriens/pyramidale lesioned (Fig. 9C and E; n = 6). Moreover, the lesions in s. oriens/pyramidale shortened the duration of afterdischarge on both of the recording sides (Fig. 9D, left; control, 19.2 ± 1.8 s (on the CA3 side of the CA1 region) and 19.2 ± 1.9 s (on its subiculum side); s.l.m.-lesioned, 19.5 ± 1.2 s (CA3 side) and 19.5 ± 1.5 s (subiculum side); s.o./s.p.-lesioned, 10.6 ± 3.1 s (CA3 side) and 13.4 ± 3.9 s (subiculum side); t test, P < (CA3 side) and P < 0.02 (subiculum side) for comparison of control versus s.o./s.p.-lesioned cases). In the s.o./s.p.- lesioned CA1 slices, the duration on the CA3 side was shorter than that on the subiculum side (paired t test, P < 0.05). Also, the frequency of afterdischarge on the CA3 side of the s.o./s.p.-lesioned slices was significantly lower than that on the subiculum side of the same slices (paired t test, P < 0.05) and that on the CA3 side of control slices (t test, P < 0.001) (Fig. 9D, right; control, 4.02 ± 0.80 Hz (on the CA3 side of the CA1 region) and 4.08 ± 0.78 Hz (on its subiculum side); s.l.m.-lesioned, 4.34 ± 1.71 Hz (CA3 side) and 4.33 ± 1.63 Hz (subiculum side); s.o./s.p.-lesioned, 2.50 ± 0.89 Hz (CA3 side) and 3.80 ± 0.95 Hz (subiculum side)). Figure 8. Synaptic transmissions in s. oriens/pyramidale during seizure-like afterdischarge A, axonal projections of recorded pyramidal cells. The branching axons (recurrent collaterals) were usually restricted to s. oriens/pyramidale. Apical dendrites are truncated. B, discharge activity during the afterdischarge in the presence (, n = 14) or absence (, n = 13) of dendrites extending within the s. oriens/pyramidale which originated from well-visualized non-fs interneurones in s. lacunosum-moleculare/radiatum. C, no change in afterdischarge generation by local application of glutamate receptor antagonists to s. radiatum after tetanization (5 mm DL-AP-5 and 1 mm CNQX, filled bar). D, complete blockade of afterdischarge generation by the application of the same antagonists to s. oriens/pyramidale (filled bar).

14 974 Y. Fujiwara-Tsukamoto and others J Physiol Figure 9. Neuronal synchronization mediated through s. oriens/pyramidale A, synchronous oscillatory responses (right) at two field-potential recording sites (FP1 and FP2, 500 µm apart) in a control CA1 slice (left). The asterisk indicates the stimulating site (Stim). B, synchronous oscillatory responses (right) at two recording sites in a CA1 slice where the s. lacunosum-moleculare (and part of the s. radiatum in some cases) was lesioned by a surgical knife (left, arrow). C, desynchronized oscillatory responses (right) in a CA1 slice where s. oriens and s. pyramidale were lesioned (left, arrow). Scale bar in the photo, 200 µm for A C. D, duration (left) and frequency (right) of the afterdischarge activities on the CA3 (FP1, filled columns) and subiculum (FP2, open columns) sides of the CA1 region in control (n = 7), s.l.m.-lesioned (n = 6), and s.o./s.p.-lesioned (n = 6) slices. Columns and error bars indicate the mean and S.D., respectively. E, desynchronization of the afterdischarge in s.o./s.p.-lesioned slices. Averaged probability histograms of FP2 responses (in 1 ms bins) were aligned with the onset of FP1 responses (0 ms) during the afterdischarge in the three slice groups. F, occurrence of afterdischarge in a pyramidal cell lacking the distal portions of its apical dendrite. Left, an s.r.-lesioned mini-slice in which the middle of the s. radiatum was cut to remove the distal dendritic layers (arrow). Note that the distal apical dendritic portions of the recorded pyramidal cell (triangle, magnified in the right panel) were not visualized with intracellular biocytin staining. Scale bar, 200 µm. Right, afterdischarge response in the same pyramidal cell. Tetanic stimulation was applied in the s. oriens/pyramidale.

15 J Physiol A network mechanism of GABA/glutamatergic rhythmic synchronization 975 Finally, the afterdischarge was found to be inducible in the CA1 region even in the s.r.-lesioned mini-slices consisting of s. oriens/pyramidale and the adjacent perisomatic part of s. radiatum (Fig. 9F; duration 15.6 ± 3.2 s, n = 5), although the duration of afterdischarge responses was relatively shortened in some slices probably because of a considerable loss of living pyramidal cells. These results suggest that the interneurones in s. lacunosummoleculare and at least the distal dendritic part of s. radiatum may basically be dispensable for the afterdischarge generation, and that the distal portions of the apical dendrites of the pyramidal cells may not participate in the GABA/glutamate-dependent generation of afterdischarge activity. Thus, the neuronal interactions within s. oriens/pyramidale are likely to be most critical for synchronization, maintenance, and, perhaps, rhythm generation of the seizure-like afterdischarge activity in the CA1 region. Discussion In the present study, we have shown that (1) interneurones in s. oriens/pyramidale, such as basket/chandelier cells and bistratified cells, display prominent spiking during the GABAergic/glutamatergic, seizure-like afterdischarge, whereas interneurones in s. lacunosummoleculare/radiatum discharge much less or not at all; (2) the spike onset in FS interneurones in s. oriens/ pyramidale often precedes that in pyramidal cells in individual afterdischarge cycles; (3) no interneurones exibit anti-phasic activity at the interval of pyramidal cell firing during the afterdischarge; (4) the pyramidal cells fire synchronously with directly connected interneurones during the afterdischarge; (5) synaptic transmissions in s. oriens/pyramidale are essential for neuronal synchronization in the afterdischarge. Based on these results, we propose a hypothetical model for local afterdischarge generation (Fig. 10). In our model, the pyramidal cells and interneurones are strongly excited during tetanization, which causes extracellular K + accumulation to stimulate neighbouring neurones synergistically (Kaila et al. 1997; Smirnov et al. 1999). The excited interneurones activate GABA A receptorsinthe pyramidal cells, leading to massive Cl influx and HCO 3 efflux (Isomura et al. 2003b). Then, Cl accumulation and subsequent HCO 3 redistribution in the pyramidal cells make their GABA A reversal potentials much higher than their resting membrane potentials (Staley et al. 1995; Staley & Proctor, 1999). Consequently, the posttetanic depolarization, often carrying gamma oscillations (Whittington et al. 1997; Bracci et al. 1999), and subsequent afterdischarge are expressed in the local network. There are only a few direct recurrent connections among the CA1 pyramidal cells (Knowles & Schwartzkroin, 1981a; Amaral & Witter, 1995), but their recurrent collaterals innervate many interneurones in s. oriens/pyramidale (Knowles & Schwartzkroin, 1981a; Lacaille et al. 1987; Freund & Buzsáki, 1996). Accordingly, it is conceivable that local GABAergic interneurones in s. oriens/pyramidale synchronously excite the glutamatergic pyramidal cells, which, in turn, activate the interneurones of origin. Thus, these neurones may form a layer-specific positive feedback circuit to express the afterdischarge. The locally generated afterdischarge activity will propagate into other regions where GABAergic excitation no longer occurs (Isomura et al. 2003a). GABA A responses are normally more depolarizing in the distal apical dendrites than in the somata of hippocampal pyramidal cells (Andersen et al. 1980; Gulledge & Stuart, 2003). However, our results suggest that excitatory GABAergic inputs into somatic or perisomatic (proximal dendritic/axonal) portions may be most crucial for epileptic afterdischarge. Actually, depolarizing GABA responses became largest near the somata during the afterdischarge (authors unpublished observations), and somatic Cl accumulation responsible for GABAergic depolarization seems to be mediated by local s. oriens/pyramidale interneurones (Isomura et al. 2003b). Such layer specificity in the afterdischarge may be caused by the spatially limited distribution of recurrent collaterals in s. oriens/pyramidale, and by the Figure 10. A hypothetical network model for the generation of seizure-like afterdischarge in the hippocampal CA1 region Positive feedback circuit formed by pyramidal cells and interneurones in s. oriens/pyramidale to express neuronal synchronization in the seizure-like afterdischarge. See Discussion for details. s.l.m., s. lacunosum-moleculare; s.r., s. radiatum; s.p., s. pyramidale; s.o., s. oriens; alv., alveus.

16 976 Y. Fujiwara-Tsukamoto and others J Physiol abundance of highly active (FS) interneurones in those layers. Moreover, the layer specificity can depend on the density of recurrent collateral synapses and their proximity to the somata of postsynaptic interneurones. In this way, the synaptic interactions between the pyramidal cells and interneurones within the s. oriens/pyramidale might be essential and sufficient for neuronal synchronization of the afterdischarge. The candidates for local interneurones participating in the neuronal synchronization are likely to include the basket, chandelier, bistratified and trilaminar cells (see Figs 2, 3, 6 and 7; see also Sik et al. 1995; Freund & Buzsáki, 1996). In fact, these interneurones are directly connected with the pyramidal cells in a reciprocal fashion (Buhl et al. 1994a; Halasy et al. 1996; Ali & Thomson, 1998; Ali et al. 1998; Maccaferri et al. 2000; Pawelzik et al. 2002). In particular, parvalbumin-positive hippocampal interneurones, most of which are basket cells with FS properties, are concentrated in the s. oriens/pyramidale (Kawaguchi et al. 1987; Pawelzik et al. 2002), and they receive excitatory synaptic input much more intensely than other types of interneurones (Gulyás et al. 1999). Although the mechanism of synchrony in the afterdischarge can be accounted for by a cooperative action of these interneurones and the pyramidal cells, the mechanism of its rhythmicity still remains a mystery in our network model. It seems unlikely that intrinsic membrane potential oscillations in the LM interneurones (Chapman & Lacaille, 1999) or slow GABA A transmission by these LM interneurones (Banks et al. 2000; cf. Bertrand & Lacaille, 2001) may contribute to rhythm generation in the seizure-like afterdischarge, because the present results have clearly demonstrated that most LM interneurones are not so active during the afterdischarge (see Figs 1 and 6). The data obtained from our lesion experiments imply that a rhythm generator probably exists in s. oriens/pyramidale, but not in s. lacunosum-moleculare. Although we cannot as yet exclude the possibility that interneurones in s. lacunosum-moleculare/radiatum may partly contribute to the synchrony of the afterdischarge, they appear to be just slaves following other active neurones, and not to be essential for the rhythm generation. It is also unlikely that a rebound depolarization following strong IPSP may drive the next oscillatory cycle in the pyramidal cells (cf. Cobb et al. 1995), because such strong IPSPs were not observed in the pyramidal cells recorded during the afterdischarge. Hence, it should be hypothesized that intrinsic membrane potential oscillations in a population of pyramidal cells (Leung & Yim, 1991) or unidentified interneurones underlie the rhythm generation of the afterdischarge. The synchronization mechanism of the tetanusinduced seizure-like afterdischarge might be partly similar to that of seizure-like afterdischarge induced in a low Mg 2+ condition, in that depolarizing GABAergic responses appear to contribute to the generation of low Mg 2+ -induced epileptiform activity (Köhling et al. 2000; Perez Velazquez, 2003). In contrast, these synchronizing events of seizure-like afterdischarge are apparently different from that of disinihibition-dependent afterdischarge which is produced by application of GABA A antagonists such as bicuculline and picrotoxin (Borck & Jefferys, 1999). The direct glutamatergic recurrent connections among the pyramidal cells dominantly contribute to the epileptogenesis in a bicuculline-induced disinhibitory condition (Crépel et al. 1997), as well as in a collateral-sprouting condition after chronic kainate treatment (Smith & Dudek, 2001, 2002). The tetanusinduced seizure-like afterdischarge is also different from hippocampal theta oscillations in that GABA naturally acts as an inhibitory (hyperpolarizing) mediator; the basket and chandelier cells discharge at the interval of pyramidal cell firing, whereas the bistratified and O-LM cells discharge synchronously with the pyramidal cells during the theta oscillations (Ylinen et al. 1995; Klausberger et al. 2003, 2004). It remains unknown, however, what functional roles the bistratified and O-LM cells might play in the generation of seizure-like afterdischarge. Taken together, there would be several different mechanisms underlying the rhythmic synchronizations such as seizure-like activities and theta oscillations in the same (CA1) region of the hippocampus, and distinct types of GABAergic interneurones might play a key role in controlling these synchronous oscillatory phenomena. Our positive feedback circuit hypothesis for the rhythmic synchronization may be practically useful to understand the seizure activity in human temporal lobe epilepsy. It has long been believed that reduced GABAergic inhibition (disinhibition) would lead to the hyperexcitability of glutamatergic neurones in the cerebral cortex of epilepsy patients. However, GABAergic interneurones and their terminals containing glutamate decarboxylase are still preserved in the human epileptic hippocampus (Babb et al. 1989), and GABA, as well as glutamate, is massively released in the hippocampus during spontaneous seizures of temporal lobe epilepsy (During & Spencer, 1993). Although an abnormal loss of GABA transporters could decrease non-synaptic GABA release by GABA transporter reversal (During et al. 1995), hippocampal GABA transporters are also preserved in temporal lobe epilepsy patients (Mathern et al. 1999).