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Glutamate Controls the Induction of GABA-Mediated Giant Depolarizing Potentials Through AMPA Receptors in Neonatal Rat Hippocampal Slices SONIA BOLEA,1 ELENA AVIGNONE,2 NICOLA BERRETTA,2 JUAN V. SANCHEZ-ANDRES,1 AND ENRICO CHERUBINI2 1 Departamento de Fisiologia, Instituto de Bioingenieria, Universidad Miguel Hernandez, Campus de San Juan, 03550 San Juan, Alicante, Spain; and 2Neuroscience Program and Istituto Nazionale Fisica della Materia Unit, International School for Advanced Studies, 34014 Trieste, Italy INTRODUCTION A peculiar characteristic of the hippocampus of neonatal rats is the presence of spontaneous network-driven GABA-mediated oscillatory events, the so-called giant depolarizing potentials (GDPs) (Ben Ari et al. 1989; Xie et al. 1994). These appear to be synchronous within the entire hippocampus (Menendez de la Prida et al. 1998; Strata et al. 1997) and are generated by GABA (acting on GABAA receptors) that at this early developmental stage is depolarizing and excitatory (Cherubini et al. 1991). GABA-induced depolarization represents a general feature of postnatal development (Ben Ari et al. 1989; Chen et al. 1996; Cherubini et al. 1991; Kaneda et al. 1996; Owens et al. 1996; Rohrbough and Spitzer 1996; Serafini et al. 1995; Wu et al. 1992). GABA, through its depolarizing action, activates voltagedependent calcium channels, thereby increasing the levels of [Ca21]i (Leinekugel et al. 1995). Therefore GDPs are strictly associated with spontaneous calcium oscillations occurring in groups of neighboring cells (Garaschuk et al. 1998). These highly correlated calcium signals are thought to be essential for consolidation of synaptic connections and development of the adult neuronal network (Shatz 1990). However, GDPs need a glutamatergic drive for their expression. In previous studies from this (Strata et al. 1995) and other laboratories (Ben Ari et al. 1989; Gaiarsa et al. 1991) it has been shown that GDPs can be modulated by activation of both ionotropic and metabotropic glutamate receptors. More recently, it was suggested that synchronization is determined by cooperation of excitatory GABAergic connections between interneurons and glutamatergic connections presumably from pyramidal cells to interneurons (Khazipov et al. 1997). Hence in interneurons a glutamatergic component of the GDP could be revealed after intracellular blockade of GABAA receptors with an internal solution containing fluoride (Khazipov et al. 1997). In particular, it was suggested that the main contribution to GDP generation is provided by N-methyl-D-aspartate (NMDA) receptors that would be activated after GABA-induced depolarization, whereas AMPA receptors would play only a minor role (Ben Ari et al. 1997; Leinekugel et al. 1997; McLean et al. 1995). The current experiments were undertaken to further elucidate the role of glutamatergic receptors in GDP generation. In contrast to previously published data (Khazipov et al. 1997; Leinekugel et al. 1997) it was found that AMPA receptors play a major role in neuronal synchronization. Part of this work was reported in preliminary form (Bolea et al. 1998). METHODS Slice preparation The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Experiments were performed on hippocampal slices obtained from postnatal P1–P6 Wistar rats (P0 is the day of birth) according to the 0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society 2095 Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 15, 2017 Bolea, Sonia, Elena Avignone, Nicola Berretta, Juan V. SanchezAndres, and Enrico Cherubini. Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. J. Neurophysiol. 81: 2095–2102, 1999. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 – 40 mM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 mM) or (RS)-a-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 mM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50 –100 mM). In the presence of GYKI 53655, both kainic or domoic acid (0.1–1 mM) were unable to induce GDPs. In contrast, bath application of D-(2)-2-amino-5-phosphonopentanoic acid (50 mM) or (1)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 mM) produced only a 37 6 9% (SE) and 36 6 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 6 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KClfilled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals. 2096 BOLEA, AVIGNONE, BERRETTA, SANCHEZ-ANDRES, AND CHERUBINI nous in pair of CA3 recordings, 3) they are reversibly blocked by a high-magnesium, low-calcium solution, and 4) they are abolished by TTX. These events are GABAA mediated because they are reversibly blocked by bicuculline. In a first set of experiments, the role of ionotropic glutamate receptors in GDP generation was reexamined, taking also advantage of new pharmacological tools. Then, in a second series of experiments, the conductances activated during GDPs in pyramidal cells were studied after intracellular blockade of GABAA-receptor– evoked responses with fluoride. Patch-clamp whole cell recordings AMPA receptor activation is necessary for GDP induction Spontaneous or evoked GDPs were recorded in the whole cell configuration of the patch-clamp technique (current-clamp mode) with a standard amplifier (Axoclamp 2B, Axon Instruments; Foster City, CA). Patch electrodes had a resistance of 3– 6 MV when filled with an intracellular solution containing (in mM) 140 KCl or KF, 1 MgCl2, 1 NaCl, 1 EGTA, 5 HEPES, and 2 K2ATP; pH was adjusted to 7.3 with KOH. GDPs were evoked with bipolar twisted NiCrinsulated electrodes (50 mm OD) placed in the hilus. Pulses of variable amplitude (5–15 V, 100-ms duration) were applied. Membrane input resistance was measured from the amplitude of small hyperpolarizations (300-ms duration) evoked by passing current pulses across the cell membrane. To test the role of AMPA receptors in GDPs induction, the selective AMPA– kainate receptor antagonist CNQX (10 – 40 mM) was applied (n 5 11). As reported earlier (Gaiarsa et al. 1991), this compound completely blocked spontaneous GDPs (data not shown), even in rats as young as P2. A full recovery was obtained 10 –15 min after wash. However, in the presence of CNQX, it was still possible to re-induce the appearance of spontaneous GDPs with GABA (20 mM) or AMPA (5 mM) and to evoke GDPs by focal stimulation. The effect of AMPA was usually associated to a 5- to 12-mV membrane depolarization. Both GDPs as well as membrane depolarization disappeared when AMPA was applied in the presence of bicuculline (10 mM, data not shown), suggesting that both phenomena were due to the release of GABA after an incomplete block of AMPA receptors with CNQX. Similar results were obtained with kynurenic acid (2 mM, n 5 8), DNQX (20 mM, n 5 2), and GYKI 52466 (20 –100 mM, n 5 6). The possibility to synchronize again the network with AMPA suggests that AMPA receptors were not completely blocked by the selective antagonists used. To check whether this hypothesis was correct, a more potent and selective AMPA antagonist, GYKI 53655, was tested (Lerma et al. 1997). GYKI 53655 (50 –100 mM) blocked spontaneous GDPs as well as those evoked by electrical stimulation of the hilus. Moreover, AMPA (5 mM) or GABA (20 mM) application were unable to re-induce GDPs (Fig. 1). These data suggest that the glutamatergic drive via AMPA receptors is essential for GDP generation. To further examine the role of AMPA receptors in GDPs induction, experiments were performed in the presence of cyclothiazide, a selective blocker of AMPA receptor desensitization (Partin et al. 1993). In the presence of cyclothiazide (20 mM) GDPs frequency increased by 76 6 14% (n 5 6, Fig. 2). Kainate receptors were not apparently involved in GDPs induction because in the presence of GYKI 53655 both kainate and domoic acid (0.1–1 mM) failed to induce GDPs (n 5 3, data not shown). In contrast to AMPA receptor antagonists, the selective NMDA receptor antagonist D-AP5 (50 mM) applied in the bath for 10 –15 min produced only a reduction of GDP frequency (Fig. 3). Occasionally a complete blockade was observed during the first minutes of drug application. Later on, in the continuous presence of D-AP5, GDP frequency slowly returned toward control values. On average a 35% reduction in GDP frequency was measured along the time of drug application. Similar results were obtained with CPP (20 mM). No significant changes in the shape of GDPs were observed during application of CPP or D-AP5. These data suggest that, although NMDA receptors may contribute to GDP generation, they are Drugs Drugs were dissolved in ACSF and applied in the bath by changing the superfusion solution to one that differed only in its content of drug(s). The ratio of flow rate to bath volume ensured complete exchange within 1 min. Drugs used were (RS)-a-amino-3-hydroxy5-methyl-4-isoxadepropionate (AMPA), (1)-3-(2-carboxy-piperazin4-yl)-propyl-1-phosphonic acid (CPP), or D-(2)-2-amino-5-phosphonopentanoic acid (D-AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3(1H,4H)-dione (DNQX), domoic acid, kainic acid, GABA, and bicuculline (all purchased from Tocris Cookson; Bristol, UK); kynurenic acid, TTX, and 6-chloro-3,4dihydro-3-[2-norbornen-5-yl]22H-1,2– 4benzothiadiazine-7-sulfonamide 1,1-dioxide (cyclothiazide; from Sigma; Milano, Italy); GYKI 52466 (from RBI); and GYKI 53655 (from Lilly; Indianapolis, IN). If not otherwise stated, data are expressed as means 6 SE. Data acquisition and analysis Data of spontaneous and evoked GDPs were stored on a magnetic tape and transferred to a computer after digitization with an A/D converter (Digidata 1200). Data acquisition was done with pClamp (Axon Instruments; Foster City, CA), and the amplitude and frequency of GDPs were analyzed with Axoscope (Axon Instruments; Foster City, CA). RESULTS Stable whole cell recordings (in current-clamp configuration) lasting .30 min were obtained from 120 CA3 hippocampal pyramidal cells in slices from P1- to P6-old rats that exhibited spontaneous GDPs. They occurred at the frequency of 5.2 6 0.43 GDPs/min and consisted of large (30 –50 mV) depolarizing potentials lasting 400 –700 ms, which triggered action potentials, followed by an afterhyperpolarization. In previous experiments (Ben Ari et al. 1989), it was reported that GDPs are network-driven events generated by a large population of neurons firing synchronously because 1) their frequency is independent of the membrane potential, 2) they are synchro- Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 15, 2017 methods already described (Strata et al. 1995). Briefly, animals were decapitated after being anesthetized with intraperitoneal injection of urethan (2 g/kg). The brain was quickly removed from the skull, and the hippocampi were dissected free. Transverse 600-mm thick slices were cut with a tissue chopper and maintained at room temperature (22–24 °C) in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM) 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 1.3 MgCl2, 2 CaCl2, 25 NaHCO3, and 11 glucose, pH 7.3, saturated with 95% O2-5% CO2. After incubation in ACSF for $1 h, an individual slice was transferred to a submerged recording chamber, continuously superfused at 33–34°C with oxygenated ACSF at a rate of 3 ml/min. GDPS AND GLUTAMATE RECEPTORS not essential for their induction. However, in the presence of CNQX, D-AP5 was able to prevent evoked GDPs or re-induction of spontaneous oscillations by GABA and AMPA (data not shown), indicating that in particular conditions also NMDA receptors may contribute to network synchronization. Intracellular blockade of GABAA receptor with fluoride reveals an AMPA-mediated component of GDPs In a previous work aimed at understanding synchronization of interneuronal network (Khazipov et al. 1997), whole cell recordings were made from interneurons dialyzed with the poorly permeable anion F2, which produces a block of GABAA receptors (Bormann et al. 1987). We used a similar approach to see whether in CA3 pyramidal cells glutamate receptors are activated during GDPs. To this purpose we tested 1) GDP reversal potential immediately and 30 – 40 min after a full whole cell access was achieved with a F2 containing solution and 2) the pharmacological sensitivity of GDPs to GABAA and ionotropic NMDA and non-NMDA glutamate receptor antagonists. Immediately (3–5 min) after breaking into whole cell configuration, the reversal of evoked and spontaneous GDPs was 245.7 6 6.1 mV (n 5 28) and 240.8 6 6.2 mV (n 5 11), respectively (Fig. 4, A and B). Plots of amplitude–voltage relationships were always linear. The slope of the regression lines through different data points was 20.58 6 0.19 (n 5 28) for evoked and 20.67 6 0.14 (n 5 11) for spontaneous GDPs. After 40-min dialysis with an intracellular F2 containing solution a complete block of GABA-evoked responses in the presence of TTX (1 mM) was observed (Fig. 4C). The block of GABAA receptors was associated with a significant shift of the reversal toward more positive values. The reversal of the evoked and spontaneous GDPs was 214.1 6 11.4 mV (n 5 17) and 215.8 6 13.1 mV (n 5 6), respectively (Fig. 4, A and B). Changes in the reversal were associated with a reduction in the slope of the regression lines, indicating a reduction of GDP amplitude (this was 20.29 6 0.12, n 5 17, for evoked and 20.31 6 0.13, n 5 6, for spontaneous GDPs). In the majority of cells the amplitude– voltage relationship was linear, whereas in four cases a rectification at more negative potentials was observed. The shift of the reversal potential toward more positive values as well as the reduction of the slope in the amplitude–voltage relationship suggest the presence of a different component unmasked after intracellular blockade of GABAA receptors with F2. To understand the nature of this component, spontaneous and evoked GDPs were recorded at different membrane potentials in the FIG. 2. Cyclothiazide increases the frequency of GDPs. Representative tracing from a CA3 pyramidal cell recorded at P4 in control condition (A) and during bath application of cyclothiazide (B). C: GDP frequency of the cell shown in A and B is plotted against time. Each column represents the number of GDPs recorded in 1 min. Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 15, 2017 FIG. 1. (RS)-a-Amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) receptor activation is essential for Giant depolarizing potential (GDP) induction. A–D: tracings from the same cell (recorded at P5). Bath application of the selective AMPA receptor antagonist GYKI 53655 (arrow) readily blocked spontaneous and evoked GDPs. In the presence of GYKI 53655, electrical stimulation failed to evoke GDPs (see inset above the trace on an expanded time scale). In the presence of GYKY 53655, both AMPA (bar, B) and GABA (bar, C) failed to re-induce GDPs; 30 min after washing GYKI, spontaneous GDPs reappeared; GDP frequency increased during application of GABA (20 mM, bar). 2097 2098 BOLEA, AVIGNONE, BERRETTA, SANCHEZ-ANDRES, AND CHERUBINI FIG. 3. N-Methyl-D-aspartate (NMDA) receptor activation is not essential for GDP generation. A: representative tracing from a CA3 pyramidal cell recorded at P4 in control condition and during bath application of D-AP5 (bar). B: 2 GDPs from the recording in A are shown on an expanded time scale. C: GDP frequency of the cell shown in A is plotted against time. Each column represents the number of GDPs recorded in 1 min. D: normalized GDP frequency recorded during application of D-AP5 (50 mM) and CPP (20 mM). Bars indicate SE; n refers to the number of experiments et al. 1998). In early postnatal days, when the present experiments were performed, mossy fibers were not completely developed (Gaarskjaer 1986) and therefore their contribution to GDPs induction appears to be modest. Other sources of glutamate are the associative-commisural fibers coming from the controlateral hippocampus as well as from collaterals of principal cells. To examine whether GDPs can be generated locally by these fibers, in three slices, a small part of the CA3 region was isolated with a knife cut from the rest of the hippocampus, and pair recordings were performed from two CA3 adjacent pyramidal cells with two intrapipette solutions containing KF and KCl, respectively. In these conditions GDPs with characteristics similar to those already reported could be still recorded synchronously in the two cells (Fig. 7). This suggests that a local population of pyramidal cells and interneurons is sufficient to generate GDPs and that collaterals of principal cells are probably the main source of glutamatergic drive. Source of glutamatergic drive needed to synchronize GABAergic interneurons AMPA receptor activation is necessary for GDP induction Glutamate may be released from mossy fibers, known to make synaptic contacts with the proximal dendrites of CA3 pyramidal neurons and with GABAergic interneurons (Acsády DISCUSSION In line with a previous report (Ben Ari et al. 1997), the current experiments clearly show that GDPs are generated by the synergistic action of glutamate and GABA. However, in contrast with earlier findings on interneurons (Khazipov et al. 1997; Leinekugel et al. 1997), they demonstrate that the release of GABA is triggered by glutamate acting mainly on AMPA type of receptors and that the ionotropic glutamatergic component of GDPs, revealed after blockade of GABAA-evoked responses with intracellular F2, is predominantly of the nonNMDA type. AMPA receptor activation was necessary for GDP induction as proved by the observation that AMPA receptor antagonists were fully effective in blocking GDPs, whereas NMDA receptor antagonists only produced a transient reduction in their Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 15, 2017 presence of AMPA and NMDA receptor antagonists. Bath application of CNQX (10 –20 mM) completely abolished the residual component of spontaneous GDPs and in 8 of 11 completely blocked the evoked GDPs. D-AP5 (50 mM) reduced the residual component only in 3 of 11 (Fig. 5, A–C). In the presence of D-AP5 and CNQX GDPs could not be evoked. It could be that the glutamatergic component of the GDP, which remains after blockade of GABAA receptor, is amplified as the result of the interference of intracellular F2 with phosphatases (Andrews and Babior 1984), enzymes that regulate receptor desensitization (Yakel 1997). To test this hypothesis, the amplitude of AMPA responses recorded in TTX (1 mM) with a F2 containing intrapipette solution was compared with that of responses recorded with a KCl solution. As shown in Fig. 5D, in F2, AMPA responses were not significantly (P . 0.08) different from those recorded in KCl. Pair recordings performed from adjacent CA3 pyramidal cells (n 5 21) with two different intrapipette solutions showed that GDPs recorded with KF and KCl intrapipette solutions were still synchronous in the two cells, confirming their combined glutamatergic and GABAergic nature (Fig. 6). However, a clear difference in the shape of GDPs recorded with KF or KCl was observed. As depicted in Fig. 6, in KF GDPs were smaller in amplitude and exhibited a slower rising phase that often did not reach the threshold for action potential generation. Moreover, when GABA (20 mM) was applied in the presence of kynurenic acid (2 mM), known to abolish spontaneous GDPs (Strata et al. 1995), this amino acid was able to re-induce GDPs only in the cell recorded with KCl electrode (Fig. 6). GDPS AND GLUTAMATE RECEPTORS 2099 frequency (see also Gaiarsa et al. 1991). In agreement with the current data, it has been recently shown, with fluorometric calcium imaging techniques, that CNQX is able to completely block spontaneous oscillatory calcium transients, associated to GDPs, recorded in the CA1 region of the hippocampus during early postnatal development (Garaschuk et al. 1998). In previous studies a complete block of GDPs with NMDA receptors antagonists was reported. The same block was occasionally observed only during the first 2–3 min of drug application. At an early stage of development, at least in the CA1 area, glutamatergic synapses containing only NMDA receptors were described (Durand et al. 1996). However, the observations that at P2 it is already possible to block the entire network with CNQX and to increase GDPs frequency with cyclothiazide indicate that also AMPA receptors are present and functional at this early developmental period. The contribution of NMDA receptors to network synchronization is suggested by the experiments in which GDPs evoked by GABA or AMPA in the presence of CNQX were abolished by addition of D-AP5 or CPP. However, it should be stressed that in these cases AMPA responses were not completely abolished, probably because of AMPA receptors localized on basket cells, not entirely blocked by CNQX. Therefore we suggest that functional AMPA receptors would provide the depolarization sufficient to remove the Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 15, 2017 FIG. 4. Intracellular blockade of GABA receptors with fluoride reveals a novel component of evoked GDPs. A: evoked GDPs (arrows) recorded at different membrane potentials (at P4) with a patch pipette containing KF, 1 min (left) and 40 min (right) after breaking into the whole cell configuration. B: plot of GDPs (shown in A) amplitude vs. membrane potentials. Note the shift to the right of the reversal potential and the change in the slope of the amplitude-voltage plot, 40 min after dialyzing the cell with fluoride. C: pairs of recordings from adjacent CA3 pyramidal cells patched with pipettes containing KCl and KF. Application of GABA (bar) in the presence of TTX (1 mM) induced a membrane depolarization associated with an increase in membrane conductance only in the cell recorded with KCl. Downward deflections are electrotonic potentials evoked by injection of steady current pulses of the same amplitude (250-ms duration). 2100 BOLEA, AVIGNONE, BERRETTA, SANCHEZ-ANDRES, AND CHERUBINI magnesium block and to activate NMDA receptor channels present on interneurons. Further evidence in favor of the involvement of AMPA receptor in GDP induction is given by the experiment with GYKI 53655, a more-selective AMPA receptor antagonist (Lerma et al. 1997). In these cases, the impossibility to re-activate the network with GABA or AMPA suggest that network synchronization may involve AMPA receptors localized on GABAergic interneurons, having, in comparison with principal cells, distinct biophysical and pharmacological properties. Striking differences in the functional characteristics of AMPA receptors in basket and principal cells were reported (Isa et al. 1996; Koh et al. 1995; for review see Freund and Buzsaki 1996). These consist of larger singlechannel conductance, rapid desensitization kinetics (see also Livsey et al. 1993), and in the majority of the cases a higher permeability to calcium (Burnashev et al. 1995; Koh et al. 1995). This latter issue appears to be particularly interesting in view of the role that this divalent cation has in synaptic plasticity processes during development. Thus the rapid transient increase in calcium flux through AMPA receptor channel at rest would temporally summate to that entering the cell through GABA-mediated activation of voltage-dependent cal- FIG. 6. GDPs component revealed after intracellular blockade of GABAA-mediated responses with fluoride is glutamatergic. A: pairs of recordings from adjacent pyramidal cells (at P5) patched with intrapipette solutions containing either KCl or KF. GDPs are synchronous in the 2 recordings. However, GDPs recorded with KF were smaller in amplitude and exhibited slower kinetics. Two GDPs (marked with an *) are superimposed and shown on the right on an expanded time scale. B: bath application of the broad spectrum NMDA and non-NMDA antagonist kynurenic acid abolished GDPs that could be reinduced by bath application of GABA (bar) only on the cell patched with KCl. Downloaded from http://jn.physiology.org/ by 10.220.33.6 on June 15, 2017 FIG. 5. A NMDA and non-NMDA ionotropic glutamatergic component can be revealed in the evoked GDPs after block of GABAA-mediated responses with intracellular fluoride. A: GDPs evoked after 30 min breaking into whole cell configuration, in control condition, and in the presence of D-AP5 (50 mM) and CNQX (20 mM). Membrane potential 280 mV. B: plot of GDPs area vs. membrane potential in control conditions () and in the presence of D-AP5 (ƒ). C: GDPs evoked in another neuron in the same experimental conditions as in A. Note the presence of a D-AP5– sensitive component. Arrows indicate the time of stimulation; membrane potential ranging from 277 to 265 mV. D: responses to increasing concentrations of AMPA recorded in the presence of TTX (1 mM) with KCl (E) or KF (●). Each symbol is the mean of 3–7 responses obtained at P3-P6. Bars represent the SE (when not visible they are within the symbols). GDPS AND GLUTAMATE RECEPTORS cium channels. As suggested by recombinant studies (Burnashev et al. 1992), different subunit composition may underline differences in function. Interestingly, in a recent study on hippocampal slices obtained from 16- to 20-day-old rats, it has been shown that only interneurons activated by the mossy fibers bear polyamine toxin (philanthotoxin)-sensitive AMPA receptors, presumably calcium permeable (Tóth and McBain 1998). Further support to the idea that glutamate triggers the release of GABA from GABAergic interneurons through AMPA receptor subtypes is given by the experiments in which during superfusion of CNQX, AMPA-evoked membrane depolarization and associated increase in frequency of GDPs are blocked by bicuculline. Moreover the double-recording experiments (with KCl or KF), in which in the presence of kynurenic acid it was not possible to induce GDPs in the cell recorded with KF, strongly indicate that AMPA receptors on pyramidal cells are blocked. The involvement of kainate receptors in GDP generation can be excluded on the basis of the impossibility to synchronize the network when kainic or domoic acid are applied in the presence of GYKI 53655. AMPA receptor-mediated component of GDPs could be revealed after intracellular blockage of GABAA receptor with fluoride After dialyzing principal cells with F2 (see also Khazipov et al. 1997) in concomitance with a complete block of GABAAinduced responses, a residual component with slower kinetics can be revealed. This component appears to be glutamatergic in origin, as suggested by the shift in the reversal potential toward more positive values and by the change in the slope of the amplitude–voltage plot, obtained after dialyzing the cell with F2 for $30 – 40 min. The observed shift in reversal was similar to that already reported for GDPs in interneurons (Khazipov et al. 1997) and was associated, in the majority of the cases, with a linear amplitude–voltage relationship. This indicates the activation of a non-NMDA receptor type, unmasked after intracellular blockade of GABA. Further support in favor of activation of AMPA receptor type in evoked GDPs is given by pharmacological experiments in which, in 8 out of 11 cases, CNQX was able to completely block the residual component. In this respect our data differ from those reported earlier by Khazipov et al. (1997) and Leinekugel et al. (1997). However, in those reports, the assumption that the residual part of GDPs (which remains after intracellular dialysis with F2) is NMDA dependent was based only on the characteristic voltage dependency of the responses. No pharmacological tools were used to dissect out the different glutamatergic components. On the other hand, the lack of effect of CNQX in blocking spikes recorded in cell-attached configuration and supposed to represent interneuronal GDPs reported in those experiments (Khazipov et al. 1997; Leinekugel et al. 1997) has, as pointed out by the same authors, strong limitations. In the current experiments, the similarity in the dose-response curves for AMPA, obtained with intracellular solutions containing KCl and F2, respectively, allows to exclude the possibility that the residual AMPA-mediated component of GDPs is modified as the result of the interference of F2 with some phosphorylation processes (Andrews and Babior 1984). Source of glutamatergic drive needed to synchronize GABAergic interneurons As already shown by Khazipov et al. (1997), Garaschuk et al. (1998), and Menendez de la Prida et al. (1998), GDPs were still present in small CA3 islands isolated from the rest of the hippocampus, implying that a local circuit comprehensive of a relatively small population of principal cells and interneurons was sufficient to generate them. In these cases, the glutamatergic input presumably originates from collaterals of principal cells because the mossy fibers are disconnected from the granule cells of the dentate gyrus. Also the glutamatergic component revealed in pyramidal cells recorded with KF is presumably due to glutamate release from recurrent collaterals. In summary, these data show that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals. Other glutamatergic components can also be involved but do not seem to be essential. This work was partially supported by a grant from Consiglio Nazionale delle Ricerche, Ministero dell’Universita’ e Ricerca Scientifica e Tecnologica to E. Cherubini and by a grant from the Fondo de Investigacion Sanitaria to J. V. Sanchez-Andres. S. Bolea was supported by a fellowship from the Ministerio de Educacion y Cultura. N. Berretta was supported by a fellowship from Novartis Pharma. S. Bolea and E. Avignone contributed equally to this work. Present address of N. Berretta: IRCCS, S. Lucia, Via Ardeatina 306, 00178 Roma, Italy. Address for reprint requests: E. 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