R Khazipov
email: khazipov@inmed.univ-mrs.fr

Minlebaev M, Ben-Ari Y, Khazipov R. Network mechanisms of spindle-burst oscillations in the neonatal rat barrel cortex in vivo. J Neurophysiol 97: 692-700, 2007. First published November 8, 2006;doi:10.1152/jn.00759.2006. Early in development, cortical networks generate particular patterns of activity that participate in cortical development. The dominant pattern of electrical activity in the neonatal rat neocortex in vivo is a spatially confined spindle-burst. Here, we studied network mechanisms of generation of spindle-bursts in the barrel cortex of neonatal rats using a superfused cortex preparation in vivo. Both spontaneous and sensory-evoked spindle-bursts were present in the superfused barrel cortex. Pharmacological analysis revealed that spindle-bursts are driven by glutamatergic synapses with a major contribution of AMPA/kainate receptors, but slight participation of NMDA receptors and gap junctions. Although GABAergic synapses contributed minimally to the pacing the rhythm of spindleburst oscillations, surround GABAergic inhibition appeared to be crucial for their compartmentalization. We propose that local spindleburst oscillations, driven by glutamatergic synapses and spatially confined by GABAergic synapses, contribute to the development of barrel cortex during the critical period of developmental plasticity.

I N T R O D U C T I O N

During development, cortical neuronal networks generate particular patterns of activity that participate in cortical development [START_REF] Ben-Ari | Developing networks play a similar melody[END_REF][START_REF] Fox | Anatomical pathways and molecular mechanisms for plasticity in the barrel cortex[END_REF][START_REF] Katz | Synaptic activity and the construction of cortical circuits[END_REF][START_REF] Khazipov | Early patterns of electrical activity in the developing cerebral cortex of humans and rodents[END_REF][START_REF] Moody | Ion channel development, spontaneous activity, and activity-dependent development in nerve and muscle cells[END_REF][START_REF] O'donovan | The origin of spontaneous activity in developing networks of the vertebrate nervous system[END_REF]. The dominant pattern of electrical activity in the neonatal rat neocortex in vivo is a spindle-burst [START_REF] Hanganu | Retinal waves trigger spindle bursts in the neonatal rat visual cortex[END_REF]Khazipov et al. 2004b). Spindle-bursts are spatially confined spindle-shape oscillations at alpha-beta frequency, associated with phase-locked neuronal firing and activation of the glutamatergic and GABAergic synapses. Spindle-bursts constitute a self-organizing pattern that persists after deafferentation. In the intact animal, compartmentalized spindle-bursts in somatosensory cortex are triggered by sensory feedback resulting from spontaneous movements in a somatotopic manner (Khazipov et al. 2004b). In visual cortex, spindle-bursts are triggered by spontaneous retinal waves in an eye-specific manner [START_REF] Hanganu | Retinal waves trigger spindle bursts in the neonatal rat visual cortex[END_REF]. However, the network mechanisms underlying the generation of cortical spindle-bursts are poorly understood. Several patterns of correlated neuronal activity sharing some common features with the spindle-bursts in vivo were previously described in postnatal rodent neocortical and hippocampal slices and intact preparations in vitro. These include "synchronized-via-gapjunctions neuronal domains" (Kandler andKatz 1995 1998a,b;[START_REF] Yuste | Neuronal domains in developing neocortex[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex: mechanisms of coactivation[END_REF] and calcium waves [START_REF] Peinado | Traveling slow waves of neural activity: a novel form of network activity in developing neocortex[END_REF][START_REF] Peinado | Immature neocortical neurons exist as extensive syncitial networks linked by dendrodendritic electrical connections[END_REF], gap-junction-and NMDA-receptor-based cholinergic oscillations [START_REF] Dupont | Rapid developmental switch in the mechanisms driving early cortical columnar networks[END_REF], giant depolarizing potentials (GDPs), and early network oscillations driven by glutamatergic and excitatory GABAergic synapses (Agmon et al. 1996;[START_REF] Ben-Ari | Giant synaptic potentials in immature rat CA3 hippocampal neurones[END_REF][START_REF] Garaschuk | Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus[END_REF][START_REF] Garaschuk | Large-scale oscillatory calcium waves in the immature cortex[END_REF]Khazipov et al. 1997;[START_REF] Leinekugel | Ca 2ϩ oscillations mediated by the synergistic excitatory actions of GABA A and NMDA receptors in the neonatal hippocampus[END_REF][START_REF] Leinekugel | Giant depolarizing potentials: the septal pole of the hippocampus paces the activity of the developing intact septohippocampal complex in vitro[END_REF]. Consistent with the in vitro findings, generation of the early hippocampal in vivo pattern of sharp waves [START_REF] Buhl | Developmental emergence of hippocampal fast-field "ripple" oscillations in the behaving rat pups[END_REF][START_REF] Leinekugel | Correlated bursts of activity in the neonatal hippocampus in vivo[END_REF] was also shown to involve excitatory action of ␥-aminobutyric acid (GABA) [START_REF] Sipila | The cationchloride cotransporter (NKCC1) promotes sharp waves in the neonatal rat hippocampus[END_REF]). However, to what extent these developmentally regulated mechanisms contribute to generation of the neocortical in vivo pattern of spindle-bursts remains largely unknown.

In the present study, we studied the mechanisms of spindlebursts in the neonatal rat barrel cortex in vivo using a superfused neocortex preparation initially developed for the adolescent rat hippocampus [START_REF] Khazipov | Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo[END_REF]. We found that the physiological pattern of spindle-bursts is preserved in the superfused barrel cortex. Pharmacological profiling of spindle-bursts indicated that 1) generation of spindle-bursts is primarily based on glutamatergic synapses, with a major role of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a slight contribution of N-methyl-Daspartate (NMDA) receptors and gap junctions; and that 2) GABAergic synapses are not directly involved in the generation of spindle-burst oscillations, although they play an important role in their spatial compartmentalization.

M E T H O D S

This study followed INSERM guidelines on animal care, with approval from the animal care and use local committee. Wistar rats of both sexes from postnatal day (P) 1 to P7 were used. P0 was the day of birth. During the surgical procedure, rats were anesthetized with a combination of 0.5-1.5 g/kg urethane injected intraperitoneally and ice-cooling. The skin and periosteum were removed from the skull, which was then covered with a layer of dental acrylic, except for two areas about 7 mm in diameter above the right and left barrel cortices (Fig. 1A). The rat was positioned in the stereotaxic apparatus; the skull was attached to the nose (nasal bones) and ear bars (occipital bone) with dental acrylic. A 5-mm-diameter burr hole was drilled in the skull above the right barrel cortex, the dura was cut and removed, and the cortical surface was covered with 0.9% NaCl during the procedure to prevent it from drying. The perfusion chamber was prepared as described previously [START_REF] Khazipov | Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo[END_REF] with some modifications. In brief, a 2-mm-long cylinder was cut from a plastic tube (ID 3.5 mm, OD 4.5 mm) and glued to stretched nylon mesh with cyanoacrylamide glue. The chamber was positioned at the cortical surface so that the mesh gently pressed onto the cortex. The chamber was then fixed to the skull by dental acrylic. Chlorided silver wire was inserted into the cerebellum and served as a ground electrode.

During recordings, rats were heated by a thermal pad (37°C). The chamber was perfused with oxygenated (95% O 2 -5% CO 2 ) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 126 NaCl, 3.5 KCl, 2.0 CaCl 2 , 1.3 MgCl 2 , 25 NaHCO 3 , 1.2 NaH 2 PO 4 , and 11 glucose (pH 7.4 at a rate of 2 ml/min). Temperature in the chamber was kept at 35-37°C using an automatic temperature controller (TC-344B; Warner Instruments, Hamden, CT). Extracellular field potential recordings (ϫ1,000; 3-to 3,000-Hz band-pass) were performed using arrays of metal electrodes of 50 m in diameter (California Fine Wire, Grover Beach, CA). Patch-clamp recordings were performed using a Axopatch 200A amplifier (Axon Instruments, Union City, CA) using a patching technique similar to that described in vivo [START_REF] Leinekugel | Correlated bursts of activity in the neonatal hippocampus in vivo[END_REF][START_REF] Margrie | In vivo, low-resistance, whole-cell recordings from neurons in the anaesthetized and awake mammalian brain[END_REF]. The pipettes were filled with a solution of the following composition (in mM): 135 Cs-gluconate (or methylsulfate), 2 MgCl 2 , 0.1 CaCl 2 , 1 EGTA, and 10 HEPES (pH 7.25). Membrane potential values were corrected for liquid junction potential of ϩ12 mV. Afferent stimulation was performed by applying electrical pulses (60 V, 50 s, 0.03 s Ϫ1 ) through pairs of electrodes inserted into the whisker pads and glued to the skin with super glue.

Data were digitized at 10 kHz using a Digidata 1322A interface (Axon Instruments) and analyzed off-line using an Axon package (Axon Instruments), MiniAnalysis (Synaptosoft, Decatur, GA), Origin (Microcal Software, Northampton, MA), and Matlab (The Math-Works, Natick, MA). Group measures are expressed as means Ϯ SE. The statistical significance of differences was assessed with the Student's t-test. The level of significance was set at P Ͻ 0.05.

The drugs gabazine, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-2-amino-5-phosphonovaleric acid (D-APV) were purchased from Tocris Neuramin (Bristol, UK), diazepam from Roche (Basel, Switzerland), and all other compounds from Sigma (St. Louis, MO).

R E S U L T S

To study the mechanisms of spindle-bursts in the barrel cortex of neonatal rats (postnatal days P1-P7), we used a technique of superfused neocortex in vivo that was originally developed for adult hippocampus [START_REF] Khazipov | Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo[END_REF]. The photograph and the experimental setup are schematically shown in Fig. 1A. In brief, under anesthesia, a round window was cut in the scull above the barrel cortex and dura was removed. A cylinder with fine mesh at its bottom was gently pressed onto the surface of the barrel cortex (mesh prevents pulsations and allows application of drugs directly onto the cortical surface). In addition to the ease of pharma-FIG. 1. Spindle-bursts in the superfused barrel cortex of neonatal rat in vivo. A: scheme (top) and photograph (bottom) of the experimental preparation. Perfusion chamber is a cylinder with fine mesh at its bottom. It is placed at the surface of barrel cortex so that mesh gently presses on the cortex to prevent pulsations. This construction allows application of drugs directly on the cortical surface. Pair of stimulation electrodes is implanted into the whisker pads. B: local field potential recordings (3-to 3,000-Hz band-pass) from the postnatal day 4 (P4) rat superfused barrel cortex (BCx); below are shown the corresponding time-frequency analysis and multiple unit activity (MUA). Spindle-bursts are marked with asterisks. C: spindle-burst marked by asterisk on B (top trace) is shown on an expanded timescale. D: action potentials of individual cortical neuron recorded concomitantly in current-clamp mode during the spindle-burst oscillation (depth, 600 m). E-G: spindle-burst occurrence (E), duration (G), and normalized power (F) in the superfused and intact barrel cortices. Pooled data from 13 P2-P7 rats; total 1,165 spindle-bursts. cological manipulations, this technique is also convenient for placement of electrodes. The contralateral barrel cortex remained intact and served as a control.

We first determined whether the normal physiological pattern of spindle-bursts and sensory-evoked responses are preserved in the superfused barrel cortex by comparing the activity in the barrel cortex superfused with normal ACSF with the intact contralateral barrel cortex using local field potential recordings (3-to 3,000-Hz band-pass). Spontaneous activity in both superfused and intact barrel cortices was similar and characterized by highly discontinuous periods of activity. Virtually all multiple unit activity (Fig. 1, B andC) and action potentials of individual cortical neurons recorded concomitantly in current-clamp mode (n ϭ 9 cells; Fig. 1D) were synchronized to intermittent spindle-shape oscillations. These were similar to the spindle-bursts described previously in the primary somatosensory cortex for areas of body representation (Khazipov et al. 2004b) as well as in visual cortex (Hanganu et al. 2006). Spindle-bursts had similar durations of 344 Ϯ 15 and 370 Ϯ 15 ms and peak power of oscillations at 15 Ϯ 3 and 21 Ϯ 3 Hz, and reversed in polarity at about 1 mm depth (in the superfused and intact barrel cortices) (Fig. 1, F andG; n ϭ 13 rats; P2-P7). Spindle-bursts occurred more frequently in the superfused barrel cortex (4.2 Ϯ 0.4 min Ϫ1 ) than in the intact barrel cortex (2.5 Ϯ 0.4 min Ϫ1 ; P Ͻ 0.05; Fig. 1E).

Sensory potentials evoked by electrical stimulation of the contralateral whisker pad occurred with a similar delay of 38.3 Ϯ 1.4 and 39.5 Ϯ 3.5 ms and had amplitudes of 792 Ϯ 49 and 593 Ϯ 12 V in the superfused and intact barrel cortices, respectively (Fig. 2A; n ϭ 17 rats; P2-P7). Relatively long latencies of sensory responses are typical for the immature animals and human and they probably reflect the lack of myelinization (Khazipov et al. 2004b;[START_REF] Pihko | Somatosensory processing in healthy newborns[END_REF]. Transcortical current source density analysis of the sensory-evoked potentials (100-m-depth increment) in the superfused barrel cortex revealed a major sink in the middle cortical layers (Fig. 2B; n ϭ 3 rats; P4). Whole cell recordings with a low-chloride pipette solution were used to discriminate between glutamatergic and GABAergic events. At the reversal potential of the GABA A -mediated postsynaptic currents (PSCs, around Ϫ60 mV) and glutamatergic excitatory (E)PSCs (around 0 mV), sensory stimulation sequentially evoked EPSCs and GABA-PSCs (Fig. 2C; n ϭ 5 neurons, depth from 450 to 600 m). Sensory-evoked EPSCs coincided with the field potential response (delay, 44.1 Ϯ 1.9 ms), whereas the sensory-evoked GABAergic component was organized in a barrage of GABA-PSCs similar to the responses evoked by stimulation in the neonatal thalamocortical slices (Agmon et al. 1996). First, GABA-PSCs in a barrage occurred 9.8 Ϯ 1.6 ms after the EPSC (n ϭ 5 cells; P Ͻ 0.05). These results suggest that sensory stimulation evokes in cortical neurons AMPA/ kainate receptor-mediated EPSCs, probably of thalamocortical origin, and activates local interneurons that generate delayed barrage of GABA-PSCs. Sensory potentials were followed by spindle-bursts in eight of ten stimuli both in the superfused and intact barrel cortex (Fig. 3, A andB; n ϭ 17 rats). Powerspectrum (Fig. 3C) and spindle-burst duration analysis (Fig. 3D) did not reveal any difference between the stimulationevoked and spontaneous spindle-bursts in the superfused barrel cortex. Thus the physiological patterns of spindle-bursts and sensory-evoked cortical responses are preserved in the superfused barrel cortex preparation.

We further explored the pharmacological profile of spontaneous and sensory evoked activity in the superfused barrel cortex. Tetrodotoxin (TTX, 2 M), a blocker of the voltagegated sodium channels, completely and reversibly suppressed spontaneous spindle-bursts (Fig. 4, A and E), sensory-evoked potentials, and sensory-evoked spindle-bursts (Fig. 5D; n ϭ 3 rats). AMPA/kainate receptors antagonist CNQX (20 M) completely and reversibly eliminated spontaneous spindlebursts (Fig. 4, A andE), reduced sensory field potentials evoked by contralateral whisker pad stimulation to 8.8 Ϯ 0.3% of control values (Fig. 5, A andB), and blocked sensoryevoked spindle-bursts, which was also evidenced by a reduction in the power of activity at the dominant frequency of the evoked spindle-burst oscillations (18 Ϯ 2 Hz) from 49.4 Ϯ 16.4 to 0.15 Ϯ 0.03 V 2 in the time frame of 300 -1,000 ms after sensory stimulation (Fig. 5, A andD; n ϭ 10 rats; P2-P7). Spindle-bursts and sensory-evoked responses fully recovered 25-40 min after washout of CNQX (Fig. 5B). The amplitude of sensory responses in the intact barrel cortex was not affected by the CNQX applied to the superfused barrel cortex (Fig. 5B). Consistent with the extracellular data, whole cell recordings revealed complete suppression of the EPSCs and spindle-bursts (Fig. 5C; n ϭ 3 cells; depth 450 -600 m). Although spindleburst oscillations were virtually completely suppressed by CNQX, bursts of multiple units were still efficiently evoked by sensory stimulation and occurred spontaneously (Fig. 5A). This residual activity persisted in the presence of a high concentration of bath-applied CNQX (50 M; n ϭ 9) and after ipsilateral intraventricular CNQX injection (1 l of 10 mM CNQX, n ϭ 4). The mechanism underlying this residual activity at present remains unknown. The NMDA receptor antagonist D-APV (80 M) did not significantly affect sensory potentials and only modestly affected spindle-bursts. The occurrence of spontaneous spindle-bursts (5.1 Ϯ 0.8 min Ϫ1 ; n ϭ 9) and the probability of evoking a spindle-burst (0.8 Ϯ 0.1; n ϭ 5) was not significantly modified by D-APV; however, the duration of spindle-bursts was slightly shortened (from 292 Ϯ 14 to 264 Ϯ 19 ms), reduced in power (33 Ϯ 14 to 8 Ϯ 3 V 2 ), and the oscillation frequency was slightly increased (Figs. 4, B-E and5D; n ϭ 9 rats; P2-P7). Taken together, these results suggest that generation of spindle-burst oscillation is based on glutamatergic synapses with the major contribution of AMPA/ kainate receptors and slight participation of NMDA receptors.

In the next series of experiments we studied the contribution of GABAergic synapses to the generation of spindle-bursts by applying the GABA A -receptor antagonist gabazine. To control the efficacy of gabazine in the superfused cortex, we studied its action on the GABA A -PSCs recorded with low-chloride solution at 0 mV holding potential. Gabazine (40 M) completely blocked spontaneous and sensory-evoked GABA A -PSCs (n ϭ 5 neurons; depth 450 -600 m; Fig. 6B). Gabazine did not significantly change the amplitude of the early negative peak of the sensory-evoked field potentials [control: 450 Ϯ 69 V, gabazine: 496 Ϯ 78 V (110 Ϯ 6%); n ϭ 5; P Ͼ 0.05; P3-P5; Fig. 6B]. This is in keeping with the fact that the early part of the sensory-evoked potential is purely glutamatergic thalamocortical EPSP and the GABAergic conductance is delayed (Figs. 2C and 6B, *; see also above and [START_REF] Agmon | NMDA receptor-mediated currents are prominent in the thalamocortical synaptic response before maturation of inhibition[END_REF]Agmon et al. 1996;[START_REF] Moore | Spatio-temporal subthreshold receptive fields in the vibrissa representation of rat primary somatosensory cortex[END_REF]. However, gabazine induced the appearance or strongly enhanced (to 929 Ϯ 6%; n ϭ 5) the late negative peak of the sensory EPSP, which probably reflects the intracortical spread of excitation (Fig. 6B, **). Blockade of GABA A receptors by gabazine also significantly increased spontaneous cortical activity by almost doubling the occurrence of spontaneous spindle-bursts, as well as increasing the probability of evoking a spindle-burst by stimulation to 0.96 Ϯ 0.02 (n ϭ 10; P Ͻ 0.05; Fig. 6, A, E, andF). The dominant frequency of spindle-burst oscillations was not significantly modified by gabazine (Fig. 6C), althought the power increased from 16 Ϯ 5 to 24 Ϯ 6 V 2 for spontaneous and from 9 Ϯ 2 to 22 Ϯ 5 V 2 for sensory-evoked spindles, and the duration of spindle-bursts increased from 323 Ϯ 13 to 658 Ϯ 41 ms (n ϭ 10; P2-P6; P Ͻ 0.01, Fig. 6D). Superfusion with the NKCC1 antagonist bumetanide (20 M), which negatively shifts the GABA A reversal potential in neonatal cortical neurons [START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1[END_REF], did not appreciably affect the occurrence and the power-frequency characteristics of spindlebursts (n ϭ 6; Fig. 6F). Consistent with these results, systemic intraperitoneal injection of bumetanide (0.6 g/kg) did not affect spindle-bursts (n ϭ 3; data not shown). Application of the positive allosteric GABA A modulator diazepam (70 M), which transiently increases the frequency of hippocampal GDPs in vitro [START_REF] Khalilov | Dual role of GABA in the neonatal rat hippocampus[END_REF], reduced the frequency of spindle-burst occurrence by twofold without any effect on the frequency of spindle-burst oscillations and their power (Fig. 6F; n ϭ 5; P2-P6). These results suggest that GABAergic transmission is not necessarily required for pacing the rhythm of spindle-burst oscillations but that it plays an inhibitory role at the network level.

Horizontal compartmentalization is a salient feature of spindle-burst (Khazipov et al. 2004b). Therefore in the next experiment we tested a hypothesis that GABAergic synapses are involved in compartmentalization of spindle-bursts. Using four site recordings from the barrel cortex with a separation distance between the electrodes of 500 m, we found that spindlebursts are predominantly local events with a relatively low coefficient of cross-correlation of activity between the recording sites (Fig. 7A; n ϭ 5; P2-P7) in keeping with the results obtained in the body representation areas of somatosensory cortex (Khazipov et al. 2004b). In the presence of gabazine (40 M), spindle-bursts were synchronously recorded from several (and often all four) recording sites, accompanied with a strong increase in the cross-correlation between recording sites (Fig. 7B). To verify whether surface application of gabazine does not abolish the potential deep burst generators we administered gabazine intraperitoneally (0.3 mg/kg) and found a similar increase in the occurrence of spindle-bursts and their crosscorrelation over large cortical areas (n ϭ 6; data not shown). Thus although GABA A receptors are not instrumental for pacing the rhythm of spindle-bursts, they appear to play a critical role in the compartmentalization of spindle-burst activity-probably by the mechanism of surround inhibition.

Several studies using intact cortex and cortical slices in vitro indicated an important role for gap junctions in the generation of correlated neuronal activity during the early postnatal period [START_REF] Dupont | Rapid developmental switch in the mechanisms driving early cortical columnar networks[END_REF]Kandler andKatz 1995, 1998a,b;[START_REF] Peinado | Traveling slow waves of neural activity: a novel form of network activity in developing neocortex[END_REF][START_REF] Peinado | Immature neocortical neurons exist as extensive syncitial networks linked by dendrodendritic electrical connections[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex: mechanisms of coactivation[END_REF]. Therefore we studied the effect of the gap-junction blocker mefloquine (50 M) [START_REF] Cruikshank | Potent block of Cx36 and Cx50 gap junction channels by mefloquine[END_REF]) on spindle-bursts. Mefloquine increased the occurrence of spontaneous spindle-bursts to 146 Ϯ 33% (n ϭ 7; P1-P3; P Ͻ 0.05) without affecting the power and frequency of the spindle-burst oscillations (Fig. 4E) and probability of evoked spindle-bursts (Fig. 5D). An increase in the occurrence of spontaneous spindle-bursts to 157 Ϯ 14% was also observed after injection of mefloquine (25 mM, 100 nl) into the cortex (depth 1,000 m; n ϭ 4 rats; P2-P3; P Ͻ 0.05). These results suggest that spindle-bursts significantly differ in their generation mechanisms from the early patterns of cortical activity in vitro that are blocked by the gap-junction antagonists [START_REF] Dupont | Rapid developmental switch in the mechanisms driving early cortical columnar networks[END_REF]Kandler andKatz 1995, 1998a,b;[START_REF] Peinado | Traveling slow waves of neural activity: a novel form of network activity in developing neocortex[END_REF][START_REF] Peinado | Immature neocortical neurons exist as extensive syncitial networks linked by dendrodendritic electrical connections[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex: mechanisms of coactivation[END_REF].

D I S C U S S I O N

The principal findings of the present study can be summarized as follows: 1) spindle-bursts are the principal pattern of activity in the barrel cortex during the first week of postnatal life; like spindle-bursts in the body representation areas, spindle-bursts in the barrel cortex can be evoked by sensory stimulation; 2) the principal properties of spindle-bursts are preserved in the superfused cortex preparation that enables easy pharmacological manipulations and recordings; 3) generation of spindle-burst oscillations requires AMPA/kainate receptor activity at glutamatergic synapses; NMDA receptors and gap junctions are slightly involved in the generation of spindle-bursts; and 4) GABAergic synapses importantly con-FIG. 6. Effect of the GABA A -receptor-acting drugs on spindle-bursts. A: field potential recordings from superfused barrel cortex in control conditions and in the presence of the GABA A receptor antagonist gabazine (20 M). Events marked by asterisks are shown below on expanded timescale. B: sensory-evoked response in barrel cortex recorded using field potential (depth, 400 m) (*, early; **, late negative peak of the sensory evoked field potential) and whole cell patch-clamp (depth, 600 m) recordings with low-chloride pipette solution in control and in the presence of GABA A antagonist at 0 mV membrane potential. C-F: effect of gabazine on the normalized power spectrum (C), duration of spontaneous spindle-bursts (D), and the probability of the evoked spindle-bursts (E). F: summary plot on the occurrence of spontaneous spindle-bursts in the presence of the GABA A -acting drugs: antagonist gabazine (40 M), NKCC1 antagonist bumetanide (40 M), and diazepam (70 M) (*P Ͻ 0.05).

tribute to the compartmentalization of spindle-bursts by surround inhibition.

We used the novel methodological approach of superfused neocortex in vivo to provide a stable platform for electrophysiological recordings and pharmacological manipulation. Advantages of the present technique compared with conventional in vivo recordings can be summarized as follows: 1) the cortex is exposed and the recording electrodes can be placed under visual control as easily as in the intact cortex or cortical slices in vitro and 2) rapid pharmacological manipulations, including application and washout of drugs, are possible. This is a distinct advantage over other in vivo techniques that require systemic or local injections. An important finding of the present study is that the physiological pattern of spindle-bursts is preserved in the superfused neocortex in vivo. The reason for the higher frequency of occurrence of spindle-bursts in the superfused cortex is unknown, but it may be attributable to the washing out of urethane from the superfused cortex and/or some difference in the composition of ACSF from that of the physiological cerebrospinal fluid. Other than the higher occurrence, the characteristics of spindle-bursts and sensory-evoked activity in the superfused barrel cortex were not significantly different from the intact barrel cortex. Thus the preparation of the superfused neocortex combines the in vitro approaches with the in vivo situation, thus enabling one to study the physiological patterns of activity that appear to be much more complex in the intact brain than in isolated slices [START_REF] Steriade | Impact of network activities on neuronal properties in corticothalamic systems[END_REF].

Our results suggest that the generation of spindle-bursts in the neonatal barrel cortex is primarily based on synapses between glutamatergic cells, with the major contribution of the AMPA/kainate receptors. Because the glutamatergic synapses on cortical neurons are mainly provided by the intracortical connections and thalamocortical input, at least two models of spindle-burst oscillations can be proposed: intracortical and thalamocortical. In the intracortical model, the spindle-burst is a local oscillation generated in the network of interconnected cortical glutamatergic neurons. Each cycle of oscillation is set by synchronous activation of local population by mutual excitation of cortical neurons by AMPA/kainate receptors at the cortico-cortical synapses. Because the GABA A antagonists did not significantly affect the frequency of spindle-burst oscillations, neuronal inhibition probably comes from an afterhyperpolarization mediated by the voltage-and calcium-dependent potassium channels. In the thalamocortical model, spindlebursts are generated by a rhythmic thalamocortical input provided by oscillation in thalamic neurons, similar to the mechanism of adult sleep spindles [START_REF] Steriade | Thalamocortical oscillations in the sleeping and aroused brain[END_REF]. The spatial confinement of spindle-bursts in the latter model could be explained by the relative sparsity of long-range intracortical and corticothalamic connections, which are important for synchronization of sleep spindles over the entire cortex [START_REF] Contreras | Control of spatiotemporal coherence of a thalamic oscillation by corticothalamic feedback[END_REF]. This is aided by efficient shunting surround inhibition of depolarizing GABA. The two models are not incompatible and both thalamic and cortical oscillators may resonate given that their intrinsic frequencies are close.

In both the present and previous studies whole cell recordings revealed GABAergic synaptic currents phase-locked with the local field potential oscillations [START_REF] Hanganu | Retinal waves trigger spindle bursts in the neonatal rat visual cortex[END_REF]Khazipov et al. 2004b), suggesting that interneurons are activated during spindle-bursts. However, because blockade of GABA A receptors did not significantly affect the frequency of oscillation it seems that GABAergic interneurons play only a minor role in pacing of the rhythm of oscillation. This differs FIG. 7. Effect of the GABA A antagonist gabazine on the compartmentalization of spindle-bursts. A: field-potential recordings from the superfused barrel cortex with a 4-microelectrode array (500-m separation distance between the electrodes) in control conditions and (B) in the presence of gabazine (40 M). Spindle-burst marked by black asterisk on A (left) is shown on an expanded timescale in the middle. On the right, cross-correlation analysis of spindle-bursts at 4 recordings sites (pooled data from 6 rats). from the adult brain in which the major patterns of activity are significantly influenced by interneurons [START_REF] Freund | Interneurons of the hippocampus[END_REF][START_REF] Fuentealba | The reticular nucleus revisited: intrinsic and network properties of a thalamic pacemaker[END_REF]. GABAergic inhibition undergoes significant developmental changes during the first postnatal week, during which time GABA-acting by GABA A receptors-depolarizes immature neocortical neurons because of elevated intracellular [Cl Ϫ ] i [START_REF] Loturco | GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis[END_REF][START_REF] Luhmann | Postnatal maturation of the GABAergic system in rat neocortex[END_REF][START_REF] Owens | Excitatory GABA responses in embryonic and neonatal cortical slices demonstrated by gramicidin perforated-patch recordings and calcium imaging[END_REF][START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1[END_REF][START_REF] Yuste | Control of postsynaptic Ca 2ϩ influx in developing neocortex by excitatory and inhibitory neurotransmitters[END_REF]. Interestingly, in the hippocampus, GDPs in vitro and sharp waves in vivo are blocked by the NKCC1 antagonist bumetanide, which shifts the reversal potential of the GABA A -mediated responses toward negative values [START_REF] Dzhala | NKCC1 transporter facilitates seizures in the developing brain[END_REF][START_REF] Sipila | The cationchloride cotransporter (NKCC1) promotes sharp waves in the neonatal rat hippocampus[END_REF]. Although a similar effect of bumetanide on the GABA A reversal potential was found in the neocortical neonatal neurons [START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions in immature rat neocortical neurones is mediated by NKCC1[END_REF]), we did not observe any effect of bumetanide on spindlebursts. Therefore it appears that early hippocampal patterns of sharp waves and GDPs are more dependent on the depolarizing/excitatory GABA than the neocortical pattern of spindleburst, which is consistent with the observations made in vitro [START_REF] Garaschuk | Large-scale oscillatory calcium waves in the immature cortex[END_REF].

Although GABAergic interneurons are not directly involved in setting the rhythm of spindle-burst oscillations, they play important role in their horizontal compartmentalization. Blockade of GABA A receptors significantly increased the area of activation during spindle-bursts, evidenced by increases in the amplitude and power of oscillations, duration of spindle-bursts, and their horizontal spread. Thus compartmentalization of spindle-bursts is determined not only by the vertical segregation of the sensory feedback-driven essentially AMPA/kainitereceptor-mediated somatotopic excitation (Agmon et al. 1996;[START_REF] Bureau | Precise development of functional and anatomical columns in the neocortex[END_REF][START_REF] Ferezou | Visualizing the cortical representation of whisker touch: voltage-sensitive dye imaging in freely moving mice[END_REF][START_REF] Higashi | Prenatal development of neural excitation in rat thalamocortical projections studied by optical recording[END_REF]Khazipov et al. 2004b;[START_REF] Kidd | Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses[END_REF][START_REF] Petersen | Functionally independent columns of rat somatosensory barrel cortex revealed with voltage-sensitive dye imaging[END_REF] but also by surround GABAergic inhibition, which prevents the horizontal spread of activity by long-range glutamatergic cortical connections, a pattern observed in the adult neocortex [START_REF] Chagnac-Amitai | Horizontal spread of synchronized activity in neocortex and its control by GABA-mediated inhibition[END_REF][START_REF] Fox | The origin of cortical surround receptive fields studied in the barrel cortex[END_REF][START_REF] Sun | Barrel cortex microcircuits: thalamocortical feedforward inhibition in spiny stellate cells is mediated by a small number of fast-spiking interneurons[END_REF]. The inhibitory action of GABA at the network level is probably explained by the shunting mechanisms amplified by the activation of the voltage-gated potassium channels and inactivation of sodium channels [START_REF] Borg-Graham | Visual input evokes transient and strong shunting inhibition in visual cortical neurons[END_REF][START_REF] Gao | GABA-dependent firing of glutamateevoked action potentials at AMPA/kainate receptors in developing hypothalamic neurons[END_REF][START_REF] Gulledge | Excitatory actions of GABA in the cortex[END_REF][START_REF] Lu | Mixed excitatory and inhibitory GABA-mediated transmission in chick cochlear nucleus[END_REF]. These results are in general agreement with the findings that administration of GABA A antagonists induces hypersynchronous seizurelike activity in the neocortex in vivo by P3 [START_REF] Baram | Bicuculline induced seizures in infant rats: ontogeny of behavioral and electrocortical phenomena[END_REF] and in vitro by P2 [START_REF] Wells | GABAergic inhibition suppresses paroxysmal network activity in the neonatal rodent hippocampus and neocortex[END_REF].

Comparing various neonatal patterns and mechanisms of neuronal synchronization described in vitro and in the present study in vivo, it appears that the in vivo and in vitro patterns share some common features, although none of the patterns described in vitro fully matches spindle-bursts, probably because the in vitro models cannot fully reproduce the in vivo conditions [START_REF] Steriade | Impact of network activities on neuronal properties in corticothalamic systems[END_REF]. Studies using cortical preparations in vitro emphasized the role of several developmentally regulated mechanisms of neuronal synchronization in the developing cortex, including 1) gap junctions [START_REF] Dupont | Rapid developmental switch in the mechanisms driving early cortical columnar networks[END_REF]Kandler andKatz 1995, 1998a,b;[START_REF] Peinado | Traveling slow waves of neural activity: a novel form of network activity in developing neocortex[END_REF][START_REF] Peinado | Immature neocortical neurons exist as extensive syncitial networks linked by dendrodendritic electrical connections[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex[END_REF][START_REF] Yuste | Neuronal domains in developing neocortex: mechanisms of coactivation[END_REF], 2) NMDA receptors [START_REF] Ben-Ari | Giant synaptic potentials in immature rat CA3 hippocampal neurones[END_REF][START_REF] Dupont | Rapid developmental switch in the mechanisms driving early cortical columnar networks[END_REF][START_REF] Leinekugel | Ca 2ϩ oscillations mediated by the synergistic excitatory actions of GABA A and NMDA receptors in the neonatal hippocampus[END_REF], and 3) depolarizing GABA [START_REF] Ben-Ari | Giant synaptic potentials in immature rat CA3 hippocampal neurones[END_REF][START_REF] Garaschuk | Developmental profile and synaptic origin of early network oscillations in the CA1 region of rat neonatal hippocampus[END_REF][START_REF] Garaschuk | Large-scale oscillatory calcium waves in the immature cortex[END_REF][START_REF] Khazipov | Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices[END_REF]Khazipov et al. , 2004a ; ;[START_REF] Leinekugel | Ca 2ϩ oscillations mediated by the synergistic excitatory actions of GABA A and NMDA receptors in the neonatal hippocampus[END_REF][START_REF] Sipila | Depolarizing GABA acts on intrinsically bursting pyramidal neurons to drive giant depolarizing potentials in the immature hippocampus[END_REF][START_REF] Sipila | The cationchloride cotransporter (NKCC1) promotes sharp waves in the neonatal rat hippocampus[END_REF]. Our results suggest that the generation of the in vivo neocortical pattern of spindle-bursts relies on a rather "mature" mechanism based on AMPA/kainite-receptor-mediated synaptic transmission. It should be noted that AMPA/ kainate antagonists were also efficient in suppressing some types of cortical network activity in the neonatal period, including spontaneous-but not stimulation-evoked-GDPs [START_REF] Ben-Ari | Giant synaptic potentials in immature rat CA3 hippocampal neurones[END_REF][START_REF] Bolea | Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices[END_REF][START_REF] Khazipov | Synchronization of GABAergic interneuronal network in CA3 subfield of neonatal rat hippocampal slices[END_REF][START_REF] Lamsa | Synaptic GABA(A) activation inhibits AMPA-kainate receptor-mediated bursting in the newborn (P0 -P2) rat hippocampus[END_REF], polysynaptic events in barrel cortex evoked by thalamic stimulation (Agmon et al. 1996), and neocortical early network oscillations [START_REF] Garaschuk | Large-scale oscillatory calcium waves in the immature cortex[END_REF].

In the rodent somatosensory cortex, activity-dependent cortical plasticity is maximal over an early "critical" postnatal developmental period, which is characterized by enhanced synaptic plasticity and by the potential for profound alterations of anatomical and functional organization of the barrel cortex by manipulation of the sensory input [START_REF] Crair | A critical period for long-term potentiation at thalamocortical synapses[END_REF][START_REF] Erzurumlu | Neural activity: sculptor of "barrels" in the neocortex[END_REF][START_REF] Feldman | Long-term depression at thalamocortical synapses in developing rat somatosensory cortex[END_REF][START_REF] Feldman | Synaptic plasticity at thalamocortical synapses in developing rat somatosensory cortex: LTP, LTD, and silent synapses[END_REF][START_REF] Fox | Anatomical pathways and molecular mechanisms for plasticity in the barrel cortex[END_REF][START_REF] Fox | A comparison of experience-dependent plasticity in the visual and somatosensory systems[END_REF][START_REF] Katz | Development of cortical circuits: lessons from ocular dominance columns[END_REF][START_REF] Katz | Synaptic activity and the construction of cortical circuits[END_REF][START_REF] Van Der Loos | Somatosensory cortex: structural alterations following early injury to sense organs[END_REF]. Glutamate receptor blockade during the first postnatal week was previously shown to disrupt the topographic refinement of thalamocortical connectivity and columnar organization of the barrel cortex [START_REF] Elias | Glutamate receptor blockade alters the development of intracortical connections in rat barrel cortex[END_REF][START_REF] Fox | Glutamate receptor blockade at cortical synapses disrupts development of thalamocortical and columnar organization in somatosensory cortex[END_REF] and impairs formation of the intracortical connectivity [START_REF] Dagnew | Glutamate receptor blockade alters the development of intracortical connections in rat barrel cortex[END_REF]. We propose that local spindle-burst oscillations, driven by glutamatergic synapses and compartmentalized by GABAergic synapses, contribute to development of the barrel cortex during the critical period of developmental plasticity.

FIG. 2 .

 2 FIG. 2. Sensory potentials evoked by electrical whisker pad stimulation. A: sensory potentials evoked by the left ant right whisker pad (WP) stimulation in the intact left and superfused right barrel cortex. B: depth profile of the sensory-evoked potentials in the superfused barrel cortex. C: sensory-evoked response in barrel cortex recorded using field potential (depth, 400 m) and whole cell patch-clamp (depth, 600 m) recordings with low-chloride solution to discriminate between the glutamatergic (Ϫ60 mV) and GABAergic (0 mV) postsynaptic currents.

FIG. 3 .

 3 FIG.3. Spindle-bursts evoked by whisker pad stimulation. A: electrical stimulation of the contralateral whisker pad (arrow) evokes a sensory potential followed by spindle-burst in the superfused P4 rat barrel cortex. Below, corresponding MUA (filtered at Ͼ200 Hz) and time-frequency analysis. B: probability of sensory-evoked spindle-bursts in the superfused (chamber) and intact barrel cortex. C: normalized power spectrum. D: duration of spontaneous and sensory-evoked spindle-bursts. Pooled data from 14 P2-P7 rats, 583 stimulations, and 499 spindle-bursts.

FIG. 4 .

 4 FIG. 4. Effect of the glutamate ionotropic receptors antagonists and tetrodotoxin (TTX) on spontaneous spindle-bursts. A: field potential recordings in control conditions and in the presence of the ␣-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 M), sodium channel blocker TTX (2 M), N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) (80 M), and gap-junction blocker mefloquine (50 M). Recordings from P4 (control, CNQX, TTX, D-APV) and P2 (mefloquine) superfused barrel cortex. B-D: effect of D-APV on the duration (B) and normalized power of spontaneous (C) and sensory-evoked (D) spindle-bursts. Pooled data from 9 P3-P6 rats and 225 spontaneous and 214 evoked spindlebursts. E: summary plot on the effect of D-APV (n ϭ 9 rats; P3-P6), TTX (n ϭ 3 rats; P4 -P6), CNQX (n ϭ 12 rats; P2-P7), and mefloquine (n ϭ 5 rats; P1-P3) on the occurrence of spontaneous spindle-bursts.

FIG. 5 .

 5 FIG. 5. Effect of AMPA/kainate receptor antagonist CNQX on the sensoryevoked spindle-bursts. A: sensory-evoked potentials and spindle-bursts in control conditions (top) and in the presence of CNQX (20 M) (bottom). B: time course of the effect of CNQX on the amplitude of the sensory-evoked potentials in the superfused (squares) and intact barrel cortex (circles). C: whole cell recordings of the sensory-evoked responses in control conditions and in the presence of CNQX (20 M). D: summary plot on the effect of D-APV (n ϭ 5 rats; P3-P6), TTX (n ϭ 3 rats; P4 -P6), CNQX (n ϭ 7 rats; P2-P7), and mefloquine (n ϭ 5 rats; P1-P3) on the probability of sensoryevoked spindle-bursts (*P Ͻ 0.05).

  A C K N O W L E D G M E N T S

Marat Minlebaev, Yehezkel Ben-Ari, and Rustem Khazipov The

  Network Mechanisms of Spindle-Burst Oscillations in the Neonatal Rat Barrel Cortex In Vivo Mediterranean Institute of Neurobiology, Institut National de la Sante ´et de la Recherche Me ´dicale U29, Universite ´Me ´diterrene ´en, Marseille, France

	Submitted 21 July 2006; accepted in final form 6 November 2006

J Neurophysiol• VOL 97 • JANUARY 2007 • www.jn.org on February 6, 2007 jn.physiology.org Downloaded from

We thank G. Buzsa ´ki, A. Sirota, M. Colonnese, M. Milh, R. Tyzio, I. Hanganu, and A. Zakharov for helpful comments on the manuscript.

G R A N T S

This work was supported by grants from the North Atlantic Treaty Organization and Universite ´Me ´diterrene ´en to M. Minlebaev, Institut National de la Sante ´et de la Recherche Me ´dicale to Y. Ben-Ari, and Fondation pour la Recherche Me ´dicale and Agence National de la Recherche to R. Khazipov.


Yehezkel Ben-Ari
email: <ben-ari@inmed.univ-mrs.fr>

Basic developmental rules and their implications for epilepsy in the immature brain

The construction of the human brain with its 10 15 synapses follows a set of complex developmentally and environmentally regulated steps. A series of sequences have been described that are instrumental, in the sense that a failure of any one of them leads to dramatic, life-long consequences. Hence the importance of determining the sequential maturation of neurons, synapses and cortical maps. It is also important to determine how networkdriven events become installed, as neuronal activity intervenes in all of these steps and modulates, for better or worse, the outcome. A fundamental consequence of these sequential events is that any disruption will have very different consequences depending on when it occurs, indeed, "when is as important as what". An obvious aspect of these general features is related to seizures. In fact, the developing brain has both a higher incidence of seizures in human and animal models, and experiences seizures that can produce long-lasting consequences that are also stage-dependent. This seminar and the series of slides presented are an introduction to these issues, summing up several studies made notably by INMED researchers during the last two decades (http://www.inmednet.com). It concentrates on four basic developmental rules: i) the generation by very immature neurons, of very large currents mediated by the activation of receptors in neurons that bear no synapses. This is due to the release of GABA that diffuses to distal sites and acts as a paracrine factor; ii) the excitatory/inhibitory shift of the actions of GABA during development because of a progressive reduction in the intracellular chloride concentration; iii) the sequential formation of GABAergic synapses and networks before glutamatergic ones, implying that, at an early stage, all the excitatory drive will be GABAergic; iv) the presence, at an early stage, of a unique, primitive pattern in all developing structures, this pattern disappears when most GABAergic synapses have shifted to their adult configuration-.Several consequences of these sequences are described including: i) a control of neuronal migration by GABA-acting drugs, and the possibility that migration disorders are also generated by environmental factors that include the effects of GABA-acting agents; ii) If GABA excites immature neurons and inhibits adult one, then GABA-acting agents will also produce different effects on the mother and the embryo; iii) early brain oscillations are generated by the periphery and propagate centrally -notably to the sensory-motor cortex, suggesting that peripherally-generated movements may provide an important signal for the formation of cortical maps, in keeping with the importance of embryonic movements; iv) "seizures beget seizures" in the developing brain. This has now been shown in a triple chamber with the two intact hippocampi that we developed, and with which it has been possible to show that only recurrent seizures that include high frequency oscillations can transform the naïve,

contralateral hippocampus to an epileptic one that seizes spontaneously. Most interestingly, at an early developmental stage, when GABA excites many neurons and the density of glutamatergic synapses is not sufficiently high, purely glutamatergic seizures cannot lead to long-term consequences, the additional excitatory drive provided by GABAergic synapses is needed. In other words, at that stage, blocking GABA synapses generates seizures, as in adults, but these do not lead to long-term consequences. The mechanisms that underlie these differences is due to the need for high frequency oscillations (> 80 Hz or so), and these can only be generated when GABA synapses are operative in the developing brain: GABA receptor antagonists are ictogenic, but not epileptogenic.To facilitate teaching purposes the paper is published together with supplemental data (as a PowerPoint presentation included in the accompanying DVD), thus allowing an overview of important developmental steps and their implications.

[Published with supplemental data on CD] Key words: neuronal migration, excitatory action of GABA, oscillation, seizure, antiepileptic drug, GABAergic interneuron There is a paradox as far as epilepsy in the developing brain is concerned. The incidence of seizures is higher early in life and seizures lead to long-term deleterious sequel; yet immature neurons are less vulnerable than adult ones to a variety of insults including anoxo-ischemic episodes and seizures. In other words, seizures are more frequent -indicating that immature networks are endowed with a capacity for high excitability and synchronized events. These observations lead to a number of conclusions. Firstly, seizures are more readily generated because one or more of the mechanisms that in adults prevent excessive activity, are not available. Secondly, seizures must produce their consequences by altering fundamental processes rather than "simply" causing neuronal lesions. This implies that understanding and treating neonatal and early seizures must incorporate a detailed analysis of the sequences of expression of the principal constituent of cortical networks in order to determine where the missing mechanisms are that fail to prevent seizure generation, and which sequence is affected by hyperactivity leading to permanent alterations of the normal sequence of events. To further complicate the issue, it is essential to take into account that the developing brain is not a small adult brain, but a unique machine with channels, molecules and mechanisms that are entirely or largely absent in adults. Thus, it is not possible to rely on mechanisms operating in adults to unravel the causes of the paradox summarized above. The central issue is to determine when during development, neuronal activity can alter developmental processes (figure 1/DVD figure 2). Studies performed mostly in Unit 29 of the French medical research council (INSERM) have unraveled several fundamental rules of brain development and have shown that these rules most likely underlie the unique properties of the developing brain as far as epileptogenesis is concerned. This work summarizes the salient features of these observations and, relying on pictures from actual experiments, shows how they explain the high incidence of seizures and their long lasting consequences on the developing brain. We shall divide the sequence into 3 steps: migration, synapse formation, network construction (figure 2/DVD figure 3).

To evaluate the impact of activity on brain maturation, we shall examine successively the role of the principal transmitters, GABA and glutamate, on essential developmental steps, and, in parallel, illustrate how this information impacts on neurological disorders. In the adult brain, glutamatergic neurons constitute the vast majority of neurons, and provide the main source of the excitatory drive. The dense networks of GABAergic interneurons that constitute 10-15% of the neurons on the other hand, provide the source of the inhibitory drive. In spite of this apparent imbalance, interneurons play a crucial role in the generation of behaviorally relevant patterns [START_REF] Freund | Interneurons of the hippocampus[END_REF]. Various types of interneurons control the fine discharge of the principal cells, and the patterns that networks will generate. In the absence of functional GA-BAergic neurons and synapses, cortical networks seize and do not generate behaviorally relevant patterns. Let us now examine whether they are operative at early stages and whether they control essential developmental processes.

Neuronal migration

In a wide range of systems, receptors develop before synapses and the obvious question is whether they exert an influence. In other words, does modulation by environmental factors, including neuronal activity, start with synapse formation or does it occur at an earlier stage when synapses are not yet operative? If transmitters are released and act on immature neurons endowed with receptors but not functional synapses, they should generate a current by activating post-synaptic receptors. With this in mind, we therefore patch-clamp recorded immature neurons in which the conventional, fast synaptic responses cannot be generated and found that indeed, electrical stimuli evoked giant currents that are several order of magnitude larger than conventional synaptic currents (figure 3; DVD figure 456). These cur-rents are mediated by the activation of GABA-and glutamate NMDA-type receptors -but not AMPA receptors that are responsible for the majority of fast acting, ionotropic synapses in the adult brain. We also found that there is a tonic release of GABA and glutamate at an early stage, since a GABA receptor antagonist generates a tonic current indicating an ongoing release of GABA [START_REF] Demarque | Paracrine intercellular communication by a Ca2+-and SNARE-independent release of GABA and glutamate prior to synapse formation[END_REF]. The release mechanisms are unique in the sense that they do not depend on essential vesicular release proteins and factors including release proteins and machinery. They can even be generated in animals in which the release machinery has been knocked out. This paracrinic release of GABA acts on distal neurons to generate large currents prior to synapse formation. At a later stage, this system disappears and is replaced by the more conventional focal action of transmitters. Therefore, neurons communicate prior to synapse formation and there is a non-canonical release of transmitter that can act diffusely on distal sites (also see below).

Transmitter release and neuronal migration

Is transmitter release functional and does it alter migration? We tested the possible role of this paracrine release on migration in vitro, using an original preparation in which two hippocampal slices are cultivated next to each other: one of the slices was obtained from a GFP transgenic mouse in which all neurons are green, allowing us to follow visually their course and migration on the other, dark slice (figure 4 evidence that GABA is a morphogenic agent that, at an early stage, modulates neuronal migration in addition to synapse formation, and neuronal growth and differentiation. Thus, transmitters, notably GABA, can modulate early developmental processes by means of actions on receptors that are, in essence, extra -synaptic (also see [START_REF] Owens | Changing properties of GABA(A) receptor-mediated signaling during early neocortical development[END_REF][START_REF] Owens | Is there more to GABA than synaptic inhibition?[END_REF][START_REF] Represa | Trophic actions of GABA in neuronal development[END_REF]. Therefore, agents acting on GABA receptors can be expected to act on neurons that possess few or no synapses at all. This type of mechanism could be involved in the formation of heterotopic masses and other migration disorders such as those produced in pregnant rats leading to the formation of aberrant connections between the hippocampus and the neocortex (Chevassus-Au-Louis et al. 1998 and figure 5; DVD figure 8). At any rate, they draw our attention to the possible deleterious actions of drugs acting on GABA receptors, notably benzodiazepines or certain anti-epileptic agents. In keeping with this, preliminary observations suggest that some AEDs indeed affect migration (Manent et al. in preparation). These actions are of the utmost importance in view of the part played by migration disorders in human epilepsies. A variety of animal models of these disorders exist, some of them such as the one produced by in utero injection of an anti-mitotic agent MAM (figure 5; DVD figure 8). Studies using this model show that the ectopic masses present in the hippocampus are, in fact, neocortical neurons that were trapped in the hippocampus because of the delayed migration. Patch recordings from both the "normal" target of these neurons and their host structure shows that they have connections both with the neocortex and the hippocampus, creating thus an aberrant bridge connecting, via one synapse, two brain structures that are not connected normally (figure 6; DVD figure 9; Chevassus- [START_REF] Chevassus-Au-Louis | Neuronal migration disorders: heterotopic neocortical neurons in CA1 provide a bridge between the hippocampus and the neocortex[END_REF]. Seizures will readily propagate from the hippocampus to the cortex.

Transmitter uptake systems and early seizures

If transmitters act at this early stage, in a paracrinic manner, then the control of their concentration may play an important role. The concentration of GABA and glutamate in the extracellular space is controlled by a variety of transporters. We therefore tested the effects of blockers of the main transporters on neuronal activity at an early developmental stage E18 to P0 (DVD figure 10). We found that GABA transporters are not active at birth -blockers of this transport do not alter currents generated by GABA. This will allow GABA to diffuse and act on distal sites (DVD figure 11). In contrast, glutamate transporters are operative early on, indicating that the control of glutamate concentration is essential at this early stage to prevent possibly excitotoxic actions of glutamate (DVD figure 12). In keeping with this, glutamate transporter blockade generates large oscillations in hippocampal and neocortical networks (DVD figure 13), and in vivo icv injections of a glutamate receptor antagonist in pups generates recurrent seizures with suppression bursts that are reminiscent of the encephalopathies [START_REF] Demarque | Glutamate transporters prevent the generation of seizures in the developing rat neocortex[END_REF]). Thus, deficiencies in glutamate transport could mediate early, severe epilepsies.

Synapse formation

The formation of synapses (figure 7; DVD figure 14) is an important step as it signals a more organized, topical mode of communication and the beginning of network formation with its generation of behaviorally relevant patterns. The two principal transmitter systems that operate are GABA and glutamate, acting on receptors permeable to anions, chloride in particular for the former, and cations, sodium, potassium and calcium for the latter (figure 8; DVD figure 15). Agents that augment the efficacy of GABAergic synapses are often anti-epileptics and anxiolytics; agents that increase the excitatory drive will induce hyperactivity and cell death (DVD figure 16). This raises the issue of how an equilibrium between these transmitters is reached throughout development (DVD figure 17). This also raises the question of how to develop cortical networks with functional activity at all stagespromote neuronal development and synapse formation yet preserve stability and prevent over excitation that will be excitotoxic, or dominant GABAergic inhibition that will retard developmental functions that are modulated by the increased intracellular calcium that results from the activation of glutamate synapses (DVD figure 17)?

In 1989, three basic developmental rules (figure 9; DVD figure 18) for the maturation of GABA and glutamate systems were unraveled. These present a possible solution to this issue: i) GABAergic synapses precede glutamate synapses (figure 10; DVD figure 19; Ben-Ari 2002; [START_REF] Tyzio | The establishment of GABAergic and glutamatergic synapses on CA1 pyramidal neurons is sequential and correlates with the development of the apical dendrite[END_REF]). In the developing hippocampus, interneurons do not develop hand-in-hand with the pyramidal neurons: GABAergic interneurons and synapses mature first and are operative at a stage when the principal cells are silent [START_REF] Ben-Ari | Interneurons set the tune of developing networks[END_REF]. Interneurons divide prior to the glutamatergic neurons, extend their axons and dendrites prior to the principal cells, establish functional synapses before principal neurons and can generate patterns when principal neurons are essentially inert. We patch-clamp recorded a large sample of CA1 pyramidal neurons at birth and reconstructed them after identifying their PSCs. We found three populations of neurons, silent ones with no PSCs that have few or no dendrites, bigger neurons with GABA but no glutamate synapses have a small, apical dendrite, neurons with both GABA and glutamate PSCs have a large, apical dendrite that reaches, for the first time, the distal lacunosum moleculare and a basal dendrite. Therefore, the first synapses are established on the apical dendrites and are GABAergic, neurons must reach the distal lacunosum moleculare to have functional glutamate synapses. Since the axons are there already, the critical factor is the degree of maturation of the post-synaptic target: GABAergic synapses are established when axons meet their post-synaptic target, but glutamatergic synapses will not be formed unless the target is more developed. This sequential expression of GABA and glutamate synapses has been confirmed in a wide range of species including primate neurons in utero (figure 10; DVD figure 19; Tyzio et al., see below,). We patch-clamped CA1 pyramidal neurons in slices obtained from macaque embryos delivered by caesarean section from mid-gestation to two weeks before full-term, and found an earlier formation of GABAergic synapses on pyramidal neurons with a small, apical dendrite and both GABA and glutamate synapses on neurons with extended apical dendrites and basal dendrites. There were no neurons with functional glutamate but no glutamate synapses confirming the earlier formation of GABAergic synapses.

ii) GABA inhibits adult neurons but excites immature ones (figure 11;DVD figure 20) This is due to a higher concentration of intracellular chloride in immature neurons, which leads to an efflux of chloride when GABA receptors are activated [START_REF] Ben-Ari | Giant synaptic potentials in immature rat CA3 hippocampal neurones[END_REF][START_REF] Tyzio | Membrane potential of CA3 hippocampal pyramidal cells during postnatal development[END_REF]). These gradients have been observed in all species and brain structures studied, indicating that they have been retained throughout evolution [START_REF] Gao | GABA, not glutamate, a primary transmitter driving action potentials in developing hypothalamic neurons[END_REF][START_REF] Owens | Changing properties of GABA(A) receptor-mediated signaling during early neocortical development[END_REF][START_REF] Ben-Ari | Excitatory actions of GABA during development: the nature of the nurture[END_REF][START_REF] Hubner | Disruption of KCC2 reveals an essential role of K-CI transport already in early synaptic inhibition[END_REF]. They are also observed in primate neurons in utero, and are thus valid for human fetuses and preterm babies [START_REF] Khazipov | Early development of neuronal activity in the primate hippocampus in utero[END_REF]). The consequence is that when GABA receptors are activated there is an efflux of chloride instead of the usual influx, and a depolarization that will generate action potentials, remove the voltage-dependent magnesium block from NMDA channels leading to an influx of calcium [START_REF] Ben-Ari | GABAA, NMDA and AMPA receptors: a developmentally regulated "ménage à trois[END_REF]). This early action of GABA reverses subsequently, with the delayed activation of a chloride co-transporter that exports chloride from the inside to the outside and install the adult concentration of chloride (figure 12; DVD figure 21; [START_REF] Rivera | The k+/Cl-co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation[END_REF]. We have suggested elsewhere that this sequence provides a solution to the problem of how to equilibrate, at all developmental stages, excitation and inhibition during brain maturation (DVD figure 22 and Ben-Ari 2002). Indeed, as the excitatory drive provided by GABAergic synapses is not toxic -as the E rev of GABA is always closer to the resting membrane potential than glutamate (E 50-60 mV as compared to 0 mV). This is also facilitated by the shunting actions of GABAergic synapses that inhibit overexcitation. Thus, GABA will provide a sufficient degree of excitatory drive to raise intracellular calcium via the activation of NMDA channels or voltagedependent calcium channels, and thus to modulate developmental processes without danger. Also, the longer kinetics of GABAergic as compared to glutamate AMPA receptor-mediated PSCs enables an accumulation of synaptic current in neurons that possess but few synapses. Therefore, during early stages, when the glutamatergic drive is limited, GABA "does the job", exciting neurons and raising intracellular calcium. When the density of glutamatergic synapses is sufficient and a strong inhibition is required to prevent seizure generation, GABA assumes its adult actions following the expression of a transporter that will reduce the intracellular concentration of chloride. This model provides a rational solution to the issue raised above (DVD figure 22, Ben-Ari 2002). If GABAergic synapses are formed first, GABAergic interneurons should also develop at an early stage. In the developing hippocampus, interneurons do not develop hand-in-hand with the pyramidal neurons: GABAergic interneurons and synapses mature first and are operative at a stage when the principal cells are silent [START_REF] Hennou | Early sequential formation of functional GABA(A) and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus[END_REF], Ben-Ari et al. 2004). Interneurons divide prior to glutamatergic neurons, extend their axons and dendrites prior to the principal cells, establish functional synapses before principal neurons and can generate patterns when principal neurons are essentially inert. Most interestingly, the rules for establishing functional GABA or glutamate synapses between an axon and its dendritic target are not identical: GABA synapses are established once the contact is established, whereas glutamate synapses can only be formed when the target neuron has reached a certain degree of maturation. Therefore, in spite of a rather long journey -interneurons are not generated in the close vicinity of the principal neurons, but in the distal ganglionic eminence -interneurons must, for some reason, be at work at an early stage. In parallel studies, we found that already in utero, patterns generated exclusively by interneurons can be recorded [START_REF] Ben-Ari | Interneurons set the tune of developing networks[END_REF].

iii) Developing networks speak only one language (figures 13, 14; DVD figures 23-24)

We discovered some time ago that the developing hippocampal network has a single pattern -which we referred to as giant depolarising potentials (GDPs) [START_REF] Ben-Ari | Giant synaptic potentials in immature rat CA3 hippocampal neurones[END_REF][START_REF] Ben-Ari | Developing networks play a similar melody[END_REF]. This pattern is characterised by recurrent bursts with large, polysynaptic currents mediated by GABA and glutamate receptors. Subsequent studies revealed that a similar pattern is present in all developing networks, in all species including the developing primate hippocampus in utero. GDPs provide almost all the activity as long as GABAergic synapses are excitatory. The shift from excitatory to inhibitory GABA signals the end of GDPs and the expression of more adult patterns; it is at that stage that a network is sufficiently complex that it can generate behaviorally relevant patterns. GDPs are generated by the combined action of excitatory GABA synapses and large NMDA receptor-mediated synaptic currents. The latter are of particularly long duration in the immature brain consequent to the presence of a subunit of NMDA receptors that confer to neurons very long kinetics. The synergistic action of GABA and NMDA receptors is the core of this pattern, the small depolarization produced by GABA is sufficient to remove the blockade from NMDA receptors and generates the long, poly-synaptic currents notably via the activation of an excitatory recurrent loopthe activation of interneurons leading, in return, to an enhancement of the excitation on principal cells and the generation of large synaptic currents. GDPs have now been recorded by a plethora of techniques -including cell-attached and whole cell recordings, as well as field and surface recordings -and preparations including slices and also cultures and in vivo. In the intact hippocampus in vitro -a preparation developed in the laboratory that has major advantages in comparison to slices -GDPs can be generated by a wide range of sites but they propagate along the developmental gradient -from rostral to caudal sites -indicating a developmental activity gradient [START_REF] Khalilov | A novel in vitro preparation: the intact hippocampal formation[END_REF][START_REF] Khalilov | In vitro formation of a secondary epiletogenic mirror focus by interhippocampal propagation of seizures[END_REF]. GDPs can be considered as a type of a primitive pattern with little information content that exists to signal activity in developing connections and to promote synapse formation and possibly provide early signals for the construction of operating functional units on the basis of neurons that fire together, wire together". A general developmental curve depicting the maturation sequences in primate hippocampal neurons is shown in figure 15 (DVD figure 25). This figure, that provides the most complete curve available today, is a series of measures (Boltzmann equations to depict the temporal matu- ration of the various functions). Thus, proliferation (taken from the literature) is followed by axon (first) and dendritic extension (then, expressed in lm per day) and spine acquisition. Pyramidal neurons of CA1 have no spines at mid-gestation, but as many as 7000 glutamate synapses a few weeks before delivery. The earlier formation of GABA synapses is described quantitatively in the last curve. GDPs disappear when the density of glutamate synapses is sufficient to generate more complex, behaviorally relevant patterns and GABA assumes its adult actions. An obvious consequence of this early expression of functional GABAergic synapses is that GABA-mimetic agents and anti-epileptic drugs will exert a particularly strong action on the activity-dependent formation of the network [START_REF] Represa | Trophic actions of GABA in neuronal development[END_REF]. The obvious implications of these gradients are that drugs targeted to a given molecular species will have complex. unexpected actions: they will exert no action on some neurons endowed with different molecular species and act only on that proportion of neurons that have shifted to adult types of molecular species. In addition, the gradient of heterogeneity will depend notably on the species: animals that have an extended period of brain development -such as monkeys and humans-will offer a picture of heterogeneity for an extended period of time thus allowing extrinsic factors to exert a stronger influence than animals in which this period is restricted to a few days.

Implications of these rules

There are several implications and consequences of these rules (figure 16; DVD figure 26).

Early activity may participate in the formation of cortical maps

Recordings in chronically implanted pups revealed the presence of sharp waves and tails that are reminiscent of GDPs (figures 17, 18; DVD figures 27-28; [START_REF] Leinekugel | Correlated bursts of activity in the neonatal hippocampus in vivo[END_REF][START_REF] Khazipov | Early motor activity drives spindle bursts in the developing somatosensory cortex[END_REF]). These patterns are generated by GABA and glutamatergic synaptic currents as revealed by whole cell recordings in non-anaesthetized, restrained pups. Most importantly, when these events are recorded in both the periphery and the cortical centrethe sensori-motor cortex -the spindles in the periphery precedes those in the center. Lesions of the spinal cord reveal a large reduction of spontaneous spindles suggesting that the peripherally-generated spindles are the generators of those in the cortical sites. These and other similar observations are in line with the concepts that developing circuits in the periphery and centres communicate via a single pattern and that peripheral structures can provide a strong source of modulation of the centres. In a wider perspective, these studies raise the possibility that the programming of the construction of central corti- cal organization, including cortical maps and other functional entities, are controlled or at least modulated by peripheral activity. In view of this, a unified pattern of activity in developing structures is perfectly able to contribute to the wiring of distant structures. The alterations produced by aberrant activities such as seizures would produce their deleterious sequelae merely by interrupting these patterns rather than by more specific informationcontaining signals.

Impact on early seizures

Immature brains have a higher incidence of seizures early on. This is, at least in part, due to the late maturation of a potent inhibitory GABAergic system and the early excitatory actions (figure 19; DVD figure 29; Holmes and Ben-Ari 1998). The peak seizure generation in the developing hippocampus (bottom curve) is attained when GABAergic excitation shifts to a dominant inhibition around P 10), glutamate excitatory drive steadily increases, and the im-portance of GDPs declines, being replaced by more sophisticated patterns that require a relatively elaborated network to function. In keeping with this, seizures are readily generated around the last trimester in primate hippocampal neurons in utero (DVD figure 30).

Seizures can lead to long lasting consequences

In vivo studies have documented extensively the possible long-term actions of seizures at an early stage. As shown earlier, seizures such as those produced by administration of kainic acid during the first week of post-natal life are not associated with brain damage [START_REF] Tremblay | Maturation of kainic acid seizure-brain damage syndrome in the rat. I. Clinical, electrographic and metabolic observations[END_REF]. Various convulsive agents lead to long-lasting consequences including persistent changes in neuronal excitability (DVD figure 31; [START_REF] Villeneuve | Neonatal seizures induced persistent changes in intrinsic properties of CA1 rat hippocampal cells[END_REF]. One way of mediating these long term actions could be a reduction in the seizure threshold after an episode of severe seizures; In other words, in this model, "seizures would beget seizures" as first suggested by Gower in the early nineties. This concept has however never received direct confirmation. We recently developed a unique preparation in which this property can be tested readily in vitro (figure 20;DVD figure 32;[START_REF] Khalilov | A novel in vitro preparation: the intact hippocampal formation[END_REF][START_REF] Khalilov | In vitro formation of a secondary epiletogenic mirror focus by interhippocampal propagation of seizures[END_REF]. The preparation consists of a triple chamber that can accommodate the two intact hippocampi and their connecting commissure in three independent compartments. It is possible to apply a convulsive agent to one hippocampus and allow a given number of seizures to propagate to the other hippocampus. We can then block the connections reversibly and determine whether the hippocampus has been transformed by seizures. We found that a series of brief seizures generated by the kainate in one hippocampus generated seizures that propagated to the other site leading to a transformation of the naive hippocampus to an epileptic one that generates spontaneous and evoked Khalilov et al. Neuron 1997;Khazipov et al. J Neurophysiol 1999. seizures (figure 21; DVD figures [33][34][35]. A number of recurrent seizures are required for this effect-five to 10. Most interestingly, the epileptogenic mirror focus is truly epileptic as even 48 hours later, it generates spontaneous ictal events, and when slices are prepared from the intact hippocampus, it generates high frequency ictal events: this is to the best of my knowledge, the only example of an ictal event generated in hippocampal slices. In contrast to interictal events that are readily generated in slicesincluding human slices obtained after resections from epileptic patients-ictal events are not generated. We therefore used this preparation both to determine what the minimal requirements for the transformation to take place are -what the mechanisms of induction are -and what the mechanisms mediating the expression of the property are? Examining the pattern of oscillations in the propagated seizures, we observed that very high frequency events are present -80 to 120 Hz or more in the kainite-treated hippocampus (DVD figure 36). Initially, the seizures that propagate to the other side generate only low frequency events. After the propagation of several, high frequency events from the other side, the naïve hippocampus also becomes progressively capable of generating high frequency events and this signals that the naïve hippocampus has become epileptogenic (DVD figure 36). If high frequency oscillations are required for "seizures to beget seizures", then conditions that prevent the generation of these oscillations should also prevent the formation of a mirror focus. We found that blocking either GABA or NMDA receptors by applications of selective antagonists exclusively in the naïve side, the seizures still propagate from the other side, but they do not contain high frequency events (DVD figure 37). Applications of kainate to one hemisphere that generate high frequency events in the treated but not the naïve side, fail to transform the naïve side to an epileptic one. In other words, functional GABAergic receptors are required for "seizures to beget seizures". Without these high frequency events, seizures are not epileptogenic, and most likely will not induce long-term consequences on target structures. Interestingly, blocking GABA receptors in the kainite-treated side also led to a blockade of the long-term effect locally: the kainite-treated side had no high frequency oscillations and did not generate seizures after repeated applications; the other side that did not receive kainate but which was stimulated by low frequency recurrent seizures coming from the treated side, did generate high frequency oscillations and became epileptic [START_REF] Khalilov | Epileptogenic actions of GABA and fast oscillations in the developing hippocampus[END_REF]. Low frequency seizures can generate high frequency events in the contralateral side provided that GABA and NMDA receptors are operative there. This transformation of a naïve hippocampus to an epileptic one is thus analogous to a form of long-term plasticity -an alteration of synaptic plasticity, the activation by high frequency currents of GABA receptors leading to the activation of NMDA receptors, and this synergistic action leads to the increased synaptic efficacy. Interestingly, patching neurons from the epileptic hippocampus, we found that GABA again excites neurons: the seizures induced a long-term transformation of the inhibitory actions of GABA to excitatory, most likely because of a loss of the capacity to remove and regulate chloride via co-transporters (figure 22; DVD figure 38). To some extent, epileptogenesis recapitulates ontogenesis (DVD figure 39, [START_REF] Khalilov | Epileptogenic actions of GABA and fast oscillations in the developing hippocampus[END_REF][START_REF] Cohen | On the origin of interictal activity in human temporal lobe epilepsy in vitro[END_REF], and seizures re-induce a return to immature neurons with an excitatory GABA -except that now with the high density of glutamate synapses, seizures will be generated. Therefore, the expression of the epileptogenic properties is, in fact, due to a persistent accumulation of chloride, the seizures producing a loss in the efficacy of neurons to remove the excess chloride produced during the seizures. This loss of efficacy of the transporters may be more readily triggered by high frequency oscillations.

In conclusion, these observations suggest (DVD figure 40) that seizures are not necessarily epileptogenic -they can occur recurrently, without sequelae, if they do not include high frequency oscillations that require both operative GABA and NMDA receptors. The most likely explanation for this requirement is that the excitatory drive provided by GABA is required at this stage as the density of glutamate synapses may not suffice: a small but decisive activation of excitatory GABA, activates NMDA receptors by removing the voltage-dependent magnesium blockade leading to a large calcium influx, and a sequence of events that leads to a permanent consequence. This observation suggests that it may be possible to evaluate the epileptogenic capacity of seizures in children by evaluating their intrinsic, dominating, oscillatory frequencies. GABA is both a pioneer and an essential actor in early development. It is operative first; it excites neurons at a time when neurons have few synapses, and it has a paracrine action on immature neurons, modulating neuronal migration. Then it acts in coordination with NMDA receptors stimulating developing networks intervening in the construction of cortical networks and exerting a wide range of trophic actions. Clearly, because of this plethora of actions and its unique properties, this transmitter is able to act very differently according to the conditions. It seems ideally suited to the job of regulating the complex functional sequences involved in brain development. And last but not least, GABA is greatly involved in early seizures, both in their induction and in their long-lasting consequences: seizures do not transform a naïve network to one that seizes unless GABA synapses are operative. Clearly, the effects of GABA-acting drugs on developing brains must be re-evaluated in the light of this information. M

Figure 1 .

 1 Figure 1. Nature and Nurture in development & epilepsies.

Figure 2 .

 2 Figure 2. Constructing a cortex.

  ; DVD figure 7; Manent et al. 2005. We found (figure 5; DVD figure 8) that blocking GABA receptors retarded neuronal migration considerably indicating that migration -an activity-dependent process (DVD figure 6; Komuro and Rakic) -is modulated by a non-focal, paracrinic action of transmitters, most notably GABA. These and other observations are in line with the extensive

Figure 3 .

 3 Figure 3. Receptors are formed before synapses. Demarque et al. Neuron 2004.

Figure 4 .

 4 Figure 4. Slice co-cultures to visualize migrating neuroblasts. Manent et al. J Neurosci 2005.

Figure 5 .

 5 Figure 5. Blockade of GABA Rs retards migration. Manent et al. J Neurosci 2005.

Figure 6 .

 6 Figure 6. Clinical relevance: migration disorders! Chevassus-Au-Louis et al. PNAS 2000.

Figure 8 .

 8 Figure 8. Inhibition and Excitation.

Figure 7 .

 7 Figure 7. Constructing a cortex. C. Hammond, Molecular and Cellular Neurobiology, Elsevier.

Figure 9 .

 9 Figure 9. Three rules preserved throughout evolution! Ben-Ari et al. J Physiol, 1989

Figure 12 .

 12 Figure 12. Higher [Cl -] i : a question of pumps & transporters.Figure 11. Second Rule: More Chloride inside immature neurons! Ben-Ari et al. J Physiol 1989.

Figure 11 .

 11 Figure 12. Higher [Cl -] i : a question of pumps & transporters.Figure 11. Second Rule: More Chloride inside immature neurons! Ben-Ari et al. J Physiol 1989.

Figure 13 .

 13 Figure 13. Three rules preserved throughout evolution!

Figure 15 .

 15 Figure 15. Developmental sequences in Macaques. Khazipov et al. J Neuroscience 2002.

Figure 16 .

 16 Figure 16. Implications of these rules.

Figure 17 .

 17 Figure 17. Spindles in the developing cortex. Leinekugel et al. Science 2003 ; Khazipov et al. Nature 2004.

Figure 18 .

 18 Figure 18. Pattern oscillators: from spinal cord to centre.

Figure 19 .

 19 Figure19. Incidence on ictogenesis.Khazipov et al, 2003.

Figure 20 .

 20 Figure20. A novel preparation: interconnected intact hippocampi in vitro.Khalilov et al. Neuron 1997; Khazipov et al. J Neurophysiol 1999.

Figure 21 .

 21 Figure 21. Formation of an epileptogenic mirror focus.Figure 22. Chloride shifts during maturation and after seizures. Ben-Ari et al. J Physiol 1989 ; Khalilov et al., Nature Neurosccience, 2003.

Figure 22 .

 22 Figure 21. Formation of an epileptogenic mirror focus.Figure 22. Chloride shifts during maturation and after seizures. Ben-Ari et al. J Physiol 1989 ; Khalilov et al., Nature Neurosccience, 2003.

Epileptic Disord Vol. 8, No. 2, June 2006

Basic developmental rules and their implications for epilepsy in the immature brain