Director, Yehezkel Ben-Ari
email: ben-ari@inmed.univ-mrs.fr

Epilepsies and neuronal plasticity: for better or for worse?

Keywords: temporal lobe epilepsy, sprouting, novel synapse

etworks are endowed with the capacity to generate a large repertoire of behaviorally relevant patterns and oscillations relying on the intrinsic properties of assemblies of interconnected neurons. These oscillations are not only involved in integrative programs, but also play an important role in the induction of long-lasting modifications of synapse efficacy. [START_REF] Freund | Interneurons of the hippocampus[END_REF][START_REF] Ylinen | Sharp wave associated high-frequency oscillation (200Hz) in the intact hippocampus: network and intracellular mechanisms[END_REF][START_REF] Buzsaki | Rhythms of the Brain[END_REF][START_REF] Khazipov | Early motor activity drives spindle bursts in the developing somatosensory cortex[END_REF][START_REF] Leinekugel | Correlated bursts of activity in the neonatal hippocampus in vivo[END_REF][START_REF] Bartos | Fast synaptic inhibition promotes synchronized gamma oscillations in hippocampal interneuron networks[END_REF][START_REF] Fuentealba | The reticular nucleus revisited: intrinsic and network properties of a thalamic pacemaker[END_REF][START_REF] Destexhe | Mechanisms underlying the synchronizing action of corticothalamic feedback through inhibition of thalamic relay cells[END_REF][START_REF] Cunningham | A role for fast rhythmic bursting neurons in cortical gamma oscillations in vitro[END_REF] Recurrent activation of synapses and interconnected networks leads to various forms of neuronal plasticity that constitute the main mechanisms on which networks rely to augment or reduce the response to incoming information. [START_REF] Buzsaki | Rhythms of the Brain[END_REF][START_REF] Isomura | Integration and segregation of activity in entorhinal-hippocampal subregions by neocortical slow oscillations[END_REF] These mechanisms have been extensively investigated, relying on protocols that induce short-and long-term potentiation and depression of synaptic efficacy. [START_REF] Bliss | Long-term potentiation in commissural and Schaffer projections to hippocampal CA1 cells: an in vivo study in the rat[END_REF][START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF][START_REF] Malenka | Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission[END_REF][START_REF] Nicoll | Expression mechanisms underlying NMDA receptor-dependent long-term potentiation[END_REF] They are usually triggered by a rise in [Ca 2+ ] i that initiates a cascade of molecular events and more permanent changes associated with the formation of novel synapses. These mechanisms constitute the core of the modulation by activity and experience of network activity and the essence of memory processes. [START_REF] Bliss | Long-term potentiation in commissural and Schaffer projections to hippocampal CA1 cells: an in vivo study in the rat[END_REF][START_REF] Bliss | A synaptic model of memory: long-term potentiation in the hippocampus[END_REF][START_REF] Malenka | Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission[END_REF][START_REF] Nicoll | Expression mechanisms underlying NMDA receptor-dependent long-term potentiation[END_REF] Epilepsies are associated with episodes of altered/enhanced activity, and therefore they are expected to also produce long-term alterations in synaptic efficacy and network reorganizations. Studies performed in the last 2 to 3 decades have shown that "seizures beget seizures," with a cascade of events triggered by seizures that transform a naive network into one that generates seizures. [START_REF] Lothman | Epileptogenic effects of status epilepticus[END_REF][START_REF] Ari | Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy[END_REF][START_REF] Ari | Kainate, a double agent that generates seizures: two decades of progress[END_REF][START_REF] Ari | Effects of seizures on developmental processes in the immature brain[END_REF][START_REF] Represa | Kainate binding sites in the hippocampal mossy fibers: localization and plasticity[END_REF][START_REF] Ben-Ari | Long-lasting modification of the synaptic properties of rat CA3 hippocampal neurones induced by kainaic acid[END_REF] The sequence starts with an inaugurating event/insult, followed by a cascade of molecular and biochemical events that produce the quasi-permanent shift. They culminate with a reactive 75 B a s i c r e s e a r c h N Extensive experimental investigations have confirmed that "seizures beget seizures." Thus, in adults, limbic seizures lead to cell loss, followed by the formation of novel excitatory synapses that contribute to generating further seizures. The triggering signal is an enhancement of synaptic efficacy, followed by a molecular cascade that triggers axonal sprouting. New synapses are aberrant, since they are formed in regions in which they are not present in controls. They also involve receptors that are not present in controls, and this facilitates the generation of seizures. Therefore, an aberrant form of reactive neuronal plasticity provides a substrate for the long-lasting sequelae of seizures. Since these events take place in brain structures involved in integrative and mnemonic functions, they will have an important impact. Reactive plasticity is documented for other insults and disorders, and may be the basis for the long-term progression of neurodegenerative disorders.

plasticity associated with sprouting of axons and the formation of novel synapses that appear to play a central role in the generation of seizures. These novel synapses are aberrant, since they are formed on neurons that do not possess these inputs in naive conditions. They are also unique to epileptic tissues, as they use receptors and signals that do not operate in controls (reactive plasticity). These important features have several important fundamental and applied consequences. This paper will discuss these issues, stress the similarities and differences between physiological and pathogenic forms of neuronal plasticity, and ask the following questions: Do seizures lead to similar alterations as episodes of enhanced activity engaged in memory processes? If not, how do they differ? Are physiologically relevant mechanisms altered to leave a trace of the original insult, leading to the pathogenic generation of abnormal patterns and their neurological correlates? Since alterations of neuronal activity are observed in other disorders, is reactive plasticity also involved in nonepileptic types of disorders? Finally, the general implications of these observations will be briefly discussed. Since seizures also induce long-lasting sequelae in developing brain structures, but by quite different means, the relationship between seizures and epilepsies in the developing brain will also be briefly addressed.

The hippocampal circuitry: a target for studies on various forms of neuronal plasticity

Most of our understanding of the mechanisms of neuronal plasticity and their link with seizures comes from studies devoted to the hippocampus. This "simple" cortical structure is composed of a principal layer-the pyramidal and granule cell layer-and molecular layers with basal and apical dendrites of principal neurons-the stratum oriens and stratum radiatum respectively. These principal neurons are excitatory, using glutamate as a transmitter with primarily α-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA) receptors to generate the fast-acting excitatory postsynaptic currents (EPSCs). Powerful stimulation can also activate Nmethyl-D-aspartic acid (NMDA) receptors-a calciumpermeable receptor channel complex that is blocked in physiological conditions by Mg++ ions in a voltagedependent manner. [START_REF] Nowak | Magnesium gates glutamate-activated channels in mouse central neurones[END_REF] The increase in the intracellular concentration of calcium leads to a cascade of molecular events that will produce long-lasting quasi-permanent changes in synaptic efficacy, primarily by means of postsynaptic mechanisms. [START_REF] Nicoll | Expression mechanisms underlying NMDA receptor-dependent long-term potentiation[END_REF][START_REF] Malenka | Synaptic plasticity in the hippocampus: LTP and LTD[END_REF] In addition, a plethora of inhibitory γ-aminobutyric acid-(GABA)ergic neurons and synapses provide a highly specific pattern of innervations that modulate neuronal excitability and thus control the entire network and the generation of behaviorally relevant oscillations. The basic circuit is organized around a trisynaptic excitatory circuit, with a massive input originating in the entorhinal cortex that innervates the apical dendrites of the granule cells, followed by the axons of granule cells that establish giant mossy fiber synapses with the proximal segment of the CA3 pyramidal neurons and finally the axons of the latter-the Schaeffer collateral-that innervate the apical dendrites of CA1 pyramidal neurons. CA3 pyramidal neurons also send out an extensive network of recurrent collaterals that innervate via other CA3 pyramidal neurons, facilitating the generation of synchronized activities and seizures. The CA3 pyramidal system is amongst the brain regions the most susceptible to seizures, triggering events, and drugs, mostly because of an abundant excitatory recurrent collateral net of synapses between pyramidal neurons that will facilitate the emergence of synchronized activities.

Physiological and pathological forms of long-term potentiation of synaptic efficacy

High-frequency stimuli of synapses generate a long-term potentiation of synaptic transmission with long-lasting enhanced EPSCs. The induction is generated by an influx of calcium, mainly mediated by NMDA receptors, and the expression is mediated by a persistent increase in the density of AMPA receptors. [START_REF] Malenka | Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission[END_REF][START_REF] Nicoll | Expression mechanisms underlying NMDA receptor-dependent long-term potentiation[END_REF] In most protocols, NMDA receptors play an important role in the induction but not the expression of the augmented signals, ie, AMPA not NMDA receptor-mediated EPSCs are augmented by the protocol. Brief seizures produced for instance by brief applications of the powerful convulsive agent kainate (see below) also induce a long-term potentiation (LTP) of EPSCs. [START_REF] Ben-Ari | Long-lasting modification of the synaptic properties of rat CA3 hippocampal neurones induced by kainaic acid[END_REF] Like physiological LTP, the induction of this process is mediated by the activation of NMDA receptors. However, the expression of this "epileptic LTP" also involves NMDA receptor-mediated EPSCs that are persistently facilitated. [START_REF] Aniksztejn | Expression of LTP by AMPA and/or NMDA receptors is determined by the extent of NMDA receptors activation during the tetanus[END_REF][START_REF] Ben-Ari | A united theory for the multiple forms of LTP[END_REF] Thus, seizures produce long-lasting alterations of synapse efficacy, but the underlying mechanisms are different. Several mechanisms have been suggested to explain the persistent increase of NMDA receptor-mediated EPSCs, including alterations of regulating sites of the receptor channel complex. [START_REF] Aniksztejn | Expression of LTP by AMPA and/or NMDA receptors is determined by the extent of NMDA receptors activation during the tetanus[END_REF][START_REF] Ben-Ari | A united theory for the multiple forms of LTP[END_REF] Interestingly, other insults also produce long-term alterations of synaptic efficacy, including brief anoxic insults, suggesting that pathological forms of LTP may constitute a general mechanism involved in translating their deleterious sequelae into alterations of synapse efficacy. [START_REF] Crepel | A selective LTP of NMDA receptor-mediated currents induced by anoxia in CA1 hippocampal neurons[END_REF][START_REF] Crepel | Synaptic plasticity in ischemia: role of NMDA receptors[END_REF] Therefore, episodes of augmented activity or other insults do lead to persistent changes of synaptic efficacy, somehow deviating physiological processes.

Kainic acid: an analogue of glutamate that plays an important role in neuronal synchronizations

Purified-form marine algae, kainic acid, is an analogue of glutamic acid that produces a long-lasting excitation of neurons via a subclass of glutamate receptors. [START_REF] Ben-Ari | Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy[END_REF][START_REF] Lerma | Glutamate receptors of the kainate type and synaptic transmission[END_REF] These receptors are enriched, notably on mossy fiber synapses established on the proximal apical dendrites of CA3 pyramidal neurons (stratum lucidum). [START_REF] Represa | Kainate binding sites in the hippocampal mossy fibers: localization and plasticity[END_REF] More recent studies have shown kainate receptor-mediated synapses confirming their role in physiological conditions. Thus, "pure" kainate receptor-mediated EPSCs are recorded in CA3 pyramidal neurons. [START_REF] Cossart | Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons[END_REF][START_REF] Castillo | Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons[END_REF] These EPSCs differ from AMPA receptor-mediated EPSCs by their longer kinetics and their smaller amplitude. In addition, mixed AMPA/kainate receptor-mediated EPSCs are also recorded, indicating that both receptor types are involved in mediating fast glutamatergic synaptic activity. Kainate receptors are also enriched on GABAergic interneurons, where they provide almost half the ongoing glutamatergic activity. [START_REF] Cossart | Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons[END_REF][START_REF] Cossart | Activation of GluR5 receptors in interneurons increases tonic GABAergic inhibition of pyramidal neurons[END_REF] A direct association between mossy fiber terminals and "kainatergic" synapses has also been shown, confirming the close relationship between kainate signaling and the CA3 mossy fiber terminals. [START_REF] Cossart | Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons[END_REF] Kainatergic postsynaptic currents (PSCs) have a slower and smaller amplitude than the conventional AMPA receptor-mediated currents, suggesting that they will play different tunes in the generation of synchronized oscillations.

Several subclasses of kainate receptors have been cloned, including the GluR5 subunits that are enriched on interneurons, and the GluR 6 subunits enriched on mossy fiber synapses. [START_REF] Coussen | Kainate receptor-interacting proteins and membrane trafficking[END_REF][START_REF] Hollmann | Cloning by functional expression of a member of the glutamate receptor family[END_REF][START_REF] Bettler | Cloning of a putative glutamate receptor: a low affinity kainate-binding subunit[END_REF] The GluR 6 subunits are enriched on mossy fiber terminals, and have been associated with seizures. The threshold for seizure generation is reduced in transgenic animals depleted of this subunit. [START_REF] Ari | Kainate, a double agent that generates seizures: two decades of progress[END_REF][START_REF] Mulle | Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice[END_REF][START_REF] Gaïarsa | Neonatal irradiation prevents the formation of hippocampal mossy fibers and the epileptic action of kainate on rat CA3 pyramidal neurons[END_REF] On the other hand, GluR 5 subunits are enriched on interneurons where they provide as much as half of the excitatory inputs. [START_REF] Ari | Kainate, a double agent that generates seizures: two decades of progress[END_REF][START_REF] Cossart | GluR5 kainate receptor activation in interneurons increases tonic inhibition of pyramidal cells[END_REF] Therefore, kainate signaling will both facilitate paroxysmal activity by means of GluR 6 receptor-containing synapses, and prevent seizures by means of GluR 5 receptor-containing synapses and the augmented inhibition that they produce. [START_REF] Cossart | Activation of GluR5 receptors in interneurons increases tonic GABAergic inhibition of pyramidal neurons[END_REF][START_REF] Khalilov | Paradoxical anti-epileptic effects of a GluR5 agonist of kainate receptors[END_REF] The kainate animal model generates a seizure and brain damage syndrome that mimics human temporal lobe epilepsies Systemic or intracerebral injections of kainate generate long-lasting limbic seizures, followed by a pattern of lesions in the hippocampus and other limbic structures. [START_REF] Ari | Kainate, a double agent that generates seizures: two decades of progress[END_REF][START_REF] Ben-Ari | Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy[END_REF][START_REF] Lothman | Functional anatomy of hippocampal seizures[END_REF][START_REF] Ben-Ari | The role of epileptic activity in hippocampal and 'remote' cerebral lesions induced by kainic acid[END_REF] The electrographic pattern and the subsequent lesions have a distribution that is quite similar to that observed in patients suffering from temporal lobe epilepsy (TLE) with a preferential involvement of the CA3 and CA1 regions, GABAergic interneurons, and the pyriform cortex, as well as other limbic cortices and subcortical regions. The amygdala plays a central role in the sequence, as suggested by the powerful actions of very low concentrations of kainate injected in that region and the clinical and electrographic signs that imply the involvement of that structure (ref?). Extensive investigations clearly indicate that the neuronal cell loss is at least in part due to the excessive activation of certain glutamatergic fibers, and in particular the mossy fiber terminals that produce a lesion of the neurons that they innervate.

From cell loss to neosynapse formation

Neurons adjacent to, or even linked to, damaged neurons do not remain idle. Studies in the early 1970s had already shown that in the hippocampus, lesions of the perforant pathway leads to a reorganization of other inputs, with aberrant connections being formed. [START_REF] Nadler | Selective reinnervation of hippocampal area CA1 and the fascia dentata after destruction of CA3-CA4 afferents with kainic acid[END_REF] Sprouting after an episode of status epilepticus has been extensively illustrated with kainate and other models of seizures. In the early 1980s, studies relying on the unique feature of mossy fibers-the high density of zing-positive Timm stain and of high-affinity kainate receptors-showed that, after seizures, an additional band of mossy fiber terminals is formed (Figures 1 to 3). [START_REF] Represa | Kainate binding sites in the hippocampal mossy fibers: localization and plasticity[END_REF][START_REF] Nadler | Kainic acid as a tool for the study of temporal lobe epilepsy[END_REF][START_REF] Lothman | Basic mechanisms of the epilepsies[END_REF][START_REF] Esclapez | Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy[END_REF][START_REF] Represa | Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha-tubulin[END_REF] This has been extensively confirmed in a variety of animal models of epilepsies, and in human temporal lobe epileptics and other types of epilepsies. [START_REF] Represa | Sprouting of mossy fibers in the hippocampus of epileptic human and rat[END_REF] The formation of novel synapses was also shown by electron microscopy, relying on the unique features of mossy fiber terminals that form the largest complex of postsynaptic excrescences readily identified with certainty. [START_REF] Represa | Epilepsy induced collateral sprouting of hippocampal mossy fibers: does it induce the development of ectopic synapses with granule cell dendrites?[END_REF] Therefore, new synapses are formed after status epilepticus, and many of these synapses are aberrant, ie, present on target neurons that are not innervated by these neurons in naive networks. Studies using molecular biology tools indicate that after a status epilepticus as many as 1000 genes are activated. [START_REF] Nedivi | Numerous candidate plasticity-related genes revealed by differential cDNA cloning[END_REF][START_REF] Pollard | Seizureinduced molecular changes, sprouting and synaptogenesis of hippocampal mossy fibers[END_REF][START_REF] Ben-Ari | Brief seizure episodes induce long-term potentiation and mossy fibre sprouting in the hippocampus[END_REF][START_REF] Khrestchatisky | Molecular correlates between reactive and developmental plasticity in the rat hippocampus[END_REF] These occur with a progressive time course-from immediate early genes to growth factors (hours after the event) to receptors and cell adhesion molecules days later, providing a likely scenario of molecular cascades B a s i c r e s e a r c h set in motion by the episode of hyperactivity. The actual sprouting is most likely terminated within 1 to 2 weeks. The functionality of these novel synapses was confirmed with electrophysiological recordings. Thus, recordings of pyramidal neurons after a status epilepticus showed an increased frequency of glutamatergic EPSCs, in keeping with the increased density of glutamatergic terminals (Figure 4, also see below). [START_REF] Esclapez | Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy[END_REF][START_REF] Bernard | What is GABAergic inhibition? How is it modified in epilepsy?[END_REF][START_REF] Epsztein | Ongoing epileptiform activity in the post-ischemic hippocampus is associated with a permanent shift of the excitatory-inhibitory synaptic balance in CA3 pyramidal neurons[END_REF] Since GABAergic interneurons also degenerate, inhibitory currents were also expected to be affected.

Earlier studies suggested that a reduction in GABAergic inhibition precedes the initiation of ongoing seizures by the epileptic circuit. [START_REF] Kapur | Evidence for a chronic loss of inhibition in the hippocampus after kindling: electrophysiological studies[END_REF][START_REF] Mody | The balance between excitation and inhibition in dentate granule cells and its role in epilepsy[END_REF] Recordings made more recently from a variety of interneurons and the somata and dendrites of principal cells showed a massive reduction of the frequency of GABAergic PSCs in the dendrites of the principal cells, in keeping with the preferential degeneration of somatostatin-positive interneurons that innervate selectively the dendrites of pyramidal neurons. [START_REF] Hirsch | Deficit in quantal release of GABA in experimental models of temporal lobe epilepsy[END_REF][START_REF] Cossart | Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy[END_REF] The frequency of GABAergic PSCs recorded in the somata of principal neurons was not significantly different from controls (ref?). Therefore, a dual consequence of seizures on inhibition will occur with a loss of dendritic inhibition but a preservation of somatic inhibition. Interestingly, whereas somatic inhibition controls the generation of spikes and the output of the system, dendritic inhibition controls the currents generated by incoming EPSCs and therefore the inputs of the network. [START_REF] Miles | Differences between somatic and dendritic inhibition in the hippocampus[END_REF] Therefore, although the threshold of seizures is reduced in epileptic networks, the control of somatic activity still operates and prevents an ongoing status epilepticus. Most importantly, these observations suggest that reactive plasticity does not occur in GABAergic neurons, since the loss of dendrite inhibition is permanent. This difference between anatomic plasticity and reaction to damage may constitute a major property of networks in neurodegenerative disorders.

The operation of epileptic and naive networks: similar but different

Newly formed synapses are aberrant anatomically, but also in their operation. Thus, in controls, granule cells operate exclusively by means of AMPA (and NMDA) receptor-mediated channels. In contrast, weeks after seizures, when novel synapses have been formed, they also display pure kainate receptor-mediated EPSCs. [START_REF] Epsztein | Recurrent mossy fibers establish aberrant kainate receptor-operated synapses on granule cells from epileptic rats[END_REF] Therefore, the sprouting of mossy fibers imposes the formation of aberrant kainatergic synapses in the novel targets of mossy fibers (Figure 3). [START_REF] Represa | Kainate binding sites in the hippocampal mossy fibers: localization and plasticity[END_REF] These new synapses will have important consequences on the operation of this essential input of the hippocampal formation. Indeed, AMPA receptor-mediated EPSCs are brief, with a short latency and a time-locked precise response, therefore generating large synchronized currents. In contrast, kainite-operated currents are of small amplitude, long duration, and there are probably more opportunities for summations and alterations of synapse efficacy. To sum up, epileptic tissues have epileptic synapses that are unique to this tissue, in that they have an aberrant distribution and a signaling profile that promotes synchronized activities. This important observation implies clearly that even in the absence of seizures, "epileptic networks" operate differently from control ones. It also shows also why the link between kainate receptors, seizures, and epilepsies is instrumental.

Another illustration of the differences between naive and genuine epileptic networks comes from studies in humans: surgically removed hippocampal slices in patients suffering from TLE. Electrophysiological recordings made in these slices show that in a significant per- centage of neurons, GABA has a depolarizing action because of an accumulation of chloride in neurons and thus a different gradient for chloride. [START_REF] Cohen | On the origin of interictal activity in human temporal lobe epilepsy in vitro[END_REF] Indeed, GABA excites neurons in the epileptic network; this is expected to lead to major changes in the operation of the network, since inhibitory GABA plays a central role in the generation of oscillations. This property also illustrates yet another property of networks in degenerative disorders: the return to immature properties. Indeed, in all developing brain structures and animal species, there is a higher [Cl -] i , a property that appears to have been preserved throughout evolution. [START_REF] Benari | Giant synaptic potentials in immature rat Ca3 hippocampal-neurons[END_REF][START_REF] Ari | Excitatory actions of GABA during development: The nature of the nurture[END_REF] The consequence is that GABA excites immature neurons, generating sodium and calcium action potentials and producing a large calcium influx that underlies the trophic actions of GABA on developing neurons. [START_REF] Represa | Trophic actions of GABA on neuronal development[END_REF] There are several indications that after insults of different types, the neurons recuperate or return to their immature situation, at least as far as some signals are concerned, with the expression of various factors only found during development:"epileptogenesis recapitulates ontogenesis." These effects are due to a loss of a chloride cotransporter that acts to remove chloride from neurons. [START_REF] Rivera | The K+/Cl-co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation[END_REF][START_REF] Rivera | Mechanism of activitydependent downregulation of the neuron-specific K-Cl cotransporter KCC2[END_REF][START_REF] Fiumelli | Modulation of GABAergic transmission by activity via postsynaptic Ca2+-dependent regulation of KCC2 function[END_REF] Here again, the genuinely epileptic tissue has unique features not found in naive networks.

Anoxic insults lead to similar post-traumatic alterations

Reactive plasticity is not restricted to epilepsies. Indeed, it has long been recognized that ischemic insult augments the occurrence of seizures and late-onset epilepsy in humans and in animal models. [START_REF] Congar | Permanent reduction of seizure threshold in post-ischemic CA3 pyramidal neurons[END_REF][START_REF] Lossius | Poststroke epilepsy: occurrence and predictors -a long-term prospective controlled study (Akershus stroke study)[END_REF][START_REF] Cocito | Epileptic seizures in cerebral arterial occlusive disease[END_REF] A remodeling of neuronal networks also often follows the cell loss produced in CA1 pyramidal neurons after a four-vessel occlusion model. [START_REF] Pulsinelli | Selective neuronal vulnerability: morphological and molecular characteristics[END_REF][START_REF] Schmidt-Kastner | Selective vulnerability of the hippocampus in brain ischemia[END_REF] The damage includes various GABAergic interneurons, and is associated with long-term hyperexcitability. [START_REF] Epsztein | Ongoing epileptiform activity in the post-ischemic hippocampus is associated with a permanent shift of the excitatory-inhibitory synaptic balance in CA3 pyramidal neurons[END_REF][START_REF] Congar | Permanent reduction of seizure threshold in post-ischemic CA3 pyramidal neurons[END_REF] Electrophysiological recordings of CA3 pyramidal neurons suggest that important morphofunctional reorganization has occurred, and that this is irreversible. [START_REF] Crepel | Synaptic plasticity in ischemia: role of NMDA receptors[END_REF] Sprouting of mossy fibers has also been documented after ischemic insults, [START_REF] Congar | Permanent reduction of seizure threshold in post-ischemic CA3 pyramidal neurons[END_REF] as well as an increased glutamatergic activity manifested by a dramatic enhancement of both the frequency of spontaneous glutamatergic EPSCs and that of miniature synaptic currents, suggesting an enhanced quantal release of glutamate. Therefore, cell loss produces massive modifications of the entire circuit, including neurons that are afferent to the damage and are associated with reorganization of net-works. These alterations may then lead to hyperactivity and seizures in the postischemic network, in keeping with the extensive clinical data suggesting postischemic hyperactivity.

General implications of these observations

The first implication of these observations is that seizures beget seizures. This is not acknowledged by many clinicians, primarily concerning temporal lobe seizures, since surgery often alleviates the ongoing seizures. Had alterations occurred, and if seizures begot seizures, then this would not be the case. However, it may be worth stressing that antiepileptic regimens are usually pursued after successful surgery; this is not readily compatible with a completely focal origin and restriction of the events. In addition, the removal in successful operations of large samples of the presumed focus may reflect the need to remove generators other than the identified focus, possibly because of generalization of seizures and alterations of sites distal to the focus. In other terms, we do not know at present whether the morphological substrate of the focus corresponds exactly to the electrical pacemaker cell assembly. I suggest that this is not the case, and that an ensemble of neurons outside the focus -in the vicinity of, or in distal regions connected to the focus-contribute to the damage and the seizures. However, additional experiments are clearly needed to clarify how many seizures lead to synapse reorganizations, and how this contributes to the formation of distal independent pacemaker cell assemblies (see also below). We need to compare the extent of the damage after a few seizures to that observed after a long period of ongoing seizures. My predictions are that these will differ significantly.

The second implication is that epileptic networks may well operate differently from naive ones, independently of the epileptogenicity. Thus, if aberrant synapses operate with different receptors, the generation of behaviorally relevant oscillations by the network will be affected. The kinetics of epileptic kainite-mediated synaptic currents is much longer than the naive AMPA currents, and thus the generation of high-frequency oscillations and the integrative properties are expected to be affected. Since epileptic networks use similar ensembles to those that generate important integrative functions, they are expected to impact these functions. In keeping with this, place cells operate differently in naive hippocampi and in epileptic ones. [START_REF] Shatskikh | Electrical induction of spikes in the hippocampus impairs recognition capacity and spatial memory in rats[END_REF]

B a s i c r e s e a r c h

The third implication is that reactive plasticity should be taken more into account in our understanding of epilepsies, and possibly also neurodegenerative disorders. If synapses reorganize, new ones are formed, and a fortiori if these operate by different receptors and intracellular signals, then it may be worth using genuine epileptic networks to understand the underlying mechanisms and develop new antiepileptic drugs and regimens. Using a naive network that seizes acutely under the influence of a convulsive agent is to a large extent irrelevant. This approach, which is by far the most used to study basic mechanisms of epilepsies and develop new drugs, simply does not take into account the salient feature of genuine epilepsies, namely the aberrant synapses and signals as well as the unique networks formed a few weeks after the inaugurating event. Stated differently, it is important to study the chronic aspects of genuine epilepsies and not convulsions. An anticonvulsant drug may not be an effi-cient antiepileptic one. The following section illustrates this for the developing brain, which operates differently from the adult brain, yet still provides interesting elements for the debate.

In the developing brain, seizures also beget seizures, but differently Do seizures also beget seizures in the developing brain? This is important in view of the extensive experimental data suggesting that immature neurons are more resistant to insults in terms of neuronal cell loss than adult ones. Thus, anoxic episodes require much longer durations and severity to produce cell loss early in life, in contrast to adults. [START_REF] Cherubini | Anoxia produces smaller changes in synaptic transmission, membrane potential, and input resistance in immature rat hippocampus[END_REF][START_REF] Crepel | Developmental and regional differences in the vulnerability of rat hippocampal slices to lack of glucose[END_REF] Also, administration of kainate to pups until the end of the second postnatal week triggers seizures but no brain damage, and little if any apparent signs of damage. [START_REF] Ben-Ari | Limbic seizure and brain damage produced by kainic acid: mechanisms and relevance to human temporal lobe epilepsy[END_REF] Therefore, a long-lasting status epilepticus can be generated in pups without neuronal damage and reactive plasticity. Yet, these seizures can lead to long-lasting consequences as shown by a lower threshold for seizure generation and major alterations of several intrinsic excitability in adults. [START_REF] Villeneuve | Neonatal seizures induced persistent changes in intrinsic properties of CA1 rat hippocampal cells[END_REF] A parsimonious explanation for this paradox is that seizures in immature networks produce their long-lasting consequences by different mechanisms than in adults, and notably without producing damage, but rather by altering activity and developmental programs.

We developed a unique in vitro preparation to determine the consequences of seizures on immature networks (Figure 5). [START_REF] Khalilov | In vitro formation of a secondary epileptogenic mirror focus by interhippocampal propagation of seizures[END_REF] The two intact hippocampi are dissected intact from immature rodent brains and placed in a three-chamber compartments in which each chamber can accommodate one hippocampus and the commissural interhemispheric connections. 77,78 Applications of a convulsant agent to one hippocampus generated seizures that propagated to the other hippocampus. After one seizure, interruption of the propagation does not transform the naive side to an epileptic one that generates spontaneous seizures. In contrast, after recurrent seizures, [START_REF] Bartos | Fast synaptic inhibition promotes synchronized gamma oscillations in hippocampal interneuron networks[END_REF][START_REF] Fuentealba | The reticular nucleus revisited: intrinsic and network properties of a thalamic pacemaker[END_REF][START_REF] Destexhe | Mechanisms underlying the synchronizing action of corticothalamic feedback through inhibition of thalamic relay cells[END_REF][START_REF] Cunningham | A role for fast rhythmic bursting neurons in cortical gamma oscillations in vitro[END_REF][START_REF] Isomura | Integration and segregation of activity in entorhinal-hippocampal subregions by neocortical slow oscillations[END_REF] the naive side generates seizures when disconnected from the treated hippocampus; recurrent seizures have formed an epileptogenic mirror focus (Figure 5). Using this preparation, it is possible to determine the conditions needed to form a mirror focus and those that define an epileptic network. It was found that only seizures that include high-frequency oscillations (HFOs, above 40 Hz) transform a naive network into an epileptic one; lower frequency events can occur with little consequences. The conditions required to generate HFOs include operative NMDA receptors, since applications of an antagonist of these receptors to the naive side blocked the HFO components of the propagated seizures and prevented the formation of a mirror focus. [START_REF] Khalilov | Epileptogenic actions of GABA and fast oscillations in the developing hippocampus[END_REF][START_REF] Van | The dark side of high-frequency oscillations in the developing brain[END_REF] Since NMDA receptors play an important role in triggering persistent changes of synaptic efficacy, these observations illustrate the concept of pathological changes of synaptic efficacy that rely on mechanisms identified to play a role in physiological integrative and memory processes. In other words, the formation of epileptogenic foci occurs in regions known to play an active role in plasticity, and relies on alterations of synaptic efficacy triggered by HFO components that are constitutive of inaugurating seizures.

In further relevance to the abovementioned issues, recordings in the established mirror focus show a stable increase in [Cl -] i , and depolarizing and excitatory actions of GABA. [START_REF] Khalilov | Epileptogenic actions of GABA and fast oscillations in the developing hippocampus[END_REF] Therefore, recurrent seizures trigger a persistent accumulation of chloride in neurons, in keeping with the unique properties of epileptogenic networks and the importance of taking into account the differences between acute seizures and epilepsies.

Conclusion

In conclusion, these observations call for a re-examination of the study of epilepsies and other lifelong neurological disorders by examining the properties of chronically affected neurons and networks, rather than acute models. The importance of reactive plasticity and the unique features and signals of pathological networks suggest that alterations that occur during the "maturation" process of the focus contribute to the disorder, and must be taken into account in its treatment. The nervous system is extremely "plastic" and reacts to a lesion or a disorder by forming new synapses and new signaling cascades that impact the operation of the structure in ongoing conditions, independently of the pathological events. ❏ Numerosas investigaciones experimentales han confirmado que "las convulsiones engendran convulsiones". En adultos, las convulsiones límbicas producen pérdida de células, proceso que es seguido por la formación de nuevas sinapsis excitatorias, las cuales contribuyen a generar nuevas convulsiones. La señal gatillo es un aumento de la eficacia sináptica, seguida de una cascada molecular que genera brotes axonales. Las nuevas sinapsis son aberrantes y se forman en regiones que en los sujetos controles no presentan este tipo de sinapsis. Ellas también incluyen receptores que tampoco están presentes en los controles, y esto facilita la generación de convulsiones. Por lo tanto, una forma aberrante de plasticidad neuronal reactiva provee un sustrato para tener convulsiones como una forma de secuelas a largo plazo. Estos acontecimientos tendrán un importante impacto ya que ocurren en estructuras cerebrales involucradas en funciones de integración y mnémicas. La plasticidad reactiva está documentada por otros daños y trastornos, y puede ser la base de la progresión a largo plazo de trastornos neurodegenerativos.

B a s i c r e s e a r c h 76 Selected

 76 abbreviations and acronyms AMPA Ͱ-amino-3-hydroxy-5-methylisoxazole-4-propionic acid EPSC excitatory postsynaptic current GABA Ͳ-aminobutyric acid HFO high-frequency oscillations LTP long-term potentiation NMDA N-methyl-D-aspartic acid

Figure 1 .

 1 Figure 1. Sprouting of fibers in the hippocampus of epileptic rats. Morphological reconstruction of CA1 hippocampal neurons intracellularly injected with dyes weeks after an inaugurating status epilepticus. Note the extensive sprouting of axons, including to layers (the molecular layers) that are not innervated by pyramidal axons in controls. O, stratum oriens; P, stratum pyramidale; R: stratum radiatum; S, xxxxxx Reproduced from ref 44: Esclapez M, Hirsch JC, Ben Ari Y, Bernard C. Newly formed excitatory pathways provide a substrate for hyperexcitability in experimental temporal lobe epilepsy. J Comp Neurol. 1999;408:449-460. Copyright © Wiley-Liss 1999

Figure 2 . 2 Formation of ectopic mossy fiber synapses with granule cell dendrites in epileptic

 22 Figure 2. Electron microscopic identification of aberrant mossy fiber terminals on granule cells weeks after an inaugurating status. Note the formation of synapses in regions that are not innervated by mossy fibers in controls. These aberrant synapses have all the typical unique features of mossy fiber terminals, notably the large size and the enrichment in vesicles. Reproduced from ref 45: Represa A, Pollard H, Moreau J, Ghilini G, Khrestchatisky M, Ben-Ari Y. Mossy fiber sprouting in epileptic rats is associated with a transient increased expression of alpha-tubulin. Neurosci Lett. 1993;156:149-152. Copyright © Elsevier Scientific Publishers Ireland 1993

79 Figure 3 .

 793 Figure 3. Increased kainate receptors binding in regions innervated by aberrant mossy fibers. There is an increased density of receptors thirty days, but not 12 days, after an inaugurating status. The latency correlates with the time needed to form novel synapses. Reproduced from ref 19: Represa A, Tremblay E, Ben-Ari Y. Kainate binding sites in the hippocampal mossy fibers: localization and plasticity. Neuroscience. 1987;20:739-748. Copyright © Elsevier Science 1987

  Reactive plasticity in neurological disorders -Ben-AriDialogues in Clinical Neuroscience -Vol 10 . No. 1 . 2008 81

Figure 4 .CFigure 5 .

 45 Figure 4. Enhanced glutamatergic activity in epileptic hippocampus weeksafter the inaugurating event. Slices were prepared from rats 4 weeks after the status produced by a single injection of pilocarpine. Note the considerable increase of spontaneous glutamatergic activity in the epileptic hippocampus (right side) versus a control slice (left). The bottom left column illustrates the increase quantitatively (in frequency of glutamate EPSCs). The right columns show a similar increase in the miniature PSCs recorded after applications of tetrodoxin (TTX) to block activitydependent currents. This suggests a long-lasting increase in quantal release of transmitter in the epileptic network.[START_REF] Epsztein | Ongoing epileptiform activity in the post-ischemic hippocampus is associated with a permanent shift of the excitatory-inhibitory synaptic balance in CA3 pyramidal neurons[END_REF] EPSCs, excitatory postsynaptic currents; PSCs, postsynaptic currents

B a s i c r e s e a r c h 82

 82 Reactive plasticity in neurological disorders -Ben-Ari Dialogues in ClinicalNeuroscience -Vol 10 . No. 1 . 2008 83 Epilepsias y plasticidad neuronal: ¿para mejor o para peor?

© 2008, LLS SASDialogues Clin Neurosci. 2008;10:000-000.

Reactive plasticity in neurological disorders -Ben-AriDialogues in Clinical Neuroscience -Vol 10 .No. 1 . 2008


Sylvain Rheims

Marat Minlebaev

Anton Ivanov

Alfonso Represa

Rustem Khazipov

Gregory L Holmes

Yehezkel Ben-Ari

Yuri Zilberter

Excitatory GABA in Rodent Developing Neocortex In Vitro

Rheims S, Minlebaev M, Ivanov A, Represa A, Khazipov R, Holmes GL, Ben-Ari Y, Zilberter Y. Excitatory GABA in rodent developing neocortex in vitro. J Neurophysiol 100: 609 -619, 2008. First published May 21, 2008;doi:10.1152/jn.90402.2008. GABA depolarizes immature cortical neurons. However, whether GABA excites immature neocortical neurons and drives network oscillations as in other brain structures remains controversial. Excitatory actions of GABA depend on three fundamental parameters: the resting membrane potential (E m ), reversal potential of GABA (E GABA ), and threshold of action potential generation (V thr ). We have shown recently that conventional invasive recording techniques provide an erroneous estimation of these parameters in immature neurons. In this study, we used noninvasive single N-methyl-D-aspartate and GABA channel recordings in rodent brain slices to measure both E m and E GABA in the same neuron. We show that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). However, GABA generates action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells, since L5/6 pyramidal cells have more depolarized resting potentials and more hyperpolarized V thr . The excitatory GABA transiently drives oscillations generated by L5/6 pyramidal cells and interneurons during development (P5-P12). The NKCC1 co-transporter antagonist bumetanide strongly reduces [Cl Ϫ ] i, GABA-induced depolarization, and network oscillations, confirming the importance of GABA signaling. Thus a strong GABA excitatory drive coupled with high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapsedriven oscillatory activity in the rodent neocortex in vitro. In the companion paper, we show that the excitatory GABA drives layer-specific seizures in the immature neocortex.

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

Immature neurons readily generate network driven patterns in a wide range of brain structures and animal species (Adelsberger et al. 2005;Ben-Ari et al. 1989;Corlew et al. 2004;Galli and Maffei 1988;Garaschuk et al. 2000;Khazipov et al. 2001;Meister et al. 1991;Owens and Kriegstein 1998;[START_REF] Yuste | Neuronal domains in developing neocortex[END_REF]. Several factors converge to faciltiate the generation of early oscillations including the depolarizing and excitatory actions of GABA (Ben-Ari 2002;Ben-Ari et al. 1989, 2007), high-input resistance of immature neuron and, possibly, high intrinsic excitability of immature neurons (Ben-Ari 2002;Ben-Ari et al. 2007;Moody and Bosma 2005). Understanding how immature networks generate oscillations is conditionned by a detailled description of the developmental shifts of intrinsic and synapse driven properties and identification of the neuronal generators. Since the developing brain has a higher incidence to seizures and synchronized activity patterns [START_REF] Tyzio | Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery[END_REF], this issue also directly related to epilepsies.

Like other brain regions, immature neocortical neurons generate oscillations that modulate the construction of functional maps (Cang et al. 2005;Kandler and Gillespie 2005;Katz and Shatz 1996;Khazipov et al. 2004b). These patterns are intrinsic since they occur in vitro (Aguilo et al. 1999;Corlew et al. 2004;Dupont et al. 2006;Garaschuk et al. 2000;[START_REF] Yuste | Neuronal domains in developing neocortex[END_REF]) and are generated when neocortex is deprived of its sensory inputs (Hanganu et al. 2006;Khazipov et al. 2004b;[START_REF] Tyzio | Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery[END_REF]. Although GABA depolarizes neocortical neurons (Daw et al. 2007b;Luhmann and Prince 1991;Owens et al. 1996Owens et al. , 1999;;[START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions inimmature rat neocortical neurones is mediated by NKCC1[END_REF], cortical patterns reported to date (Aguilo et al. 1999;Corlew et al. 2004;Garaschuk et al. 2000;[START_REF] Yuste | Neuronal domains in developing neocortex[END_REF] are not generated by GABA (Garaschuk et al. 2000;McCabe et al. 2006). The reasons for this discrepancy and a possible contribution of intrinsic excitability properties of neurons (Burgard and Hablitz 1993;Daw et al. 2007b;Franceschetti et al. 1998;McCormick and Prince 1987;[START_REF] Bahrey | Early development of voltage-gated ion currents and firing properties in neurons of the mouse cerebral cortex[END_REF][START_REF] Reboreda | Development of cholinergic modulation and graded persistent activity in layer v of medial entorhinal cortex[END_REF][START_REF] Zhang | Maturation of layer V pyramidal neurons in the rat prefrontal cortex: intrinsic properties and synaptic function[END_REF][START_REF] Zhou | Postnatal development of membrane properties of layer I neurons in rat neocortex[END_REF]) are yet unknown.

In this study, we describe the intrinsic and synapse driven properties of pyramidal neurons and interneurons in deep and superficial cortical layers during maturation and show that these properties underlie the generation of a layer specific pattern equivalent to the giant depolarizing potentials (GDPs) reported in many cortical and subcortical structures (Ben-Ari et al. 2007). We exclusively used the noninvasive techniques (single channel recordings) for measuring neuronal properties since, as we have shown previously [START_REF] Tyzio | Membrane potential of CA3 hippocampal pyramidal cells during postnatal development[END_REF], the invasive recording techniques (including perforated patch) result in significant errors in the estimation of E m and E GABA of immature neurons. We therefore used cell-attached recordings of N-methyl-D-aspartate (NMDA) and GABA channels to determine E m and driving force of GABA (DF GABA ) , respectively, and, importantly, measured both parameters in the same neuron thus identifying the genuine E GABA . We report the laminar and neuron type-specific distribution of excitatory action of GABA and intrinsic excitability that results in the generation of layer-specific network-driven oscillations. The impact of these properties on seizure generation is described in a companion paper [START_REF] Tyzio | Postnatal change in somatic GABA signaling in the rat hippocampus[END_REF].

Address for reprint requests and other correspondence: S. Rheims, Institut de Neurobiologie de la Mediterranee, INSERM U901, Universite ´de la Me ´diterrane ´e, Route de Luminy, Marseille, France (E-mail: sylvain.rheims@ free.fr).

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.

J Neurophysiol 100: 609 -619, 2008. First published May 21, 2008;doi:10.1152/jn.90402.2008

Slices preparation

Brain slices were prepared from postnatal day (P)1 to P15 Swiss mice of both sexes. P0 was the day of birth. All animal protocols conformed to the French Public Health Service policy and the INSERM guidelines on the use of laboratory animals. Animals were decapitated, and brains were removed. Sagittal slices (300 -400 m) were cut using a Microm tissue slicer (International) with ice-cold oxygenated modified artificial cerebrospinal fluid (mACSF), with 0.5 mM CaCl 2 and 7 mM MgSO 4 , in which Na ϩ was replaced by an equimolar concentration of choline. Slices were transferred into oxygenated normal ACSF containing (in mM) 126 NaCl, 3.5 KCl, 1.2 NaH 2 PO 4 , 26 NaHCO 3 , 1.3 MgCl 2 , 2.0 CaCl 2 , and 10 D-glucose, pH 7.4, at room temperature (20 -22°C) for Ն1 h before use.

Electrophysiology

For recordings, slices were placed into a conventional fully submerged chamber superfused with ACSF (32-34°C) at a rate of 2-3 ml/min. Cell types were identified by IR-DIC video microscopy. Patch-clamp recordings were performed using dual EPC-9 or EPC-10 amplifiers (HEKA Elektronik). Pipettes (resistance of 3.5-8 MOhm) were pulled from borosilicate glass capillaries.

Patch-clamp recordings in cell-attached configuration were performed using the following pipette solutions (in mM): for recordings of single GABA channels, 140 NaCl, 2.5 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 HEPES, and 0.01 GABA, pH adjusted to 7.3 by NaOH; and for recordings of single NMDA channels: 140 NaCl, 2.5 KCl, 2 CaCl 2 , 10 HEPES, 0.01 NMDA, 0.01 and glycine, pH adjusted to 7.3 by NaOH. Analysis of currents through single channels and current-voltage relationships were performed using Clampfit 9.2 (Axon Instruments, Union City, CA) as described previously [START_REF] Tyzio | Membrane potential of CA3 hippocampal pyramidal cells during postnatal development[END_REF][START_REF] Tyzio | Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery[END_REF].

To estimate reliability of this technique for E m measurements, we performed an experiment using dissociated cultures of rat hippocampus at 16 -21 days in vitro. We first estimated E m in the intact cell using cell-attached recordings of NMDA channels that gave a value of Ϫ81 Ϯ 3 mV (n ϭ 3 for dual recordings; n ϭ 8 for single cellattached recordings). Keeping cell-attached recordings, the cell was patched with a second pipette and recorded in the whole cell mode. Under these recording conditions, E m deduced from cell-attached recordings of NMDA channels shifted to a more depolarized value of Ϫ54 Ϯ 4 mV (n ϭ 3) that was close to the E m measured directly in whole cell recordings (Ϫ56 Ϯ 3 mV; n ϭ 72), and the difference between these two values was within Ϯ3 mV (0.9 Ϯ 1.3 mV; n ϭ 3) [START_REF] Tyzio | Postnatal change in somatic GABA signaling in the rat hippocampus[END_REF].

In addition, because the composition of solution for cell-attached recordings differs from ACSF, it may introduce a systematic error in measurements of DF GABA . This error was estimated experimentally in two experimental sets. First, we determined the reversal potential of currents via GABA channels in outside-out patches. To prevent rundown of GABA currents, 2 mM Mg-ATP and 5 mM EGTA were added to the pipette solution. Brief pressure application of isoguvacine (100 M, 100 ms, 2-8 psi) to the outside-out or nucleated patches evoked GABA A receptor (GABA A R)-mediated currents that were completely suppressed by the GABA A R antagonist bicuculline (20 M; n ϭ 2, data not shown). Current-voltage relationships showed that these responses have a reversal potential of 2.1 Ϯ 0.4 mV (n ϭ 7 for outside-out and n ϭ 1 for nucleated patches). In a second series of experiments, we measured DF GABA in cell-attached configuration (1-month-old rats) using the pipette solution containing ACSF and GABA (2 M). To maintain pH at a physiological level, the pipette ACSF was continuously oxygenated with 95% O 2 -5% CO 2 via the patch pipette holder. The value of DF GABA with the ACSF-based pipette solution was of 4.0 Ϯ 0.6 mV (n ϭ 9) compared with 5.4 Ϯ 1.3 mV (n ϭ 4) obtained using the pipette solution for GABA channel recordings. The difference of 1.6 mV was close to that obtained with outside-out patches (2.1 mV) [START_REF] Tyzio | Postnatal change in somatic GABA signaling in the rat hippocampus[END_REF].

Analyzing GABA-activated single channel recordings, we used a linear fit since a function theoretically appropriate for fitting currentvoltage relationships is not known. We compared reversal potentials obtained with the linear or polynomial fit in 14 randomly selected cells (P2-P15). With the linear fit, DF GABA was more depolarizing by 0.6 Ϯ 0.78 mV.

Patch-clamp recordings in the whole cell configuration were performed using the following pipette solution (in mM): 115 potassium gluconate, 20 KCl, 4 ATP-Mg, 10 Na-phosphocreatine, 0.3 GTP-Na, 10 HEPES, and 0.5% biocytin, pH 7.3, adjusted by NaOH. A similar solution was used for recordings of action potentials (APs).

Field potentials were recorded using electrodes made from borosilicate glass capillaries filled with ACSF. Signals were amplified using DAM8A (World Precision Instruments).

Continuous recordings were digitized (10 kHz) on-line using a Digidata 1322 (Axon Instruments) and analyzed off-line with Clamfit 9.0 (Axon Instruments). Noncontinuous recordings were digitized (10 or 50 KHz) on-line and analyzed off-line (Igor Wavemetrics, Lake Oswego, OR).

Measurements of V thr

Cells were excited by random synaptic stimulation (L2/3 and L4) or spontaneous oscillations (L5/6). During random synaptic stimulation, nerve fibers were stimulated each 10 s via a bipolar metal extracellular electrode (50 m) positioned in L1, L4, or white matter. Each stimulus consisted of a random sequence of 0.2-ms stimuli (minimal interpulse interval, 10 ms) for 200 ms. Detection of the AP activation threshold was performed off-line using the custom made software (IGOR Pro, WaveMetrics). Only the first AP in each sequence was analyzed. V thr was detected by measuring the maximum of a second derivative of membrane potential. The average potential slope was measured by averaging the first derivative of membrane potential in the time range starting from the set deviation from E m and up to V thr .

Since the input resistance of neurons was large [522 Ϯ 159 (SD) M⍀ for L2/3 pyramids; 734 Ϯ 324 M⍀ for L2/3 interneurons; 560 Ϯ 96 M⍀ for L4 stellate cells; 430 Ϯ 149 M⍀ for L5/6 pyramids; 491 Ϯ 128 M⍀ for L5/6 interneurons at P7-P8], it was necessary to correct the measured potentials [START_REF] Tyzio | Membrane potential of CA3 hippocampal pyramidal cells during postnatal development[END_REF] using the following formula

V ϭ V* ϫ R seal /͑R seal Ϫ R in ͒
where V* is the measured potential; R seal is the seal resistance; and R in is the input resistance.

Pharmacology

Drugs used were purchased from Tocris (gabazine, NBQX, D-APV), Sigma (bumetanide, biocytin, picrotoxin), and Roche Pharmaceutical Division (diazepam).

Histological processing

To show biocytin-injected cells, slices were immerged in a fixative solution of paraformaldehyde (4%) and glutaraldehyde (0.2%) overnight at 4°C after electrophysiological recordings. To increase penetration of the reagents used for biocytin detection, slices were quickly frozen on dry ice and thawed in a phosphate buffer. Slices were rinsed in 0.05 M Tris-buffered saline (TBS), pH 7.4, containing 0.3% Triton X-100 for 30 min and incubated overnight at 4°C in an avidin-biotinperoxidase solution prepared in TBS according to the manufacturer recommendation (Vectastain Elite ABC, Vector Laboratories, Burlingame, CA). After a 30-min wash in TBS and a 10-min rinse in Tris buffer (TB), pH 7.6, slices were processed for 15 min in 0.06% 3-3-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide diluted in TB. The slices were rinsed in TB for 30 min, mounted on gelatin-coated slides, dehydrated, and coverslipped with permount. Stained cells were reconstructed for morphometric analysis using the Neurolucida system (MicroBrightField, Colchester, VT).

Statistical analysis

Group measures are expressed as means Ϯ SE; error bars also indicate SE. The statistical significance was assessed with the Student's t-test or ANOVA. The level of significance was set at P Ͻ 0.05.

R E S U L T S

Strongly depolarizing GABA signaling over the first postnatal week

To determine the effects of GABA, it is essential to measure correctly the basic electrical properties of immature neurons, in particular, E m and E GABA . We relied exclusively on noninvasive recordings and measured E m and E GABA in the same target neurons by analyzing I-V relations for NMDA-and GABAactivated single channels with a dual patch recording protocol (Fig. 1A). Recordings in the same neuron enabled measuring both values in absolute potentials. At P7-P8 (Fig. 1B), E m of neurons in L2/3 and L4 was not statistically different (P ϭ 0.38 by ANOVA) for L2/3 pyramidal cells (Ϫ84.5 Ϯ 1.2 mV; n ϭ 35), for L2/3 interneurons (Ϫ82 Ϯ 1.5 mV; n ϭ 18), and for L4 spiny stellate cells (Ϫ82 Ϯ 1 mV; n ϭ 13). In contrast, E m of both pyramidal and interneurons of L5/6 were significantly more depolarized than L2/3 neurons [Ϫ73.9 Ϯ 0.7 mV (n ϭ 23) for pyramidal cells; and Ϫ69.2 Ϯ 3 mV for interneurons (n ϭ 9), P Ͻ 0.001 by ANOVA]. Therefore deep layer neurons have ϳ10 mV more depolarized E m than superficial layer neurons and are potentially more liable for excitation.

Figure 1C shows E GABA values in different cell types and layers at P7-P8. The mean E GABA values were Ϫ53.8 Ϯ 2 mV for L2/3 pyramidal cells (n ϭ 10); Ϫ47.5 Ϯ 2.1 mV for L2/3 interneurons (n ϭ 9); Ϫ52.7 Ϯ 3.3 mV for L4 stellate cells (n ϭ 13); Ϫ49.6 Ϯ 2.1 mV for L5/6 pyramidal cells (n ϭ 18); and Ϫ46.5 Ϯ 2 mV for L5/6 interneurons (n ϭ 9). E GABA values deviated in cells of the same type, presumably reflecting variability of [Cl Ϫ ] i . Nevertheless, a considerable difference in the E GABA and E m values (Fig. 1D) allows to conclude that, for all layers and cell types, GABAergic transmission provide a strong depolarizing drive.

Figure 2A shows the developmental profile of E m and E GABA in pyramidal cells of L2/3 and L5/6. The profiles suggest that a switch of GABA actions occurs after P10. Before this age, E GABA gradually increases in all layers. Interestingly, E m gradually hyperpolarizes with age reaching a steady-state level after P5 (P Ͻ 0.001 by ANOVA for E m at ϽP5 and ϾP5 for both L2/3 and L5/6 pyramidal cells). E m of pyramidal cells in L2/3 and L5/6 do not differ significantly at P9 -P14 [Ϫ80.4 Ϯ 1.1 mV (n ϭ 31) and Ϫ78.7 Ϯ 1.4 (n ϭ 25), respectively; P ϭ 0.34, by ANOVA].

To verify that the positive values of E GABA are related to a high [Cl Ϫ ] i , we blocked activity of the NKCC1 transporter by 10 M bumetanide [START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions inimmature rat neocortical neurones is mediated by NKCC1[END_REF]). Single channel I-V relations were measured in control and after application of bumetanide in the same L5/6 pyramidal cells (Fig. 2B). At P7-P8, bumetanide induced a small but significant hyperpo-larization (E m ϭ Ϫ73.9 Ϯ 1 mV in control and Ϫ78.6 Ϯ 1.3 mV after bumetanide; n ϭ 15; P Ͻ 0.01, paired t-test). Bumetanide also induced a strong reduction in [Cl Ϫ ] i and corresponding E GABA (E GABA ϭ Ϫ52.8 Ϯ 3.5 mV in control and Ϫ76.4 Ϯ 3.5 mV after bumetanide; n ϭ 6; P Ͻ 0.01, paired t-test). Interestingly, the effect of bumetanide on E m was more pronounced at P4 (Fig. 2B): Ϫ68.2 Ϯ 2 mV in control and Ϫ78.7 Ϯ 3.2 mV after bumetanide (n ϭ 7). In presence of bumetanide, the E m values did not differ significantly at P4 and P7-P8 (P ϭ 0.97), suggesting that the age-dependent E m depolarization directly relates to [Cl Ϫ ] i . The mechanism underlying this effect was not investigated further.

Layer 5/6 pyramidal neurons have a more negative action potential threshold than L2/3 pyramidal neurons Another major factor that underlies neuronal excitability is the AP threshold (V thr ). Since V thr is defined by co-activation of voltage-sensitive channels during synaptic input, we measured V thr in spontaneously generated APs or in APs induced by random extracellular synaptic stimulation (see METHODS).

Figure 3A shows the dependence of V thr on the average potential slope in APs induced in three L2/3 interneurons (P7-P8) by random synaptic stimulation (see inset). All interneurons (coded by different colors) were filled out with biocytin and reconstructed afterward. Representative reconstructions of interneurons are shown in Fig. 3A. Scattering of the V thr values reflects the heterogeneity of interneuron population. The mean V thr was Ϫ47.6 Ϯ 4 mV (n ϭ 9). Note, however, that some interneurons subtypes (e.g., in blue on Fig. 3A) have a V thr at about Ϫ60 mV, indicating that the corresponding interneurons are highly excitable. Measurements in L5/6 interneurons (Fig. 3B) also showed pronounced variability of the V thr values and corresponding heterogeneity of interneuron subtypes. Primary morphological characterization of interneurons is provided in Supplementary Materials. 1Similar measurements in L2/3 pyramidal and L4 stellate cells at P7-P8 showed much smaller variability of the V thr values within each neuron type (Fig. 3C). The mean V thr values were Ϫ36.8 Ϯ 0.46 mV at neocortical pyramids (12 cells) and Ϫ37.4 Ϯ 0.4 mV at stellate cells (11 cells). Thus V thr in both neuron types are very positive, indicating that voltage deviations from E m of Ͼ40 mV are required for activating these neurons (Fig. 3D). L5/6 pyramidal cells at P7-P8 had more negative V thr (P Ͻ 0.001 for L2/3 and L5/6 pyramids), Ϫ42 Ϯ 1.9 mV (16 cells), with large variability between neurons (Fig. 3C). Importantly, plotting the mean V thr values relative to the corresponding mean E m values (Fig. 3D) clearly shows that L5/6 neurons are potentially more excitable than neurons in other layers. For pyramidal cells, this conclusion remains valid at P14 animals (Fig. 3, E and F), although V thr is getting significantly more negative for both L2/3 (Ϫ48.3 Ϯ 1.2 mV, P Ͻ 0.001) and L5/6 (Ϫ54.7 Ϯ 3.7 mV, P Ͻ 0.001) pyramids. Neuron spiking could be induced by synaptic stimulation at earlier (ϽP5) ages. Unfortunately, a very high membrane resistance (Ͼ1 GOhm) of neurons prevented the accurate estimation of V thr at these ages.

Therefore deep layer pyramidal neurons are more excitable than superficial ones because of a lower V thr .

GABAergic transmission generates APs in L5/6 but not L2/3 pyramidal neurons

The results above suggest that, at P7-P8, GABAergic transmission is depolarizing and presumably strong enough to initiate cell firing. To directly test this hypothesis, we blocked glutamatergic transmission (5 M NBQX ϩ 50 M APV) and stimulated slice by the extracellular electrode (Davies et al. 1990). In most interneurons tested in L2/3 (10 of 11), extra-cellular stimulation induced APs (Fig. 4A, cell-attached recordings). This was prevented by application of 10 M gabazine (data not shown). In contrast, such stimulation failed to induce APs in L2/3 pyramidal cells (n ϭ 5; Fig. 4A). In L5/6, extracellular stimulation reliably induced APs in both pyramidal cells and interneurons (Fig. 4A). Figure 4B represents a summary of these experiments, and Fig. 4C shows deviations between the mean values of E GABA and V thr in each cell type. Note, that APs are generated in a fraction of L5/6 neurons, although their mean E GABA is more negative than mean V thr . This can be explained by the large deviations of V thr in neuron populations (from Ϫ35 to Ϫ60 mV for interneurons and from Ϫ31 to Ϫ58 mV for pyramids). It is also because of the activation of Na channels that boost postsynaptic potentials (PSPs), thereby facilitating the generation of APs (Ha ¨usser et al. 2001;[START_REF] Williams | Voltage-and site-dependent control of the somatic impact of dendritic IPSPs[END_REF]. Indeed, Fig. 4D shows whole cell recordings in an L2/3 pyramidal cell and an interneuron. The pipette solution contained 20 mM Cl Ϫ that corresponded to an E GABA of about Ϫ49 mV (shown by dash lines on the figure). Nevertheless, GABAergic PSP values recorded at the soma exceeded E GABA and readily triggered APs.

Thus GABAergic synapses are able to initiate APs in the intact L2/3 interneurons but not in pyramidal cells. L5/6 pyramidal cells seem to be more excitable than the L2/3 ones since they have been readily activated by GABAergic transmission.

Recurrent GABA driven oscillations in deep layer neurons

At P6 -P8, field and cell-attached recordings showed recurrent oscillations (each 20 s in average; Ͻ1-s duration) in the majority of slices (68/76; see Fig. 5A). These oscillations are generated by deep layer (L5/6) neurons since 1) simultaneous field recordings show that they are most powerful in L5/6, much smaller in L4 and nearly absent in L2/3 (Fig. 5B); (4 similar experiments; 13 experiments with simultaneous L2/3 and L5/6 recordings); 2) combined field and dual cell-attached recordings showed that pyramidal cells and interneurons of L2/3 are quiescent during oscillations (Fig. 5, C and D; 12/14 interneurons were silent and 21/21 pyramids); field oscillations in L4 are not associated with firing of stellate or pyramidal cells (Fig. 5, C and D; 7 cells); oscillations are always synchronous with firing of most pyramidal cells and interneurons in L5/6 (Fig. 5, A and D; 23 pyramidal cells and 23 interneurons); 3) oscillations always sustain after resection of all subcortical regions (n ϭ 4; not shown); and 4) a mini-slice, prepared from a L5/6 slice region originally showing oscillations (Supplementary Fig. S2), is still highly active (6 similar experiments). In contrast, surgically isolated superficial layers did not show any activity (3 experiments; data not shown). These results are summarized in Fig. 5D. Therefore the dominant activity pattern at that developmental stage is generated by deep cortical layer neurons.

The following observations suggest that these oscillations, referenced as cortical GDPs (cGDPs), resemble the classical GDPs recorded in hippocampus and other developing brain structures (Ben-Ari et al. 1989; for review, see Ben-Ari 2002; Feller 1999). First the oscillations are controlled by GABAergic signaling: 1) the cGDP frequency is significantly reduced by low doses of gabazine (100 nM, n ϭ 3; Fig. 6, A andB), a selective GABA A R antagonist; and by low doses of picrotoxin (3 M, n ϭ 4; Fig. 6B), a specific GABA channel blocker; 2) the cGDP frequency is highly increased by diazepam (5 M, n ϭ 3; Fig. 6, A andB), a benzodiazepine efficiently enhancing GABAergic currents; and 3) bumetanide (10 M, n ϭ 9; Fig. 6, A andB), which reduces the intracellular concentration of chloride ([Cl Ϫ ] i ) by blocking activity of NKCC1 (Dzhala et al. 2005;Hannaert et al. 2002;[START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions inimmature rat neocortical neurones is mediated by NKCC1[END_REF]), significantly suppresses cGDPs. Second, cGDPs require operative glutamatergic synapses since they are suppressed by APV (50 M, n ϭ 3; Fig. 6B) and abolished by NBQX (5 M, n ϭ 2; Fig. 6B). Third, like GDPs of the hippocampus (Ben-Ari et al. 1989;Khazipov et al. 2004a), cGDPs are present only transiently during maturation. As shown in Fig. 6C, the peak occurrence of cGDPs is within P6 -P10 and they disappear by the end of second postnatal week.

To ensure that the network oscillations are not animal strain specific, we also tested the presence of cGDPs in P5-P8 rat slices and observed oscillations in all experiments (n ϭ 7). Therefore cGDPs are generated by a mutual excitatory activity of pyramidal cells and interneurons in deep cortical layers.

D I S C U S S I O N

Using noninvasive single channel recordings, we showed that GABA robustly depolarizes immature neocortical neurons. However, although strong GABAergic depolarizing drive was observed in all cortical layers, spontaneous activity of neurons in deep layers was generally associated with oscillations. This is because of important intrinsic differences between E m and V thr in neuronal populations. E m and V thr are cell type specific, and the membrane voltage deviations required for neuron activation by synaptic input vary in different neuron typesfrom ϳ20 to Ͼ50 mV-suggesting major differences in intrinsic excitability. Strong excitatory GABA signaling in combination with high intrinsic excitability of deep layer neurons generates a coherent network-driven activity in the immature neocortex. This convergence seems to be the main conditioning factor for selective generation of cGDPs by layer 5/6 neurons. Oscillations typically remain confined to deep neocortical layers, requiring recurrent excitatory loops of pyramidal neurons and interneurons.

GABAergic transmission is strongly depolarizing over the first postnatal week

To directly determine the action of GABA, we recorded NMDA-and GABA-activated channels in the same neuron (Fig. 1) that provide E GABA in absolute values. With this technique, we can compare alterations in DF GABA across neuronal populations and different developmental stages. We found that E GABA is Ͼ20 mV more depolarized than E m at P7-P8 in neurons of all cortical layers. Thus the GABAergic transmission provides depolarizing drive to quiescent neurons. For pyramidal neurons, the developmental profile of E GABA showed gradual depolarization at earlier ages (Fig. 2A), and GABA was still depolarizing at the end of second postnatal week. The shift in the mode of GABA action occurs most likely within the second week of life.

After the first postnatal week, E m of principal cells in all layers did not differ significantly and stabilized at a negative level of about Ϫ80 mV (see Fig. 2A). In contrast to hippocampal neurons recorded with the same techniques [START_REF] Tyzio | Membrane potential of CA3 hippocampal pyramidal cells during postnatal development[END_REF][START_REF] Tyzio | Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery[END_REF], E m of cortical neurons showed a developmentally regulated trend to more hyperpolarized potentials in both L5/6 and L2/3 pyramidal cells (Fig. 2A). This shift is chloride dependent, since bumetanide, which reduces [Cl Ϫ ] i by blocking activity of NKCC1 (Dzhala et al. 2005;Hannaert et al. 2002), mimicked the age-dependent hyperpolarization: E m was hyperpolarized by ϳ10 mV at P4 and by ϳ5 mV at P7/8 (Fig. 2B). Although further experiments are required to explain the underlying mechanism, it is possible that GABA-induced tonic current modulates E m in immature neurons as it regulates pyramidal cell excitability in adult somatosensory cortex [START_REF] Salin | Spontaneous GABAA receptor-mediated inhibitory currents in adult rat somatosensory cortex[END_REF][START_REF] Yamada | Molecular basis for the GABAA receptor-mediated tonic inhibition in rat somatosensory cortex[END_REF].

Earlier studies have measured DF GABA using the gramicidin perforated-patch technique and reported values of Ͻ15 mV (Owens et al. 1996;[START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions inimmature rat neocortical neurones is mediated by NKCC1[END_REF] or even hyperpolarizing (Daw et al. 2007b;[START_REF] Yamada | Cl-uptake promoting depolarizing GABA actions inimmature rat neocortical neurones is mediated by NKCC1[END_REF]) before the end of first postnatal week. The differences with these observations may be explained by significant errors introduced by invasive recording techniques. Indeed, the correct estimation of E m is of a primary importance for showing the mode of GABA action, be it inhibitory or excitatory. The high-input resistance of immature neurons introduces significant errors in measurements of E m in whole cell or gramicidin perforated-patch recordings [START_REF] Tyzio | Membrane potential of CA3 hippocampal pyramidal cells during postnatal development[END_REF]. We tested these effects directly in neocortical neurons by performing a dual recording from the same neuron with cell-attached single channel and whole cell pipettes. E m measured with single NMDA channels was about Ϫ80 mV, but once an additional whole cell recording electrode was added, E m depolarized by Ͼ20 mV and reached a value of 56 mV [START_REF] Tyzio | Postnatal change in somatic GABA signaling in the rat hippocampus[END_REF]. Clearly, E m of immature neocortical neurons differs when measured by noninvasive or invasive techniques. This also confirms the validity of single channel recordings that provides a reliable estimation of E m. An alternative invasive recording technique-the gramicidin perforated patch-avoids the direct dialysis of internal chloride but may indirectly change [Cl Ϫ ] i via modification of [Na ϩ ] i and [K ϩ ] i . It is worth noting that [Cl Ϫ ] i depends on the activity of NKCC1 and KCC2 co-transporters, which in turn are sensitive to alterations in [Na ϩ ] i or [K ϩ ] i (Brumback and Staley 2008; for review, see Adragna et al. 2004;[START_REF] Payne | Cation-chloride co-transporters in neuronal communication, development and trauma[END_REF].

Deep layer pyramidal neurons are more excitable than superficial ones

Despite its generally strong depolarizing action, the GABAergic transmission generates APs in some interneuron subtypes and in deep but not superficial layer pyramidal neurons (Fig. 4). We observed significant variations of E m and V thr across different cell types. At P7-P8, concomitant with the maximum intensity of cGDPs, the voltage deviation required for activating L2/3 pyramidal or L4 stellate cells is 15 mV larger than that for L5/6 pyramidal cells (Fig. 3D). Also, the V thr values for some subtypes of interneurons in L2/3 and L5/6 are very negative (Fig. 3, A andB) making these cells highly excitable. This is also the case for most L5/6 pyramidal cells compared with those in L2/3. Therefore although L5/6 interneurons are able to create excitatory loops with pyramidal cells, in L2/3, this seems not to be possible. It is important to stress that, although E GABA is often not more negative than the spike threshold, its driving force is sufficient to generate action potentials because of activation of voltage-gated currents (Fig. 4D).

Results also indicated that V thr is progressively shifted during maturation to more hyperpolarized values. Similar developmental shift has been reported previously in L5 pyramidal neurons of the medial entorhinal cortex [START_REF] Reboreda | Development of cholinergic modulation and graded persistent activity in layer v of medial entorhinal cortex[END_REF]) and prefrontal cortex [START_REF] Zhang | Maturation of layer V pyramidal neurons in the rat prefrontal cortex: intrinsic properties and synaptic function[END_REF]). The underlying mechanisms are not known but are consistent with the progressive maturation of Na ϩ and K ϩ channels (Picken Bahrey and Moody 2003; for review, see Moody and Bosma 2005). Interestingly, despite the large driving force of GABA in the very immature neurons (P3), the excitatory action of GABA was not observed. Indeed, synaptic stimulation failed to initiate APs in pyramidal cells at P3 (n ϭ 5) and activated only one of four interneurons tested. This is most likely because of a small density of sodium channels and corresponding low excitability of these neurons. In fact, we could not determine the threshold of action potential generation in these neurons since the input resistance is very high (Ͼ1 Gohm).

Therefore the developmental and cell type variations of E m and V thr underlie higher excitability of deep layer neurons.

Deep cortical layers generate GABA-driven cGDPs

A variety of spontaneous patterns have been observed in neocortical slices (Aguilo et al. 1999;Corlew et al. 2004;Dupont et al. 2006;Garaschuk et al. 2000;[START_REF] Yuste | Neuronal domains in developing neocortex[END_REF]., However, cGDPs differ from activity patterns reported in the developing neocortex previously, in their temporal/spatial parameters and generating mechanisms. Thus cGDPs dominate neocortical activity around the end of first postnatal week, contrasting with earlier patterns that peak at birth and disappear shortly after, like the synapse-independent coherent activity (Corlew et al. 2004;[START_REF] Yuste | Neuronal domains in developing neocortex[END_REF] or the layer 1-correlated activity (Aguilo et al. 1999).

In addition, cGDPs differ from the early cortical networks oscillations (ENOs, Adelsberger et al. 2005;Garaschuk et al. 2000) since (1) ENOs peak at P1-P2 and disappear at P5-P6, whereas cGDPs are not observed before P4, peak at P7-P9, and disappear at about P13; (2) ENOs occur at very low rates (approximately once per 1-12 min), whereas cGDPs are observed once per 6 -40 s at P7-P9; (3) ENOs involve the entire network of neurons whereas cGDPs are generated by deep cortical layers; and (4) GABA A Rs are not involved in the ENOs initiation; in contrast, cGDPs are inhibited by selective GABA A R antagonists and bumetanide and are enhanced by diazepam (Fig. 6). CGDPs also differ from the recently described carbachol-induced oscillations (Dupont et al. 2006) that have a much longer duration, a propagation to the entire cortical mantle, and, across all layers, an initial dependence on gap junctions and subsequently on NMDA but not GABA receptors.

The sequential maturation of central oscillations has been systematically studied recently in the developing hippocampus by fast dynamic imaging techniques (Crepel et al. 2007). Around delivery, the gap junction interconnected small cell assemblies generate nonsynaptically induced calcium signals [synchronous plateau assemblies (SPA)] followed, at the end of first postnatal week, by synapse-driven GDPs (Crepel et al. 2007). SPAs coexist with GDPs during a transient developmental stage. However, an increase in occurrence of GDPs correlates with the decrease in SPA activity. Therefore the initial synapse-driven oscillations in hippocampus are preceded by nonsynapse driven patterns. Further studies are required to find out whether a similar developmentally regulated sequence exists in the immature neocortex as well.

The relevance of cGDPs to in vivo patterns remains to be determined. In the neonatal rat neocortex in vivo, the dominant pattern of electrical activity is spindle bursts. This transient coordinated activity controls the organization and shaping of sensory and motor systems (Dupont et al. 2006;Khazipov et al. 2004b;[START_REF] Tyzio | Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery[END_REF]). Delta-brushes, observed in human preterm neonates, may represent the alike pattern in humans playing a similar role during formation of sensorimotor maps (Milh et al. 2007). Although cGDPs and spindle bursts share certain common features, it is difficult to directly test this hypothesis in the motor cortex since functional thalamocortical connections are needed. Thalamocortical slices are available for the barrel, visual, auditory, and anterior cingulate cotices (Agmon and Connors 1991;Cruikshank et al. 2002;Daw et al. 2007a,b;Lee et al. 2007;MacLean et al. 2006) but not the motor cortex. Interestingly, we found that glutamate puffs in thalamocortical slices of the barrel cortex (Agmon and Connors 1991) generate spindle-burst-like oscillations (P7 mice; data not shown). Thus thalamic input can initiate oscillations in deep neocortical layers, at least in the barrel cortex.

Thus our results indicate that GABA depolarizes and excites neocortical neurons in an age-, layer-, and type-specific manner and that excitatory GABA plays an important role in generation of endogenous pattern of cGDPs in the developing neocortical networks. This is in agreement with our more general hypothesis that, early in development, neuronal networks generate specific patterns of activity driven by depolarizing GABA (Ben-Ari 2002;Ben-Ari et al. 2007). We suggest that cGDPs, which provide coordinated activation of neocortical neurons and thus stimulate synaptic plasticity, participate in the activity-dependent formation of neocortical circuitry.

FIG. 1 .

 1 FIG. 1. E m and E GABA in neonatal neurons measured by cell-attached recordings of single N-methyl-D-aspartate (NMDA)-and GABA-activated channels. A: current-voltage relationships of single NMDA-and GABA-activated channels. Inset: examples of single NMDA (left) and GABA (right) channel activity at various pipette potentials. Recordings from the same P2 cortical plate pyramidal cell (left) and from the same P9 layer 2/3 pyramidal cell (right). B: E m in various cell types and layers of mouse P7-P8 neocortical slices (97 neurons). C: values of E GABA . D: GABA driving force (DF GABA ) in different cell types and layers of mouse neocortical slices at P7-P8 (59 neurons).

FIG. 2 .FIG. 3 .

 23 FIG.2. Developmental profile of E m and E GABA in pyramidal cells. A: developmental changes in E m and E GABA in layer 2/3 and 5/6 pyramidal cells (pooled data from 166 cells). B: bumetanide, a NKCC1 chloride co-transporter antagonist, induces hyperpolarization of E m in P4 (7 cells) and P7/8 layer 5/6 pyramidal cells (15 cells) and strongly decreases E GABA in P7/8 layer 5/6 pyramidal cells (6 cells).

  FIG.4. Cell type and layer specificity of the excitatory GABA action in the immature neocortex. A: examples of responses to synaptic activation of GABA A receptors (GABA A Rs; stimulation in the presence of NBQX and APV) in different cell types recorded in a cell-attached mode. B: summary plot of the proportion of cells excited by GABA in different cell types at P7 (36 cells). C: summary plot of the excitatory force for GABA (i.e., the difference between E GABA and V thr ) in different cell types at P7 (54 cells). D: whole cell recordings in a L2/3 pyramidal cell and an interneuron. The pipette solution contained 20 mM Cl Ϫ that corresponded to E GABA of about Ϫ49 mV (shown by dash lines on the figure). Nevertheless, GABAergic postsynaptic potential (PSP) values recorded at the soma considerably exceeded E GABA and readily induced action potentials (APs).

FIG. 5 .

 5 FIG. 5. Cortical giant depolarizing potentials (GDPs)-the principal pattern of activity in the neonatal neocortex in vitro is generated by deep cortical layers. A: L5 local field potential (top) and cell-attached recordings from an L5 pyramidal cell (middle) and an interneuron (bottom) in a P8 mouse neocortical slice. cortical GDP (cGDP) outlined with a dashed line is shown on the right panel at expanded time scale. B: simultaneous field recordings at different layers indicate that oscillations are most powerful in L5/6 (top), much smaller in L4 (middle), and nearly absent in L2/3 (bottom). C: dual cell-attached recordings in combination with field ones in L2/3 (left) and L4 (right). Cells in L2/3 and L4 are quiescent during cGDPs. Field recordings in B and C are low-pass filtered at 100 Hz. D: proportion of active cells during cGDPs in different layers at P7-P8.

  FIG.6. Excitatory GABA is instrumental for generation of cGDPs. A: L5 local field potential recordings in control (top trace) and in the presence of GABAergic transmission modulators: the GABA A R antagonist gabazine (100 nM), the positive GABA A channel effector diazepam (5 M), and the NKCC1 co-transporter antagonist bumetanide (10 M). B: effects of GABA A R and GluR acting drugs on the occurrence (left) and amplitude (right) of cGDPs (pooled data from 26 P7-P8 neocortical slices). C: frequency (left) and amplitude (right, 100-Hz low pass) of cGDPs as a function of age (pooled data from 64 slices).

J Neurophysiol• VOL 100 • AUGUST 2008 • www.jn.org

The online version of this article contains supplemental data.

We thank I. Jorquera for technical assistance in histological processing.