Accepted Manuscript Neuroscience forefront review The GABA excitatory /inhibitory developmental sequence: a personal journey Y. Ben-Ari PII: DOI: Reference:

S0306-4522(14)00641-1 http://dx.doi.org/10.1016/j.neuroscience.2014.08.001 NSC 15608

To appear in:

Neuroscience

Accepted Date:

1 August 2014

Please cite this article as: Y. Ben-Ari, The GABA excitatory /inhibitory developmental sequence: a personal journey, Neuroscience (2014), doi: http://dx.doi.org/10.1016/j.neuroscience.2014.08.001

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The GABA excitatory /inhibitory developmental sequence: a personal journey Y Ben-Ari Emeritus director of research INSERM Founder and honorary director of INMED, Leader of the Neuroarcheology group CEO Neurochlore Address Y. Ben-Ari The neuroarcheology group and Neurochlore at INMED Inserm U901, campus scientifique de luminy, 163 route de luminy, 13273,Marseilles Cedex 09, France

Key words: GABA, polarity, intracellular chloride, embryo, delivery, postnatal, mice, rats, humans, primates, network activities, GDPs, plasticity, excitation to inhibition shift, developmental sequences,

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Abstract The developing brain is talkative but its language is not that of the adult. Most if not all voltage and transmitter gated ionic currents follow a developmental sequence and network driven patterns differ in immature and adult brains. This is best illustrated in studies engaged almost 3 decades ago where we observed elevated intracellular chloride (Cl-)i levels and excitatory GABA early during development and a perinatal excitatory /inhibitory shift. This sequence is observed in a wide range of brain structures and animal species suggesting that it has been conserved throughout evolution. It is mediated primarily by a developmentally regulated expression of the NKCC1 and KCC2 choride importer and exporter respectively. The GABAergic depolarisation acts in synergy with NMDA receptor mediated and voltage gated calcium currents to enhance intracellular calcium exerting trophic effects on neuritic growth, migration and synapse formation. These sequences can be deviated in utero by genetic or environmental insults leading to a persistence of immature features in the adult brain. This “neuroarcheology” concept paves the way to novel therapeutic perspectives based on the use of drugs that block immature but not adult currents. This is illustrated notably with the return to immature high levels of chloride and excitatory actions of GABA observed in many pathological conditions. This is due to as in the immature brain a down regulation of KCC2 and an up regulation of NKCC1. Here, I present a personal history of how an unexpected observation led to novel concepts in developmental neurobiology and putative treatments of autism and other developmental disorders. Being a personal account, this review is neither exhaustive nor provides an update of this topic with all the studies that have contributed to this evolution. We all rely on previous inventors to allow science to advance. Here, I present a personal summary of this topic primarily to illustrate why we often fail to comprehend the implications of our own observations. They remind us – and policy deciders- why Science cannot be programmed, requiring time, risky investigations that raise interesting questions before being translated from bench to bed. Discoveries are always on side ways, never on highways.

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Introduction GABAergic signals play crucial role in the generation of behaviourally relevant oscillations by means of specific synaptic connections. The network of GABAergic interneurons is particularly diversified enabling a fine tuning of the activity that principal neurons generate (Buzsaki and Draguhn, 2004; Freund and Buzsaki, 1996). Classically, GABA is presented as the prototype of inhibitory transmitter acting by means of a hyperpolarisation and shunting inhibition to reduce on-going activity. This however does not give enough credit to GABAergic currents that are endowed with the unique capacity to inhibit or excite neurons depending on activity levels and intrinsic or extrinsic factors. No other fast acting transmitter shares this feature of GABA- and its twin brother glycine. This is a consequence of the permeability of GABA receptors channels to chloride and other anions and the exquisite capacity of intracellular chloride levels to change and reverse the polarity of the current. As many other researchers, I have been intrigued in the mid seventies by the mechanisms and pathological implications of this feature. One obvious line of research was the role of chloride regulation in epilepsies as many antiepileptic agents act by reinforcing GABAergic inhibition and conversely blocking GABA receptors generate seizures. For me as for many other researchers, understanding epilepsies was an excellent way to better understand how the inhibition operates in physiological conditions: brain pathology has always been an excellent school to understand brain operation provided that these studies are done in parallel. My interest to developmental neurobiology shifted when I moved to head a new laboratory in Paris located in a maternity hospital. Obviously, this was time to look at the GABA/(Cl-)i/network activity links in health and disease in a developmental perspective since the incidence of seizures is highest early in life. It turned out rapidly that GABA signals and immature networks had been largely ignored. With my colleague E Cherubini and our students J-L Gaiarsa and E Corradeti, we therefore started from scratch in 1987 performing intracellular recordings of hundreds of hippocampal neurons from foetal to post natal life{ (Ben-Ari et al., 1989; 2007; Krnjevic and Ben-Ari, 1989). This led to a series of unexpected observations that required lore than two decades to comprehend and put in a wider frame. Indeed, as often in science, it became apparent that the GABA sequence is but the visible part of the iceberg, the submerged one pertains to fundamental issues related to the maturation process, the role of activity in brain development, the timing of shifts from immature to adult networks etc. The present account illustrates the difficulties of going beyond one’s expertise, crossing across disciplines and taking time to think and elaborate global concepts. This is more difficult today than it was in the past because of publish or perish agenda, the short-term financial support and grant systems that are incompatible with conceptualisation. Thinking « Big » is not fashionable at times when speed is more important than content, technology prevails over depth oriented research and “big data collection” over concept oriented research. Yet, having then a relatively stable recurrent public financial support, I was offered favourable conditions to take my time to investigate issues that were initially neither meant to be published in “high profile” journals like Nature or Science nor to novel “translatable” observations with therapeutic perspectives. Surprises as always come from leaving aside the obvious.

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To set the scene, I shall first describe briefly the various classical observations made by the pioneers of GABA research before describing the evolution of the questions and hesitations I had during this procedure and how they led me very slowly across 3 decades to a fascinating direction that included the development of the concepts of “Checkpoint” (Ben-Ari and Spitzer, 2010) and « Neuroarcheology » (Ben-Ari, 2008) and a novel treatment of autism and other developmental disorders (Lemonnier et al., 2012) (Lemonnier et al., 2013). I. Pioneering pre-historical times: the invention of GABA At early times, the GABA saga resulted from a conjugated series of observations made primarily in New York, Montreal, Camberra and Harvard. Florey and colleagues isolated GABA from brain extract and Grundfest, Purpura, Kuffler and colleagues showed depressive actions of GABA in various preparations (Purpura et al., 1959; 1957) (Kuffler, 1960). The effects of GABA on the spinal cord were described first by Curtis and colleagues in Camberra and synthetized analogues and antagonists in collaboration with J Watkins (CURTIS et al., 1961; CURTIS and WATKINS, 1965) . During these early times, when Journals like Nature were keen to publish fundamental studies in neurophysiology and not primarily short term genetic stories, K Krnjevic and colleagues reported the actions of various molecules on cortically evoked inhibition in rabbits, cats or monkeys (Krnjevic and PHILLIS, 1963; Krnjevic and Schwartz, 1966). Using the newly developed micro-iontophoretic applications of drugs, K Krnjevic revealed the inhibitory actions of an agent isolated from cortical material identified as GABA. The description of these crude observations and the depth of deductions made are worth quoting at times when eloquence is not a dominating quality of papers being replaced by sterilized, impersonal style less accounts: « In view of the possibility that GABA is the chemical agent mediating cortical inhibition, an attempt was made to find a selective antagonist of its depressant action on cortical neurones. None of the agents listed above, nor any other of the substances tested, were able to block this action. It was concluded that cortical inhibition differs from spinal inhibition in its pharmacological properties; and that our observations are consistent with the possibility that GABA is the cortical inhibitory transmitter (whereas glycine is for the spinal chord) »(Kelly and Krnjevic, 1968). To confirm the crucial role of GABA in cortical inhibition, a selective antagonist was indeed much needed and this was achieved by D Curtis and colleagues « Bicuculline, a specific GABA antagonist, diminishes basket cell inhibition of hippocampal pyramidal neurones, an inhibition which is not affected by strychnine. The inhibitory transmitter released by basket cells is thus probably GABA » (CURTIS et al., 1961; CURTIS and WATKINS, 1965). It became rapidly apparent that the antagonists of GABA are also epileptic agents leading to the still dominant concept of the excitatory /inhibitory balance needed to avoid seizures and hyperactivity. This was to be further reinforced by the discovery of the antiepileptic actions of pro-GABA drugs like the benzodiazepines and phenobarbital. This was the basis for the long lasting and still dominating Excitatory/inhibitory imbalance suggested to be THE central cause of epilepsies and many pathological conditions. Although used here as well, this simplified view fails to fully account to the plethora of roles that GABAergic systems fulfil. II. A personal pilgrimage to GABA research: Adult GABAergic inhibition in health and disease

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a) An introduction to GABA: The Medical Research Council unit of Cambridge Finishing my PhD in France (« Unitary cellular plasticity in the amygdala in vivo using a Pavlovian sensory conditioning »)(Ben-Ari, 1972; Ben-Ari and Le Gal La Salle, 1972), I decided to make my Post doc in Cambridge. Without really programming it, I started to work on GABAergic signals. Having worked for some times in the amygdala, I started to examine the distribution of GAD and other markers making the first detailed description of GABAergic signals in the various nuclei of the amygdala. We used a dissection method, which would seem genuinely prehistoric today- intra-cardiac perfusion of the heart with cold ACSF and dissection of the fresh material in the cold room…well dressed to that occasion. Using this method and a methylene blue stain of the surface of fresh slices, we could dissect with R Zigmond and assay GAD even the optic tract or the stria terminalis and its bed nucleus. We provided a rather complete description of the concentrations of GABAergic markers in amygdaloid nuclei (Ben-Ari, Kanazawa, and Zigmond, 1976; BenAri, Lewis, et al., 1976; Zigmond and Ben-Ari, 1976). In parallel, with JS Kelly, we examined the effects of microiontophoretic applications of dopamine and GABA on amygdaloid neurons using the recently developed technique of iontophoresis of agents with brief currents. We relied on stereotaxically positioned guide tube, sealed to the skull in order to obtain stable recording conditions. Then, the stereotaxic position of the cells was determined with the position of the microelectrode tracks determined histologically. Dopamine exerted inhibitory actions on the spontaneous activity of neurons but interestingly, neuroleptics like alpha-flupenthixol or pimozide had no significant effect on the dopamine or GABA sensitivity of neurons. We suggested that the effects of neuroleptics on animal behaviour may not be explicable simply by a general blockade of dopamine receptors at post-synaptic sites (Ben-Ari and Kelly, 1976; 1974). With Kanazawa and Dingledine, we compared the actions of micro-iontophoretic applications of GABA and acethylcholine on neurons of the reticular and ventrobasal nuclei of the thalamus(Ben-Ari, Dingledine, et al., 1976; Ben-Ari, Kanazawa, and Kelly, 1976) . Using again in vivo recordings, we found that acethylcholine inhibited reticular neurons but excited ventro-basal neurons selectively, an important observation considering the control by the reticular nucleus of the inflow of information to the specific nuclei of the thalamus. Intravenous perfusion with atropine or picrotoxin blocked respectively the cholinergic and GABAergic inhibitions. The cat’s brains were perfused in Cambridge and the formalin containers brought with me during my monthly trips to France for histology (leading to interesting animated discussions with custom officers). There were clearly opposite effects of acetylcholine in reticular and ventrobasal neurons in keeping with their involvements in the sleep waking cycle that were to be uncovered by Steriade and colleagues (Contreras and Steriade, 1997; Steriade, 1997). b) Investigating the complex links between GABA and on-going activity in Montreal: la belle province Already at these early times, it became obvious that as chloride is the main anion player, alterations of (Cl-)i could shift the polarity of GABA actions and contribute to epilepsies. Going to learn with K Krnjevic the roles of GABA in relation to epilepsies was an obvious choice. Krecho or KK as called then was one of the genuine experts of GABAergic signals. Having worked with Eccles and Miledi on presynaptic inhibition in the end of the fifties (ECCLES et al., 1959); KK made some of the main descriptions of central

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inhibition. KK was known for his incredibly hard working regimen, days starting at 8 and finishing seldom before midnight. My task was to elaborate an in vivo preparation in order to perform intracellular recordings of hippocampal neurons in anaesthetised rats. Recording neurons for 10-15 minutes preferably with a reasonable resting membrane potential (negative to -50mV) was celebrated as this was no simple task. Hippocampal GABAergic IPSCs were found to be extremely labile, rapidly reduced by recurrent synaptic activation leading to the conclusion that the plasticity of inhibition is a major factor in epilepsies (Ben-Ari, Krnjevic, et al., 1980) (Ben-Ari, Krnjevic, et al., 1981). The reversal of the chloride gradient was readily observed in these conditions, after short episodes of hyperactivity, the IPSP became an « EPSP ». Our paper entitled « Lability of synaptic inhibition of hippocampal pyramidal neurons » published by the Canadian journal of physiology and pharmacology was to the best of my knowledge one of the first descriptions of this fragility of inhibition, stressing the possible links of this property with hyperactivity and seizures. The complex mechanisms underlying the shift of GABA polarity with recurrent activation had to await more appropriate in vitro preparation and molecular approaches: this was achieved 2 decades later. The Montreal work inaugurated a series of investigations on the basic mechanisms of epilepsies. C ) Kainate : the dragon of the epilepsies Returning to France, I decided to work more on the amygdala and trace some pathways to and from the amygdala. An ingenious method had been developed at these early days to trace functional connections between brain structures. It consisted in the destruction of cell bodies while sparing axons en passant by injecting kainic acid, a powerful excitatory agent in the structure investigated (references in (Ben-Ari, 1985). I was interested in tracing amygdala connections by injecting dyes, determining their transport while avoiding the en passant fibers. I was struck already during the first experiment how convulsive was kainate when injected even at homeopathic doses in the amygdala. Long lasting recurrent seizures and status epilepticus were generated leading to the discovery of a useful model of temporal lobe epilepsies (Ben-Ari and Lagowska, 1978; Ben-Ari et al., 1979). This was followed by a systematic investigation of the seizures generated by central or systemic administrations of kainic acid (Ben-Ari, Tremblay, et al., 1980) (Ben-Ari, 1985; Ben-Ari, Riche, et al., 1981; Nadler, 1979). It was also in accordance with human data pointing to the amygdala as nodal in this form of epilepsies. These studies that led to thousands of investigations using kainic acid to study epileptogenesis (2657 references as of january 2014) have been reviewed elsewhere and need not further development here. Suffice it to stress the contributions of this model in gaining better understanding of the importance of seizures in neuronal loss, the progressive maturation of seizures induced brain damage or the classical seizures induced sprouting of mossy fibres that remained the first and most compelling model of reactive plasticity induced by seizures. This provided a direct demonstration of the Jacksonian concept of « seizures beget seizures » and how this operates. The concepts initiated in these early investigations have been summarized in my highly quoted 1985 review (Ben-Ari, 2010; 1985). These studies made well before the receptors were cloned, provided most of present understanding of basic mechanisms of temporal lobe epilepsies. The demonstration decades later of the shift in granule cells from purely AMPA to mixed AMPA/Kainate receptor mediated synaptic EPSCs after seizures provided a vivid illustration of the importance of reactive plasticity in epilepsies (Epsztein et al., 2005; 2010)… and why important observations require decades to be substantiated. GABAergic signalling were never far from our

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preoccupations. We and many other groups showed how recurrent seizures in vivo and in vitro led to specific loss of GABAergic interneurons notably the somatostatin containing dendritic projecting neurons and the relevance of this failure of inhibition in epilepsies(Cossart et al., 2001; Dinocourt et al., 2003; Hirsch et al., 1999). Interestingly, it turned out that kainatergic synapses also innervate preferentially certain types of neurons underlying their vulnerability to seizures (Cossart et al., 1998) (Cossart et al., 2001) (Ben-Ari and Cossart, 2000). Working on epilepsies is bound to lead you gently to investigate GABAergic signals (also see below). III. Discovering the GABA developmental sequence in Paris a) Three unexpected observations In January 1986, I was nominated head of a medical research council unit (INSERM) at the Port-Royal maternity (Paris). This implied shifting my research to brain development and the effects of early insults. A bibliographic search revealed that the developing brain was not much a topic of interest in terms of neuronal activity, we knew next to nothing on the electrical properties of intrinsic and transmitter gated currents in embryonic and early post-natal neurons. It was assumed then that they should not differ from adults in keeping with the pharmaco-therapeutic approaches that were then similar in young and adults. Probably the first suggestion of a developmentally regulated shift of GABA actions was made by Obata et al. (Obata, 1972) in spinal cord neurons. Applications of GABA or glycine depolarized 6-day-old chick spinal neurons in culture and hyperpolarized 10day-old embryos. The authors suggested an ingenious developmental sequence with GABA present first on the somata prior to dendrites as they develop (Obata et al., 1978). Purpura, Schwartzkroin Prince and colleagues made pioneering studies at this period. Intracellular recordings of kitten hippocampus in vivo – an achievements at these early times- showed that inhibition was the predominant form of synaptic activity early on, i.e. that inhibition measured by the polarity of the PSPs preceded excitatory PSPs (Purpura et al., 1968). In contrast, subsequent in vitro studies suggested that excitatory synaptic events were more common in hippocampal tissue from young kittens, and that inhibitory synaptic activity was fairly late in developing in slices of rabbit (Schwartzkroin, 1981; Schwartzkroin and Kunkel, 1982) and rat (Dunwiddie, 1981; Harris and Teyler, 1983) hippocampus (also see (Schwartzkroin and Altschuler, 1977). Hoever, these studies raise a number of issues. The resting membrane potential was strongly depolarising in these early investigations, this will necessarily impact the determination of the genuine driving force of GABA (see also below and (Tyzio et al., 2003; 2008). Thus, using intracellular recordings, Schwartzkroin and colleagues found depolarizing responses to somatic GABA application and depolarizing GABAergic PSCs in neonatal (P6–10) rabbit hippocampal CA1 pyramidal neurons. The Em was of _ 53 mV, and the reversal potentials of GABAergic postsynaptic potentials were of -36 and -46 mV, respectively (Mueller et al., 1983; 1984), although a more negative value of - 54 mV of the somatic EGABA was also reported (Mueller et al., 1983). The authors suggested that depolarizing GABA inhibits ongoing activity via shunting mechanisms and that these developmental changes are due to two types of GABA receptors/channels: a hyperpolarizing type permeable to chloride and a depolarizing type permeable to

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sodium and/or calcium in addition to chloride. An alternative hypothesis was that GABAergic signals differ in dendrites and somata in keeping with parallel investigations (Alger and Nicoll, 1979; Andersen et al., 1980; Mueller et al., 1984). Another study by the same group (Mueller et al., 1984)showed that the depolarizing potential produced in CAI neurones by stimulation of the stratum radiatum is partially mediated by GABA, which is depolarizing at this stage of development. In sum, there was no consensus on the actions of GABA on immature neurons. To resolve this controversy, we decided to record neurons starting from late gestation the end of the 2nd postnatal week. In this aim, we developed slicing and recording procedures to enable stable recordings of intrinsic parameters from embryonic or very immature neurons to wean and adult rodents. Already on our first recordings in 1987(Ben-Ari et al., 1989), we encountered 3 unexpected observations (fig 1A&B ) : i) Immature neurons generated a bizarre network driven pattern that we called « Giant Depolarising Potentials (GDPs) » that seemed similar to inter-ictal activities (Fig 1A); ii) But, GDPs like currents evoked by the GABA analog isoguvacine reversed at membrane potentials that were close to the reversal of GABAergic and transiently blocked by GABA antagonists suggesting that they are mediated by GABA receptors (Fig 1-B) iii) In very immature slices, GABA receptor antagonists blocked all on-going currents (Fig 1A) before generating seizures, in contrast to adult neurons where they triggered recurrent seizures directly (Fig1 A). . Synaptic currents like those evoked by exogenous GABA had a similar physiological and pharmacological profile excluding a mixed receptor permeable to cations and anions (Fig 1B). Parallel studies revealed that GDPs like the depolarising actions of GABA followed a developmental sequence shifting earlier in older structures than younger ones (CA3 than CA1). Collectively, these observations suggested that GABAergic signals came first, excited neurons before shifting to inhibition because of a reduction of intracellular chloride (Cl-)i and that depolarising GABA is instrumental in the generation of a unique pattern of activity that disappears when the shift has taken place. The presence of GDPs was observed in thousands of recordings since then in neonatal slices and in fact their absence usually signified that the slice was in bad conditions. Our reaction to these unexpected observations was a mixture of scepticism and conviction that there must be an artefact somewhere that we have underestimated. We made hundreds of recordings, altering the experimental conditions, the recordings tools, the ACSF and almost everything that could be considered … but the results were « disappointing »: GDPs were consistently observed as well as depolarising/excitatory GABA. In addition, the frequency of GDPs was age dependent suggesting a developmentally regulated sequence. As GDPs were also reduced and even blocked by NMDA receptor antagonists, we thought that somehow glycine - then discovered by P Ascher and colleagues to be essential for the operation of NMDA currents (Nowak et al., 1984)- could be involved in this response. In favour of this hypothesis, GDPs was highly sensitive to NMDA receptor blockers and the voltage dependent Mg++ block of NMDA currents –that determines the contribution of NMDA currents to ongoing activity – was less operative in immature than adult neurons (Ben-Ari et al., 1988) raising the possibility of a more substantial contribution of NMDA currents to immature patterns than adults (see below). We nevertheless tested this possibility with various

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approaches including glycine receptor antagonists and other manipulations (Gaiarsa et al., 1990). Although glycinergic signals were later found to be more active in the immature hippocampus than the adult one, this possibility had to be discarded. We concluded that our results must be genuine but were still far from understanding the biological advantage of such a complex sequence. To convince ourselves that these observations are not due to an artefact, with K Krnjevic and E Cherubini, we asked P Ascher, a highly respected expert in ionic currents to evaluate them. We met with an even harder scepticism than ours « this is obviously an artefact because you are not patch clamping the neurons ». Only modern tools are valid, the older ones are for old-fashioned scientists not capable of adapting to modern life. We were to discover much later that his opposition was permanent, as even the use of perforated patch or single channels failed to alter his convictions, for reasons that we still fail to understand! We therefore decided to submit our paper to the Journal of Physiology after hesitating for over 1 year (Ben-Ari et al., 1989). 3 messages stood from this paper: GABAergic currents mature before glutamatergic ones, immature networks generate a single common pattern rather than the plethora of patterns observed later and GABA provides initially the main excitatory inputs of developing neurons possibly mediating the trophic actions that GABA suggested by many investigations. Two decades were needed to confirm these suggestions and more importantly to unify them in a single concept that would turn out to be a basic rule of developmental neurobiology. But this required time, impact from a wide range of investigations and better understanding of developmental processes that at that time was not our domain of competence. Making an interesting observation is one thing, putting it in a conceptual frame is far more complex. To examine the implications of these observations, we directly tested the 3 rules that seemed to emerge from our recordings. b) GABA a pioneer early operating transmitter Does GABA operate before glutamate? Does it really provide the early tonus of activity required to organise early patterns? Our observations were indirect and could be due to other actions of bicuculline–which indeed was found subsequently to be the case. I decided therefore that the only way to prove or disprove this hypothesis was to study the maturation of GABAergic currents in morphologically reconstructed neurons: if GABA is operative first, then the first functional synapses in poorly developed neurons must be GABAergic. At these early days, the sophisticated genetic tools to birth and fate date neurons were not available. We recorded in slices prepared a few hours after birth (P0) hundreds of pyramidal neurons in CA3 or CA1 pyramidal hippocampal neurons and determined their synaptic currents, filled them with biocytine and reconstructed them post hoc. The results were remarkably clear cut (Tyzio et al., 1999) illustrating how old fashioned experiments can be highly informative. There were 3 types of neurons (Fig 2 A): i) Neurons with no synaptic currents at all, these pyramidal neurons had a small soma and no apical or basal dendrites. They correspond to recently born neurons and constitute at birth the vast majority of pyramidal neurons (80%).

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ii) Neurons with only GABAergic PSCs, these were endowed with a larger soma, and only a small apical dendrite that has not reached the distal lacunosum moleculare. These neurones correspond to intermediate developmental stage. iii) Neurons with both GABA and glutamate PSCs endowed with both an apical dendrite that reaches the lacunosum moleculare and a basal dendrite seen for the first time (Fig 2A). These correspond to the “oldest” neurons of the population. Several hundreds of recordings failed to reveal a single pyramidal neuron with glutamate but not GABA currents at birth indicating that GABAergic signals must be operative first: GABAergic synapses require a small apical dendrite to operate whereas glutamatergic PSCs operate when the apical dendrites have reached the distal molecular layer. Therefore, GABAergic synapses are formed first, requiring no specific conditions, in contrast to glutamatergic synapses that require more mature targets. Experiments in culture were to confirm this scenario with an early formation of GABAergic synapses. Interestingly, the axons always started to grow at 6 o’clock and dendrites at 12 challenging the idea that neurites grow in all directions and the one growing fastest will become a dendrite. This study paved the way to other experiments required to confirm the sequence and to determine whether this sequence is restricted to pyramidal neurons or occurs also in GABAergic interneurons(Gozlan and Ben-Ari, 2003; Hennou et al., 2002). The results were again clear-cut; at P0, there were 3 types of interneurons: i) Interneurons with no synaptic current and a limited extension ii) Interneurons with GABA but not glutamate synaptic currents endowed with more extended dendritic and axonal arbours iii) Mature interneurons with GABA and glutamate currents with extended dendrites and axons Therefore, this sequence applies to glutamatergic and GABAergic neurons (Gozlan and Ben-Ari, 2003; Hennou et al., 2002). There was however an important difference: at birth 80 % of interneurons had operative GABA and glutamate synapses whereas less than 20% of pyramidal neurons where in that situation. Indeed, extending this approach to embryonic neurons revealed that at E19/20 almost none of the pyramidal neurons had functional synapses, contrasting with the relatively high percentage of interneurons (80 %) endowed with both GABA and glutamate signals. Interestingly, dendritic targeted interneurons appeared to operate before somatic oriented ones suggesting an additional developmental sequence within the interneuronal population. This concept was to be generalised by Cossart ‘s group in the lab with more modern genetic fate mapping tools to show that early born GABAergic interneurons are also Hub generating neurons (Picardo et al., 2011) (see below). Clearly, hippocampal GABAergic interneurons mature first, innervate pyramidal neurons and other interneurons providing most if not all the synaptic currents initially. Interestingly, these observations also suggest that pyramidal neurons innervate GABAergic interneurons before innervating other pyramidal neurons. In keeping with this, although rare, early embryonic GDPs are exclusively mediated by GABAergic receptors. This developmental sequence was also observed in CA1 neurons but with a later onset in keeping with the protracted maturation of these neurons. Therefore, this sequence is developmentally regulated depending on the birth date of the neuron in

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keeping with basic principles of developmental neurobiology. Recent studies using more sophisticated methods have confirmed this property in some but not all brain structures. Therefore, at least in the hippocampus, in spite of the “long journey” of interneurons (Marín and Rubenstein, 2001), interneurons are first come, first served. Subsequent observations showed that newly born neurons in the adult brain have also immature features including early GABAergic depolarising actions (Ben-Ari et al., 2007; Mejia-Gervacio et al., 2011; Tao et al., 2012). b) A similar sequence in primate in utero Are these observations restricted to rodents? We tested this in macaques relying on Csections in pregnant macaques, slice preparations, physiological recordings and anatomical reconstructions. We succeeded to have access to 8 pregnant macaques and to perform this experimental design that provided a reasonable illustration of the events occuring between mid-gestation to a few weeks before delivery. The results were quite striking confirming the presence of 3 neuronal populations (Khazipov et al., 2001) (Fig 2B): i) Pyramidal neurons with no synaptic currents and no apical or basal dendrites. ii) Pyramidal neurons with only GABAergic PSCs endowed with only a small apical dendrite that has not reached the distal lacunosum moleculare. iii) Pyramidal neurons with GABA and glutamate PSCs endowed with both an apical dendrite that reaches the lacunosum moleculare and a basal dendrite seen for the first time (Fig.2B). The morphological features of neurons were determined in detail including counting all the spines as an indication of the density of glutamatergic synapses. A quantification of these results using Bolzmann equations revealed that at mid gestation, neurons had no spines (and no glutamatergic PSCs) and over 7000 spines and presumably functional synapses a few weeks before birth. GABAergic currents are recorded before glutamatergic ones in poorly arborized immature neurons, the presence of glutamatergic EPSCs requiring long apical dendrites reaching the distal lacunosum moleculare. GDPs are present roughly from mid gestation but are absent shortly before delivery presumably when networks generate more complex patterns (see below). Therefore, the developmental sequences are similar in rodents and primates but at different ages. Pair recordings of interneurons and pyramidal cells revealed that in both rodents (Fig 3A-B) and macaque (Fig 3 C-D), interneurons and pyramidal neurons are synchronised during GDPs (see below). It is of particular interest to note that embryonic primate networks operate primarily by means of GABAergic neurons initially. This has implications for the use of drugs and neuroactive agents notably on GABAegic signals during pregnancy in humans as they might have different effects on the mother’s brain and that of her embryo (see below). Collectively, these observations provided a detailed scheme of the maturation of synaptic connections at least in the hippocampus where the data available is far more substantial than other brain structures. Subsequent studies were to extend some of these events –particularly the excitation /inhibition shift to a wide range of animal species from worms, turtles to chicken and frogs suggesting that it corresponds to a general evolution conserved property(Ben-Ari et al., 2007). I neither imply that this sequence is identical in all brain structures considering the enormous variability of

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animal species and brain structures, nor do I imply that all neurons belonging to the same population have the same time course shifts. Interestingly, if one assumes that in some neurons glutamatergic but not GABAergic currents are functional in utero, it might be interesting to determine which type of inhibition prevents the excitotoxicity that induced by an exclusively glutamatergic excitatory drive. c) Elevated (Cl-)i and depolarising actions of GABA In our initial observations, relying on hundreds of intracellular recordings, we reported that the reversal potential of the principal network driven pattern recorded in immature pyramidal neurons was close to that of GABA (Ben-Ari et al., 1989). We also showed a developmental sequence with a progressive reduction of the reduced during the first post-natal week reaching adult values by the end of the second postnatal week. Electrical stimulation of slices evoked synaptic currents that reversed like the currents generated by applications of GABA agonists indicating that they are mediated by GABAergic receptors. Because of the considerable importance and plasticity of intracellular chloride, these results could not be sustained without compelling quantitative non invasive measures of (Cl-)i and the resting membrane potential (V rest) in immature neurons as developmental alterations of the latter could explain the apparent differences of the former. We started by using various whole cell recordings configurations including the recently developed perforated patch clamp recording technique to measure (Cl-)i and DF GABA. It became rapidly apparent that these techniques are inadequate to measure V rest as the high input capacitance of immature neurons endowed with few operating voltage and transmitter gated currents produced important leak currents and deviations from the expected values by over 15-20mV(BenAri et al., 2007; Tyzio et al., 2003). We therefore decided to implement dual single channel recordings from the same neuron – single NMDA channels to determine V rest - and single GABA channels to determine the driving force of GABA (DF GABA): these two values enable then to calculate precisely and unambiguously E GABA. Indeed, NMDA currents reverse at 0mV, hence by injecting current, it is possible to determine V rest relying on the current injected to reverse NMDA currents. Using this technique, we found that DF GABA shifts progressively during development –whereas V rest showed a small alteration. The use of other recording or imaging techniques do not provide such unambiguous results; indeed, with all invasive recording techniques including perforated patch, there is a significant shift in the resting membrane potential (Tyzio et al., 2003) and imaging techniques are far from reliable in their estimations of (Cl-)i levels (see below). We used single GABA or GABA & NMDA channel recordings in other brain structures with quite similar results (Dehorter et al., 2012) (Tyzio et al., 2006) (Rheims, Minlebaev, et al., 2008). It bears stressing that these are average values that are strongly dependent on the amount of activity immediately preceding the measurement: there is to some extent no correct value of (Cl-)i levels as they might be altered by a single burst (also see {Marchetti:2005kx}{Yamada:2004hj} {Achilles:2007dd}). However, when large numbers of measures are performed, more elevated levels will be found in immature than adult neurons belonging to the same neuronal population and performed in the same experimental conditions. This sequence then received considerable support and its mechanistic substrate substantiated with the discovery by Rivera, Kaila, Delpire and many others of the

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developmental sequence of chloride co-transporters: NKCC1 –the importer found to operate early in development – and KCC2 –the exporter that operates later (Fig 4) (Huberfeld et al., 2007; Khirug et al., 2010; Li et al., 2007; Lu et al., 1999; Rivera et al., 2002; 1999) (Lu et al., 1999). The use of selective blockers most notably the diuretic NKCC1 selective antagonist bumetanide (Feit, 1981)that reduces intracellular chloride levels also shifted DF GABA (Rheims, Minlebaev, et al., 2008) (Dzhala et al., 2005) (Huberfeld et al., 2007; Rohrbough and Spitzer, 1996) (Nardou, Yamamoto, Chazal, et al., 2011) (Glykys et al., 2009) (Khirug et al., 2010; Rivera et al., 2002; Tao et al., 2012) {Yamada:2004hj} {Achilles:2007dd}. Other investigations showed the importance of KCC2 in specific types of neurons and how these correlate with the actions of GABA (Gulácsi et al., 2003). Collectively, these observations provided a formidable support to the developmental sequence of [Cl-]i levels and GABA polarity. Interestingly, newly born granule cells in adults have also high [Cl-]i levels and depolarising GABA and have GABAergic synaptic currents before glutamatergic ones {Ge:2005ga} also see {Carleton:2003ct}{Bordey:2011hq}{Duveau:2011tc} . We have recently reviewed extensively the large number of similar observations made using various recordings or imaging techniques, preparations and brain structures (Ben-Ari et al., 2007) . Here, I shall only stress a number of observations that are particularly relevant to better comprehend frequently raised issues and discuss how these investigations led to novel rules in developmental neurobiology. d) GDPs: a primitive synaptic network pattern The Giant Depolarising Potentials (GDPs) are probably the most unexpected observation made during these experiments (Fig 1). GDPs are clearly network-driven, synaptic events that engage large number of neurons in a synchronous discharge. In an intact neonatal hippocampus preparation (Khalilov, Esclapez, et al., 1997), GDPs propagate from the hippocampus to the septum (Leinekugel et al., 1998). In a triple chamber preparation composed of the two intact hippocampi and their connecting commissures (Khazipov et al., 1999), GDPs propagate from one hippocampus to the other (Khalilov et al., 1999; 2005). GDPs are developmentally regulated appearing a few days after birth, peaking by the end of the first week and disappearing subsequently. Cell-attached and whole-cell dual recordings of interneurons revealed that interneurons fire bursts of spikes triggered by periodical large inward currents during GDPs, (Fig 3A-B). These are blocked by TTX and by high divalent cations (6mM Mg++ and 4mM Ca++) and can be evoked in an all-or-none manner by electrical stimulation in different regions of the hippocampus (Khalilov, Khazipov, et al., 1997) (Khazipov et al., 1997). GDPs can be generated by isolated CA3 subfield of the slice confirming their local generation by small set of interneurons and pyramidal neurons.

(i)

(ii) (iii)

(iv)

GDPs are mediated by GABAA and glutamate receptors, since: Their developmental time course paralleled that of the excitatory actions of GABA both in terms of the maturation of the neuronal population and the brain region (CA3 before CA1). They are readily blocked by glutamate receptor antagonists but at least initially by GABA receptor antagonists Their reversal potential strongly depends on [Cl-]i and includes at the reversal potential of GABAA an inward component having the same kinetics as a-amino-3-hydroxy-5methylisoxazole-4-propionate (AMPA) receptor-mediated EPSCs When GABAA receptors are blocked by intracellular dialysis with MgATP-free solution,

13

(v)

(vi)

(vii)

the remaining component of GDPs reversed near at 0mV and rectified at membrane potentials more negative than -20 mV, suggesting an important contribution of NMDA receptors in addition to AMPA receptors. When AMPA receptors are blocked, GABAA receptor-mediated depolarization enabled the activation of NMDA receptors presumably via attenuation of their voltage-dependent magnesium block and together generate GDPs like events (see below). In spite of their apparent similarity to interictal events, comparative studies showed that GDPs have a more hyperpolarised reversal potential than interictal events in keeping with a more significant contribution of glutamatergic currents {BenAri:2012cj} {Khalilov:1999wj}. Extensive investigations particularly by Cherubini and colleagues showed a wide range of molecules and systems that modulate and control the expression of GDPs and showed elegantly how these also control /modulate synapse formation {Mohajerani:2007fc} {Voronin:2004wk}{Safiulina:2005bm}{Safiulina:2008wp}. GDPs differ from other patterns notably the Early Network Oscillations (ENOs) in their reversal potential that is less depolarising suggesting a more important contribution of GABAergic signals {BenAri:2012cj} {Allene:2008ci}. GDPs are also readily blocked by anoxic conditions whereas ENOs are enhanced by these events {Allene:2008ci}. The model proposed suggested a synchronous activation of SR-CA3 interneurons by the co-operation of excitatory GABAergic connections between interneurons and glutamatergic connections to interneurons originating from pyramidal cells that would trigger GDPs (Figure 5, A &B) (Ben-Ari, 2002; 2001). The slower rise time of GABAergic than AMPA receptor mediated currents will synchronise neurons with a dispersed kinetics in a population event in comparison to the seizures generated by GABA receptor antagonists and mediated by AMPA receptors. GDP equivalent patterns are observed in vivo(Leinekugel et al., 2002) and in a variety of other in vitro preparations including the intact hippocampi and neuronal cultures (references in {BenAri:2007jw}). Similar patterns have also been observed in many immature brain structures including the spinal chord, neocortex, brain stem structures, retina, inner ear (reviewed in (Ben-Ari et al., 2007). A picture started to emerge with immature networks endowed with similar singular features that disappear with a well-determined time course to adult time locked activities. I coined this developmental sequence stating that « developing networks play a similar melody » (Ben-Ari, 2001) implying that in spite of their differences, they share the fact that they have a slow kinetic, are long lasting and have a transient expression and function. Other patterns restricted to immature neurons like the retinal waves have been shown to play important roles in the construction of functional units (Ackman and Crair, 2014; Torborg and Feller, 2005) (Ford et al., 2012; Mooney et al., 1996; Xu et al., 2011) and to shift timely to enable vision {Hanganu:2006fn} {Colonnese:2010ju} {Colonnese:2012ec}. In addition, GABAergic currents play an active role in the generation of retinal waves and their functional consequences (Chabrol et al., 2012). The underlying concept is that brain development required synchronised activities in order to enable heterogeneous neurons to fire and connect together. The developing brain neither needs nor can generate a large repertoire of patterns; GDPS and similar primitive patterns are poorly informative and are not behaviourally relevant unlike their adult counterparts. Studies in preterm babies and immature rodents suggested that immature patterns propagate primarily from the periphery to the centres, do not code sensory information but rather provide an important source of information required to activity dependently modulate the formation of neuronal ensembles (Milh et

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al., 2007) (Khazipov et al., 2004) (Colonnese et al., 2010) {Colonnese:2012ec}{Hanganu:2006fn} {Milh:2007bp}. These straightforward electrophysiological experiments were to be confirmed subsequently with more sophisticated tools providing more details on their underlying mechanisms without altering the fundamental scheme. In sum, the 3 properties of developing neurons and patterns were confirmed and extended in these investigations. A schematic diagram of the development of neurons and patterns in rodents and macaque is presented in Fig 6. GDPs are depicted in A and their relation to neurogenesis, dendritic growth and spine formation are shown in relation to development from embryos to adults. In B, this is presented in macaque with Boltzman integrated values from roughly E 40 to a few weeks before delivery. Note that at mid gestation, neurogenesis in the hippocampus is terminated; axons develop prior to dendrites, and GABA before glutamate. Spines acquisition –is almost nil at mid gestation- but reaches 7000 on a single pyramidal neuron before birth. GDPs are present from mid gestation to a few weeks before delivery. This is also a vulnerable time to epileptogenesis. We then decided to examine in more details the maturation of brain sequences. Indeed, during evolution, a large variety of communication devices have been used prior to synapses suggesting that GDPs might not be the first pattern in the developing brain. e) A tri-phasic developmental sequence of brain patterns This approach became possible when the development of suitable tools enabled to measure with imaging techniques the activity of large neuronal ensembles in embryonic and early post-natal slices. Indeed, using a two photons system, with Cossart and her team, we began to investigate the mechanisms underlying the generation of GDPs and the type of activities that preceded their occurrence. The development of suitable mathematical tools by several ingenious experts(Cossart et al., 2003) (Crépel et al., 2007) (Bonifazi et al., 2009), enabled the measure of the activity of hundreds of neurons simultaneously while labelling many neurons in the slice and then performing targeted patch recordings from neurons suspected to play a central role in their generation. We discovered a developmentally regulated tri-phasic sequence during development (Crépel et al., 2007), GDPs constituting the final stage of the maturation of patterns (Fig 7 A). Initially at an early embryonic stage, neurons only bear voltage gated non-synaptic calcium channels. These are intrinsic and unaltered by a cocktail of synapse and transmitter antagonists or blockers. Similar calcium currents are generated at an early stage in a wide range of animal species and brain structures and their frequency and parameters have been shown to control the phenotype of neurons {Borodinsky:2004cb} {Spitzer:2008bq}. Subsequently, during the delivery period, the first synchronised patterns are observed under the form of Synchronised Plateaux in small cell Assemblies (SPAs). These events are non-synapse intrinsic large calcium plateaux generated by gap junctions and also insensitive to synapse or transmitter gated antagonists. Then, GDPs are observed, these are the first synapse driven pattern in the developing hippocampus (Crépel et al., 2007). These 3 patterns have a well-defined developmental sequence, shifting from one to the other and during transitional periods (Fig 7B), GDPs and Spas can be recorded in the same neuron, the former inhibits the latter {Crepel:2007jx}.There is a developmental sequence, with functional synapses most likely inhibiting the earlier intrinsic non-synapse driven pattern; this is initially transient but this shift becomes permanent after repeated GDPs, possibly by shutting off gap junctions. A similar sequence was then observed in other brain structures including the neocortex (Allene et

15

al., 2008), hypothalamus (Gao and van den Pol, 2001), striatum (Dehorter, Michel, Marissal, Rotrou, Matrot, Lopez, Humphries, and Hammond, 2011a) and early calcium currents also in the substantia nigra (Ferrari et al., 2012) associated with an early release of dopamine. Extensive investigations on the mechanisms of generation and propagation of GDPs (58 references on GDPs in pubmed – notably by Cherubini, Kaila and co-workers (references in our review (Ben-Ari et al., 2007). The development of genetic fate mapping devices enabled to better define the type of neuron involved in synchronising the early patterns. Indeed, an intriguing issue is that of the behaviour of neurons who develop first: are these programed to play an important synchronizing role, a sort of chief conductor of developing symphonies? In an attempt to determine these issues, Cossart and colleagues investigated the fine patterns of neurons involved in the generation of GDPs and noted that GABAergic interneurons play a role of Hub generators, their activity orchestrating that of the entire network (Bonifazi et al., 2009) (Picardo et al., 2011). Then, they showed that these orchestrating neuron are always GABAergic and born earlier than other neurons (interneurons or pyramidal cells) with other specific features. Collectively, these and other studies of emerging networks indicate that the developmental sequences of ionic currents and brain networks include also an age and fate dependent maturation of neuronal types. Neurons are not equal during maturation and here again GABAergic interneurons appear to serve a role on orchestrating patterns that they will also have later. Therefore, the excitatory/inhibitory GABA shifts are associated with a complex timing of events that stands at the core of the development of functional neuronal units. When evaluating the importance of the GABA depolarising actions, it is important to take into account all these features that collectively draw the picture of a global coherent scheme of the succession of events taking place. Without the sequential development of other ionic currents, brain patterns and chloride co-transporters, this observation is a curious one; with these additional properties, this becomes a fundamental feature of brain development. IV. Confirming and extending the developmental sequences As often in science, the general developmental scheme suggested by these investigations raised important questions including the possible biological significance of the sequences, their underlying consequences on the firing of immature neurons, the alternative sources of inhibition operating in the absence of inhibitory GABAergic tone and their general biological relevance. Light was as often would come from other basic experiments. a) Delivery is associated with an abrupt E to I shift mediated by oxytocin This remains probably the most unexpected, compelling and direct proof of the fundamental role and biological relevance of excitatory GABA in developmental neurobiology. Using the single channel recordings techniques, we –in particular R Tyzio who developed the double single channel recording techniques from the same neuron (Tyzio et al., 2003)- started to reconstruct the entire developmental sequence of (Cl-)i levels starting from embryos to adults. We expected to find a progressive possibly exponential decline of (Cl-)i levels from embryos to adults. Indeed, we observed elevated (Cl-)i in embryonic neurons and depolarising DF GABA that declined to adults with an asymptotic curve. However, when we examined in more detail the sequence, we discovered an abrupt and dramatic shift of DF GABA and E GABA during the delivery period

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roughly a couple of hours before and after delivery (Fig 8). This shift was dramatic as such (Cl-)i levels were never seen before or after that stage and transient as the levels expected from the developmental curve were reached a few hours after delivery. This curiosity required a biological explanation to be validated and understood. We reasoned that as delivery is associated with the release of a plethora of hormones and other signals that control essential processes, one of these might trigger this abrupt shift for reasons that had to be understood. Oxytocin was a reasonable choice as in addition to triggering labor, oxytocin exerts important roles in social communication between the mother and her baby and even analgesic effects that may imply GABAergic mechanisms (see below). We tested the effects of a selective antagonist of oxytocin receptors (developed to delay early labour) and the results were amazingly straightforward: administration of the antagonist in vivo (to the mother) or in vitro (to the newly born slice) blocked completely the chloride shift and the excitatory actions of GABA (Tyzio et al., 2006)! The oxytocin receptor antagonist had no effects on neurons 2 days after delivery indicating that the endogenous oxytocin levels in the slice are significant during the peak of the hormonal effects. Stated differently, in addition to triggering labour, oxytocin reduces dramatically and transiently the levels of neuronal intracellular chloride in newly born pups for a short period. Most intriguingly, oxytocin also modulated the generation of network oscillations activating the large calcium plateaux –the SPAs - present during delivery. Clearly, delivery is associated with a set of mechanisms dedicated to enable a smooth transition of brain activities during this complex event. We examined the biological significance of this abrupt shift: why do neurons need strong inhibitory GABA and low levels of chloride during the delivery period? We reasoned that strong inhibitory GABA would lead to reduced network activity and enhance neuronal resistance to anoxic episodes as many other and we had shown in various experimental conditions. In my earlier experiments with K Krnjevic and E Cherubini in France, we had shown that during the delivery period, neurons are extremely resistant to anoxic episodes; long lasting durations of anoxic and reduced glycaemic conditions barely affect neuronal properties (Krnjevic and Ben-Ari, 1989; Krnjevic et al., 1989). Specifically, when an adult and an immature hippocampal slice are placed in the same chamber, oxygen deprivation abolished synaptic transmission in 5 mins in the former and over 1 hour in the latter. Yet, applying the oxytocin receptor antagonist strongly reduced this resistance thereby rendering neurons more susceptible to damage (Fig 8 B)(Tyzio et al., 2006) . Therefore, the hormone is also neuroprotective. This was not all. Elegant studies by Lagercrantz and colleagues (Bergqvist et al., 2009)have shown that babies delivered by C-Sections felt more pain than vaginal delivered ones raising the possibility that oxytocin –known to have also analgesic actions- might be involved in this reaction. Curiously, pain reactions of the newborn during delivery had not been investigated although they might be of some importance. We therefore tested the hypothesis that the reduction of (Cl-)i levels and associated decrease of on-going activity (see below) might impact pain threshold (Mazzuca et al., 2011). Using a thermal tail-flick assay, we observed that pain sensitivity is two fold lower in new-borns than 2 days later. Oxytocin receptor antagonist strongly enhanced pain in new-borns but not 2 days later whereas the hormone reduced pain sensitivity at both ages suggesting an

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endogenous analgesia by oxytocin restricted to the delivery period. Pain vocalisation produced by whisker pad stimuli were also attenuated in de-cerebrated animals. Oxytocin reduced intracellular calcium levels and depolarising actions of GABA in trigeminal neurons, suggesting that these effects are mediated by (Cl-)i levels. The diuretic and specific NKCC1 chloride importer antagonist bumetanide that reduces (Cl-)i levels exerted the same effects on pain and intracellular chloride in pain pathways indicating that the effects of the hormone are indeed mediated by the polarity of GABA actions. Therefore, in addition to triggering labour and delivery, oxytocin also acts as a painkiller during this highly vulnerable period and its actions are mediated by the excitatory/inhibitory developmental sequence. A plethora of reactions unique to that day take place to accommodate the massive alterations that must occur within a few hours. This abrupt shift during delivery and the amazing mechanisms elaborated by mother Nature not only validated our model but stressed unambiguously its biological relevance. An incredible unexpected illustration of the importance of the delivery shift was to come later at the end of this journey with our studies on autism. b) The GABA/NMDA cooperation: a ménage à trois of receptors Does GABA also activate calcium currents and by this mechanism also exert trophic actions? Indeed, it has long been known that GABA exerts trophic actions often mediated by molecular cascades triggered by a rise of intracellular calcium (Represa and Ben-Ari, 2005) (Meier et al., 1983). The next step was obviously to better investigate whether and how would depolarizing GABAergic signals be involved in these actions. GABA could increase intracellular calcium by two classical mechanisms: activation of NMDA receptor/channels and/or voltage dependent calcium currents. We found that they both operate (Fig 9B). Indeed, using a wide range of imaging and physiological recordings, we reported that the depolarisation produced by GABA is more than sufficient to remove the Voltage dependent Mg++ block of NMDA channels thereby producing large intracellular calcium influx (Leinekugel et al., 1997) (Leinekugel et al., 1995). In fact, the combination of this depolarisation and the large and long lasting NMDA mediated EPSCs are the main generating mechanism of GDPs and other immature oscillations: the developing brain talks a lot primarily because of a combined depolarising GABA AND long lasting NMDA currents initiating synaptic plasticity (see below) and the generation of oscillations that modulate activity dependently the formation of functional units (Safiulina et al., 2010) (Safiulina et al., 2006). This may turn out to be more important than spike generation as was shown later with the demonstration that the threshold of spike generation differed in different neuronal populations stressing the importance of intrinsic currents in immature neurons (Rheims, Minlebaev, et al., 2008) (Rheims, Represa, et al., 2008). We also found that the depolarising GABA also activated voltage gated calcium currents although the nature of the events in which NMDA or Calcium currents are involved deserves more investigation. In a review, I called this situation a “ménage a trois of transmitters” speaking of GABA, AMPA and NMDA currents in the developing brain acting together to excite (Ben-Ari et al., 1997). This stands in contrast to the adult situation where activation of GABAergic currents reduces the likelihood of activating NMDA currents, synaptic plasticity and long-term potentiation. The cover page of this TINS review included following a suggestion of the chief editor of TINS a picture taken from the film of the French film director François Truffaut entitled Jules et Jim with a vivid and

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genuine “ménage a trois”. Therefore, the depolarising GABA currents combined with long lasting NMDA currents serve as crucial role in the synchronisation of immature neurons to fire together and wire together. The GABA excitatory /inhibitory sequence is but one of the multiple facets of the maturation of brain activities. c) Dual actions of GABA: the yin and the yang Does the depolarising action of GABA also lead to the generation, of action potentials? Is GABA genuinely “excitatory”? The threshold of spike generation is not readily reached by the depolarisation produced by GABA. Indeed a simple calculation shows that even when depolarising in many neurons, a stronger depolarisation is needed. It remained therefore to determine whether and how the depolarising actions of GABA are sufficient to generate action potentials. In many neurons, cell attached evoked responses were partly blocked in immature neurons by glutamatergic receptor antagonists and fully blocked when GABA receptor antagonists were added (Fig 9A). We investigated these issues in neocortical neurons comparing the spike threshold and GABA currents in neurons of layers 5/6 and layers 3 to take into account the different developmental stages of these neurons, the former being older than the latter (Rheims, Minlebaev, et al., 2008) (Rheims, Represa, et al., 2008). Using non-invasive single N-methyl-d-aspartate and GABA channel recordings to measure both Em and DF GABA in the same neuron, we found that GABA strongly depolarizes pyramidal neurons and interneurons in both deep and superficial layers of the immature neocortex (P2-P10). Yet, GABA generated action potentials in layer 5/6 (L5/6) but not L2/3 pyramidal cells consequently to a more depolarizing resting potentials of L5/6 pyramidal cells and more hyperpolarized spike threshold. Interestingly, GABA generated more readily action potentials in interneurons of both layers because of a more suitable Em and spike threshold. GABA transiently drove oscillations generated by L5/6 pyramidal cells and interneurons that propagated to more superficial layers. These were readily blocked by the NKCC1 co-transporter antagonist bumetanide that reduced (Cl-)i levels, GABA-induced depolarization, and network oscillations. Therefore, the high intrinsic excitability of L5/6 pyramidal neurons and interneurons provide a powerful mechanism of synapse-driven oscillatory activity in the rodent neocortex generated by GABAergic depolarisation. The dual role of GABA has also been investigated in relation to neuronal migration acting as "go" and "stop" signals and in relation to epilepsies {Heck:2007to} {Colonnese:2010ju, Fukuda:2013gc} {Kanold:2010bm} {Hanganu:2002wf} {Lapray:2010cf}. Interestingly, the depolarisation produced by GABA can also activate the sodium noninactivating current (I Nap) leading to the generation of bursts. This activation is of particular interest considering the strong dependence of this current on the kinetic of the depolarising event (Valeeva et al., 2010). These observations suggest that the activation of depolarising GABA AND voltage gated currents can indeed generate action potentials. Indeed, in parallel studies on kainatergic synapses in the epileptic hippocampus (Artinian et al., 2011; Epsztein et al., 2010), this current was found to be activated by currents with a slow rise time kinetics like the ones present in immature GABAergic currents. Collectively, these observations stress the importance the sequential maturation of voltage and transmitter gated ionic currents. These are not only neuronal type and age but also sex dependent (see below). In an elegant study, Van den pol and colleagues used outside out single patches as sensors of the response to GABA and showed that growth cones release packets of GABA

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that can be « seen » at some distance illustrating the importance of this paracrine mode of communication (Gao and van den Pol, 2000).This group also showed the importance of the timing of GABA and glutamate interactions and how GABA could either block or augment the excitatory glutamatergic drive in a time dependent manner {Gao:2001tn}. Collectively, these observations stress the importance of taking into account the different agenda of developing networks and the heterogeneity of neurons at early developmental stages. This implies a different conceptual investigation of developing neurons incorporating the fact that contrary to adult neurons, two neurons belonging to the same population meant to act together later differ completely at an early stage. Also, the actions of GABA are highly plastic depending ultimately on the amount of activity preceding the measure: a single burst might alter a strongly hyperpolarising event to a depolarising and even excitatory one. Pushing a little bit this notion, one might say that measurements of resting (Cl-)i levels is not possible as this is too volatile! Therefore, determination of the dynamic control of (Cl-)i levels in relation to activity is instrumental in order to understand the GABA developmental sequence. This parameter is not readily measured. The most straightforward way is to augment artificially (Cl-)i levels and then determine the speed with which control (pre-stimulation) levels are restored as an indication of the relative efficacy of NKCC1 and KCC2. We developed a technique based on perforated patch recordings with repeated focal applications of GABA to measure every few minutes DF GABA and the ongoing activity of the cotransporters; then a large depolarising pulse is applied to augment (Cl-)i levels and the time course of the recuperation to pre-stimulation applications of GABA is determined. . We found (Nardou, Yamamoto, Chazal, et al., 2011) that the recuperation to prestimulation levels took several minutes in immature neurons and tens of seconds in adult ones in the same conditions. Similarly, in epileptic neurons where KCC2 is clearly degraded, tens of minutes were required (Nardou, Yamamoto, Chazal, et al., 2011)( and see below). Therefore, immature neurons require longer periods than adults to regulate (Cl-)i levels and return to pre-stimulation levels. d) Is there a stop signal to organise the shift? In the model proposed, immature neurons require large network driven patterns to interconnect and construct functional units. Yet, at some well-defined stage, these must shift to enable the generation of behaviourally relevant patterns with their time locked currents. But how and when are they interrupted to shift? C. Hammond and her team in the lab decided to answer to this question relying on the most appropriate brain structure to that effect: the striatum. In adults, the GABAergic Medium Spiny Neurons (MSNs) compose over 95% of the striatal population and are usually quite silent with a highly hyperpolarised resting membrane potential (over -85). This is required in order to enable the generation of coherent targeted movements by the incoming motor cortex signals. Indeed, in Parkinson disease, the striatum is highly active generating synchronised currents that are thought to play an important role in akinesia and other symptoms {RivlinEtzion:2010ez} {Hammond:2007bz} {Brown:2006ex}. Therefore, the striatum constitutes a suitable target to investigate this issue as immature MSNs are expected to generate GDPs and be silenced timely to enable targeted movements. Using a two photons microscope, enhanced oscillatory activity was found in immature striatal slices that are reminiscent of GDP until P8-10 (Dehorter, Michel, Marissal, Rotrou, Matrot, Lopez, Humphries, and Hammond, 2011a); these disappeared abruptly consequently to the activation of voltage-gated currents and a reduction of the NMDA

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immature currents (Fig 10A). Interestingly, this is also the stage when the pups shift abruptly from immobility or crawling to targeted movements (Fig 10B) (Dehorter et al., 2012). Therefore, the timing of the shift is programmed specifically to occur when most needed. It is likely that other systems have similar time dependence to accommodate development and more adult requirements. Indeed, similar abrupt changes have been reported in various sensory systems such as the inner ear (Wong et al., 2013) and the retina with the classical retinal waves (Chabrol et al., 2012) (Mooney et al., 1996; Torborg and Feller, 2005; Xu et al., 2011). Summing up, the GABA developmental sequence is associated with other alterations that converge to generate a depolarising-occasionally excitatory- signal, activating NMDA receptors and calcium currents leading to large calcium influxes and various forms of synaptic plasticity. In sum, the unique property of GABA (and glycine signals) to shift polarity in a variety of conditions is particularly suited for developing neurons that require synchronised activities in large neuronal ensembles. V. Challenging the GABA E to I sequence: much ado about nothing! Science advances by trial and errors and challenging fundamental discoveries can fuel novel concepts raising more questions. Our developmental sequence remained undisputed for over 2 decades before being curiously and severely challenged by 3 former INMED students (C Bernard, P Bregestovski and Y Zilberter) and by K Staley and colleagues. a) The Ketone bodies challenge by Zilberter, Bregestovski and Bernard In Marseille, P Bregestovski (Brest), Y and T Zilberter were by far the most vehement claiming that the GABA depolarisation in slices is «an artefact » for metabolic reasons. The concept was that as maternal milk is enriched in ketone bodies, observations obtained in glucose perfused immature slices are due to energy deprivation since glucose (10mM) cannot replace ketonergic metabolism (Rheims et al., 2009) (Holmgren et al., 2010) . While still at INMED, the Zilberter’s, Brest and colleagues found that GABA did not depolarise immature neurons in the presence of a ketone body metabolite BHB, lactate or pyruvate -in addition to glucose- (Holmgren et al., 2010; Rheims et al., 2009). As repeated internal discussions failed to convince them of the limitations of their results, we resolved to repeat these experiments using the same setups, drugs, animals and experimental conditions and a wide range of tools extending from single channel recordings to dynamic two-photons microscopy. This large cooperative work with many different INMED teams infirmed completely their results: ketone bodies metabolites had no effects on the actions of GABA (Tyzio et al., 2011) and the GABA dependent maturation of hippocampal networks. Then, using a wide range of preparations, techniques, animal species and brain structures, 8 other independent laboratories infirmed their observations that to the best of our knowledge have not been confirmed by a single other team casting severe doubt on their validity (references in (Ben-Ari et al., 2012) also see (Gomez-Lira et al., 2011)). In addition, the Zilberter’s and colleagues used abnormally high concentrations of pyruvate that are only found in pathological conditions and provide in fact a signature of damage and inflammation (hundreds of references in pubmed, see (Ben-Ari et al., 2012)). Using reliable measures- notably mitochondrial pH- Kaila and co-workers demonstrated that these effects are due to acidosis (Ruusuvuori et al., 2010) in contrast to the indirect evaluations made by

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Bregestovski and colleagues(Bregestovski and Bernard, 2012). Clearly, this criticism was a dead end based on irreproducible results and intrinsic contradictions. b) The injury challenge by Staley and co-workers Although they contributed to some important initial observations on the roles of chloride co-transporters and the depolarising actions of GABA, K Staley and co-workers generated a second front suggesting that the GABA depolarisation is due to injury in the preparation of young slices (Dzhala et al., 2012). Using glucose perfused slices and primarily chlomeleon imaging techniques, they suggested that in immature neurons « increases in [Cl-]i correlated with disruption of neural processes and biomarkers of cell injury… These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures ». The damage underlies the depolarising actions of GABA in slices, less so in intact preparations and is largely restricted to the surface of slices. Interestingly, one of the leading co-authors of this publication – R Khazipov –published another paper (Valeeva et al., 2013) showing that “the excitatory actions of GABA in hippocampal slices during the first post-natal days are not due to neuronal injury during slice preparation, and the trauma-related excitatory GABA actions at the slice surface are a fundamentally different phenomenon observed during the second post-natal week”. So the challenge concerns not the embryonic, delivery and early post-natal period but only the exact date at which this shift takes place (rather after P5 not P7 or else). In a developmental neurobiology perspective, the debate on the exact date of the shift is futile considering the variability of experimental conditions, neuronal type and heterogeneity of neurons etc. It is quite difficult to see why would a vibratome damage neurons starting from P5 and not a couple of hours before or after! Here also, there is a wide range of observations that cannot be reconciled with the injury explanation (Ben-Ari et al., 2007) (Tyzio et al., 2014) {DeFazio:2002gd}including the fact that depolarising GABA has been observed in intact hippocampal preparations, in various adult preparations and present in adult slices of autistic animal models but not if these have been treated with a diuretic during the delivery period (ibid and below). It is important to stress that the chlolemeleon technique is primarily a pH measure providing at best a ratiometric qualitative indication of chloride levels (Ben-Ari et al., 2012) and requiring elementary controls that were not performed by Staley and coworkers (Glykys et al., 2009) (Dzhala et al., 2012) (Glykys et al., 2014). The heterogeneity of chloride levels (from 1 to 120mM) with this technique is an order of magnitude above that observed with more reliable electrical techniques in slices prepared by expert teams. The imaging scanning speed is hundred times slower than that assessed with electrical recordings and the separation of YFP and CFP signals with a band pass for yellow (510-540nm) is debatable, differing from the original one used by Kuner and Augustine (Kuner and Augustine, 2000). It is manifest that the validity of challenging the observations made with direct electrical recordings with an imaging technique is questionable (also see below). c) The slice artefact explanation of Bernard and Bregestovski This study was nevertheless welcomed by Bernard and Bregestovski who rushed to publish a review edited by their team member Y. Zilberter entitled: « Excitatory GABA:

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how a correct observation may turn out to be an experimental artefact » (Bregestovski and Bernard, 2012) . This review amalgamates irreproducible results, ignore ones that cannot be reconciled with their suggestions, refers to observations that are incompatible with one another and attest a limited understanding of developmental processes. For example, Bregestovski and Bernard rely on Staley’s suggestions of an injury explanation to challenge the GABA excitatory actions (Dzhala et al., 2012) ignoring that these experiments were made in glucose perfused « slices » that according to them are artefact suffering from insufficient metabolic supply, raising the paradox of two incompatible explanations for a single phenomenon. Bernard and Bregestovski also rely on the Wang and Kriegstein (Wang and Kriegstein, 2011) who showed that in utero and post-natal administration of bumetanide leads to important malformations in offsprings suggesting that the diuretic ought not to be administered during pregnancy. Relying on this study, Bregetovski and Bernard conclude that the GABA developmental sequence is valid until delivery –but not after. Yet, had they read carefully the study of Wang & Kriegstein, they would have noticed that the most dramatic effects of bumetanide are observed when the diuretic is injected during the first week post natal suggesting that the post natal depolarising actions of GABA are essential. Curiously, Bernard and colleagues have in almost parallel studies stressed the importance of GABA excitatory sequence actions (Quilichini et al., 2012)! The paper of Bernard and Bregestovski (Bregestovski and Bernard, 2012)was heavily publicised leading to a paradoxical situation where referees of publications dealing with GABA signalling in developing neurons imposed to discuss this challenge. Grants of studies based on excitatory actions of GABA on immature neurons were rejected! With 12 other experts in studying GABA signaling, we decided that a detailed reply was mandatory (Ben-Ari et al., 2012). We refuted point by point these allegations that failed to meet the minimal requirements required for a scientific debate, namely reproducible observations, reliable suggestions and correct literature reviewing. The conclusion of this disagreeable moment is that scientific debates are useful only when relying on serious investigations, reproducible results, convincing arguments and if possible a novel concept. This was clearly not the case here. d) The Impermeant Anion challenge by Staley and co-workers! But the story did not end there! Staley and workers pursued their fascination by the chlomeleon Technicolor technique to change 180° their views on the actions of GABA and the roles of (Cl-)i levels (Glykys et al., 2014). Staley and co-workers infirmed their numerous pioneering observations on depolarising GABA in immature neurons and the effects of bumetanide (Dzhala and Staley, 2003) (Dzhala et al., 2005) (Dzhala et al., 2008) (Dzhala et al., 2010; Glykys et al., 2009), modeling KCC2 functions and NKCC1 stoichiometry (Brumback and Staley, 2008) and the role of slice damage (Dzhala et al., 2012) (Staley and Proctor, 1999). Now, slices are not damaged and NKCC1 and KCC2 have little impact on (Cl-)i levels: the polarity of GABA actions is determined by vaguely defined intracellular impermeant anions (Glykys et al., 2014). This is an amazing study with no experimental controls, investigations relying exclusively on “specific” KCC2 antagonists that remain to be thoroughly investigated and lack of effects of bumetanide –that previously was found by the same group to have a major effect on (Cl-)i levels of immature neurons. There are other major caveats in this study. Thus, the authors described the lack of effects of bumetanide on neurons that have low (Cl-)I omitting to stress that in these neurons bumetanide has anyhow no effect. Comparing (Cl-)i levels

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before and after 30 min applications of bumetanide is inadequate as the changes have plenty of time to fade. Astonishingly, the authors neither compared deep and superficial layers nor even referred to their own paper published 2 years earlier where elevated (Cl-)i and excitatory GABA were primarily due to surface injury (Dzhala et al., 2012). Contrary to the earlier study, slices are now not damaged and the reasons for these alterations are not explained. Refuting the extensive direct recordings with the clear cut regulation of GABAergic synaptic currents by chloride co-transporter blockers relying on a pH qualitative imaging technique with a poor, low and slow sensitivity to chloride is doomed to fail. Ad minima, the authors should have compared in real time in a set of neurons the alterations of (Cl-)i with their imaging and more reliable single GABA channel recordings. Without these comparisons, these observations remain a distorted, reflection of the dynamic synaptic events occurring at a fast time course in real life. Summing up, the GABA developmental sequence is now widely accepted because of the convergence of observations obtained in a wide range of issues and preparations. Indeed, it is important in this debate to take into account these apparently disparate observations that converge including the sequence itself with the oxytocin mediated abrupt shift during delivery, the neuronal type & age dependence of the sequence, the alterations according to the sex of the animal, the fact that GABA usually does not depolarise adult neurones that are far more susceptible to lack of energy supply, the presence of the sequence in chicken, insects and frogs that to the best of our knowledge do not rely on maternal milk, the confirmation of the sequence in intact preparations, neuronal cultures and in vivo, the chloride co-transporter sequential development that provide a superb mechanistic substrate to the concept, (Ben-Ari et al., 2007) the difference between normal and albino rodents (Barmashenko et al., 2005) and the long term effects of bumetanide in animal models of autism where this shift is abolished (see below). Admittedly, several Whys and Hows are not clear but the model is validated and its conservation during evolution is still an interesting issue to be determined. VI. Pathological implications of the sequence The developing brain differs from the adult one in its susceptibility to insults. Thus, the incidence of seizures is highest early in life raising the possibility that the lack of a powerful GABAergic inhibitory element contributes to that feature. Anoxic insults during the delivery period issue are also an important issue as there are amongst the most frequent cause of severe life long deleterious sequels. Curiously, immature post natal neurons are during the first 2-3 days after delivery extremely resistant to anoxic episodes; recordings made form adult and immature slices placed in the same chamber showed a dramatic difference in the duration of anoxic episodes required to abolish synaptic transmission (Cherubini et al., 1989) (Crepel et al., 1992). As in addition, a plethora of neurological and psychiatric disorders originate during the embryonic and/or early post natal period, it seemed important to determine the mechanisms underlying these differences: Are the unique features of the developing brain and notably the excitatory actions of GABA a contributing factor? We set to investigate these issues somewhat returning to our early-preferred domain of interest: epilepsies. a) The GABA shift and epilepsies It was obvious from very early studies that we performed in vivo (see above) that recurrent activation of synaptic inputs to pyramidal neurons shifted the actions of GABA from inhibitory to excitatory because of an accumulation of chloride and a failure of

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neurons to cope with the large fluxes of chloride associated with high frequency GABAergic PSCs. Other observations indicate that KCC2 is highly susceptible to seizures, readily inactivated and internalized by recurrent –not single (Khirug et al., 2010)– seizures(Woo et al., 2002) (Nardou, Yamamoto, Chazal, et al., 2011) (Lee et al., 2011; 2007) and partial knock out of KCC2 also reduces the threshold of seizures (Khalilov et al., 2011) also see (Huberfeld et al., 2007) {Pellegrino:2011tp}. We used the triple chamber that we had developed to investigate these issues (Khazipov et al., 1999) (Khalilov, Esclapez, et al., 1997) (Fig11). This chamber composed of the two intact neonatal hippocampi interconnected with the commissures that are placed in 3 independent chambers has several unique advantages versus other in vitro preparations. Indeed, here it is possible to perfuse the 2 hippocampi with different liquids enabling to generate seizures with a convulsive agent and check the consequences of the recurrent seizures on the other hippocampi that has not received the convulsive agent. It is also the only in vitro preparation in which the 2 hippocampi can be reversibly disconnected after a predetermined number of propagated seizures in order to determine the consequences of a defined number of seizures on the electrical properties of the naive hemisphere. Using this preparation, Khalilov and colleagues showed that after a single seizure, the contralateral neurons remain free of spontaneous seizures after the disconnection (Khalilov et al., 2005; 2003; Nardou, Yamamoto, Chazal, et al., 2011). In contrast, the propagation of many recurrent seizures from the stimulated to the naïve hippocampus transforms it to an epileptogenic structure that generates seizures after being disconnected from the kainate treated hippocampus: it has become epileptic. This preparation, also allows to investigate in good conditions the alterations required by a network to become spontaneously epileptic. These were found to include elevated [Cl-]i , excitatory GABA and an internalised KCC2 suggesting a return to an immature state. Using our dynamic approach method (see above) to measure the dynamic of [Cl-]I levels regulation, we found that epileptic neurons had difficulties to export chloride as they required several tens of minutes to return to pre-stimulation values in comparison to the tens of seconds of naive age matched neurons (also see (Barmashenko et al., 2011)). Using this preparation, we also determined the minimal requirements that must be met in propagated seizures in order to transform naive neurons to epileptic ones. It turned out that the seizures must contain elevated frequencies –above 40Hz- to produce their effects (Khalilov et al., 2003) (Nardou, Yamamoto, Chazal, et al., 2011) (Khalilov et al., 2005). Interestingly, GABAergic signals are essential for the generation of elevated frequencies and the formation by seizures of an epileptogenic mirror focus. This was directly shown by applications of GABAzine or bicuculline to the naïve hippocampus that prevented both the occurrence of high frequencies and the transformation of the naive hippocampus to an epileptic one. Therefore, the hyperactivity produced by blocking GABA signals is in fact not epileptogenic because GABAergic currents are essential for the generation of high frequency events that are indispensable for the longterm effects of seizures. b) Bumetanide, chloride co-transporters and epilepsies: a complex story If GABA excites epileptic neurons, reducing [Cl-]i levels to restore hyperpolarising actions of GABA seemed a reasonable antiepileptic strategy. Activating KCC2 or reducing NKCC1 to respectively enhance its removal or reduce its import can achieve this. Selective KCC2 agonists were not available then (Gagnon et al., 2013) and the

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inactivation and internalization of KCC2 by hyperactivity suggested that this approach might not be efficient. We shifted to investigate the antiepileptic properties of the highly selective NKCC1 antagonist bumetanide (Feit, 1981). This drug has been used for several decades to treat oedema and hypertension with limited side effects –essentially a diuresis and hypokalemia. In vitro studies confirmed its powerful actions in restoring strong hyperpolarising and inhibitory actions. The effects of bumetanide in different animal models yielded contradictory results. In some, bumetanide reduced acute and chronic seizures in others it failed (Dzhala et al., 2005) (Kilb et al., 2007). Using the triple chamber, we decided to compare its actions on the transformation by propagated seizures of a naive network to an epileptic one and on a mirror focus (Nardou et al., 2009; Nardou, Yamamoto, Chazal, et al., 2011). Applying bumetanide to one hippocampus and kainate to the other did not prevent the generation of kainateinduced seizures, their propagation to the contralateral hippocampus, and the formation of an epileptogenic mirror focus. However, bumetanide reduced DF GABA, the excitatory action of GABA in epileptic neurons and blocked spontaneous epileptiform activity in the mirror focus. Therefore, although bumetanide does not prevent formation of the epileptogenic mirror focus suggesting that in addition to NKCC1, other mechanisms contribute to increase DF GABA in epileptic neurons. Koyama and colleagues showed an interesting illustration of the developmental links between the actions of GABA and epilepsies (Tao et al., 2012). Using a model of febrile seizures to elicit temporal lobe epilepsies later in life, they showed that the aberrant migration of neonatal-generated granule cells persists into adulthood. Bumetanide like RNAi-mediated knockdown of NKCC1 prevented this aberrant migration, rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, febrile seizures produce persistent immature GABAergic signals associated with an architectural signature of pathological conditions with enhanced NKCC1 acting as a triggering factor. This investigation is also in keeping with the depolarising actions of GABA on newly born granule cells again emphasizing the importance of the excitatory /inhibitory shift during development including in an adult environment. c) Phenobarbital and Diazepam to treat epilepsies: another twist If GABA excites epileptic neurons, molecules acting on GABA might produce paradoxical effects on epileptic neurons. Phenobarbital (PB) and Diazepam (DZP) are extensively used as first and second line drugs to treat acute seizures in neonates and their actions are thought to be mediated by increasing the actions of GABAergic signals. PB and DZP have been used for decades to treat epilepsies with positive effects in some types of infantile epilepsies and a failure even paradoxical aggravating actions in others types of epilepsies. This heterogeneity of actions might be due to the plasticity of the polarity of GABA that has been reported by many teams(Balena and Woodin, 2008) (Balena et al., 2010) (Khirug et al., 2010) (Blaesse et al., 2009; Huberfeld et al., 2007). Other studies have however suggested an enhanced efficacy of GABA mimetics when combined with Bumetanide (Dzhala et al., 2005) (Brandt et al., 2010). We used the triple chamber to test the role of excitatory GABA following single or repeated seizures (Nardou et al., 2009). We found that genetic invalidation of NKCC1 (NKCC1 Kos) did not prevent the formation by seizures of an epileptogenic mirror focus suggesting that NKCC1 is not required for the epileptogenic process (see preceding chapter). Also, when applied to one hippocampus from the start, phenobarbital blocked the seizure, prevented the

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formation by seizures of an epileptogenic mirror focus and the GABA polarity shift. In contrast, when applied after several propagated seizures, phenobarbital aggravated the epileptiform activity (Figure 11 A-C). Therefore, the actions of phenobarbital are conditioned by the history of seizures prior to its first application raising important conceptual and clinical issues. To get better insights in these actions, we repeated these experiments with DZP and compared them to PB (Nardou, Yamamoto, Bhar, et al., 2011). Using the threecompartment chamber, kainate was applied to one hippocampus and DZP (or PB) to the contralateral one after the formation by propagated interictal and ictal activities of an epileptogenic mirror focus. We found that in contrast to PB, DZP aggravated propagating seizures from the start, and failed to prevent the formation by propagated seizures of a mirror focus. PB reduced and DZP increased the network driven GDPs suggesting that they might exert other different actions. In keeping with this, PB but not DZP reduced field potentials generated by AMPA/kainate receptor mediated EPSCs, in the presence of GABA and NMDA receptor antagonists. The following observations suggest that PB exerts direct actions on AMPA/kainate receptors: (i) a reduction of AMPA/kainate receptor mediated currents generated by focal applications of glutamate; (ii) a reduction of the amplitude and the frequency of AMPA but not NMDA receptor mediated miniature excitatory postsynaptic currents; (iii) an increase of the number of AMPA receptor mediated EPSCs failures evoked by minimal stimulation. These effects persisted in mirror foci. Therefore, the enhanced efficacy of PB versus DZP is due to a reduction by AMPA/kainate receptors mediated EPSCs in addition to the proGABA effects. These additional actions might confer an advantage of PB over DZP in the treatment of neonatal seizures. Relying on the initial success of bumetanide in experimental conditions, a european clinical trial was initiated on 2 days old babies with encephalopathic seizures that are refractory to phenobarbital (www.nemo.eu.). This trial was stopped because of the poor effects of bumetanide and important side effects of the antibiotic treatment on the auditory system. The development of GABA (and glycine) has been investigated at depth (Milenković and Rübsamen, 2011) (Witte et al., 2014)and suggest that the immature hearing system is vulnerable to bumetanide because of the roles of NKCC1 in the endolymph at an early stage (also see www.nemo-eu). In addition, it is possible that the antibiotic regimen contributes to these side effects and/or the fact that babies were enrolled only once phenobarbital was found to have no effects indicating a possible down regulation of KCC2. If so, then the usefulness of bumetanide might be limited to older babies/children and/or early usage of the diuretic before the recurrent seizures have down regulated KCC2. d) Bumetanide : a plethora of beneficial actions but … The story did not end here. If GABA excites epileptic neurons, then a similar situation might also occur in other pathological situations. Studies using a plethora of animal models of other pathologies have confirmed a similar sequence (Boulenguez et al., 2010) (Huberfeld et al., 2007; Khirug et al., 2010; Löscher et al., 2012; Reid et al., 2013). The use of bumetanide to treat all these disorders is however challenged by complications and side effects that might occur in specific conditions including menopause women

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where long lasting treatment with bumetanide increase the renal calcium excretion and alters the diurnal rhythm of plasma Parathyroid hormone thereby affecting bone formation (Rejnmark et al., 2006). Also, continuous administration of the diuretic to the mother and pups from E18 to postnatal periods exerts pathological actions including sensory deficits and schizophrenic types behaviours stressing the importance of the depolarising actions of GABA in utero and early post-natal life and its trophic role (Wang and Kriegstein, 2011). As stressed elsewhere {BenAri:2011kv}, the use of bumetanide during pregnancy is only justified in pathological conditions when chloride levels might be elevated and then restoring normal levels might be useful. e) Epilepsies and the heterogeneity of pyramidal neurons A basic assumption of studies on pyramidal neurons of the hippocampus –say of the CA3 region- is that they are homogeneous. Yet, these neurons have different birth dates and might belong to different stem cells. Studies on epilepsies provided some indications that this is indeed the case. In adult hippocampal slices, blocking GABAergic signals generates efficiently seizures. Furthermore, in a classical experiment, Miles and Wong (Miles and Wong, 1983) also see (Wittner and Miles, 2007) (Miles et al., 1988)showed that stimulation of a single CA3 pyramidal neuron in the bicuculline treated slice triggers all or none synchronised paroxysmal events an effect mediated by the wide range of recurrent glutamatergic excitatory collaterals. Yet, curiously, a similar protocol failed to do so in an immature slice, although seizures were generated. Using the two photon microscope and targeted patch clamp recording of identified neurons, the apparent dilemma was resolved by uncovering a sub-population of early-generated glutamatergic neurons that impacts network dynamics when stimulated in the juvenile hippocampus (Marissal et al., 2012). This population displayed characteristic morphophysiological features in the juvenile and adult hippocampus. This study illustrates the heterogeneity of apparently homogeneous glutamatergic neurons rooted in their different temporal embryonic origins. In addition, although functional hub neurons are exclusively GABAergic as far as GDPs are concerned, some early born glutamatergic pyramidal neurons are capable of contributing to the generation of synchronised events in the absence of GABAergic signals. Therefore, from a developmental viewpoint, the heterogeneity of neuronal populations must be taken in consideration in epilepsies and most likely also other pathogenic conditions. VII. Autism: an incredible & unexpected confirmation of the sequence and its potentials in treatment of autism Things often happen when unexpected and not even desired. I was about to retire and was giving lectures and wide audience talks on my research. Giving a talk to the national meeting of association of parents of autistic children, I described the developmental sequence, the role of GABA/intracellular chloride and why in various pathological conditions, intracellular chloride is elevated, GABA excites and BZ can produce paradoxical reactions. The audience was highly interested but one of the doctors treating autistic kids in Brittany –Dr E Lemonnier- drew my attention to the fact that autistic children have often-paradoxical reactions to Valium and other GABA acting agents. Dr Lemonnier asked me if I would agree to help him making first a small pilot study using bumetanide to block selectively the chloride importer NKCC1. I agreed and he had the ingenious luck to find the appropriate doses of bumetanide to use. He obtained rapidly the authorisation from the ethical committee considering that this drug

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has been used for almost 4 decades including in babies with little or no side effects. He selected children and adolescents (3-11yrs old) with autism of various severities since we could not select a particular type of autism as we might loose important information. The results of this open pilot study revealed an amelioration of the severity (Lemonnier and Ben-Ari, 2010). This led to a double blind randomised investigation on 54 children with autism with a regimen of 3 months bumetanide (1.5mg daily) or placebo followed by wash out. Again the results were significant with the conventional evaluation tools but also by the parents stressing that the children are more « present » (Lemonnier et al., 2012). The side effects were restricted to the usual diuresis associated in a minority of children with hypokalemia treated readily with K+ gluconate syrup. Publishing this result was no simple matter with the usual suspects refusing to admit or even consider that a simple diuretic might work when all the sophisticated genetic driven therapeutic trials had failed or were less promising. The paper was finally accepted by Translational Psychiatry, receiving very little attention from the media, but not from parents as many –over 80 – continued to rely on this treatment some for over 3 years by now. This also led to the usual avenue when trying to develop a new drug, including search of investors, patents, start up etc. A financial investment (an investment company linked to the Simons foundation) provided us with the opportunity to initiate a large European approved multi centric randomised trial (80 children 2 to 18 years old) that is now under way to be finalised during the spring of 2015. In the meantime, pilot examinations suggested that the treatment might be useful in Fragile X (Lemonnier et al., 2013). Parallel investigations showed that in 8 Asperger adolescents, long-term bumetanide treatment ameliorated visual communication and recognition of emotive figures (hadjikhani etal). This also enhanced the activation of brain regions involved in social and emotional perception during the perception of emotional faces. These observations converge to suggest that bumetanide might have beneficial effects in the treatment of autism. Yet, whether indeed (Cl-) i levels are high in cortical neurons of patients with autism remained an open question particularly as bumetanide binds to Albumin and has a poor blood brain barrier permeability. Clearly, experimental investigations were needed to test these issues in animal models of autism. We used the Valproate in utero rat {Kuwagata:2009ko}{Williams:2001cu}{Williams:2011hz} and the Fragile x mice models, the latter being the most frequent genetic disorder associated with autism traits and a frequently used model to test therapeutic approaches to autism {Hagerman:2002tl} {Padmashri:2013ga}{Zoghbi:2012jq}{Bear:2004ht}. We first observed with single GABA channel recordings, elevated (Cl-) i levels & a depolarised GABA that with both cell attached and field recordings was converted to excitatory actions of GABA. These effects were blocked by bumetanide. But this was not all. We then investigated the entire GABA developmental sequence and found to our surprise that it was abolished already from the embryonic –delivery shift stage: neurons have the same DF GABA in embryonic and post-natal neurons (Fig 12 H). Bumetanide like the hormone oxytocin restored the correct hyperpolarised driving force of GABA. Therefore, at least hippocampal neurons “behave” as if they remained immature with bumetanide and oxytocin sensitive high (Cl-) i levels and excitatory GABA.

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Perhaps a more astonishing observation stemmed when we started to correct the (Cl-) i shift only during delivery. We asked the following question: might the oxytocin mediated delivery shift be a signal that resets the clock leading to long lasting consequences if it fails. This turned out to be instrumental. Administration of bumetanide in drinking water to the mother during a period of 24 hrs -shortly before and after delivery- restored the complete developmental sequence: DF GABA followed the naïve sequence not only during delivery but also several weeks later (Fig 12 A-F and 13 A &D). This treatment also restored GABA inhibitory actions at birth (Fig 13 G-H) and several weeks later (Fig 13 B-C &E-F). Other electrical signatures of networks in animal models of autism including enhanced glutamatergic activity and in vivo increase of brain oscillations in the gamma and higher band frequency were also attenuated (Fig 13). The treatment also ameliorated behavioural signatures of autism including vocalisation – produced in pups following separation from the mother- but also sociability and grooming tests (Tyzio et al., 2014). In parallel, we observed that the administration of an antagonist of oxytocin receptor to naïve pregnant rats during the same restricted period produced “autistic features” including depolarising and excitatory GABA and autistic behaviours. Therefore, the polarity of GABA during delivery exerts long term effects on brain and behaviour and these actions involve oxytocin signals stressing the links between GABA/oxytocin and delivery. This observation raises formidable questions that are pertinent to major public health issues. As ASD is “born” in utero (Courchesne et al., 2011; 2007), how can a manipulation during delivery correct the programmed sequels? Are the genetic effects of Fragile X also reversible by bumetanide? This issue also applies to programmed Csections, pre-term delivery and complications during delivery that are associated with a higher incidence of ASD. They also raise the fundamental conceptual issue of how in utero insults can led to protracted brain disorders and possibly an attenuation of their deficits by processes at work during and shortly after delivery. Is it possible that delivery exerts a priming effect on brain development? These issues are neither restricted to ASD nor to GABA. Since many ionic currents follow developmental sequences, it might reasonably be expected that the interruption of other sequences leads to other life long deleterious sequels that can be reduced by selective antagonists. In a more general perspective, this is another example of the persistence of “immature “ currents in neurons that have not adequately performed their assigned programs (see below). VIII. Linking genes and environmental events: the Checkpoint and Neuroarcheology concepts Initially centered on GABA developmental sequences, our investigations revealed a bigger picture that bears relevance to the links between genes and environment and to the role of activity in brain maturation. Indeed, it is clear that the developing brain is highly active, immature neurons generating ionic currents patterns that differ from adults. Stated differently, the developing brain is very talkative but its language differs from the adult’s. The brain requires enhanced activity to construct its maps and functional units and these specific requirements must timely shift to enable the generation of the behaviourally relevant oscillations. These developmental sequences provide the type of activity needed at various developmental stages and enable neurons at very different developmental stages to fire and wire together. Contrary to the

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construction of an engine (a car or a plane that are “silent until the end”), the brain is active during its construction and the activity it generates is not that which will be used once finished. The checkpoint concept (Ben-Ari and Spitzer, 2010)suggests that genes and activity operates in series and as in all biological operations; a feedback control is needed and activity does that job (Fig 14). CNS development requires intermediate stages of differentiation that provides information needed for gene expression. In this model, embryonic neuronal functions constitute a series of phenotypic signatures. In keeping with this, extensive investigations have shown that procedures and agents that perturb on going activities during maturation produce long-term effects. We have suggested that the expression of different embryonic features at different developmental stages provide that control. This has been observed at various essential developmental steps with a proliferation checkpoint, a migration checkpoint, axon guidance checkpoint and neurotransmitter specification checkpoint. An illustration is provided by the nice experiments of Spitzer and colleagues showing that immature neurons generate specific patterns of calcium currents and when these are disturbed they can modify the neurons phenotype (GABAergic instead of glutamatergic) (Borodinsky et al., 2004) (Spitzer and Borodinsky, 2008). The GABA/chloride checkpoint is yet another important event of a long chain of informative alterations that signal the developmental stage of a neuron and whether it has successfully implemented its maturation program. This is by no means restricted to GABA and chloride, parallel developmental sequences have been reported in a wide range of systems with similar alterations of ionic currents –from long lasting to shorter kinetics- and shifts of network driven patterns- such as in sensory systems (Ford et al., 2012; Milenković and Rübsamen, 2011; Mooney et al., 1996; Torborg and Feller, 2005; Xu et al., 2011; Zhang et al., 2010; Zhou and Zhao, 2000). What if things go wrong? From an architectural standpoint, errors in the construction of a building can be unraveled years later and the mechanisms underlying the damage uncovered years later by analysing the elements that have led to the collapse. The damage –the brain disorder- can be manifested long after the initial insult has taken place depending on the “use” of brain networks and a second hit/insult. Yet, it is likely that the initial insult leaves a trace of the generating event in time and space. I have suggested that neurons that fail to perform their assigned targets and are either misplaced or misconnected remain “frozen” in an immature state that corresponds to the stage at which the developmental sequences was interrupted/modified. In this neuro-archeology concept (Ben-Ari, 2008), alterations of developmental sequences lead to a persistent electrical or architectural signature of the timing of the failure. In this perspective, developmental disorders are due –at least in part- to the persistent expression of immature currents and oscillations in the adult brain that perturb the operation of well-developed functional networks. This concept has been confirmed in many migration disorders including Double cortex, heterotopic nodules, SRPX2 mutations, where misplaced neurons keep immature features thereby perturbing normo-functioning oscillations (Ackman et al., 2009; Falace et al., 2014; Salmi et al., 2013). This concept suggests that the use of specific antagonists /blockers of immature currents in an adult brain might be a useful strategy to block the perturbing activity without altering adjacent networks that have performed adequately their programs. In this model, the persistence of immature currents and aberrant activities and networks is the ultimate cause of disorder. Gene therapy –replacing the mutation by the correct

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gene- is unlikely to cure as it will not correct the aberrant activities generated by misplaced and misconnected neurons and produce in the crowded adult brain migration and reconnection with the correct tragets. Thus, introduction of the Double Cortin gene in neurons that failed to migrate following the invalidation of that gene, partly improved the defaulted migration during the first few days post natal but not later (Manent et al., 2009). Therefore, to understand and treat brain disorders, it is essential to determine how brain patterns are deviated by the mutation and environmental insult. This coupled with early diagnosis and the use of selective drugs that block the perturbing activities might provide novel therapeutic avenues. Conclusions One conclusion of this journey is that studies initiated to check how neuronal activity develops at early developmental stages unraveled a plethora of issues, observations, and novel queries and concepts. They have also led to possible innovative therapeutics to treat disorders that are at present orphan of treatments. As most if not all-ionic currents follow developmental sequences, our observations with GABAergic signals might have major implications. It is possible that antagonists of other immature voltage gated and or transmitter mediated currents will tomorrow constitute a common strategy to block immature perturbing currents without altering the normally developed adult currents. Studies on voltage gated ionic currents notably calcium currents will be useful to test this hypothesis. Therefore, to understand and treat developmental disorders, it is instrumental to understand developmental sequences and how they are deviated by genetic mutations of environmental insults. The translation of newly identified mutations to therapeutic agents is conditioned by a better understanding of the cascade of events that this mutation induces. In sum, studies on developmental disorders must embrace a genuine developmental perspective. A second conclusion that remains to be better understood is the functional advantage of this complex developmental Excitatory /Inhibitory developmental sequence. Why immature neurons need elevated intracellular chloride? One possibility is that this is an evolutionary conserved feature aimed at equilibrating a smaller sum of intracellular negative charges in developing systems. But this speculative hypothesis will have to explain why many peripheral neurons have in adulthood low levels of intracellular chloride. Finally, the fundamental issue of delivery and its impact on the developmental sequence of ionic currents remains to be better understood. This is by itself a major topic considering the complicated events occurring during delivery that have been ignored until recently as far as the impact they have on neuronal and network activities. Indeed, delivery is associated with a major stress reaction associated with elevated cathecholamine (Lagercrantz and Bistoletti, 1977) (Faxelius et al., 1983) (Greenough et al., 1987) (Hillman et al., 2012) and cortisol levels that are not met in adults even under conditions of severe stress. These are meant to enable delivery to perform its major functions including the acquisition of immunological and microbiot features –acquired during delivery – but also the shift to aerobic oxygenation. Interestingly, GABA accelerates lung maturation and promote chloride extrusion in lungs (Chintagari et al., 2010). Yet, elevated catecholamine augment intracellular chloride and produce an inhibitory to excitatory shift of the actions of GABA (Inoue et al., 2013) . A speculative but highly interesting possibility is that oxytocin and other hormones compensate the

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possible deleterious effects of catecholamine on central neurons during delivery. In this perspective, evolution has enabled both to produce stress needed for peripheral essential functions but at the same time provide the clues to avoid the deleterious actions of this stress on neurons. Whatever the correct explanation, we cannot omit from our agenda a better understanding of the alterations during delivery and their impacts. Acknowledgements I am indebted to my many student and colleagues who have worked on these programs. Financial support of INSERM – and various public agencies (ANR, EU, OSEO)- for financial support.

Figures Legends Figure 1 GDPs are polysynaptic events generated by GABA and glutamate currents. A: Spontaneous GDPs are generated by a polysynaptic circuit in which GABAergic signals play a central role. Note the spontaneous GDPs that occur at a stable frequency. Application of the GABA a receptor antagonist –bicuculline- blocked on going activity whereas a similar application in adults (traces below) generated high frequency recurrent interictal activity –from (Ben-Ari et al., 1989). B: in the continuous presence of ionotropic glutamate receptor antagonists (CNQX and APV), focal applications of the GABA a receptor agonist –Isoguvacine 10uM- generated a current that reversed at -40mV (B). C&D: Isoguvacine generated recurrent action potentials attesting to the excitatory actions of GABA that were reduced but not blocked by AMPA and NMDA receptor antagonists but fully blocked by the addition of the GABA receptor antagonist bicuculline. Figure 2 GABAergic PSCs develop before glutamatergic ones in rodent and primates. A: in rats, hippocampal CA1 pyramidal neurons were recorded at P0, their PSCs identified and then they were filled with biocytine and reconstructed post hoc. Note that the neurons can be subdivided in 3 groups: i) neurons with axons extending in stratum oriens (SO) but with almost no apical dendrites in the pyramidal (py), Stratum Radiatum (SR) or lacunosum Moleculare (lm), these neurons are silent with no PSCs at all; ii) neurons with also an apical dendrite that however is restricted to stratum radiatum (SR), these have only GABAergic PSCs, and iii) neurons (far right) that have both basal dendrites and long apical dendrites reaching the lacunosum moleculare (lm) , these have GABA and glutamate PSCS. B: similar observations in embryonic macaques. CA1 or CA3 pyramidal neurons were recorded at various embryonic ages showing a similar distribution, neurons at E85 are silent at E105 have only GABAergic PScs and at E154 have GABA and glutamate PSCs. From (Khazipov et al., 2001).

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Figure 3 GDPs are generated by the synchronized activities of interneurons and pyramidal neurons. Upper part: A : GDPs in SR-CA3 interneurons are synchronous with GDPs in CA3 pyramidal cells : 
A, dual recordings of CA3 pyramidal neuron in whole-cell mode (upper trace)and SR-CA3 interneuron in cell-attached mode (lower trace). Note that bursts of action potentials in the interneuron are synchronous with GDPs in the pyramidal cell. B, dual whole-cell recordings of CA3 pyramidal cell and SR-CA3 interneuron. Note synchronous generation of GDPs in simultaneously recorded cells. C : latency between onset of GDPs in pyramidal cells and simultaneously recorded interneurons. Each point represents one pair:SR-CA3 interneuron-CA3 pyramidalcell(n= 11 pairs) . Recordings with potassium gluconate pipette solution(1). In whole-cell recordings cells were kept at-80mV;cell-attached recordings. Reproduced from (Khazipov et al., 1997). D : Similar observations in primates. GDPs synchronize most of the macaque hippocampal neuronal activity in utero. A, Pair recordings of CA3 pyramidal cells and interneurons (E109). Pair 1, The pyramidal cell (top trace) is recorded in the whole-cell mode at the reversal potential of glutamatergic PSCs (0 mV), and the GABA(A) PSCs are outwardly directed; the hilar interneuron (bottom trace) is recorded in the cell-attached mode. Each GABA PSC detected in the pyramidal cell is shown as a bar below. Note the periodic oscillations (GDPs) synchronously generated in both neurons and associated with an increase of the GABA(A) PSC frequency in the pyramidal cell and bursts of action potentials in the interneuron. The pyramidal cell (top trace) is recorded in whole-cell mode at 0 mV, and the interneuron (bottom trace) is recorded in whole-cell mode at the reversal potential of the GABA(A) PSCs (-70 mV) so that the AMPA PSCs are inwardly directed. Each AMPA PSC detected in the interneuron is shown as a bar below. E : GDPs outlined by dashed boxes on A are shown on expanded time scale. Note an increase in the frequency of the GABA(A) and AMPA PSCs during GDPs. On the right, the distribution of GABA and AMPA PSCs is cross-correlated with the population discharge (bin size, 100 msec). Insets, Averaged GABA and AMPA PSCs. From (Khazipov et al., 2001). Figure 4 Schematic diagram of the developmental alterations of [Cl-] i levels and the polarity of the actions of GABA and the actions of chloride co-transporters. A: the intracellular [Cl- ] i levels are higher in immature than adult neurons. B: GABA depolarises and excites immature neurons and inhibits adult ones. The chloride exporter KCC2 I chloride importer is poorly active initially whereas the NKCC1 is highly active in immature neurons leading to different chloride gradients and actions of GABA. Also see (Ben-Ari et al., 2007). Figure 5 Schematic diagram of the network that generates GDPs. A : whole cell and field recording of GDPs. B : a schematic local netwrok composed of pyramidal neurons (triangles) and interneurons (circles). GABAergic depolarisation in interneurons (see chloride export) and in interneurons is sufficient to generate GDPs. See text.

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Figure 6 Schematic diagram of the maturation of the principal markers that have been measured in rodents and primates. A : GDPs are present after birth and until P10 before shifting to adult patterns, developmental synaptogenesis occurs from E20 to P30 and neurogenesis (in CA3/CA1) terminates before birth, and the peak of dendritic growth and spinogenesis are shown. B: Similar results in primate (macaque neurons in utero). Boltzmann equations to depict the rate of neurogenesis, rate of growth, first synaptic currents, spines acquisition, and network activities. Note that GDPs are present from mid gestation until shortly before delivery. (adapted from {Khazipov:2001wa}. Fig 7 A triphasic sequence of maturation of patterns in the rodent hippocampus. Using two photons dynamic imaging system, we recorded the activity of hundreds of neurons in a single sweep and quantified the activity and synchronicity in hippocampal slices at various developmental stages. In embryos, neurons only generate intrinsic calcium currents that do not propagate, after delivery, neurons generate Synchronized Plateaux potentials in small Assemblies (SPAS). These are intrinsic non synapse driven plateaux potentials ; Later these are replaced by the first synapse driven synaptic events the Giant Depolarising Potentials (GDPs). Inset :The developmental time course of these events. Note that the calcium spikes (or currents) that are the only pattern in utero, the Spas appear around birth and disappear shortly later and the GDPs appear a few day post delivery and peak at P6-10. From (Crépel et al., 2007). Figure 8 Delivery is associated with an abrupt reduction of [Cl-] i levels . A : Delivery related transient perinatal loss of the GABAA mediated excitation. (A) Responses of CA3 pyramidal cells recorded in cell-attached mode to the GABAA agonist isoguvacine. Below, summary plot of the proportion of cells excited by isoguvacine during the perinatal period. There is a transient loss of the excitatory effect of isoguvacine near term. Red corresponds to the fetuses whose mothers received SSR126768A. [E21 corresponds to the early phase of delivery (1 to 2 hours before birth); P0 is the day of birth; pooled data from 146 neurons.] B: Summary plot of the onset of Anoxic Depolarisation (AD) in control, in the presence of the oxytocin receptors antagonists atosiban (AT, 5 mM, fetal intracardial perfusion and SSR126768A (1 mg/kg to the mother), and after further addition of bumetanide (10 mM). Each circle corresponds to one hippocampus (n = 82 intact hippocampi at E21). Error bars indicate SEM. Note that the ADs occured earlier when oxytocin receptors are blocked and control levels are obtained when bumetanide is added . From (Tyzio et al., 2006) Figure 9 GABA potentiates the activity of NMDA receptors in the neonatal hippocampus. A: synaptically elicited responses in neonatal CA3 pyramidal neurons (P5) recorded in cell-attached configuration. a: In control conditions, electrical stimulation elicited a burst of five action potentials. The number of action potentials was slightly affected by AMPA receptor antagonist CNQX (10 uM) (b) and strongly reduced by further addition of the NMDA receptor antagonist APV (50 uM) (c). d: The remaining response was

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blocked by the GABAA receptor antagonist bicuculline (10 uM). B: a CA3 pyramidal neuron (P5) was loaded extracellularly with the Ca2+-sensitive dye fluo-3 AM, and the slice was continuously superfused with the voltage-gated Ca2+ channel blocker D600 (50 uM). Focal pressure ejection of a GABAA-receptor agonist, isoguvacine (100 uM), or bath application NMDA (10 uM) had no effect on [Ca2+]i fluorescence. However, a combined activation of GABAA and NMDA receptors resulted in a significant increase of [Ca2+]i fluorescence. C: Schematic presentation of the interactions between GABAA and NMDA receptors in immature neurons. From (Ben-Ari et al., 2007). Figure 10 Parallel development of the intrinsic and synaptic properties of medium spiny neurons (MSNs) of the striatum and pup motricity. A: the developmental time course of various parameters including calcium spikes, calcium plateaux, global events (GDPs like), morphology of MSNs, GABAergic, glutamatergic and intrinsic currents are depicted. Immature (light orange) and adultlike (grey) phases are separated by a transitory immature period (orange). B : pups were filmed and their contact with a glass floor quantified. Note that pups have primarily belly contacts with the basement at P2 , then they rise on their paws around p6 and exhibit targeted movements starting from P10. From (Dehorter, Michel, Marissal, Rotrou, Matrot, Lopez, Humphries, and Hammond, 2011b) (Ferrari et al., 2012) (Dehorter et al., 2012). Figure 11 The efficacy of Phenobarbital effects on seizures depends on the number of seizures that have occured before the first application. A: the triple chamber with the two intact interconnected hippocampi and the connecting commissures placed in 3 independent chambers ; this allows to apply a convulsive agent to one chamber and the anticonvulsive one in the other and detrmine the effects of the propagated epileptic activity on the naive contralateral side. B: Late application of phenobarbital fails to block the ictal-like event and gammaoscillations. Kainate (KA) was applied repeatedly (every 20 min) X15 to one hippocampus (ipsilateral = ipsi-) and artificial cerebrospinal fluid (ACSF) to the contralateral naıve hippocampus (contra-). Whereas phenobarbital (PB) applied initially to the contralateral side blocked the propagated seizures (not shown), a similar application after the 15th application of kainate to the ipsilateral side failed to block propagating ictal-like events. C : Phenobarbital enhances GABA excitation in mirror focus neurons. A mirror focus was generated by in ine hippocampus by recurrent applications of kainate to the other hippocampus . Cell attached recordings from mirror focus neurons to illustrate the increased number of action potentials generated by focal application of GABA in the presence of phenobarbital (PB) . Below : Superimposed traces from perforated patch clamp recordings of post-synaptic currents generated by focal application of GABA (every 20 s, arrowheads) in the presence of CNQX and APV before

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(black), during (red) application of phenobarbital and after wash out (blue). From (Nardou, Yamamoto, Chazal, et al., 2011) . Figure 12 Developmental excitatory-inhibitory GABA sequence is abolished in hippocampal CA3 pyramidal neurons in VPA rats and FRX mice. (A) developmental seqeucne of DF GABA changes in control and VPA rats. (B) Currentvoltage (I-V) relations of GABAAR single-channel currents at P0 in VPA rats in artificial cerebrospinal fluid (red) or bumetanide (10 mM, blue). (Inset) Single-channel openings at different holding potentials (scale bars mean 1 pA and 200 ms). (C) Bumetanide and oxytocin shifted DFGABA from depolarizing to hyperpolarizing at P0 (control rats, black; VPA, red; bumetanide application, blue; and oxytocin application, purple). (D to F) The same as in (A) to (C) for wild-type (WT, black) and FRX mice (red). (G to J) Excitatory action of the GABA receptor agonist isoguvacine (10 mM, black bars) on spontaneous spiking recorded in cell-attached configuration in VPA and FRX. (G) Control and VPA rats and (I) wild-type and FRX mice at P0 with and without isoguvacine. Time-course of spike frequency changes is shown under each trace. (H) Average values of normalized to control spike frequency for P0 and P15 control (gray) and VPA (red) rats and effect of isoguvacine application (hatched bars). (J) The same as in (H) but for P0 and P15 mice wild-type (gray) and FRX (red). Data are presented as means T SEM. *P < 0.05; **P < 0.01; ***P < 0.001. From (Tyzio et al., 2014) Figure 13 Maternal pretreatment with bumetanide before delivery switches the action of GABA from excitatory to inhibitory in offspring in VPA and FRX rodents at P15. (A) Average values of DF GABA measured in hippocampal CA3 pyramidal neurons at P15 in control (black), VPA (red), and VPA rats pretreated with bumetanide (blue). Note that pretreatment with bumetanide shifts DF GABA from depolarizing to almost isoelectric level. (B) Effects of isoguvacine (10 mM; black bars) in rats: Representative traces of spontaneous extracellular field potentials recorded in hippocampal slices at P15 in control, VPA, and VPA rats pretreated with bumetanide (BUM) shortly before and during the delivery period. Corresponding time courses of spike frequency changes are shown under each trace. (C) Average histograms of normalized spike frequency in rats. Isoguvacine (hatched bars) decreased the spikes frequency in control rats (to 38.9 T 5.1%; gray); increased it in VPA rats (to 213.5 T 16.3%; red); and decreased it in VPA rats pretreated with bumetanide (to 82.8 T 10.7%; blue). (D) The same as in (A) for mice. Wild-type mice (WT, black), FRX mice (red), and FRX mice pretreated with bumetanide (blue). (E) The same as in (B) for FRX mice. (F) : The same as in (C) for FRX mice. Wild-type mice (decreased to 67.9 T 6.1%; gray); FRX mice (increased to 165.8 T 13.5%; red); FRX mice pretreated with bumetanide (decreased to 80.8 T 8.2%; blue). Data are presented as means SEM. **P < 0.01; ***P < 0.001. Reproduced from (Tyzio et al., 2014) Figure 14 Genes and activity in brain development. The various essential steps of the construction of a functional brain are shown. It is suggested that many if not most of these steps are also “activity dependent”. It is suggested that immature neurons generate various primitive forms of activity that

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provide a substrate for the development of functional units. The checkpoint concept (Ben-Ari and Spitzer, 2010) suggests that genes and activity operate in series not in parallel, therefore it is not easy or even possible to separate one form the other. In addition, an early insult–genetic or environmental – will alter developmental cascades leading to aberrant immature activities in the adult brain that perturb the operation of normo-functioning cell assemblies. In this “neuroarcheology” concept, the identification of the malformed wrongly operating, misplaced or misconnected neurons is crucial in order to develop novel therapeutic strategies based on the use of selective antagonists of immature currents. From (Ben-Ari and Spitzer, 2010) & (Ben-Ari, 2008).

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49

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relative contribution of bicarbonate permeability of 1. GABAFigure A channels producing a delayed depolarization (587). This mechanism does not, however, operate in immature cells because of delayed expression of intracellular carbonic anhydrase (520, 533). 2. GABA triggers sodium action potentials

Depolarizing GABA is also often excitatory in immature neurons, i.e., neurons generate action potentials in response to GABA. This occurs when GABAA-mediated depolarization reaches directly or via voltage-gated conductance the threshold for the generation of action potentials. For example, synaptic activation of GABAA receptors by electrical stimulation in the presence of the glutamate receptors antagonists (CNQX and APV) evoked one or two action potentials in P2–5 CA3 pyramidal cells and interneurons recorded in the cell-attached configuration, and the response was blocked by the GABAA antagonist bicuculline (Fig. 2) (325, 326, 377). Similar excitatory responses can be recorded with gramicidin perfo-

of the GABAergic responses, also increased GDPs frequency, synaptic activity, and MUA in neonatal rats (316). Blockade of GABAA receptors reduces and increases MUA in neonatal and adult rat CA3 hippocampus neurons, respectively (126, 172), probably via suppression of background GABAergic tone (567). Thus GABA clearly excites immature pyramidal cells and interneurons (see Table 1 for excitatory actions of GABA in other brain structures). Interestingly, hyperpolarizing GABA can also act as an excitatory transmitter in adult neurons where it activates excitatory conductance and rebound firing. For example, GABAergic inhibitory postsynaptic potentials (IPSPs) are often followed by a rebound firing of the cells due to activation of the low-threshold calcium and hyperpolarization-activated cationic Ih currents in cerebellar nuclear neurons (6, 390). In thalamic relay cells of ferret dorsal lateral geniculate nucleus in vitro, spindle waves are associated with barrages of inhibitory postsynaptic potentials generated by burst firing of interneurons in the perigeniculate nucleus. These IPSPs result in rebound burst firing in thalamic relay cells triggered by activation

Downloaded from physrev.physiology.org on October 12, 2007

FIG. 1. GABAA-mediated postsynaptic responses in a P2 CA3 pyramidal cell with gramicidin perforated patch. A: responses evoked by puff of the GABAA agonist isoguvacine in voltage-clamp mode at different holding potentials. B: dependence of the peak of the isoguvacine-induced responses on the membrane potential. Note that the responses reverse at "44 mV. C: current-clamp recordings of the same neuron. Brief application of isoguvacine (left) and electrical stimulation (E.S.) of the slice in the presence of the ionotropic glutamate receptors antagonists CNQX and D-APV (right) evoke depolarization and action potentials. The responses are blocked by bicuculline (20 !M, traces below). D: in the presence of the glutamate receptors antagonists CNQX and D-APV, spontaneous GABAA-mediated postsynaptic potentials often result in action potentials. In C and D, the membrane potential is held at "80 mV. [From Tyzio et al. (625).]

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10378 J. Neurosci., December 1, 1999, 19(23):10372–10382

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Khazipov et al. • Activity in Fetal Primate Hippocampus

Figure 6. C amera lucida reconstruction of injected P0 pyramidal cells grouped accordingly to their synaptic properties. The neurons not represented here are shown in Figure 8 A. Silent cells are shown in green, GABA-only in red, and neurons with GABA and NMDA or GABA plus NMDA plus AM PA receptor-mediated PSC s are in black and blue, respectively. Note that each group of cells has a relative homogenous degree of maturation. GABA-active and GABA plus glutamate-active neurons differ essentially by the presence of an apical dendrite in the stratum lacunosum moleculare. Note also that there are no morphological differences between GABA plus NMDA and GABA plus NMDA plus AM PA-active cells. Table 2. Mean capacitance and statistical values of cells T ype

Reconstructed

Silent

A, 18.9 " 1.2 (n # 18)

B, 16.9 " 0.8 (n # 37)

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D, 28.1 " 1.3 (n # 18)

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B

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p # 0.93 p # 0.73

p # 0.98

Mean capacitance of each group of cells reconstructed and not reconstructed classified according to their synaptic properties (top table). The bottom table represents the statistical values when one group of cells was compared to another group. Asterisks indicate that the difference is significantly different ( p % 0.05; ANOVA test).

membrane potential at 40 mV revealed an outward synaptic current that was reduced by bicuculline and C NQX and completely blocked by f urther application of APV (50 !M), reflecting the participation of NMDA receptors in the synaptic response. In one neuron, glutamatergic activity was mediated by NMDA but not by AM PA receptors, i.e., the synaptic response at !70 mV was eliminated by bicuculline, whereas at 40 mV a C NQXinsensitive synaptic response was observed that was f ully blocked by APV (50 !M) (Fig. 4 B,C). The converse, AM PA but not NMDA receptor-mediated PSC s, was not observed. Neurons with GABA and glutamate PSC s had a significantly higher capacitance than the GABA-active cells (46.2 " 4.5 pF; p # 0.006). They also had significantly larger currents generated by AM PA and NMDA because in the presence of bicuculline and TTX, bath application of: (1) AM PA (5 !M, 1.5 min) generated,

at !70 mV, a mean inward current of 309 " 48 pA (n # 5; p # 0.005) with a mean slope conductance of 4.3 " 0.6 nS ( p # 0.004) (Fig. 3C); (2) NMDA (10 !M, 3 min) generated a mean inward current at !35 mV of 38 " 6.3 pA (n # 5; p # 0.009) and with a mean slope conductance measured in the linear part of the curve of 3.3 " 0.4 nS ( p # 0.004) (Fig. 3C). These cells have a higher level of morphological maturation than GABA-active cells because their apical dendrite reached the stratum lacunosum moleculare (Figs. 3A, 4 A; see Figs. 6, 8 A), and they have also basilar dendrites. However, the quantitative comparison of the morphological differences between this group and the neurons with GABA PSC s only is precluded by the limited number of neurons. We therefore used patch recordings under visual control to increase the number of neurons with synaptic currents.

Visual patch recordings confirm the differences between the two populations of active neurons Because blind patch studies suggest that active neurons have larger soma and a more extended apical dendrite, we recorded under visual control pyramidal neurons with these features. All 51 neurons recorded had PSC s, 24 had only GABA, and 27 had GABA and glutamate PSC s, including two neurons in which the synaptic activity was mediated by GABA and NMDA but not AM PA receptors. The nine additional neurons that were recorded under visual control with small soma and no apparent dendrite were, as expected, not active (Figs. 5, 6). Pooling the data from blind and visual patch recordings suggests that neurons with GABA and glutamate PSC s are more mature than neurons with GABA PSC s only (Table 1). This conclusion is supported by the following observations: (1) the apical dendrite of the neurons with GABA and glutamate PSC s reached the stratum lacunosum moleculare and arborized within this layer; in contrast the apical dendrite of neurons with GABA PSC s only were exclusively restricted to the stratum radiatum. (2) The basilar dendrite of neurons with GABA and glutamate PSC s are more developed (length, number of branching points) than neurons with GABA PSC s only, although the number of cells in which these dendrites

Figure 1. Morphological differentiation of pyramidal cells in the cynomolgus monkey hippocampus during the second half of gestation. Reconstruction of biocytin-filled pyramidal cells in CA1 and CA3 hippocampal subfields at various ages in utero (E85–E154). Note an intensive growth of the pyramidal cells that reach a high level of morphological differentiation at E134, 1 month before birth. O, Stratum oriens; P, pyramidal cell layer; R , stratum radiatum; LM, stratum lacunosum moleculare; the dashed lines indicate the limits between layers, and the solid line indicates the hippocampal fissure. Axons ( gray), color of the dendritic arborization indicates the expression of synaptic currents (blue, silent; green, GABA-only; red, GABA " glutamate neurons). Electrophysiological recordings from neurons 1–3 are shown on Figure 3A. Scale bar, 100 !m.

A. Tyzio et al., The Journal of Neuroscience 19(23): 10372-10382, 1999.! “GABA-only” neurons had only GABAergic, but not glutamaFigure 6.

GABAergic and glutamatergic postsynaptic currents (PSC s), pyramidal cells could be divided into three groups: Synaptically “silent” neurons did not have f unctional synaptic inputs because no spontaneous activity was recorded in these cells and electrical stimulations of afferent pathways did not evoke any synaptic currents. Morphometric analysis of these neurons showed that synaptically silent neurons had short and poorly ramified axonal and dendritic processes. The dendrites were nonspiny and restricted to the pyramidal cell layer and the

tergic, synaptic inputs. All evoked and spontaneous PSC s in these neurons had a reversal potential near EC l! and were abolished by the GABA(A) receptor antagonist bicuculline (Fig. 3A). “GABA only” neurons were observed between E85 and E109 and were more differentiated than “silent” ones. They had significantly longer neuronal processes and their apical dendrites (still nonspiny) penetrated deeper into the stratum radiatum (Fig. 4C). Interestingly, at midgestation, GABAergic terminals immunola-

B. Khazipov et al., The Journal of Neuroscience 21(24): 9770-9781, 2001.! Figure 1.

Nature of GDPs in SR-CA3 interneurons Two types of synaptic inputs, GABAergic and glutamatergic, could contribute to the generation of GDPs in SR-CA3 interneurons. The contribution of a GABAA receptoractivated Cl- conductance was estimated from the dependence of the charge-voltage relationships of GDPs on [CF-]1.

neuronal GDPs.

Indeed, after dialysis with a solution containing low [Cl-]1 (4'2 mm, solution 1), when GDPs were recorded at the reversal potential of GABAA postsynaptic currents (PSCs), a clear inward component of the GDPs was revealed (n = 6). Single events that composed this inward component in some cases could be clearly resolved and the kinetics of these

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Figure 2. GDPs in SR-CA3 interneurons are synchronous with GDPs in CA3 pyramidal cells A, dual recordings of CA3 pyramidal cell in whole-cell mode (upper trace) and SR-CA3 interneuron in cellattached mode (lower trace). Note that bursts of action potentials in the interneuron are synchronous with GDPs in the pyramidal cell. B, dual whole-cell recordings of CA3 pyramidal cell and SR-CA3 interneuron. Note synchronous generation of GDPs in simultaneously recorded cells. C, latency between onset of GDPs in pyramidal cells and simultaneously recorded interneurons. Each point represents one pair: SR-CA3 interneuron-CA3 pyramidal cell (n = 11 pairs). Recordings with potassium gluconate pipette solution (1). In whole-cell recordings cells were kept at -80 mV; cell-attached recordings were performed in 'track' mode.

Downloaded from J Physiol (jp.physoc.org) by guest on April 28, 2014

D

Figure 5. Giant depolarizing potentials synchronize most of the hippocampal neuronal activity in utero. A, Pair recordings of CA3 pyramidal cells and interneurons (E109). Pair 1, The pyramidal cell (top trace) is recorded in the whole-cell mode at the reversal potential of glutamatergic PSC s (0 mV), and the GABA(A) PSC s are outwardly directed; the hilar interneuron (bottom trace) is recorded in the cell-attached mode. Each GABA(A) PSC detected in the pyramidal cell is shown as a bar below. Note the periodic oscillations (GDPs) synchronously generated in both neurons and associated with an increase of the GABA(A) PSC frequency in the pyramidal cell and bursts of action potentials in the interneuron. Pair 2, The pyramidal cell (top trace) is recorded in whole-cell mode at 0 mV, and the interneuron (bottom trace) is recorded in whole-cell mode at the reversal potential of the GABA(A) PSC s (!70 mV) so that the AM PA PSC s are inwardly directed. Each AM PA PSC detected in the interneuron is shown as a bar below. B, Lef t, GDPs outlined by dashed boxes on A are shown on expanded time scale. Note an increase in the

A and B. Khazipov et al., Journal of Physiology 498(3): 763-772, 1997.! Figure 2. Figure 6.

Blockade of GABA(A) receptors suppresses GDPs and in-

duces an epileptiform activity. A, Spontaneous GDPs are transformed C and D. Khazipov et al., The Journal of Neuroscience 21(24): 9770-9781, 2001.! into interictal-like epileptiform events after addition of the GABA(A) receptor antagonist bicuculline. Whole-cell recordings of a stratum radiaFigure 5A and B. tum interneuron (E109) in voltage-clamp mode (holding potential, !50 mV). Single GDP (*) and interictal-like events (**) are shown on an expanded time scale below. In current-clamp mode, the interictal-like event generates burst of action potentials (***, resting potential !72 mV).

Figure 4

EXCITATORY GABA IN DEVELOPMENT

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A

B

ing one Na!, one K!, and two Cl" in electroneutral cou- opmental expression of the members of the cation-Cl" pled fashion (499). Cloning and functional expression cotransporter gene family in rat brain (121). Of the instudies have identified two major isoforms of NKCC co- wardly directed cotransporters, NKCC-1, NKCC-2, and transporters that are products of distinct genes. The NCC-1, only NKCC-1 was detected at significant levels in NKCC1 isoform is the so-called secretory isoform best brain. NKCC-1 was expressed in neurons, appearing first characterized in secretory epithelial cells. NKCC1 is ex- in the cortical plate but not in the ventricular or subvenpressed in virtually all mammalian cells and is thought to tricular zone. Expression levels peaked by the third postplay a housekeeping role in cell volume homeostasis and natal week and were maintained in adults. Outwardly Ben-Ari et al., Physiol Rev 87: 1215-1284, 2007.! the common control of cytosolic ion content. NKCC1 directed cotransporters demonstrated a different time Figure 3. ATP but operates using electrochemical course of expression: KCC-1 was expressed prenatally at does not use gradient for Na! and K! produced by Na!-K!-ATPase. low levels that increased slightly over the course of deThere is considerable evidence that uptake of Cl" in velopment; KCC-2 expression appeared around birth and immature neurons is mediated by NKCC1. High expres- increased dramatically after the first week of postnatal sion of NKCC1 in immature neurons plays an important life. Using single-cell PCR, Rivera et al. (521) showed role in maintaining high intracellular Cl" (157, 173, 207, that at birth, KCC2 mRNA was barely detectable; a 383, 440, 499, 506, 528, 596, 651, 671). KCC2 is the principal transporter for Cl" extrusion steep increase in the expression was evident at P5, from neurons. KCC2 extrudes K! and Cl" using the elec- reaching adult level by P15. Interestingly, mature dorsal trochemical gradient for K!. Cl" extrusion is weak in root ganglion neurons that have depolarizing GABA immature neurons and increases with neuronal matura- (160) did not express KCC2. In contrast, KCC2 mRNA tion (330, 405, 678). The KCC2 isoform of KCC cotrans- was present in abundant amounts at embryonic day E42 porters is expressed in mature neurons, thus underlying in the hippocampus of guinea pig, a species with early the developmental changes in Cl" extrusion (400, 521, maturation. Spatiotemporal expression pattern of 562, 651, 671). K!-Cl" cotransport also contributes to the KCC2 mRNA expression follows a caudorostral gradilow [Cl"]i in mature neurons (156, 286, 450, 613– 615). ent reflecting functional maturation of various brain Additionally, the KCC1, KCC3, and KCC4 isoforms have areas. Electrophysiological recordings using Cl"-free been also found in the central nervous system but with a sharp electrodes revealed strong correlation between limited expression in neurons (499). the hyperpolarizing GABAA-mediated responses and Using ribonuclease protection analysis and in situ KCC2 mRNA expression in the pyramidal hippocampal hybridization, Clayton et al. (121) determined the devel- neurons of the rat, guinea pig, and dorsal root ganglion

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FIG. 3. Developmental changes in chloride homeostasis during development. A: during development, the intracellular chloride concentration decreases. In the immature neurons, efflux of the negatively charged chloride ions produces inward electric current and depolarization. In the mature neurons, chloride enters the cell and produces outward electric current and hyperpolarization. B: developmental change in the intracellular chloride is due to the changes in the expression of the two major chloride cotransporters, KCC2 and NKCC1. Chloride extruder KCC2 is expressed late in development, whereas NKCC1, which accumulates chloride in the cell, is more expressed in the immature neurons.

Ben-Ari et al. Figure 5

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of intrinsic membrane conductances in small groups of neurons coupled by gap junctions (Crepel and others 2007). They occur primarily during delivery and are modulated by oxytocin, in parallel with the transient shift of GABA actions (Tyzio and others 2006). As the network matures, SPAs are down-regulated possibly via CREB signaling and the activation of NMDA receptors that reduce connexins (Arumugam and others 2005). The down-regulation of SPA coincides with the appearance of GDPs. These are generated by the synergistic action of glutamate and GABA, which, as already mentioned, orchestrates neuronal ensembles via its depolarizing and excitatory action (Cherubini and others 2011; Ben-Ari 2002). GDPs disappear toward the end of the second postnatal week in concomitance with the shift of GABA from the depolarizing to the hyperpolarizing direction (Ben-Ari and others 1989). ENOs, initially thought to constitute the cortical counterpart of hippocampal GDPs, have been shown to precede and coexist during a restricted period of time with GDPs (Allene and others 2008; Allene and Cossart 2010b). ENOs are low-frequency oscillations dysplaying slow kinetics that gradually involve the entire network, whereas GDPs are recurrent oscillations that repetitively synchronize local Figure 5. Giant depolarizing potentials (GDPs) are generated neuronal assemblies. ENOs have different reversal Ben-Ari et al., The Neuroscientist 18(5): 467-486, 2012.! within a local network by the interplay between GABAergic potentials than GDPs—with a more substantial particiand Figure glutamatergic 5. neurons. (A) Concomitant gramicidinpation of NMDA receptor-driven currents (Figure 6; perforated patch clamp (upper trace) and field potentials Allene and Cossart 2010a)—and are facilitated by anoxic recordings (lower trace) from a P6 CA3 pyramidal cell. On conditions, raising the possibility that they may be relethe right, a GDP is shown on an expanded time scale. (B) Schematic drawing showing interconnected pyramidal cells vant to pathological situations (Allene and Cossart (green) and GABAergic interneurons (pink). GABA, released 2010b). Interestingly, the maturation of NMDA signalfrom GABAergic interneurons, binds and opens GABAA ing that controls immature patterns may recruit AMPA receptors (in blue), leading to an outwardly directed flux of receptors, thereby converting postsynaptically silent chloride, which depolarizes principal cells. connections into active ones (Voronin and Cherubini 2004). Interestingly, when both SPAs and GDPs are sleep, and feeding (Leinekugel and others 2002). GDPs present at intermediary developmental stages, GDPs are characterized by long-lasting recurrent depolarizations switch off SPAs (Crepel and others 2007). up to 50 mV in amplitude, giving rise to bursts of action potentials and separated by silent periods. This discontinuHow Are GDPs Generated? ity pattern is highly reminiscent of the “tracé discontinu” first described by Dreyfus-Brisac in electroencephaloGDPs require the synchronous activation of a relatively grams of immature babies (Stockard-Pope and others small number of cells. At least in the CA3 area, they can 1992). A similar pattern of activity also has been detected be still detected in small islands comprising hundreds of in the hippocampus of fetal macaque during the second neurons, isolated from the rest of the hippocampus half of gestation (R. Khazipov, unpublished data, 2001). (Khazipov and others 1997; Garaschuk and others 1998;

Figure 6 EXCITATORY GABA IN DEVELOPMENT

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B

FIG. 10. Giant depolarizing potentials in the neonatal rat hippocampus. A: gramicidin perforated patch recordings from a CA3 pyramidal cell in P6 rat hippocampus. Concomitant field potential recordings are shown on trace below. Note that GDPs in CA3 pyramidal cell coincide with the field potential population burst. One GDP is shown on expanded time scale in the right panel. B: the developmental template of the rat hippocampus. Note that GDPs are present during the phase of the intense growth of the hippocampal neurons and synaptogenesis.

cells of the fascia dentate (see also Ref. 269) where they follow the GDPs generated in CA3. These groups and others also showed that although CA3 is the most frequent pacemaker, all hippocampal regions can generate GDPs when separated from the other components, a small region containing a couple of thousand neurons will suffice (432). Therefore, the entire hippocampal network possesses the capacity to generate GDPs, but in situ some regions are more apt to impose their frequency on other regions, and the best candidate for a pacemaker region is the CA3 region that is particularly equipped to generate oscillations. CA3 pyramidal neurons mature before CA1 pyramidal neurons, and granule cells can generate synchronized patterns somewhat later (see above). As other polysynaptic events, GDPs are more sensitive than monosynaptic currents to many agents and procedures that alter neuronal excitability, including high Mg2! solutions (513), anoxoischemic episodes (142), and manipulations that affect pre- or postsynaptic efficacy including adenosine receptors (536), acetylcholine receptors that modulate the release of GABA (409), endogenous acetylcholine that enhances the release of GABA (70), glutamate metabotropic receptors that enhance the

synchronous release of GABA as in adults (509) through cAMP-dependent protein kinase (594), ATP operating via distinct P2X and P2Y receptors (537), and cannabinoid signaling that mediates depolarization induced suppression of GABA release (66). Lauri and co-workers (371, 372) suggested that kainate receptors that are highly expressed throughout the neonatal brain shift function during maturation and modulate the occurrence of GDPs. The activation of these receptors by ambient glutamate tonically reduces glutamate release. In contrast, the activation of these receptors at an early stage strongly upregulates GABAergic transmission and thus may regulate the occurrence of GDPs. Activation level of kainate receptors is finely tuned to permit synchronized network activity in the neonatal hippocampus, because both increased activation and inhibition of GluR5 kainate receptors inhibits GDPs. Apparently, these effects are dependent on distinct cellular mechanisms: activation of kainate receptors vastly increases asynchronous GABA release and thus shunts the network, whereas inhibition of the kainate receptors reduces interneuronal synchronization, therefore mitigating the build-up of network bursts (371, 372). J. Neurosci., December 15, 2001, 21(24):9770–9781 9779 GDPs are also modulated by presynaptic NMDA receptors. Thus Gaiarsa et al. (212) have shown that glycine repeatedly demonstrated that augments epileptiformthe activities the neoacting on NMDA receptors releaseinof GABA natalthe brain — often subclinical— can result in is neurological and frequency of GDPs. This action mimickedand by behavioral problems in adulthood, including reduction in the D-serine that also acts on that site and fully prevented by seizures threshold and development of temporal lobe epilepsy NMDA antagonists. Interestingly, Pooaand col(Holmesreceptor and Ben-Ari, 1998). Thus, our study raises concern leagues (384) have shown recently thatininthe thehippocampus developing about seizures that can potentially occur tadpole retinotectal system, visual generate a longalready prenatally, during the last third stimuli of gestation. Interestingly, term of GABAergic synapses mediated by prefrom depression the earliest developmental stages GABAergic interneurons inhibit the NMDA paroxysmal discharges. Thisatis that in agreement with the synaptic receptors that developmental efficacy modulate of positive the GABA(A) modulators for the treatstages releasereceptor of GABA. mentSimilar of seizures in neonates, including the premature neonates to GDPs, patterns of sharp waves and popu(Holmes, 1997). This is also in agreement with the data obtained lation bursts have also been observed in the hippocampus in neonatal rats, in which GABAergic interneurons prevent the of neonatal rats in vivo (376). Using multisite extracellular generation of paroxysmal activities from the early developmental and patch-clamp recordings in the hippocampus of freely stages via a shunting mechanism caused by the activation of moving and anesthetized pups, respectively, theinhiauGABA(A) receptors (K halilovrat et al., 1999), the presynaptic thors spontaneous bursts of synchrobition observed of glutamate release, and recurrent the postsynaptic hyperpolarizanized neuronalbyactivity thatreceptors lasted 0.5–3 s likeetGDPs and tion mediated GABA(B) (McLean al., 1996; C aillard al., 1998). in whichetGABA and glutamate PSCs contribute. Firing of CA1 pyramidal neurons occurred mostly within sharp A developmental program that maintained waves and population bursts thatiswere separated by long throughout evolution periods of silence. This electrographic pattern was reComparing early developmental events in the rat and macaque, it corded during sleep, immobility, or feeding behavior. appears that the general developmental scenario is very similar in Sharp waves are but generated by synchronous of these two species with a general shift towarddischarge fetal life in CA3 pyramidal andratare conveyed tothe CA1 via Schaffer primates (Fig. 9).cells In the hippocampus, neurogenesis of collaterals; thus CA3 during like inthevitro appears as thelife, most pyramidal cells proceeds last third of embryonic at E16 –E20 (Bayer, 1980) and in the rhesus macaque during the first likely pacemaker. A major difference between immature half of gestation, at E38 –E80 (Rakic and Nowakowski, 1981). At sharp waves and adult ones is the occurrence of fast birth (E22 in the the hippocampal cells attain a ripples that the rat), neonatal network pyramidal cannot generate and level appear of maturity andthe synaptic inputs yzio et al., 1999) compathat from end of the(T second postnatal week rable with that achieved in the monkey at midgestation. Boltz(85, 376; see also below). The developmental time course mann fits of the morphometric data obtained in the rat reveal that of ripple parallels the switch GABAday A receptorthethe rate of axonal growth peaks in at the postnatal 7 (P7)

Figure 9. Developmental template of the fetal macaque hippocampus. (Gomez-Di C esare et al., 1997) (in macaque: E109), the dendritic Hippocampal neurons are generated during the first half of gestation, with Physiol Rev •cells VOL grow 87 • OCTOBER 2007 •peaks www.prv.org growth at P9 (Minkwitz, 1976) (in macaque: E120), and the a peak at E55. During the second half of gestation, pyramidal intensively with the maximal rate of axonal and dendritic growth attained spinogenesis peaks at P16 (Englisch et al., 1974; Minkwitz, 1976; at !E105 and E120 (200 and 400 !m /d), respectively. Already at midBannister and Larkman, 1995) (in macaque: E125). GDPs are gestation (E85), half of the pyramidal cells receive GABAergic synaptic present in rat hippocampal slices from birth to P12–P16 (Ben-Ari inputs. Glutamatergic synaptic currents appear later, and their expression et al., 1989; Garaschuk et al., 1998) whereas in the macaque, they coincides with the appearance of the first dendritic spines. The acquisition of glutamatergic synapses (as deduced from the number of dendritic dominate during the last third of gestation and decrease before spines) proceeds with a maximal rate of !200 synapses formed every day birth. Epileptiform activities emerge in rat hippocampus during on a pyramidal cell at !E125. During the phase of intensive neuronal the first postnatal week, and the peak of epileptogenesis falls in growth and synaptogenesis most of the neuronal activity is synchronized the second and third postnatal weeks (K halilov et al., 1999; in a particular pattern of spontaneous network activity—the GDPs. By Swann et al., 1999; Wells et al., 2000), whereas in the macaque, the last third of gestation, the hippocampal network also becomes capable of generating epileptiform activities. Data for the neurogenesis were epileptiform activity builds up during the last third of gestation taken from Rakic and Nowakowski (1981) and fitted with a Gaussian

A. Ben-Ari et al., Physiol Rev 87: 1215-1284, 2007.! Figure 10.

B. Khazipov et al., The Journal of Neuroscience 21(24): 9770-9781, 2001.! Figure 9.

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Khazipov et al. • Activity in Fetal Primate Hippocampus

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n = 5893 events, significantly different from calcium spikes in terms of duration, Kolmogorov-Smirnov, p < 0.001; Figures 1 and 2). These plateaus produced recurrent burst discharges when cells were ecorded at Vrest, as shown by targeted wholecell recordings (Vm = !61 ± 1 mV, DV = 12 ± 4 mV, 0.5 ± 0.2 Hz, n = 12 cells, Figures 1 and 4). n contrast to calcium spikes, the onsets of calcium plateaus were significantly synchronized between neurons within a 200 ms time window (7.8% pairs of calcium plateaus correlated versus 1.3% calcium spikes pairs, p < 0.05, Figures 6 and 8). On average, synchronous calcium plateaus involved 3.0 ± 0.2 neurons; per movie, the largest synchronous events comprised 7.4 ± 1.9 neurons (n = 11 slices, 384 cells producing plateaus). Synchronous events occurred on average 7 ± 1 times per min (n = 11 slices). The synchronous occurrence of calcium plateaus in a subpopulation of neurons represents he first coherent activity pattern in the hippocampus. We propose to refer to this primary coherent activity, characterized by the occurrence of synchronous calcium plateaus in neuronal assemblies as SPA (synchronous plateau assemblies). At later postnatal stages (P6–P10), a majority of cells (64% ± 4%) were active in a strongly coordinated manner (21.1% pairs significantly correlated, peaks of synchronous activation involving up to 80% of imaged cells, n = 30 slices, p < 0.05; Figure 1, see Movie S2). This activity corresponds to he well-described GDPs (Ben Ari et al., 1989), as evealed by current-clamp recordings (Figure 1) and by their blockade by GABAA and glutamate eceptor antagonists (Figure 3).

onclude that three patterns, differing both by their dynamics and their electrophysiological correominate early spontaneous activity in the CA1 rencorrelated isolated spikes, synchronized calcium s nesting recurrent burst discharges (SPAs), and rn corresponding to the synapse-driven GDPs. velopmental incidence of calcium spikes, SPAs, DPs also differed: uncorrelated spikes were the nt pattern at embryonic stages (E16–E19) and en progressively replaced by SPAs and GDPs that ed by the end of the first postnatal week (Figure 2). PAs and GDPs disappear after the second postnaek (Figure 2). Therefore, population coherence s at birth in the hippocampus in the transient

Figure 7 A

B

Figure 2. Dynamics and Developmental Features of the Three Dominant Forms of Primary Activity in the CA1 Region (A1) Distribution plot of the durations of calcium transients for the three types of primary activities reveals three distinct populations (Kolmogorov-Smirnov, p < 0.001). (A2) Representative calcium fluorescence A.calcium Modified Crepel et al., inNeuron 54,dif105-120, traces for spikes,from two and three measured neurons from ferent movies. Note1B.! that first two and bottom three highly synchronized Figure: GDP traces are from the same movie. B. Crepel et al., Neuron 54, 105-120, 2007.! (B) Graph indicates the fraction of calcium-spike cells (black), SPA cells Figures: 2B. of active cells, as well as the frequency of (red) relative to the number GDPs (blue), for six successive age groups. Error bars indicate SEMs.

form of SPAs involving bursting neuronal assemblies. We next investigated the cellular mechanisms responsible for the generation of the two dominating patterns from E16 until birth, calcium spikes and SPAs. Intrinsic Voltage-Gated Ionic Conductances Generate Calcium Spikes and SPAs Sporadic calcium spikes were intrinsically generated by high-threshold calcium conductances with a contribution of sodium channels since (1) L-type calcium (10 mM nifedipine) and sodium (1 mM TTX) channel antagonists

2007.!

Figure 8

*To whom correspondence should be addressed. E-mail: [email protected]

A

Fig. 4. Blockade of oxytocin receptors decreases fetal brain resistance to anoxia-aglycemia at birth. (A) Representative traces to illustrate the effects of anoxic-aglycemic solution on extracellular field potential recordings from E21 intact hippocampi. Arrows indicate terminal AD that marks neuronal death. AD occurs earlier in the presence of SSR126768A (middle trace). Addition of bumetanide (10 mM) occludes the effects of the antagonist and restores the initial delay (bottom trace). (B) Summary plot of the onset of AD in control, in the presence of the oxytocin receptors antagonists atosiban (AT, 5 mM, fetal intracardial perfusion and SSR126768A (1 mg/kg to the mother), and after further addition of bumetanide (10 mM). Each circle corresponds to one hippocampus (n = 82 intact hippocampi at E21). Error bars indicate SEM.

56 ± 3%, n values of 7 movies and 931 cells (Fig. 2, B to D)]. In contrast, at E21 to P0, [Ca2+]i increased in only 31 ± 6% (n values of 10 movies and 1546 cells, P < 0.01) cells. Isoguvacine also decreased the frequency of spontaneous calcium

B

Fig. 1. Transient perinatal loss of the GABAAmediated excitation. (A) Responses of CA3 pyramidal cells recorded in cell-attached mode to the GABAA agonist isoguvacine. Below, summary plot of the proportion of cells excited by isoguvacine during the perinatal period. There is a transient loss of the excitatory effect of isoguvacine near term. Red corresponds to the fetuses whose mothers received SSR126768A. [E21 corresponds to the early phase of delivery (1 to 2 hours before birth); P0 is the day of birth; pooled data from 146 neurons.] (B) Cellattached recordings of single GABAA channels with 1 mM of GABA in patch pipette (top trace); the channels were not observed in the presence of the GABAA antagonist picrotoxin (100 mM; bottom trace). (C) I-V relationships of the currents through GABAA channels in two cells www.sciencemag.org SCIENCE VOL 314 15 DECEMBER 1791 at E212006 and E18; their reversal potential corresponds to DFGABA. (D) Summary plot of the age dependence of DFGABA inferred from Tyzio et al., Science 314(5806): 1788-1792, single 2006.! GABAA channels recordings [mean ± SEM; 209 CA3 pyramidal cells (○) and 17 Figures 1D and 4B. neocortical pyramidal cells (□); 6 to 24 patches for each point]. Red indicates pretreatment with SSR126768A (n values are 25 hippocampal and 9 neocortical patches). (E) Age dependence of the resting membrane potential (Em) of CA3 pyramidal cells inferred from the reversal of single NMDA channels recorded in cell-attached mode (n = 84 cells; 4 to 12 patches for each point). (F) Age dependence of the GABAA reversal potential (EGABA = Em + DFGABA). There is a transient hyperpolarizing shift of EGABA near birth.

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switching to a hyperpolarizing value of –8.4 ± 2.5 mV at term [mean ± SEM, n = 34 (Fig. 1D)]. Thus, the disappearance of GABA-mediated excitation at term (Fig. 1A) coincides with a switch in polarity of GABA signals.

Downlo

U29, Université de la Méditerranée, Campus Scientifique de Luminy, Boite Postale 13, 13273 Marseille Cedex 09, France. 2Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, 20251 Hamburg, Germany.

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Figure 9 EXCITATORY GABA IN DEVELOPMENT

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Ben-Ari et al., Physiol Rev 87: 1215-1284, 2007.! Figure 2.

channels as it persists when sodium channels are blocked and is suppressed by calcium channel blockers. Interestingly, blockade of GABAA receptors induces a significant decrease in [Ca2!]i, indicating that tonic release of GABA is sufficient to increase Ca2! in immature neurons (484). Connor et al. (132) were among the first to use digital imaging of the Ca2! to study the depolarizing responses of developing granule cells in culture to GABA. Application of GABA induced a transient increase in membrane conductance and caused [Ca2!]i increase that outlasted the exposure to GABA by several minutes. Glutamate or kainate also elevated [Ca2!]i, but unlike GABA, this [Ca2!]i response reversed rapidly upon removal of the transmitter. In keeping with these results, GABA and glycine increased [Ca2!]i in a number of embryonic and neonatal neurons including neocortex (223, 385, 399, 675), hippocampus (222, 377, 378), spinal cord (359), dorsal horn neurons (515, 653), hypothalamus, olfactory bulb, cortex, medulla, striatum, thalamus, hippocampus, and colliculus (484) (see Table 1). Synaptically released GABA also increases intracellular calcium in immature neurons. Obrietan and van den Physiol Rev • VOL

Pol (484) have shown that addition of bicuculline to monosynaptically connected hypothalamic neurons decreased [Ca2!]i, indicating that hypothalamic neurons were secreting GABA at an early age of development, and that sufficient GABA was released to elicit an increase in [Ca2!]i. This effect was seen even after blocking all glutamatergic activity with glutamate receptor antagonists (484). Leinekugel et al. (378) have demonstrated that electrical stimulation of afferent fibers induces a transient increase in [Ca2!]i in neonatal pyramidal cells and interneurons (P5). This elevation of [Ca2!]i was reversibly blocked by bicuculline but not by glutamate receptor antagonists. During simultaneous electrophysiological recording in current-clamp mode and [Ca2!]i monitoring from P5 pyramidal cells, electrical stimulation of afferent fibers, in the presence the glutamate receptors antagonists, caused synaptic depolarization accompanied by a few action potentials and a transient increase in [Ca2!]i. In voltage-clamp mode, however, there was no increase in [Ca2!]i following synaptic stimulation, showing that it is depolarization dependent (378).

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FIG. 2. GABA potentiates the activity of NMDA receptors in the neonatal hippocampus. A: synaptically elicited responses in neonatal CA3 pyramidal neurons (P5) recorded in cell-attached configuration. a: In control conditions, electrical stimulation elicited a burst of five action potentials. The number of action potentials was slightly affected by AMPA receptor antagonist CNQX (10 !M) (b) and strongly reduced by further addition of the NMDA receptor antagonist APV (50 !M) (c). d: The remaining response was blocked by the GABAA receptor antagonist bicuculline (10 !M). B: a CA3 pyramidal neuron (P5) was loaded extracellularly with the Ca2!-sensitive dye fluo-3 AM, and the slice was continuously superfused with the voltage-gated Ca2! channel blocker D600 (50 !M). Focal pressure ejection of a GABAA-receptor agonist, isoguvacine (100 !M), or bath application NMDA (10 !M) had no effect on [Ca2!]i fluorescence. However, a combined activation of GABAA and NMDA receptors resulted in a significant increase of [Ca2!]i fluorescence. C: scheme of the interactions between GABAA and NMDA receptors in immature neurons. [From Leinekugel et al. (377).]

Dehorter et al.

Immature patterns in the striatum

Figure 10 A

B

FIGURE 11 | Medium spiny neurons have entered the adult-like phase before pups start quadruped locomotion. (A–E) Immature (light orange) and adult-like (gray) phases are separated by a transitory immature period (orange). Development of MSN intrinsic and synaptic properties from experiments of Figures 1–7. (F) Photos of representative postnatal mice showing the number and extent of pressure points (in blue) at P2, P6, and P10 (top). Body motion of the same pups in the open field (calibration bars 3 cm) during a 1-min test (bottom). (G) Mean percentage (±SEM) of belly contact time (n = 16, top) and total distance traveled along straight line segments (n = 14, bottom) as a function of postnatal age (see Movies S1–S3 in Supplementary Material).

Dehorter et al., Frontiers in Cellular Neuroscience ! November 2011, volume 5, article 24.! Figure 11.

DISCUSSION Our results show that MSNs, the dominant neuronal population of the striatum, generate immature patterns of activity at embryonic and early postnatal stages that are reminiscent of the patterns observed in developing cortical structures (Garaschuk et al., 2000; Corlew et al., 2004; Allene et al., 2008). This confirms the similarity between developmental activities of networks independently of their neuronal structure and final function (Ben-Ari, 2001). In the middle of the second postnatal week, MSNs shift to an adultlike pattern characterized by little activity in vitro (Carrillo-Reid et al., 2008), just before pups lift their body and begin to walk.

of MSNs and striatal network activity parallels the development of locomotor structures and pathways (Grillner et al., 2005). Two features of immature MSNs emerge as central players in this progression. (i) Intrinsic voltage-gated Ca2+ currents: we propose that the reduced K+ currents and the consequent depolarized resting membrane potential allow spontaneous opening of N and L-type voltage-gated Ca2+ channels, then quieted near P10 by resting membrane potential hyperpolarization and/or developmental changes of Ca2+ channel properties; (ii) The transient NR2C/Dmediated cationic current: the expression of long lasting NMDA EPSCs mediated by the NR2C/D receptor subunit is a general feature of different developing brain structures (Monyer et al., 1994; Nansen et al., 2000; Logan et al., 2007; Dravid et al., 2008). NMDA receptor-mediated EPSCs are conspicuous in cortico-striatal neurons as early as P2 (see Hurst et al., 2001) for contradictory results). Using a dose of PPDA (100 nM) that preferentially antagonizes NR2C/D subunits (KD = 0.096 and 0.130 µM, respectively; Traynelis et al., 2010), we demonstrate that the window of operation of NR2C/D-mediated events is highly restricted to P5–P8 (Dunah et al., 1996). Therefore, as in other brain structures, immature neurons first generate long lasting synapse-driven patterns of activity that include large NMDA receptor driven currents. These currents, together with voltage-gated Ca2+ currents, trigger the large calcium fluxes needed for a wide range of essential developmental functions including neuronal growth, synapse formation, and the formation of neuronal ensembles (Spitzer, 2006). Indeed, we demonstrated that during this P5–P8 window, network-wide changes in calcium event statistics correlated with the reliable formation of neural ensembles. Our observations also provide interesting insights concerning the generation of GDPs that have been observed in a wide range of brain networks but investigated primarily in cortical structures (Ben-Ari et al., 2007). GDPs are generated both by depolarizing GABAergic and glutamatergic notably NMDA receptors-mediated currents. The striatum is an interesting site to investigate the debated role of glutamate in GDPs generation because it has in contrast with other investigated structures no internal glutamatergic neurons. Clearly, the maturation of the glutamatergic cortico-striatal inputs is instrumental in the emergence of GDPs and particularly the long lasting NR2C/D component. Adesnik et al. (2008) suggested that modest activity through NMDA receptors prevents the constitutive trafficking of AMPA receptors to the postsynaptic density via an LTD type mechanism. This ensures that synapses become functional only after strong or correlated activity, when enough calcium entry through these NMDA receptors overrides the inhibitory pathway and drives AMPA receptor insertion. This surge is provided by bursts of action potentials during GDPs and NMDA-mediated corticostriatal EPSPs as shown here. From this perspective the elimination of the long lasting NR2C/D component in cortico-striatal EPSPs would constitute a gating device to induce the expression of AMPAergic currents in MSNs. Therefore, our results suggest an intrinsic program that switches MSN activity from an immature low threshold activation state to a high threshold state in the adult with a low activity

Figure 11 A

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Figure 1 Phenobarbital prevents the formation by ictal-like events of an epileptogenic mirror focus. (A–C) The scheme depicts the triple chamber preparation with the two intact hippocampi and their connecting inter-hemispheric commissure in independent chambers (left). Field recordings were made in the two hippocampi. (A) Kainate (KA) applied to one hippocampus (ipsilateral = ipsi-, grey) generated seizure activity referred to as ictal-like events that propagated to the contralateral naı¨ve hippocampus (contra-). The ictal-like events included g-oscillations (440 Hz) as shown in the time frequency analysis in the right side of the figure. (B) Application of phenobarbital (PB) toetcontralateral hippocampus (100 mM, during 15 min) before second application of kainate decreased the ictal-like event and almost A. Nardou al., Brain 134: 987-1002, 2011.! completely blocked g-oscillations. (C) After 15 transient applications of kainate on the ipsilateral side with continuous application of Figure 2. phenobarbital on contralateral side, disconnection of the two hippocampi by applications of tetrodotoxin to the commissural chamber revealed that the contralateral side generated neither spontaneous nor evoked (by electrical stimulation = stim) ictal-like events. (D) Power B. Nardou etshowing al., Brain 134: 987-1002, 2011.! Figure Late application failsside to block thewith ictal-like event and(PB) g-oscillations. Kainate (KA) was applied repeatedly (every spectra decreased amplitude of the2 population activityofofphenobarbital the contralateral treated phenobarbital during application 20 min) " 15 tocerebrospinal one hippocampus the ipsilateral side. ACSF = artificial fluid. (ipsilateral = ipsi-) and artificial cerebrospinal fluid (ACSF) to the contralateral naı¨ve hippocampus Figure of 1Bkainate andon 2B. (contra-). (A) Fourteenth ictal-like event is generated on the ipsilateral side (grey) and propagated to the contralateral side (black). The Figure 5 Phenobarbital enhances GABA excitation in mirror focus neurons. (A) Cellanalysis attached recordings from mirror focusshows neurons to (onand the right,hippocampus hippocampus = hippo-) g-oscillations (in the 490 side), and recorded the activity fromtime bothfrequency the kainate treated (Khalilov et al., 2003, 2005). We refer to Hz thisband). as a (B) phenobarbital (PB) applied to illustrate the increased number ofC. action potentials generated by focal987-1002, application of GABA inmin thebefore presence of phenobarbital (PB) (Aa–Ac). Nardou et al., Brain 134: 2011.! the contralateral side 15 the 15th application of kainate to the ipsilateral side failed to block propagating ictal-like events (red). the other hippocampus naı¨ve for kainate (referred to as the newly formed epileptogenic mirror focus. Using this paradigm, we Quantified group data are shownFigure in Ad, **P 5 0.01. (Ba) from perforated clamp (C) recordings of post-synaptic Thetraces time frequency also showspatch g-oscillations. Superimposed ictal-like events (from A and B) recorded from the contralateral side before 5Aa, andinInSuperimposed 5Bb. Figure 5 Phenobarbital enhances GABA5Ab excitation mirror focuswith neurons. Cell attached recordingscompared from mirror neurons tosituations: contralateral side). keeping our (A) earlier observations twofocus experimental (black) andpresence after phenobarbital application (red). Phenobarbital produced a small increase of the amplitude and duration of ictal-like currents generated by focal application of GABA (every 20 s, arrowheads) in the of CNQX and APV before (black), during (red) illustrate the increased number of action potentials generated by focal application of GABA the presence of phenobarbital (PB) (Aa–Ac). (Khalilov et al., 2003, 2005), kainate generated an in ictal-like events. magnification (D) Superimposed ictal-like events generated spontaneously in the contralateral side after formation of mirror focus before (black) application of Quantified phenobarbital after wash out (blue). (Bb) Traces at higher from Ba (asterisks), to illustrate the increased group and data are shown in Ad, **P 5 0.01. (Ba) Superimposed traces from perforated patch clamp recordings of post-synaptic (i) When applied from the beginning on the initial ictal-like event with large-amplitude (1–2 mV) (Fig. 1A, seeapplication. Phenobarbital also increased the amplitude and discharges after (red) phenobarbital and duration of spontaneous ictal-like events. generated by focal application of GABA (every 20 arrowheads) in the presence of CNQX and APV before (black), during (red) amplitude andcurrents duration of post-synaptic currents generated bys,focal applications of propagated GABA in the presence of phenobarbital. events, phenobarbital prevents the formation of (from a mirror also Khalilov et al., 2003). These ictal-like events to (E) Power spectra of spontaneous ictal-like events before and after phenobarbital treatment D). (F) Average power histogram of application of phenobarbital and after wash (blue). Traces5 at0.001). higher magnification from Ba (asterisks), to illustrate the increased (Bc) Histogram representing the quantifications of out area (n =(Bb) 4, ***P focus.phenobarbital. Application ofPhenobarbital phenobarbitalapplication (100 mM) significantly to the contrathe contralateral hippocampus wherespontaneous they generated similar activ-before and after ictal-like events increased power of ictal-like events amplitude and duration of post-synaptic currents generated by focal applications of GABA in the presence of phenobarbital. lateral hippocampus reduced the total power of ictal-like ities. In keeping with our earlier observations (Khalilov al.,0.01). 24.2 ! 7.3%, n = 4,etP 5 (Bc) Histogram representing the quantifications of area (n = 4, ***P(by 5 0.001). events and the occurrence of g-oscillations in every experi2003, 2005), the duration of each ictal-like event increased after ment (Fig. 1B and D, n = 6). g-Oscillations shifted from repeated applications of kainate (from 82.2 ! 9.6 to 102.7 ! 7.8 s, 460 Hz recorded prior to phenobarbital application to P 5 0.01, n = 23). In further agreement with these earlier studies, 1557.2 ! 14.3 ms (P 5 0.001). The rise time (10–90%) was not interictal-like events were generated by slices prepared from the Hz prepared in presence of the phenobarbital. This effect is a good ictal-like events included was g-oscillations (40–120 Hz) that were alsogenerated by520 1557.2 ! 14.3 ms (P 5 0.001). The rise time (10–90%) not interictal-like events were slices from mirror focus hippocampus (Fig. 6Cb). Therefore, this cotransporter changed significantly (from 523 ! 18.6 to 562.8 ! 10.3with ms, indicator of progressively applications of kainate to mirror focus hippocampus (Fig. 6Cb). Therefore, thisphenobarbital cotransporteraction as g-oscillations constitutes changed significantly (from 523 ! 18.6 toincreased 562.8 ! 10.3 recurrent ms, isetisnot necessary forthe theapformation ofsignature a mirror P = 0.53). Therefore, phenobarbital augments GABAGABA signals in of focus. severe seizures and their sites of origin (Bragin the ipsilateral (Khalilov al., After not 2005). necessary for"15 formation of aamirror focus. P = 0.53). Therefore, phenobarbital augmentshippocampus signals in Since in immature neurons, invasive recording techniques not Jacobs et al., 2008a). et al., 1999; Khalilov plications, the contralateral hippocampus generated spontaneous mirror focus neurons possibly by a chronic increase of intracellular Since in immature neurons, invasive recording techniques do et notal.,do2005; mirror focus neurons possibly by a chronic increase of intracellular When the two hippocampi were disconnected after ictal-like events (n = 23) when disconnected from the treated provide a reliable estimate of resting membrane potential (V ) provide a reliable estimate of resting membrane potential (V chloride. We chloride. next examined the roles of chloride cotransporters We next examined the roles of chloride cotransporters rest) rest

Figure 2 Late application of phenobarbital fails to block the ictal-like event and g-oscillations. Kainate (KA) was applied repeatedly (e

(Tyzioetetal., 2003), we single N-MethylD-aspartic acid andacid and (Tyzio 2003), weused used single N-MethylD-aspartic (ipsilateral = ipsi-) andal., artificial cerebrospinal fluid (ACSF) to the contralateral naı¨ve hippocampus GABA(A) channel channel recordings to to determine V and GABA GABA(A) recordings determine Vresttheand the GABA drivingon forcethe (DF ipsilateral ), respectively,side from which the reversal po(contra-). (A) Fourteenth ictal-like event is generated (grey) and propagated to the contralateral side (black). driving force (DF ), respectively, from which the reversal poGABA NKCC1 is preserved and KCC2 is tential of GABA-induced currents (E = DF + V ) can be NKCC1 is preserved and KCC2 is time frequency analysis (on the right, = hippo-) showscurrents g-oscillations (in the Hz band). (B) phenobarbital (PB) applie of GABA-induced (EGABA = DFneurons, + mean Vrest490 ) can be GABA calculated (Fig. 7A) (Tyzio et al., 2007). In control downregulated in mirror focushippocampus neurons tential

NKCC1inand KCC2 in these changes. NKCC1 and KCC2 changes. 20 min) " 15 tothese one hippocampus

rest

GABA

GABA

GABA

rest

icantly decreased [Cl–]i and DFGABA at P0 in neurons recorded in VPA rats and FRX mice (Fig. 1, B, C, E, and F; and table S2). Therefore, the GABA developmental sequence is abolished in two animal models of autism, with GABA ex-

Figure 12

(10–12). KCC2 was down-regulated in the hippocampi of juvenile VPA rats and FRX mice (fig. S1, A to C, and table S3). In addition, as in epileptic neurons (10), there was a shift of KCC2 labeling from the membrane to the cytoplasm in neurons

actions of GABA were associated with neuronal excitation. In naïve animals, the specific GABAAR agonist isoguvacine (10 mM) inhibited or did not affect spike frequency in cell-attached recordings at P0 (Fig. 1, G to J, and table S5) and P15 (fig. S2,

Fig. 1. Developmental excitatory-inhibitory GABA sequence is abol- (WT, black) and FRX mice (red). (G to J) Excitatory action of the GABAAR ished in hippocampal CA3 pyramidal neurons in VPA rats and FRX mice. agonist isoguvacine (10 mM, black bars) on spontaneous spiking recorded in (A) Age-dependence of DFGABA in control and VPA rats. (B) Current-voltage (I-V) cell-attached configuration in VPA and FRX. (G) Control and VPA rats and (I) relations of GABAAR single-channel currents at P0 in VPA rats in artificial cerebro- wild-type and FRX mice at P0 with and without isoguvacine. Time-course of spinal fluid (red) or bumetanide (10 mM, blue). (Inset) Single-channel openings spike frequency changes is shown under each trace. (H) Average values of at different holding potentials (scale bars mean 1 pA and 200 ms). (C) normalized to control spike frequency for P0 and P15 control (gray) and VPA Tyzio al.,DFGABA Science 343(6171): 675-679, 2014.! (red) rats and effect of isoguvacine application (hatched bars). (J) The same Bumetanide and oxytocinet shifted from depolarizing to hyperpolarizing at P0 (control rats, black; VPA, red; bumetanide application, blue; and oxy- as in (H) but for P0 and P15 mice wild-type (gray) and FRX (red). Data are Figure 1. tocin application, purple). (D to F) The same as in (A) to (C) for wild-type mice presented as means T SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

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Figure 13

REPORTS

aternal pretreatment with bumetanide before delivery switches the GABA from excitatory to inhibitory in offspring in VPA and FRX at P15. (A) Average values of DFGABA measured in hippocampal CA3 neurons at P15 in control (black), VPA (red), and VPA rats pretreated with de (blue). Note that pretreatment with bumetanide shifts DFGABA from ng to almost isoelectric level. (B) Effects of isoguvacine (10 mM; black ats: Representative traces of spontaneous extracellular field potentials in hippocampal slices at P15 in control, VPA, and VPA rats pretreated etanide (BUM). Corresponding time courses of spike frequency changes n under each trace. (C) Average histograms of normalized spike frerats. Isoguvacine (hatched bars) decreased the spikes frequency in control 8.9 T 5.1%; gray); increased it in VPA rats (to 213.5 T 16.3%; red); and it in VPA rats pretreated with bumetanide (to 82.8 T 10.7%; blue). (D) as in (A) for mice. Wild-type mice (WT, black), FRX mice (red), and FRX eated with bumetanide (blue). (E) The same as in (B) for FRX mice. (F) as in (C) for FRX mice. Wild-type mice (decreased to 67.9 T 6.1%; X mice (increased to 165.8 T 13.5%; red); FRX mice pretreated with de (decreased to 80.8 T 8.2%; blue). Data are presented as means T < 0.01; ***P < 0.001.

Spontaneous activity is increased in VPA and nts at P15 and restored to control values by pretreatment with bumetanide. Whole-cell amp recordings of sEPSCs at –70 mV from individcampal CA3 pyramidal neurons in acute brain slices VPA rats or FRX mice and respective control and de or SSR126768A pretreated animals. (A and C) ative traces of sEPSCs recorded from rats (A) and Note that maternal pretreatment of animals with de decreases sEPSCs frequency in both models, eatment with SSR126768A increases spontaneous f neuronal networks in rats and mice. (B) Avers of sEPSCs frequencies in rats: Control rats (gray) ats (red), VPA rats with maternal pretreatment with de (blue) and SSR126768A-treated rats (orange). me as (B) for mice. Wild-type mice (gray), FRX mice mice with maternal pretreatment with bumetanide d SSR126768A-treated mice (orange). One-way analance (ANOVA) Fisher’s least significant difference as test. Data are presented as means T SEM. *P < 0.05;

Tyzio et al., Science 343(6171): 675-679, 2014.! Figure 2.

Figure 14 TINS-650; No of Pages 11

Review

Trends in Neurosciences Vol.xxx No.x

Figure 1. Schematic illustration to depict the impact of the environment and genetic mutations on all developmental stages. (a) Proliferation, (b) differentiation and migration, (c) guidance, (d) growth or synapse formation and (e) network elaboration are modulated by genetic and environmental factors. Alterations of these steps due to mutations and/or environmental factors can lead to developmental-stage-dependent malformations that will be associated with inappropriate proliferation, migration, guidance, differentiation, growth or synapse formation.

and, in primates by E112, visual cortex neurons possess a dense network of synapses indicating that the system is ready even before visual experience [7,8]. Retinal waves that are generated early [9–11] modulate the construction of the visual system [12,13], and in utero enucleation in primates enhances the size of the projection areas in cortex [8,14]. Visual maturation is experience dependent [15–17] with identified inducing factors [18,19]. Activity regulates essential developmental processes including the cell cycle [20], axonal growth [21], phenotype selection [22] and cell outgrowth [11,17,23–26]. Synapse formation follows a programmed sequence, with GABAergic synapses formed before glutamatergic ones in rodents and primates [27,28]. The universal developmental shift in the actions of GABA vividly illustrates the differences between adult and immature brains. GABA (the main inhibitory transmitter in adults) excites immature neurons because of an evolutionarily conserved higher intracellular Cl! concentration ([Cl!]i) [26,29–31] (Box 1 Figure I). GABA generates sodium and calcium currents and removes the voltage-dependent Mg2+ block from NMDA channels, thereby increasing intracellular Ca2+ concentration and exerting trophic actions on developing neurons [32–34]. Delivery in rodents is preceded by an oxytocin-triggered transient excitatory-to-inhibitory shift of the action of GABA that protects neurons from deliveryrelated anoxic damage [35]. Artificially shifting GABA actions from excitatory to inhibitory increases the formation of GABAergic synapses [36–38]. The data indicating that immature neurons use different ionic and receptor-mediated channels than those used

by mature neurons is overwhelming. All receptors and receptor channels investigated (sodium and calcium channels, NMDA and AMPA [a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid] or kainate receptors, acetylcholine, K+ channels etc), and even extracellular matrix molecules, are different in immature and adult systems and these differences have an important role in development [1,39–47]. Usually, the subunits of immature neurons tend to have slow kinetics (e.g. NMDA receptor subunit shift from NR2B to NR2A) [33,39,48,49], in keeping with the ‘clumsy’ properties of developing activity. In addition, and perhaps more striking, immature neurons use receptor signals that are subsequently eliminated during development. Thus, vertebrate embryonic neuromuscular junctions express functional GABA, glutamate and glycine receptors, in addition to the acetylcholine receptors that will remain into maturity [50,51]. Spinal cord neurons that are glycinergic in adults express functional GABA receptors first; these are eliminated during development [52]. Similarly, GABAergic synaptic responses in the trapezoid body precede the glycinergic responses of adults [53] (for review, see Ref. [29]). Neuronal activity also plays a crucial part in the selection of the adequate phenotype, reflecting the importance of the interactions between genetic programs and environmental information [51,54,55]. Whether these receptor change-overs are evolutionary remnants or adaptive is, at present, not clear. Nevertheless, this implies that drugs might exert dramatic actions on developing neurons while having little or no effect in the adult. Developing neurons are highly excitable and have an intrinsic tendency to oscillate [56–58]. In utero they

Ben-Ari et al., Trends Neurosci 31(12): 626-636, 2008.! Figure 1.

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Highlights 1) The GABA excitatory/inhibitory developmental sequence is evolutionary conserved 2) Excitatory GABA plays a fundamental role on many developmental processes 3) The oxytocin mediated GABA shift during delivery is abolished in autism 4) Immature currents including E GABA persist in pathological conditions 5) Therapeutic strategies using selective antagonists of immature currents in adults

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