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Since their discovery in the 1960s, the term paroxysmal depolarization shift (PDS) has been applied to a wide variety of reinforced neuronal discharge patterns. Occurrence of PDS as cellular correlates of electrographic spikes during latent phases of insult-induced rodent epilepsy models and their resemblance to giant depolarizing potentials (GDPs) nourished the idea that PDS may be involved in epileptogenesis. Both GDPs and – in analogy – PDS may lead to progressive changes of neuronal properties by generation of pulsatile intracellular Ca2+ elevations. Herein, a key element is the gating of L-type voltage gated Ca2+ channels (LTCCs, Cav1.x family), which may convey Ca2+ signals to the nucleus. Accordingly, the present study investigates various insult-associated neuronal challenges for their propensities to trigger PDS in a LTCC-dependent manner. Our data demonstrate that diverse disturbances of neuronal function are variably suited to induce PDS-like events, and the contribution of LTCCs is essential to evoke PDS in rat hippocampal neurons that closely resemble GDPs. These PDS appear to be initiated in the dendritic sub-compartment. Their morphology critically depends on the position of recording electrodes and on their rate of occurrence. These results provide novel insight into induction mechanisms, origin, variability, and co-existence of PDS with other discharge patterns and thereby pave the way for future investigations regarding the role of PDS in epileptogenesis.

In the developing brain, microglial cells play an important role in shaping neuronal circuits. These immune cells communicate with neurons through fractalkine (CX3CL1), a neuronal cytokine that acts on microglial CX3CR1 receptor. Among various functions, this signaling pathway has been implicated in the postnatal maturation of glutamatergic synapses. Although microglial cells are present in the neonate hippocampus when GABA receptor-mediated synaptic transmission and synchronized oscillatory events take place, it remains unknown whether microglial cells tune the establishment of these activities. Using CX3CR1-deficient mice and electrophysiological means, we investigated in CA3 pyramidal neurons the role of the fractalkine signaling in the maturation of GABAA receptor-mediated synaptic currents and giant depolarizing potentials (GDPs), a network activity important for shaping synaptic connections. In CX3CR1-deficient mice, GABAergic currents were slightly altered, whereas the developmental changes of these currents were comparable with wild-type animals. Despite these minor changes in GABAergic transmission, the GDP frequency was strikingly reduced in CX3CR1-deficient mice compared to wild-type, with no change in the GDP shape and ending period. Collectively, it emerges that, in the neonate hippocampus, the fractalkine signaling pathway tunes GDP activities and is marginally involved in the maturation of GABAergic synapses, suggesting that microglial cells have distinct impact on maturing GABAergic, glutamatergic, and network functions.

Homocysteine is a sulfur-containing amino acid, which at high concentrations induces neurotoxic effects and causes impairments in the development of the nervous system. In the blood homocysteine is rapidly oxidized, forming disulfide bonds with proteins and low-molecular thiols, and is also converted into homocysteine-thiolactone. Therefore during chronic exposure, homocysteine neurotoxicity is mediated by its derivatives. Our work aimed to study the effects of homocysteine, homocystine, and homocysteine-thiolactone on the network spontaneous activity of rat hippocampal neurons during the first postnatal week. Giant depolarizing potentials (GDP) and multiple-unit activities (MUA) were recorded using the extracellular electrode in the CA3 zone of the hippocampus. It was shown that all three thiol compounds induced an increase of the frequency of GDP and MUA in concentrations of 100 and 500 μM, while homocystine exerted the most profound effects on the network activity. Inhibition of NMDA and AMPA receptors completely prevented the effects of homocysteine and its derivatives on the spontaneous neuronal activity. Thus, homocysteine and its derivatives lead to the increase of network activity of hippocampal neurons of neonatal rats, which may impair the formation of hippocampal neuronal networks in chronic hyperhomocysteinemia; and may cause hyperexcitability and the risk of epilepsy development in the postnatal period.

Significance The transition from placental to lung-based oxygen supply at mammalian birth involves an obligatory period of asphyxia, which is further aggravated by complications during delivery. This oxygen deprivation is a major threat to the fetal brain, and, under such conditions, hormonal and cardiovascular mechanisms are activated to enhance brain perfusion. Our work now demonstrates an intrinsic mechanism in the fetal brain whereby vasopressin activates hippocampal interneurons, leading to desynchronization and suppression of neuronal network activity in species (rat and guinea pig) that are born at widely different stages of brain maturation. Silencing of synchronous neuronal activity by vasopressin is expected to decrease neuronal energy demand and prevent maladaptive synaptic plasticity, thus acting as a pan-mammalian neuroprotective mechanism during birth., During birth in mammals, a pronounced surge of fetal peripheral stress hormones takes place to promote survival in the transition to the extrauterine environment. However, it is not known whether the hormonal signaling involves central pathways with direct protective effects on the perinatal brain. Here, we show that arginine vasopressin specifically activates interneurons to suppress spontaneous network events in the perinatal hippocampus. Experiments done on the altricial rat and precocial guinea pig neonate demonstrated that the effect of vasopressin is not dependent on the level of maturation (depolarizing vs. hyperpolarizing) of postsynaptic GABAA receptor actions. Thus, the fetal mammalian brain is equipped with an evolutionarily conserved mechanism well-suited to suppress energetically expensive correlated network events under conditions of reduced oxygen supply at birth.

The effects of ionotropic &gamma, aminobutyric acid receptor (GABA-A, GABAA) activation depends critically on the Cl&minus, gradient across neuronal membranes. Previous studies demonstrated that the intracellular Cl&minus, concentration ([Cl&minus, ]i) is not stable but shows a considerable amount of activity-dependent plasticity. To characterize how membrane properties and different molecules that are directly or indirectly involved in GABAergic synaptic transmission affect GABA-induced [Cl&minus, ]i changes, we performed compartmental modeling in the NEURON environment. These simulations demonstrate that GABA-induced [Cl&minus, ]i changes decrease at higher membrane resistance, revealing a sigmoidal dependency between both parameters. Increase in GABAergic conductivity enhances [Cl&minus, ]i with a logarithmic dependency, while increasing the decay time of GABAA receptors leads to a nearly linear enhancement of the [Cl&minus, ]i changes. Implementing physiological levels of HCO3&minus, conductivity to GABAA receptors enhances the [Cl&minus, ]i changes over a wide range of [Cl&minus, ]i, but this effect depends on the stability of the HCO3&minus, gradient and the intracellular pH. Finally, these simulations show that pure diffusional Cl&minus, elimination from dendrites is slow and that a high activity of Cl&minus, transport is required to improve the spatiotemporal restriction of GABA-induced [Cl&minus, ]i changes. In summary, these simulations revealed a complex interplay between several key factors that influence GABA-induced [Cl]i changes. The results suggest that some of these factors, including high resting [Cl&minus, ]i, high input resistance, slow decay time of GABAA receptors and dynamic HCO3&minus, gradient, are specifically adapted in early postnatal neurons to facilitate limited activity-dependent [Cl&minus, ]i decreases.

The effects of ionotropic γ-aminobutyric acid receptor (GABA-A, GABAA) activation depends critically on the Cl−-gradient across neuronal membranes. Previous studies demonstrated that the intracellular Cl−-concentration ([Cl−]i) is not stable but shows a considerable amount of activity-dependent plasticity. To characterize how membrane properties and different molecules that are directly or indirectly involved in GABAergic synaptic transmission affect GABA-induced [Cl−]i changes, we performed compartmental modeling in the NEURON environment. These simulations demonstrate that GABA-induced [Cl−]i changes decrease at higher membrane resistance, revealing a sigmoidal dependency between both parameters. Increase in GABAergic conductivity enhances [Cl−]i with a logarithmic dependency, while increasing the decay time of GABAA receptors leads to a nearly linear enhancement of the [Cl−]i changes. Implementing physiological levels of HCO3−-conductivity to GABAA receptors enhances the [Cl−]i changes over a wide range of [Cl−]i, but this effect depends on the stability of the HCO3− gradient and the intracellular pH. Finally, these simulations show that pure diffusional Cl−-elimination from dendrites is slow and that a high activity of Cl−-transport is required to improve the spatiotemporal restriction of GABA-induced [Cl−]i changes. In summary, these simulations revealed a complex interplay between several key factors that influence GABA-induced [Cl]i changes. The results suggest that some of these factors, including high resting [Cl−]i, high input resistance, slow decay time of GABAA receptors and dynamic HCO3− gradient, are specifically adapted in early postnatal neurons to facilitate limited activity-dependent [Cl−]i decreases.

Ethanol exerts multiple adverse effects in the developing hippocampus resulting in the life-long neurological and behavioral deficits. However, the early disturbances in the hippocampal network function after exposure to ethanol remain largely unknown. Here, we examined the properties of the neonatal CA3 hippocampal network-driven giant depolarizing potentials using extracellular recordings of the local field potential and multiple units from the hippocampal slices prepared from the 5–11-day-old rats treated by ethanol in vivo (6 g/kg, intraperitoneally), 12 h before the slice preparation. Activity in hippocampal slices from the ethanol-treated animals displayed several abnormalities including a threefold increase in overall neuronal firing and profound, by nearly sevenfold, reduction of synchronization of CA3 units in giant depolarizing potentials. Thus, alterations in the hippocampal network function emerge shortly after the ethanol exposure and manifest in the enhanced excitation and severe impairments to the giant depolarizing potentials.

Direct electrical coupling between neurons through gap junctions is prominent during development, when synaptic connectivity is scarce, providing the additional intercellular connectivity. However, functional studies of gap junctions are hampered by the unspecificity of pharmacological tools available. Here we have investigated gap-junctional coupling between CA3 pyramidal cells in neonatal hippocampus and its contribution to early network activity. Four different gap junction inhibitors, including the general blocker carbenoxolone, decreased the frequency of network activity bursts in CA3 area of hippocampus of P3-6 rats, suggesting the involvement of electrical connections in the generation of spontaneous network activity. In CA3 pyramidal cells, spikelets evoked by local stimulation of stratum oriens, were inhibited by carbenoxolone, but not by inhibitors of glutamatergic and GABAergic synaptic transmission, signifying the presence of electrical connectivity through axo-axonic gap junctions. Carbenoxolone also decreased the success rate of firing antidromic action potentials in response to stimulation, and changed the pattern of spontaneous action potential firing of CA3 pyramidal cells. Altogether, these data suggest that electrical coupling of CA3 pyramidal cells contribute to the generation of the early network events in neonatal hippocampus by modulating their firing pattern and synchronization.

Spontaneously generated network activity is a hallmark of developing neural circuits, and plays an important role in the formation of synaptic connections. In the rodent hippocampus, this activity is observedin vitroas giant depolarizing potentials (GDPs) during the first postnatal week. Interneurons importantly contribute to GDPs, due to the depolarizing actions of GABA early in development. While they are highly diverse, cortical interneurons can be segregated into two distinct groups based on their embryonic lineage from either the medial or caudal ganglionic eminences (MGE and CGE). There is evidence suggesting CGE-derived interneurons are important for GDP generation; however, their contribution relative to those from the MGE has never been directly tested. Here, we optogenetically inhibited either MGE- or CGE-derived interneurons in a region-specific manner in mouse neonatal hippocampusin vitro. In CA1, where interneurons are the primary source of recurrent excitation, we found that those from the MGE strongly and preferentially contributed to GDP generation. Furthermore, in dual whole-cell patch recordings in neonatal CA1, MGE interneurons formed synaptic connections to and from neighboring pyramidal cells at a much higher rate than those from the CGE. These MGE interneurons were commonly perisomatic targeting, in contrast to those from the CGE, which were dendrite targeting. Finally, inhibiting MGE interneurons in CA1 suppressed GDPs in CA3 and vice versa; conversely, they could also trigger GDPs in CA1 that propagated to CA3 and vice versa. Our data demonstrate a key role for MGE-derived interneurons in both generating and coordinating GDPs across the hippocampus.SIGNIFICANCE STATEMENTDuring nervous system development, immature circuits internally generate rhythmic patterns of electrical activity that promote the establishment of synaptic connections. Immature interneurons are excitatory rather than inhibitory and actively contribute to the generation of these spontaneous network events, referred to as giant depolarizing potentials (GDPs) in the hippocampus. Interneurons can be generally separated into two distinct groups based on their origin in the embryo from the medial or caudal ganglionic eminences (MGE and CGE). Here we show that MGE interneurons play a dominant role in generating GDPs compared with their CGE counterparts. They accomplish this due to their high synaptic connectivity within the local circuitry. Finally, they can control network activity across large regions of the developing hippocampus.

Paroxysmal depolarization shifts (PDS) have been described by epileptologists for the first time several decades ago, but controversy still exists to date regarding their role in epilepsy. In addition to the initial view of a lack of such a role, seemingly opposing hypotheses on epileptogenic and anti-ictogenic effects of PDS have emerged. Hence, PDS may provide novel targets for epilepsy therapy. Evidence for the roles of PDS has often been obtained from investigations of the multi-unit correlate of PDS, an electrographic spike termed “interictal„ because of its occurrence during seizure-free periods of epilepsy patients. Meanwhile, interictal spikes have been found to be associated with neuronal diseases other than epilepsy, e.g., Alzheimer’s disease, which may indicate a broader implication of PDS in neuropathologies. In this article, we give an introduction to PDS and review evidence that links PDS to pro- as well as anti-epileptic mechanisms, and to other types of neuronal dysfunction. The perturbation of neuronal membrane voltage and of intracellular Ca2+ that comes with PDS offers many conceivable pathomechanisms of neuronal dysfunction. Out of these, the operation of L-type voltage-gated calcium channels, which play a major role in coupling excitation to long-lasting neuronal changes, is addressed in detail.

Correction Volume: 143 Pages: 349-350 DOI: 10.1016/j.neuropharm.2018.10.012 Based on the potential role of Na-K-Cl cotransporters (NKCCs) in epileptic seizures, the loop diuretic bumetanide, which blocks the NKCC1 isoforms NKCC1 and NKCC2, has been tested as an adjunct with phenobarbital to suppress seizures. However, because of its physicochemical properties, bumetanide only poorly penetrates through the blood-brain barrier. Thus, concentrations needed to inhibit NKCC1 in hippocampal and neocortical neurons are not reached when using doses (0.1-0.5 mg/kg) in the range of those approved for use as a diuretic in humans. This prompted us to search for a bumetanide derivative that more easily penetrates into the brain. Here we show that bumepamine, a lipophilic benzylamine derivative of bumetanide, exhibits much higher brain penetration than bumetanide and is more potent than the parent drug to potentiate phenobarbital's anticonvulsant effect in two rodent models of chronic difficult-to-treat epilepsy, amygdala kindling in rats and the pilocarpine model in mice. However, bumepamine suppressed NKCC1-dependent giant depolarizing potentials (GDPs) in neonatal rat hippocampal slices much less effectively than bumetanide and did not inhibit GABA-induced Ca2+ transients in the slices, indicating that bumepamine does not inhibit NKCC1. This was substantiated by an oocyte assay, in which bumepamine did not block NKCC1a and NKCC1b after either extra- or intracellular application, whereas bumetanide potently blocked both variants of NKCC1. Experiments with equilibrium dialysis showed high unspecific tissue binding of bumetanide in the brain, which, in addition to its poor brain penetration, further reduces functionally relevant brain concentrations of this drug. These data show that CNS effects of bumetanide previously thought to be mediated by NKCC1 inhibition can also be achieved by a close derivative that does not share this mechanism. Bumepamine has several advantages over bumetanide for CNS targeting, including lower diuretic potency, much higher brain permeability, and higher efficacy to potentiate the anti-seizure effect of phenobarbital.

International audience; Ethanol-induced neuroapoptosis in the developing brain has been suggested to involve suppression of neuronal activity. However, ethanol acts as a potent stimulant of neuro-nal activity by increasing the frequency of depolarizing GABA dependent giant depolarizing potentials in the neonatal rat hippocampal slices in vitro. Here, we show that ethanol strongly inhibits, in a dose-dependent manner (1-6 g/kg), sharp waves and multiple unit activity in the hippocampus of neonatal (postnatal days P4-6) rats in vivo. Thus, the effects of ethanol on the developing hippocampal network activity cardinally differ in vitro (stimulation) and in vivo (inhibition).

OBJECTIVE Phenobarbital is the most commonly utilized drug for the treatment of neonatal seizures. The use of phenobarbital continues despite growing evidence that it exerts suboptimal seizure control and is associated with long-term alterations in brain structure, function, and behavior. Alterations following neonatal phenobarbital exposure include acute induction of neuronal apoptosis, disruption of synaptic development in the striatum, and a host of behavioral deficits. These behavioral deficits include those in learning and memory mediated by the hippocampus. However, the synaptic changes caused by acute exposure to phenobarbital that lead to lasting effects on brain function and behavior remain understudied. METHODS Postnatal day (P)7 rat pups were treated with phenobarbital (75 mg/kg) or saline. On P13-14 or P29-37, acute slices were prepared and whole-cell patch-clamp recordings were made from CA1 pyramidal neurons. RESULTS At P14 we found an increase in miniature inhibitory postsynaptic current (mIPSC) frequency in the phenobarbital-exposed as compared to the saline-exposed group. In addition to this change in mIPSC frequency, the phenobarbital group displayed larger bicuculline-sensitive tonic currents, decreased capacitance and membrane time constant, and a surprising persistence of giant depolarizing potentials. At P29+, the frequency of mIPSCs in the saline-exposed group had increased significantly from the frequency at P14, typical of normal synaptic development; at this age the phenobarbital-exposed group displayed a lower mIPSC frequency than did the control group. Spontaneous inhibitory postsynaptic current (sIPSC) frequency was unaffected at either P14 or P29+. SIGNIFICANCE These neurophysiological alterations following phenobarbital exposure provide a potential mechanism by which acute phenobarbital exposure can have a long-lasting impact on brain development and behavior.

It is generally thought that hippocampal neurons of perinatal rats and mice lack transport-functional K-Cl cotransporter KCC2, and that chloride regulation is dominated by Cl- uptake on the Na-K-2Cl cotransporter NKCC1. Here we demonstrate robust enhancement of spontaneous hippocampal network events (Giant Depolarizing Potentials; GDPs) induced by the KCC2 inhibitor VU0463271 in neonatal rats and late gestation wild-type mouse embryos, but not in KCC2 null littermates. Our data indicate that KCC2 is instrumental in restraining cellular and network excitability during perinatal GDP activity by mediating dendritic extrusion of Cl- in CA3 pyramidal cells. That dendrites are the main targets of GABAergic interneurons at this stage of development readily explains the functionally inhibitory nature of these early KCC2-dependent synaptic GABA actions.

Spontaneous network activity (SNA) emerges in the spinal cord (SC) before the formation of peripheral sensory inputs and central descending inputs. SNA is characterized by recurrent giant depolarizing potentials (GDPs). Because GDPs in motoneurons (MNs) are mainly evoked by prolonged release of GABA, they likely necessitate sustained firing of interneurons. To address this issue we analyzed, as a model, embryonic Renshaw cell (V1(R)) activity at the onset of SNA (E12.5) in the embryonic mouse SC (both sexes). V1(R) are one of the interneurons known to contact MNs, which are generated early in the embryonic SC. Here, we show that V1(R) already produce GABA in E12.5 embryo, and that V1(R) make synaptic-like contacts with MNs and have putative extrasynaptic release sites, while paracrine release of GABA occurs at this developmental stage. In addition, we discovered that V1(R) are spontaneously active during SNA and can already generate several intrinsic activity patterns including repetitive-spiking and sodium-dependent plateau potential that rely on the presence of persistent sodium currents (I(Nap)). This is the first demonstration that I(Nap) is present in the embryonic SC and that this current can control intrinsic activation properties of newborn interneurons in the SC of mammalian embryos. Finally, we found that 5 μm riluzole, which is known to block I(NaP), altered SNA by reducing episode duration and increasing inter-episode interval. Because SNA is essential for neuronal maturation, axon pathfinding, and synaptogenesis, the presence of I(NaP) in embryonic SC neurons may play a role in the early development of mammalian locomotor networks. SIGNIFICANCE STATEMENT The developing spinal cord (SC) exhibits spontaneous network activity (SNA) involved in the building of nascent locomotor circuits in the embryo. Many studies suggest that SNA depends on the rhythmic release of GABA, yet intracellular recordings of GABAergic neurons have never been performed at the onset of SNA in the SC. We first discovered that embryonic Renshaw cells (V1(R)) are GABAergic at E12.5 and spontaneously active during SNA. We uncover a new role for persistent sodium currents (I(NaP)) in driving plateau potential in V1(R) and in SNA patterning in the embryonic SC. Our study thus sheds light on a role for INaP in the excitability of V1(R) and the developing SC.

Synchronized neuronal activity occurring at different developmental stages in various brain structures represents a hallmark of developmental circuits. This activity, which differs in its specific patterns among animal species may play a crucial role in de novo formation and in shaping neuronal networks. In the rodent hippocampus in vitro, the so-called giant depolarizing potentials or GDPs constitute a primordial form of neuronal synchrony preceding more organized forms of activity such as oscillations in the theta and gamma frequency range. GDPs are generated at the network level by the interaction of the neurotransmitters glutamate and GABA which, immediately after birth, exert both a depolarizing and excitatory.action on their targets. GDPs are triggered by GABAergic interneurons, which in virtue of their extensive axonal branching operate as functional hubs to synchronize large ensembles of cells. Intrinsic bursting activity, driven by a persistent sodium conductance and facilitated by the low expression of Kv7.2 and Kv7.3 channel subunits, responsible for IM , exerts a permissive role in GDP generation. Here, we discuss how GDPs are generated in a probabilistic way when neuronal excitability within a local circuit reaches a certain threshold and how GDP-associated calcium transients act as coincident detectors for enhancing synaptic strength at emerging GABAergic and glutamatergic synapses. We discuss the possible in vivo correlate of this activity. Finally, we debate recent data showing how, in several animal models of neuropsychiatric disorders including autism, a GDPs dysfunction is associated to morphological alterations of neuronal circuits and behavioral deficits reminiscent of those observed in patients.

International audience; During development, hippocampal CA3 network generates recurrent population bursts, so-called Giant Depolarizing Potentials (GDPs). GDPs are characterized by synchronous depolarization and firing of CA3 pyramidal cells followed by afterhyperpolarization (GDP-AHP). Here, we explored the properties of GDP-AHP in CA3 pyramidal cells using gramicidin perforated patch clamp recordings from neonatal rat hippocampal slices. We found that GDP-AHP occurs independently of whether CA3 pyramidal cells fire action potentials (APs) or remain silent during GDPs. However, the amplitude of GDP-AHP increased with the number of APs the cells fired during GDPs. The reversal potential of the GDP-AHP was close to the potassium equilibrium potential. During voltage-clamp recordings, current-voltage relationships of the postsynaptic currents activated during GDP-AHP were characterized by reversal near the potassium equilibrium potential and inward rectification, similar to the responses evoked by the GABA(B) receptor agonists. Finally, the GABA(B) receptor antagonist CGP55845 strongly reduced GDP-AHP and prolonged GDPs, eventually transforming them to the interictal and ictal-like discharges. Together, our findings suggest that the GDP-AHP involves two mechanisms: (i) postsynaptic GABA(B) receptor activated potassium currents, which are activated independently on whether the cell fires or not during GDPs; and (ii) activity-dependent, likely calcium activated potassium currents, whose contribution to the GDP-AHP is dependent on the amount of firing during GDPs. We propose that these two complementary inhibitory postsynaptic mechanisms cooperate in the termination of GDP.

Although functional glycinergic synapses have not been identified in the hippocampus, neurons in this area express Cl(−) permeable extrasynaptic glycine receptors (GlyRs). In experiments on CA3 pyramidal neurons on postnatal day 0–6 rat hippocampal slices, we detected robust GlyR activity as a tonic current and as single-channel events. Glycine release was independent of neuronal activity or extracellular Ca(2+). The endogenous GlyR activity was strongly enhanced by inhibition of the glycine-transporter-1 (GlyT1). Blockade of GlyT1 also caused a profound increase in the baseline current induced by exogenous glycine. Inhibition of GlyT1 reduced the frequency of spontaneous network events known as field giant depolarizing potentials (fGDPs) and of the unit activity in the absence of synaptic transmission. This inhibitory action on fGDPs was mimicked by applying 2 μm glycine or 0.1 μm isoguvacine, a GABA(A)-receptor agonist. Furthermore, 2 μm glycine suppressed unit spiking in the absence of synaptic transmission. Hence, despite the well known depolarizing Cl(−) equilibrium potential of neonatal hippocampal neurons, physiologically relevant extracellular glycine concentrations can exert an inhibitory action. The present data show that, akin to GABA uptake, GlyT1 exerts a powerful modulatory action on network events in the newborn hippocampus.

Many mammals are born in a very immature state and develop their rich repertoire of behavioral and cognitive functions postnatally. This development goes in parallel with changes in the anatomical and functional organization of cortical structures which are involved in most complex activities. The emerging spatiotemporal activity patterns in multi-neuronal cortical networks may indeed form a direct neuronal correlate of systemic functions like perception, sensorimotor integration, decision making or memory formation. During recent years, several studies – mostly in rodents – have shed light on the ontogenesis of such highly organized patterns of network activity. While each local network has its own peculiar properties, some general rules can be derived. We therefore review and compare data from the developing hippocampus, neocortex and – as an intermediate region – entorhinal cortex. All cortices seem to follow a characteristic sequence starting with uncorrelated activity in uncoupled single neurons where transient activity seems to have mostly trophic effects. In rodents, before and shortly after birth, cortical networks develop weakly coordinated multineuronal discharges which have been termed synchronous plateau assemblies (SPAs). While these patterns rely mostly on electrical coupling by gap junctions, the subsequent increase in number and maturation of chemical synapses leads to the generation of large-scale coherent discharges. These patterns have been termed giant depolarizing potentials (GDPs) for predominantly GABA-induced events or early network oscillations (ENOs) for mostly glutamatergic bursts, respectively. During the third to fourth postnatal week, cortical areas reach their final activity patterns with distinct network oscillations and highly specific neuronal discharge sequences which support adult behavior. While some of the mechanisms underlying maturation of network activity have been elucidated much work remains to be done in order to fully understand the rules governing transition from immature to mature patterns of network activity.

International audience; Hydrogen sulfide (H 2 S) is an endogenous gaso-transmitter with neuroprotective properties that participates in the regulation of transmitter release and neuronal excitability in various brain structures. The role of H 2 S in the growth and maturation of neural networks however remains unclear. The aim of the present study is to reveal the effects of H 2 S on neuronal spontaneous activity relevant to neuronal maturation in hippocampal slices of neonatal rats. Sodium hydrosulfide (NaHS) (100 lM), a classical donor of H 2 S produced a biphasic effect with initial activation and subsequent concentration-dependent suppression of network-driven giant depolarizing potentials (GDPs) and neuronal spiking activity. Likewise, the substrate of H 2 S synthesis L-cysteine (1 mM) induced an initial increase followed by an inhibition of GDPs and spiking activity. Our experiments indicate that the increase in initial discharge activity by NaHS is evoked by neuronal depolarization which is partially mediated by a reduction of outward K + currents. The subsequent decrease in the neuronal activity by H 2 S appears to be due to the rightward shift of activation and inactivation of voltage-gated Na + currents, thus preventing network activity. NaHS also reduced N-methyl-D-aspartate (NMDA)-mediated currents, without essential effect on AMPA/kainate or GABA A-mediated currents. Finally, H 2 S abolished the interictal-like events induced by bicuculline. In summary, our results suggest that through the inhibitory action on voltage-gated Na + channels and NMDA receptors, H 2 S prevents the enhanced neuronal excitability typical to early hippocampal networks. Ó

© 2016 IBROHydrogen sulfide (H2S) is an endogenous gasotransmitter with neuroprotective properties that participates in the regulation of transmitter release and neuronal excitability in various brain structures. The role of H2S in the growth and maturation of neural networks however remains unclear. The aim of the present study is to reveal the effects of H2S on neuronal spontaneous activity relevant to neuronal maturation in hippocampal slices of neonatal rats. Sodium hydrosulfide (NaHS) (100 μM), a classical donor of H2S produced a biphasic effect with initial activation and subsequent concentration-dependent suppression of network-driven giant depolarizing potentials (GDPs) and neuronal spiking activity. Likewise, the substrate of H2S synthesis L-cysteine (1 mM) induced an initial increase followed by an inhibition of GDPs and spiking activity. Our experiments indicate that the increase in initial discharge activity by NaHS is evoked by neuronal depolarization which is partially mediated by a reduction of outward K+ currents. The subsequent decrease in the neuronal activity by H2S appears to be due to the rightward shift of activation and inactivation of voltage-gated Na+ currents, thus preventing network activity. NaHS also reduced N-methyl-D-aspartate (NMDA)-mediated currents, without essential effect on AMPA/kainate or GABAA-mediated currents. Finally, H2S abolished the interictal-like events induced by bicuculline. In summary, our results suggest that through the inhibitory action on voltage-gated Na+ channels and NMDA receptors, H2S prevents the enhanced neuronal excitability typical to early hippocampal networks.

The expression of Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is responsible for high intracellular Cl(-) resulting in the excitatory action of GABA(A) receptor activation in the developing brain. Giant depolarizing potentials (GDPs) are spontaneous network oscillations that involve GABA(A) receptors and are thought to be important in establishing neuronal circuit wiring. Earlier work established that seizure susceptibility in the GABA(A) γ2(R43Q) epilepsy mouse is impacted by developmental consequences of impaired GABA(A) receptor function. We investigated the potential mechanism of the developmental influence by recording GDPs in the CA3 pyramidal neurons from brain slices of the neonatal GABA(A) γ2(R43Q) mouse. Interestingly, the number of GPDs was significantly lower in slices from mutant mouse compared with wild-type control, suggesting an involvement in setting seizure susceptibility. To test this idea we blocked NKCC1 with bumetanide in neonatal mice and reduced the number of GDPs to a level similar to that seen in the mutant mice. We found that neonatal treatment with bumetanide resulted in a similar level of susceptibility to thermally induced seizures as described for the GABA(A) γ2(R43Q) mouse. These results provide evidence that a human GABA(A) receptor epilepsy mutation exerts a developmental influence by modulating the number of GDPs. It also draws attention to the potential risk of early treatment with bumetanide.

During the last two decades, it has become widely accepted that GABA, the main inhibitory neurotransmitter in mammalian nervous system, exhibits excitatory action at the early stages of postnatal development. This results from a high intracellular Cl− concentration at these stages of the development and is associated with spontaneous synchronized network discharges known as Giant Depolarizing Potentials (GDPs). It has been hypothesized that the excitatory action of GABAergic system stimulates synaptogenesis and the development of neuronal networks. However, accumulating recent observations challenge this view. Here we present a brief review of the current concepts and problems they face in the light of new data.

Summary It is generally thought that hippocampal neurons of perinatal rats and mice lack transport-functional K-Cl cotransporter KCC2, and that Cl− regulation is dominated by Cl− uptake via the Na-K-2Cl cotransporter NKCC1. Here, we demonstrate a robust enhancement of spontaneous hippocampal network events (giant depolarizing potentials [GDPs]) by the KCC2 inhibitor VU0463271 in neonatal rats and late-gestation, wild-type mouse embryos, but not in their KCC2-null littermates. VU0463271 increased the depolarizing GABAergic synaptic drive onto neonatal CA3 pyramidal neurons, increasing their spiking probability and synchrony during the rising phase of a GDP. Our data indicate that Cl− extrusion by KCC2 is involved in modulation of GDPs already at their developmental onset during the perinatal period in mice and rats., Graphical Abstract, Highlights • Transport-functional KCC2 is present in perinatal CA3 pyramidal neurons • KCC2 restrains depolarizing synaptic GABAergic drive onto CA3 pyramidal neurons • KCC2-mediated Cl− extrusion regulates pyramidal neuron spiking and synchronization • KCC2 regulates GDPs already at their developmental onset, Immature hippocampal pyramidal neurons are thought to lack chloride extrusion mediated by K-Cl cotransporter KCC2. Spoljaric et al. demonstrate that KCC2 restrains the depolarizing GABAergic synaptic drive onto CA3 pyramidal neurons in perinatal mice and rats and thereby regulates spontaneous hippocampal network events (GDPs) from their developmental onset.

J. Neurochem. (2011) 116, 316–321. Abstract Several energy substrates complementary to glucose, including lactate, pyruvate and β-hydroxybutyrate, serve as a fuel for neurons. It was reported recently that these substrates can substantially modulate cortical excitability in neonatal slices. However, complementary energy substrates (CES) can also induce an intracellular acidification when added exogenously. Therefore, action of CES on the neuronal properties governing excitability in neonatal brain slices may be underlain by a change in the cell energy status or by intracellular acidification, or both. Here, we attempt to elucidate these possibilities in neonatal hippocampus by recording neuronal population activity and monitoring intracellular pH. We show that a spontaneous network activity pattern, giant depolarizing potentials (GDPs), characteristic for the neonatal hippocampal slices exposed to artificial cerebrospinal fluid, is strongly inhibited by CES and this effect is unlikely to be caused by a subtle intracellular acidification induced by these compounds. Indeed, a much stronger intracellular acidification in the HCO3-free solution inhibited neither the GDP frequency nor the GDP amplitude. Therefore, modulation of neuronal energy homeostasis is the most likely factor underlying the effect of lactate, pyruvate and β-hydroxybutyrate on network excitability in neonatal brain slices.

Early in postnatal life γ-aminobutyric acid (GABA), the primary inhibitory transmitter in adults, excites targeted neurons by an outwardly directed flux of chloride which results from the unbalance between the cation-chloride cotransporters NKCC1 and KCC2, involved in chloride uptake and extrusion, respectively. This effect contributes to generate synchronized network activity or giant depolarizing potentials (GDPs) in the developing hippocampus. Here, we review some recent data concerning the mechanisms by which GDPs are generated and their functional role in enhancing synaptic efficacy at poorly developed GABAergic and glutamatergic synapses. In adulthood, reshaping neuronal circuits due to changes in chloride homeostasis and to the shift of GABA from hyperpolarizing to depolarizing, has been implicated in several neurological disorders, including epilepsy. Evidence has been recently provided that in chronically nerve growth factor-deprived mice expressing a progressive age-dependent neurodegenerative pathology resembling that observed in patients with Alzheimer's disease, the reduced expression of mRNA encoding for the Kcc2 gene and the depolarizing action of GABA lead to the reorganization of the neuronal hippocampal network. This may represent a novel mechanism by which GABAergic signaling counterbalances the loss of synaptic activity in neurodegenerative diseases.

While the ultimate dependence of brain function on its energy supply is evident, how basic neuronal parameters and network activity respond to energy metabolism deviations is unresolved. The resting membrane potential (E(m)) and reversal potential of GABA-induced anionic currents (E(GABA)) are among the most fundamental parameters controlling neuronal excitability. However, alterations of E(m) and E(GABA) under conditions of metabolic stress are not sufficiently documented, although it is well known that metabolic crisis may lead to neuronal hyper-excitability and aberrant neuronal network activities. In this work, we show that in slices, availability of energy substrates determines whether GABA signaling displays an inhibitory or excitatory mode, both in neonatal neocortex and hippocampus. We demonstrate that in the neonatal brain, E(m) and E(GABA) strongly depend on composition of the energy substrate pool. Complementing glucose with ketone bodies, pyruvate or lactate resulted in a significant hyperpolarization of both E(m) and E(GABA), and induced a radical shift in the mode of GABAergic synaptic transmission towards network inhibition. Generation of giant depolarizing potentials, currently regarded as the hallmark of spontaneous neonatal network activity in vitro, was strongly inhibited both in neocortex and hippocampus in the energy substrate enriched solution. Based on these results we suggest the composition of the artificial cerebrospinal fluid, which bears a closer resemblance to the in vivo energy substrate pool. Our results suggest that energy deficits induce unfavorable changes in E(m) and E(GABA), leading to neuronal hyperactivity that may initiate a cascade of pathological events.

Recent years have witnessed a steep increase in studies on the diverse roles of neuronal cation-chloride cotransporters (CCCs). The versatility of CCC gene transcription, posttranslational modification, and trafficking are on par with what is known about ion channels. The cell-specific and subcellular expression patterns of different CCC isoforms have a key role in modifying a neuron's electrophysiological phenotype during development, synaptic plasticity, and disease. While having a major role in controlling responses mediated by GABA(A) and glycine receptors, CCCs also show close interactions with glutamatergic signaling. A cross-talk among CCCs and trophic factors is important in short-term and long-term modification of neuronal properties. CCCs appear to be multifunctional proteins that are also involved in shaping neuronal structure at various stages of development, from stem cells to synaptogenesis. The rapidly expanding work on CCCs promotes our understanding of fundamental mechanisms that control brain development and functions under normal and pathophysiological conditions.

Early in development, network activity in the hippocampus is characterized by recurrent synchronous bursts, whose cellular correlates are giant depolarizing potentials (GDPs). The propensity for generating GDPs is attributed to GABAergic synaptic transmission being depolarizing and excitatory in neonatal neurons. However, developmental regulation of intrinsic conductances may also influence GDPs generation. A likely candidate is the non-inactivating, low-threshold, muscarinic-sensitive K+ current (M current; Im), which down-regulates intrinsic bursting activity in adult hippocampal pyramidal neurons. Western blot analysis of homogenates of the CA3 hippocampal region showed that expression of the Kv7.2 subunit, one of the constituents of neuronal M channels, is weak in neonatal neurons, and markedly increases after the first postnatal week. Likewise, the density of Im was very low in neonatal CA3 pyramidal cells and increased later on. Spontaneously occurring intrinsic bursts in neonatal neurons were longer and more robust, and recurred more regularly, than in juvenile neurons. The Im blocker linopirdine only mildly affected intrinsic bursting in neonatal neurons, but strongly facilitated and regularized it in juvenile neurons. We conclude that the low expression of Kv7/M channels and the depolarizing action of GABA early after birth enhance intrinsic bursting and neuronal synchronization leading to generation of GDPs within the hippocampal network.

© 2016, Springer Science+Business Media New York.Ethanol exerts multiple adverse effects in the developing hippocampus resulting in the life-long neurological and behavioral deficits. However, the early disturbances in the hippocampal network function after exposure to ethanol remain largely unknown. Here, we examined the properties of the neonatal CA3 hippocampal network-driven giant depolarizing potentials using extracellular recordings of the local field potential and multiple units from the hippocampal slices prepared from the 5–11-day-old rats treated by ethanol in vivo (6 g/kg, intraperitoneally), 12 h before the slice preparation. Activity in hippocampal slices from the ethanol-treated animals displayed several abnormalities including a threefold increase in overall neuronal firing and profound, by nearly sevenfold, reduction of synchronization of CA3 units in giant depolarizing potentials. Thus, alterations in the hippocampal network function emerge shortly after the ethanol exposure and manifest in the enhanced excitation and severe impairments to the giant depolarizing potentials.

In the adult hippocampus, two different forms of GABAA receptor-mediated inhibition have been identified: phasic and tonic. The first is due to the activation of GABAA receptors facing the presynaptic releasing sites, whereas the second is due to the activation of receptors localized away from the synapses. Because of their high affinity and low desensitization rate, extrasynaptic receptors are persistently able to sense low concentrations of GABA. Here we show that, early in postnatal life, between postnatal day (P) 2 and P6, CA1 and CA3 pyramidal cells but not stratum radiatum interneurons, express a tonic GABAA-mediated conductance. Block of the neuronal GABA transporter GAT-1 slightly enhanced the persistent GABA conductance in principal cells but not in GABAergic interneurons. However, in adulthood, a tonic GABAA-mediated conductance could be revealed in stratum radiatum interneurons, indicating that the ability of these cells to sense ambient GABA levels is developmentally regulated. Pharmacological analysis of the tonic conductance in principal cells demonstrated the involvement of β2/β3, α5 and γ2 GABAA receptor subunits. Removal of the tonic depolarizing action of GABA with picrotoxin, reduced the excitability and the glutamatergic drive of principal cells but did not modify the excitability of stratum radiatum interneurons. The increased cell excitability and synaptic activity following the activation of extrasynaptic GABAA receptors by ambient GABA would facilitate the induction of giant depolarizing potentials.

Tonic activation of GABA(A) receptors takes place before the development of functional synapses in cortical structures. We studied whether inefficient GABA uptake might explain the presence of a tonic GABA(A)-mediated current (I(GABA-A)) in early postnatal hippocampal pyramidal neurons. The data show, however, that the tonic I(GABA-A) is enhanced by the specific blocker of GABA transporter-1 (GAT-1), NO-711 (1-[2-[[(Diphenylmethyleneimino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), at birth in rat CA3 pyramidal neurons. NO-711 also prolonged the duration of GABA transients during endogenous hippocampal network events (known as giant depolarizing potentials) at postnatal day 0. The endogenous tonic I(GABA-A) was seen and it was enhanced by NO-711 in the presence of tetrodotoxin, which itself had only a minor effect on the holding current under control conditions. This indicates that the source of interstitial GABA is largely independent of action-potential activity. The tonic I(GABA-A) in neonatal CA3 pyramidal neurons was increased by zolpidem, indicating that at least a proportion of the underlying GABA(A) receptors contain gamma2 and alpha1-alpha3 subunits. The present data point to a significant role for GAT-1 in the control of the excitability of immature hippocampal neurons and networks.

The study of Kirmse et al. (2015) provides long-awaited evidence that GABA acts as an inhibitory neurotransmitter in the neonatal brain—the same way it acts in the adult brain. To recap, the concept of GABA acting as an excitatory neurotransmitter in the neonates dominated research for a long time. Based almost exclusively on the in vitro studies (Zilberter et al., 2010; Bregestovski and Bernard, 2012), extrapolations have leads to the theory of excitatory-to-inhibitory switch of GABA action and its central role in brain maturation (Ben-Ari et al., 2007, 2012). In anesthetized P3-4 mice, Kirmse and colleges first showed that GABA application induced a dramatic, 16-fold drop in cortical plate (CP) cell membrane resistance. Then, using non-invasive patch recordings (cell-attached), the authors found that applied GABA did not generate action potentials (APs) in any of the cells. To avoid confounding factors such as anesthetics and brain damage, the authors employed Ca2+ imaging on N2O-sedated animals keeping the dura-mater intact. In line with electrophysiological data, afferent electrical stimulation readily evoked AP-generated Ca2+ transients in the majority of cells while the cells were nonresponsive in the presence of glutamate receptor blockers, indicating that GABAergic transmission failed to induce APs. In line with these results, the epidural GABA applications failed to induce detectable Ca2+ transients in the vast majority of CP cells in P1 and P3-4 mice. To clarify the mode of GABA action on network activity, the authors simultaneously recorded correlated field and fluorescence (recorded in some cases through the intact skull in the absence of sedation) responses to spontaneous activity in the intact brain. Recordings revealed spindle-like oscillations resembling spindle burst activity previously described in the visual cortex of neonatal rats (Hanganu et al., 2006; Colonnese et al., 2010). Interestingly, the oscillations were not affected by bumetanide (NKCC1 transporter antagonist which modifies the driving force for Cl− currents via GABAA receptor channels) confirming the absence of GABAergic contribution to the spontaneous network excitation. However, diazepam (benzodiazepine strongly increasing the GABAA receptor channel open time) and gabazine (antagonist of GABAA receptors) strongly modified spontaneous activity inducing its inhibition and amplification, respectively. These results are in contrast to those in slices showing inhibition of spontaneous activity by GABAA antagonists [e.g., blockade of so called giant depolarizing potentials, GDPs, by bicuculine and picrotoxine in the original study (Ben-Ari et al., 1989)]. Altogether, the paper results show convincingly that GABAergic transmission provides efficient inhibition of network activity in neonatal mice—definitively the major message of the Kirmse and colleges study. Regarding the mechanism of inhibition, the authors demonstrated powerful shunting induced by GABA in glutamatergic cells. Another potential factor in observed GABA action is a shift in membrane potential. Indeed, in the process of membrane depolarization during neuronal excitation, GABAergic transmission becomes progressively hyperpolarizing due to increasing driving force for Cl− inward currents. In neonatal slices, however, the values for reversal potential of GABA-induced currents have been found to be so positive in respect to the resting potential (Ben-Ari et al., 2007), that GABA application in neurons could both induce generation of APs and efficiently promote spontaneous network activity (GDPs). The concept of excitation/inhibition switch in the GABA action during early development was created exactly on this background. Kirmse and colleges showed that in vivo, the GABA-induced shift in the membrane potential is either hyperpolarizing or depolarizing (in the majority of cells). The depolarization, however, was of a very small value since neither GABA application nor afferent stimulation evoked detectable Ca2+ transients in the vast majority of cells. Therefore, in vivo GABA-induced depolarization is radically different from that found in slices and does not contribute to the cell excitation. Meanwhile, based on the authors' data, it is not possible to conclude that GABA action in P3-4 differs qualitatively from that in P1 or P25-27. Indeed, the larger number of GABA-induced Ca2+ transients at P1 vs. P3-4 (11/117 vs. 6/204) can be easily explained just by a higher membrane resistance of P1 cells, in contrast to the author's suggestion of higher intracellular Cl− concentration at P1. As compared with mature cells, hyperpolarization was found in only one cell from 10 in P25-27 vs. 2/15 in P3-4, making any age difference unconvincing. In addition, comparison of the potential shift values does not have any particular meaning since this method (cell-attached patch) does not provide quantitative measurements (Mason et al., 2005). The evident common feature of all mouse groups was that the potential shifted by GABA was very close to the membrane potential at rest. Therefore, the data provided by the authors so far support neither the hypothesis of the depolarization/hyperpolarization nor the high [Cl−]i/low [Cl−]i developmental switch. Taking into account a small value of GABA-induced potential shift at rest and the absence of significant difference in GABA action between age groups, it is surprising that the authors focused on the issue of “depolarizing GABA.” A shift of few mV above or below membrane potential at rest provided by GABA has an unclear physiological implication and, as the authors agreed, is unlikely to make any significant contribution to the cell excitability. Therefore, association of this shift with the very concept of depolarizing GABA is misleading since the accurate term for neonatal GABA action has always been “excitatory GABA” (Ben-Ari et al., 2007). The excitatory GABA concept has been seriously questioned in several studies (Rheims et al., 2009; Holmgren et al., 2010; Dzhala et al., 2012), which attempted to find out the reason for such “unusual” GABA behavior in slices. These studies suggested that the increased neuronal [Cl−]i in slices may be the consequence of acute injury of neuronal processes or/and inappropriate metabolic conditions. They showed that correction of these abnormalities [e.g., intact hippocampal preparation used by Dzhala and colleges or enhanced energy metabolism in Rheims et al. (2009) and Holmgren et al. (2010)] resulted in GABA-induced inhibition of spontaneous network activity. Importantly, in respect to the resting potential, GABA was slightly depolarizing in the majority of neurons (e.g., Holmgren et al., 2010) and therefore the categorization of these studies as opposing “depolarizing” (Kirmse et al., 2015) instead of “inhibitory” GABA is factually incorrect. To conclude, the study by Kirmse and colleges convincingly demonstrated GABA to be an inhibitory neurotransmitter in both developing and mature animals, which should have a big impact in the field of developmental neuroscience. Many important questions are still left for future studies, e.g., what is the developmental profile of [Cl−]i and how it relates to the resting membrane potential. However, citing the co-authors' previous publication on the subject, “…an absence of GABA-mediated excitation (Rheims et al., 2009; Holmgren et al., 2010) could have major implications for a central hypothesis of developmental neurobiology” (Kirmse et al., 2010). Now, the authors give us the opportunity to reconsider this central hypothesis.