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Brain pH is a critical factor for determining neuronal activity, with alkalosis increasing and acidosis reducing excitability. Acid shifts in brain pH through the breathing of carbogen (5% CO2/95% O2) reduces seizure susceptibility in animal models and patients. The molecular mechanisms underlying this seizure protection remain to be fully elucidated. Here, we demonstrate that male and female mice exposed to carbogen are fully protected from thermogenic-triggered seizures. Whole-cell patch-clamp recordings revealed that acid shifts in extracellular pH (pHo) significantly reduce action potential firing in CA1 pyramidal neurons but did not alter firing in hippocampal inhibitory interneurons. In real-time dynamic clamp experiments, acidification reduced simulated action potential firing generated in hybrid model neurons expressing the excitatory neuron predominant NaV1.2 channel. Conversely, acidification had no effect on action potential firing in hybrid model neurons expressing the interneuron predominant NaV1.1 channel. Furthermore, knockdown ofScn2amRNAin vivousing antisense oligonucleotides reduced the protective effects of carbogen on seizure susceptibility. Both carbogen-mediated seizure protection and the reduction in CA1 pyramidal neuron action potential firing by low pHo were maintained in anAsic1aknock-out mouse ruling out this acid-sensing channel as the underlying molecular target. These data indicate that the acid-mediated reduction in excitatory neuron firing is mediated, at least in part, through the inhibition of NaV1.2 channels, whereas inhibitory neuron firing is unaffected. This reduction in pyramidal neuron excitability is the likely basis of seizure suppression caused by carbogen-mediated acidification.SIGNIFICANCE STATEMENTBrain pH has long been known to modulate neuronal excitability. Here, we confirm that brain acidification reduces seizure susceptibility in a mouse model of thermogenic seizures. Extracellular acidification reduced excitatory pyramidal neuron firing while having no effect on interneuron firing. Acidification also reduced dynamic clamp firing in cells expressing the NaV1.2 channel but not in cells expressing NaV1.1 channels.In vivoknockdown of Scn2a mRNA reduced seizure protection of acidification. In contrast, acid-mediated seizure protection was maintained in the Asic1a knock-out mouse. These data suggest NaV1.2 channel as an important target for acid-mediated seizure protection. Our results have implications on how natural variations in pH can modulate neuronal excitability and highlight potential antiseizure drug development strategies based on the NaV1.2 channel.

Objective: Birth asphyxia is a major cause of hypoxic-ischemic encephalopathy (HIE) in neonates and often associated with mortality, neonatal seizures, brain damage, and later life motor, cognitive, and behavioral impairments and epilepsy. Preclinical studies on rodent models are needed to develop more effective therapies for preventing HIE and its consequences. Thus far, the most popular rodent models have used either exposure of intact animals to hypoxia-only, or a combination of hypoxia and carotid occlusion, for the induction of neonatal seizures and adverse outcomes. However, such models lack systemic hypercapnia, which is a fundamental constituent of birth asphyxia with major effects on neuronal excitability. Here, we use a recently developed noninvasive rat model of birth asphyxia with subsequent neonatal seizures to study later life adverse outcome. Methods: Intermittent asphyxia was induced for 30 min by exposing male and female postnatal day 11 rat pups to three 7 + 3-min cycles of 9% and 5% O-2 at constant 20% CO2. All pups exhibited convulsive seizures after asphyxia. A set of behavioral tests were performed systematically over 14 months following asphyxia, that is, a large part of the rat's life span. Video-electroencephalographic (EEG) monitoring was used to determine whether asphyxia led to the development of epilepsy. Finally, structural brain alterations were examined. Results: The animals showed impaired spatial learning and memory and increased anxiety when tested at an age of 3-14 months. Video-EEG at similar to 10 months showed an abundance of spontaneous seizures, which was paralleled by neurodegeneration in the hippocampus and thalamus, and by aberrant mossy fiber sprouting. Significance: The present model of birth asphyxia recapitulates several of the later life consequences associated with human HIE. This model thus allows evaluation of the efficacy of novel therapies designed to prevent HIE and seizures following asphyxia, and of how such therapies might alleviate long-term adverse consequences.

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.

Aim To study brain-sparing physiological responses in a rodent model of birth asphyxia which reproduces the asphyxia-defining systemic hypoxia and hypercapnia. Methods Steady or intermittent asphyxia was induced for 15-45 min in anesthetized 6- and 11-days old rats and neonatal guinea pigs using gases containing 5% or 9% O2 plus 20% CO2 (in N2). Hypoxia and hypercapnia were induced with low O2 and high CO2, respectively. Oxygen partial pressure (PO2) and pH were measured with microsensors within the brain and subcutaneous (?body?) tissue. Blood lactate was measured after asphyxia. Results Brain and body PO2 fell to apparent zero with little recovery during 5% O2 asphyxia and 5% or 9% O2 hypoxia, and increased more than twofold during 20% CO2 hypercapnia. Unlike body PO2, brain PO2 recovered rapidly to control after a transient fall (rat), or was slightly higher than control (guinea pig) during 9% O2 asphyxia. Asphyxia (5% O2) induced a respiratory acidosis paralleled by a progressive metabolic (lact)acidosis that was much smaller within than outside the brain. Hypoxia (5% O2) produced a brain-confined alkalosis. Hypercapnia outlasting asphyxia suppressed pH recovery and prolonged the post-asphyxia PO2 overshoot. All pH changes were accompanied by consistent shifts in the blood-brain barrier potential. Conclusion Regardless of brain maturation stage, hypercapnia can restore brain PO2 and protect the brain against metabolic acidosis despite compromised oxygen availability during asphyxia. This effect extends to the recovery phase if normocapnia is restored slowly, and it is absent during hypoxia, demonstrating that exposure to hypoxia does not mimic asphyxia.

© 2014 Macmillan Publishers Limited. All rights reserved. Electrical activity in neurons requires a seamless functional coupling between plasmalemmal ion channels and ion transporters. Although ion channels have been studied intensively for several decades, research on ion transporters is in its infancy. In recent years, it has become evident that one family of ion transporters, cation-chloride cotransporters (CCCs), and in particular K + -Cl â ' cotransporter 2 (KCC2), have seminal roles in shaping GABAergic signalling and neuronal connectivity. Studying the functions of these transporters may lead to major paradigm shifts in our understanding of the mechanisms underlying brain development and plasticity in health and disease.

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.

Concepts of epilepsy, based on a simple change in neuronal excitation/inhibition balance, have subsided in face of recent insights into the large diversity and context-dependence of signaling mechanisms at the molecular, cellular and neuronal network level. GABAergic transmission exerts both seizure-suppressing and seizure-promoting actions. These two roles are prone to short-term and long-term alterations, evident both during epileptogenesis and during individual epileptiform events. The driving force of GABAergic currents is controlled by ion-regulatory molecules such as the neuronal K-Cl cotransporter KCC2 and cytosolic carbonic anhydrases. Accumulating evidence suggests that neuronal ion regulation is highly plastic, thereby contributing to the multiple roles ascribed to GABAergic signaling during epileptogenesis and epilepsy.

Seizures are a common manifestation of acute neurologic insults in neonates and are often resistant to the standard antiepileptic drugs that are efficacious in children and adults. The paucity of evidence-based treatment guidelines, coupled with a rudimentary understanding of disease pathogenesis, has made the current treatment of neonatal seizures empiric and often ineffective, highlighting the need for novel therapies. Key developmental differences in γ-aminobutyric acid (GABA)ergic neurotransmission between the immature and mature brain, and trauma-induced alterations in the function of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2, probably contribute to the poor efficacy of standard antiepileptic drugs used in the treatment of neonatal seizures. Although CCCs are attractive drug targets, bumetanide and other existing CCC inhibitors are suboptimal because of pharmacokinetic constraints and lack of target specificity. Newer approaches including isoform-specific NKCC1 inhibitors with increased central nervous system penetration, and direct and indirect strategies to enhance KCC2-mediated neuronal chloride extrusion, might allow therapeutic modulation of the GABAergic system for neonatal seizure treatment. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Objective There is considerable interest in using bumetanide, a chloride importer Na-K-Cl cotransporter antagonist, for treatment of neurological diseases, such as epilepsy or ischemic and traumatic brain injury, that may involve deranged cellular chloride homeostasis. However, bumetanide is heavily bound to plasma proteins (∼98%) and highly ionized at physiological pH, so that it only poorly penetrates into the brain, and chronic treatment with bumetanide is compromised by its potent diuretic effect. Methods To overcome these problems, we designed lipophilic and uncharged prodrugs of bumetanide that should penetrate the blood–brain barrier more easily than the parent drug and are converted into bumetanide in the brain. The feasibility of this strategy was evaluated in mice and rats. Results Analysis of bumetanide levels in plasma and brain showed that administration of 2 ester prodrugs of bumetanide, the pivaloyloxymethyl (BUM1) and N,N-dimethylaminoethylester (BUM5), resulted in significantly higher brain levels of bumetanide than administration of the parent drug. BUM5, but not BUM1, was less diuretic than bumetanide, so that BUM5 was further evaluated in chronic models of epilepsy in mice and rats. In the pilocarpine model in mice, BUM5, but not bumetanide, counteracted the alteration in seizure threshold during the latent period. In the kindling model in rats, BUM5 was more efficacious than bumetanide in potentiating the anticonvulsant effect of phenobarbital. Interpretation Our data demonstrate that the goal of designing bumetanide prodrugs that specifically target the brain is feasible and that such drugs may resolve the problems associated with using bumetanide for treatment of neurological disorders. ANN NEUROL 2014;75:550–562

Network activity in the brain is associated with a transient increase in extracellular K(+) concentration. The excess K(+) is removed from the extracellular space by mechanisms proposed to involve Kir4.1-mediated spatial buffering, the Na(+)/K(+)/2Cl(-) cotransporter 1 (NKCC1), and/or Na(+)/K(+)-ATPase activity. Their individual contribution to [K(+)]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na(+)/K(+)-ATPase and to resolve their involvement in clearance of extracellular K(+) transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K(+)]o increases above basal levels. Increased [K(+)]o produced NKCC1-mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K(+) clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K(+) removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K(+)]o increase. In contrast, inhibition of the different isoforms of Na(+)/K(+)-ATPase reduced post-stimulus clearance of K(+) transients. The astrocyte-characteristic α2β2 subunit composition of Na(+)/K(+)-ATPase, when expressed in Xenopus oocytes, displayed a K(+) affinity and voltage-sensitivity that would render this subunit composition specifically geared for controlling [K(+)]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na(+)/K(+)-ATPase accounted for the stimulus-induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity-induced extracellular K(+) recovery in native hippocampal tissue while Kir4.1 and Na(+)/K(+)-ATPase serve temporally distinct roles.

In cortical and hippocampal neurons, cation-chloride cotransporters (CCCs) control the reversal potential ( E GABA ) of GABA A receptor-mediated current and voltage responses and, consequently, they modulate the efficacy of GABAergic inhibition. Two members of the CCC family, KCC2 (the major neuron-specific K–Cl cotransporter; KCC isoform 2) and NKCC1 (the Na–K–2Cl cotransporter isoform 1 which is expressed in both neurons and glial cells) have attracted much interest in studies on GABAergic signaling under both normal and pathophysiological conditions, such as epilepsy. There is tentative evidence that loop diuretic compounds such as furosemide and bumetanide may have clinically relevant antiepileptic actions, especially when administered in combination with conventional GABA-mimetic drugs such as phenobarbital. Furosemide is a non-selective inhibitor of CCCs while at low concentrations bumetanide is selective for NKCCs. Search for novel antiepileptic drugs (AEDs) is highly motivated especially for the treatment of neonatal seizures which are often resistant to, or even aggravated by conventional AEDs. This review shows that the antiepileptic effects of loop diuretics described in the pertinent literature are based on widely heterogeneous mechanisms ranging from actions on both neuronal NKCC1 and KCC2 to modulation of the brain extracellular volume fraction. A promising strategy for the development of novel CCC-blocking AEDs is based on prodrugs that are activated following their passage across the blood–brain barrier. This article is part of the Special Issue entitled ‘New Targets and Approaches to the Treatment of Epilepsy’.

Birth asphyxia is often associated with a high seizure burden that is predictive of poor neurodevelopmental outcome. The mechanisms underlying birth asphyxia seizures are unknown. Using an animal model of birth asphyxia based on 6-day-old rat pups, we have recently shown that the seizure burden is linked to an increase in brain extracellular pH that consists of the recovery from the asphyxia-induced acidosis, and of a subsequent plateau level well above normal extracellular pH. In the present study, two-photon imaging of intracellular pH in neocortical neurons in vivo showed that pH changes also underwent a biphasic acid-alkaline response, resulting in an alkaline plateau level. The mean alkaline overshoot was strongly suppressed by a graded restoration of normocapnia after asphyxia. The parallel post-asphyxia increase in extra- and intracellular pH levels indicated a net loss of acid equivalents from brain tissue that was not attributable to a disruption of the blood-brain barrier, as demonstrated by a lack of increased sodium fluorescein extravasation into the brain, and by the electrophysiological characteristics of the blood-brain barrier. Indeed, electrode recordings of pH in the brain and trunk demonstrated a net efflux of acid equivalents from the brain across the blood-brain barrier, which was abolished by the Na/H exchange inhibitor, N-methyl-isobutyl amiloride. Pharmacological inhibition of Na/H exchange also suppressed the seizure activity associated with the brain-specific alkalosis. Our findings show that the post-asphyxia seizures are attributable to an enhanced Na/H exchange-dependent net extrusion of acid equivalents across the blood-brain barrier and to consequent brain alkalosis. These results suggest targeting of blood-brain barrier-mediated pH regulation as a novel approach in the prevention and therapy of neonatal seizures.

Patterned neuronal activity such as spindle bursts in the neonatal cortex is likely to promote the maturation of cortical synapses and neuronal circuits. Previous work on cats has shown that removal of subplate neurons, a transient neuronal population in the immature cortex, prevents the functional maturation of thalamocortical and intracortical connectivity. Here we studied the effect of subplate removal in the neonatal rat primary somatosensory cortex (S1). Using intracortical EEG we show that after selective removal of subplate neurons in the limb region of S1, endogenous and sensory evoked spindle burst activity is largely abolished. Consistent with the reducedin vivoactivity in the S1 limb region, we find byin vitrorecordings that thalamocortical inputs to layer 4 neurons are weak. In addition, we find that removal of subplate neurons in the S1 barrel region prevents the development of the characteristic histological barrel-like appearance. Thus, subplate neurons are crucially involved in the generation of particular types of early network activity in the neonatal cortex, which are an important feature of cortical development. The altered EEG pattern following subplate damage could be applicable in the neurological assessment of human neonates.

SUMMARY Purpose: Febrile seizures (FS) are the most common type of convulsive events in children. FS are suggested to result from a combination of genetic and environmental factors. However, the pathophysiologic mechanisms underlying FS remain unclear. Using an animal model of experimental FS, it was demonstrated that hyperthermia causes respiratory alkalosis with consequent brain alkalosis and seizures. Here we examine the acid–base status of children who were admitted to the hospital for FS. Children who were admitted because of gastroenteritis (GE), a condition known to promote acidosis, were examined to investigate a possible protective effect of acidosis against FS. Methods: We enrolled 433 age-matched children with similar levels of fever from two groups presented to the emergency department. One group was admitted for FS (n = 213) and the other for GE (n = 220). In the FS group, the etiology of fever was respiratory tract infection (74.2%), otitis media (7%), GE (7%), tonsillitis (4.2%), scarlet fever (2.3%) chickenpox (1.4%), urinary tract infection (1.4%), postvaccination reaction (0.9%), or unidentified (1.4%). In all patients, capillary pH and blood Pco2 were measured immediately on admission to the hospital. Key Findings: Respiratory alkalosis was found in children with FS (pH 7.46 ± 0.04, [mean ± standard deviation] Pco2 29.5 ± 5.5 mmHg), whereas a metabolic acidosis was seen in all children admitted for GE (pH 7.31 ± 0.03, Pco2 37.7 ± 4.3 mmHg; p < 0.001 for both parameters). No FS were observed in the latter group. A subgroup (n = 15; 7%) of the patients with FS had GE and, notably, their blood pH was more alkaline (pH 7.44 ± 0.04) than in the GE-admitted group. During the enrollment period, eight of the patients were admitted on separate occasions because of FS or GE. Consistent with the view that generation of FS requires a genetic susceptibility in addition to acute seizure triggering factors, each of these patients had an alkalotic blood pH when admitted because of FS, whereas they had an acidotic pH (and no FS) when admitted because of GE (pH 7.47 ± 0.05 vs. pH 7.33 ± 0.03, p < 0.005). Significance: The results show that FS are associated with a systemic respiratory alkalosis, irrespective of the severity of the underlying infection as indicated by the level of fever. The lack of FS in GE patients is attributable to low pH, which also explains the fact that children with a susceptibility to FS do not have seizures when they have GE-induced fever that is associated with acidosis. The present demonstration of a close link between FS and respiratory alkalosis may pave the way for further clinical studies and attempts to design novel therapies for the treatment of FS by controlling the systemic acid–base status.

During neuronal maturation, the neuron-specific K–Cl co-transporter KCC2 lowers the intracellular chloride and thereby renders GABAergic transmission hyperpolarizing. Independently of its role as a co-transporter, KCC2 plays a crucial role in the maturation of dendritic spines, most probably via an interaction with the cytoskeleton-associated protein 4.1N. In this study, we show that neural-specific overexpression of KCC2 impairs the development of the neural tube- and neural crest-related structures in mouse embryos. At early stages (E9.5–11.5), the transgenic embryos had a thinner neural tube and abnormal body curvature. They displayed a reduced neuronal differentiation and altered neural crest cell pattern. At later stages (E11.5–15.5), the transgenic embryos had smaller brain structures and a distinctive cleft palate. Similar results were obtained using overexpression of a transport-inactive N-terminal-deleted variant of KCC2, implying that the effects were not dependent on KCC2′s role as a K–Cl co-transporter. Interestingly, the neural tube of transgenic embryos had an aberrant cytoplasmic distribution of 4.1N and actin. This was corroborated in a neural stem cell line with ectopic expression of KCC2. Embryo phenotype and cell morphology were unaffected by a mutated variant of KCC2 which is unable to bind 4.1N. These results point to a role of KCC2 in neuronal differentiation and migration during early development mediated by its direct structural interactions with the neuronal cytoskeleton.

GABAergic excitatory [K+]o transients can be readily evoked in the mature rat hippocampus by intense activation of GABAA receptors (GABAARs). Here we show that these [K+]o responses induced by high-frequency stimulation or GABAA agonist application are generated by the neuronal K+–Cl− cotransporter KCC2 and that the transporter-mediated KCl extrusion is critically dependent on the bicarbonate-driven accumulation of Cl− in pyramidal neurons. The mechanism underlying GABAergic [K+]o transients was studied in CA1 stratum pyramidale using intracellular sharp microelectrodes and extracellular ion-sensitive microelectrodes. The evoked [K+]o transients, as well as the associated afterdischarges, were strongly suppressed by 0.5–1 mm furosemide, a KCl cotransport inhibitor. Importantly, the GABAAR-mediated intrapyramidal accumulation of Cl−, as measured by monitoring the reversal potential of fused IPSPs, was unaffected by the drug. It was further confirmed that the reduction in the [K+]o transients was not due to effects of furosemide on the Na+-dependent K+-Cl− cotransporter NKCC1 or on intraneuronal carbonic anhydrase activity. Blocking potassium channels by Ba2+ enhanced [K+]o transients whereas pyramidal cell depolarizations were attenuated in further agreement with a lack of contribution by channel-mediated K+ efflux. The key role of the GABAAR channel-mediated anion fluxes in the generation of the [K+]o transients was examined in experiments where bicarbonate was replaced with formate. This anion substitution had no significant effect on the rate of Cl− accumulation, [K+]o response or afterdischarges. Our findings reveal a novel excitatory mode of action of KCC2 that can have substantial implications for the role of GABAergic transmission during ictal epileptiform activity.

In the majority of neurons, the intracellular Cl(-) concentration is set by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the K(+)-Cl(-) cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments. In contrast, KCC2 was found in the insoluble fraction as well as in the soluble fraction, where it co-distributed with the non-raft marker transferrin receptor. Both KCC2 populations displayed a mature glycosylation pattern. Disrupting membrane rafts with methyl-beta-cyclodextrin (MbetaCD) increased the solubility of KCC2, yet had no effect on NKCC1. In human embryonic kidney-293 cells, KCC2 was strongly activated by a combined treatment with MbetaCD and sphingomyelinase, while NKCC1 was inhibited. These data indicate that membrane rafts render KCC2 inactive and NKCC1 active. In agreement with this, inactive KCC2 of the perinatal rat brainstem largely partitioned into membrane rafts. In addition, the exposure of the transporters to MbetaCD and sphingomyelinase showed that the two transporters differentially interact with the membrane rafts. Taken together, membrane raft association appears to represent a mechanism for co-ordinated regulation of chloride transporter function.

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.

SummaryThe neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl− transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

The major part of the brain's energy budget ( approximately 60%-80%) is devoted to its communication activities. While inhibition is critical to brain function, relatively little attention has been paid to its metabolic costs. Understanding how inhibitory interneurons contribute to brain energy consumption (brain work) is not only of interest in understanding a fundamental aspect of brain function but also in understanding functional brain imaging techniques which rely on measurements related to blood flow and metabolism. Herein we examine issues relevant to an assessment of the work performed by inhibitory interneurons in the service of brain function.

An activation study of the human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes VII and XIV using a small library of natural/non-natural amino acids and aromatic/heterocyclic amines is reported. hCA VII was efficiently activated by L-/D-His, dopamine and serotonin (K(A)s of 0.71-0.93 microM). The best hCA XIV activators were histamine (K(A) of 10 nM), L-Phe, L-/D-His and 4-amino-L-Phe (K(A)s of 0.24-2.90 microM). In view of the significant expression levels of CA VII and CA XIV in the brain, selective activation of these isoforms may be useful when developing pharmacologic agents for the management of major disorders such as epilepsy and Alzheimer's disease.

After having been in routine use for about half a century, neonatal EEG is currently facing unprecedented challenges in assessing and monitoring brain function during intensive care of preterm babies. It has therefore become increasingly important to understand the neurophysiological processes underlying EEG activity, as well as to identify those features of brain activity that are essential for brain development. By integrating the existing literature from basic neuroscience to neonatal EEG, the present review proposes a simple, neurophysiologically and neuroanatomically based framework for neonatal EEG interpretation. This is composed of two developmental trajectories: one related to discrete spontaneous activity transients (SAT) and the other to the ongoing, apparently oscillatory EEG activity. This framework can readily be applied to clinical use. It may open novel avenues to automated analysis in EEG monitoring and, moreover, it may facilitate genuine translational research.

Febrile seizures are frequent during early childhood, and prolonged (complex) febrile seizures are associated with an increased susceptibility to temporal lobe epilepsy. The pathophysiological consequences of febrile seizures have been extensively studied in rat pups exposed to hyperthermia. The mechanisms that trigger these seizures are unknown, however. A rise in brain pH is known to enhance neuronal excitability. Here we show that hyperthermia causes respiratory alkalosis in the immature brain, with a threshold of 0.2–0.3 pH units for seizure induction. Suppressing alkalosis with 5% ambient CO2 abolished seizures within 20 s. CO2 also prevented two long-term effects of hyperthermic seizures in the hippocampus: the upregulation of the Ih current and the upregulation of CB1 receptor expression. The effects of hyperthermia were closely mimicked by intraperitoneal injection of bicarbonate. Our work indicates a mechanism for triggering hyperthermic seizures and suggests new strategies in the research and therapy of fever-related epileptic syndromes.

Earlier studies indicate a crucial role for the interconnected network of intrinsically bursting CA3 pyramidal neurons in the generation of in vivo hippocampal sharp waves (SPWs) and their proposed neonatal in vitro counterparts, the giant depolarizing potentials (GDPs). While mechanisms involving ligand- and voltage-gated channels have received lots of attention in the generation of CA3 network events in the immature hippocampus, the contribution of ion-transport mechanisms has not been extensively studied. Here, we show that bumetanide, a selective inhibitor of neuronal Cl− uptake mediated by the Na+–K+–2Cl− cotransporter isoform 1 (NKCC1), completely and reversibly blocks SPWs in the neonate (postnatal days 7–9) rat hippocampus in vivo, an action also seen on GDPs in slices (postnatal days 1–8). These findings strengthen the view that GDPs and early SPWs are homologous events. Gramicidin-perforated patch recordings indicated that NKCC1 accounts for a large (∼10 mV) depolarizing driving force for the GABAA current in the immature CA3 pyramids. Consistent with a reduction in the depolarization mediated by endogenous GABAA-receptor activation, bumetanide inhibited the spontaneous bursts of individual neonatal CA3 pyramids, but it slightly increased the interneuronal activity as seen in the frequency of spontaneous GABAergic currents. An inhibitory effect of bumetanide was seen on the in vitro population events in the absence of synaptic GABAA receptor-mediated transmission, provided that a tonic GABAA receptor-mediated current was present. Our work indicates that NKCC1 expressed in CA3 pyramidal neurons promotes network activity in the developing hippocampus.

The CA3 area of the mature hippocampus is known for its ability to generate intermittent network activity both in physiological and in pathological conditions. We have recently shown that in the early postnatal period, the intrinsic bursting of interconnected CA3 pyramidal neurons generates network events, which were originally called giant depolarizing potentials (GDPs). The voltage-dependent burst activity of individual pyramidal neurons is promoted by the well-known depolarizing action of endogenous GABA on immature neurons. In the present work, we show that a persistent Na + current, I-Nap, accounts for the slow regenerative depolarization that triggers the intrinsic bursts in the neonatal rat CA3 pyramidal neurons (postnatal day 3-6), while a slow Ca 2+ -activated K + current, sl-K(Ca), is primarily responsible for the postburst slow afterhyperpolarization and consequent burst termination. In addition, we exploited pharmacological data obtained from intracellular recordings to study the mechanisms involved in network events recorded with field potential recordings. The data as a whole indicate that I-Nap and sl-K(Ca) are involved in the initiation and termination, respectively, of the pyramidal bursts and consequent network events underlying GDPs.

A hallmark in the development of GABAergic neurotransmission is the switch in GABA A -mediated responses from depolarizing to hyperpolarizing. This occurs due to a gradual decrease in the intracellular concentration of chloride caused by the functional expression of the neuron-specific K-Cl cotransporter KCC2. Whether a mere increase in the amount of KCC2 protein is the rate-limiting step in vivo, or a further activation of the otherwise nonfunctional cotransporter is required, is not clear. Imposing a fixed Cl - load via patch pipette we measured the resultant somato-dendritic gradients in reversal potential of GABAergic currents to determine the time course of functional maturation of KCC2-mediated Cl - extrusion in two preparations: cultured mouse hippocampal neurons plated at embryonic day 17 and CA1 pyramidal cells in acute slices. We found that in immature neurons in both preparations the gradient is initially small or not detectable. It undergoes an abrupt increase at around days 13-14 in culture, while a more gradual increase occurs between postnatal days 5-14 in slices. Consistent with the presence of a nonfunctional form of KCC2 in immature hippocampal neurons grown in culture, application of the broad-spectrum kinase inhibitor staurosporine produces a rapid and potent up-regulation of KCC2 function in these cultured neurons, but not in neonatal slices. Taken together with our previously published data, these results indicate that the functional activity of KCC2 in vivo parallels the developmental expression of the protein, whereas cultured neurons require an additional activation step (mimicked by staurosporine) for KCC2 to become functional.

The inhibition of a newly cloned human carbonic anhydrase (CA, EC 4.2.1.1), isozyme VII (hCA VII), has been investigated with a series of aromatic and heterocyclic sulfonamides, including some of the clinically used derivatives (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide and benzolamide), as well as the sulfamate antiepileptic drug topiramate. Inhibition data for the the other physiologically relevant cytosolic isoforms hCA I, hCA II and mCA XIII are also provided for comparison. hCA VII shows a high catalytic activity for the CO(2) hydration reaction, with a k(cat) of 9.5 x 10(5)s(-1) and k(cat)/K(m) of 8.3 x 10(7)M(-1)s(-1) at pH7.5 and 20 degrees C. A very interesting inhibition profile against hCA VII with this series of 32 sulfonamides/sulfamates was observed. hCA VII shows high affinity for all the investigated compounds, with inhibition constants in the range of 0.45-210 nM. Topiramate, ethoxzolamide and benzolamide showed subnanomolar hCA VII inhibitory activity, whereas acetazolamide, methazolamide, dorzolamide and brinzolamide showed K(I)-s in the range of 2.1-3.5 nM. Dichlorophenamide was slightly less active (K(I) of 26.5 nM). A number of heterocyclic or bicyclic aromatic sulfonamides also showed excellent hCA VII inhibitory properties (K(I)-s in the range of 4.3-7.0 nM) whereas many monosubstituted or disubstituted benzenesulfonamides were less active (K(I)-s in the range of 45-89 nM). The least active hCA VII inhibitors were some substituted benzene-1,3-disulfonamides as well as some halogenated sulfanilamides (K(I)-s in the range of 100-210 nM). The inhibition profile of hCA VII is rather different of that of the other cytosolic isozymes, providing thus a possibility for the design of more selective, hCA VII-specific inhibitors. In addition, these data furnish further evidence that hCA VII is the isozyme responsible for the anticonvulsant/antiepileptic activity of sulfonamides and sulfamates.

We studied the mechanisms underlying CO2-dependent DC potential shifts, using epicranial, epidural, epicortical, intraventricular, and intraparenchymal (intraneuronal, intraglial, and field) recordings in ketamine–xylazine-anesthetized cats. DC shifts were elicited by changes in artificial ventilation, causing end-tidal CO2variations within a 2–5% range. Hypercapnia was consistently associated with negative scalp DC shifts (average shift −284.4 μV/CO2%, range −216 to −324 μV/CO2%), whereas hypocapnia induced positive scalp DC shifts (average shift 307.8 μV/CO2%, range 234 to 342 μV/CO2%) in all electrodes referenced versus the nasium bone. The former condition markedly increased intracranial pressure (ICP), whereas the latter only slightly reduced ICP. Breakdown of the blood–brain barrier (BBB) resulted in a positive DC shift and drastically reduced subsequent DC responses to hypo-/hypercapnia. Thiopental and isoflurane also elicited a dose-dependent positive DC shift and, at higher doses, hypo-/hypercapnia responses displayed reverted polarity. As to the possible implication of neurons in the production of DC shifts, no polarity reversal was recorded between scalp, various intracortical layers, and deep brain structures. Moreover, the membrane potential of neurons and glia did not show either significant or systematic variations in association with the scalp-recorded CO2-dependent DC shifts. Pathological activities of neurons during spike-wave seizures produced DC shifts of significantly smaller amplitude than those generated by hyper-/hypocapnia. DC shifts were still elicited when neuronal circuits were silent during anesthesia-induced burst-suppression patterns. We suggest that potentials generated by the BBB are the major source of epicortical/cranial DC shifts recorded under conditions affecting brain pH and/or cerebral blood flow.

The human brain spontaneously generates large-scale network oscillations at around 10 and 20 Hz. The amplitude envelope of these oscillations fluctuates intermittently and was recently reported to exhibit power-law decay of the autocorrelation for hundreds of seconds. This indicates that the underlying networks are in a dynamic state resembling the self-organized critical state known to exist in many complex systems. Based on the mechanism of how correlations emerge in these systems, we hypothesized that the physiological basis of long-range power-law correlations is the buildup of a memory of past activity by a continuous modification of the network's functional connectivity by the ongoing oscillations. In this framework, exogenous perturbations of ongoing oscillations would degrade or abolish this dynamic network memory. We investigated the sensitivity of the temporal correlations in sensorimotor 10- and 20-Hz oscillations to median nerve stimulation that is known to have immediate effects on ongoing oscillations. Our results show that the amplitude fluctuations of these oscillations were effectively modulated by the somatosensory stimuli but still exhibited long-range temporal correlations and power-law scaling behaviour. The magnitude of the temporal correlations was, however, attenuated and the power-law exponents were decreased. This implies that the stimuli indeed degraded the network's memory of its past.

Summary: Purpose: To study whether respiratory alteration caused by vagal nerve stimulation (VNS) can change end-tidal carbon dioxide (EtCO2) levels. Methods: We performed polygraphic recordings including capnographic monitoring during daytime sleep on adults with VNS therapy. Results: Ten of 13 patients showed VNS-induced alterations in the frequency or amplitude of respiration. Five patients had a consistent increase in respiratory rate with a simultaneous, consistent and significant decrease (p < 0.01; 5–22%) in EtCO2 during VNS. Three subjects showed occasional decreases in EtCO2 during VNS, and two showed no clearly detectable VNS-related EtCO2 changes. Conclusions: Our findings suggest that VNS may alter brain CO2 levels through changes in respiration. Because carbon dioxide (CO2) has potent effects on various brain functions, it is possible that these transient CO2 changes may have an effect on the state transitions between interictal and preictal states.

Objective : To study whether hemodynamic changes in human brain generate scalp-EEG responses. Methods : Direct current EEG (DC-EEG) was recorded from 12 subjects during 5 non-invasive manipulations that affect intracranial hemodynamics by different mechanisms: bilateral jugular vein compression (JVC), head-up tilt (HUT), head-down tilt (HDT), Valsalva maneuver (VM), and Mueller maneuver (MM). DC shifts were compared to changes in cerebral blood volume (CBV) measured by near-infrared spectroscopy (NIRS). Results : DC shifts were observed during all manipulations with highest amplitudes (up to 250 μV) at the midline electrodes, and the most pronounced changes (up to 15 μV/cm) in the DC voltage gradient around vertex. In spite of inter-individual variation in both amplitude and polarity, the DC shifts were consistent and reproducible for each subject and they showed a clear temporal correlation with changes in CBV. Conclusions : Our results indicate that hemodynamic changes in human brain are associated with marked DC shifts that cannot be accounted for by intracortical neuronal or glial currents. Instead, the data are consistent with a non-neuronal generator mechanism that is associated with the blood–brain barrier. Significance : These findings have direct implications for mechanistic interpretation of slow EEG responses in various experimental paradigms.