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In a study using a heated chamber for induction of experimental febrile seizures (eFS) in rat pups, ictal activity was shown to be precipitated by a respiratory alkalosis (Schuchmann et al., 2006). In sharp contrast to this, in a recent review Dubé et al., (2007) suggest that the respiratory alkalosis is model specific, and that no increase in respiratory rate is observed in the widely used "hair dryer model" of eFS. The data in the present work, based on well-established techniques for measuring respiratory rates in rat pups, show a pronounced increase in the "hair dryer model" with values that are slightly higher than those recorded in the heated chamber model. Hence, a temperature-evoked increase in respiration is a common feature of these two models of eFS.

It is well established that voluntary hyperventilation (HV) slows down electroencephalographic (EEG) rhythms. Little information is available, however, on the effects of HV on cortical responses elicited by sensory stimulation. In the present study, we recorded auditory evoked potentials (AEPs) and magnetic fields (AEFs), and somatosensory evoked magnetic fields (SEFs) from healthy subjects before, during, and after a 3- to 5-min period of voluntary HV. The effectiveness of HV was verified by measuring the end-tidal CO2 levels. Long-latency (100–200 ms) AEPs and long-latency AEFs originating at the supratemporal auditory cortex, as well as long-latency SEFs from the primary somatosensory cortex (SI) and from the opercular somatosensory cortex (OC), were all reduced during HV. The short-latency SEFs from SI were clearly less modified, there being, however, a slight reduction of the earliest cortical excitatory response, the N20m deflection. A middle-latency SEF deflection from SI at about 60 ms (P60 m) was slightly increased. For AEFs and SEFs, the center-of-gravity locations of the activated neuronal populations were not changed during HV. All amplitude changes returned to baseline levels within 10 min after the end of HV. The AEPs were not altered when the subjects breathed 5% CO2 in air in a hyperventilation-like manner, which prevented the development of hypocapnia. We conclude that moderate HV suppresses long-latency evoked responses from the primary projection cortices, while the early responses are less reduced. The reduction of long-latency responses is probably mediated by hypocapnia rather than by other nonspecific effects of HV. It is suggested that increased neuronal excitability caused by HV-induced hypocapnia leads to spontaneous and/or asynchronous firing of cortical neurones, which in turn reduces stimulus-locked synaptic events.

Synaptic activation of GABAA receptors induces neuronal uptake of Ca2+ in adult rat hippocampal slices. Synaptically evoked transmembrane movements of Ca2+ in the adult CNS have almost exclusively been attributed to activation of glutamate receptor channels and the consequent triggering of voltage-gated calcium channels (VGCCs). Using microelectrodes for measuring free extracellular Ca2+([Ca2+]o) and extracellular space (ECS) volume, we show here for the first time that synaptic stimulation of γ-aminobutyric acid-A (GABAA) receptors can result in a decrease in [Ca2+]o in adult rat hippocampal slices. High-frequency stimulation (100–200 Hz, 0.4–0.5 s) applied in stratum radiatum close (≤0.5 mm) to the recording site induced a 0.1- to 0.3-mM transient fall in [Ca2+]o from a baseline level of 1.6 mM. Concomitantly, a 30–40% decrease in the ECS volume was seen. Exposure of drug-naı̈ve slices to the GABAA receptor antagonist picrotoxin (100 μM) first attenuated and only thereafter augmented the Ca2+ shifts. Application of ionotropic glutamate receptor antagonists resulted in a monotonic reduction of the Ca2+ response, but a large Ca2+ shift persisted (60–70% of the original), which was attenuated by a subsequent application of picrotoxin or bicuculline. In the absence of ionotropic glutamatergic transmission, pentobarbital sodium (100 μM), an up-modulator of the GABAA receptor, strongly enhanced the activity-evoked changes in [Ca2+]o. We suggest that the underlying mechanism of GABA-induced Ca2+ transients is the activation of VGCCs by bicarbonate-dependent GABA-mediated depolarizing postsynaptic potentials. Accordingly, stimulation-evoked Ca2+ shifts were inhibited by the membrane-permeant inhibitor of carbonic anhydrase, ethoxyzolamide (50 μM) or in N-2-hydroxyethylpiperazine- N′-2-ethanesulfonic acid (HEPES)–buffered HCO3-free solution. Neuronal Ca2+ uptake caused by intense synaptic activation of GABAA receptors may prove to be an important mechanism in the modulation of activity-dependent neuronal plasticity, epileptogenesis, and cell survival in the adult brain.

The gamma-aminobutyric acid (GABA) receptor rho subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABA(C). In this work we have examined the distribution of rho1, rho2 and rho3 subunits, and found expression of all three transcripts in several regions of the rat nervous system. In situ hybridization revealed expression of rho2 in the adult rat retina and some other parts of the visual pathways. A high local rho2 expression was seen in the superficial grey layer of the superior colliculus, and in the dorsal lateral geniculate nucleus. Expression was also detected in the 6th layer of visual cortex and in the CA1 pyramidal cell layer of hippocampus. With reverse transcriptase-polymerase chain reaction, expression of rho1 was mainly seen in the adult rat retina and dorsal root ganglia, as well as, at a significantly lower level, in the superior colliculus, hippocampus, brain stem, thalamus, postnatal day 8 (P8) superior colliculus and P8 hippocampus. Expression pattern of rho3 mRNA was clearly different from that of rho1 and rho2, being strongest in the hippocampus, and significantly lower in the retina, dorsal root ganglia and cortex. No rho3 expression was observed in adult or P8 superior colliculus or in P8 hippocampus. The present results clearly demonstrate that expression of GABA receptor rho subunits is not restricted to the retina, but significant expression can also be detected in many other brain regions, especially in those belonging to the visual pathways. The expression pattern of the rho subunits may be helpful in solving the functional significance of the receptors formed from these subunits.

This unit presents an improved method for quantitative analysis of surface expression of membrane proteins utilizing a cold-adapted trypsin. Preservation of the proteolytic activity of the enzyme at 0° to 4°C allows cleavage of surface-expressed membrane proteins at temperatures at which trafficking of the mammalian plasmalemmal proteins is blocked. This provides an important advantage over established trypsin-cleavage protocols since it can be applied to membrane proteins with a fast turnover rate of the membrane pool and a fast recycling rate. Compared to surface biotinylation, the method is less time consuming. Curr. Protoc. Protein Sci. 73:3.11.1-3.11.12. © 2013 by JohnWiley & Sons, Inc. Keywords: membrane protein; surface expression; trafficking; trypsin; biotinylation; BS3; surface analysis

H(+) ions are remarkably efficient modulators of neuronal excitability. This renders brain functions highly sensitive to small changes in pH which are generated "extrinsically" via mechanisms that regulate the acid-base status of the whole organism; and "intrinsically", by activity-induced transmembrane fluxes and de novo generation of acid-base equivalents. The effects of pH changes on neuronal excitability are mediated by diverse, largely synergistically-acting mechanisms operating at the level of voltage- and ligand-gated ion channels and gap junctions. In general, alkaline shifts induce an increase in excitability which is often intense enough to trigger epileptiform activity, while acidosis has the opposite effect. Brain pH changes show a wide variability in their spatiotemporal properties, ranging from long-lasting global shifts to fast and highly localized transients that take place in subcellular microdomains. Thirteen catalytically-active mammalian carbonic anhydrase isoforms have been identified, whereof 11 are expressed in the brain. Distinct CA isoforms which have their catalytic sites within brain cells and the interstitial fluid exert a remarkably strong influence on the dynamics of pH shifts and, consequently, on neuronal functions. In this review, we will discuss the various roles of H(+) as an intra- and extracellular signaling factor in the brain, focusing on the effects mediated by CAs. Special attention is paid on the developmental expression patterns and actions of the neuronal isoform, CA VII. Studies on the various functions of CAs will shed light on fundamental mechanisms underlying neuronal development, signaling and plasticity; on pathophysiological mechanisms associated with epilepsy and related diseases; and on the modes of action of CA inhibitors used as CNS-targeting drugs.

H+ ions are remarkably efficient modulators of neuronal excitability. This renders brain functions highly sensitive to small changes in pH which are generated “extrinsically” via mechanisms that regulate the acid–base status of the whole organism; and “intrinsically”, by activity-induced transmembrane fluxes and de novo generation of acid–base equivalents. The effects of pH changes on neuronal excitability are mediated by diverse, largely synergistically-acting mechanisms operating at the level of voltage- and ligand-gated ion channels and gap junctions. In general, alkaline shifts induce an increase in excitability which is often intense enough to trigger epileptiform activity, while acidosis has the opposite effect. Brain pH changes show a wide variability in their spatiotemporal properties, ranging from long-lasting global shifts to fast and highly localized transients that take place in subcellular microdomains. Thirteen catalytically-active mammalian carbonic anhydrase isoforms have been identified, whereof 11 are expressed in the brain. Distinct CA isoforms which have their catalytic sites within brain cells and the interstitial fluid exert a remarkably strong influence on the dynamics of pH shifts and, consequently, on neuronal functions. In this review, we will discuss the various roles of H+ as an intra- and extracellular signaling factor in the brain, focusing on the effects mediated by CAs. Special attention is paid on the developmental expression patterns and actions of the neuronal isoform, CA VII. Studies on the various functions of CAs will shed light on fundamental mechanisms underlying neuronal development, signaling and plasticity; on pathophysiological mechanisms associated with epilepsy and related diseases; and on the modes of action of CA inhibitors used as CNS-targeting drugs.

Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate-driven GABAergic excitation during intense GABAA-receptor activation. P13–14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13–P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis-dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age-dependent neuronal pH regulation with consequent effects on generation of FS., Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures Carbonic anhydrase VII plays a key role in age-dependent neuronal pH regulation, promoting excitatory GABA transmission and febrile seizures.

The K–Cl cotransporter KCC2 establishes the low intraneuronal Cl – levels required for the hyperpolarizing inhibitory postsynaptic potentials mediated by ionotropic γ-aminobutyric acid receptors (GABA A Rs) and glycine receptors (GlyRs). Decreased KCC2-mediated Cl – extrusion and impaired hyperpolarizing GABA A R- and/or GlyR-mediated currents have been implicated in epilepsy, neuropathic pain, and spasticity. Recent evidence suggests that the intrinsic ion transport rate, cell surface stability, and plasmalemmal trafficking of KCC2 are rapidly and reversibly modulated by the (de)phosphorylation of critical serine, threonine, and tyrosine residues in the C terminus of this protein. Alterations in KCC2 phosphorylation have been associated with impaired KCC2 function in several neurological diseases. Targeting KCC2 phosphorylation directly or indirectly via upstream regulatory kinases might be a novel strategy to modulate GABA- and/or glycinergic signaling for therapeutic benefit.

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.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.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.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.

We have studied the effect of extracellular pH (pHo) on the GABAA receptor-mediated chloride conductance in acutely isolated pyramidal neurons from area CA1 of the rat hippocampus under whole-cell voltage clamp in bicarbonate-free solutions. The conductance evoked by saturating or near-saturating concentrations (200-1000 microM) of GABA showed a marked sensitivity to variations of pHo around 7.4. A decrease in pHo between 8.4 and 6.4 increased the GABAA receptor-mediated chloride conductance by about two-fold per pH unit. In contrast, when evoked by a low agonist concentration (1-10 microM) the conductance showed an equally marked decrease upon a decrease in pHo. The half-time for desensitization of the conductance induced by 500 microM GABA was around 900 ms at pHo 6.4 and 7.4, but decreased to 650 ms at pHo 8.4. A fall in pHo decreased the amount of desensitization of the conductance evoked by a 5 s application of 5 microM, but not of 500 microM, GABA. The concentration-response relationship of the GABA-induced conductance showed a local plateau between 50 and 100 microM of GABA, which was particularly evident at high pHo. Assuming two receptor populations with a high and a low affinity for GABA, the effect of H+ on the GABAA receptors could be explained as an increase in the EC50 of the high affinity receptor, and an apparently non-competitive potentiation of both the high and the low affinity receptors. The GABAA receptor-mediated conductance was markedly inhibited by 20-50 microM Zn2+. In addition, Zn2+ reverted the down-modulation by H+ observed at low GABA concentrations to up-modulation. Diazepam (1-10 microM) had only a marginal effect on the GABA-gated conductance. Taken together, the results suggest the coexistence in individual hippocampal neurons of two distinct GABAA receptor populations having differential sensitivities to H+. In the light of the inhibitory action of Zn2+ and the virtual absence of an effect of diazepam it is probable that a significant fraction of the GABAA receptors lack the gamma 2 subunit. The observation that an elevated pH has a strong suppressing effect on the conductance evoked by high concentrations of GABA may at least partly explain why an extracellular alkalosis leads to neuronal hyperexcitability.

It is generally known that hyperventilation produces an increase in neuronal excitability. However, the mechanism whereby a change in CO2 partial pressure (Pco2) leads to changes in neural excitability is not known. We have studied this phenomenon in rat hippocampal slices using double-barrelled microelectrodes for simultaneous recording of field excitatory postsynaptic potentials (EPSPs) and extracellular pH in stratum radiatum of area CAI. A drop in Pco2 from the control level, 36 mmHg to 7 mmHg, produced an increase in extracellular pH of 0.4–0.6 pH units and a transient increase in EPSP slope by about 20–30%. Despite the stable extracellular alkalosis, the EPSP reverted back to its original level within 10 min. Switching back to 36 mmHg Pco2 restored the original extracellular pH and caused a transient decrease in the EPSP slope. Pharmacological blockade of NMDA receptor and/or GABAA receptor had no influence on the effects of C02. An increase in PCO2 to 145 mmHg led to a stable fall in extracellular pH by 0.6 units and to a transient 30–50% decrease in EPSP slope. The above results indicate that the CO2-induced changes in neuronal excitability were not caused by changes in extracellular pH but they might have been mediated by changes in intracellular pH. Indeed, exposing the slices to the permeant weak base, trimethylamine (20 mM), which is known to produce a rise in intracellular pH, increased the EPSP slope by 50–70%. Application of 20 mM propionate (a permeant weak acid) decreased the EPSP slope by 40–60%. We conclude that the transient changes in the EPSP seen in response to changes in Pco2 are mediated by in intracellular pH.

Background The developmental transition from depolarizing to hyperpolarizing γ-aminobutyric acid-mediated neurotransmission is primarily mediated by an increase in the amount of the potassium-chloride cotransporter KCC2 during early postnatal life. However, it is not known whether early neuronal activity plays a modulatory role in the expression of total KCC2 mRNA and protein in the immature brain. As general anaesthetics are powerful modulators of neuronal activity, the purpose of this study was to explore how these drugs affect KCC2 expression during the brain growth spurt. Methods Wistar rat pups were exposed to either a single dose or 6 h of midazolam, propofol, or ketamine anaesthesia at postnatal days 0, 5, 10, or 15. KCC2 expression was assessed using immunoblotting, immunohistochemistry, or quantitative polymerase chain reaction analysis up to 3 days post-exposure in the medial prefrontal cortex. Results There was a progressive and steep increase in the expression of KCC2 between birth and 2 weeks of age. Exposure to midazolam, propofol, or ketamine up to 6 h at any investigated stages of the brain growth spurt did not influence the expression of this cotransporter protein. Conclusion I.V. general anaesthetics do not seem to influence developmental expression of KCC2 during the brain growth spurt.

Several investigations have suggested that sphingosine (SP) derivatives are potent inhibitors of protein kinase C. In GH4C1 cells, protein kinase C is a potent modulator of voltage-operated calcium channels (VOCCs). The aim of the present study was to investigate whether SP derivatives could modify calcium entry via VOCCs. Using fura-2-loaded cells and 45Ca2+ flux studies, we show that several SPs potently and rapidly inhibit depolarization-evoked calcium entry in a dose-dependent manner. The effect was not due to an enhanced efflux of calcium from the cells, as the depolarization-evoked entry of Ba2+ was inhibited by the SPs. A similar inhibition was observed with 1,2-dioctanoylglycerol, an activator of sphingomyelinase in GH3 cells. Phorbol myristate acetate and 1-oleyl-2-acetylglycerol had only a modest inhibitory effect. Furthermore, whole cell patch-clamp experiments showed that sphingosinephosphorylcholine (SPC) potently attenuated calcium entry via VOCCs. In experiments using cells grown on coverslips, we found that the inhibitory effect of SPC on calcium entry was reversible. The addition of sphingomyelinase or hexanoyl ceramide, a cell-permeable ceramide, only modestly inhibited the depolarization-evoked entry of calcium, whereas arachidonic acid and phosphatidic acid had no effect. The SP metabolite sphingosine-1-phosphate had no effect on the entry of calcium. The results suggest that the effects of the SPs were probably not the result of a conversion to ceramide or of the production of other lipid second messengers. In cells with down-regulated protein kinase C, SPC, SP, and 1,2-dioctanoylglycerol inhibited depolarization-evoked calcium entry, suggesting that the inhibition was independent of an action mediated via protein kinase C. The SPs per se did not induce any changes in intracellular free calcium, and they did not inhibit the TRH-evoked release of sequestered calcium in the cells. However, TRH-evoked calcium entry was inhibited. The results suggest that SPs are potential regulators of calcium entry mediated by VOCCs in GH4C1 cells.

There has been considerable interest recently in trying to elucidate the roles of metabotropic glutamate receptors (mGluRs) in the induction of long-term potentiation (LTP) in area CA1 of rat hippocampal slices. This has come about principally because of the development of specific mGluR agonists and antagonists. Recently we reported that the competitive mGluR antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG) blocks the induction of LTP but not short-term potentiation (STP). We describe here the dose-dependency of the MCPG block; there is no effect at 100 μM while at 200 μM the block of LTP is normally complete but STP is spared. A higher concentration of MCPG (500 μM) has the same effect as 200 μM. We have also reported recently that high-frequency (tetanic) stimulation conditions a pathway such that MCPG fails to block the induction of subsequent LTP. We illustrate here that the conditioning effect of a tetanus lasts at least 6 h. We show how the pathway can be conditioned, without any persistent change in the synaptic response, by delivering tetanic stimulation in the presence of the specific NMDA receptor antagonist d-2-amino-5-phosphonopentanoate (AP5). The pathway can subsequently be deconditioned by delivering low-frequency stimulation (900 shocks at 2 Hz) so that MCPG blocks the induction of subsequent LTP. We also have reported that the specific mGluR agonist 1-aminocyclopentane-(1S,3R)-dicarboxylate (ACPD) can induce LTP without the need for STP. We now report the surprising observation that ACPD-induced LTP is prevented reversibly under conditions in which the slice is submerged below the bathing medium. One consequence of submerging the slice, which might account for the difference, is acidification of the tissue.

The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl−concentration ([Cl−]i) that forms the basis for hyperpolarizing GABAAreceptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg2+-free solution led to a fast reduction in KCC2-mediated Cl−transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and monitoring of brain function in preterm and term babies. Most common indications are the diagnosis of epileptic seizures, assessment of brain maturity, and recovery from hypoxic-ischemic events. EEG recording techniques and the understanding of neonatal EEG signals have dramatically improved, but these advances have been slow to penetrate through the clinical traditions. The aim of this presentation is to bring theory and practice of advanced EEG recording available for neonatal units. In the theoretical part, we will present animations to illustrate how a preterm brain gives rise to spontaneous and evoked EEG activities, both of which are unique to this developmental phase, as well as crucial for a proper brain maturation. Recent animal work has shown that the structural brain development is clearly reflected in early EEG activity. Most important structures in this regard are the growing long range connections and the transient cortical structure, subplate. Sensory stimuli in a preterm baby will generate responses that are seen at a single trial level, and they have underpinnings in the subplate-cortex interaction. This brings neonatal EEG readily into a multimodal study, where EEG is not only recording cortical function, but it also tests subplate function via different sensory modalities. Finally, introduction of clinically suitable dense array EEG caps, as well as amplifiers capable of recording low frequencies, have disclosed multitude of brain activities that have as yet been overlooked. In the practical part of this video, we show how a multimodal, dense array EEG study is performed in neonatal intensive care unit from a preterm baby in the incubator. The video demonstrates preparation of the baby and incubator, application of the EEG cap, and performance of the sensory stimulations.

Little is known about the physiological roles of aquaporin-4 (AQP4) in the central nervous system. AQP4 water channels are concentrated in endfeet membranes of astrocytes but also localize to the fine astrocytic processes that abut central synapses. Based on its pattern of expression, we predicted that AQP4 could be involved in controlling water fluxes and changes in extracellular space (ECS) volume that are associated with activation of excitatory pathways. Here, we show that deletion of Aqp4 accentuated the shrinkage of the ECS that occurred in the mouse hippocampal CA1 region during activation of Schaffer collateral/commissural fibers. This effect was found in the stratum radiatum (where perisynaptic astrocytic processes abound) but not in the pyramidal cell layer (where astrocytic processes constitute but a minor volume fraction). For both genotypes the ECS shrinkage was most pronounced in the pyramidal cell layer. Our data attribute a physiological role to AQP4 and indicate that this water channel regulates extracellular volume dynamics in the mammalian brain.

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.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 Pco(2) were measured immediately on admission to the hospital.Respiratory alkalosis was found in children with FS (pH 7.46 ± 0.04, [mean ± standard deviation] Pco(2) 29.5 ± 5.5 mmHg), whereas a metabolic acidosis was seen in all children admitted for GE (pH 7.31 ± 0.03, Pco(2) 37.7 ± 4.3 mmHg; p0.001 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, p0.005).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.

1. The ionic mechanism underlying the fast, GABAA receptor-mediated inhibitory postsynaptic potential (IPSPA) was examined in rat neocortical neurones using intracellular recording techniques. Synaptic responses were evoked by orthodromic stimulation applied to the subcortical white matter or to the pial surface. All experiments were carried out at a constant extracellular Cl- concentration. 2. The resting membrane potential was -76.2 +/- 1.0 mV (mean +/- S.E.M., n = 32) and in most cells IPSPA was depolarizing. The reversal potential of IPSPA (EIPSP-A) was -70.2 +/- 0.9 mV (n = 32) and that of a more slowly developing hyperpolarizing response (IPSPB) was -91.4 +/- 1.3 mV (n = 28). 3. An examination of the temporal relationships between excitatory postsynaptic potentials (EPSPs) and IPSPAs in different cells suggested that, despite partial overlap of these responses, EPSPs had little influence on the measured values of EIPSP-A. 4. Application of 20 mM trimethylamine (TriMA), a membrane-permeant weak base which is expected to produce a rise in pHi (and hence in intracellular HCO3-), induced a reversible positive shift in EIPSP-A of up to +9.0 mV (mean + 4.2 mV) at an extracellular pH (pHo) of 7.4. In some experiments, the shift in reversal potential was associated with a change in the polarity of IPSPA from hyperpolarizing to depolarizing. 5. Application of 20 mM lactate (a membrane-permeant weak acid which is expected to produce a fall in pHi and hence in intracellular HCO3-) at pHo 7.0 produced a hyperpolarizing shift in EIPS-A of up to -7.5 mV (mean -5.6 mV). In some experiments, exposure to lactate changed the polarity of IPSPA from depolarizing to hyperpolarizing. 6. Changes in pHo from 7.4 to 7.0 reduced the effect of TriMA and augmented that of lactate on EIPSP-A, as could be expected on the basis of the pHo-dependent change in the fraction of membrane permeable non-charged weak base or acid. 7. Under control conditions, a change in pHo from 7.4 to 7.0 produced a slight positive shift (< +2 mV) in EIPSP-A. In the presence of TriMA, a similar change in pHo gave rise to a negative shift (-1.8 to -2.7 mV). 8. The results obtained indicate that HCO3- ions contribute significantly to the IPSPA, thereby making EIPSP-A more positive than the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 400 WORDS)

In two recent papers (Rheims et al., 2009; Holmgren et al., 2010), Zilberter and coworkers argue that the well known depolarizing GABA actions that take place at the cellular and network level in the neonatal hippocampus and neocortexin vitroare pathophysiological phenomena, attributable to deficient mitochondrial energy metabolism. In their experiments, supplementing the glucose-containing solution with weak-acid substrates of mitochondrial energy metabolism (such as β-hydroxy-butyrate, lactate, or pyruvate) abolished the spontaneous network events (giant depolarizing potentials; GDPs) and the underlying depolarizing actions of GABA. In this study, we made electrophysiological recordings of GDPs and monitored the mitochondrial membrane potential (Ψm) and intracellular pH (pHi) in CA3 neurons in neonatal rat hippocampal slices. Supplementing the standard physiological solution withl-lactate did not produce a change in Ψm, whereas withdrawal of glucose, in the presence or absence ofl-lactate, was followed by a pronounced depolarization of Ψm. Furthermore,d-lactate (a poor substrate of mitochondrial metabolism) caused a prompt inhibition in GDP frequency which was similar to the effect ofl-lactate. The suppression of GDPs was strictly proportional to the fall in pHicaused by weak carboxylic acids (l-lactate,d-lactate, or propionate) or by an elevated CO2. The main conclusions of our work are that the inhibitory effect ofl-lactate on GDPs is not mediated by mitochondrial energy metabolism, and that glucose at its standard 10 mmconcentration is an adequate energy substrate for neonatal neuronsin vitro. Notably, changes in pHiappear to have a very powerful modulatory effect on GDPs.

Functional expression of the K-Cl cotransporter KCC2 in developing central neurons is crucial for the maturation of Cl−-dependent, GABAAreceptor-mediated inhibitory responses. In pyramidal neurons of the rodent hippocampus, GABAergic postsynaptic responses are typically depolarizing and often excitatory during the first postnatal week. Here, we show that a single neonatal seizure episode induced by kainate injection during postnatal days 5–7 results in a fast increase in the Cl−extrusion capacity of rat hippocampal CA1 neurons, with a consequent hyperpolarizing shift of the reversal potential of GABAA-mediated currents (EGABA). A significant increase in the surface expression of KCC2 as well as the α2 subunit of the Na-K-ATPase parallels the seizure-induced increase in the Cl−extrusion capacity. Exposing hippocampal slices to kainate resulted in a similar increase in the neuronal Cl−extrusion and in the surface expression of KCC2. Both effects were blocked by the kinase inhibitor K252a. Hence, in the neonatal hippocampus the overall KCC2 expression level is high enough to promote a rapid functional activation of K-Cl cotransport and a consequent negative shift in EGABAclose to the adult level. The activity-dependent regulation of KCC2 function and its effect on GABAergic transmission may represent an intrinsic antiepileptogenic mechanism.

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, phenols, and coumarins. Polyamines such as spermine, spermidine, and many synthetic congeners are described to constitute a novel class of CA inhibitors (CAIs), interacting with the different CA isozymes with efficiency from the low nanomolar to millimolar range. The main structure-activity relationship for these CAIs have been delineated: the length of the molecule, number of amine moieties, and their functionalization are the main parameters controlling activity. The X-ray crystal structure of the CA II-spermine adduct allowed understanding of the inhibition mechanism. Spermine anchors to the nonprotein zinc ligand through a network of hydrogen bonds. Its distal amine moiety makes hydrogen bonds with residues Thr200 and Pro201, which further stabilize the adduct. Spermine binds differently compared to sulfonamides, phenols, or coumarins, rendering possible to develop CAIs with a diverse inhibition mechanism, profile, and selectivity for various isoforms.

Objective: The mechanisms whereby birth asphyxia leads to generation of seizures remain unidentified. To study the possible role of brain pH changes, we used a rodent model that mimics the alterations in systemic CO2 and O2 levels during and after intrapartum birth asphyxia. Methods: Neonatal rat pups were exposed for 1 hour to hypercapnia (20% CO2 in the inhaled gas), hypoxia (9% O2), or both (asphyxic conditions). CO2 levels of 10% and 5% were used for graded restoration of normocapnia. Seizures were characterized behaviorally and utilizing intracranial electroencephalography. Brain pH and oxygen were measured with intracortical microelectrodes, and blood pH, ionized calcium, carbon dioxide, oxygen, and lactate with a clinical device. The impact of the postexposure changes in brain pH on seizure burden was assessed during 2 hours after restoration of normoxia and normocapnia. N-methyl-isobutyl-amiloride, an inhibitor of Na þ /H þ exchange, was given intraperitoneally. Results: Whereas hypercapnia or hypoxia alone did not result in an appreciable postexposure seizure burden, recovery from asphyxic conditions was followed by a large seizure burden that was tightly paralleled by a rise in brain pH, but no change in brain oxygenation. By graded restoration of normocapnia after asphyxia, the alkaline shift in brain pH and the seizure burden were strongly suppressed. The seizures were virtually blocked by preapplication of N-methyl-isobutyl-amiloride. Interpretation: Our data indicate that brain alkalosis after recovery from birth asphyxia plays a key role in the triggering of seizures. We question the current practice of rapid restoration of normocapnia in the immediate postasphyxic period, and suggest a novel therapeutic strategy based on graded restoration of normocapnia. ANN NEUROL 2011;69:493–500

The presence and properties of membrane-bound carbonic anhydrases have been difficult to establish with conventional enzymological and immunohistochemical techniques. We have therefore studied carbonic anhydrase (CA) activity in single intact crayfish muscle fibres by superfusing them alternately with a 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffered and a 5% CO2/HCO3−-buffered solution (pH of both solutions 7.4) while recording the intracellular pH (pHi) and extracellular surface pH (pHs) with H+-selective microelectrodes. In order to prevent regulation of pHi, Na+ ions were replaced with N-methyl-D-glucamine. Application of the CO2-containing solution produced a fast fall in pHi coupled with a marked (0.5–0.8 pH units) transient increase in pHs. Submicromolar concentrations of acetazolamide (AA) and benzolamide (BA) immediately blocked the pHs transients. A concentration of 8×10−8 M (both compounds) reduced the response by 50%. A more prolonged application of BA and AA at concentrations of 10−7 M and higher slowed the CO2-induced fall in pHi, which attained a rate corresponding to uncatalysed intracellular CO2 hydration at an AA concentration of 10−4 M. The effect of BA and AA on the pHi changes developed with a time constant of 25±4 min and 7.6±1.5 min respectively, indicating that BA is less permeant than AA. CNO− ions (5×10−4 M) had little effect on the CO2-induced pHs and pHi changes. The results are consistent with the view that the muscle fibres are equipped with an intracellular CA and with a sarcolemmal CA which has its active site at the extracellular surface. The pharmacological inhibition pattern of the latter is not fully compatible with any CA isozyme identified so far.

A two-microelectrode current-voltage clamp and Cl(-)-selective microelectrodes were used to examine the effects of gamma-aminobutyric acid (GABA) on membrane potential, current and intracellular Cl- activity (aiCl) in the crayfish stretch receptor neurone. All experimental solutions were CO2-HCO3- free. 2. GABA (500 microM) produced a mono- or biphasic depolarization (amplitude < or = 10 mV), often with a prominent initial depolarizing component followed by a transient shift to a more negative level. In some neurones, an additional depolarizing phase was seen upon washout of GABA. Receptor desensitization, being absent, played no role in the multiphasic actions of GABA. 3. The pronounced increase in membrane conductance evoked by GABA (500 microM) was associated with an increase in aiCl which indicates that the depolarizing action was not due to a current carried by Cl- ions. 4. The currents activated by GABA under voltage clamp conditions were inwardly directed when recorded at the level of the resting membrane potential, and they often revealed a biphasic character. The reversal potential of peak currents activated by pulses of 500 microM-GABA (EGABA) was 9-12 mV more positive than the reversal potential of the simultaneously measured net Cl- flux (ECl). ECl was 2-7 mV more negative than the resting membrane potential. 5. EGABA (measured using pulses of 500 microM-GABA) was about 10 mV more positive than the reversal potential of the current activated by 500 microM-muscimol, a GABA agonist that is a poor substrate of the Na(+)-dependent GABA uptake system. 6. In the absence of Na+, the depolarization and inward current caused by 500 microM-GABA were converted to a hyperpolarization and to an outward current. Muscimol produced an immediate outward current both in the presence and absence of Na+. 7. Following block of the inhibitory channels by picrotoxin (100-200 microM), the depolarizing effect of 500 microM-GABA was enhanced and the transient hyperpolarizing shifts were abolished. 8. In the presence of picrotoxin, GABA (> or = 2 microM) produced a concentration-dependent monophasic inward current which had a reversal potential of +30 to +60 mV. This current was inhibited in the absence of Na+ and by the GABA uptake blocker, nipecotic acid. Unlike the channel-mediated current, the picrotoxin-insensitive current was activated without delay also at low (2-10 microM) concentrations of GABA.(ABSTRACT TRUNCATED AT 400 WORDS)

Although the requirement for a strict regulation of pH in the brain is frequently emphasized, recent studies indicate that neuronal activity gives rise to significant changes in intracellular and extracellular pH. Given the sensitivity of many ion channels to hydrogen ions, this modulation of local pH might influence brain function, particularly where pH shifts are sufficiently large and rapid. Studies using pH-sensitive microelectrodes have demonstrated marked cellular and regional variability of activity-dependent pH shifts, and have begun to uncover several of their underlying mechanisms. Accumulating evidence suggests that regional and subcellular pH dynamics are governed by the respective localization of glial cells, ligand-gated ion channels, and extracellular and intracellular carbonic anhydrase.

Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatalNKCC1−/−mice that lack the Na–K–2Cl cotransporter isoform 1. Strikingly,NKCC1−/−slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABAAagonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K–Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in theNKCC1−/−hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.

Febrile seizures (FS) are the most common type of convulsive events in children. FS have been extensively studied using animal models, where rat and mice pups are placed in a hyperthermic environment. Such work has largely focused on the consequences rather than on the mechanisms of experimental febrile seizures (eFS). We have recently shown that eFS are preceded by a dramatic rise in the rate of respiration. The consequent respiratory alkalosis affecting the brain and increasing neuronal excitability is a direct cause of the eFS [1]. If a similar mechanism contributes to human FS and other fever-related epileptiform syndromes, a number of factors operating at the molecular, cellular and systems level that have not been previously thought to be involved in their etiology must be considered. These include physiological and pathophysiological factors affecting CO(2) chemosensitivity as well as cellular and systemic mechanisms of acid-base regulation. Furthermore, a critical role for brain pH in FS points to novel types of susceptibility genes, which include genes coding pH-sensitive target proteins (e.g. neuronal ion channels) and pH-regulatory proteins. We will discuss these novel ideas and putative therapies based on them.

GABAergic terminals of axo-axonic cells (AACs) are exclusively located on the axon initial segment (AIS) of cortical principal neurons, and they are generally thought to exert a powerful inhibitory action. However, recent work (Szabadics et al., 2006) indicates that this input from AACs can be depolarizing and even excitatory. Here, we used local photolysis of caged GABA to measure reversal potentials (EGABA) of GABAAreceptor-mediated currents and to estimate the local chloride concentration in the AIS compared with other cellular compartments in dentate granule cells and neocortical pyramidal neurons. We found a robust axo-somato-dendritic gradient in which theEGABAvalues from the AIS to the soma and dendrites become progressively more negative. Data fromNKCC1−/−and bumetanide-exposed neurons indicated that the depolarizingEGABAat the AIS is set by chloride uptake mediated by the Na–K–2Cl cotransporter NKCC1. Our findings demonstrate that spatially distinct interneuronal inputs can induce postsynaptic voltage responses with different amplitudes and polarities as governed by the subcellular distributions of plasmalemmal chloride transporters.

1. The mode of action of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) and surface pH (pHs) was studied in crayfish muscle fibres using H(+)-selective microelectrodes. The extracellular HCO3- concentration was varied (0-30 mM) at constant pH (7.4). 2. GABA (5 x 10(-6)-10(-3) M) produced a reversible fall in pHi which showed a dependence on the concentrations of both GABA and HCO3-. The fall in pHi was associated with a transient increase in pHs and it was inhibited by a K(+)-induced depolarization. 3. In the presence of 30 mM-HCO3-, a near-saturating concentration of GABA (0.5 mM) produced a mean fall in pHi of 0.43 units. This change in pHi accounted for about two-thirds of the GABA-induced decrease (from -66 to -29 mV) in the sarcolemmal H+ driving force, while the rest was due to the simultaneous depolarization. 4. The apparent net efflux of HCO3- (JHCO3e) produced by a given concentration of GABA was estimated on the basis of the instantaneous rate of change of pHi. In the presence of 30 mM-HCO3-, JHCO3e following exposure to 0.5 mM-GABA had a mean value of 8.0 mmol l-1 min-1. Under steady-state conditions (at plateau acidosis), the intracellular acid load produced by 0.5 mM-GABA was about 25% of that seen at the onset of the application. 5. The GABA-induced HCO3- permeability, calculated on the basis of the flux data, showed a concentration dependence similar to that of the GABA-activated conductance described in previous work. 6. The GABA-induced increase in pHs was immediately blocked by both a membrane-permeant inhibitor of carbonic anhydrase (acetazolamide, 10(-6) M) and by a poorly permeant inhibitor (benzolamide, 10(-6) M). 7. Application of acetazolamide (10(-4) M) for 5 min or more produced a decrease of up to 60% in the maximum rate of fall of pHi at GABA concentrations higher than 20 microM. 8. The recovery of the GABA-induced acidosis was associated with a fall in pHs. The recovery was completely blocked in solutions devoid of Na+ or of Cl-, as well as by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, 10(-5) M). This indicates that the maintenance of a non-equilibrium H+ gradient at plateau acidosis and the recovery of pHi are attributable to Na(+)-dependent Cl(-)-HCO3- exchange. 9. We conclude that the effects of GABA on pHi and pHs are due to electrodiffusion of HCO3- across postsynaptic anion channels.(ABSTRACT TRUNCATED AT 400 WORDS)

The dependence of intracellular free calcium ([Ca2+]i) and tension on membrane potential and intracellular pH (pHi) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm2). In resting fibres, pHi had a mean value of 7.06. Contractions evoked by an increase extracellular potassium [( K+]0) produced a fall in pHi of 0.01-0.05 units. The lowest measured levels of resting [Ca2+]i corresponded to a pCai (= -log [Ca2+]i) of 6.8. Intracellular Ca2+ transients recorded during K(+)-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca2+]i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pHi (quantified as -dT/dpHi) showed a progressive increase during a fall in pHi within the range examined (pHi 6.2-7.5). The pHi/[Ca2+]i and pHi/tension relationships were monotonic throughout the multiphasic pHi change caused by NH4Cl. A fall of 0.5-0.6 units in pHi did not produce a detectable shift in the pCai/tension relationship at low levels of force development. The results indicate that resting [Ca2+]i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pHi in crayfish muscle fibres is attributable to a direct H+ and Ca2+ interaction at the level of Ca2+ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca2+ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.

H(+)-selective microelectrodes and a two- or three-microelectrode voltage clamp were used to examine the influence of weak-acid, carboxylate anions on the actions of GABA on postsynaptic intracellular pH, surface pH and on membrane potential in fibres of the crayfish leg opener muscle. Substitution of 30 mM Cl- by formate or acetate promoted a GABA-induced decrease in intracellular pH, which was coupled to an increase in surface pH and to a depolarization. Such effects were not seen in the presence of an equivalent amount of lactate, methanesulphonate or glucuronate. Both the GABA-induced depolarization and the fall in internal pH promoted by formate and acetate were blocked by picrotoxin, and the fall in pH was reversibly inhibited by a K(+)-induced depolarization. The rate of the fall in intracellular pH produced by GABA (0.2 mM) was about 0.02 pH units/min in the presence of formate and 0.03 pH units/min in the presence of acetate. Under steady-state conditions, both 30 mM formate and acetate (but not lactate) induced a positive shift in the reversal potential of GABA-activated current, which was accounted for by a relative permeability vs Cl- of formate and acetate of 0.5 and 0.15, respectively. The conductance sequence of the anions was identical to the permeability sequence, i.e. Cl- greater than formate greater than acetate greater than lactate approximately equal to 0. This sequence is strictly correlated to the Stokes diameter of the anions. The relative permeabilities of the anions indicate that the effective diameter of the GABA-gated channel is about 0.5 nm. The fact that the GABA-induced acidosis was slower in the presence of formate than in the presence of acetate suggests that, in the former case, the rate-limiting step in the fall in internal pH is the entry of non-dissociated formic acid. All the above results are consistent with a scheme where GABA induces a channel-mediated efflux of permeant weak-acid anions, which gives rise to an inward (depolarizing) current and to an intracellular acidosis. A comparison of the permeability properties of crayfish and vertebrate GABA-gated channels suggests that effects similar to those seen in this work are likely to occur in mammalian and other vertebrate neurons in the presence of permeant weak-acid anions.

Cation chloride cotransporters have been reported to be expressed in neurons in the hippocampus and to regulate intracellular Cl − concentration. The neuron-specific K-Cl cotransporter 2 (KCC2) is necessary for maintaining the low intracellular chloride concentration required for the hyperpolarizing actions of GABA. In this study we examined the vulnerability of KCC2-containing neurons as well as the changes in the pattern of KCC2 distribution in the rat hippocampus following 15 min ischemia induced by four-vessel occlusion. Immunostaining for the 72 kDa heat shock protein (HSP-72) was used to investigate the extent of damage in neuronal populations previously shown to be vulnerable to ischemia. At 6–24 h after ischemia, when the pyramidal cells in the CA1 (subfield of cornu Ammonis) region showed no morphological signs of damage, a small rise of KCC2 immunoreactivity was already observed. After 2 days, when the CA1 pyramidal cells started to degenerate, a progressive downregulation of the KCC2 protein was visible. Interestingly, in the same areas, the parvalbumin containing interneurons showed no signs of ischemic damage, and KCC2 immunoreactivity was retained on their membrane surface. In CA1 pyramidal cells, the reduction in KCC2 expression may lead to an elevation of intracellular Cl − concentration, which causes a shift in equilibrium potential toward more positive levels. Consequently, the reduction of the inhibitory action of GABA through downregulation of KCC2 function may be involved in the pathomechanisms of delayed neuronal death in the CA1 subfield.

Changes in chloride (Cl−) homeostasis may be involved in the generation of some epileptic activities. In this study, we asked whether Cl−homeostasis, and thus GABAergic signaling, is altered in tissue from patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis. Slices prepared from this human tissue generated a spontaneous interictal-like activity that was initiated in the subiculum. Records from a minority of subicular pyramidal cells revealed depolarizing GABAAreceptor-mediated postsynaptic events, indicating a perturbed Cl−homeostasis. We assessed possible contributions of changes in expression of the potassium–chloride cotransporter KCC2. Doublein situhybridization showed that mRNA for KCC2 was absent from ∼30% of CaMKIIα (calcium/calmodulin-dependent protein kinase IIα)-positive subicular pyramidal cells. Combining intracellular recordings with biocytin-filled electrodes and KCC2 immunochemistry, we observed that all cells that were hyperpolarized during interictal events were immunopositive for KCC2, whereas the majority of depolarized cells were immunonegative. Bumetanide, at doses that selectively block the chloride-importing potassium–sodium–chloride cotransporter NKCC1, produced a hyperpolarizing shift in GABAAreversal potentials and suppressed interictal activity. Changes in Cl−transporter expression thus contribute to human epileptiform activity, and molecules acting on these transporters may be useful antiepileptic drugs.

Functional magnetic resonance imaging (fMRI) is a valuable method for probing postnatal circuit refinement and plasticity. However, its use during early development has been hindered by uncertainty as to the nature of neurovascular coupling in young individuals. Here we used somatosensory stimulation in rats to determine age-related parameters of the blood oxygenation level-dependent (BOLD) signal from its apparent inception on postnatal day 13 to adulthood. By comparing fMRI measurements with electrophysiological recordings, we determined that the regional BOLD response in these animals undergoes a systematic decline in latency and growth in amplitude over this period. We found no evidence of negative BOLD at any age. Maturation of hemodynamic responses correlated with age-dependent increases in susceptibility to inhibition of carbonic anhydrase. With knowledge of the infant BOLD response characteristics, we showed that interhemispheric and higher-order cortical stimulus responses are enhanced during the first several weeks after birth.

The neuronal K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the hyperpolarizing actions of inhibitory neurotransmitters gamma-aminobutyric acid and glycine in the central nervous system. This study shows that the mammalian KCC2 gene (alias Slc12a5) generates two neuron-specific isoforms by using alternative promoters and first exons. The novel KCC2a isoform differs from the only previously known KCC2 isoform (now termed KCC2b) by 40 unique N-terminal amino acid residues, including a putative Ste20-related proline alanine-rich kinase-binding site. Ribonuclease protection and quantitative PCR assays indicated that KCC2a contributes 20-50% of total KCC2 mRNA expression in the neonatal mouse brain stem and spinal cord. In contrast to the marked increase in KCC2b mRNA levels in the cortex during postnatal development, the overall expression of KCC2a remains relatively constant and makes up only 5-10% of total KCC2 mRNA in the mature cortex. A rubidium uptake assay in human embryonic kidney 293 cells showed that the KCC2a isoform mediates furosemide-sensitive ion transport activity comparable with that of KCC2b. Mice that lack both KCC2 isoforms die at birth due to severe motor defects, including disrupted respiratory rhythm, whereas mice with a targeted disruption of the first exon of KCC2b survive for up to 2 weeks but eventually die due to spontaneous seizures. We show that these mice lack KCC2b but retain KCC2a mRNA. Thus, distinct populations of neurons show a differential dependence on the expression of the two isoforms: KCC2a expression in the absence of KCC2b is presumably sufficient to support vital neuronal functions in the brain stem and spinal cord but not in the cortex.

Endogenous activity is a characteristic feature of developing neuronal networks. In the neonatal rat hippocampus, spontaneously occurring network events known as “Giant Depolarizing Potentials” (GDPs) are seen in vitro at a stage when GABAergic transmission is depolarizing. GDPs are triggered by the CA3 region and they are seen as brief recurrent events in field-potential recordings, paralleled by depolarization and spiking of pyramidal neurons. In the light of current data, GDPs are triggered by the glutamatergic pyramidal neurons which act as conditional pacemakers, while the depolarizing action of GABA plays a permissive role for the generation of these events in in vitro preparations. From an in vivo perspective, GDPs appear to be an immature form of hippocampal sharp waves.

Endogenous activity is a characteristic feature of developing neuronal networks. In the neonatal rat hippocampus, spontaneously occurring network events known as "Giant Depolarizing Potentials" (GDPs) are seen in vitro at a stage when GABAergic transmission is depolarizing. GDPs are triggered by the CA3 region and they are seen as brief recurrent events in field-potential recordings, paralleled by depolarization and spiking of pyramidal neurons. In the light of current data, GDPs are triggered by the glutamatergic pyramidal neurons which act as conditional pacemakers, while the depolarizing action of GABA plays a permissive role for the generation of these events in in vitro preparations. From an in vivo perspective, GDPs appear to be an immature form of hippocampal sharp waves.

GABA(A) receptors mediate fast synaptic inhibition in the CNS. Whilst this is undoubtedly true, it is a gross oversimplification of their actions. The receptors themselves are diverse, being formed from a variety of subunits, each with a different temporal and spatial pattern of expression. This diversity is reflected in differences in subcellular targetting and in the subtleties of their response to GABA. While activation of the receptors leads to an inevitable increase in membrane conductance, the voltage response is dictated by the distribution of the permeant Cl(-) and HCO(3)(-) ions, which is established by anion transporters. Similar to GABA(A) receptors, the expression of these transporters is not only developmentally regulated but shows cell-specific and subcellular variation. Untangling all these complexities allows us to appreciate the variety of GABA-mediated signalling, a diverse set of phenomena encompassing both synaptic and non-synaptic functions that can be overtly excitatory as well as inhibitory.

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.

GABA(A) receptors mediate fast synaptic inhibition in the CNS. Whilst this is undoubtedly true, it is a gross oversimplification of their actions. The receptors themselves are diverse, being formed from a variety of subunits, each with a different temporal and spatial pattern of expression. This diversity is reflected in differences in subcellular targetting and in the subtleties of their response to GABA. While activation of the receptors leads to an inevitable increase in membrane conductance, the voltage response is dictated by the distribution of the permeant Cl(-) and HCO(3)(-) ions, which is established by anion transporters. Similar to GABA(A) receptors, the expression of these transporters is not only developmentally regulated but shows cell-specific and subcellular variation. Untangling all these complexities allows us to appreciate the variety of GABA-mediated signalling, a diverse set of phenomena encompassing both synaptic and non-synaptic functions that can be overtly excitatory as well as inhibitory.

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 ( approximately 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.

An inhibition study of the cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isozyme VII (hCA VII) with anions has been conducted. Cyanate, cyanide, and hydrogensulfite were weak hCA VII inhibitors (KIs in the range of 7.3–15.2 mM). Cl− and HCO 3 - showed good inhibitory activity against hCA VII (KIs of 0.16–1.84 mM), suggesting that this enzyme is not involved in metabolons with anion exchangers or sodium bicarbonate cotransporters. The best inhibitors were sulfamate, sulfamide, phenylboronic, and phenylarsonic acid (KIs of 6.8–12.5 μM).

Spontaneous periodic network events are a characteristic feature of developing neuronal networks, and they are thought to play a crucial role in the maturation of neuronal circuits. In the immature hippocampus, these types of events are seen in intracellular recordings as giant depolarizing potentials (GDPs) during the stage of neuronal development when GABAA-mediated transmission is depolarizing. However, the precise mechanism how GABAergic transmission promotes GDP occurrence is not known. Using whole-cell, cell-attached, perforated-patch, and field-potential recordings in hippocampal slices, we demonstrate here that CA3 pyramidal neurons in the newborn rat generate intrinsic bursts when depolarized. Furthermore, the characteristic rhythmicity of GDP generation is not based on a temporally patterned output of the GABAergic interneuronal network. However, GABAergic depolarization plays a key role in promoting voltage-dependent, intrinsic pyramidal bursting activity. The present data indicate that glutamatergic CA3 neurons have an instructive, pacemaker role in the generation of GDPs, whereas both synaptic and tonic depolarizing GABAergic mechanisms exert a temporally nonpatterned, facilitatory action in the generation of these network events.

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.