Eva Ruusuvuori

The K+/Cl-co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation

GABA (γ-aminobutyric acid) is the main inhibitory transmitter in the adult brain, and it exerts its fast hyperpolarizing effect through activation of anion (predominantly Cl−)-permeant GABAA receptors. However, during early neuronal development, GABA A-receptor-mediated responses are often depolarizing,, which may be a key factor in the control of several Ca2+ −dependent developmental phenomena, including neuronal proliferation, migration and targeting. To date, however, the molecular mechanism underlying this shift in neuronal electrophysiological phenotype is unknown. Here we show that, in pyramidal neurons of the rat hippocampus, the ontogenetic change in GABAA-mediated responses from depolarizing to hyperpolarizing is coupled to a developmental induction of the expression of the neuronal Cl−-extruding K+/Cl − co-transporter, KCC2 (ref. 7). Antisense oligonucleotide inhibition of KCC2 expression produces a marked positive shift in the reversal potential of GABAA responses in functionally mature hippocampal pyramidal neurons. These data support the conclusion that KCC2 is the main Cl− extruder to promote fast hyperpolarizing postsynaptic inhibition in the brain.

The K+–Cl− cotransporter KCC2 promotes GABAergic excitation in the mature rat hippocampus

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.

Carbonic Anhydrase Isoform VII Acts as a Molecular Switch in the Development of Synchronous Gamma-Frequency Firing of Hippocampal CA1 Pyramidal Cells

Identification of the molecular mechanisms that enable synchronous firing of CA1 pyramidal neurons is central to the understanding of the functional properties of this major hippocampal output pathway. Using microfluorescence measurements of intraneuronal pH, in situ hybridization, as well as intracellular, extracellular, and K+-sensitive microelectrode recordings, we show now that the capability for synchronous gamma-frequency (20–80 Hz) firing in response to high-frequency stimulation (HFS) emerges abruptly in the rat hippocampus at approximately postnatal day 12. This was attributable to a steep developmental upregulation of intrapyramidal carbonic anhydrase isoform VII, which acts as a key molecule in the generation of HFS-induced tonic GABAergic excitation. These results point to a crucial role for the developmental expression of intrapyramidal carbonic anhydrase VII activity in shaping integrative functions, long-term plasticity and susceptibility to epileptogenesis.

Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Slc4a10 gene, which encodes the Na+-coupled Cl−–HCO3− exchanger Slc4a10 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4a10 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4a10 also was expressed in neurons. Within the hippocampus, the Slc4a10 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure.

GABA actions and ionic plasticity in epilepsy

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.

Pharmacotherapeutic targeting of cation-chloride cotransporters in neonatal seizures

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.

Spontaneous network events driven by depolarizing GABA action in neonatal hippocampal slices are not attributable to deficient mitochondrial energy metabolism.

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 neocortex in vitro are 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 with l-lactate did not produce a change in Ψm, whereas withdrawal of glucose, in the presence or absence of l-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 of l-lactate. The suppression of GDPs was strictly proportional to the fall in pHi caused 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 of l-lactate on GDPs is not mediated by mitochondrial energy metabolism, and that glucose at its standard 10 mm concentration is an adequate energy substrate for neonatal neurons in vitro. Notably, changes in pHi appear to have a very powerful modulatory effect on GDPs.

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures

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.

Acid extrusion via blood–brain barrier causes brain alkalosis and seizures after neonatal asphyxia

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.

Vasopressin excites interneurons to suppress hippocampal network activity across a broad span of brain maturity at birth.

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.