Volodymyr Dzhala

NKCC1 transporter facilitates seizures in the developing brain

During development, activation of Cl−-permeable GABAA receptors (GABAA-R) excites neurons as a result of elevated intracellular Cl− levels and a depolarized Cl− equilibrium potential (ECl). GABA becomes inhibitory as net outward neuronal transport of Cl− develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na+-K+-2Cl− cotransporter (NKCC1) facilitates the accumulation of Cl− in neurons. The NKCC1 blocker bumetanide shifted ECl negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABAA-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl−-extruding transporter (KCC2) in human and rat cortex showed that Cl− transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.

Bumetanide enhances phenobarbital efficacy in a neonatal seizure model.

High levels of expression of the Na+‐K+‐2Cl− (NKCC1) cotransporter in immature neurons cause the accumulation of intracellular chloride and, therefore, a depolarized Cl− equilibrium potential (ECl). This results in the outward flux of Cl− through GABAA channels, the opposite direction compared with mature neurons, in which GABAA receptor activation is inhibitory because Cl− flows into the cell. This outward flow of Cl− in neonatal neurons is excitatory and contributes to a greater seizure propensity and poor electroencephalographic response to GABAergic anticonvulsants such as phenobarbital and benzodiazepines. Blocking the NKCC1 transporter with bumetanide prevents outward Cl− flux and causes a more negative GABA equilibrium potential (EGABA) in immature neurons. We therefore tested whether bumetanide enhances the anticonvulsant action of phenobarbital in the neonatal brain

Mechanisms of Fast Ripples in the Hippocampus

Hippocampal fast ripples (FRs) have been associated with seizure onset in both human and experimental epilepsy. To characterize the mechanisms underlying FR oscillations (200-600 Hz), we studied activity of single neurons and neuronal networks in rat hippocampal slices in vitro. The correlation between the action potentials of bursting pyramidal cells and local field potential oscillations suggests that synchronous onset of action potential bursts and similar intrinsic firing patterns among local neurons are both necessary conditions for FR oscillations. Increasing the fidelity of individual pyramidal cell spike train timing by blocking accommodation dramatically increased FR amplitude, whereas blockade of potassium conductances decreased the fidelity of action potential timing in individual pyramidal cell action potential bursts and decreased FR amplitude. Blockade of ionotropic glutamate receptors desynchronized onset of action potential bursts in individual pyramidal cells and abolished fast ripples. Thus, synchronous burst onset mediated by recurrent excitatory synaptic transmission and similar intrinsic spike timing mechanisms in neighboring pyramidal cells are necessary conditions for FR oscillations within the hippocampal network.

Progressive NKCC1-Dependent Neuronal Chloride Accumulation during Neonatal Seizures

Seizures induce excitatory shifts in the reversal potential for GABAA-receptor-mediated responses, which may contribute to the intractability of electro-encephalographic seizures and preclude the efficacy of widely used GABAergic anticonvulsants such as phenobarbital. We now report that, in intact hippocampi prepared from neonatal rats and transgenic mice expressing Clomeleon, recurrent seizures progressively increase the intracellular chloride concentration ([Cl−]i) assayed by Clomeleon imaging and invert the net effect of GABAA receptor activation from inhibition to excitation assayed by the frequency of action potentials and intracellular Ca2+ transients. These changes correlate with increasing frequency of seizure-like events and reduction in phenobarbital efficacy. The Na+–K+–2Cl− (NKCC1) cotransporter blocker bumetanide inhibited seizure-induced neuronal Cl− accumulation and the consequent facilitation of recurrent seizures. Our results demonstrate a novel mechanism by which seizure activity leads to [Cl−]i accumulation, thereby increasing the probability of subsequent seizures. This provides a potential mechanism for the early crescendo phase of neonatal seizures.

Differences in Cortical versus Subcortical GABAergic Signaling: A Candidate Mechanism of Electroclinical Uncoupling of Neonatal Seizures

Electroclinical uncoupling of neonatal seizures refers to electrographic seizure activity that is not clinically manifest. Uncoupling increases after treatment with Phenobarbital, which enhances the GABA(A) receptor (GABA(A)R) conductance. The effects of GABA(A)R activation depend on the intracellular Cl(-) concentration ([Cl(-)](i)) that is determined by the inward Cl(-) transporter NKCC1 and the outward Cl(-) transporter KCC2. Differential maturation of Cl(-) transport observed in cortical versus subcortical regions should alter the efficacy of GABA-mediated inhibition. In perinatal rat pups, most thalamic neurons maintained low [Cl(-)](i) and were inhibited by GABA. Phenobarbital suppressed thalamic seizure activity. Most neocortical neurons maintained higher [Cl(-)](i), and were excited by GABA(A)R activation. Phenobarbital had insignificant anticonvulsant responses in the neocortex until NKCC1 was blocked. Regional differences in the ontogeny of Cl(-) transport may thus explain why seizure activity in the cortex is not suppressed by anticonvulsants that block the transmission of seizure activity through subcortical networks.

Local Impermeant Anions Establish the Neuronal Chloride Concentration

Neuronal intracellular chloride concentration [Cl(-)](i) is an important determinant of γ-aminobutyric acid type A (GABA(A)) receptor (GABA(A)R)-mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl(-) across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl(-)](i). Measurement of [Cl(-)](i) in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A](i)) and polyanionic extracellular matrix glycoproteins ([A](o)) constrain the local [Cl(-)]. CCC inhibition had modest effects on [Cl(-)](i) and neuronal volume, but substantial changes were produced by alterations of the balance between [A](i) and [A](o). Therefore, CCCs are important elements of Cl(-) homeostasis, but local impermeant anions determine the homeostatic set point for [Cl(-)], and hence, neuronal volume and the polarity of local GABA(A)R signaling.Causing Chloride Changes Because intracellular chloride concentrations largely determine the direction and magnitude of current flow through GABAA channels, the stability of intracellular chloride concentration is important to maintain consistent synaptic inhibition. Glykys et al. (p. 670) examined the mechanisms by which chloride gradients in neurons are established, using chloride imaging with transgenically expressed clomeleon dye. Surprisingly, intracellular chloride was not primarily determined by transporters. Instead, subcellular gradients of immobile anions generated inverse chloride gradients. Imaging of a fluorescent chloride indicator reveals a role for impermeant anions in setting intraneuronal chloride levels. Neuronal intracellular chloride concentration [Cl–]i is an important determinant of γ-aminobutyric acid type A (GABAA) receptor (GABAAR)–mediated inhibition and cytoplasmic volume regulation. Equilibrative cation-chloride cotransporters (CCCs) move Cl– across the membrane, but accumulating evidence suggests factors other than the bulk concentrations of transported ions determine [Cl–]i. Measurement of [Cl–]i in murine brain slice preparations expressing the transgenic fluorophore Clomeleon demonstrated that cytoplasmic impermeant anions ([A]i) and polyanionic extracellular matrix glycoproteins ([A]o) constrain the local [Cl–]. CCC inhibition had modest effects on [Cl–]i and neuronal volume, but substantial changes were produced by alterations of the balance between [A]i and [A]o. Therefore, CCCs are important elements of Cl– homeostasis, but local impermeant anions determine the homeostatic set point for [Cl–], and hence, neuronal volume and the polarity of local GABAAR signaling.

Dual Role of GABA in the Neonatal Rat Hippocampus

The effects of modulators of GABA-A receptors on neuronal network activity were studied in the neonatal (postnatal days 0–5) rat hippocampus in vitro. Under control conditions, the physiological pattern of activity of the neonatal hippocampal network was characterized by spontaneous network-driven giant depolarizing potentials (GDPs). The GABA-A receptor agonist isoguvacine (1–2 μM) and the allosteric modulator diazepam (2 μM) induced biphasic responses: initially the frequency of GDPs increased 3 to 4 fold followed by blockade of GDPs and desynchronization of the network activity. The GABA-A receptor antagonists bicuculline (10 μM) and picrotoxin (100 μM) blocked GDPs and induced glutamate (AMPA and NMDA)-receptor-mediated interictal- and ictal-like activities in the hippocampal slices and the intact hippocampus. These data suggest that at early postnatal ages GABA can exert a dual – both excitatory and inhibitory – action on the network activity.

Traumatic Alterations in GABA Signaling Disrupt Hippocampal Network Activity in the Developing Brain

Severe head trauma causes widespread neuronal shear injuries and acute seizures. Shearing of neural processes might contribute to seizures by disrupting the transmembrane ion gradients that subserve normal synaptic signaling. To test this possibility, we investigated changes in intracellular chloride concentration ([Cl−]i) associated with the widespread neural shear injury induced during preparation of acute brain slices. In hippocampal slices and intact hippocampal preparations from immature CLM-1 mice, increases in [Cl−]i correlated with disruption of neural processes and biomarkers of cell injury. Traumatized neurons with higher [Cl−]i demonstrated excitatory GABA signaling, remained synaptically active, and facilitated network activity as assayed by the frequency of extracellular action potentials and spontaneous network-driven oscillations. These data support a more inhibitory role for GABA in the unperturbed immature brain, demonstrate the utility of the acute brain slice preparation for the study of the consequences of trauma, and provide potential mechanisms for both GABA-mediated excitatory network events in the slice preparation and early post-traumatic seizures.

Seizures accelerate anoxia‐induced neuronal death in the neonatal rat hippocampus

Seizures occurring in infants with hypoxia are frequently associated with an ominous prognosis. There is, however, no direct evidence that seizures are involved in the pathogenesis of hypoxia‐induced neuronal damage. Here, we report that seizures significantly aggravate the hypoxic state by accelerating rapid anoxic depolarization (AD) and associated neuronal death in preparations of the intact hippocampus of neonatal rats in vitro. Under control conditions, prolonged episodes of anoxia/aglycemia induced rapid suppression of synaptic activity followed sequentially by brief bursts of epileptiform activity and then by rapid AD. AD was associated with irreversible neuronal damage manifested by irreversible loss of the membrane potential, synaptic responses, and neuronal degeneration. Aggravation of electrographic seizure activity during anoxic episodes by the adenosine A1 receptor antagonists DPCPX and caffeine or the γ‐aminobutyric acid‐A receptor antagonist bicuculline or pretreatment with 4‐aminopyridine accelerated AD and associated neuronal death by up to twofold, whereas blockade of seizure activity by the glutamate receptor antagonists or tetrodotoxin significantly delayed the onset of AD. This report provides direct evidence for the need to prevent seizures during neonatal brain hypoxia. Ann Neurol 2000;48:632–640

Generation and propagation of 4-AP-induced epileptiform activity in neonatal intact limbic structures in vitro.

We examined the generation, propagation and pharmacology of 4‐aminopyridine (4‐AP)‐induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0–7) rat in vitro. Whole‐cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4‐AP‐induced EA as early as P0–1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal cortex. Starting from P3–4, EA propagated from CA3 to the entorhinal cortex. Along the CA3 septo‐temporal axis, EA arose predominantly from the septal pole and spread towards the temporal site. Whereas the onset of 4‐AP‐induced EA decreased with age from 21.2 ± 1.6 min at P0–1 to 4.7 ± 0.63 min at P6–7, the seizure duration increased in the same age groups from 98 ± 14 s to 269.4 ± 85.9 s, respectively. The EA was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) but not by dl‐2‐amino‐5‐phosphonovaleric acid (APV), (+)‐MK‐801 hydrogen maleate (MK‐801) or (±)‐alpha‐methyl‐4‐carboxyphenylglycine (MCPG), suggesting that they were mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptor activation. We conclude that: (i) the septal pole of the hippocampal CA3 region plays a central role in the generation of EA in the neonatal limbic system; and (ii) AMPA/kainate receptor‐mediated EA can be generated in CA3 already at birth. Therefore, the recurrent collateral synapses and circuits required for the generation of EA are developed earlier than previously suggested on the basis of studies on hippocampal slices.