Joseph Glykys

Which GABA(A) receptor subunits are necessary for tonic inhibition in the hippocampus

GABAA receptors (GABAARs) assembled of different subunits mediate tonic and phasic inhibition in hippocampal neurons. CA1/CA3 pyramidal cells (PCs) predominantly express α5 subunits whereas dentate gyrus granule cells (DGGCs) and molecular layer (ML) interneurons predominantly express δ subunits. Both α5- and δ-containing GABAARs mediate tonic inhibition. We have shown previously that mice lacking α5 subunits (Gabra5−/−) have a residual tonic current in CA1/CA3 PCs because of an upregulation of δ subunits, but the basis of the residual tonic current in DGGCs and ML interneurons of mice lacking the δ subunit (Gabrd−/−) is still unknown. We now show that wild-type DGGCs have a small tonic current mediated by α5 subunit-containing GABAARs responsible for ∼29% of the total tonic current. To better identify the GABAARs mediating tonic inhibition in hippocampal neurons, we generated mice lacking both α5 and δ subunits (Gabra5/Gabrd−/−). Recordings from CA1/CA3 PCs, DGGCs, and ML interneurons in these mice show an absence of tonic currents without compensatory changes in spontaneous IPSCs (sIPSCs), sEPSCs, and membrane resistance. The absence of tonic inhibition results in spontaneous gamma oscillations recordable in vitro in the CA3 pyramidal layer of these mice, which can be mimicked in wild-type mice by blocking α5 subunit-containing GABAARs with 50 nm L-655,708. In conclusion, depending on the cell type, the α5 and δ subunits are the principal GABAAR subunits responsible for mediating the lions share of tonic inhibition in hippocampal neurons.

A new naturally occurring GABA A receptor subunit partnership with high sensitivity to ethanol

According to the rules of GABAA receptor (GABAAR) subunit assembly, α4 and α6 subunits are considered to be the natural partners of δ subunits. These GABAARs are a preferred target of low, sobriety-impairing concentrations of ethanol. Here we demonstrate a new naturally occurring GABAAR subunit partnership: δ subunits of hippocampal interneurons are coexpressed and colocalized with α1 subunits, but not with α4, α6 or any other α subunits. Ethanol potentiates the tonic inhibition mediated by such native α1/δ GABAARs in wild-type and in α4 subunit–deficient (Gabra4−/−) mice, but not in δ subunit–deficient (Gabrd−/−) mice. We also ruled out any compensatory upregulation of α6 subunits that might have accounted for the ethanol effect in Gabra4−/− mice. Thus, α1/δ subunit assemblies represent a new neuronal GABAAR subunit partnership present in hippocampal interneurons, mediate tonic inhibitory currents and are highly sensitive to low concentrations of ethanol.

The main source of ambient GABA responsible for tonic inhibition in the mouse hippocampus

The extracellular space of the brain contains γ‐aminobutyric acid (GABA) that activates extrasynaptic GABAA receptors mediating tonic inhibition. The source of this GABA is uncertain: it could be overspill of vesicular release, non‐vesicular leakage, reverse transport, dying cells or glia. Using a novel approach, we simultaneously measured phasic and tonic inhibitory currents and assessed their correlation. Enhancing or diminishing vesicular GABA release in hippocampal neurons caused highly correlated changes in the two inhibitions. During high‐frequency phasic inhibitory bursts, tonic current was also enhanced as shown by simulating the summation of IPSCs and by recordings in knockout mice devoid of tonic inhibitory current. When vesicular release was reduced by blocking action potentials or the vesicular GABA transporter, phasic and tonic currents decreased in a correlated fashion. Our results are consistent with most of hippocampal tonic inhibitory current being mediated by GABA released from the very vesicles responsible for activating phasic inhibition.

Seizures and enhanced cortical GABAergic inhibition in two mouse models of human autosomal dominant nocturnal frontal lobe epilepsy.

Selected mutations in the human α4 or β2 neuronal nicotinic acetylcholine receptor subunit genes cosegregate with a partial epilepsy syndrome known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To examine possible mechanisms underlying this inherited epilepsy, we engineered two ADNFLE mutations (Chrna4S252F and Chrna4+L264) in mice. Heterozygous ADNFLE mutant mice show persistent, abnormal cortical electroencephalograms with prominent delta and theta frequencies, exhibit frequent spontaneous seizures, and show an increased sensitivity to the proconvulsant action of nicotine. Relative to WT, electrophysiological recordings from ADNFLE mouse layer II/III cortical pyramidal cells reveal a >20-fold increase in nicotine-evoked inhibitory postsynaptic currents with no effect on excitatory postsynaptic currents. i.p. injection of a subthreshold dose of picrotoxin, a use-dependent γ-aminobutyric acid receptor antagonist, reduces cortical electroencephalogram delta power and transiently inhibits spontaneous seizure activity in ADNFLE mutant mice. Our studies suggest that the mechanism underlying ADNFLE seizures may involve inhibitory synchronization of cortical networks via activation of mutant α4-containing nicotinic acetylcholine receptors located on the presynaptic terminals and somatodendritic compartments of cortical GABAergic interneurons.

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.

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.

Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation

Summary. Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [3H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced.

Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential

Pharmacoresistant seizures and cytotoxic cerebral edema are serious complications of ischemic and traumatic brain injury. Intraneuronal Cl- concentration ([Cl-]i) regulation impacts on both cell volume homeostasis and Cl--permeable GABAA receptor-dependent membrane excitability. Understanding the pleiotropic molecular determinants of neuronal [Cl-]i - cytoplasmic impermeant anions, polyanionic extracellular matrix (ECM) glycoproteins, and plasmalemmal Cl- transporters - could help the identification of novel anticonvulsive and neuroprotective targets. The cation/Cl- cotransporters and ECM metalloproteinases may be particularly druggable targets for intervention. We establish here a paradigm that accounts for recent data regarding the complex regulatory mechanisms of neuronal [Cl-]i and how these mechanisms impact on neuronal volume and excitability. We propose approaches to modulate [Cl-]i that are relevant for two common clinical sequela of brain injury: edema and seizures.