Astrocyte chloride, excitatoryinhibitory balance and epilepsy

Verena Untiet, Maiken Nedergaard, Alexei Verkhratsky

Excitation and inhibition are at the core of brain function and malfunction.To sustain the activity of neuronal networks over time and space, glutamatergic excitation is balanced by GABAergic inhibition.The equipoise of excitation and inhibition, known as the excitation/inhibition (E/I) balance, is crucial for proper brain function.The E/I balance is highly dynamic and shifts across different brain states: wakefulness primarily augments excitatory activity, while sleep promotes a decrease in excitation and an increase in inhibition (Bridi et al., 2020).Neuronal activity during various brain states is primarily regulated by neurotransmitters (Schiemann et al., 2015),alongside non-synaptic mechanisms that operate on a slower timescale.The non-synaptic mechanisms are many, with the ionic composition of the extracellular space playing a significant role; altering extracellular ion concentrations affects sleep, arousal,electroencephalogram patterns, and behavioral states(Ding et al., 2016).

Chloride ion is the major anion in the body, and it mediates ionotropic inhibition in the central nervous system.The inhibitory neurotransmitter GABA activates ligand-gated anion channels, known as GABAAreceptors (GABAAR).Neurons in the mature brain have low (～5 mM) intracellular Cl–concentration([Cl–]i) and hence the opening of GABAAR generates Cl–influx leading to hyperpolarization.Sustained inhibitory activity depletes extracellular Cl–, which is replenished by astrocytes acting as a dynamic Cl–reservoir in the central nervous system (Untiet et al.,2023).Astrocytes, which cover synapses with their terminal leaflets thus creating astroglial synaptic cradle (Verkhratsky and Nedergaard, 2014), in contrast to neurons have high [Cl–]iwhich may reach the level of 20–50 mM (Engels et al., 2021; Untiet et al., 2023).Astrocytic Cl–reservoir dynamically changes in various brain states: during sleep [Cl–]iis higher and it is stable (Untiet et al., 2017); during wakefulness,it decreases and fluctuates in parallel to sensory stimulation and locomotion (Untiet et al., 2023).Astrocytic leaflets ensheathing inhibitory synapses are rich in GABAAR, which, when opened, generate Cl–efflux into the synaptic cleft thus maintaining inhibitory transmission.Sustained inhibition therefore relies on both astrocytic GABAAR and astrocytic[Cl–]iwhereas reduction of either may shift the E/I balance towards excitation thus potentially promoting seizures.Astrocyte supply of Cl–also depends on gap junctions and astroglial syncytium so that Cl–may intracellularly diffuse towards sites of inhibitory activity; pharmacological inhibition of astrocytic gap junctions in brain slices accelerates the decline of inhibitory transmission (Egawa et al., 2013; Figure 1).In epilepsy, the aberrant E/I balance is likely to be the central biophysical mechanism.Excitatory responses to GABA were observed in approximately 30% of neurons from brain slices of epilepsy patients(Cohen et al., 2002).This phenomenon has also been replicatedin vitrousing various convulsive agents and related procedures (Yamada et al., 2004).Increased neuronal [Cl–]iwas linked to seizures, spinal cord lesions, and other pathological conditions.Most studies analyzing the excitatory effects of GABA focus on neuronal [Cl–]iand the associated regulatory mechanisms involving Na+–K+–Cl–cotransporter 1(NKCC1/SLC12A2) and K+–Cl–cotransporter 2/SLC12A5(KCC2/Slc12A5) (Ben-Ari et al., 2012).However, recent demonstration of astrocytic Cl–directly modulating neuronal activity highlights the role of astroglial Cl–regulation in the pathophysiology of seizures.Dynamic fluctuations of [Cl–] in the synaptic cleft dictate activity-dependent disinhibition, which can differ between different neuronal compartments or be globally regulated by astrocytes.

Increased anion conductance of astroglia leading to a decrease in [Cl–]itriggers apoptotic loss of these cells (Kovermann et al., 2020).Such astroglial loss is observed in ataxia 6, the genetic disorder manifested by ataxia and epileptic seizures, suggesting that depletion of astroglial Cl–reservoir is a significant pathophysiological factor.When astrocytes are unable to provide sufficient Cl–, GABAergic inhibition can mutate into excitation thus instigating seizures and epileptiform pathology.

Seizures are closely influenced by sleep patterns.Sleep deprivation exacerbates epileptic seizures and interictal epileptiform discharges.Concurrently,astrocytic [Cl–]idisplays a brain state-dependent pattern.Earlier studies have described lower neuronal activity in the cortex during sleep compared to wakefulness.This reduced demand for astrocytic Cl–release during sleep potentially contributes to an increase in astrocytic [Cl–]i.High demand for Cl–during wakefulness could be a limiting factor facilitating epileptic seizures, especially in the background of sleep deprivation.

Equilibrium potential for Cl–(which is the function of Cl gradient, consisting of [Cl–]iand [Cl–]o(Alfonsa et al., 2022)) that defines the direction of GABAARs currents (EGABAA) in pyramidal neurons depends on the time of day, shifting towards hyperpolarization during sleep and towards depolarization during wakefulness.Sleep deprivation upregulates NKCC1, leading to Cl–accumulation in neurons.This effect can be countered by inhibiting NKCC1 with bumetanide (Alfonsa et al., 2022).However,EGABAAis also influenced by [Cl–]oin the synaptic cleft, which is likely controlled by astrocytic [Cl–]i.Elevated astrocytic [Cl–]ilevels during sleep supply Cl–to maintain a hyperpolarizing EGABAA.Conversely, lower Cl–levels during wakefulness limit Cl–availability, making GABAAR depolarizing and excitatory.Consequently, astrocytic [Cl–]imodulates neuronal GABAergic inhibition in a brain state-dependent manner.Aberrant astrocytic Cl–supply therefore can be considered as a relevant mechanism explaining the generation of seizures, and hence astrocytic Cl–homeostasis can represent a valid target for anti-epileptic therapies.Epilepsy is a major burden to our society with available treatments being mainly symptomatic.Astrocytic [Cl–]ihomeostasis and regulation represent a novel paradigm and therapeutic target that could potentially translate into novel therapeutic strategies.

Figure 1 ｜Astrocytic Cl– modulates E/I balance thus potentially contributing to pathogenesis of seizures and epilepsy.Inhibitory synaptic transmission is mediated by GABAAR.Synaptic GABA release also opens GABAAR in presynaptic astrocytic leaflets leading to Cl– efflux.Astrocytic Cl– affects neuronal inhibition and pathophysiological low levels in [Cl–]i lead to reduced inhibition and seizure activity.Modified from Untiet et al.(2023) using Abode Illustrator.[Cl–]i:Intracellular chloride concentration; EEG: electroencephalogram; E/I balance: excitation/inhibition balance; IPSP:inhibitory postsynaptic potential.

Verena Untiet*, Maiken Nedergaard,Alexei Verkhratsky

Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, University of Copenhagen, Copenhagen, Denmark (Untiet V,Nedergaard M, Verkhratsky A)Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, Department of Neurosurgery, University of Rochester Medical Center,Rochester, NY, USA (Nedergaard M)Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK(Verkhratsky A)Achucarro Centre for Neuroscience, IKERBASQUE,Basque Foundation for Science, Bilbao, Spain(Verkhratsky A)

*Correspondence to:Verena Untiet, Dr.,verena@sund.ku.dk.

https://orcid.org/0000-0002-1888-6378(Verena Untiet)

Date of submission: August 22, 2023

Date of decision: September 18, 2023

Date of acceptance: October 20, 2023

Date of web publication: December 15, 2023

https://doi.org/10.4103/1673-5374.390981

How to cite this article:Untiet V, Nedergaard M,

Verkhratsky A (2024) Astrocyte chloride, excitatoryinhibitory balance and epilepsy.Neural Regen Res

19(9):1887.

Open access statement:This is an open access

journal, and articles are distributed under the terms of the Creative Commons AttributionNonCommercial-ShareAlike 4.0 License, which allows others to remix,tweak, and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.