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Molecular mechanisms of antiseizure drug activity at GABAA receptors

Open ArchivePublished:May 16, 2013DOI:https://doi.org/10.1016/j.seizure.2013.04.015

      Abstract

      The GABAA receptor (GABAAR) is a major target of antiseizure drugs (ASDs). A variety of agents that act at GABAARs s are used to terminate or prevent seizures. Many act at distinct receptor sites determined by the subunit composition of the holoreceptor. For the benzodiazepines, barbiturates, and loreclezole, actions at the GABAAR are the primary or only known mechanism of antiseizure action. For topiramate, felbamate, retigabine, losigamone and stiripentol, GABAAR modulation is one of several possible antiseizure mechanisms. Allopregnanolone, a progesterone metabolite that enhances GABAAR function, led to the development of ganaxolone. Other agents modulate GABAergic “tone” by regulating the synthesis, transport or breakdown of GABA. GABAAR efficacy is also affected by the transmembrane chloride gradient, which changes during development and in chronic epilepsy. This may provide an additional target for “GABAergic” ASDs. GABAAR subunit changes occur both acutely during status epilepticus and in chronic epilepsy, which alter both intrinsic GABAAR function and the response to GABAAR-acting ASDs. Manipulation of subunit expression patterns or novel ASDs targeting the altered receptors may provide a novel approach for seizure prevention.

      Keywords

      Seizures frequently result from an imbalance of excitation and inhibition due to a failure of inhibitory neurotransmission. Most agents that enhance GABAA receptor (GABAAR) function have antiseizure properties due to their ability to increase inhibitory neurotransmitter tone. The evidence linking epilepsy with dysfunction of GABAergic inhibition is substantial, and has been extensively reviewed
      • Treiman D.M.
      GABAergic mechanisms in epilepsy.
      • Olsen R.W.
      • DeLorey T.M.
      • Gordey M.
      • Kang M.H.
      GABA receptor function and epilepsy.
      • Ben-Ari Y.
      Seizures beget seizures: the quest for GABA as a key player.
      • Sperk G.
      • Furtinger S.
      • Schwarzer C.
      • Pirker S.
      GABA and its receptors in epilepsy.
      .

      1. GABAARs and epilepsy

      GABAARs are pharmacologically complex, with binding sites for benzodiazepines (BZs), barbiturates, neurosteroids, general anesthetics, loreclezole, and the convulsant toxins, picrotoxin and bicuculline. Protein subunits from seven different subunit families
      • Macdonald R.L.
      • Olsen R.W.
      GABAA receptor channels.
      assemble to form pentameric
      • Nayeem N.
      • Green T.P.
      • Martin I.L.
      • Barnard E.A.
      Quaternary structure of the native GABAA receptor determined by electron microscopic image analysis.
      transmembrane chloride channels (Fig. 1). In mammals, 16 subunit subtypes have been cloned, including 6 α, 3 β and 3 γ subtypes, as well as δ, π
      • Hedblom E.
      • Kirkness E.F.
      A novel class of GABAA receptor subunit in tissues of the reproductive system.
      , ɛ
      • Davies P.A.
      • Hanna M.C.
      • Hales T.G.
      • Kirkness E.F.
      Insensitivity to anesthetic agents conferred by a class of GABAA receptor subunit.
      and θ
      • Bonnert T.P.
      • McKernan R.M.
      • Le Bourdelles B.
      • Smith D.W.
      • Hewson L.
      • Rigby M.R.
      • Sirinathsinghji D.J.
      • Brown N.
      • Wafford K.
      • Whiting P.J.
      Θ, a novel γ-aminobutyric acid type A subunit.
      and alternatively spliced variants of the β2 and γ2 subtypes. Subunit expression is regulated by region, cell type
      • Wisden W.
      • Laurie D.J.
      • Monyer H.
      • Seeburg P.H.
      The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon.
      and developmental stage
      • Brooks-Kayal A.R.
      • Jin H.
      • Price M.
      • Dichter M.A.
      Developmental expression of GABAA receptor subunit mRNAs in individual hippocampal neurons in vitro and in vivo.
      , reducing the number of isoforms expressed in specific brain regions and individual neurons. The most common composition includes two α1, two β2 and a single γ2 subunit; the δ subunit is found instead of γ in receptors expressed extrasynaptically. The subunits are arranged around a central water-filled pore, which gates to conduct Cl ions when GABA is bound (Fig. 1). Individual subunit subtypes confer different sensitivities to GABAAR modulators including BZs
      • Pritchett D.B.
      • Sontheimer H.
      • Shivers B.D.
      • Ymer S.
      • Kettenmann H.
      • Schofield P.R.
      • Seeburg P.H.
      Importance of a novel GABAA subunit for benzodiazepine pharmacology.
      , loreclezole
      • Wingrove P.B.
      • Wafford K.A.
      • Bain C.
      • Whiting P.J.
      The modulatory action of loreclezole at the γ-aminobutyric acid type A receptor is determined by a single amino acid in the β2 and β3 subunit.
      and zinc ions
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      • Verdorn T.A.
      • Ewert M.
      • Seeburg P.H.
      • Sakmann B.
      Functional and molecular distinction between recombinant rat GABAA receptor subtypes by Zn2+.
      (Table 1).
      Figure thumbnail gr1
      Fig. 1Model of a GABAA receptor in the plasma membrane. A. A space-filling model of the pentomer in side view (A) and top view (B) based on the high sequence homology with the nicotinic acetylcholine receptor. There are two binding sites for GABA, between α and β subunits (bent arrows), and one for BZs between the alpha and gamma subunits (arrow). C. A schematic view shows the topology of each subunit with a large extracellular loop containing a cysteine loop and 4 transmembrane domains (M1-M4), the second of which forms the chloride ion channel. Binding of GABA allows the channel to open and conduct Cl ions, resulting in the fast inhibitory post-synaptic potential (IPSP). D. Putative arrangement of 5 subunits to form a pentamer with central chloride channel lined by the M2 subunit. [Derived from the published structure: RCSB PDB Database ·PDB ID: 2BG9 from Unwin, N. (2005)
      • Unwin N.
      Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
      . Images modified from en.wikipedia.org/wiki/GABA_A_receptor, used with permission (public domain)].
      Table 1Antiseizure Drugs and their GABAAR Effects.
      Anti-seizure drugSubunit specificitySite of actionGABAAR actionOther mechanisms
      Benzodiazepinesα1–3, α5; γα/γ interface, α1(H101)Left shift of GABA C/R curve, ↑ open frequencyPossible increased GABAAR channel conductance
      Zolpidemα1 > α2,3; γα/γ interface
      Barbituratesβ subunits?M3 residues?↑ channel open time & burst durationUse-dependent Na+ channel block
      Loreclezoleβ2, β3B2(N289)Barbiturate-like?inhibits GABAAR at ↑ concentration
      Ganaxoloneα, β, δ, ↓ with ɛα1(Q241)Barbiturate-like, also ↑ open frequencyOpen channel block at high conc.
      Topiramateα6 > α4 > α1-α2?unknownEnhanced GABA currentsNa+/Ca2+ channel block, AMPA/kainate receptor block
      FelbamateunknownunknownBarbiturate-likeBlocks NMDA receptor currents
      RetigabineunknownunknownIncreased GABA IPSCsOpens KCNQ2/3 potassium channels
      LosigamoneunknownunknownEnhanced GABA receptor currentUse-dependent Na+ channel block
      Stiripentolα3, ↓ with β1, ɛunknownBarbiturate-likeCYP450 inhibition
      VigabatrinGABA transaminaseincreases [GABA]
      Gabapentin, pregabalinGlutamic acid decarboxylase↑ GABA synthesis?α2-δ subunit of voltage-gated Ca2+ channel
      Valproic AcidGlutamic acid decarboxylase↑ GABA synthesis?block of Na+, T-type Ca2+ channels
      TiagabineGAT-1 (GABA transporter)Blocks GABA reuptake
      GABAARs are the target not only of the BZs, but other ASDs including barbiturates and agents like tiagabine and vigabatrin
      • Treiman D.M.
      GABAergic mechanisms in epilepsy.
      that increase GABA concentration at the synapse. Bicuculline and picrotoxin, which are GABAAR antagonists, induce seizures in animals and epileptiform activity in brain slice preparations. Several animal models of epilepsy have altered GABAAR number or function
      • Treiman D.M.
      GABAergic mechanisms in epilepsy.
      • Olsen R.W.
      • DeLorey T.M.
      • Gordey M.
      • Kang M.H.
      GABA receptor function and epilepsy.
      • Jones-Davis D.M.
      • Macdonald R.L.
      GABA(A) receptor function and pharmacology in epilepsy and status epilepticus.
      . GABAAR subunit expression is altered in the hippocampi of experimental animals with recurrent seizures
      • Kokaia M.
      • Pratt G.D.
      • Elmer E.
      • Bengzon J.
      • Fritschy J.M.
      • Kokaia Z.
      • Lindvall O.
      • Mohler H.
      Biphasic differential changes of GABAA receptor subunit mRNA levels in denate gyrus granule cells following recurrent kindling-induced seizures.
      and in patients with temporal lobe epilepsy
      • Brooks-Kayal A.R.
      • Shumate M.D.
      • Jin H.
      • Rikhter T.Y.
      • Coulter D.A.
      Selective changes in single cell GABAA receptor subunit expression and function in temporal lobe epilepsy.
      • Loup F.
      • Weiser H.G.
      • Yonekawa Y.
      • Aguzzi A.
      • Fritschy J.M.
      Selective alterations in GABAA receptor subtypes in human temporal lobe epilepsy.
      . Angelman's syndrome is a human neurodevelopmental disorder associated with severe mental retardation and epilepsy, which is linked to a deletion mutation on chromosome 15q11-13
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      • Kumagai T.
      • Miura K.
      • Miyazaki S.
      • Hayakawa C.
      • Yamanaka T.
      Epilepsy in Angelman syndrome associated with chromosome 15q deletion.
      in a region encoding the GABAAR β3 subunit
      • DeLorey T.M.
      • Handforth A.
      • Anagnostaras S.G.
      • Homanics G.E.
      • Minassian B.A.
      • Asatourian A.
      • Fanselow M.S.
      • Delgado-Escueta A.
      • Ellison G.D.
      • Olsen R.W.
      Mice lacking the beta3 subunit of the GABAA receptor have the epilepsy phenotype and many of the behavioral characteristics of Angelman syndrome.
      . Two mutations in the γ2 subunit that impair GABAAR function
      • Bianchi M.T.
      • Song L.
      • Zhang H.
      • Macdonald R.L.
      Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans.
      , K289 M
      • Baulac S.
      • Huberfeld G.
      • Gourfinkel-An I.
      • Mitropoulou G.
      • Beranger A.
      • Prud’homme J.-F.
      • Baulac M.
      • Brice A.
      • Bruzzone R.
      • LeGuern E.
      First genetic evidence of GABAA receptor dysfunction in epilepsy: a mutation in the γ2-subunit gene.
      and R43Q
      • Wallace R.H.
      • Marini C.
      • Petrou S.
      • Harkin L.A.
      • Bowser D.N.
      • Panchal R.G.
      • Williams D.A.
      • Sutherland G.R.
      • Mulley J.C.
      • Scheffer I.E.
      • Berkovic S.F.
      Mutant GABA(A) receptor gamma2-subunit in childhood absence epilepsy and febrile seizures.
      , have been linked to a human syndrome of childhood absence epilepsy and febrile seizures, and a loss-of-function mutation in the α1 subunit causes autosomal dominant Juvenile Myoclonic Epilepsy
      • Cossette P.
      • Liu L.
      • Brisebois K.
      • Dong H.
      • Lortie A.
      • Vanasse M.l.
      • Saint-Hilaire J.M.
      • Carmant L.
      • Verner A.
      • Lu W.Y.
      • Wang Y.T.
      • Rouleau G.A.
      Mutation of GABRA1 in an autosomal dominant form of juvenile myoclonic epilepsy.
      . The R43Q mutation in the γ2 subunit reduces BZ sensitivity
      • Bowser D.N.
      • Wagner D.A.
      • Czajkowski C.
      • Cromer B.A.
      • Parker M.W.
      • Wallace R.H.
      • Harkin L.A.
      • Mulley J.C.
      • Marini C.
      • Berkovic S.F.
      • Williams D.A.
      • Jones M.V.
      • Petrou S.
      Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit mutation [gamma 2(R43Q)] found in human epilepsy.
      by altering GABAAR assembly
      • Frugier G.
      • Coussen F.
      • Giraud M.F.
      • Odessa M.F.
      • Emerit M.B.
      • Boue-Grabot E.
      • Garret M.
      A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface.
      • Hales T.G.
      • Tang H.
      • Bollan K.A.
      • Johnson S.J.
      • King D.P.
      • McDonald N.A.
      • Cheng A.
      • Connolly C.N.
      The epilepsy mutation, gamma2(R43Q) disrupts a highly conserved inter-subunit contact site, perturbing the biogenesis of GABAA receptors.
      • Sancar F.
      • Czajkowski C.
      A GABAA receptor mutation linked to human epilepsy (gamma2R43Q) impairs cell surface expression of alphabetagamma receptors.
      and trapping the receptor in the endoplasmic reticulum
      • Kang J.Q.
      • Macdonald R.L.
      The GABAA receptor gamma2 subunit R43Q mutation linked to childhood absence epilepsy and febrile seizures causes retention of alpha1beta2gamma2S receptors in the endoplasmic reticulum.
      . Other point mutations in GABAAR subunits have also been associated with generalized epilepsies (see Fig. 2). Hence, GABAAR modulation by GABAergic ASDs is likely critical to their antiseizure activity.
      Figure thumbnail gr2
      Fig. 2Model of a prototype GABAAR subunit (based on α1 subunit diagram from Olsen & Tobin, 1990)
      • Olsen R.W.
      • Tobin A.J.
      Molecular biology of GABAA receptors.
      showing approximate locations of point mutations associated with generalized epilepsies (see also Macdonald et al., 2012

      Macdonald RL, Kang JQ, Gallagher MJ. GABAA receptor subunit mutations and genetic epilepsies, 2012.

      ) in black, and locations of point mutations associated with ASD sites of action. See text for details.

      2. Benzodiazepines

      BZs were initially developed as anxiolytic agents in the 1950's. Chlordiazepoxide was introduced in 1960, followed by diazepam
      • Sternbach L.H.
      • Reeder E.
      Quinazolines and 1, 4-benzodiazepines, IV: transformations of 7-chloro-2-methylamino-5-phenyl-3H-1,4-benzodiazepine-4-oxide.
      and nitrazepam.
      • Sternbach L.H.
      • Fryer R.I.
      • Keller O.
      • et al.
      Quinazolines and 1, 4-benzodiazepines, X: nitro-substituted 5-phenyl-1,4-benzodiazepine derivatives.
      In 1965, diazepam was first used to treat status epilepticus in humans.
      • Gastaut H.
      • Naquet R.
      • Poire R.
      • Tassarini C.H.
      Treatment of status epilepticus with diazepam (valium).
      Clonazepam was introduced in the 1970's primarily as an ASD,
      • Sato S.
      Benzodiazepines, clonazepam.
      and clobazam, a 1,5 benzodiazepine, was later developed as an ASD with less sedative effect.
      • Chapman A.G.
      • Horton R.W.
      • Meldrum B.S.
      Anticonvulsant action of a 1,5-benzodiazepine, clobazam, in reflex epilepsy.
      However, the induction of tolerance limits their use as ASDs. The 1,5 BZs may produce less tolerance than the 1,4 BZs, but tolerance to 1,5 BZs does occur.
      • Munn R.
      • Farrell K.
      Open study of clobazam in refractory epilepsy.
      BZ activity at GABAARs is a function of the drug's affinity for the BZ binding site and its intrinsic allosteric effect on the GABAAR. The efficacy of individual compounds varies widely. Most BZs in clinical use are full agonists that maximally enhance GABAAR activity. Flumazenil, a competitive antagonist used to reverse BZ-induced sedation,
      • Shannon M.
      • Albers G.
      • Burkhardt K.
      Safety and efficacy of flumazenil in the reversal of benzodiazepine-induced conscious sedation.
      binds to the BZ site without affecting GABAAR function. Abecarnil,
      • Turski L.
      • Stephens D.N.
      • Jensen L.H.
      • Petersen E.N.
      • Meldrum B.S.
      • Patel S.
      • Hansen J.B.
      • Loscher W.
      • Schneider H.H.
      • Schmiechen R.
      Anticonvulsant action of the beta-carboline abecarnil: studies in rodents and baboon, Papio papio.
      imidazenil,
      • Zanotti A.
      • Mariot R.
      • Contarino A.
      • Lipartiti M.
      • Giusti P.
      Lack of anticonvulsant tolerance and benzodiazepine receptor downregulation with imidazenil in rats.
      and bretazenil
      • Rundfeldt C.
      • Wlaz P.
      • Honack D.
      • Loscher W.
      Anticonvulsant tolerance and withdrawal characteristics of benzodiazepine receptor ligands in different seizure models in mice. Comparison of diazepam, bretazenil and abecarnil.
      are “partial agonists” at the BZ site which have antiseizure efficacy in animal models and appear less prone to tolerance.
      • Hernandez T.D.
      • Heninger C.
      • Wilson M.A.
      • Gallager D.W.
      Relationship of agonist efficacy to changes in GABA sensitivity and anticonvulsant tolerance following chronic benzodiazepine ligand exposure.
      Several β-carbolines are “inverse BZ agonists” that inhibit GABA binding
      • Haefely W.
      • Kyburz E.
      • Gerecke M.
      • Mohler H.
      Recent advances in the molecular pharmacology of benzodiazepine receptors and in the structure-activity relationships of their agonists and antagonists.
      and can induce seizures or anxiety.
      • Polc P.
      Electrophysiology of benzodiazepine receptor ligands: multiple mechanisms and sites of action.
      Antiseizure Activity. BZs are effective against most experimental seizure types, but individual drugs vary in their potency/efficacy in specific seizure models and their other clinical effects.
      • Randall L.O.
      • Kappell B.
      Pharmacological activity of some benzodiazepines and their metabolites.
      BZs are particularly effective against convulsive seizures induced by pentylenetetrazol
      • Rogawski M.A.
      • Porter R.J.
      Antiepileptic drugs: pharmacological mechanisms and clinical efficacy with consideration of promising developmental stage compounds.
      and less effective against tonic seizures induced by maximal electroshock.
      • Swinyard E.A.
      • Castellion A.W.
      Anticonvulsant properties of some benzodiazepines.
      BZs also slow the development of kindling.
      • Albertson T.E.
      • Stark L.G.
      • Derlet R.W.
      Modification of amygdaloid kindling by diazepam in juvenile rats.
      GABAAR Subunits and BZ Pharmacology. BZ augmentation of GABAAR currents requires a γ subunit, and the selectivity of BZ responsiveness is determined by which α subunits are present.
      • Macdonald R.L.
      • Olsen R.W.
      GABAA receptor channels.
      • Verdoorn T.A.
      • Draguhn A.
      • Ymer S.
      • Seeburg P.H.
      • Sakmann B.
      Functional properties of recombinant rat GABAA receptors depend upon subunit composition.
      The BZ binding site is located in a cleft between the extracellular amino termini of the α and γ subunits.
      • Smith G.B.
      • Olsen R.W.
      Functional domains of GABAA receptors.
      The α1 subunit results in a receptor with high affinity for the hypnotic, zolpidem, defining the “BZ-1” (or Ω-1) receptor type.
      • Lüddens H.
      • Korpi E.R.
      • Seeburg P.
      GABAA/benzodizaepine receptor heterogeneity: neurophysiological implications.
      The α2 and α3 subunits result in receptors with moderate zolpidem affinity, termed BZ-2 receptors. GABAARs containing the α5 subunit and/or the γ3 subunit are sensitive to diazepam but not to zolpidem and are termed BZ-3 receptors. GABAARs with the α4 or α6 subunits are insensitive to most BZs.
      The subunit composition not only determines the affinity for particular BZs, but also the clinical/behavioral effect of the BZ at that receptor. The role of the α subunits in BZ pharmacology was revealed by the discovery of a single histidine (H) residue found in all BZ-sensitive α subunits (H101 in the rat α1 subunit), but not in the BZ-insensitive α4 or α6 subunits, which instead have a charged arginine (R) residue. This H residue was discovered in a strain of “alcohol-non-tolerant” rats, which were found to have a spontaneous point mutation in the α6 subunit (R100Q) that made their α6-containing GABAARs (found mostly in the cerebellum) diazepam-sensitive, accounting for their ethanol and BZ intolerance.
      • Korpi E.R.
      • Kleingoor C.
      • Kettenmann H.
      • Seeburg P.H.
      Benzodiazepine-induced motor impairment linked to point mutation in cerebellar GABAA receptor.
      Mutation of R100 to H in α6 dramatically increased BZ binding in this normally insensitive subunit, while mutation of H101 to R in α1 reduced BZ sensitivity.
      • Dunn S.M.J.
      • Davies M.
      • Muntoni A.L.
      • Lambert J.J.
      Mutagenesis of the rat α1 subunit of the γ-aminobutyric acidA receptor reveals the importance of residue 101 in determining the allosteric effects of benzodiazepine site ligands.
      BZ-insensitive α subunit mutations were subsequently “knocked-in” to identify BZ actions at receptors containing that subunit. In homozygous α1(H101R) knock-in mice, the anxiolytic effect was intact, but BZs were not protective against pentylenetetrazol-induced convulsions and did not produce sedation or amnesia, suggesting that binding to the (wild type) α1 subunit is responsible for sedative, amnestic and antiseizure actions.
      • McKernan R.M.
      • Rosahl T.W.
      • Reynolds D.S.
      • Sur C.
      • Wafford K.A.
      • Atack J.R.
      • Farrar S.
      • Myers J.
      • Cook G.
      • Ferris P.
      • Garrett L.
      • Bristow L.
      • Marshall G.
      • Macaulay A.
      • Brown N.
      • Howell O.
      • Moore K.W.
      • Carling R.W.
      • Street L.J.
      • Castro J.L.
      • Ragan C.I.
      • Dawson G.R.
      • Whiting P.J.
      Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAA receptor α1 subunit.
      Moreover, the sedative-hypnotic, zolpidem, showed no sedative effect in α1(H101R) mice.
      • Crestani F.
      • Martin J.R.
      • Mohler H.
      • Rudolph U.
      Mechanism of action of the hypnotic zolpidem in vivo.
      Unfortunately, these findings underscore the association between sedative and antiseizure efficacy at α1-containing GABAARs. Similarly, the anxiolytic
      • Low K.
      • Crestani F.
      • Keist R.
      • Benke D.
      • Brunig I.
      • Benson J.A.
      • Fritschy J.M.
      • Rulicke T.
      • Bluethmann H.
      • Mohler H.
      • Rudolph U.
      Molecular and neuronal substrate for the selective attenuation of anxiety.
      and myorelaxant
      • Crestani F.
      • Low K.
      • Keist R.
      • Mandelli M.
      • Mohler H.
      • Rudolph U.
      Molecular targets for the myorelaxant action of diazepam.
      properties of BZs appear to derive from α2- and α3-containing GABAARs, while the α5 subunit was critical for amnestic effects.
      • Crestani F.
      • Keist R.
      • Fritschy J.M.
      • Benke D.
      • Vogt K.
      • Prut L.
      • Bluthmann H.
      • Mohler H.
      • Rudolph U.
      Trace fear conditioning involves hippocampal alpha5 GABA(A) receptors.
      BZs may also have a true analgesic effect independent of their sedative and anxiolytic actions, associated with the α2 and α3 more than α5 subunits.
      • Knabl J.
      • Zeilhofer U.B.
      • Crestani F.
      • Rudolph U.
      • Zeilhofer H.U.
      Genuine antihyperalgesia by systemic diazepam revealed by experiments in GABAA receptor point-mutated mice.
      Since there is no evidence of biophysically distinct effects of BZs on receptors composed of different α subunits, the different behavioral effects are likely due to the brain regions and neuronal populations expressing these specific GABAAR isoforms. New α2/α3 subunit-selective BZs appear to be anxiolytic but not sedating,
      • Dias R.
      • Sheppard W.F.
      • Fradley R.L.
      • Garrett E.M.
      • Stanley J.L.
      • Tye S.J.
      • Goodacre S.
      • Lincoln R.J.
      • Cook S.M.
      • Conley R.
      • Hallett D.
      • Humphries A.C.
      • Thompson S.A.
      • Wafford K.A.
      • Street L.J.
      • Castro J.L.
      • Whiting P.J.
      • Rosahl T.W.
      • Atack J.R.
      • McKernan R.M.
      • Dawson G.R.
      • Reynolds D.S.
      Evidence for a significant role of alpha 3-containing GABAA receptors in mediating the anxiolytic effects of benzodiazepines.
      and non-sedating antiseizure BZs that do not induce tolerance
      • Rosenberg H.C.
      • Tietz E.I.
      • Chiu T.H.
      Tolerance to anticonvulsant effects of diazepam, clonazepam, and clobazam in amygdala-kindled rats.
      may also be possible.
      BZ Actions at GABAARs. BZs increase the amplitude
      • Macdonald R.
      • Barker J.L.
      Benzodiazepines specifically modulate GABA-mediated postsynaptic inhibition in cultured mammalian neurones.
      or decay time
      • Tietz E.I.
      • Zeng X.J.
      • Chen S.
      • Lilly S.M.
      • Rosenberg H.C.
      • Kometiani P.
      Antagonist-induced reversal of functional and structural measures of hippocampal benzodiazepine tolerance.
      of GABA-mediated inhibitory post-synaptic potentials (IPSPs). Current noise fluctuation analysis
      • Study R.E.
      • Barker J.L.
      Diazepam and (−)-pentobarbital: fluctuation analysis reveals different mechanisms for potentiation of γ-aminobutyric acid responses in cultured central neurons.
      and single channel studies
      • Rogers C.J.
      • Twyman R.E.
      • Macdonald R.L.
      Benzodiazepine and beta-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.
      demonstrated that the BZs increased the opening frequency of the GABAAR chloride channel. In patch-clamp recordings of CNS neurons, BZs produce a leftward shift of the concentration-response curve for GABA,
      • Twyman R.E.
      • Rogers C.J.
      • Macdonald R.L.
      Differential regulation of γ-aminobutyric acid receptor channels by diazepam and phenobarbital.
      due to an increase in the affinity for GABA at its binding site, with no change in the kinetics of channel gating
      • Rogers C.J.
      • Twyman R.E.
      • Macdonald R.L.
      Benzodiazepine and beta-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.
      or single channel conductance. In contrast, an inverse agonist at the BZ site reduced the channel opening frequency for a given GABA concentration. These findings are consistent with binding studies showing the allosteric interaction between the BZ and GABA binding sites. By increasing the affinity of the receptor for GABA through slowing the unbinding rate, the BZs increase the current produced by low GABA concentrations, but not by high GABA concentrations at which receptor binding is saturated, as observed in the synaptic cleft
      • Bianchi M.T.
      • Botzolakis E.J.
      • Lagrange A.H.
      • Macdonald R.L.
      Benzodiazepine modulation of GABA(A) receptor opening frequency depends on activation context: a patch clamp and simulation study.
      . Thus, BZs generally do not increase the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) from individual synapses, but instead prolong the mIPSC decay phase
      • Edwards F.A.
      • Konnerth A.
      • Sakmann B.
      Quantal analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study.
      by slowing the dissociation of GABA from the receptor.
      • Otis T.S.
      • Mody I.
      Modulation of decay kinetics and frequency of GABAA receptor-mediated spontaneous inhibitory post-synaptic currents in hippocampal neurons.
      Prolongation of the mIPSC increases temporal and spatial summation of multiple synaptic inputs, which in turn increases the amplitude of stimulus-evoked polysynaptic IPSCs. The BZs thus increase the inhibitory “tone” of GABAergic synapses, which reduces the hypersynchronous firing of neuron populations that underlies seizures.
      • Treiman D.M.
      GABAergic mechanisms in epilepsy.
      An alternative mechanism for BZ enhancement of GABAAR currents has been proposed based on the controversial finding that BZs progressively increase GABAAR “single channel” conductance.
      • Eghbali M.
      • Curmi J.P.
      • Birnir B.
      • Gage P.W.
      Hippocampal GABA(A) channel conductance increased by diazepam.
      Unlike prior reports of a 27 pS main conductance level and a 19 pS subconductance level,
      • Rogers C.J.
      • Twyman R.E.
      • Macdonald R.L.
      Benzodiazepine and beta-carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.
      Eghbali et al.
      • Eghbali M.
      • Curmi J.P.
      • Birnir B.
      • Gage P.W.
      Hippocampal GABA(A) channel conductance increased by diazepam.
      found conductance levels ranging from 8 to 53 pS in response to GABA alone, and 70–80 pS in the presence of diazepam, with up to 7-fold increase in conductance observed when diazepam was added. The increases in conductance were seen predominantly in cell-attached patches onto neurons expressing native receptors, but were also observed in outside-out patches. They found similar increases in channel conductance induced by pentobarbital,
      • Eghbali M.
      • Birnir B.
      • Gage P.W.
      Conductance of GABAA channels activated by pentobarbitone in hippocampal neurons from newborn rats.
      neuroactive steroids,
      • Gaul S.
      • Ozsarac N.
      • Liu L.
      • Fink R.H.
      • Gage P.W.
      The neuroactive steroids alphaxalone and pregnanolone increase the conductance of single GABAA channels in newborn rat hippocampal neurons.
      propofol,
      • Eghbali M.
      • Gage P.W.
      • Birnir B.
      Effects of propofol on GABAA channel conductance in rat-cultured hippocampal neurons.
      and GABA itself.
      • Birnir B.
      • Eghbali M.
      • Cox G.B.
      • Gage P.W.
      GABA concentration sets the conductance of delayed GABAA channels in outside-out patches from rat hippocampal neurons.
      The same group
      • Everitt A.B.
      • Luu T.
      • Cromer B.
      • Tierney M.L.
      • Birnir B.
      • Olsen R.W.
      • Gage P.W.
      Conductance of recombinant GABA (A) channels is increased in cells co-expressing GABA(A) receptor-associated protein.
      subsequently found that high conductance (>40 pS) GABAAR channels were not observed in recombinant α1-β1-γ2 GABAARs expressed in L929 cells, nor was conductance increased by diazepam unless the GABAAR-associated protein (GABARAP) was also co-expressed, apparently facilitating clustering of GABAAR proteins through its interaction with the cytoplasmic loop of the γ2 subunit as occurs at synapses with native receptors. Since peptides mimicking the intracellular γ2 loop (γ2 381–403) self-associate, and application of this peptide to the cytoplasmic surface of inside-out patches prevented the diazepam-induced increase in conductance, they hypothesized that the apparent single channel conductance changes induced by BZs are due to synchronized openings of multichannel clustered GABAARs via concerted action through the interacting cytoplasmic loops of conjoined receptors. Such interactions might occur through shared transmembrane domains between interacting receptors, as observed in G-protein-coupled receptors.
      • Breitwieser G.E.
      G protein-coupled receptor oligomerization: implications for G protein activation and cell signaling.
      This hypothesis could be addressed using concatenated subunits, chimeras and other strategies. However, synchronization of multiple identical channels by such a mechanism should still require a discrete 9 pS unitary conductance of which the larger conductance states are integer multiples, which has not been reported.

      3. Barbiturates

      Phenobarbital was synthesized by Emil Fischer at Bayer in 1911 and introduced as an ASD by Alfred Hauptmann in 1912. Its tendency to produce sedation and cognitive slowing or confusion, as well as paradoxical hyperactivity in children, has curtailed its use in favor of more modern alternatives.
      • Kwan P.
      • Brodie M.J.
      Phenobarbital for the treatment of epilepsy in the 21st century: a critical review.
      In addition to their enhancement of GABAAR currents, barbiturates also inhibit repetitive action potential firing at neuronal sodium channels
      • Macdonald R.L.
      • McLean M.J.
      Anticonvulsant drugs: mechanisms of action.
      by reducing fractional open time and shifting the potential of half-maximal opening towards hyperpolarized potentials.
      • Rehberg B.
      • Duch D.S.
      • Urban B.W.
      The voltage-dependent action of pentobarbital on batrachotoxin-modified human brain sodium channels.
      These actions contribute to both their antiseizure action and their adverse effect profile.
      When co-applied with low concentrations of GABA, barbiturates increased the mean open time, but had no effect on the lengths of 3 distinct open state durations.
      • Macdonald R.L.
      • Rogers C.J.
      • Twyman R.E.
      Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture.
      The increase in mean open time resulted from fewer openings in the shorter two durations (O1 and O2) and more long duration (O3) openings. There was also an increase in long burst durations, but no change in the single channel opening frequency or in the closed frequency duration histogram. The increase in channel open time results in greater chloride current flux and increased likelihood that channel openings will summate, producing larger inhibitory currents. In the absence of GABA, high concentrations of pentobarbital (EC50 0.33 mM) and phenobarbital (EC50 3 mM) directly activated GABAAR chloride currents with lower efficacy (smaller maximal currents) than GABA
      • Rho J.M.
      • Donevan S.D.
      • Rogawski M.A.
      Direct activation of GABAA receptors by barbiturates in cultured rat hippocampal neurons.
      . Barbiturate-activated GABAAR currents were blocked by bicuculline and picrotoxin, and at high concentrations both phenobarbital and pentobarbital produced open channel block that rapidly terminated the induced currents. The concentrations involved in both direct activation and open channel block are far higher than usual therapeutic levels, thus the main antiseizure mechanism is likely the GABAAR allosteric effect in concert with synaptic and extrasynaptic GABA.
      Unlike the BZs, barbiturate sensitivity does not require a specific subunit composition. Homomeric β1 receptors expressed in Xenopus oocytes were directly activated by pentobarbital even though they were not responsive to GABA; these currents were blocked by picrotoxin or penicillin but not bicuculline.
      • Krishek B.J.
      • Moss S.J.
      • Smart T.G.
      Homomeric beta 1 gamma-aminobutyric acid A receptor-ion channels: evaluation of pharmacological and physiological properties.
      Pentobarbital also induced current in both α1-β3 and β3 homomeric GABAARs.
      • Davies P.A.
      • Kirkness E.F.
      • Hales T.G.
      Modulation by general anaesthetics of rat GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in human embryonic kidney 293 cells.
      At saturating GABA concentrations, pentobarbital markedly potentiated α1-β3-δ currents, increasing desensitization and single channel open duration,
      • Feng H.J.
      • Bianchi M.T.
      • Macdonald R.L.
      Pentobarbital differentially modulates alpha1beta3delta and alpha1beta3gamma2L GABAA receptor currents.
      suggesting that barbiturates may be particularly effective at enhancing tonic GABAAR currents at extrasynaptic sites activated by low GABA concentrations. The ɛ subunit, which increases spontaneous GABAAR channel openings, significantly reduced
      • Davies P.A.
      • Hanna M.C.
      • Hales T.G.
      • Kirkness E.F.
      Insensitivity to anaesthetic agents conferred by a class of GABA(A) receptor subunit.
      but did not completely eliminate
      • Neelands T.R.
      • Fisher J.L.
      • Bianchi M.
      • Macdonald R.L.
      Spontaneous and gamma-aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by epsilon subunit-containing isoforms.
      barbiturate responsiveness when co-expressed with α and β subunits. These findings suggest that barbiturate sensitivity does not require either an α or a γ subunit as it is present in β homomeric receptors; however, presence of the δ or ɛ subunits that substitute for γ can dramatically modify barbiturate responsiveness by altering the efficacy of GABA for activating the channel.
      Chimera and mutagenesis studies have identified domains and residues that contribute to barbiturate action, though in less detail than for the BZs. Mutation of the proline (P228A) in the first transmembrane domain of the β1 subunit reduced barbiturate enhancement of GABA-evoked currents in α1-β1-γ2L GABAARs while increasing apparent GABA affinity, without otherwise altering single channel kinetics.
      • Greenfield Jr., L.J.
      • Zaman S.H.
      • Sutherland M.L.
      • Lummis S.C.
      • Niemeyer M.I.
      • Barnard E.A.
      • Macdonald R.L.
      Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement.
      A chimera study using constructs created from the amino terminal end of the β3 subunit and the carboxyl terminal end of the ρ1 subunit, which forms homomeric GABAC receptors that are insensitive to barbiturates, found that residues of the β3 subunit involved in pentobarbital binding to GABAARs are located downstream from the middle of the M2 region.
      • Serafini R.
      • Bracamontes J.
      • Steinbach J.H.
      Structural domains of the human GABAA receptor 3 subunit involved in the actions of pentobarbital.
      Similarly, mutation of a ρ1 tryptophan (W328) in the third transmembrane domain to a hydrophobic residue produced both allosteric and direct channel activation by pentobarbital.
      • Amin J.
      A single hydrophobic residue confers barbiturate sensitivity to gamma-aminobutyric acid type C receptor.

      4. Loreclezole

      Loreclezole has antiseizure activity in a variety of seizure models, acting more like a barbiturate than a BZ in that the increase in seizure threshold produced by loreclezole was potentiated rather than blocked by the BZ antagonist, flumazenil.
      • Ashton D.
      • Fransen J.
      • Heeres J.
      • Clincke G.H.
      • Janssen P.A.
      In vivo studies on the mechanism of action of the broad spectrum anticonvulsant loreclezole.
      In the hippocampal slice, loreclezole, potentiated paired pulse inhibition
      • Zhang C.L.
      • Heinemann U.
      Effects of the triazole derivative loreclezole (R72063) on stimulus induced ionic and field potential responses and on different patterns of epileptiform activity induced by low magnesium in rat entorhinal cortex-hippocampal slices.
      and inhibited epileptiform discharges induced by low Ca2+ or low Mg2+. Loreclezole strongly potentiated recombinant GABAARs containing a β2 or β3 subunit but did not enhance currents from β1-containing receptors.
      • Wafford K.A.
      • Bain C.J.
      • Quirk K.
      • McKernan R.M.
      • Wingrove P.B.
      • Whiting P.J.
      • Kemp J.A.
      A novel allosteric modulatory site on the GABAA receptor beta subunit.
      A single asparagine residue (β2(N289) or β3(N290)) at the cytoplasmic end of the 2nd transmembrane domain confers sensitivity to loreclezole; this amino acid is a serine in the β1 subunit.
      • Wingrove P.B.
      • Wafford K.A.
      • Bain C.
      • Whiting P.J.
      The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.
      Mutation of β1S290 to N conferred loreclezole sensitivity to β1-containing GABAARs, while mutation of β2N289 or β3N290 to S eliminated loreclezole enhancement. When both β1 and β3 were co-expressed in the same receptor, loreclezole sensitivity was abolished, suggesting a dominant effect of the β1 subunit.
      • Fisher J.L.
      • Macdonald R.L.
      Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes.
      Coexpression with different alpha subunits altered the degree of loreclezole potentiation (α1 = α2 = α3 > α5 > α4);
      • Smith A.J.
      • Alder L.
      • Silk J.
      • Adkins C.
      • Fletcher A.E.
      • Scales T.
      • Kerby J.
      • Marshall G.
      • Wafford K.A.
      • McKernan R.M.
      • Atack J.R.
      Effect of alpha subunit on allosteric modulation of ion channel function in stably expressed human recombinant gamma-aminobutyric acid(A) receptors determined using (36)Cl ion flux.
      expression of α5 and β3 with the π subunit reduced loreclezole potentiation,
      • Neelands T.R.
      • Macdonald R.L.
      Incorporation of the pi subunit into functional gamma-aminobutyric Acid(A) receptors.
      while α1-β3-ɛ receptors showed normal loreclezole enhancement.
      • Neelands T.R.
      • Fisher J.L.
      • Bianchi M.
      • Macdonald R.L.
      Spontaneous and gamma-aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by epsilon subunit-containing isoforms.
      At higher concentrations (> 6 μM) loreclezole caused concentration-dependent inhibition of GABAAR currents by enhancing the rate of apparent desensitization.
      • Donnelly J.L.
      • Macdonald R.L.
      Loreclezole enhances apparent desensitization of recombinant GABAA receptor currents.
      The effect was inconsistent with open channel block as it was voltage independent, non-competitive, and increased with increasing GABA concentration. The BZ site was not involved as the inhibition was not antagonized by flumazenil and did not require a γ subunit. This finding has important clinical implications, since inadvertent high levels of drug could inhibit rather than enhance GABAAR function and potentially trigger seizure activity. Loreclezole inhibited α1-β1-γ2L GABAAR currents that were not potentiated by low concentrations of loreclezole, suggesting separate sites for enhancement and inhibition. At the single channel level, high loreclezole concentrations decreased α1-β1-γ2L mean open time by decreasing the average durations of the open states, and also increased the occurrence of a 20 ms closed state. Loreclezole inhibition was equally effective when applied to the intracellular side of the receptor, suggesting that its inhibitory binding site was accessible from both sides of the membrane, and pre-application of loreclezole prior to GABA inhibited the subsequent GABAAR current, indicating that binding did not require an open channel. Loreclezole was also found to inhibit ρ1 homomeric GABAC receptors with an IC50 of 0.5 μM, which was proposed as a rapid means of pharmacological identification of these receptors.
      • Thomet U.
      • Baur R.
      • Dodd R.H.
      • Sigel E.
      Loreclezole as a simple functional marker for homomeric rho type GABA(C) receptors.

      5. Ganaxolone

      Ganaxolone (3α-OH-3β-methyl-5α-pregnan-20-one) is the 3β-methylated synthetic analog of the neurosteroid, allopregnanolone, a natural metabolite of progesterone that allosterically enhances GABAAR current. It has a broad range of antiseizure activity in animal epilepsy models
      • Carter R.B.
      • Wood P.L.
      • Wieland S.
      • Hawkinson J.E.
      • Belelli D.
      • Lambert J.J.
      • White H.S.
      • Wolf H.H.
      • Mirsadeghi S.
      • Tahir S.H.
      • Bolger M.B.
      • Lan N.C.
      • Gee K.W.
      Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.
      including seizures induced by bicuculline, t-butylbicyclophosphorothionate (TBPS, a high affinity ligand for the GABAAR picrotoxin site), aminophylline and corneal kindling, and is well tolerated and effective against seizures in humans. Ganaxolone is currently under evaluation for partial onset seizures. It may have special utility in women with catamenial epilepsy who have increased seizures during periods of low progesterone in the menstrual cycle, as well as in children with infantile spasms.
      • Monaghan E.P.
      • Mcauley J.W.
      • Data J.L.
      Ganaxolone: a novel positive allosteric modulator of the GABA(A) receptor complex for the treatment of epilepsy.
      The antiseizure effect of neurosteroids is not due to action at the progesterone receptor (PR), as exogenous allopregnanolone prevented seizures in PR knockout mice.
      • Reddy D.S.
      • Castaneda D.C.
      • O’Malley B.W.
      • Rogawski M.A.
      Anticonvulsant activity of progesterone and neurosteroids in progesterone receptor knockout mice.
      Moreover, progesterone's antiseizure effect requires metabolism to allopregnanolone, as it was blocked by finasteride, a 5α-reductase inhibitor that blocks allopregnanolone synthesis from progesterone. Hence, the antiseizure effect of allopregnanolone (and ganaxolone) is mediated by GABAARs.
      Specific neurosteroids can allosterically modulate GABAAR activity, either positively or negatively, via distinct binding sites on GABAARs.
      • Reddy D.S.
      Pharmacology of endogenous neuroactive steroids.
      Since a variety of GABAAR-interacting steroid hormones are synthesized in the brain,
      • Kimoto T.
      • Tsurugizawa T.
      • Ohta Y.
      • Makino J.
      • Tamura H.
      • Hojo Y.
      • Takata N.
      • Kawato S.
      Neurosteroid synthesis by cytochrome p450-containing systems localized in the rat brain hippocampal neurons: N-methyl-D-aspartate and calcium-dependent synthesis.
      these agents represent endogenous modulators of GABAAR function. Allopregnanolone is synthesized in two steps from progesterone: 5α-reductase converts progesterone to 5α-dihydroprogesterone, then 3-α–hydroxysteroid oxidoreductase reversibly converts 5α-dihydroprogesterone to 3α-OH-5α-pregnan-20-one (allopregnanolone). Addition of the 3β-methyl group on ganaxolone does not alter binding to GABAARs, but prevents further metabolism of the 3α-OH group and thus prolongs its GABAAR-modulating actions.
      • Carter R.B.
      • Wood P.L.
      • Wieland S.
      • Hawkinson J.E.
      • Belelli D.
      • Lambert J.J.
      • White H.S.
      • Wolf H.H.
      • Mirsadeghi S.
      • Tahir S.H.
      • Bolger M.B.
      • Lan N.C.
      • Gee K.W.
      Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.
      Ganaxolone enhanced GABA and BZ binding via positive allosteric modulation of GABAAR activity, and at nanomolar concentrations potentiated GABA-evoked currents at α1-β1-γ2L, α2-β1-γ2L or α3-β1-γ2L GABAARs expressed in Xenopus oocytes, while direct activation of chloride flux occurred to a limited extent only at micromolar concentrations.
      • Carter R.B.
      • Wood P.L.
      • Wieland S.
      • Hawkinson J.E.
      • Belelli D.
      • Lambert J.J.
      • White H.S.
      • Wolf H.H.
      • Mirsadeghi S.
      • Tahir S.H.
      • Bolger M.B.
      • Lan N.C.
      • Gee K.W.
      Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.
      The effects of GABAAR-enhancing neurosteroids on single channel GABAAR currents were studied using androsterone (5α-androstan-3α-ol-17-one) and pregnanolone (5β-pregnan-3α-ol-20-one) but likely apply to other positive steroid GABAAR modulators including ganaxolone. Like the barbiturates and loreclezole, these agents increased the proportion of openings to the two longer open states (O2 and O3) without altering the intrinsic durations of those states, and produced longer burst durations.
      • Twyman R.E.
      • Macdonald R.L.
      Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture.
      There was no change in single channel conductance. Unlike the barbiturates, however, the neurosteroids also increased single channel opening frequency and reduced closed time durations in all but the shortest closed time distributions (thought to represent intraburst closures). At high concentrations (10 μM), both agents reduced open channel durations consistent with open channel (“flickering”) block.
      The subunit selectivity of GABAAR-enhancing neurosteroids was initially controversial. An early study
      • Zhu W.J.
      • Wang J.F.
      • Krueger K.E.
      • Vicini S.
      Delta subunit inhibits neurosteroid modulation of GABAA receptors.
      of recombinant receptors expressed from combinations of α1, α6, β3, γ2 and δ showed loss of neurosteroid responsiveness with combinations containing the delta subunit. There was also reduced sensitivity of cerebellar granule neurons to neurosteroids later in in vitro development, when δ subunit expression increases. These findings were interpreted as showing inhibition of neurosteroid responsiveness by inclusion of the δ subunit. However, mice in which the δ subunit was knocked out showed decreased behavioral sensitivity to neurosteroids.
      • Mihalek R.M.
      • Banerjee P.K.
      • Korpi E.R.
      • Quinlan J.J.
      • Firestone L.L.
      • Mi Z.P.
      • Lagenaur C.
      • Tretter V.
      • Sieghart W.
      • Anagnostaras S.G.
      • Sage J.R.
      • Fanselow M.S.
      • Guidotti A.
      • Spigelman I.
      • Li Z.
      • DeLorey T.M.
      • Olsen R.W.
      • Homanics G.E.
      Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice.
      Moreover, in receptors composed of α1 or α6, β3 and either γ2L or δ, the greatest potentiation by tetrahydrodeoxycorticosterone (THDOC) was seen in α1-β3-δ GABAARs.
      • Wohlfarth K.M.
      • Bianchi M.T.
      • Macdonald R.L.
      Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit.
      At high concentrations (1 μM), THDOC inhibited this isoform. There is currently consensus that δ subunit-containing receptors are more sensitive to neurosteroid enhancement, though other subunits are also involved in mediating neurosteroid actions. Presence of the α6 subunit reduced neurosteroid sensitivity.
      • Puia G.
      • Ducic I.
      • Vicini S.
      • Costa E.
      Does neurosteroid modulatory efficacy depend on GABAA receptor subunit composition?.
      THDOC (1 μM) enhanced α1-β3-δ more than α6-β3-δ currents, but increased the extent of desensitization and prolonged deactivation for both receptor isoforms; α1-α6 and α6-α1 chimeras (spliced in transmembrane domain M1) suggested that differences in deactivation rate and its voltage-dependence correlated with N-terminal domains, while the extent of desensitization and its voltage-dependence correlated with C-terminal domains.
      • Bianchi M.T.
      • Haas K.F.
      • Macdonald R.L.
      Alpha1 and alpha6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABA(A) receptors containing the delta subunit.
      In dentate granule cells from epileptic animals, an increase in α4 subunit expression was associated with decreased neurosteroid enhancement.
      • Sun C.
      • Mtchedlishvili Z.
      • Erisir A.
      • Kapur J.
      Diminished neurosteroid sensitivity of synaptic inhibition and altered location of the alpha4 subunit of GABA(A) receptors in an animal model of epilepsy.
      In receptors composed of one α subunit (from α2 through α5), β2 and γ2S, neurosteroid potentiation was dependent on the conserved glutamine residue (α1(Q241)) in the first transmembrane domain of the respective α subunit.
      • Hosie A.M.
      • Clarke L.
      • da S.H.
      • Smart T.G.
      Conserved site for neurosteroid modulation of GABA A receptors.
      The δ subunit did not affect neurosteroid binding but likely influenced the efficacy of neurosteroid potentiation. Mutation of α1Q241 to L abolished neurosteroid potentiation, while mutation to W mimicked the effect of steroids and prevented further augmentation; the neighboring S240 residue also participated in steroid binding.
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor alpha1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      A subsequent study using concatenated subunits demonstrated that a single functional binding site (not disrupted by the Q241 mutation) was sufficient for neurosteroid modulation.
      • Bracamontes J.R.
      • Steinbach J.H.
      Steroid interaction with a single potentiating site is sufficient to modulate GABA-A receptor function.

      6. GABAAR effects of other ASDs

      6.1 Topiramate

      Topiramate is a heterotricyclic sulfamate with several mechanisms of action including inhibition of sodium and calcium channels, inhibition of carbonic anhydrase, and augmentation of GABA-evoked currents. Topiramate's effects on GABAARs may contribute both to its antiseizure efficacy and its side effect profile including memory problems, fatigue and psychomotor slowing. It is approved for partial and generalized seizures, the Lennox Gastaut syndrome, and migraine.
      Topiramate inhibited voltage-gated sodium channels, with a left shift of the steady state inactivation curve
      • Zona C.
      • Ciotti M.T.
      • Avoli M.
      Topiramate attenuates voltage-gated sodium currents in rat cerebellar granule cells.
      resulting in intermittent blockade of sustained repetitive action potential firing during prolonged depolarization.
      • McLean M.J.
      • Bukhari A.A.
      • Wamil A.W.
      Effects of topiramate on sodium-dependent action-potential firing by mouse spinal cord neurons in cell culture.
      It also blocked repetitive firing in hippocampal CA3 neurons from spontaneously epileptic rats, and reduced excitatory post-synaptic potentials and responses to bath-applied glutamate, suggesting blockade of post-synaptic glutamate receptors,
      • Hanaya R.
      • Sasa M.
      • Ujihara H.
      • Ishihara K.
      • Serikawa T.
      • Iida K.
      • Akimitsu T.
      • Arita K.
      • Kurisu K.
      Suppression by topiramate of epileptiform burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro.
      affecting kainate-evoked but not NMDA-evoked currents.
      • Gibbs III, J.W.
      • Sombati S.
      • DeLorenzo R.J.
      • Coulter D.A.
      Cellular actions of topiramate: blockade of kainate-evoked inward currents in cultured hippocampal neurons.
      Topiramate also inhibited both L-type and non L-type high voltage activated calcium currents
      • Zhang X.
      • Velumian A.A.
      • Jones O.T.
      • Carlen P.L.
      Modulation of high-voltage-activated calcium channels in dentate granule cells by topiramate.
      and reduced bicarbonate production via inhibition of carbonic anhydrase,
      • Mirza N.
      • Marson A.G.
      • Pirmohamed M.
      Effect of topiramate on acid-base balance: extent, mechanism and effects.
      which may contribute to its antiseizure effect.
      Topiramate (10 μM) enhanced chloride flux into cerebellar granule neurons stimulated by 10 μM GABA, but did not significantly increase chloride influx alone.
      • White H.S.
      • Brown S.D.
      • Woodhead J.H.
      • Skeen G.A.
      • Wolf H.H.
      Topiramate enhances GABA-mediated chloride flux and GABA-evoked chloride currents in murine brain neurons and increases seizure threshold.
      It also enhanced GABA-evoked currents in cultured cortical neurons that were insensitive to the BZ, clonazepam, and clonazepam-potentiated GABA currents in topiramate-insensitive neurons, confirming that topiramate's site of action on GABAARs is independent of the BZ site.
      • White H.S.
      • Brown S.D.
      • Woodhead J.H.
      • Skeen G.A.
      • Wolf H.H.
      Topiramate modulates GABA-evoked currents in murine cortical neurons by a nonbenzodiazepine mechanism.
      Subunit selectivity studies have been inconsistent. Topiramate (1–100 μM) reversibly inhibited Cl currents evoked by 1–10 μM GABA in Xenopus oocytes expressing α1-β2-γ2S and α2-β2-γ2S GABAARs, and reduced the apparent desensitization (“current-fading rate”) in α1-β2-γ2S-expressing oocytes, but potentiated GABA-evoked Cl currents and increased the desensitization rate in α6-β2-γ2S GABAARs, with no effect on α4-β2-γ2S receptors or mixed population GABAARs expressed from rat brain mRNA.
      • Gordey M.
      • DeLorey T.M.
      • Olsen R.W.
      Differential sensitivity of recombinant GABA(A) receptors expressed in Xenopus oocytes to modulation by topiramate.
      In contrast, another study found that topiramate could both potentiate and directly activate β2 or β3-containing heteromeric GABAARs, with greatest effect on α4-β3-γ2S;
      • Simeone T.A.
      • Wilcox K.S.
      • White H.S.
      Subunit selectivity of topiramate modulation of heteromeric GABA(A) receptors.
      positive or negative effects on β1-containing GABAARs depended on the co-expressed alpha subunit. Additional studies are needed to clarify the site and mechanism of action.

      6.2 Felbamate

      Felbamate was launched as a promising novel ASD in 1993 with an uncertain mechanism of action, but its use was curtailed after early reports of aplastic anemia
      • Pennell P.B.
      • Ogaily M.S.
      • Macdonald R.L.
      Aplastic anemia in a patient receiving felbamate for complex partial seizures.
      and hepatic failure.
      • O’Neil M.G.
      • Perdun C.S.
      • Wilson M.B.
      • McGown S.T.
      • Patel S.
      Felbamate-associated fatal acute hepatic necrosis.
      Felbamate inhibited binding of the GABA antagonist [3H]T-BOB with a regional pattern different from that produced by GABA agonists, bicuculline, zinc or neurosteroids,
      • Kume A.
      • Greenfield Jr., L.J.
      • Macdonald R.L.
      • Albin R.L.
      Felbamate inhibits [3H]t-butylbicycloorthobenzoate (TBOB) binding and enhances Cl- current at the gamma-aminobutyric AcidA (GABAA) receptor.
      and enhanced GABA-elicited Cl currents in cultured cortical neurons. Felbamate enhancement was not blocked by flumazenil, and felbamate did not affect pentobarbital potentiation or PTX inhibition of GABA-evoked currents, suggesting an independent site of action. It prolonged the mean burst duration of GABA-activated single channel currents, suggesting a barbiturate-like effect.
      • Rho J.M.
      • Donevan S.D.
      • Rogawski M.A.
      Barbiturate-like actions of the propanediol dicarbamates felbamate and meprobamate.
      Felbamate also blocked N-methyl-D-aspartate (NMDA) receptor currents, an alternative possible antiseizure mechanism.
      • Subramaniam S.
      • Rho J.M.
      • Penix L.
      • Donevan S.D.
      • Fielding R.P.
      • Rogawski M.A.
      Felbamate block of the N-methyl-D-aspartate receptor.
      Derivative compounds including fluorofelbamate and carisbamate that are not associated with hematopoietic or hepatic toxicity may revive interest in felbamate–like agents.
      • Landmark C.J.
      • Johannessen S.I.
      Modifications of antiepileptic drugs for improved tolerability and efficacy.

      6.3 Ezogabine

      Ezogabine (also known as retigabine) is a novel ASD effective in a variety of animal models. Its primary effect is enhanced activation of heteromeric potassium channels composed of the KCNQ2 and KCNQ3 subunits
      • Rundfeldt C.
      • Netzer R.
      Investigations into the mechanism of action of the new anticonvulsant retigabine. Interaction with GABAergic and glutamatergic neurotransmission and with voltage gated ion channels.
      which underlie the “M current” that is a major determinant of resting membrane potential and neuronal excitability. However, ezogabine also dose-dependently and reversibly potentiated GABAAR-dependent IPSC peak amplitude, decay times and total charge transfer.
      • Otto J.F.
      • Kimball M.M.
      • Wilcox K.S.
      Effects of the anticonvulsant retigabine on cultured cortical neurons: changes in electroresponsive properties and synaptic transmission.
      EPSCs were unaffected, and paired pulse depression was unchanged, suggesting a post-synaptic effect at GABAARs. Ezogabine potentiated GABA-induced currents in rat cortical neurons in a concentration-dependent fashion at 10 μM and above.
      • Rundfeldt C.
      • Netzer R.
      Investigations into the mechanism of action of the new anticonvulsant retigabine. Interaction with GABAergic and glutamatergic neurotransmission and with voltage gated ion channels.
      This action was not antagonized by flumazenil, indicating a non-BZ site of action. Subunit dependence and binding site are not known.

      6.4 Losigamone

      Losigamone is a novel ASD which inhibited the persistent component of sodium currents in hippocampal neurons at depolarized potentials, suppressed sustained repetitive firing
      • Gebhardt C.
      • Breustedt J.M.
      • Noldner M.
      • Chatterjee S.S.
      • Heinemann U.
      The antiepileptic drug losigamone decreases the persistent Na+ current in rat hippocampal neurons.
      and decreased the frequency of spontaneous action potentials without affecting miniature post-synaptic current amplitudes.
      • Draguhn A.
      • Jungclaus M.
      • Sokolowa S.
      • Heinemann U.
      Losigamone decreases spontaneous synaptic activity in cultured hippocampal neurons.
      Losigamone stimulated 36Cl influx into spinal cord neurons in the absence of GABA, and potentiated 36Cl influx stimulated by submaximal GABA concentrations; both effects were blocked by bicuculline or picrotoxin.
      • Dimpfel W.
      • Chatterjee S.S.
      • Noldner M.
      • Ticku M.K.
      Effects of the anticonvulsant losigamone and its isomers on the GABAA receptor system.
      Losigamone did not affect the specific binding of [3H]GABA, [3H]flunitrazepam, or [35S]t-butyl-bicyclophosphorothionate (TBPS) to their receptors, and there was no difference in the effect of the + or –stereoisomers or the racemic mixture. The site of action at GABAARs is unknown.

      6.5 Stiripentol

      Stiripentol is as an adjunct ASD which was thought to act by inhibiting cytochrome P450 enzymes involved in metabolism of conventional ASDs. Recently, stiripentol was found to enhance recombinant GABAAR currents, with greater potentiation of α3-containing receptors and reduced potentiation with the β1 or ɛ subunits.
      • Fisher J.L.
      The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator.
      It caused a leftward shift in the GABA concentration-response relationship without increasing maximal GABA-evoked currents, and did not involve sites associated with neurosteroid or loreclezole potentiation.
      • Fisher J.L.
      The anti-convulsant stiripentol acts directly on the GABA(A) receptor as a positive allosteric modulator.
      Saturating barbiturate sites with pentobarbital occluded stiripentol enhancement, and stiripentol increased the duration but not the frequency of opening of GABAAR channels, suggesting a barbiturate-like mechanism.
      • Quilichini P.P.
      • Chiron C.
      • Ben Ari Y.
      • Gozlan H.
      Stiripentol, a putative antiepileptic drug, enhances the duration of opening of GABA-A receptor channels.

      6.6 GABA-enhancing agents

      An additional mechanism for enhancing GABAAR-mediated inhibition involves increasing the concentration or duration of GABA in the synaptic cleft and perisynaptic/extrasynaptic sites. Enhancing GABA synthesis or blocking its reuptake or catabolism could prolong GABA IPSPs and increase perisynaptic spill-over, increasing tonic/extrasynaptic GABA currents which likely play a major role in seizure prevention.

      6.7 Gabapentin and pregabalin

      Gabapentin was designed as a GABA analog, and some studies have suggested that it modulates the action of the GABA synthetic enzyme, glutamic acid decarboxylase (GAD) and the glutamate synthesizing enzyme, branched-chain amino acid transaminase, resulting in increased GABA synthesis.
      • Taylor C.P.
      Mechanisms of action of gabapentin.
      Gabapentin increases non-synaptic GABA responses from neuronal tissues in vitro and increases GABA levels in brain.
      • Loscher W.
      • Honack D.
      • Taylor C.P.
      Gabapentin increases aminooxyacetic acid-induced GABA accumulation in several regions of rat brain.
      Its other (likely primary) mode of action involves binding to the α2-δ binding site on L- or P/Q-type presynaptic voltage-gated calcium channels, presumably inhibiting excessive neurotransmitter release
      • Dooley D.J.
      • Taylor C.P.
      • Donevan S.
      • Feltner D.
      Ca2+ channel alpha2delta ligands: novel modulators of neurotransmission.
      by interfering with calcium channel functional expression or trafficking
      • Hendrich J.
      • Van Minh A.T.
      • Heblich F.
      • Nieto-Rostro M.
      • Watschinger K.
      • Striessnig J.
      • Wratten J.
      • Davies A.
      • Dolphin A.C.
      Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin.
      . Pregabalin, which binds to the α2-δ calcium channel subunit with higher potency, may have similar mechanisms of action. Most studies emphasize the calcium channel as the primary site,
      • Sills G.J.
      The mechanisms of action of gabapentin and pregabalin.
      though a contributory effect on GABA metabolism is possible.
      • Nemeroff C.B.
      The role of GABA in the pathophysiology and treatment of anxiety disorders.

      6.8 Valproic acid

      Valproic acid may affect GABA production, among other ASD mechanisms. Valproate caused non-significant increases in cerebral GABA levels but elevated brain GAD activity significantly.
      • Loscher W.
      Anticonvulsant and biochemical effects of inhibitors of GABA aminotransferase and valproic acid during subchronic treatment in mice.
      Valproic acid increased GABA synthesis and release in brain regions including substantia nigra,
      • Loscher W.
      Valproate enhances GABA turnover in the substantia nigra.
      which is thought to be involved in the control of seizure generation and propagation.
      • Loscher W.
      Valproate: a reappraisal of its pharmacodynamic properties and mechanisms of action.
      It also reduced the release of gamma-hydroxybutyric acid and attenuated neuronal excitation induced by NMDA-type glutamate receptors.
      • Loscher W.
      Valproate: a reappraisal of its pharmacodynamic properties and mechanisms of action.
      Additional ASD mechanisms include enhancing sodium channel inactivation (like phenytoin) and reducing T-type Ca2+ channel currents (like ethosuximide),
      • Macdonald R.L.
      • Kelly K.M.
      Antiepileptic drug mechanisms of action.
      perhaps explaining its utility against both partial onset and absence seizures.

      6.9 Tiagabine

      Tiagabine is a derivative of nipecotic acid that binds to the presynaptic GAT-1 GABA transporter and blocks GABA reuptake, without acting as a “false neurotransmitter.” In control hippocampal slices, tiagabine alone induced a significant chloride conductance, suggesting that GAT-1 activity controls the basal level of extracellular GABA.
      • Frahm C.
      • Engel D.
      • Draguhn A.
      Efficacy of background GABA uptake in rat hippocampal slices.
      Tiagabine prolonged hippocampal IPSC duration in a lamina-specific fashion, with greater effect in stratum radiatum (167%) than stratum oriens (115%).
      • Engel D.
      • Schmitz D.
      • Gloveli T.
      • Frahm C.
      • Heinemann U.
      • Draguhn A.
      Laminar difference in GABA uptake and GAT-1 expression in rat CA1.
      Tiagabine reduced the frequency of epileptiform discharges in hippocampal slices exposed to low Mg2+ or 4-aminopyridine.
      • Richter D.
      • Luhmann H.J.
      • Kilb W.
      Intrinsic activation of GABA receptors suppresses epileptiform activity in the cerebral cortex of immature mice.
      The ability of tiagabine to prolong GABA currents was preserved in hippocampal slices from pilocarpine-treated epileptic rats, hence the GAT-1 transporter remains a functional target for regulating GABA levels in this model of temporal lobe epilepsy.
      • Frahm C.
      • Stief F.
      • Zuschratter W.
      • Draguhn A.
      Unaltered control of extracellular GABA-concentration through GAT-1 in the hippocampus of rats after pilocarpine-induced status epilepticus.
      Tiagabine was effective as an adjunct agent against partial onset seizures, though its prolonged titration period, requirement for multiple daily doses and CNS side effect profile (dizziness, tremor, somnolence, mood disturbances and rare psychotic symptoms) have limited its use.
      • Adkins J.C.
      • Noble S.
      Tiagabine. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the management of epilepsy.

      6.10 Vigabatrin

      Vigabatrin (γ-vinyl-GABA) has a unique mechanism of action, functioning as a “suicide inhibitor” of GABA transaminase, the primary enzyme for GABA catalysis. This leads to permanent inhibition of the affected enzyme, requiring synthesis of new GABA transaminase and hence prolonging the biological half-life beyond its pharmacokinetic half-life of 8 hours.
      • Richens A.
      Pharmacology and clinical pharmacology of vigabatrin.
      It is effective against complex partial seizures,
      • Gram L.
      • Klosterskov P.
      Dam M. gamma-vinyl GABA: a double-blind placebo-controlled trial in partial epilepsy.
      Lennox–Gastaut syndrome and West Syndrome, for which it has become an alternative to ACTH
      • Riikonen R.
      ACTH therapy of West syndrome: Finnish views.
      particularly in children with tuberous sclerosis.
      • Parisi P.
      • Bombardieri R.
      • Curatolo P.
      Current role of vigabatrin in infantile spasms.
      Vigabatrin increases the extracellular concentrations of GABA both in vitro
      • Gram L.
      • Larsson O.M.
      • Johnsen A.H.
      • Schousboe A.
      Effects of valproate, vigabatrin and aminooxyacetic acid on release of endogenous and exogenous GABA from cultured neurons.
      and in vivo.
      • Petroff O.A.
      • Behar K.L.
      • Mattson R.H.
      • Rothman D.L.
      Human brain gamma-aminobutyric acid levels and seizure control following initiation of vigabatrin therapy.
      The anti-seizure effects of increasing GABA concentration may be complex. Biphasic responses to vigabatrin administration have been observed, with an early proconvulsant effect.
      • Stuchlik A.
      • Kubova H.
      • Mares P.
      Single systemic dose of vigabatrin induces early proconvulsant and later anticonvulsant effect in rats.
      A biphasic effect was also seen on after-discharge duration with an early facilitation of the limbic pattern of epileptiform discharge and later suppression at higher doses.
      • Mares P.
      • Slamberova R.
      Biphasic action of vigabatrin on cortical epileptic after-discharges in rats.
      Elevated GABA concentrations may increase neuronal synchronization, particularly of thalamic neurons in absence epilepsy, and there have been several reports of vigabatrin worsening absence seizures or precipitating absence status epilepticus.
      • Panayiotopoulos C.P.
      • Agathonikou A.
      • Sharoqi I.A.
      • Parker A.P.
      Vigabatrin aggravates absences and absence status.
      A major limitation to the use of vigabatrin is the relatively high incidence of vision disturbance associated with chronic use. In 21 of 30 children treated with vigabatrin who were screened for vision disturbances despite no report of visual symptoms, 4 had visual field constrictions, which did not improve after discontinuation of the drug.
      • Iannetti P.
      • Spalice A.
      • Perla F.M.
      • Conicella E.
      • Raucci U.
      • Bizzarri B.
      Visual field constriction in children with epilepsy on vigabatrin treatment.
      One study associated visual disturbances with infantile spasms rather than the drug.
      • Hammoudi D.S.
      • Lee S.S.
      • Madison A.
      • Mirabella G.
      • Buncic J.R.
      • Logan W.J.
      • Snead O.C.
      • Westall C.A.
      Reduced visual function associated with infantile spasms in children on vigabatrin therapy.
      The prevalence of vision problems ranged from 10 to 40% of pediatric and adult patients exposed to vigabatrin.
      • Wild J.M.
      • Ahn H.S.
      • Baulac M.
      • Bursztyn J.
      • Chiron C.
      • Gandolfo E.
      • Safran A.B.
      • Schiefer U.
      • Perucca E.
      Vigabatrin and epilepsy: lessons learned.
      This adverse effect may be directly related to elevated GABA concentrations, as hippocampal neurons grown in depolarizing conditions (25 mM KCl) showed 20% loss of MAP-2-positive neurons in the presence of vigabatrin, which was mimicked by GABA (100 μM) and blocked by bicuculline or picrotoxin.
      • Lukasiuk K.
      • Pitkanen A.
      GABA(A)-mediated toxicity of hippocampal neurons in vitro.
      The cellular mechanism of this toxicity is not entirely clear, but in situations in which GABA is depolarizing, excessive GABA could promote calcium entry through L-type (slowly desensitizing) voltage-gated calcium channels,
      • Lorsignol A.
      • Taupignon A.
      • Dufy B.
      Short applications of gamma-aminobutyric acid increase intracellular calcium concentrations in single identified rat lactotrophs.
      which in turn could result in activation of caspases and cell death.
      • Kahraman S.
      • Zup S.L.
      • McCarthy M.M.
      • Fiskum G.
      GABAergic mechanism of propofol toxicity in immature neurons.

      7. Excitatory GABAA currents

      Early in CNS development, neurons express the Na+/K+/Cl cotransporter, NKCC1, rather than the K+/Cl cotransporter, KCC2, which is expressed in adult neurons. NKCC1 increases intracellular Cl resulting in a depolarizing Cl reversal potential, while KCC2 exports Cl yielding the hyperpolarizing Cl reversal potential found in adult neurons.
      • Staley K.J.
      • Soldo B.L.
      • Proctor W.R.
      Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors.
      As a result, activation of GABAARs may be excitatory during early development.
      • Brumback A.C.
      • Staley K.J.
      Thermodynamic regulation of NKCC1-mediated Cl- cotransport underlies plasticity of GABA(A) signaling in neonatal neurons.
      Excitatory GABAAR currents may play a trophic role in neuronal migration and connectivity,
      • Kriegstein A.R.
      • Owens D.F.
      GABA may act as a self-limiting trophic factor at developing synapses.
      but may also contribute to epileptogenesis.
      • Dzhala V.I.
      • Talos D.M.
      • Sdrulla D.A.
      • Brumback A.C.
      • Mathews G.C.
      • Benke T.A.
      • Delpire E.
      • Jensen F.E.
      • Staley K.J.
      NKCC1 transporter facilitates seizures in the developing brain.
      Endogenous GABA appears to be proconvulsant in early postnatal rat hippocampal slices, as GABAA antagonists blocked epileptiform activity induced by depolarization with high external [K+]
      • Dzhala V.I.
      • Staley K.J.
      Excitatory actions of endogenously released GABA contribute to initiation of ictal epileptiform activity in the developing hippocampus.
      . However, BZ and barbiturate antiseizure efficacy appear to be intact, likely because persistent opening of GABAAR channels (in the presence of allosteric agents) may reduce the depolarizing chloride reversal potential, resulting in “shunt” inhibition. Alternatively, subthreshhold GABA-evoked depolarization may inactivate sodium channels and prevent action potential firing.
      • Zhang S.J.
      • Jackson M.B.
      GABAA receptor activation and the excitability of nerve terminals in the rat posterior pituitary.
      The current through GABAAR channels can also be altered by changes in intracellular bicarbonate, [HCO3],
      • Staley K.J.
      • Soldo B.L.
      • Proctor W.R.
      Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors.
      which can flow through the channel.
      • Bormann J.
      • Hamill O.P.
      • Sakmann B.
      Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones.
      Changes in [HCO3] may underlie reduced synaptic GABA currents during development of BZ tolerance.
      • Zeng X.J.
      • Tietz E.I.
      Role of bicarbonate ion in mediating decreased synaptic conductance in benzodiazepine tolerant hippocampal CA1 pyramidal neurons.
      Depolarizing GABAAR currents may also be a source of interictal spike activity, as observed in epileptic subiculum neurons in hippocampal brain slices from patients with temporal lobe epilepsy.
      • Cohen I.
      • Navarro V.
      • Clemenceau S.
      • Baulac M.
      • Miles R.
      On the origin of interictal activity in human temporal lobe epilepsy in vitro.
      Changes in the GABA current reversal potential might also explain why diazepam can be less effective in children with epileptic encephalopathies,
      • Shorvon S.D.
      Status epilepticus. Its clinical features and treatment in children and adults.
      and rarely can cause status epilepticus in patients with the Lennox Gastaut syndrome.
      • Tassinari C.A.
      • Dravet C.
      • Roger J.
      • Cano J.P.
      • Gastaut H.
      Tonic status epilepticus precipitated by intravenous benzodiazepine in five patients with Lennox-Gastaut syndrome.
      Bumetanide, an inhibitor of NKCC-1 used clinically as a potent loop diuretic, has been proposed as an adjunctive agent to reduce intraneuronal chloride accumulation and reverse the depolarizing chloride gradient that makes GABAARs excitatory, particularly in the neonatal setting.
      • Brumback A.C.
      • Staley K.J.
      Thermodynamic regulation of NKCC1-mediated Cl- cotransport underlies plasticity of GABA(A) signaling in neonatal neurons.
      Bumetanide improved the responsiveness of hippocampal slices to phenobarbital in reducing epileptiform discharges induced by low Mg2+,
      • Dzhala V.I.
      • Brumback A.C.
      • Staley K.J.
      Bumetanide enhances phenobarbital efficacy in a neonatal seizure model.
      and also suppressed electrographic seizures in neonatal rats.
      • Dzhala V.I.
      • Talos D.M.
      • Sdrulla D.A.
      • Brumback A.C.
      • Mathews G.C.
      • Benke T.A.
      • Delpire E.
      • Jensen F.E.
      • Staley K.J.
      NKCC1 transporter facilitates seizures in the developing brain.
      Depolarizing GABA currents may occur in cortical more than subcortical brain regions, possibly explaining the electroclinical uncoupling that occurs with phenobarbital in neonatal seizures.
      • Glykys J.
      • Dzhala V.I.
      • Kuchibhotla K.V.
      • Feng G.
      • Kuner T.
      • Augustine G.
      • Bacskai B.J.
      • Staley K.J.
      Differences in cortical versus subcortical GABAergic signaling: a candidate mechanism of electroclinical uncoupling of neonatal seizures.
      Bumetanide has been used successfully in a single reported human neonate with seizures,
      • Kahle K.T.
      • Barnett S.M.
      • Sassower K.C.
      • Staley K.J.
      Decreased seizure activity in a human neonate treated with bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(−) cotransporter NKCC1.
      and a clinical trial (NCT00830531) is in progress. However, elimination of the depolarizing Cl gradient during development may have adverse consequences; a permanent reduction in AMPAR-mediated excitatory neurotransmission and sensorimotor gating deficits were observed after bumetanide exposure in neonatal rats.
      • Wang D.D.
      • Kriegstein A.R.
      Blocking early GABA depolarization with bumetanide results in permanent alterations in cortical circuits and sensorimotor gating deficits.
      Such potential adverse effects will have to be weighed against the benefits associated with seizure termination or prevention.

      8. GABAAR subunit changes and ASD efficacy in epilepsy and status epilepticus

      There is considerable evidence that GABAAR composition and function change with epilepsy, both acutely in the setting of status epilepticus and in chronic epilepsy. During status epilepticus, there is a rapid change in GABAAR function that reduces BZ sensitivity,
      • Kapur J.
      • Macdonald R.L.
      Rapid seizure-induced reduction of benzodiazepine and Zn2+ sensitivity of hippocampal dentate granule cell GABAA receptors.
      apparently due to activity-dependent internalization of GABAARs and replacement with BZ-insensitive receptors.
      • Goodkin H.P.
      • Sun C.
      • Yeh J.L.
      • Mangan P.S.
      • Kapur J.
      GABA(A) receptor internalization during seizures.
      Kainic acid-induced status epilepticus in young (postnatal day 9) rats altered the normal developmental pattern of GABAAR subunit maturation,
      • Lauren H.B.
      • Lopez-Picon F.R.
      • Korpi E.R.
      • Holopainen I.E.
      Kainic acid-induced status epilepticus alters GABA receptor subunit mRNA and protein expression in the developing rat hippocampus.
      preventing the normal switch from α2 to α1-containing GABAARs. These changes correlate with the finding in animal models
      • Jones-Davis D.M.
      • Macdonald R.L.
      GABA(A) receptor function and pharmacology in epilepsy and status epilepticus.
      • Kapur J.
      • Macdonald R.L.
      Rapid seizure-induced reduction of benzodiazepine and Zn2+ sensitivity of hippocampal dentate granule cell GABAA receptors.
      • Feng H.J.
      • Mathews G.C.
      • Kao C.
      • Macdonald R.L.
      Alterations of GABA A-receptor function and allosteric modulation during development of status epilepticus.
      and humans
      • Alldredge B.K.
      • Gelb A.M.
      • Isaacs S.M.
      • Corry M.D.
      • Allen F.
      • Ulrich S.
      • Gottwald M.D.
      • O’Neil N.
      • Neuhaus J.M.
      • Segal M.R.
      • Lowenstein D.H.
      A comparison of lorazepam, diazepam, and placebo for the treatment of out-of-hospital status epilepticus.
      • Treiman D.M.
      • Meyers P.D.
      • Walton N.Y.
      • Collins J.F.
      • Colling C.
      • Rowan A.J.
      • Handforth A.
      • Faught E.
      • Calabrese V.P.
      • Uthman B.M.
      • Ramsay R.E.
      • Mamdani M.B.
      A comparison of four treatments for generalized convulsive status epilepticus. Veterans Affairs Status Epilepticus Cooperative Study Group.
      that BZs are effective early in the course of status epilepticus but lose their potency and efficacy for termination of status with longer seizure duration. These findings increase the urgency for treatment of status epilepticus early in its course, and also suggest the need to find alternative treatments that retain efficacy in refractory status epilepticus, or methods to prevent or reverse the GABAAR changes that occur with prolonged seizures.
      In the pilocarpine model of temporal lobe epilepsy, the α1 subunit in dentate granule neurons was downregulated and α4 subunit was upregulated,
      • Brooks-Kayal A.R.
      • Shumate M.D.
      • Jin H.
      • Rikhter T.Y.
      • Coulter D.A.
      Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy.
      while in younger (postnatal day 20) animals, the α1 subunit was increased after status epilepticus.
      • Raol Y.H.
      • Zhang G.
      • Lund I.V.
      • Porter B.E.
      • Maronski M.A.
      • Brooks-Kayal A.R.
      Increased GABA(A)-receptor alpha1-subunit expression in hippocampal dentate gyrus after early-life status epilepticus.
      GABAAR subunit changes after pilocarpine-induced status are associated with altered physiological properties including reduced neurosteroid sensitivity and an increase in α4-containing receptors colocalized with synapses rather than extracellular sites.
      • Sun C.
      • Mtchedlishvili Z.
      • Erisir A.
      • Kapur J.
      Diminished neurosteroid sensitivity of synaptic inhibition and altered location of the alpha4 subunit of GABA(A) receptors in an animal model of epilepsy.
      Such changes might contribute to the propensity for spontaneous seizures or alternatively represent a functional adaptation to reduce seizure expression. Substitution of α1 with α4 might reduce synaptic (phasic) but increase extrasynaptic (tonic) inhibition, and BZ sensitivity would be lost in α4-containing receptors. Using an adenovirus vector to insert the α1 subunit gene driven by the α4 promoter resulted in a decrease in seizure frequency, suggesting that the GABAAR subunit changes were contributory toward epileptogenesis.
      • Raol Y.H.
      • Lund I.V.
      • Bandyopadhyay S.
      • Zhang G.
      • Roberts D.S.
      • Wolfe J.H.
      • Russek S.J.
      • Brooks-Kayal A.R.
      Enhancing GABA(A) receptor alpha 1 subunit levels in hippocampal dentate gyrus inhibits epilepsy development in an animal model of temporal lobe epilepsy.
      Hence, altering GABAAR subunit expression in brain regions critical to seizure development may provide a new therapeutic strategy for seizure prevention. Alternatively, novel ASDs targeting the specific epilepsy-associated subunit composition might provide a less invasive approach toward seizure control.
      Long term exposure to the GABAAR allosteric agents themselves can alter GABAAR composition and function. In rat pups exposed to therapeutic concentrations of diazepam or phenobarbital from postnatal day 10 through 40, then tapered for 2 weeks and euthanized on postnatal day 90 showed increased mRNA expression in dentate granule neurons for GAT-1, 3 and 4, GABAAR subunits α4, α6, β3, δ and θ and GABAB receptor subunit R1, and decreased mRNA expression for GAD65, GAD67 and GABAAR subunits α1 and α3.
      • Raol Y.H.
      • Zhang G.
      • Budreck E.C.
      • Brooks-Kayal A.R.
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      • Kang I.
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      • Chen S.
      • Huang X.
      • Zeng X.J.
      • Sieghart W.
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      Benzodiazepine-mediated regulation of alpha1, alpha2, beta1-3 and gamma2 GABA(A) receptor subunit proteins in the rat brain hippocampus and cortex.
      and barbiturates
      • Tseng Y.T.
      • Wellman S.E.
      • Ho I.K.
      In situ hybridization evidence of differential modulation by pentobarbital of GABAA receptor alpha 1- and beta 3-subunit mRNAs.
      • Klein R.L.
      • Harris R.A.
      Regulation of GABAA receptor structure and function by chronic drug treatments in vivo and with stably transfected cells.
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