Molecular Mechanisms of Antiseizure Drug Activity at GABAA Receptors

Home , Abecarnil, Carisbamate, GABAA receptor, Ganaxolone, Imidazenil, Loreclezole

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Seizure 22 (2013) 589–600

Contents lists available at SciVerse ScienceDirect

Seizure

jou rnal homepage: www.elsevier.com/locate/yseiz

Review

Molecular mechanisms of antiseizure drug activity at GABAA receptors

L. John Greenﬁeld Jr.*

Dept. of Neurology, University of Arkansas for Medical Sciences, 4301W. Markham St., Slot 500, Little Rock, AR 72205, United States

A R T I C L E I N F O A B S T R A C T

Article history: The GABAA receptor (GABAAR) is a major target of antiseizure drugs (ASDs). A variety of agents that act at

Received 6 February 2013

GABAARs s are used to terminate or prevent seizures. Many act at distinct receptor sites determined by

Received in revised form 16 April 2013

the subunit composition of the holoreceptor. For the benzodiazepines, barbiturates, and loreclezole,

Accepted 17 April 2013

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

Keywords:

antiseizure mechanisms. Allopregnanolone, a progesterone metabolite that enhances GABAAR function,

Inhibition

led to the development of ganaxolone. Other agents modulate GABAergic ‘‘tone’’ by regulating the

Epilepsy

synthesis, transport or breakdown of GABA. GABAAR efﬁcacy is also affected by the transmembrane

Antiepileptic drugs

chloride gradient, which changes during development and in chronic epilepsy. This may provide an

GABA receptor

Seizures additional target for ‘‘GABAergic’’ ASDs. GABAAR subunit changes occur both acutely during status

Chloride channel 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.

Seizures frequently result from an imbalance of excitation and central water-ﬁlled pore, which gates to conduct Cl ions when

inhibition due to a failure of inhibitory neurotransmission. Most GABA is bound (Fig. 1). Individual subunit subtypes confer

agents that enhance GABAA receptor (GABAAR) function have different sensitivities to GABAAR modulators including BZs ,

13 14

antiseizure properties due to their ability to increase inhibitory loreclezole and zinc ions (Table 1).

neurotransmitter tone. The evidence linking epilepsy with GABAARs are the target not only of the BZs, but other ASDs

dysfunction of GABAergic inhibition is substantial, and has been including barbiturates and agents like tiagabine and vigabatrin

1–4

extensively reviewed . that increase GABA concentration at the synapse. Bicuculline and

picrotoxin, which are GABAAR antagonists, induce seizures in

1. GABAARs and epilepsy

animals and epileptiform activity in brain slice preparations.

Several animal models of epilepsy have altered GABAAR number or

GABAARs are pharmacologically complex, with binding sites for 1,2,15

function . GABAAR subunit expression is altered in the

benzodiazepines (BZs), barbiturates, neurosteroids, general anes- 16

hippocampi of experimental animals with recurrent seizures

thetics, loreclezole, and the convulsant toxins, picrotoxin and 17,18

and in patients with temporal lobe epilepsy . Angelman’s

bicuculline. Protein subunits from seven different subunit fami-

5 6 syndrome is a human neurodevelopmental disorder associated

lies assemble to form pentameric transmembrane chloride

with severe mental retardation and epilepsy, which is linked to a

channels (Fig. 1). In mammals, 16 subunit subtypes have been 20

7 8 deletion mutation on chromosome 15q11-13 in a region

cloned, including 6 a, 3 b and 3 g subtypes, as well as d, p , e and 21

9 encoding the GABAAR b3 subunit . Two mutations in the g2

u and alternatively spliced variants of the b2 and g2 subtypes. 22 23 24

10 subunit that impair GABAAR function , K289 M and R43Q ,

Subunit expression is regulated by region, cell type and

11 have been linked to a human syndrome of childhood absence

developmental stage , reducing the number of isoforms

epilepsy and febrile seizures, and a loss-of-function mutation in

expressed in speciﬁc brain regions and individual neurons. The

the a1 subunit causes autosomal dominant Juvenile Myoclonic

most common composition includes two a1, two b2 and a single 25

Epilepsy . The R43Q mutation in the g2 subunit reduces BZ

g2 subunit; the d subunit is found instead of g in receptors 26 27–29

sensitivity by altering GABAAR assembly and trapping the

expressed extrasynaptically. The subunits are arranged around a 30

receptor 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

* Tel.: +1 501 686 7236; fax: +1 501 686 7850.

E-mail address: ljgreenﬁ[email protected] ASDs is likely critical to their antiseizure activity.

590 L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600

Fig. 1. Model 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 a and b 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

198

Database PDB ID: 2BG9 from Unwin, N. (2005) . Images modiﬁed from en.wikipedia.org/wiki/GABA_A_receptor, used with permission (public domain)].

2. Benzodiazepines and nitrazepam. In 1965, diazepam was ﬁrst used to treat status

epilepticus in humans. Clonazepam was introduced in the 1970’s

BZs were initially developed as anxiolytic agents in the 1950’s. primarily as an ASD, and clobazam, a 1,5 benzodiazepine, was

31 35

Chlordiazepoxide was introduced in 1960, followed by diazepam later developed as an ASD with less sedative effect. However, the

Table 1

Antiseizure Drugs and their GABAAR Effects.

Anti-seizure drug Subunit speciﬁcity Site of action GABAAR action Other mechanisms

Benzodiazepines a1–3, a5; g a/g interface, Left shift of GABA Possible increased

a1(H101) C/R curve, " GABAAR channel conductance

open frequency

Zolpidem a1 > a2,3; g a/g interface ‘‘ ’’

Barbiturates b subunits? M3 residues? " channel open time & Use-dependent Na channel block

burst duration

Loreclezole b2, b3 B2(N289) Barbiturate-like? inhibits GABAAR at " concentration

Ganaxolone a, b, d, # with e a1(Q241) Barbiturate-like, also " Open channel block at high conc.

open frequency

+ 2+

Topiramate a6 > a4 > a1-a2? unknown Enhanced GABA currents Na /Ca channel block, AMPA/kainate

receptor block

Felbamate unknown unknown Barbiturate-like Blocks NMDA receptor currents

Retigabine unknown unknown Increased GABA IPSCs Opens KCNQ2/3 potassium channels

Losigamone unknown unknown Enhanced GABA Use-dependent Na channel block

receptor current

Stiripentol a3, # with b1, e unknown Barbiturate-like CYP450 inhibition

Vigabatrin GABA transaminase increases [GABA] –

Gabapentin, Glutamic acid decarboxylase " GABA synthesis? a2-d subunit of voltage-gated Ca channel pregabalin

+ 2+

Valproic Acid Glutamic acid decarboxylase " GABA synthesis? block of Na , T-type Ca channels

Tiagabine GAT-1 (GABA transporter) Blocks GABA reuptake

L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600 591

199

Fig. 2. Model of a prototype GABAAR subunit (based on a1 subunit diagram from Olsen & Tobin, 1990) showing approximate locations of point mutations associated with

200

generalized epilepsies (see also Macdonald et al., 2012 ) in black, and locations of point mutations associated with ASD sites of action. See text for details.

induction of tolerance limits their use as ASDs. The 1,5 BZs may receptor with high afﬁnity for the hypnotic, zolpidem, deﬁning the

produce less tolerance than the 1,4 BZs, but tolerance to 1,5 BZs ‘‘BZ-1’’ (or V-1) receptor type. The a2 and a3 subunits result in

does occur. receptors with moderate zolpidem afﬁnity, termed BZ-2 receptors.

BZ activity at GABAARs is a function of the drug’s afﬁnity for the GABAARs containing the a5 subunit and/or the g3 subunit are

BZ binding site and its intrinsic allosteric effect on the GABAAR. The sensitive to diazepam but not to zolpidem and are termed BZ-3

efﬁcacy of individual compounds varies widely. Most BZs in receptors. GABAARs with the a4 or a6 subunits are insensitive to

clinical use are full agonists that maximally enhance GABAAR most BZs.

activity. Flumazenil, a competitive antagonist used to reverse BZ- The subunit composition not only determines the afﬁnity for

induced sedation, binds to the BZ site without affecting GABAAR particular BZs, but also the clinical/behavioral effect of the BZ at

39 40 41

function. Abecarnil, imidazenil, and bretazenil are ‘‘partial that receptor. The role of the a subunits in BZ pharmacology was

agonists’’ at the BZ site which have antiseizure efﬁcacy in animal revealed by the discovery of a single histidine (H) residue found in

models and appear less prone to tolerance. Several b-carbolines all BZ-sensitive a subunits (H101 in the rat a1 subunit), but not in

are ‘‘inverse BZ agonists’’ that inhibit GABA binding and can the BZ-insensitive a4 or a6 subunits, which instead have a

induce seizures or anxiety. charged arginine (R) residue. This H residue was discovered in a

Antiseizure Activity. BZs are effective against most experimental strain of ‘‘alcohol-non-tolerant’’ rats, which were found to have a

seizure types, but individual drugs vary in their potency/efﬁcacy in spontaneous point mutation in the a6 subunit (R100Q) that made

speciﬁc seizure models and their other clinical effects. BZs are their a6-containing GABAARs (found mostly in the cerebellum)

particularly effective against convulsive seizures induced by diazepam-sensitive, accounting for their ethanol and BZ intoler-

46 53

pentylenetetrazol and less effective against tonic seizures ance. Mutation of R100 to H in a6 dramatically increased BZ

induced by maximal electroshock. BZs also slow the develop- binding in this normally insensitive subunit, while mutation of

48 52

ment of kindling. H101 to R in a1 reduced BZ sensitivity. BZ-insensitive a subunit

GABAAR Subunits and BZ Pharmacology. BZ augmentation of mutations were subsequently ‘‘knocked-in’’ to identify BZ

GABAAR currents requires a g subunit, and the selectivity of BZ actions at receptors containing that subunit. In homozygous

5,49

responsiveness is determined by which a subunits are present. a1(H101R) knock-in mice, the anxiolytic effect was intact, but

The BZ binding site is located in a cleft between the extracellular BZs were not protective against pentylenetetrazol-induced

amino termini of the a and g subunits. The a1 subunit results in a convulsions and did not produce sedation or amnesia, suggesting

592 L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600

that binding to the (wild type) a1 subunit is responsible for surface of inside-out patches prevented the diazepam-induced

sedative, amnestic and antiseizure actions. Moreover, the increase in conductance, they hypothesized that the apparent

sedative-hypnotic, zolpidem, showed no sedative effect in single channel conductance changes induced by BZs are due to

a1(H101R) mice. Unfortunately, these ﬁndings underscore synchronized openings of multichannel clustered GABAARs via

the association between sedative and antiseizure efﬁcacy at a1- concerted action through the interacting cytoplasmic loops of

containing GABAARs. Similarly, the anxiolytic and myorelax- conjoined receptors. Such interactions might occur through

ant properties of BZs appear to derive from a2- and a3- shared transmembrane domains between interacting receptors,

containing GABAARs, while the a5 subunit was critical for as observed in G-protein-coupled receptors. This hypothesis

amnestic effects. BZs may also have a true analgesic effect could be addressed using concatenated subunits, chimeras and

independent of their sedative and anxiolytic actions, associated other strategies. However, synchronization of multiple identical

with the a2 and a3 more than a5 subunits. Since there is no channels by such a mechanism should still require a discrete 9 pS

evidence of biophysically distinct effects of BZs on receptors unitary conductance of which the larger conductance states are

composed of different a subunits, the different behavioral effects integer multiples, which has not been reported.

are likely due to the brain regions and neuronal populations

expressing these speciﬁc GABAAR isoforms. New a2/a3 subunit- 3. Barbiturates

selective BZs appear to be anxiolytic but not sedating, and non-

sedating antiseizure BZs that do not induce tolerance may also Phenobarbital was synthesized by Emil Fischer at Bayer in 1911

be possible. and introduced as an ASD by Alfred Hauptmann in 1912. Its

BZ Actions at GABAARs. BZs increase the amplitude or decay tendency to produce sedation and cognitive slowing or confusion,

time of GABA-mediated inhibitory post-synaptic potentials as well as paradoxical hyperactivity in children, has curtailed its

64 77

(IPSPs). Current noise ﬂuctuation analysis and single channel use in favor of more modern alternatives. In addition to their

studies demonstrated that the BZs increased the opening enhancement of GABAAR currents, barbiturates also inhibit

frequency of the GABAAR chloride channel. In patch-clamp repetitive action potential ﬁring at neuronal sodium channels

recordings of CNS neurons, BZs produce a leftward shift of the by reducing fractional open time and shifting the potential of half-

66 79

concentration-response curve for GABA, due to an increase in the maximal opening towards hyperpolarized potentials. These

afﬁnity for GABA at its binding site, with no change in the kinetics actions contribute to both their antiseizure action and their

of channel gating or single channel conductance. In contrast, an adverse effect proﬁle.

inverse agonist at the BZ site reduced the channel opening When co-applied with low concentrations of GABA, barbitu-

frequency for a given GABA concentration. These ﬁndings are rates increased the mean open time, but had no effect on the

consistent with binding studies showing the allosteric interaction lengths of 3 distinct open state durations. The increase in mean

between the BZ and GABA binding sites. By increasing the afﬁnity open time resulted from fewer openings in the shorter two

of the receptor for GABA through slowing the unbinding rate, the durations (O1 and O2) and more long duration (O3) openings.

BZs increase the current produced by low GABA concentrations, There was also an increase in long burst durations, but no change

but not by high GABA concentrations at which receptor binding is in the single channel opening frequency or in the closed

saturated, as observed in the synaptic cleft . Thus, BZs generally frequency duration histogram. The increase in channel open

do not increase the amplitude of miniature inhibitory postsynaptic time results in greater chloride current ﬂux and increased

currents (mIPSCs) from individual synapses, but instead prolong likelihood that channel openings will summate, producing larger

the mIPSC decay phase by slowing the dissociation of GABA from inhibitory currents. In the absence of GABA, high concentrations

the receptor. Prolongation of the mIPSC increases temporal and of pentobarbital (EC50 0.33 mM) and phenobarbital (EC50 3 mM)

spatial summation of multiple synaptic inputs, which in turn directly activated GABAAR chloride currents with lower efﬁcacy

increases the amplitude of stimulus-evoked polysynaptic IPSCs. (smaller maximal currents) than GABA . Barbiturate-activated

The BZs thus increase the inhibitory ‘‘tone’’ of GABAergic synapses, GABAAR currents were blocked by bicuculline and picrotoxin,

which reduces the hypersynchronous ﬁring of neuron populations and at high concentrations both phenobarbital and pentobarbi-

that underlies seizures. tal produced open channel block that rapidly terminated the

An alternative mechanism for BZ enhancement of GABAAR induced currents. The concentrations involved in both direct

currents has been proposed based on the controversial ﬁnding activation and open channel block are far higher than usual

that BZs progressively increase GABAAR ‘‘single channel’’ conduc- therapeutic levels, thus the main antiseizure mechanism is likely

tance. Unlike prior reports of a 27 pS main conductance level the GABAAR allosteric effect in concert with synaptic and

65 70

and a 19 pS subconductance level, Eghbali et al. found extrasynaptic GABA.

conductance levels ranging from 8 to 53 pS in response to GABA Unlike the BZs, barbiturate sensitivity does not require a

alone, and 70–80 pS in the presence of diazepam, with up to 7-fold speciﬁc subunit composition. Homomeric b1 receptors expressed

increase in conductance observed when diazepam was added. The in Xenopus oocytes were directly activated by pentobarbital even

increases in conductance were seen predominantly in cell- though they were not responsive to GABA; these currents were

attached patches onto neurons expressing native receptors, but blocked by picrotoxin or penicillin but not bicuculline.

were also observed in outside-out patches. They found similar Pentobarbital also induced current in both a1-b3 and b3

71 84

increases in channel conductance induced by pentobarbital, homomeric GABAARs. At saturating GABA concentrations,

72 73 74

neuroactive steroids, propofol, and GABA itself. The same pentobarbital markedly potentiated a1-b3-d currents, increasing

75 85

group subsequently found that high conductance (>40 pS) desensitization and single channel open duration, suggesting

GABAAR channels were not observed in recombinant a1-b1-g2 that barbiturates may be particularly effective at enhancing tonic

GABAARs expressed in L929 cells, nor was conductance increased GABAAR currents at extrasynaptic sites activated by low GABA

by diazepam unless the GABAAR-associated protein (GABARAP) concentrations. The e subunit, which increases spontaneous

was also co-expressed, apparently facilitating clustering of GABAAR channel openings, signiﬁcantly reduced but did not

GABAAR proteins through its interaction with the cytoplasmic completely eliminate barbiturate responsiveness when co-

loop of the g2 subunit as occurs at synapses with native receptors. expressed with a and b subunits. These ﬁndings suggest that

Since peptides mimicking the intracellular g2 loop (g2 381–403) barbiturate sensitivity does not require either an a or a g subunit

self-associate, and application of this peptide to the cytoplasmic as it is present in b homomeric receptors; however, presence of

L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600 593

the d or e subunits that substitute for g can dramatically modify found to inhibit r1 homomeric GABAC receptors with an IC50 of

barbiturate responsiveness by altering the efﬁcacy of GABA for 0.5 mM, which was proposed as a rapid means of pharmacological

activating the channel. identiﬁcation of these receptors.

Chimera and mutagenesis studies have identiﬁed domains and

residues that contribute to barbiturate action, though in less detail 5. Ganaxolone

than for the BZs. Mutation of the proline (P228A) in the ﬁrst

transmembrane domain of the b1 subunit reduced barbiturate Ganaxolone (3a-OH-3b-methyl-5a-pregnan-20-one) is the

enhancement of GABA-evoked currents in a1-b1-g2L GABAARs 3b-methylated synthetic analog of the neurosteroid, allopregna-

while increasing apparent GABA afﬁnity, without otherwise nolone, a natural metabolite of progesterone that allosterically

altering single channel kinetics. A chimera study using enhances GABAAR current. It has a broad range of antiseizure

100

constructs created from the amino terminal end of the b3 subunit activity in animal epilepsy models including seizures induced

and the carboxyl terminal end of the r1 subunit, which forms by bicuculline, t-butylbicyclophosphorothionate (TBPS, a high

homomeric GABAC receptors that are insensitive to barbiturates, afﬁnity ligand for the GABAAR picrotoxin site), aminophylline and

found that residues of the b3 subunit involved in pentobarbital corneal kindling, and is well tolerated and effective against

binding to GABAARs are located downstream from the middle of seizures in humans. Ganaxolone is currently under evaluation for

the M2 region. Similarly, mutation of a r1 tryptophan (W328) in partial onset seizures. It may have special utility in women with

the third transmembrane domain to a hydrophobic residue catamenial epilepsy who have increased seizures during periods of

produced both allosteric and direct channel activation by low progesterone in the menstrual cycle, as well as in children with 90 101

pentobarbital. infantile spasms.

The antiseizure effect of neurosteroids is not due to action at the

4. Loreclezole progesterone receptor (PR), as exogenous allopregnanolone pre-

102

vented seizures in PR knockout mice. Moreover, progesterone’s

Loreclezole has antiseizure activity in a variety of seizure antiseizure effect requires metabolism to allopregnanolone, as it

models, acting more like a barbiturate than a BZ in that the was blocked by ﬁnasteride, a 5a-reductase inhibitor that blocks

increase in seizure threshold produced by loreclezole was allopregnanolone synthesis from progesterone. Hence, the antisei-

potentiated rather than blocked by the BZ antagonist, ﬂumaze- zure effect of allopregnanolone (and ganaxolone) is mediated by

nil. In the hippocampal slice, loreclezole, potentiated paired GABAARs.

pulse inhibition and inhibited epileptiform discharges induced Speciﬁc neurosteroids can allosterically modulate GABAAR

2+ 2+

by low Ca or low Mg . Loreclezole strongly potentiated activity, either positively or negatively, via distinct binding sites

104

recombinant GABAARs containing a b2 or b3 subunit but did on GABAARs. Since a variety of GABAAR-interacting steroid

93 105

not enhance currents from b1-containing receptors. A single hormones are synthesized in the brain, these agents represent

asparagine residue (b2(N289) or b3(N290)) at the cytoplasmic endogenous modulators of GABAAR function. Allopregnanolone is

end of the 2nd transmembrane domain confers sensitivity to synthesized in two steps from progesterone: 5a-reductase

loreclezole; this amino acid is a serine in the b1 subunit. converts progesterone to 5a-dihydroprogesterone, then 3-a–

Mutation of b1S290 to N conferred loreclezole sensitivity to b1- hydroxysteroid oxidoreductase reversibly converts 5a-dihydro-

containing GABAARs, while mutation of b2N289 or b3N290 to S progesterone to 3a-OH-5a-pregnan-20-one (allopregnanolone).

eliminated loreclezole enhancement. When both b1 and b3 were Addition of the 3b-methyl group on ganaxolone does not alter

co-expressed in the same receptor, loreclezole sensitivity was binding to GABAARs, but prevents further metabolism of the 3a-

95 100

abolished, suggesting a dominant effect of the b1 subunit. OH group and thus prolongs its GABAAR-modulating actions.

Coexpression with different alpha subunits altered the degree of Ganaxolone enhanced GABA and BZ binding via positive allosteric

loreclezole potentiation (a1 = a2 = a3 > a5 > a4); expression modulation of GABAAR activity, and at nanomolar concentrations

of a5 and b3 with the p subunit reduced loreclezole potentia- potentiated GABA-evoked currents at a1-b1-g2L, a2-b1-g2L or

tion, while a1-b3-e receptors showed normal loreclezole a3-b1-g2L GABAARs expressed in Xenopus oocytes, while direct

enhancement. activation of chloride ﬂux occurred to a limited extent only at

100

At higher concentrations (> 6 mM) loreclezole caused micromolar concentrations.

concentration-dependent inhibition of GABAAR currents by The effects of GABAAR-enhancing neurosteroids on single

enhancing the rate of apparent desensitization. The effect channel GABAAR currents were studied using androsterone

was inconsistent with open channel block as it was voltage (5a-androstan-3a-ol-17-one) and pregnanolone (5b-pregnan-

independent, non-competitive, and increased with increasing 3a-ol-20-one) but likely apply to other positive steroid

GABA concentration. The BZ site was not involved as the GABAAR modulators including ganaxolone. Like the barbitu-

inhibition was not antagonized by ﬂumazenil and did not require rates and loreclezole, these agents increased the proportion of

a g subunit. This ﬁnding has important clinical implications, since openings to the two longer open states (O2 and O3) without

inadvertent high levels of drug could inhibit rather than enhance altering the intrinsic durations of those states, and produced

106

GABAAR function and potentially trigger seizure activity. longer burst durations. There was no change in single

Loreclezole inhibited a1-b1-g2L GABAAR currents that were channel conductance. Unlike the barbiturates, however, the

not potentiated by low concentrations of loreclezole, suggesting neurosteroids also increased single channel opening frequency

separate sites for enhancement and inhibition. At the single and reduced closed time durations in all but the shortest closed

channel level, high loreclezole concentrations decreased a1-b1- time distributions (thought to represent intraburst closures).

g2L mean open time by decreasing the average durations of the At high concentrations (10 mM), both agents reduced open

open states, and also increased the occurrence of a 20 ms closed channel durations consistent with open channel (‘‘ﬂickering’’)

state. Loreclezole inhibition was equally effective when applied block.

to the intracellular side of the receptor, suggesting that its The subunit selectivity of GABAAR-enhancing neurosteroids

107

inhibitory binding site was accessible from both sides of the was initially controversial. An early study of recombinant

membrane, and pre-application of loreclezole prior to GABA receptors expressed from combinations of a1, a6, b3, g2 and d

inhibited the subsequent GABAAR current, indicating that showed loss of neurosteroid responsiveness with combinations

binding did not require an open channel. Loreclezole was also containing the delta subunit. There was also reduced sensitivity of

594 L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600

cerebellar granule neurons to neurosteroids later in in vitro topiramate’s site of action on GABAARs is independent of the BZ

123

development, when d subunit expression increases. These ﬁndings site. Subunit selectivity studies have been inconsistent.

were interpreted as showing inhibition of neurosteroid respon- Topiramate (1–100 mM) reversibly inhibited Cl currents evoked

siveness by inclusion of the d subunit. However, mice in which the by 1–10 mM GABA in Xenopus oocytes expressing a1-b2-g2S and

d subunit was knocked out showed decreased behavioral a2-b2-g2S GABAARs, and reduced the apparent desensitization

108

sensitivity to neurosteroids. Moreover, in receptors composed (‘‘current-fading rate’’) in a1-b2-g2S-expressing oocytes, but

of a1 or a6, b3 and either g2L or d, the greatest potentiation by potentiated GABA-evoked Cl currents and increased the desensi-

tetrahydrodeoxycorticosterone (THDOC) was seen in a1-b3-d tization rate in a6-b2-g2S GABAARs, with no effect on a4-b2-g2S

109

GABAARs. At high concentrations (1 mM), THDOC inhibited this receptors or mixed population GABAARs expressed from rat brain

124

isoform. There is currently consensus that d subunit-containing mRNA. In contrast, another study found that topiramate could

receptors are more sensitive to neurosteroid enhancement, though both potentiate and directly activate b2 or b3-containing

125

other subunits are also involved in mediating neurosteroid actions. heteromeric GABAARs, with greatest effect on a4-b3-g2S;

110

Presence of the a6 subunit reduced neurosteroid sensitivity. positive or negative effects on b1-containing GABAARs depended

THDOC (1 mM) enhanced a1-b3-d more than a6-b3-d currents, on the co-expressed alpha subunit. Additional studies are needed

but increased the extent of desensitization and prolonged to clarify the site and mechanism of action.

deactivation for both receptor isoforms; a1-a6 and a6-a1

chimeras (spliced in transmembrane domain M1) suggested that 6.2. Felbamate

differences in deactivation rate and its voltage-dependence

correlated with N-terminal domains, while the extent of desensi- Felbamate was launched as a promising novel ASD in 1993 with

tization and its voltage-dependence correlated with C-terminal an uncertain mechanism of action, but its use was curtailed after

111 126 127

domains. In dentate granule cells from epileptic animals, an early reports of aplastic anemia and hepatic failure.

increase in a4 subunit expression was associated with decreased Felbamate inhibited binding of the GABA antagonist [ H]T-BOB

112

neurosteroid enhancement. In receptors composed of one a with a regional pattern different from that produced by GABA

128

subunit (from a2 through a5), b2 and g2S, neurosteroid agonists, bicuculline, zinc or neurosteroids, and enhanced

potentiation was dependent on the conserved glutamine residue GABA-elicited Cl currents in cultured cortical neurons. Felbamate

(a1(Q241)) in the ﬁrst transmembrane domain of the respective a enhancement was not blocked by ﬂumazenil, and felbamate did

113

subunit. The d subunit did not affect neurosteroid binding but not affect pentobarbital potentiation or PTX inhibition of GABA-

likely inﬂuenced the efﬁcacy of neurosteroid potentiation. Muta- evoked currents, suggesting an independent site of action. It

tion of a1Q241 to L abolished neurosteroid potentiation, while prolonged the mean burst duration of GABA-activated single

129

mutation to W mimicked the effect of steroids and prevented channel currents, suggesting a barbiturate-like effect. Felba-

further augmentation; the neighboring S240 residue also partici- mate also blocked N-methyl-D-aspartate (NMDA) receptor cur-

114 130

pated in steroid binding. A subsequent study using concatenat- rents, an alternative possible antiseizure mechanism. Derivative

ed subunits demonstrated that a single functional binding site (not compounds including ﬂuorofelbamate and carisbamate that are

disrupted by the Q241 mutation) was sufﬁcient for neurosteroid not associated with hematopoietic or hepatic toxicity may revive

115 131

modulation. interest in felbamate–like agents.

6. GABAAR effects of other ASDs 6.3. Ezogabine

6.1. Topiramate Ezogabine (also known as retigabine) is a novel ASD effective in

a variety of animal models. Its primary effect is enhanced

Topiramate is a heterotricyclic sulfamate with several mecha- activation of heteromeric potassium channels composed of the

132

nisms of action including inhibition of sodium and calcium KCNQ2 and KCNQ3 subunits which underlie the ‘‘M current’’

channels, inhibition of carbonic anhydrase, and augmentation of that is a major determinant of resting membrane potential and

GABA-evoked currents. Topiramate’s effects on GABAARs may neuronal excitability. However, ezogabine also dose-dependently

contribute both to its antiseizure efﬁcacy and its side effect proﬁle and reversibly potentiated GABAAR-dependent IPSC peak ampli-

133

including memory problems, fatigue and psychomotor slowing. It tude, decay times and total charge transfer. EPSCs were

is approved for partial and generalized seizures, the Lennox unaffected, and paired pulse depression was unchanged, suggest-

Gastaut syndrome, and migraine. ing a post-synaptic effect at GABAARs. Ezogabine potentiated

Topiramate inhibited voltage-gated sodium channels, with a GABA-induced currents in rat cortical neurons in a concentration-

116 132

left shift of the steady state inactivation curve resulting in dependent fashion at 10 mM and above. This action was not

intermittent blockade of sustained repetitive action potential antagonized by ﬂumazenil, indicating a non-BZ site of action.

117

ﬁring during prolonged depolarization. It also blocked repetitive Subunit dependence and binding site are not known.

ﬁring in hippocampal CA3 neurons from spontaneously epileptic

rats, and reduced excitatory post-synaptic potentials and 6.4. Losigamone

responses to bath-applied glutamate, suggesting blockade of

118

post-synaptic glutamate receptors, affecting kainate-evoked Losigamone is a novel ASD which inhibited the persistent

119

but not NMDA-evoked currents. Topiramate also inhibited both component of sodium currents in hippocampal neurons at

120 134

L-type and non L-type high voltage activated calcium currents depolarized potentials, suppressed sustained repetitive ﬁring

and reduced bicarbonate production via inhibition of carbonic and decreased the frequency of spontaneous action potentials

121 135

anhydrase, which may contribute to its antiseizure effect. without affecting miniature post-synaptic current amplitudes.

Topiramate (10 mM) enhanced chloride ﬂux into cerebellar Losigamone stimulated Cl inﬂux into spinal cord neurons in the

granule neurons stimulated by 10 mM GABA, but did not absence of GABA, and potentiated Cl inﬂux stimulated by

122

signiﬁcantly increase chloride inﬂux alone. It also enhanced submaximal GABA concentrations; both effects were blocked by

136

GABA-evoked currents in cultured cortical neurons that were bicuculline or picrotoxin. Losigamone did not affect the speciﬁc

3 3 35

insensitive to the BZ, clonazepam, and clonazepam-potentiated binding of [ H]GABA, [ H]ﬂunitrazepam, or [ S]t-butyl-bicyclo-

GABA currents in topiramate-insensitive neurons, conﬁrming that phosphorothionate (TBPS) to their receptors, and there was

L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600 595

no difference in the effect of the + or –stereoisomers or the racemic hippocampal slices, tiagabine alone induced a signiﬁcant

mixture. The site of action at GABAARs is unknown. chloride conductance, suggesting that GAT-1 activity controls

149

the basal level of extracellular GABA. Tiagabine prolonged

6.5. Stiripentol hippocampal IPSC duration in a lamina-speciﬁc fashion, with

greater effect in stratum radiatum (167%) than stratum oriens

150

Stiripentol is as an adjunct ASD which was thought to act by (115%). Tiagabine reduced the frequency of epileptiform

inhibiting cytochrome P450 enzymes involved in metabolism of discharges in hippocampal slices exposed to low Mg or 4-

151

conventional ASDs. Recently, stiripentol was found to enhance aminopyridine. The ability of tiagabine to prolong GABA

recombinant GABAAR currents, with greater potentiation of a3- currents was preserved in hippocampal slices from pilocarpine-

containing receptors and reduced potentiation with the b1 or e treated epileptic rats, hence the GAT-1 transporter remains a

137

subunits. It caused a leftward shift in the GABA concentration- functional target for regulating GABA levels in this model of

152

response relationship without increasing maximal GABA-evoked temporal lobe epilepsy. Tiagabine was effective as an adjunct

currents, and did not involve sites associated with neurosteroid or agent against partial onset seizures, though its prolonged

137

loreclezole potentiation. Saturating barbiturate sites with titration period, requirement for multiple daily doses and CNS

pentobarbital occluded stiripentol enhancement, and stiripentol side effect proﬁle (dizziness, tremor, somnolence, mood dis-

153

increased the duration but not the frequency of opening of GABAAR turbances and rare psychotic symptoms) have limited its use.

138

channels, suggesting a barbiturate-like mechanism.

6.10. Vigabatrin

6.6. GABA-enhancing agents

Vigabatrin (g-vinyl-GABA) has a unique mechanism of action,

An additional mechanism for enhancing GABAAR-mediated functioning as a ‘‘suicide inhibitor’’ of GABA transaminase, the

inhibition involves increasing the concentration or duration of primary enzyme for GABA catalysis. This leads to permanent

GABA in the synaptic cleft and perisynaptic/extrasynaptic sites. inhibition of the affected enzyme, requiring synthesis of new

Enhancing GABA synthesis or blocking its reuptake or catabolism GABA transaminase and hence prolonging the biological half-life

154

could prolong GABA IPSPs and increase perisynaptic spill-over, beyond its pharmacokinetic half-life of 8 hours. It is effective

155

increasing tonic/extrasynaptic GABA currents which likely play a against complex partial seizures, Lennox–Gastaut syndrome

major role in seizure prevention. and West Syndrome, for which it has become an alternative to

156 157

ACTH particularly in children with tuberous sclerosis.

6.7. Gabapentin and pregabalin Vigabatrin increases the extracellular concentrations of GABA

158 159

both in vitro and in vivo. The anti-seizure effects of

Gabapentin was designed as a GABA analog, and some studies increasing GABA concentration may be complex. Biphasic

have suggested that it modulates the action of the GABA synthetic responses to vigabatrin administration have been observed, with

160

enzyme, glutamic acid decarboxylase (GAD) and the glutamate an early proconvulsant effect. A biphasic effect was also seen on

synthesizing enzyme, branched-chain amino acid transaminase, after-discharge duration with an early facilitation of the limbic

139

resulting in increased GABA synthesis. Gabapentin increases pattern of epileptiform discharge and later suppression at higher

161

non-synaptic GABA responses from neuronal tissues in vitro and doses. Elevated GABA concentrations may increase neuronal

140

increases GABA levels in brain. Its other (likely primary) mode of synchronization, particularly of thalamic neurons in absence

action involves binding to the a2-d binding site on L- or P/Q-type epilepsy, and there have been several reports of vigabatrin

presynaptic voltage-gated calcium channels, presumably inhibit- worsening absence seizures or precipitating absence status

141 162

ing excessive neurotransmitter release by interfering with epilepticus.

142

calcium channel functional expression or trafﬁcking . Pregaba- A major limitation to the use of vigabatrin is the relatively high

lin, which binds to the a2-d calcium channel subunit with higher incidence of vision disturbance associated with chronic use. In 21

potency, may have similar mechanisms of action. Most studies of 30 children treated with vigabatrin who were screened for vision

143

emphasize the calcium channel as the primary site, though a disturbances despite no report of visual symptoms, 4 had visual

144

contributory effect on GABA metabolism is possible. ﬁeld constrictions, which did not improve after discontinuation of

163

the drug. One study associated visual disturbances with

164

6.8. Valproic acid infantile spasms rather than the drug. The prevalence of vision

problems ranged from 10 to 40% of pediatric and adult patients

165

Valproic acid may affect GABA production, among other ASD exposed to vigabatrin. This adverse effect may be directly

mechanisms. Valproate caused non-signiﬁcant increases in cere- related to elevated GABA concentrations, as hippocampal neurons

145

bral GABA levels but elevated brain GAD activity signiﬁcantly. grown in depolarizing conditions (25 mM KCl) showed 20% loss of

Valproic acid increased GABA synthesis and release in brain MAP-2-positive neurons in the presence of vigabatrin, which was

146

regions including substantia nigra, which is thought to be mimicked by GABA (100 mM) and blocked by bicuculline or

147 166

involved in the control of seizure generation and propagation. It picrotoxin. The cellular mechanism of this toxicity is not

also reduced the release of gamma-hydroxybutyric acid and entirely clear, but in situations in which GABA is depolarizing,

attenuated neuronal excitation induced by NMDA-type glutamate excessive GABA could promote calcium entry through L-type

147 167

receptors. Additional ASD mechanisms include enhancing (slowly desensitizing) voltage-gated calcium channels, which in

168

sodium channel inactivation (like phenytoin) and reducing T-type turn could result in activation of caspases and cell death.

2+ 148

Ca channel currents (like ethosuximide), perhaps explaining

its utility against both partial onset and absence seizures. 7. Excitatory GABAA currents

+ +

6.9. Tiagabine Early in CNS development, neurons express the Na /K /Cl

cotransporter, NKCC1, rather than the K /Cl cotransporter, KCC2,

Tiagabine is a derivative of nipecotic acid that binds to the which is expressed in adult neurons. NKCC1 increases intracellular

presynaptic GAT-1 GABA transporter and blocks GABA reuptake, Cl resulting in a depolarizing Cl reversal potential, while KCC2

without acting as a ‘‘false neurotransmitter.’’ In control exports Cl yielding the hyperpolarizing Cl reversal potential

596 L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600

169

found in adult neurons. As a result, activation of GABAARs may In the pilocarpine model of temporal lobe epilepsy, the a1

170

be excitatory during early development. Excitatory GABAAR subunit in dentate granule neurons was downregulated and a4

190

currents may play a trophic role in neuronal migration and subunit was upregulated, while in younger (postnatal day 20)

171 172 191

connectivity, but may also contribute to epileptogenesis. animals, the a1 subunit was increased after status epilepticus.

Endogenous GABA appears to be proconvulsant in early postnatal GABAAR subunit changes after pilocarpine-induced status are

rat hippocampal slices, as GABAA antagonists blocked epileptiform associated with altered physiological properties including reduced

+ 173

activity induced by depolarization with high external [K ] . neurosteroid sensitivity and an increase in a4-containing recep-

112

However, BZ and barbiturate antiseizure efﬁcacy appear to be tors colocalized with synapses rather than extracellular sites.

intact, likely because persistent opening of GABAAR channels (in Such changes might contribute to the propensity for spontaneous

the presence of allosteric agents) may reduce the depolarizing seizures or alternatively represent a functional adaptation to

chloride reversal potential, resulting in ‘‘shunt’’ inhibition. reduce seizure expression. Substitution of a1 with a4 might

Alternatively, subthreshhold GABA-evoked depolarization may reduce synaptic (phasic) but increase extrasynaptic (tonic)

174

inactivate sodium channels and prevent action potential ﬁring. inhibition, and BZ sensitivity would be lost in a4-containing

The current through GABAAR channels can also be altered by receptors. Using an adenovirus vector to insert the a1 subunit gene

169

changes in intracellular bicarbonate, [HCO3 ], which can ﬂow driven by the a4 promoter resulted in a decrease in seizure

175

through the channel. Changes in [HCO3 ] may underlie reduced frequency, suggesting that the GABAAR subunit changes were

176 192

synaptic GABA currents during development of BZ tolerance. contributory toward epileptogenesis. Hence, altering GABAAR

Depolarizing GABAAR currents may also be a source of interictal subunit expression in brain regions critical to seizure development

spike activity, as observed in epileptic subiculum neurons in may provide a new therapeutic strategy for seizure prevention.

hippocampal brain slices from patients with temporal lobe Alternatively, novel ASDs targeting the speciﬁc epilepsy-associated

177

epilepsy. Changes in the GABA current reversal potential might subunit composition might provide a less invasive approach

also explain why diazepam can be less effective in children with toward seizure control.

178

epileptic encephalopathies, and rarely can cause status epilep- Long term exposure to the GABAAR allosteric agents

179

ticus in patients with the Lennox Gastaut syndrome. themselves can alter GABAAR composition and function. In rat

Bumetanide, an inhibitor of NKCC-1 used clinically as a potent pups exposed to therapeutic concentrations of diazepam or

loop diuretic, has been proposed as an adjunctive agent to reduce phenobarbital from postnatal day 10 through 40, then tapered

intraneuronal chloride accumulation and reverse the depolarizing for 2 weeks and euthanized on postnatal day 90 showed

chloride gradient that makes GABAARs excitatory, particularly in increased mRNA expression in dentate granule neurons for

170

the neonatal setting. Bumetanide improved the responsiveness GAT-1, 3 and 4, GABAAR subunits a4, a6, b3, d and u and GABAB

of hippocampal slices to phenobarbital in reducing epileptiform receptor subunit R1, and decreased mRNA expression for GAD65,

2+ 180 193

discharges induced by low Mg , and also suppressed electro- GAD67 and GABAAR subunits a1 and a3. Tolerance- and

172

graphic seizures in neonatal rats. Depolarizing GABA currents dependence-associated changes in GABAAR subunit composition

194,195 196,197

may occur in cortical more than subcortical brain regions, possibly with chronic use of BZs and barbiturates have been

explaining the electroclinical uncoupling that occurs with pheno- reported, and may reﬂect tolerance to either the antiseizure or

181

barbital in neonatal seizures. adverse CNS effects. However, these ﬁndings suggest that not

Bumetanide has been used successfully in a single reported only seizures, but their prolonged treatment with GABAergic

182

human neonate with seizures, and a clinical trial agents, can alter inhibitory neuronal function for extended

(NCT00830531) is in progress. However, elimination of the periods or even permanently, with possible cognitive and

depolarizing Cl gradient during development may have adverse behavioral consequences that must be considered when

consequences; a permanent reduction in AMPAR-mediated contemplating chronic GABAergic ASD use.

excitatory neurotransmission and sensorimotor gating deﬁcits

183

were observed after bumetanide exposure in neonatal rats. Such References

potential adverse effects will have to be weighed against the

beneﬁts associated with seizure termination or prevention. 1. Treiman DM. GABAergic mechanisms in epilepsy. Epilepsia 2001;42(Suppl

3):8–12.

2. Olsen RW, DeLorey TM, Gordey M, Kang MH. GABA receptor function and

8. GABA R subunit changes and ASD efﬁcacy in epilepsy and

A epilepsy. Advances in Neurology 1999;79:499–510.

status epilepticus 3. Ben-Ari Y. Seizures beget seizures: the quest for GABA as a key player. Critical

Reviews in Neurobiology 2006;18:135–44.

4. Sperk G, Furtinger S, Schwarzer C, Pirker S. GABA and its receptors in epilepsy.

There is considerable evidence that GABA R composition and

A Advances in Experimental Medicine and Biology 2004;548:92–103.

function change with epilepsy, both acutely in the setting of status 5. Macdonald RL, Olsen RW. GABAA receptor channels. Annual Reviews of Neuro-

science 1994;17:569–602.

epilepticus and in chronic epilepsy. During status epilepticus, there

184 6. Nayeem N, Green TP, Martin IL, Barnard EA. Quaternary structure of the native

is a rapid change in GABAAR function that reduces BZ sensitivity,

GABAA receptor determined by electron microscopic image analysis. Journal of

apparently due to activity-dependent internalization of GABAARs Neurochemistry 1994;62:815–8.

185

7. Hedblom E, Kirkness EF. A novel class of GABA receptor subunit in tissues of

and replacement with BZ-insensitive receptors. Kainic acid- A

the reproductive system. Journal of Biological Chemistry 1997;272:15346–50.

induced status epilepticus in young (postnatal day 9) rats altered

8. Davies PA, Hanna MC, Hales TG, Kirkness EF. Insensitivity to anesthetic agents

the normal developmental pattern of GABAAR subunit matura- conferred by a class of GABAA receptor subunit. Nature 1997;385:820–3.

186

9. Bonnert TP, McKernan RM, Le Bourdelles B, Smith DW, Hewson L, Rigby MR,

tion, preventing the normal switch from a2 to a1-containing

Sirinathsinghji DJ, Brown N, Wafford K, Whiting PJ. Q, a novel g-aminobutyric

GABAARs. These changes correlate with the ﬁnding in animal

acid type A subunit. Proceedings of the National Academy of Sciences of the United

15,184,187 188,189

models and humans that BZs are effective early in States of America 1999;96:9891–6.

the course of status epilepticus but lose their potency and efﬁcacy 10. Wisden W, Laurie DJ, Monyer H, Seeburg PH. The distribution of 13 GABAA

receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon,

for termination of status with longer seizure duration. These

mesencephalon. Journal of Neuroscience 1992;12:1040–62.

ﬁndings increase the urgency for treatment of status epilepticus

11. Brooks-Kayal AR, Jin H, Price M, Dichter MA. Developmental expression of

early in its course, and also suggest the need to ﬁnd alternative GABAA receptor subunit mRNAs in individual hippocampal neurons in vitro

and in vivo. Journal of Neurochemistry 1998;70:1017–28.

treatments that retain efﬁcacy in refractory status epilepticus, or

12. Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H, Schoﬁeld PR,

methods to prevent or reverse the GABA R changes that occur with

A Seeburg PH. Importance of a novel GABAA subunit for benzodiazepine phar-

prolonged seizures. macology. Nature 1989;338:582–5.

L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600 597

13. Wingrove PB, Wafford KA, Bain C, Whiting PJ. The modulatory action of 40. Zanotti A, Mariot R, Contarino A, Lipartiti M, Giusti P. Lack of anticonvulsant

loreclezole at the g-aminobutyric acid type A receptor is determined by a tolerance and benzodiazepine receptor downregulation with imidazenil in

single amino acid in the b2 and b3 subunit. Proceedings of the National Academy rats. British Journal of Pharmacology 1996;117:647–52.

of Sciences of the United States of America 1994;91:4569–73. 41. Rundfeldt C, Wlaz P, Honack D, Loscher W. Anticonvulsant tolerance and

14. Draguhn A, Verdorn TA, Ewert M, Seeburg PH, Sakmann B. Functional and withdrawal characteristics of benzodiazepine receptor ligands in differ-

molecular distinction between recombinant rat GABAA receptor subtypes by ent seizure models in mice. Comparison of diazepam, bretazenil and

Zn . Neuron 1990;5:781–8. abecarnil. Journal of Pharmacology and Experimental Therapy 1995;275:

15. Jones-Davis DM, Macdonald RL. GABA(A) receptor function and pharmacology 693–702.

in epilepsy and status epilepticus. Current Opinion in Pharmacology 2003;3: 42. Hernandez TD, Heninger C, Wilson MA, Gallager DW. Relationship of agonist

12–8. efﬁcacy to changes in GABA sensitivity and anticonvulsant tolerance following

16. Kokaia M, Pratt GD, Elmer E, Bengzon J, Fritschy JM, Kokaia Z, Lindvall O, chronic benzodiazepine ligand exposure. European Journal of Pharmacology

Mohler H. Biphasic differential changes of GABAA receptor subunit mRNA 1989;170(3):145–55.

levels in denate gyrus granule cells following recurrent kindling-induced 43. Haefely W, Kyburz E, Gerecke M, Mohler H. Recent advances in the molecular

seizures. Molecular Brain Research 1994;23:323–32. pharmacology of benzodiazepine receptors and in the structure-activity rela-

17. Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, Coulter DA. Selective changes tionships of their agonists and antagonists. Advances in Drug Research

in single cell GABAA receptor subunit expression and function in temporal lobe 1985;14:165–322.

epilepsy. Nature Medicine 1998;4:1166–72. 44. Polc P. Electrophysiology of benzodiazepine receptor ligands: multiple mech-

18. Loup F, Weiser HG, Yonekawa Y, Aguzzi A, Fritschy JM. Selective alterations in anisms and sites of action. Progress in Neurobiology 1988;31:349–423.

GABAA receptor subtypes in human temporal lobe epilepsy. Journal of Neuro- 45. Randall LO, Kappell B. Pharmacological activity of some benzodiazepines and

science 2000;20(14):5401–19. their metabolites. In: Garattini S, Mussini E, Randall LO, editors. The benzo-

20. Matsumoto A, Kumagai T, Miura K, Miyazaki S, Hayakawa C, Yamanaka T. diazepines. New York: Raven Press; 1973. p. 27–51.

Epilepsy in Angelman syndrome associated with chromosome 15q deletion. 46. Rogawski MA, Porter RJ. Antiepileptic drugs: pharmacological mechanisms

Epilepsia 1992;33:1083–90. and clinical efﬁcacy with consideration of promising developmental stage

21. DeLorey TM, Handforth A, Anagnostaras SG, Homanics GE, Minassian BA, compounds. Pharmacological reviews 1990;42(3):223–86.

Asatourian A, Fanselow MS, Delgado-Escueta A, Ellison GD, Olsen RW. Mice 47. Swinyard EA, Castellion AW. Anticonvulsant properties of some benzodiaze-

lacking the beta3 subunit of the GABAA receptor have the epilepsy phenotype pines. Journal of Pharmacology and Experimental Therapeutics 1966;151:

and many of the behavioral characteristics of Angelman syndrome. Journal of 369–75.

Neuroscience 1998;18:8505–14. 48. Albertson TE, Stark LG, Derlet RW. Modiﬁcation of amygdaloid kindling by

22. Bianchi MT, Song L, Zhang H, Macdonald RL. Two different mechanisms of diazepam in juvenile rats. Brain Research Developmental Brain Research

disinhibition produced by GABAA receptor mutations linked to epilepsy in 1990;51(2):249–52.

humans. Journal of Neuroscience 2002;22(13):5321–7. 49. Verdoorn TA, Draguhn A, Ymer S, Seeburg PH, Sakmann B. Functional proper-

23. Baulac S, Huberfeld G, Gourﬁnkel-An I, Mitropoulou G, Beranger A, Prud’- ties of recombinant rat GABAA receptors depend upon subunit composition.

homme J-F, Baulac M, Brice A, Bruzzone R, LeGuern E. First genetic evidence of Neuron 1990;4:919–28.

GABAA receptor dysfunction in epilepsy: a mutation in the g2-subunit gene. 50. Lu¨ ddens H, Korpi ER, Seeburg P. GABAA/benzodizaepine receptor heterogene-

Nature Genetics 2001;28:46–8. ity: neurophysiological implications. Neuropharmacology 1995;34(3):245–54.

24. Wallace RH, Marini C, Petrou S, Harkin LA, Bowser DN, Panchal RG, Williams 51. Smith GB, Olsen RW. Functional domains of GABAA receptors. Trends in

DA, Sutherland GR, Mulley JC, Scheffer IE, Berkovic SF. Mutant GABA(A) Pharmacological Sciences 1995;16(5):162–8.

receptor gamma2-subunit in childhood absence epilepsy and febrile seizures. 52. Dunn SMJ, Davies M, Muntoni AL, Lambert JJ. Mutagenesis of the rat a1

Nature Genetics 2001;28:49–52. subunit of the g-aminobutyric acidA receptor reveals the importance of

25. Cossette P, Liu L, Brisebois K, Dong H, Lortie A, Vanasse Ml, Saint-Hilaire JM, residue 101 in determining the allosteric effects of benzodiazepine site

Carmant L, Verner A, Lu WY, Wang YT, Rouleau GA. Mutation of GABRA1 in an ligands. Molecular Pharmacology 1999;56:768–74.

autosomal dominant form of juvenile myoclonic epilepsy. Nature Genetics 53. Korpi ER, Kleingoor C, Kettenmann H, Seeburg PH. Benzodiazepine-induced

2002;31:184–9. motor impairment linked to point mutation in cerebellar GABAA receptor.

26. Bowser DN, Wagner DA, Czajkowski C, Cromer BA, Parker MW, Wallace RH, Nature 1993;361:356–9.

Harkin LA, Mulley JC, Marini C, Berkovic SF, Williams DA, Jones MV, Petrou S. 54. McKernan RM, Rosahl TW, Reynolds DS, Sur C, Wafford KA, Atack JR, Farrar S,

Altered kinetics and benzodiazepine sensitivity of a GABAA receptor subunit Myers J, Cook G, Ferris P, Garrett L, Bristow L, Marshall G, Macaulay A, Brown N,

mutation [gamma 2(R43Q)] found in human epilepsy. Proceedings of the Howell O, Moore KW, Carling RW, Street LJ, Castro JL, Ragan CI, Dawson GR,

National Academy of Sciences of the United States of America 2002;99:15170–5. Whiting PJ. Sedative but not anxiolytic properties of benzodiazepines are

27. Frugier G, Coussen F, Giraud MF, Odessa MF, Emerit MB, Boue-Grabot E, Garret mediated by the GABAA receptor a1 subunit. Nature Neuroscience

M. A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA 2000;3(6):529–30.

receptor assembly and modiﬁes subunit composition on the cell surface. 55. Crestani F, Martin JR, Mohler H, Rudolph U. Mechanism of action of the

Journal of Biological Chemistry 2007;282:3819–28. hypnotic zolpidem in vivo. British Journal of Pharmacology

28. Hales TG, Tang H, Bollan KA, Johnson SJ, King DP, McDonald NA, Cheng A, 2000;131(7):1251–4.

Connolly CN. The epilepsy mutation, gamma2(R43Q) disrupts a highly con- 56. Low K, Crestani F, Keist R, Benke D, Brunig I, Benson JA, Fritschy JM, Rulicke T,

served inter-subunit contact site, perturbing the biogenesis of GABAA recep- Bluethmann H, Mohler H, Rudolph U. Molecular and neuronal substrate for the

tors. Molecular and Cellular Neuroscience 2005;29:120–7. selective attenuation of anxiety. Science 2000;290:131–4.

29. Sancar F, Czajkowski C. A GABAA receptor mutation linked to human epilepsy 57. Crestani F, Low K, Keist R, Mandelli M, Mohler H, Rudolph U. Molecular targets

(gamma2R43Q) impairs cell surface expression of alphabetagamma receptors. for the myorelaxant action of diazepam. Molecular Pharmacology 2001;59:

Journal of Biological Chemistry 2004;279:47034–9. 442–5.

30. Kang JQ, Macdonald RL. The GABAA receptor gamma2 subunit R43Q mutation 58. Crestani F, Keist R, Fritschy JM, Benke D, Vogt K, Prut L, Bluthmann H, Mohler H,

linked to childhood absence epilepsy and febrile seizures causes retention of Rudolph U. Trace fear conditioning involves hippocampal alpha5 GABA(A)

alpha1beta2gamma2S receptors in the endoplasmic reticulum. Journal of receptors. Proceedings of the National Academy of Sciences of the United States of

Neuroscience 2004;24:8672–7. America 2002;99:8980–5.

31. Sternbach LH, Reeder E. Quinazolines and 1, 4-benzodiazepines, IV: transfor- 59. Knabl J, Zeilhofer UB, Crestani F, Rudolph U, Zeilhofer HU. Genuine antihyper-

mations of 7-chloro-2-methylamino-5-phenyl- H-1,4-benzodiazepine-4-ox- algesia by systemic diazepam revealed by experiments in GABAA receptor

ide. Journal of Organic Chemistry 1961;26:4936–41. point-mutated mice. Pain 2009;141:233–8.

32. Sternbach LH, Fryer RI, Keller O, et al. Quinazolines and 1, 4-benzodiazepines, 60. Dias R, Sheppard WF, Fradley RL, Garrett EM, Stanley JL, Tye SJ, Goodacre S,

X: nitro-substituted 5-phenyl-1,4-benzodiazepine derivatives. Journal of Me- Lincoln RJ, Cook SM, Conley R, Hallett D, Humphries AC, Thompson SA, Wafford

dicinal Chemistry 1963;6:261–5. KA, Street LJ, Castro JL, Whiting PJ, Rosahl TW, Atack JR, McKernan RM, Dawson

33. Gastaut H, Naquet R, Poire R, Tassarini CH. Treatment of status epilepticus GR, Reynolds DS. Evidence for a signiﬁcant role of alpha 3-containing GABAA

with diazepam (valium). Epilepsia 1965;6:167–82. receptors in mediating the anxiolytic effects of benzodiazepines. Journal of

34. Sato S. Benzodiazepines, clonazepam. In: Levy RH, Mattson RH, Meldrum BS, Neuroscience 2005;25:10682–8.

editors. Antiepileptic drugs. New York: Raven Press; 1995 . p. 725–34. 61. Rosenberg HC, Tietz EI, Chiu TH. Tolerance to anticonvulsant effects of diaze-

35. Chapman AG, Horton RW, Meldrum BS. Anticonvulsant action of a pam, clonazepam, and clobazam in amygdala-kindled rats. Epilepsia

1,5-benzodiazepine, clobazam, in reﬂex epilepsy. Epilepsia 1978;19: 1989;30:276–85.

293–9. 62. Macdonald R, Barker JL. Benzodiazepines speciﬁcally modulate GABA-medi-

36. Munn R, Farrell K. Open study of clobazam in refractory epilepsy. Pediatric ated postsynaptic inhibition in cultured mammalian neurones. Nature

Neurology 1993;9:465–9. 1978;271:563–4.

37. Shannon M, Albers G, Burkhardt K. Safety and efﬁcacy of ﬂumazenil in the 63. Tietz EI, Zeng XJ, Chen S, Lilly SM, Rosenberg HC, Kometiani P. Antagonist-

reversal of benzodiazepine-induced conscious sedation. Journal of Pediatrics induced reversal of functional and structural measures of hippocampal ben-

1997;131:582–6. zodiazepine tolerance. Journal of Pharmacology and Experimental Therapeutics

39. Turski L, Stephens DN, Jensen LH, Petersen EN, Meldrum BS, Patel S, 1999;291:932–42.

Hansen JB, Loscher W, Schneider HH, Schmiechen R. Anticonvulsant 64. Study RE, Barker JL. Diazepam and ()-pentobarbital: ﬂuctuation analysis

action of the beta-carboline abecarnil: studies in rodents and baboon, reveals different mechanisms for potentiation of g-aminobutyric acid

Papio papio. Journal of Pharmacology and Experimental Therapeutics responses in cultured central neurons. Proceedings of the National Academy

1990;253:344–52. of Sciences of the United States of America 1981;78:7180–4.

598 L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600

65. Rogers CJ, Twyman RE, Macdonald RL. Benzodiazepine and beta-carboline 93. Wafford KA, Bain CJ, Quirk K, McKernan RM, Wingrove PB, Whiting PJ, Kemp

regulation of single GABAA receptor channels of mouse spinal neurones in JA. A novel allosteric modulatory site on the GABAA receptor beta subunit.

culture. Journal of Physiology (London) 1994;475:69–82. Neuron 1994;12:775–82.

66. Twyman RE, Rogers CJ, Macdonald RL. Differential regulation of g-aminobu- 94. Wingrove PB, Wafford KA, Bain C, Whiting PJ. The modulatory action of

tyric acid receptor channels by diazepam and phenobarbital. Annals of Neu- loreclezole at the gamma-aminobutyric acid type A receptor is determined

rology 1989;25:213–20. by a single amino acid in the beta 2 and beta 3 subunit. Proceedings of the

67. Bianchi MT, Botzolakis EJ, Lagrange AH, Macdonald RL. Benzodiazepine mod- National Academy of Sciences of the United States of America 1994;91:

ulation of GABA(A) receptor opening frequency depends on activation con- 4569–73.

text: a patch clamp and simulation study. Epilepsy Research 2009;85:212–20. 95. Fisher JL, Macdonald RL. Functional properties of recombinant GABA(A)

68. Edwards FA, Konnerth A, Sakmann B. Quantal analysis of inhibitory synaptic receptors composed of single or multiple beta subunit subtypes. Neurophar-

transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp macology 1997;36:1601–10.

study. Journal of Physiology 1990;430:213–49. 96. Smith AJ, Alder L, Silk J, Adkins C, Fletcher AE, Scales T, Kerby J, Marshall G,

69. Otis TS, Mody I. Modulation of decay kinetics and frequency of GABAA Wafford KA, McKernan RM, Atack JR. Effect of alpha subunit on allosteric

receptor-mediated spontaneous inhibitory post-synaptic currents in hippo- modulation of ion channel function in stably expressed human recombinant

campal neurons. Proceedings of the National Academy of Sciences of the United gamma-aminobutyric acid(A) receptors determined using (36)Cl ion ﬂux.

States of America 1992;78:7180–4. Molecular Pharmacology 2001;59:1108–18.

70. Eghbali M, Curmi JP, Birnir B, Gage PW. Hippocampal GABA(A) channel 97. Neelands TR, Macdonald RL. Incorporation of the pi subunit into functional

conductance increased by diazepam. Nature 1997;388:71–5. gamma-aminobutyric Acid(A) receptors. Molecular Pharmacology

71. Eghbali M, Birnir B, Gage PW. Conductance of GABAA channels activated by 1999;56:598–610.

pentobarbitone in hippocampal neurons from newborn rats. Journal of Physi- 98. Donnelly JL, Macdonald RL. Loreclezole enhances apparent desensitization of

ology 2003;552:13–22. recombinant GABAA receptor currents. Neuropharmacology 1996;35:

72. Gaul S, Ozsarac N, Liu L, Fink RH, Gage PW. The neuroactive steroids alphax- 1233–41.

alone and pregnanolone increase the conductance of single GABAA channels in 99. Thomet U, Baur R, Dodd RH, Sigel E. Loreclezole as a simple functional marker

newborn rat hippocampal neurons. The Journal of Steroid Biochemistry and for homomeric rho type GABA(C) receptors. European Journal of Pharmacology

Molecular Biology 2007;104:35–44. 2000;408:R1–2.

73. Eghbali M, Gage PW, Birnir B. Effects of propofol on GABAA channel conduc- 100. Carter RB, Wood PL, Wieland S, Hawkinson JE, Belelli D, Lambert JJ, White HS,

tance in rat-cultured hippocampal neurons. European Journal of Pharmacology Wolf HH, Mirsadeghi S, Tahir SH, Bolger MB, Lan NC, Gee KW. Characteriza-

2003;468:75–82. tion of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-

74. Birnir B, Eghbali M, Cox GB, Gage PW. GABA concentration sets the conduc- hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-afﬁnity,

tance of delayed GABAA channels in outside-out patches from rat hippocam- steroid modulator of the gamma-aminobutyric acid(A) receptor. Journal of

pal neurons. Journal of Membrane Biology 2001;181:171–83. Pharmacology and Experimental Therapeutics 1997;280:1284–95.

75. Everitt AB, Luu T, Cromer B, Tierney ML, Birnir B, Olsen RW, Gage PW. 101. Monaghan EP, Mcauley JW, Data JL. Ganaxolone: a novel positive allosteric

Conductance of recombinant GABA (A) channels is increased in cells co- modulator of the GABA(A) receptor complex for the treatment of epilepsy.

expressing GABA(A) receptor-associated protein. Journal of Biological Chemis- Expert Opinion on Investigational Drugs 1999;8:1663–71.

try 2004;279:21701–6. 102. Reddy DS, Castaneda DC, O’Malley BW, Rogawski MA. Anticonvulsant activity

76. Breitwieser GE. G protein-coupled receptor oligomerization: implications for of progesterone and neurosteroids in progesterone receptor knockout mice.

G protein activation and cell signaling. Circulation Research 2004;94: Journal of Pharmacology and Experimental Therapeutics 2004;310:230–9.

17–27. 104. Reddy DS. Pharmacology of endogenous neuroactive steroids. Critical Reviews

77. Kwan P, Brodie MJ. Phenobarbital for the treatment of epilepsy in the 21st in Neurobiology 2003;15:197–234.

century: a critical review. Epilepsia 2004;45:1141–9. 105. Kimoto T, Tsurugizawa T, Ohta Y, Makino J, Tamura H, Hojo Y, Takata N,

78. Macdonald RL, McLean MJ. Anticonvulsant drugs: mechanisms of action. In: Kawato S. Neurosteroid synthesis by cytochrome p450-containing systems

Delgado-Escueta AV, Ward Jr AA, Woodbury DM, Porter RJ, editors. Advances in localized in the rat brain hippocampal neurons: N-methyl-D-aspartate and

neurology. New York: Raven Press; 1986. p. 713–36. calcium-dependent synthesis. Endocrinology 2001;142:3578–89.

79. Rehberg B, Duch DS, Urban BW. The voltage-dependent action of pentobarbital 106. Twyman RE, Macdonald RL. Neurosteroid regulation of GABAA receptor

on batrachotoxin-modiﬁed human brain sodium channels. Biochimica et Bio- single-channel kinetic properties of mouse spinal cord neurons in culture.

physica Acta 1994;1194:215–22. Journal of Physiology 1992;456:215–45.

80. Macdonald RL, Rogers CJ, Twyman RE. Barbiturate regulation of kinetic prop- 107. Zhu WJ, Wang JF, Krueger KE, Vicini S. Delta subunit inhibits neurosteroid

erties of the GABAA receptor channel of mouse spinal neurones in culture. modulation of GABAA receptors. Journal of Neuroscience 1996;16:6648–56.

Journal of Physiology 1989;417:483–500. 108. Mihalek RM, Banerjee PK, Korpi ER, Quinlan JJ, Firestone LL, Mi ZP, Lagenaur C,

82. Rho JM, Donevan SD, Rogawski MA. Direct activation of GABAA receptors by Tretter V, Sieghart W, Anagnostaras SG, Sage JR, Fanselow MS, Guidotti A,

barbiturates in cultured rat hippocampal neurons. Journal of Physiology Spigelman I, Li Z, DeLorey TM, Olsen RW, Homanics GE. Attenuated sensitivity

1996;497(Pt 2):509–22. to neuroactive steroids in gamma-aminobutyrate type A receptor delta sub-

83. Krishek BJ, Moss SJ, Smart TG. Homomeric beta 1 gamma-aminobutyric acid A unit knockout mice. Proceedings of the National Academy of Sciences of the

receptor-ion channels: evaluation of pharmacological and physiological prop- United States of America 1999;96:12905–10.

erties. Molecular Pharmacology 1996;49:494–504. 109. Wohlfarth KM, Bianchi MT, Macdonald RL. Enhanced neurosteroid potentia-

84. Davies PA, Kirkness EF, Hales TG. Modulation by general anaesthetics of rat tion of ternary GABA(A) receptors containing the delta subunit. Journal of

GABAA receptors comprised of alpha 1 beta 3 and beta 3 subunits expressed in Neuroscience 2002;22:1541–9.

human embryonic kidney 293 cells. British Journal of Pharmacology 110. Puia G, Ducic I, Vicini S, Costa E. Does neurosteroid modulatory efﬁcacy

1997;120:899–909. depend on GABAA receptor subunit composition? Receptors Channels

85. Feng HJ, Bianchi MT, Macdonald RL. Pentobarbital differentially modulates 1993;1:135–42.

alpha1beta3delta and alpha1beta3gamma2L GABAA receptor currents. Mo- 111. Bianchi MT, Haas KF, Macdonald RL. Alpha1 and alpha6 subunits specify

lecular Pharmacology 2004;66:988–1003. distinct desensitization, deactivation and neurosteroid modulation of

86. Davies PA, Hanna MC, Hales TG, Kirkness EF. Insensitivity to anaesthetic agents GABA(A) receptors containing the delta subunit. Neuropharmacology

conferred by a class of GABA(A) receptor subunit. Nature 1997;385:820–3. 2002;43:492–502.

87. Neelands TR, Fisher JL, Bianchi M, Macdonald RL. Spontaneous and gamma- 112. Sun C, Mtchedlishvili Z, Erisir A, Kapur J. Diminished neurosteroid sensitivity

aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by of synaptic inhibition and altered location of the alpha4 subunit of GABA(A)

epsilon subunit-containing isoforms. Molecular Pharmacology 1999;55: receptors in an animal model of epilepsy. Journal of Neuroscience

168–78. 2007;27:12641–50.

88. Greenﬁeld Jr LJ, Zaman SH, Sutherland ML, Lummis SC, Niemeyer MI, Barnard 113. Hosie AM, Clarke L, da SH, Smart TG. Conserved site for neurosteroid modu-

EA, Macdonald RL. Mutation of the GABAA receptor M1 transmembrane lation of GABA A receptors. Neuropharmacology 2009;56:149–54.

proline increases GABA afﬁnity and reduces barbiturate enhancement. Neuro- 114. Akk G, Li P, Bracamontes J, Reichert DE, Covey DF, Steinbach JH. Mutations of

pharmacology 2002;42:502–21. the GABA-A receptor alpha1 subunit M1 domain reveal unexpected complex-

89. Seraﬁni R, Bracamontes J, Steinbach JH. Structural domains of the human ity for modulation by neuroactive steroids. Molecular Pharmacology

GABAA receptor 3 subunit involved in the actions of pentobarbital. Journal of 2008;74:614–27.

Physiology 2000;524(Pt 3):649–76. 115. Bracamontes JR, Steinbach JH. Steroid interaction with a single potentiating

90. Amin J. A single hydrophobic residue confers barbiturate sensitivity to gam- site is sufﬁcient to modulate GABA-A receptor function. Molecular Pharmacol-

ma-aminobutyric acid type C receptor. Molecular Pharmacology 1999;55: ogy 2009;75:973–81.

411–23. 116. Zona C, Ciotti MT, Avoli M. Topiramate attenuates voltage-gated sodium

91. Ashton D, Fransen J, Heeres J, Clincke GH, Janssen PA. In vivo studies on the currents in rat cerebellar granule cells. Neuroscience Letters 1997;231:123–6.

mechanism of action of the broad spectrum anticonvulsant loreclezole. Epi- 117. McLean MJ, Bukhari AA, Wamil AW. Effects of topiramate on sodium-depen-

lepsy Research 1992;11:27–36. dent action-potential ﬁring by mouse spinal cord neurons in cell culture.

92. Zhang CL, Heinemann U. Effects of the triazole derivative loreclezole (R72063) Epilepsia 2000;41(Suppl 1):S21–4.

on stimulus induced ionic and ﬁeld potential responses and on different 118. Hanaya R, Sasa M, Ujihara H, Ishihara K, Serikawa T, Iida K, Akimitsu T, Arita K,

patterns of epileptiform activity induced by low magnesium in rat entorhinal Kurisu K. Suppression by topiramate of epileptiform burst discharges in

cortex-hippocampal slices. Naunyn-Schmiedebergs Archives of Pharmacology hippocampal CA3 neurons of spontaneously epileptic rat in vitro. Brain

1992;346:581–7. Research 1998;789:274–82.

L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600 599

119. Gibbs III JW, Sombati S, DeLorenzo RJ, Coulter DA. Cellular actions of topir- 150. Engel D, Schmitz D, Gloveli T, Frahm C, Heinemann U, Draguhn A. Laminar

amate: blockade of kainate-evoked inward currents in cultured hippocampal difference in GABA uptake and GAT-1 expression in rat CA1. Journal of

neurons. Epilepsia 2000;41(Suppl 1):S10–6. Physiology 1998;512(Pt 3):643–9.

120. Zhang X, Velumian AA, Jones OT, Carlen PL. Modulation of high-voltage- 151. Richter D, Luhmann HJ, Kilb W. Intrinsic activation of GABA receptors sup-

activated calcium channels in dentate granule cells by topiramate. Epilepsia presses epileptiform activity in the cerebral cortex of immature mice. Epilepsia

2000;41(Suppl 1):S52–60. 2010.

121. Mirza N, Marson AG, Pirmohamed M. Effect of topiramate on acid-base 152. Frahm C, Stief F, Zuschratter W, Draguhn A. Unaltered control of extracellular

balance: extent, mechanism and effects. British Journal of Clinical Pharmacology GABA-concentration through GAT-1 in the hippocampus of rats after pilocar-

2009;68:655–61. pine-induced status epilepticus. Epilepsy Research 2003;52:243–52.

122. White HS, Brown SD, Woodhead JH, Skeen GA, Wolf HH. Topiramate enhances 153. Adkins JC, Noble S. Tiagabine. A review of its pharmacodynamic and phar-

GABA-mediated chloride ﬂux and GABA-evoked chloride currents in murine macokinetic properties and therapeutic potential in the management of

brain neurons and increases seizure threshold. Epilepsy Research epilepsy. Drugs 1998;55:437–60.

1997;28:167–79. 154. Richens A. Pharmacology and clinical pharmacology of vigabatrin. Journal of

123. White HS, Brown SD, Woodhead JH, Skeen GA, Wolf HH. Topiramate mod- Child Neurology 1991;Suppl 2:S7–10.

ulates GABA-evoked currents in murine cortical neurons by a nonbenzodia- 155. Gram L, Klosterskov P. Dam M. gamma-vinyl GABA: a double-blind placebo-

zepine mechanism. Epilepsia 2000;41(Suppl 1):S17–20. controlled trial in partial epilepsy. Annals of Neurology 1985;17:262–6.

124. Gordey M, DeLorey TM, Olsen RW. Differential sensitivity of recombinant 156. Riikonen R. ACTH therapy of West syndrome: Finnish views. Brain and

GABA(A) receptors expressed in Xenopus oocytes to modulation by topira- Development 2001;23:642–6.

mate. Epilepsia 2000;41(Suppl 1):S25–9. 157. Parisi P, Bombardieri R, Curatolo P. Current role of vigabatrin in infantile

125. Simeone TA, Wilcox KS, White HS. Subunit selectivity of topiramate modula- spasms. The European Journal of Paediatric Neurology 2007;11:331–6.

tion of heteromeric GABA(A) receptors. Neuropharmacology 2006;50:845–57. 158. Gram L, Larsson OM, Johnsen AH, Schousboe A. Effects of valproate, vigabatrin

126. Pennell PB, Ogaily MS, Macdonald RL. Aplastic anemia in a patient receiving and aminooxyacetic acid on release of endogenous and exogenous GABA from

felbamate for complex partial seizures. Neurology 1995;45:456–60. cultured neurons. Epilepsy Research 1988;2:87–95.

127. O’Neil MG, Perdun CS, Wilson MB, McGown ST, Patel S. Felbamate-associated 159. Petroff OA, Behar KL, Mattson RH, Rothman DL. Human brain gamma-ami-

fatal acute hepatic necrosis. Neurology 1996;46:1457–9. nobutyric acid levels and seizure control following initiation of vigabatrin

128. Kume A, Greenﬁeld Jr LJ, Macdonald RL, Albin RL. Felbamate inhibits [3H]t- therapy. Journal of Neurochemistry 1996;67:2399–404.

butylbicycloorthobenzoate (TBOB) binding and enhances Cl- current at the 160. Stuchlik A, Kubova H, Mares P. Single systemic dose of vigabatrin induces early

gamma-aminobutyric AcidA (GABAA) receptor. Journal of Pharmacology and proconvulsant and later anticonvulsant effect in rats. Neuroscience Letters

Experimental Therapeutics 1996;277:1784–92. 2001;312:37–40.

129. Rho JM, Donevan SD, Rogawski MA. Barbiturate-like actions of the propane- 161. Mares P, Slamberova R. Biphasic action of vigabatrin on cortical epileptic after-

diol dicarbamates felbamate and meprobamate. Journal of Pharmacology and discharges in rats. Naunyn-Schmiedebergs Archives of Pharmacology

Experimental Therapeutics 1997;280:1383–91. 2004;369:305–11.

130. Subramaniam S, Rho JM, Penix L, Donevan SD, Fielding RP, Rogawski MA. 162. Panayiotopoulos CP, Agathonikou A, Sharoqi IA, Parker AP. Vigabatrin aggra-

Felbamate block of the N-methyl-D-aspartate receptor. Journal of Pharmacol- vates absences and absence status. Neurology 1997;49:1467.

ogy and Experimental Therapeutics 1995;273:878–86. 163. Iannetti P, Spalice A, Perla FM, Conicella E, Raucci U, Bizzarri B. Visual ﬁeld

131. Landmark CJ, Johannessen SI. Modiﬁcations of antiepileptic drugs for im- constriction in children with epilepsy on vigabatrin treatment. Pediatrics

proved tolerability and efﬁcacy. Perspectives in Medicinal Chemistry 2000;106:838–42.

2008;2:21–39. 164. Hammoudi DS, Lee SS, Madison A, Mirabella G, Buncic JR, Logan WJ, Snead OC,

132. Rundfeldt C, Netzer R. Investigations into the mechanism of action of the new Westall CA. Reduced visual function associated with infantile spasms in

anticonvulsant retigabine. Interaction with GABAergic and glutamatergic children on vigabatrin therapy. Investigative Ophthalmology & Visual Science

neurotransmission and with voltage gated ion channels. Arzneimittel-For- 2005;46:514–20.

schung 2000;50:1063–70. 165. Wild JM, Ahn HS, Baulac M, Bursztyn J, Chiron C, Gandolfo E, Safran AB,

133. Otto JF, Kimball MM, Wilcox KS. Effects of the anticonvulsant retigabine on Schiefer U, Perucca E. Vigabatrin and epilepsy: lessons learned. Epilepsia

cultured cortical neurons: changes in electroresponsive properties and syn- 2007;48:1318–27.

aptic transmission. Molecular Pharmacology 2002;61:921–7. 166. Lukasiuk K, Pitkanen A. GABA(A)-mediated toxicity of hippocampal neurons in

134. Gebhardt C, Breustedt JM, Noldner M, Chatterjee SS, Heinemann U. The vitro. Journal of Neurochemistry 2000;74:2445–54.

antiepileptic drug losigamone decreases the persistent Na+ current in rat 167. Lorsignol A, Taupignon A, Dufy B. Short applications of gamma-aminobutyric

hippocampal neurons. Brain Research 2001;920:27–31. acid increase intracellular calcium concentrations in single identiﬁed rat

135. Draguhn A, Jungclaus M, Sokolowa S, Heinemann U. Losigamone decreases lactotrophs. Neuroendocrinology 1994;60:389–99.

spontaneous synaptic activity in cultured hippocampal neurons. European 168. Kahraman S, Zup SL, McCarthy MM, Fiskum G. GABAergic mechanism of

Journal of Pharmacology 1997;325:245–51. propofol toxicity in immature neurons. Journal of Neurosurgical Anesthesiology

136. Dimpfel W, Chatterjee SS, Noldner M, Ticku MK. Effects of the anticonvulsant 2008;20:233–40.

losigamone and its isomers on the GABAA receptor system. Epilepsia 169. Staley KJ, Soldo BL, Proctor WR. Ionic mechanisms of neuronal excitation by

1995;36:983–9. inhibitory GABAA receptors. Science 1995;269:977–81.

137. Fisher JL. The anti-convulsant stiripentol acts directly on the GABA(A) receptor 170. Brumback AC, Staley KJ. Thermodynamic regulation of NKCC1-mediated Cl-

as a positive allosteric modulator. Neuropharmacology 2009;56:190–7. cotransport underlies plasticity of GABA(A) signaling in neonatal neurons.

138. Quilichini PP, Chiron C, Ben Ari Y, Gozlan H. Stiripentol, a putative antiepileptic Journal of Neuroscience 2008;28:1301–12.

drug, enhances the duration of opening of GABA-A receptor channels. Epilepsia 171. Kriegstein AR, Owens DF. GABA may act as a self-limiting trophic factor at

2006;47:704–16. developing synapses. Science’s STKE 2001;2001:E1.

139. Taylor CP. Mechanisms of action of gabapentin. Revue Neurologique (Paris) 172. Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC, Benke TA, Delpire

1997;153(Suppl 1):S39–45. E, Jensen FE, Staley KJ. NKCC1 transporter facilitates seizures in the developing

140. Loscher W, Honack D, Taylor CP. Gabapentin increases aminooxyacetic acid- brain. Nature Medical 2005;11:1205–13.

induced GABA accumulation in several regions of rat brain. Neuroscience 173. Dzhala VI, Staley KJ. Excitatory actions of endogenously released GABA

Letters 1991;128:150–4. contribute to initiation of ictal epileptiform activity in the developing hippo-

141. Dooley DJ, Taylor CP, Donevan S, Feltner D. Ca channel alpha2delta ligands: campus. Journal of Neuroscience 2003;23:1840–6.

novel modulators of neurotransmission. Trends in Pharmacological Sciences 174. Zhang SJ, Jackson MB. GABAA receptor activation and the excitability of nerve

2007;28:75–82. terminals in the rat posterior pituitary. Journal of Physiology 1995;483(Pt

142. Hendrich J, Van Minh AT, Heblich F, Nieto-Rostro M, Watschinger K, Striessnig 3):583–95.

J, Wratten J, Davies A, Dolphin AC. Pharmacological disruption of calcium 175. Bormann J, Hamill OP, Sakmann B. Mechanism of anion permeation through

channel trafﬁcking by the alpha2delta ligand gabapentin. Proceedings of the channels gated by glycine and gamma-aminobutyric acid in mouse cultured

National Academy of Sciences of the United States of America 2008;105:3628–33. spinal neurones. Journal of Physiology 1987;385:243–86.

143. Sills GJ. The mechanisms of action of gabapentin and pregabalin. Current 176. Zeng XJ, Tietz EI. Role of bicarbonate ion in mediating decreased synaptic

Opinion in Pharmacology 2006;6:108–13. conductance in benzodiazepine tolerant hippocampal CA1 pyramidal neu-

144. Nemeroff CB. The role of GABA in the pathophysiology and treatment of rons. Brain Research 2000;868:202–14.

anxiety disorders. Psychopharmacology Bulletin 2003;37:133–46. 177. Cohen I, Navarro V, Clemenceau S, Baulac M, Miles R. On the origin of interictal

145. Loscher W. Anticonvulsant and biochemical effects of inhibitors of GABA activity in human temporal lobe epilepsy in vitro. Science 2002;298:1418–21.

aminotransferase and valproic acid during subchronic treatment in mice. 178. Shorvon SD. Status epilepticus. Its clinical features and treatment in children

Biochemical Pharmacology 1982;31:837–42. and adults. New York: Cambridge University Press; 1994.

146. Loscher W. Valproate enhances GABA turnover in the substantia nigra. Brain 179. Tassinari CA, Dravet C, Roger J, Cano JP, Gastaut H. Tonic status epilepticus

Research 1989;501:198–203. precipitated by intravenous benzodiazepine in ﬁve patients with Lennox-

147. Loscher W. Valproate: a reappraisal of its pharmacodynamic properties and Gastaut syndrome. Epilepsia 1972;13:421–35.

mechanisms of action. Progress in Neurobiology 1999;58:31–59. 180. Dzhala VI, Brumback AC, Staley KJ. Bumetanide enhances phenobarbital

148. Macdonald RL, Kelly KM. Antiepileptic drug mechanisms of action. Epilepsia efﬁcacy in a neonatal seizure model. Annals of Neurology 2008;63:

1995;36(Suppl 2):S2–12. 222–35.

149. Frahm C, Engel D, Draguhn A. Efﬁcacy of background GABA uptake in rat 181. Glykys J, Dzhala VI, Kuchibhotla KV, Feng G, Kuner T, Augustine G, Bacskai BJ,

hippocampal slices. Neuroreport 2001;12:1593–6. Staley KJ. Differences in cortical versus subcortical GABAergic signaling:

600 L.J. Greenﬁeld Jr. / Seizure 22 (2013) 589–600

a candidate mechanism of electroclinical uncoupling of neonatal seizures. 191. Raol YH, Zhang G, Lund IV, Porter BE, Maronski MA, Brooks-Kayal AR.

Neuron 2009;63:657–72. Increased GABA(A)-receptor alpha1-subunit expression in hippocampal

182. Kahle KT, Barnett SM, Sassower KC, Staley KJ. Decreased seizure activity in a dentate gyrus after early-life status epilepticus. Epilepsia 2006;47:

human neonate treated with bumetanide, an inhibitor of the Na(+)-K(+)- 1665–73.

2Cl() cotransporter NKCC1. Journal of Child Neurology 2009;24:572–6. 192. Raol YH, Lund IV, Bandyopadhyay S, Zhang G, Roberts DS, Wolfe JH, Russek SJ,

183. Wang DD, Kriegstein AR. Blocking early GABA depolarization with bumeta- Brooks-Kayal AR. Enhancing GABA(A) receptor alpha 1 subunit levels in

nide results in permanent alterations in cortical circuits and sensorimotor hippocampal dentate gyrus inhibits epilepsy development in an animal model

gating deﬁcits. Cerebral Cortex 2010. of temporal lobe epilepsy. Journal of Neuroscience 2006;26:11342–6.

184. Kapur J, Macdonald RL. Rapid seizure-induced reduction of benzodiazepine 193. Raol YH, Zhang G, Budreck EC, Brooks-Kayal AR. Long-term effects of diaze-

and Zn sensitivity of hippocampal dentate granule cell GABAA receptors. pam and phenobarbital treatment during development on GABA receptors,

Journal of Neuroscience 1997;17:7532–40. transporters and glutamic acid decarboxylase. Neuroscience 2005;132:

185. Goodkin HP, Sun C, Yeh JL, Mangan PS, Kapur J. GABA(A) receptor internaliza- 399–407.

tion during seizures. Epilepsia 2007;48(Suppl 5):109–13. 194. Kang I, Miller LG. Decreased GABAA receptor subunit mRNA concentrations

186. Lauren HB, Lopez-Picon FR, Korpi ER, Holopainen IE. Kainic acid-induced status following chronic lorazepam administration. British Journal of Pharmacology

epilepticus alters GABA receptor subunit mRNA and protein expression in the 1991;103:1285–7.

developing rat hippocampus. Journal of Neurochemistry 2005;94: 1384–94. 195. Chen S, Huang X, Zeng XJ, Sieghart W, Tietz EI. Benzodiazepine-mediated

187. Feng HJ, Mathews GC, Kao C, Macdonald RL. Alterations of GABA A-receptor regulation of alpha1, alpha2, beta1-3 and gamma2 GABA(A) receptor subunit

function and allosteric modulation during development of status epilepticus. proteins in the rat brain hippocampus and cortex. Neuroscience 1999;93:

Journal of Neurophysiology 2008;99:1285–93. 33–44.

188. Alldredge BK, Gelb AM, Isaacs SM, Corry MD, Allen F, Ulrich S, Gottwald MD, 196. Tseng YT, Wellman SE, Ho IK. In situ hybridization evidence of differential

O’Neil N, Neuhaus JM, Segal MR, Lowenstein DH. A comparison of lorazepam, modulation by pentobarbital of GABAA receptor alpha 1- and beta 3-subunit

diazepam, and placebo for the treatment of out-of-hospital status epilepticus. mRNAs. Journal of Neurochemistry 1994;63:301–9.

New England Journal of Medicine 2001;345:631–7. 197. Klein RL, Harris RA. Regulation of GABAA receptor structure and function by

189. Treiman DM, Meyers PD, Walton NY, Collins JF, Colling C, Rowan AJ, Handforth chronic drug treatments in vivo and with stably transfected cells. Japanese

A, Faught E, Calabrese VP, Uthman BM, Ramsay RE, Mamdani MB. A compari- Journal of Pharmacology 1996;70:1–15.

son of four treatments for generalized convulsive status epilepticus. Veterans 198. Unwin N. Reﬁned structure of the nicotinic acetylcholine receptor at 4A

Affairs Status Epilepticus Cooperative Study Group. New England Journal of resolution. Journal of Molecular Biology 2005;346:967–89.

Medicine 1998;339(12):792–8. 199. Olsen RW, Tobin AJ. Molecular biology of GABAA receptors. FASEB Journal

190. Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, Coulter DA. Selective changes 1990;4:1469–80.

in single cell GABA(A) receptor subunit expression and function in temporal 200. Macdonald RL, Kang JQ, Gallagher MJ. GABAA receptor subunit mutations and

lobe epilepsy. Nature Medicine 1998;4:1166–72. genetic epilepsies, 2012.