Possible alterations in GABAA receptor signaling that

underlie benzodiazepine-resistant seizuresTarek Z. Deeb, Jamie Maguire, and Stephen J. Moss

Department of Neuroscience, Tufts University School of Medicine, Boston, Massachusetts, U.S.A.

SUMMARY

Benzodiazepines have been used for decades as

first-line treatment for status epilepticus (SE). For

reasons that are not fully understood, the efficacy

of benzodiazepines decreases with increasing

duration of seizure activity. This often forces clini-

cians to resort to more drastic second- and third-

line treatments that are not always successful. The

antiseizure properties of benzodiazepines are

mediated by c-aminobutyric acid type A (GABAA)

receptors. Decades of research have focused on

the failure of GABAergic inhibition after seizure

onset as the likely cause of the development ben-

zodiazepine resistance during SE. However, the

details of the deficits in GABAA signaling are still

largely unknown. Therefore, it is necessary to

improve our understanding of the mechanisms of

benzodiazepine resistance so that more effective

strategies can be formulated. In this review we dis-

cuss evidence supporting the role of altered

GABAA receptor function as the major underlying

cause of benzodiazepine-resistant SE in both

humans and animal models. We specifically

address the prevailing hypothesis, which is based

on changes in the number and subtypes of GABAA

receptors, as well as the potential influence of per-

turbed chloride homeostasis in the mature brain.

KEY WORDS: Diazepam, KCC2, Receptors, Refrac-

tory, Seizures, Trafficking.

Soon after their discovery in the late 1950s, benzodiaze-pines have been used as anticonvulsant therapies (forreview see Goodkin & Kapur, 2009; Neligan & Shorvon,2009). Benzodiazepines offered substantial advantagesover previous medications, as noted by the early clini-cians, including high efficacy, rapid onset of action, andlow toxicity. Benzodiazepines such as diazepam andlorazepam are now the standard first-line treatments forstatus epilepticus (SE). However, mounting clinical obser-vations soon revealed that the effectiveness of ben-zodiazepines decreased substantially with increasingduration of seizures (Treiman et al., 1998; Mayer et al.,2002). These diazepam-resistant seizures are defined asrefractory seizures (Shorvon, 2011). This therapeutic phe-nomenon has persisted to this day, and the mechanisticunderpinnings are somewhat unclear (Remy & Beck,2006). The reduction of diazepam efficacy suggests thatseizures incur changes in the benzodiazepine-sensitivec-aminobutyric acid (GABA)A receptor system, and it is

possible that these alterations could contribute to themaintenance of seizures during SE and the recurrence ofseizures at later stages. Therefore, understanding exactlyhow diazepam’s efficacy is reduced could reveal impor-tant clues about the mechanisms of seizures and provideinsight into potentially more effective treatments. In thisreview, we attempt to clarify what is actually known aboutrefractory seizures within the context of the associatedchanges in benzodiazepine-sensitive GABAA receptors aswell as the theoretical contribution of altered Cl) homeo-stasis in humans and animal models (Fig. 1).

Benzodiazepine-Sensitive

GABAA Receptors

GABAA receptors belong to the large family of penta-meric Cysteine-loop receptors, which were formerlyknown as the nicotinic-acetylcholine family of receptors(Alexander et al., 2011). There are 19 GABAA receptorsubunits in humans: a1-6, b1-3, c 1-3, d, e, p, h, and q1-3. GABAA receptor subunit composition dictates thesubcellular localization, kinetics, and pharmacology ofthese receptors. Receptors containing c subunits aresensitive to benzodiazepines, with the c2 subunit beingthe major c subunit expressed in the brain. Furthermore,

Address correspondence to Stephen J. Moss, PhD, Department ofNeuroscience, Tufts University School of Medicine, 136 Harrison Ave.,Boston, MA 02111, U.S.A. E-mail: stephen.moss@tufts.edu

Wiley Periodicals, Inc.ª 2012 International League Against Epilepsy

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the benzodiazepine-binding site is formed at the extra-cellular interface of a c2 and adjacent a1-3 or a5 subunit;the a4 and a6 subunits do not support benzodiazepinebinding (Puia et al., 1991; Benson et al., 1998; Rudolph& Knoflach, 2011). Most of these benzodiazepine-sensi-tive receptors have a low affinity for GABA and aretherefore exclusively functional in the sub-synaptic

space where GABA concentrations are high. The excep-tions to this rule are the a5-containing receptors, whichare largely extrasynaptic and mediate a portion of persis-tent Cl) leak currents (tonic inhibition) in specific brainareas (Farrant & Nusser, 2005; Jacob et al., 2008). Thesespecialized subunit-dependent biophysical propertiessuggest that benzodiazepines largely exert their inhibi-tory efficacy by acting on synaptic GABAergic signaling(phasic inhibition). Although the precise intramolecularmechanism that enables benzodiazepine binding to alterGABAA function is still under investigation, most evi-dence indicates that benzodiazepines increase the affin-ity of GABA (Vicini et al., 1987; Rogers et al., 1994;Goldschen-Ohm et al., 2010). The ultimate effect of ben-zodiazepine binding is to increase the amplitude of tonicor prolong the duration of phasic GABAA-mediated cur-rents (Farrant & Nusser, 2005). These effects reduceexcitability by decreasing the probability that excitatorypostsynaptic potentials will trigger action potentials.

The Ionic Permeability of

GABAA Receptors

The ionic flux through the GABAA receptor is the maindeterminant of its physiologic role. GABAA receptors areanion permeable receptors, in which the anion selectivityis largely determined by residues that form the ion porelining the second transmembrane helix (Keramidas et al.,2004). Chloride is the third most abundant ion and themost abundant anion in the central nervous system.GABAA receptors are therefore predominantly Cl) chan-nels, although bicarbonate makes a significant contribu-tion to GABAA-mediated currents under certainconditions (Bormann et al., 1987; Kaila & Voipio, 1987;Fatima-Shad & Barry, 1993; Wotring et al., 1999; Viita-nen et al., 2010). For some GABAA receptors, the relativepermeability of Cl) to HCO�3 is roughly 3–1. However,unlike the cation-permeable subdivision of Cysteine-loopreceptors where the relative permeability of Ca2+ to Na+ iswell studied (e.g., Imoto et al., 1988; Livesey et al., 2011),similar studies of the molecular determinants and GABAA

subunit dependence of the Cl) to HCO�3 relative perme-ability are limited.

The direction that Cl) and HCO�3 flow through theGABAA receptor is entirely determined by their respec-tive electrochemical gradients, which rely on the functionof ion transporters and enzymes (Kaila, 1994; Farrant &Kaila, 2007). Active intracellular pH regulation results ina HCO�3 reversal potential of nearly )10 to )15 mV.Therefore, under nearly all circumstances, the HCO�3 cur-rent will depolarize the neuron. Chloride however is morecomplex. Typically the actions of GABA are hyperpolar-izing due to the inward flux of chloride upon GABA-acti-vated channel opening. However, under certainconditions, GABAA receptors can mediate net Cl) efflux

A

B

Figure 1.

Potential mechanisms of reduced GABAA receptor

function and benzodiazepine resistance. The GABAA-

mediated shunt current is equal to the product of the

number of open GABAA receptors (conductance) and

the electrochemical driving force (voltage) on the per-

meant ions (in this case Cl)). (A) The prevailing theory

of benzodiazepine resistance is based on a reduction in

conductance, that is, lower number of open benzodiaz-

epine-sensitive GABAA receptors due to increased lat-

eral diffusion from the subsynaptic space and

internalization. (B) A hypothetical mechanism based on

an elevated intracellular Cl) concentration, which

reduces or reverses the electrochemical driving force

and the GABAA-mediated current. The figure depicts a

polarity reversal of the GABAA-mediated current. Red

circles are GABA molecules, and black circles are ben-

zodiazepine molecules.

Epilepsia ILAE

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T. Z. Deeb et al.

that depolarizes neurons, such as in embryonic and imma-ture neurons (Ben-Ari et al., 2007), as well as in some sub-sets of mature neurons (e.g. Michelson & Wong, 1991;Verheugen et al., 1999; Song et al., 2011; Sarkar et al.,2011). The canonical hyperpolarizing Cl) currents exhib-ited by neurons are largely due to the expression and activ-ity of the potassium-chloride cotransporter type 2, KCC2(Thompson & G�hwiler, 1989; Payne, 1997). KCC2 is amember of a small family of cation-chloride cotransport-ers (SLC12), four of which (KCC1-4) are potassiumdependent and mediate K-Cl extrusion, and is the onlymember that exhibits constitutive transporter activity(Alexander et al., 2011). Even if a Cl) load did not resultin excitatory GABAergic responses, it would depolarizeEGABA, thereby reducing the electrochemical drivingforce on GABAA-mediated Cl) currents. It is thereforereasonable to propose that alterations in KCC2 functionwould impair GABAA receptor function, as well as theefficacy of compounds that modulate GABAA receptoractivity, such as benzodiazepines.

Evidence in Humans of Reduced

Benzodiazepine Efficacy and

Benzodiazepine Binding Sites

Although clinical experience accumulated over thecourse of decades suggested that prolonged seizures andSE are more difficult to control than brief seizure epi-sodes, two case studies in particular confirmed the earlyobservations (Treiman et al., 1998; Mayer et al., 2002).Patients experiencing SE are more likely to develop resis-tance to first-line antiseizure drugs and require second-and third-line interventions (Kwan & Sperling, 2009).However, because of the time constraints of emergencyroom admittance, prior exposure to benzodiazepines, thevarious etiologies, and of course ethical considerations, itis difficult to determine the exact point at which the effi-cacy of a first-time exposure to benzodiazepines begins towane in humans.

The simplest explanation for reduced benzodiazepineefficacy is a reduction in the number of benzodiazepinereceptors. We will briefly review some of the dataobtained from patients with temporal lobe epilepsies(TLEs). Patients exhibiting drug-resistant forms of TLEhave a reduced number of benzodiazepine binding sites inthe hippocampus that cannot be accounted for by the lossof neurons in sclerotic areas (Savic et al., 1988; Koeppet al., 1997; Hand et al., 1997; Loup et al., 2000). Furtheranalysis indicated that the affinity of the benzodiazepinebinding sites changed in some areas, suggesting a biophys-ical change in the types of benzodiazepine-sensitiveGABAA receptors. In addition, positron emission tomog-raphy (PET) scans of benzodiazepine binding sites havesuggested that deficits in the number of GABAA receptors

are a useful indicator of the epileptogenic focus prior tosurgery (Ryvlin et al., 1998; Bouvard et al., 2005). Thesedata indicate that forms of chronic epilepsies exhibit a lossof benzodiazepine-sensitive receptors. However, thesecases do not necessarily shed light on SE in patients withdifferent histories or induced refractory SE in animals.

Reduced Benzodiazepine

Efficacy in Rodent Models

Pilocarpine-induced seizures result in a progressivechange in electroencephalography (EEG) patterns thatresemble those observed in humans experiencing general-ized convulsive SE, although the patients in this study hadvarious histories including some with epilepsy (Treimanet al., 1990; Treiman, 1990). The survival rate of ratsgiven diazepam decreases if it is administered after pilo-carpine-induced seizures compared to prior administration(Morrisett et al., 1987). Walton and Treiman (1988) thencorrelated temporal changes in the EEG patterns after sei-zure induction with the efficacy of the same first-time dos-age of diazepam (20 mg/kg), thereby ruling out tolerance.Indeed, SE caused a sharp decline in diazepam efficacybetween 10 and 15 min after the first seizure that wors-ened over the next 30–180 min. Similar studies have sup-ported these findings (Kapur & Macdonald, 1997; Rice &DeLorenzo, 1999; Gao et al., 2007) and have demon-strated a rightward shift in the therapeutic potency of diaz-epam by an order of magnitude between 0 and 10 minafter the first stage 3 seizure (Jones et al., 2002). Theseexperiments clearly demonstrate that pilocarpine-inducedSE in rats causes a progressive reduction in diazepam’stherapeutic efficacy.

Evidence of Altered GABAA

Receptor Trafficking and

Benzodiazepine Efficacy within

an Hour of Seizure Induction

For this section we will discuss chemical-induced sei-zures in rodents at several time points after seizure induc-tion (Lçscher, 2002). An early demonstration of a reducedGABAergic efficacy was performed in freely moving rats.Systemic kainate injections ablated paired-pulse depres-sion in the dentate gyrus, suggesting a failure of inhibitionafter 25 min of kainate-induced seizures (Milgram et al.,1991). In mechanically isolated CA1 neurons, a 45-minperiod of pilocarpine-induced SE reduced the potency ofexogenous GABA applications (Kapur & Coulter, 1995).At the same time point, the modulatory efficacy of diaze-pam, but not pentobarbital, was also reduced in mechani-cally isolated dentate granule cells (Kapur & Macdonald,1997). Recordings performed on dentate granule cells inslices obtained from seizing rats revealed several changes

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GABAA Signaling and Refractory Seizures

during the acute phase (Feng et al., 2008) (Table 1). Thesedata were obtained from rats undergoing pilocarpine-induced seizures that were not terminated by benzodiaze-pines, but the slices were incubated for an hour for recovery.Immediately after the first stage 3 seizure (0 min group),the minimum inhibitory postsynaptic current (mIPSC)amplitudes were smaller and the decay times faster com-pared to sham controls, whereas the mIPSCs recordedfrom a separate group obtained 30 min after the first stage3 seizure were of the same amplitude but had slower decaytimes compared to controls. The results from this studydid not report any change in the baseline frequency of mI-PSCs of either group compared to sham controls. mIPSCsfrom all three groups (sham, or 0 and 30 min after the firststage 3 seizure) were sensitive to modulation by diaze-pam, and the percent increase of the decay times fromeach group were not different compared to sham. How-ever, the percent increase of the decay was significantlygreater for the 0 min group when compared to the 30 mingroup. These data indicated that GABAA receptors of den-tate granule cells exhibit two distinct phases that shouldcontribute to the rapid reduction in the therapeutic effi-cacy of diazepam. The initial phase occurs after the firststage 3 seizure and entails a reduction of receptor number,whereas the next phase occurs over the next 30 min andinvolves a rebound in receptor number to control levelsbut with a reduction in the ability of diazepam to prolong

the mIPSC, suggesting that a portion of synaptic receptorswere internalized and replaced with a receptor subtypethat was less sensitive to diazepam. It should be noted thatmeasurements of mIPSC parameters do not necessarilyreflect the amount of inhibition. Nevertheless, to the bestof our knowledge, this is the only report that demonstrateda change in GABAA-mediated currents at these early timepoints in slices obtained from seizing animals.

Several groups then further investigated altered traf-ficking of GABAA subunits in slices obtained 1 h after thefirst stage 5 seizure, which is well into the development ofdiazepam resistance observed behaviorally, followed bydissection under halothane anesthesia and a 30–60 minperiod of slice incubation (Table 1). Immunohistochemi-cal data indicated that the b2/3 and c2 subunits were inter-nalized (Naylor et al., 2005), and this was later supportedby surface biotinylation assays in slices (Terunuma et al.,2008; Goodkin et al., 2008). The analysis also revealedthat surface levels of a1, a2, and a4 subunits were signifi-cantly reduced, but that the a5 and d subunits wereincreased (Terunuma et al., 2008). The mechanism bywhich the b3- and c2-containing synaptic receptors wereinternalized was dependent on PKC phosphorylation ofthe b3 subunit and AP2-mediated clathrin-dependentendocytosis. The mIPSC amplitudes were also reduced inCA1 and dentate granule cells at the 1 h time point(Goodkin et al., 2008; Terunuma et al., 2008), but the abil-ity of diazepam to prolong the decay of mIPSCs in dentategranule cells was not significantly different compared tocontrols (Naylor et al., 2005). Furthermore, SE decreasedthe frequency of mIPSCs in dentate granule cells (Nayloret al., 2005; Goodkin et al., 2008), but did not alter fre-quency in the CA1 (Terunuma et al., 2008). These dataindicate that approximately 1 h of SE reduces the numberof diazepam-sensitive synaptic GABAA receptors in twodifferent populations of neurons with concurrent changesin specific extrasynaptic subtypes.

Evidence of Long-Term Altered

GABAA Receptor Trafficking

and Benzodiazepine Efficacy

after Seizure Induction

In contrast to the data obtained in acute preparations,data obtained post-SE at 24 h and onward are more vari-able, possibly due to differences in the allowed duration ofSE and whether or not spontaneous seizures were moni-tored just prior to tissue preparation. We will neverthelessattempt to review them in order to chronicle short- andlong-term changes in GABAA receptors after chemicallyinduced seizures. Mechanically isolated dentate granuleneurons obtained 24 h after pilocarpine-induced SEexhibited reduced a1- and b1-subunit messenger RNA(mRNA) levels, whereas the a4-, d-, and e-subunit mRNA

Table 1. Changes in GABAA receptor

subunits and mIPSCs in dentate granule cells

at various times after initiation of seizure

activity

0 min after

Stage 3

30 min after

Stage 3

60 min after

SE onset

GABAA R subunit

a1 – – Lower

a2 – – Lower

a4 – – Lower

a5 – – Higher

b2/3 – – Lower

c2 – – Lower

d – – Higher

mIPSC parameter

Amplitude Smaller NS Smaller

Decay Faster Slower Slower, NS

Charge transfer Less Greater Less

Frequency NS NS Less, NSa

% Increase of decay

by diazepam

NS NS, <0 min

groupb

NS

NS, not statistically significant compared to controls; –, not

tested.aData were obtained from distinct populations of neurons

(see text).bData were obtained from two separate statistical compari-

sons from the same article (Feng et al., 2008).

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levels were increased (Brooks-Kayal et al., 1998). Thechange in the expression of GABAA receptor subunits wassustained during the chronic stage (1–4 months), and cellsalso exhibited a significant reduction in zolpidem sensitiv-ity. In agreement, in situ hybridization analysis revealedthat the mRNA level of the a1 subunit was also reduced inthe CA3 and CA4 areas at the 24 h point (Friedman et al.,1994). In contrast, flumazenil autoradiographic analysisshowed an increase in binding in all hippocampal areas atthe 24 h point, which was followed by a decrease in someof these areas that was sustained into the chronic phase(Vivash et al., 2011). However, this study used low-dosekainate injections to induce SE that was terminated bydiazepam after 4 h. Another pilocarpine study showed anincrease of mIPSC amplitudes at the 24–48 h time pointand 3–5 months later in dentate granule cells in slices(Leroy et al., 2004). Further analysis revealed that mIPSCswere less sensitive to diazepam modulation at the 24–48 htime point and that they eventually lost all sensitivity todiazepam 3–5 months later, but mIPSCs from both popu-lations were sensitive to modulation by flumazenil. Itshould be noted that this study did not terminate SE withany antiseizure drugs.

During the latent period (6–8 days), mIPSC amplitudesin dentate granule cells were reduced, but at later stages,the mIPSCs had larger amplitudes and were insensitive tozolpidem (Cohen et al., 2003). The expression of the c2subunit was decreased but not significantly at 4 daysbefore significantly increasing 60 days after SE (Penget al., 2004). The a4 subunit was also decreased at 4 daysbefore rising beyond control values at 30 days after SE.Further analysis revealed an apparent switch in expressionof the d subunit; it decreased in the dendrites of dentategranule cells but increased in the local interneuronpopulation. These changes are associated with increasedexcitability, as assessed by medial perforant path stimula-tion.

In addition, in mechanically isolated neurons fromchronic epileptic rats (�6 weeks), clonazepam sensitivityincreased in dentate granule cells but decreased in CA1neurons, whereas zolpidem sensitivity decreased in den-tate granule cells, suggesting a change in the relativeexpression of a subunits (Gibbs et al., 1997). The samestudy found increased current density values in dentategranule cells, but decreased values in CA1 neurons withincreased GABA potency values. In situ hybridizationanalysis of mRNA levels during the chronic phase(2 months) indicated that the a2 and a5 transcripts weredecreased throughout Ammon’s horn, but the expressionof the a5 subunit increased in the granule cell layer of thedentate gyrus (Rice et al., 1996). This study also showedincreased excitability in the CA1 region following Schaeffercollateral stimulation 2 months after SE induction. Thesestudies provided evidence of synaptic and extrasynapticGABAA receptor plasticity that lasted for prolonged

periods after SE induction, which could further affect thetherapeutic efficacy of benzodiazepines during SE devel-opment in patients with a history of epilepsy.

In Vitro Analysis of Rapid

Alterations of GABAA

Receptors

Several studies have also utilized hyperexcitable condi-tions in cultured neurons and organotypic slices to analyzethe immediate effects of epileptiform activity. The zero-Mg2+ model produces benzodiazepine-resistant activity incultured neurons (Sombati & Delorenzo, 1995; Deshpan-de et al., 2007). Exposures for longer than 10 minincreased internalization rates of the b2/3 subunits utiliz-ing an antibody feeding technique (Goodkin et al., 2005,2007), with changes in the kinetic parameters and chargetransfer values of mIPSCs that were reported for only the2–3 h time points. Changes in mIPSC properties uponexposure to zero-Mg2+ were stated to occur between 10and 60 min; however, no values were actually reported(Goodkin et al., 2007). It should also be noted that the anti-body feeding technique does not account for changes inthe insertion rate of GABAA receptors. Leaving open thepossibility that zero-Mg2+ increased the overall turnoverrate without altering the actual number of surface recep-tors, which is why the lack of reported functional values atthe earliest time points is critical. In contrast, it is notknown if there are changes in GABAA receptor traffickingin the zero-Mg2+ or 4-AP models of benzodiazepineresistance in the organotypic or acute-slice preparation(Albus et al., 2008; Wahab et al., 2010). However, expo-sure to high [K]o + N-methyl-D-aspartate (NMDA) for1 h decreased the surface levels of the c2 subunit but didnot alter the d subunit in organotypic slices (Goodkinet al., 2008), although it is unclear if this treatment proto-col produces benzodiazepine-resistant epileptiform activity.

Several studies have utilized single particle trackingmethods to analyze how GABAA receptors behave underhyperexcitable conditions in cultured neurons. Treatmentwith 4-AP increased the lateral mobility of endogenousGABAA receptors after just 3 min and decreased theimmunolabeled size of GABAA clusters (Bannai et al.,2009). These data were supported by an electrophysiolog-ic analysis that utilized direct current injection protocolsto generate hyperexcitability, which decreased the ampli-tudes of mIPSCs. It is unclear if these conditions producediazepam-resistant activity, although one could predictthat such effects would reduce diazepam’s inhibitory effi-cacy. In contrast, 4-AP did not increase the mobility oftransfected c2-tagged subunits at a lower temperature of29�C (Bouthour et al., 2012). It is likely that both thetransfection and lower temperature could account for thissmall discrepancy. Indeed, another group found that 4-APtreatment increased the lateral diffusion of c2-containing

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GABAA Signaling and Refractory Seizures

receptors within 2.5 min, which correlated with a reduc-tion in immunolabeled GABAA clusters (Niwa et al.,2012). Tracking experiments utilizing glutamate orNMDA applications also demonstrated that GABAA

receptors become more mobile within 10 min (Muir et al.,2010; Niwa et al., 2012). Again, it is not evident that theseconditions cause a reduction in the inhibitory efficacy ofdiazepam. Nevertheless, these single-particle trackingstudies suggest that GABAA receptors exhibit increasedmigratory behavior during hyperexcitable states, therebyraising the likelihood that receptors reach an endocyticzone and are internalized (Smith et al., 2012). Futureexperiments should reveal the precise temporal correla-tions between the onset of diazepam-resistant epileptiformactivity with reductions in the function of GABAergicsynapses and GABAA receptor trafficking in cultured neu-rons, and acute and organotypic slice preparations. Suchinvestigations would support the therapeutic value oftargeting the mechanisms that regulate the moleculardeterminants of the formation, size, and stability ofsynaptic GABAA receptor clusters (Saiepour et al., 2010;Papadopoulos & Soykan, 2011; Mukherjee et al., 2011).

Chloride Homeostasis and

Refractory Seizures

Antiseizure compounds such as benzodiazepines, barbi-turates, and propofol potentiate GABAA receptor activity.However, GABAA receptors have a dual role in neurons.As discussed above, GABAA receptors can either depolar-ize or hyperpolarize a neuron, which is well documentedduring the course of neuronal maturation. As mentionedearlier, the canonical inhibitory hyperpolarizing effect ofGABAA-mediated currents is largely determined by Cl)

homeostasis and KCC2 function. If KCC2 activity isreduced and Cl) homeostasis perturbed, the efficacy ofthese antiseizure drugs would also theoretically be altered.In fact, it has been demonstrated that the Cl) gradient hasa significant impact on the efficacy of GABAA modulators(Staley, 1992). To the best of our knowledge, this is theonly article to date that has explored this relationship.However, this investigation utilized the whole-cell patch-clamp technique, which dictates the reversal potential ofGABAA-mediated currents (EGABA) and therefore did notinvestigate any changes in endogenous Cl) gradientsunder pathophysiologic conditions. It should also be notedthat in this study high intracellular Cl) concentrationsreduced the inhibitory efficacy of the benzodiazepineflunitrazepam and the barbiturate pentobarbital. Althoughrefractory SE is by definition poorly responsive to ben-zodiazepines, it is sensitive to the anesthetics pentobarbi-tal and propofol (Shorvon, 2011). Nevertheless, thisdiscovery could be vitally important to the mechanismsunderlying changes in the efficacy of benzodiazepinesduring SE. Curiously, no single investigation has yet

attempted to demonstrate that altered Cl) homeostasiscontributes to the reduced inhibitory or therapeuticefficacy of benzodiazepines under pathophysiologicconditions, although our research groups are currentlyundertaking this task.

We briefly discuss some of the articles on altered cat-ion-chloride cotransporters in patients with chronic epi-lepsy, although several comprehensive reviews havealready summarized this literature (Payne et al., 2003;Galanopoulou, 2007; Kahle et al., 2008; Blaesse et al.,2009; Lçscher et al., 2012). KCC2 function was found tobe decreased in patients with temporal lobe epilepsy(Palma et al., 2006; Munoz et al., 2007; Eichler et al.,2008). Subsets of neurons in the subiculum of resectedhuman epileptic brain tissue exhibit depolarizing GABAresponses (Cohen et al., 2002), which is attributable todecreased KCC2 expression (Huberfeld et al., 2007). Fur-ther analysis revealed that the type 1 Na+/K+/Cl) cotrans-porter, NKCC1, inhibitor bumetanide blocked theinterictal epileptiform activity. This suggests that per-turbed Cl) homeostasis directly contributed to interictalevents, although the pathophysiologic role of these eventswas unclear in these experiments (Staley et al., 2005;Avoli et al., 2006). Although not investigated directly onthese particular tissue samples, these forms of epilepsy aregenerally refractory to benzodiazepines. So it is possiblethat the altered Cl) homeostasis contributes to the reducedtherapeutic efficacy of these compounds in the chronicphase. Even though patients with a history of epilepsy candevelop refractory SE (Treiman et al., 1990), changes inCl) homeostasis might occur at later stages, and thereforethese data do not shed light on the initial loss of diazepamefficacy observed during chemically induced refractoryseizures in animals or humans.

There is evidence in rodents of altered cation-chloridecotransporter expression and/or perturbed Cl) homeostasisafter chemically induced seizures in adults (Lçscher et al.,2012). To the best of our knowledge, the first study thatdemonstrated a positive shift in EGABA after SE inductionwas performed by Kapur and Coulter (1996). This studyused the whole-cell patch configuration and was performedon mechanically isolated CA1 neurons after 45 min of SE.To date, this is the earliest time point after SE induction thatEGABA values have been characterized. Pilocarpine-induced SE that proceeded for an hour after the first stage 5seizure, caused a marked reduction in KCC2 surface andtotal levels in the hippocampus after 1 h of seizures, with aconcurrent enhancement of tyrosine phosphorylation ofKCC2 residues Y903 and Y1087 (Lee et al., 2010). Block-ing of all tyrosine-dependent phosphorylation in culturedneurons decreased KCC2 surface activity and clusteringwithout altering the surface or total protein levels (Watana-be et al., 2009), indicating that dynamic regulation offunctional activity at the cell surface is important. Indeed,KCC2 and NKCC1 are dynamically regulated by

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phosphorylation. These processes can have rapid effects onCl) homeostasis, which can be either transient or sustainedand are potentially more relevant than total or even surfaceprotein levels, thereby adding an extra degree of complex-ity for this system (for review see Kahle et al., 2010; Cham-ma et al., 2012).

In the chemically induced models of SE, there is somedisagreement in the literature at time points greater than1 h after SE induction. Kainate-induced SE in micecaused a marked increase in KCC2 total protein levels at2, 6, and 24 h prior to returning to basal levels at the 48-htime point in the hippocampus (Shin et al., 2012). Thisstudy also found a small decrease in the expression ofNKCC1 at 6 h. Similarly, pilocarpine-induced SE causeda transient up-regulation of KCC2 mRNA levels at the 6-htime point (Zhu et al., 2012). In sharp contrast, severalstudies indicated a shift in the Cl) homeostatic mechanismthat would favor a depolarizing shift in EGABA values,although some measurements were obtained at differenttime points. Pilocarpine-induced SE resulted in anincrease in NKCC1 protein expression in the subiculumand dentate gyrus at the 24-h time point (Brandt et al.,2010). Pilocarpine-induced SE depolarized EGABA valuesin dentate granule cells at the 24-h and 1-week time points,with a concurrent reduction in KCC2 total protein (Pathaket al., 2007). Similarly, pilocarpine produced a positiveshift in EGABA values in dentate granule cells, subiculumneurons, and CA1 neurons between 7 and 14 days afterSE induction, with a concurrent reduction in the ratio ofmRNA expression of KCC2 relative to NKCC1 (Bar-mashenko et al., 2011). Pilocarpine-induced SE caused amarked increase in NKCC1 and a decrease in KCC2 pro-tein and mRNA levels at the 1-, 14-, and 45-day timepoints in the CA1 region, although the reduction in KCC2protein levels were only significant at 14 and 45 days (Liet al., 2008). Similarly, pilocarpine-induced SE increasedNKCC1 and decreased KCC2 protein and mRNA levels atthe 2- and 3-week time points in the entorhinal cortex(Bragin et al., 2009). Pilocarpine also caused a reductionof KCC2 mRNA levels and depolarized EGABA values inthe subiculum at the 4-month time point (De Guzmanet al., 2006). In summary, the bulk of data supports areduction in the Cl) extrusion capacity in multiple subre-gions of the hippocampus and associated areas beginningat 1 h after SE induction that lasted weeks. Future studiesof Cl) homeostasis will need to entail a more detailedanalysis of the biochemical properties of the cation-chlo-ride cotransporters at time points during the earliest obser-vable seizures in order to test if Cl) homeostasis has animpact, if any at all, on the progressive reduction of diaze-pam’s efficacy during SE.

From a technical standpoint, protocols usually have a1-h incubation period to allow the brain slices to recoverfrom the acute slice preparation; it is therefore possiblethat short term, virtually instantaneous changes in EGABA

are missed. Chloride plasticity, or fluctuations in EGABA,following intense neuronal activity can occur even in thepresence of functional KCC2, and these alterations canrecover quickly within minutes to baseline values if thestimulation or insult is terminated (Kaila, 1994; Riveraet al., 2005; Buzs�ki et al., 2007). Therefore, in vitrostudies on neurons and brain tissue have the potential toyield far greater detail in terms of the temporal correla-tion between the inhibitory efficacy of benzodiazepinesand changes in EGABA or the biochemistry of the Cl)

homeostatic mechanism. To date, several models of phar-macoresistant epileptiform activity have been established(Wahab et al., 2010). For example, prolonged exposureto Mg2+-free solution caused the development of laterecurrent discharges that were resistant to a number ofantiseizure drugs including benzodiazepines in acutehippocampal-entorhinal brain slices (Zhang et al., 1995;Drier et al., 1998) and organotypic slices (Albus et al.,2008). Of interest, zero-Mg2+ protocols caused a reduc-tion in KCC2 protein expression and Cl) extrusioncapacity in acute slice preparations (Rivera et al., 2004)and a depolarizing shift of EGABA in organotypic slicepreparations (Ilie et al., 2012). The latest evidence sug-gests that the molecular underpinnings of these zero-Mg2+–induced events involve calpain-dependent degrada-tion of KCC2 with a concurrent reduction in Cl) extru-sion capacity (Puskarjov et al., 2012). In terms of thehypothesized role of Cl) homeostasis, this molecularmechanism could also explain the refractoriness ofresected human brain tissue, which exhibited increasedlevels of calpain activity (Feng et al., 2011; Das et al.,2012). Future in vitro analyses must perform a moredetailed analysis of the potential influence of Cl) homeo-stasis on diazepam-resistant activity.

Concluding Remarks

The bulk of the research on the development of benzodi-azepine-resistant seizures has focused on changes inGABAA receptors in both humans and animals. However,we believe that Cl) homeostasis may play a significant rolein the development of diazepam-resistant seizures. Furtherresearch on this possibility is imperative to at least rule itout. Furthermore, changes in GABAA receptor traffickingand perturbed Cl) homeostasis are not mutually exclusive.It is possible that both aspects contribute to reduce diaze-pam’s efficacy, and experimentally, both are certainly capa-ble. Future work will undoubtedly shed more light on therole of GABAA receptors and Cl) homeostasis in this wide-spread and costly debilitating disorder.

Acknowledgments

This work was supported in part by U.S. National Institutes of Health/National Institute of Neurological Disorders and Stroke grant NS036296awarded to SJM; JM is funded by NS073574.

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Disclosure

None of the authors has any conflict of interest to disclose.

We confirm that we have read the Journal’s position on issues involved inethical publication and affirm that this report is consistent with thoseguidelines.

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