pharmacological plasticity of gabaa receptors at dentate gyrus

J Physiol 557.2 (2004) pp 473–487 473

Pharmacological plasticity of GABAA receptors at dentategyrus synapses in a rat model of temporal lobe epilepsy

Claire Leroy1, Pierrick Poisbeau2, A. Florence Keller2 and Astrid Nehlig1

1Psychopathologie et Pharmacologie de la cognition, INSERM U405, Strasbourg, France2Neurophysiologie Cellulaire et Integree, ULP/CNRS UMR 7519, Strasbourg, France

In the lithium–pilocarpine model (Li-pilocarpine) of temporal lobe epilepsy, GABAA receptor-mediated inhibitory postsynaptic currents (GABAA IPSCs) were recorded in dentate gyrusgranule cells (GCs) from adult rat hippocampal slices. The properties of GABAA IPSCs werecompared before and after superfusion of modulators in control conditions (Li-saline rats) andin Li-pilocarpine rats 24–48 h and 3–5 months (epileptic rats) after status epilepticus (SE). Themean peak amplitude of GABAA IPSCs increased by about 40% over Li-saline values in GCs24–48 h after SE and remained higher in epileptic rats. In Li-pilocarpine rats, studied at 24–48 hafter SE, diazepam (1 µm) lost 65% of its effectiveness at increasing the half-decay time (T 50%)of GABAA miniature IPSCs (mIPSCs). Diazepam had no effects on mIPSC T 50% in epileptic rats.The benzodiazepine ligand flumazenil (1 µM), acting as an antagonist in Li-saline rats, exhibiteda potent inverse agonistic effect on GABAA mIPSCs of GCs from Li-pilocarpine rats 24–48 hand 3–5 months after SE. The neurosteroid allopregnanolone (100 nM), which considerablyprolonged GABAA mIPSCs in Li-saline rats, totally lost its effect in rats studied 24–48 h afterSE. However, this decrease in effectiveness was transient and was totally restored in epilepticrats. In addition to the up-regulation in the number of receptors at individual GC synapses,we propose that these ‘epileptic’ GABAA receptors possess benzodiazepine binding sites withaltered allosteric properties. The failure of benzodiazepine and neurosteroid to potentiateinhibition early after SE may be a critical factor in the development of epileptogenesis andoccurrence of seizures.

(Resubmitted 5 December 2003; accepted after revision 18 March 2004; first published online 19 March 2004)Corresponding author A. Nehlig: INSERM U405, Faculty of Medicine, 11, rue Humann, 67085 Strasbourg, France.Email : [email protected]

Mesial temporal lobe epilepsy (MTLE) is a common formof drug-refractory epilepsy (Engel, 1998). Hippocampalsclerosis, the main neuropathological feature of MTLE,is characterized by massive neuronal loss and gliosis inCA1, CA3 and the hilus, while most granule cells ofthe dentate gyrus are preserved. It is associated withaxon and synaptic reorganization of surviving neuro-nes. Reorganized axons include sprouting of excitatorymossy fibres, inhibitory GABAergic neurones, and fibreswith other neurotransmitters such as neuropeptide Yand somatostatin (Mathern et al. 1997). Occurrence ofepileptic seizures is the consequence of an imbalancein the neurotransmission systems in favour of neuro-nal hyperexcitation. The plasticity of inhibitory GABAA

receptor-mediated synaptic inhibition is considered to

Claire Leroy and Pierrick Poisbeau contributed equally to the workreported here.

contribute to hyperexcitability in the dentate gyrusgranule cells layer in MTLE patients. Indeed, the dentategyrus critically regulates seizures by making synchronousactivity from entorhinal cortex less able to invade thehippocampus. However, a failure in the gating functionof the dentate gyrus can generate robust paroxysmaldischarges (Lothman et al. 1992; Behr et al. 1998).

Alterations in GABA neurotransmission associated withMTLE in dentate gyrus granule cells have been observedpre- and postsynaptically. At the neuronal level, strongup-regulations of the GABA neuronal marker glutamatedecarboxylase (GAD) have been observed during the latentand chronic phases of lithium–pilocarpine and pilocarpinemodels of MTLE (Brooks-Kayal et al. 1998; Andre et al.2001). At the postsynaptic level, an increased density ofGABAA receptors has been noted in the dentate gyrus ofhuman MTLE patients (Brooks-Kayal et al. 1998; Loupet al. 2000) and in the kindling (Otis et al. 1994; Nusser

C© The Physiological Society 2004 DOI: 10.1113/jphysiol.2003.059246

474 C. Leroy and others J Physiol 557.2

et al. 1998), pilocarpine (Gibbs et al. 1997; Fritschy et al.1999) and kainate (Schwarzer et al. 1997) models. On theother hand, a decrease in the number of GABA trans-porters occurs in human MTLE patients and in the kainatemodel (Patrylo et al. 2001). These molecular changesfavour a higher amount of GABA in the synaptic cleftmaking GABA more available for postsynaptic receptors.However, the reinforcement of GABAA receptor currentsdoes not prevent the breakdown of the dentate gyrus’sgating function in epileptic patients or animals.

The GABAA receptor is the result of the assembly intopentameres of several types of subunits many of whichconfer unique properties on the receptors. Many changesin the subunit composition of GABAA receptors havebeen reported in human cases (Loup et al. 2000) and inexperimental models of MTLE (Rice et al. 1996; Brooks-Kayal et al. 1998; Fritschy et al. 1999). Changes in thesubunit composition of the granule cell GABAA receptorshave led to changes in their pharmacological properties.Thus, while GABAA receptors from the dentate gyrus ofcontrol animals are virtually insensitive to zinc, it has beenfound that zinc enhances the effectiveness of the blockadeon GABAA receptors in human MTLE (Shumate et al.1998) and animal models (Buhl et al. 1996; Gibbs et al.1997; Mtchedlishvili et al. 2001). Moreover, experimentaland human data in MTLE suggest a rapid change inthe effects of benzodiazepine on granule cell GABAA

receptors. Benzodiazepines such as diazepam, whichenhance the binding of GABA to the GABAA receptorrepresent the initial treatment of status epilepticus (SE).However, SE of longer duration becomes increasingly lessresponsive to benzodiazepines and progressively refractoryto treatment (Rice et al. 1996; Kapur & Macdonald,1997; Jones et al. 2002). The mechanisms underlying thedeclining effectiveness of benzodiazepines, which may berelated to changes in the subunit composition of GABAA

receptors, have not yet been clarified. Finally, the GABAA

receptor is also modulated by neuroactive steroids suchas allopregnanolone (Lambert et al. 1995, 1996). Physio-logical nanomolar concentrations of this steroid have anti-convulsant effects (Kokate et al. 1998; Frye & Scalize,2000). Recent studies reported that allopregnanoloneenhances GABA-evoked currents less potently in dentategyrus granule cells of epileptic rats than in controls(Mtchedlishvili et al. 2001).

Thus, the objective of the present study was to evaluatealterations of GABAA-mediated inhibitory transmission indentate gyrus granule cells occurring at different times inthe epileptogenic process, i.e. during early epileptogenesis(24–48 h after SE) and after the occurrence of numerousspontaneous recurrent seizures (3–5 months after SE) in

the lithium–pilocarpine model of MTLE. This modelreproduces the temporal development of the disease,i.e. an initial event – here SE – followed by a latentseizure-free phase characterized by the genesis of neuronaldamage and the development of a hyperexcitable circuitunderlying the occurrence of spontaneous recurrentseizures (Cavalheiro, 1995). As in humans, the neuro-pathology includes hippocampal sclerosis (Andre et al.2001; Roch et al. 2002). In this model, we characterizedthe properties of GABAA-receptor-generated IPSCs andtheir changes in sensitivity to benzodiazepines and neuro-steroids by whole-cell patch-clamp recording in granulecells from hippocampal slices of Li-pilocarpine- and Li-saline-treated rats.

Methods

Animals and status epilepticus induction

Adult male Sprague–Dawley rats from Janvier BreedingCenter (Le Genest-St-Isle, France) were maintained understandard laboratory conditions on a 12 : 12 h light–dark cycle (lights on at 07.00 h). All experiments wereconducted in conformity with the rules set by the ECCouncil Directive (86/69/EEC) of November 24, 1986and French Department of Agriculture (License no. 67-97 issued to A.N. and 67-116 to P.P.).

Status epilepticus (SE) was induced in rats weighing250–300 g. Lithium chloride (3 mequiv kg−1, Sigma, StLouis, MO, USA) was administrated i.p. to all rats 18–20 h before a s.c. injection of pilocarpine (25 mg kg−1,Sigma) in the experimental groups or saline in the controlgroup. All animals were given a subcutaneous injectionof methylscopolamine (1 mg kg−1, Sigma) 30 min beforepilocarpine or saline. Methylscopolamine is used toreduce the peripheral consequences of the convulsant, i.e.the pilocarpine. Lithium chloride potentiates theconvulsive effect of pilocarpine reducing the amount ofpilocarpine that has to be injected.

The behavioural characteristics of SE were similar tothose observed in previous studies (Roch et al. 2002).Within 5 min of pilocarpine injection, rats developeddiarrhoea, piloerection and other signs of cholinergicstimulation. Over the following 15–20 min, they exhibitedhead bobbing, scratching, masticatory automatisms, wet-dog shakes and exploratory behaviour. Episodes of headand bilateral forelimb myoclonic movements with rearingand falling started at around 20–25 min and progressed toSE at about 50 min after pilocarpine administration. SEwas characterized by long-lasting, sustained clonic seizureactivity accompanied by episodes of rearing and falling. A

C© The Physiological Society 2004

J Physiol 557.2 Pharmacological plasticity of synaptic GABAA receptors in epilepsy 475

total of 75 rats, 25 Li-saline and 50 Li-pilocarpine rats wereused for this study. Within the Li-pilocarpine-injectedrats, three rats did not develop SE. These rats survivedbut were excluded from this study. Moreover, nine ratsthat developed SE died during the course of SE becauseof cardio-respiratory complications mainly caused bypulmonary hypersecretion during the sustained seizures.After the SE phase, the 38 surviving rats experienced alatent seizure-free phase of a mean duration of 11 ± 4 days,after which all animals exhibited two to three spontaneousrecurrent seizures per week until kill (Roch et al. 2002).Occurrence of seizures was video-recorded for 10 h per day.Li-pilocarpine-treated rats were used either early, i.e. 24 or48 h after the onset of SE or late, i.e. about 3–5 monthsafter SE after they had developed repeated spontaneousrecurrent seizures for several months.

Thus, 63 rats were available for electrophysiologicalrecordings. Only three epileptic rats were excluded fromthe study after slice preparation because the hippocampuswas too slightly damaged or extremely damaged, bothconditions being considered outside the range of usualhippocampal sclerosis. Within the remaining 60 rats, wewere unable to monitor electrophysiological activity from19 rats (13 Li-saline and 6 Li-pilocarpine) because cellrecordings were too poor, unstable or unsuccessful. Finally,a total of 41 rats could be used for electrophysiologicalrecordings: 12 Li-saline rats, 11 rats studied 24–48 h afterLi-pilocarpine-induced SE and 18 epileptic rats examined3–5 months after SE. Rats used at both 24–48 h and 3–5 months after Li-saline injection composed the Li-salinegroup.

Electrophysiological study

Brain dissection and slice preparation. Rats were deeplyanaesthetized by intraperitoneal injection of a mixture ofketamine (100 mg kg−1, Merial, Lyon, France) and xylazine(5 mg kg−1, Rompun, Bayer, Germany). Animals weredecapitated and brains were rapidly removed from theskull while being continuously refreshed with oxygenatedice-cold (−4◦C) sucrose–artificial CSF (S-aCSF). S-aCSFcontained 248 mm sucrose, 11 mm glucose, 2 mmNaHCO3, 2 mm KCl, 1.25 mm KH2PO4, 2 mm CaCl2,

2 mm MgSO4 (pH 7.35 ± 0.05). S-aCSF was bubbledcontinuously with 95% O2–5% CO2. Meningeas wererapidly and delicately removed from the brain immersedin S-aCSF. The brain region containing the hippocampuswas isolated and glued using cyanoacrylate cement appliedto the dorsal side of the brain (enabling horizontal slicing)to the platform of a Vibratome chamber filled withoxygenated ice-cold S-aCSF. Slices (300–350 µm thick)

were cut in the horizontal plane and were incubatedin normal aCSF at room temperature (23–28◦C) for atleast 1 h before electrophysiological recordings. They werecontinuously perfused with oxygenated normal aCSF inwhich 125 mm NaCl was substituted for sucrose. Lastly,they were transferred to a recording chamber under a ZeissAxioscope equipped with infrared differential interferencecontrast (IR-DIC) and water immersion objectives capableof visualizing neurones in thick living tissue.

Electrophysiological recordings and data acquisition. Allrecordings were performed at room temperature. Patchpipettes were obtained by pulling borosilicate glasscapillaries with inner filament using a horizontal laserpuller (P-2000, Sutter Instruments, Novato, CA, USA).The pipettes were filled with a solution containing 130 mmCsCl, 2 mm MgCl2, 10 mm Hepes (pH 7.3, adjusted usingCsOH). Biocytine was added to the intracellular solutionfor neuronal labelling. A typical granule cell labelled usingbiocytine is shown in Fig. 1.

Whole-cell patch-clamp recordings were obtained usingan Axopatch 200B amplifier (Axon Instruments, FosterCity, CA) with > 80% series resistance compensation.Recordings were low-pass filtered (5–10 kHz), digitized,and stored on videotape. Off-line, recordings were filteredat 2 kHz and digitized at 4–10 kHz on an Intel Pentium-based computer. Data were analysed using the Strathclydeelectrophysiology software (Dr J. Dempster, University of

Figure 1. Illustration of a typical dentate gyrus granule cellrecorded from a Li-saline ratThe cellular morphology was reconstructed with confocal microscopyusing biocytine immunocytochemistry. Scale bar: 0–100 µM.



Strathclyde, UK) and in-house software kindly suppliedby Dr Y. De Koninck. Detection of individual mIPSCswas performed using a software trigger described in detailin previous studies (Poisbeau et al. 1997, 1999). Over95% of events satisfying the trigger criteria were detected,even during compound mIPSCs. For each experiment,all detected events were examined and any noises whosecompliance with trigger specification was spurious wererejected.

Statistical analysis. The mean amplitudes, total rise times,half-decay times (T50%), and frequency of occurrence ofmIPSCs were compared between groups using one-way(saline/pilocarpine/epileptic) or two-way ANOVA withrepeated measures (with or without drug), followed byTukey’s post hoc multiple comparison test (Sigmastat 2.0).Statistical significance was considered as P < 0.05. For eachcondition analysis, 4–12 rats and 7–32 cells were used. Innone of the experimental group was there any statisticallysignificant difference between rats.

In the figures, decay time constants of mIPSCs werefitted using non-linear least square methods, and goodnessof fit was evaluated by fitting subsets of points drawnfrom the whole set of data points, the assessment ofthe reduced χ 2 values, and the change in the F valuescalculated from the sum of squared differences from thefitted line. The half-decay time values (T50%) of mIPSCsare also represented graphically in cumulative probabilityplots. The Kolmogorov-Smirnov (KS) test was used tocompare two cumulative probability distributions to eachother and statistical significance was set at P < 0.01. Allother numerical data are expressed as means ± s.e.m.

Drug application

For all IPSC recordings, slices were continuallyperfused with oxygenated aCSF containing 2 mmkynurenic acid (Fluka, Neu-Ulm, Germany). For mIPSCrecordings, 0.5 µm tetrodotoxin (TTX, Sigma) was added.Modulations of GABAA receptors were tested using 1 µmdiazepam (Sigma), 1 and 10 µm flumazenil (gift of Roche,Basel, Switzerland) and 100 nm allopregnanolone (Sigma).All drugs were prepared as 1000 times concentrated frozenstock solution aliquots. Diazepam and allopregnanolonewere diluted in 96% ethanol whereas all other drugs wereprepared in distilled water.

Results

In the present study, the development of hippocampalsclerosis was very progressive. At 24 h after SE, there was

already some neuronal loss, which was marked in thehilus and more moderate in Ammon’s horn, as has beenshown previously (Roch et al. 2002). At 3–5 months afterSE, neuronal loss had worsened and there was a majoratrophy of the hippocampus. By this time, massive neuro-nal loss had occurred in CA3, CA1 and the hilus of thedentate gyrus, while CA2 pyramidal cells survived. Thegreat majority of cells were also preserved in the granulecell layer of the dentate gyrus which was subject to a degreeof dispersion.

Characteristics of GABAA receptor-mediated sIPSCsand mIPSCs recorded from dentate gyrus granule cellsin lithium–saline- and lithium–pilocarpine-treated rats

We began by analysing the spontaneous and miniatureGABAA receptor-mediated synaptic currents (GABAA

sIPSCs and GABAA mIPSCs, respectively) recorded fromthe dentate gyrus granule cells (GCs) of Li-saline- andLi-pilocarpine-treated rats. Recordings were performedat a holding potential of −60 mV and in the presenceof 2 mm kynurenic acid, an antagonist of ionotropicglutamate receptors. To record GABAA mIPSCs, steady-state concentrations of tetrodotoxin (0.5 µm) were addedto the perfusion medium.

Table 1 summarizes IPSC characteristics obtained inthe three animal groups, i.e. Li-saline and Li-pilocarpinerats at 24–48 h and 3–5 months after SE. For both sIPSCsand mIPSCs, the total rise time and half-decay timewere similar in all groups of rats (Table 1). However, asearly as 24–48 h after SE, IPSCs increased significantly inamplitude compared to the Li-saline condition (+44%and +25% for sIPSCs and mIPSCs, respectively). Therewas no further change in sIPSC and mIPSC amplitudes inthe epileptic Li-pilocarpine group (+59% and +33% forsIPSCs and mIPSCs, respectively, compared to Li-salinerats). Conversely, at 24–48 h after SE, the frequency of GCsIPSCs and mIPSCs was unchanged compared to Li-salinerats. At 3–5 months after SE, the frequency of mIPSCs wasstill similar to that recorded in Li-saline and Li-pilocarpineanimals studied at 24–48 h after SE, while the frequencyof sIPSCs was increased by a factor of 2–2.5 compared toLi-saline and Li-pilocarpine animals studied at 24–48 hafter SE (Table 1). Representative GC recordings obtainedfrom Li-saline and epileptic Li-pilocarpine animals at3–5 months after SE shown in Fig. 2, together with anaveraged trace obtained from 10 isolated GABAA sIPSCs,illustrate the increase in frequency and amplitude ofsIPSCs in epileptic Li-pilocarpine compared to Li-salinerats.



Table 1. Characteristics of spontaneous and miniature GABAA receptor-mediated IPSCs recorded from thehippocampal GCs of Li-saline and Li-pilocarpine rats examined 24–48 h and 3–5 months after SE

Amplitude RT T50% Frequency n(pA) (ms) (ms) (Hz)

Spontaneous GABAA receptor-mediated IPSCsLi-saline −40.3 ± 3.4 1.21 ± 0.09 10.8 ± 0.6 0.75 ± 0.21 11Li-Pilo 24–48 h −58.0 ± 4.8∗ 1.36 ± 0.09 11.1 ± 0.6 0.60 ± 0.11 11Li-Pilo 3–5 months −63.9 ± 3.5∗∗ 1.30 ± 0.09 10.9 ± 0.7 1.59 ± 0.25∗+ 18

Miniature GABAA receptor-mediated IPSCsLi-saline −35.6 ± 1.2 1.31 ± 0.04 8.9 ± 0.3 0.57 ± 0.06 32Li-Pilo 24–48 h −44.5 ± 1.9∗∗ 1.23 ± 0.06 9.2 ± 0.2 0.59 ± 0.07 22Li-Pilo 3–5 months −47.3 ± 2.2∗∗ 1.38 ± 0.05 9.5 ± 0.2 0.55 ± 0.04 29

Mean peak amplitudes, total rise time (RT), half-decay time (T50%) and frequency of occurrence wereanalysed. Values are expressed as means ± S.E.M. of the number of cells (n). ∗P < 0.05, ∗∗P < 0.005,statistically significant differences from Li-saline rats; +P < 0.05, statistically significant difference fromLi-pilo rats, 24–48 h.

Changes of pharmacological properties of synapticGABAA receptors after lithium–pilocarpine-induced SE

To test the change in pharmacological properties ofsynaptic GABAA receptors, their sensitivity to diazepam,flumazenil and allopregnanolone on mIPSCs was studiedin Li-saline and Li-pilocarpine rats, early and late after SE.Table 2 summarizes the amplitude, total rise time, half-decay time (T50%) and frequency of mIPSCs obtainedbefore and after drug perfusion in the three groups, i.e. Li-saline and Li-pilocarpine rats at 24–48 h and 3–5 monthsafter SE.

Modulation of GABAA receptor-mediated mIPSCs bybenzodiazepines. Perfusion of diazepam (1 µm), aclassical benzodiazepine agonist used clinically to blockepileptic seizures, had no significant effects on the meanpeak amplitude and frequencies of mIPSCs in Li-salineor Li-pilocarpine rats (Table 2). Conversely, the effectof diazepam on the total rise time and the mean half-decay time (T50%) of mIPSCs could be observed. In Li-saline rats, diazepam increased the T50% of all GABAA

mIPSCs. The increase in T50% appears as a rightward shiftof cumulative probability (Fig. 3A), with a prolongationof the mean T50% by 46% (Fig. 3D) and an increase intotal rise time of 21% (Fig. 3E). Washout experimentsindicated that the effect of diazepam perfusion on mIPSCsis reversible (Fig. 3F). In Li-pilocarpine rats, 24–48 hafter SE, diazepam was already significantly (62%) lesseffective at potentiating T50% compared to Li-salinerats (Fig. 3D, Table 2). This decrease in the effectivenessof diazepam was found throughout the whole mIPSCpopulation and led to a diminished shift of the cumulativeprobability (Fig. 3B). In epileptic Li-pilocarpine animals,

no significant potentiating effect of diazepam was observed(Table 2 and Fig. 3D). This led to a strong match betweenthe cumulative probability of the T50% of mIPSCs beforeand after diazepam perfusion (Fig. 3B), illustrating theGCs’ progressive loss of sensitivity to diazepam over thecourse of the epileptogenic process. The effects of diazepamon GABAA sIPSCs in GCs from Li-saline (data not shown,n = 3) and epileptic Li-pilocarpine animals (data notshown, n = 7) were similar to those reported here formIPSCs.

To find out more about the changes in the propertiesof the GABAA receptor benzodiazepine binding site, weperfused flumazenil, a benzodiazepine site antagonist,onto the GABAA receptors. During our preliminaryinvestigations, we tested two concentrations of flumazenil,1 µm and 10 µm. Since the effect of flumazenil was similarat both concentrations, all the data presented here wereobtained using 1 µm of fumazenil. As previously reported,flumazenil had no effect on GABAA mIPSCs in Li-salineanimals (Table 2). Neither the mean rise time nor T50% ofthe mIPSCs was affected during perfusion of flumazenil(Fig. 4A, D and E). Conversely, this was not the case forrecordings performed in Li-pilocarpine GCs. Flumazenilreduced the GABAA mIPSC T50% significantly, by 17–18%,in all GCs recorded from Li-pilocarpine animals 24–48 hor 3–5 months after SE (Fig. 4D). This decrease in T50%

led to a leftward shift of the curves in both groups of Li-pilocarpine rats (Fig. 4B and C). In contrast to diazepam,flumazenil did not produce any potentiation of the totalrise time of mIPSCs recorded either from Li-saline or Li-pilocarpine rats (Fig. 4E). Washout experiments indicatedthat the effect of flumazenil perfusion on Li-pilocarpinerats is reversible (Fig. 4F).



Modulation of GABAA receptor-mediated mIPSCs byallopregnanolone

In GCs from Li-saline rats, perfusion of allopregnanolone(AP, 100 nm) significantly increased the GABAA mIPSCmean T50% and rise time, while the amplitude andfrequency of the mIPSCs were not affected (Table 2).The effect of AP on T50% corresponded to a significantprolongation of GABAA mIPSCs (Fig. 5A and D).However, in Li-pilocarpine rats, the sensitivity of GABAA

mIPSCs to AP changed according to the time that hadelapsed between SE and GC recordings. Surprisingly,

Figure 2. Typical recordings of GABAA receptor-mediated sIPSCsexhibited by hippocampal dentate gyrus granule cells from oneLi-saline rat (top traces) and one epileptic rat, i.e. 3–5 monthsafter Li-pilocarpine-induced SE (bottom traces)As shown in the recordings, ‘epileptic’ granule cells displayed GABAA

sIPSCs with greater amplitudes and frequencies than those of Li-salinegranule cells. The individual trace was obtained by averaging 10representative individual sIPSCs. The GABAA mIPSC deactivation phasewas best described by a monoexponential function and a decay timeconstant (τ ) of about 16 ms. Note that GABAA sIPSCs only differed bytheir maximal peak amplitude while their time to peak (rise time) andmonoexponential time constant (τ ) were similar in both groups.

at 24–48 h after SE, perfusion of 100 nm AP did notproduce any significant modulation of GABAA mIPSCs(Table 2). The lack of effect on T50% can be seen bysuperimposing both cumulative curves, before and afterAP perfusion (Fig. 5B). This insensitivity was only trans-ient, as AP strongly potentiated the effect on both thetotal rise time and mean T50% in epileptic Li-pilocarpinerats (Fig. 5D and E). The prolonging effect of T50%

is illustrated by a rightward shift of the cumulativecurves (Fig. 5C). The difference between Li-saline andepileptic Li-pilocarpine rats in terms of the extent towhich AP modulated T50% was not statistically significant(P = 0.17). As with benzodiazepine modulation, washoutexperiments indicated that the effects of AP perfusion onLi-saline and epileptic Li-pilocarpine rats are reversible(Fig. 5F).

Discussion

The present data reflect drastic changes in GABAA-mediated inhibition within dentate gyrus GCs in the Li-pilocarpine model of MTLE. Changes occur in both thebiophysical properties and sensitivity of GABAA receptorsto allosteric modulators. We show that synaptic GABAA

receptors become progressively insensitive to diazepam.Conversely, flumazenil has no effect in Li-saline rats anddisplays inverse agonist properties in Li-pilocarpine rats asearly as 24 h after SE. Likewise, we report for the first timethat synaptic GABAA receptors temporarily loose theirsensitivity to neurosteroids early after SE.

Enhanced GABAA-mediated inhibition inlithium–pilocarpine-treated rats

In accordance with previous findings in human andvarious models of TLE (Otis et al. 1994; Buhl et al. 1996;Gibbs et al. 1997; Shumate et al. 1998; Cohen et al. 2003),the present data demonstrate an increase in GABAA-mediated inhibition in the dentate gyrus of epilepticLi-pilocarpine rats. From the recordings of sIPSCs andmIPSCs, this potentiation appears to be both pre- andpostsynaptic.

At the postsynaptic level, our data show an increasein the mean peak amplitude of mIPSCs and sIPSCs inLi-pilocarpine rats studied at 24–48 h and 3–5 monthsafter SE compared to Li-saline rats. This most probablyreflects an increased number of GABAA receptor channelsat individual synapses, reflecting an adaptive process aimedat the reinforcement of inhibitory neurotransmissionoccurring early during epileptogenesis and persisting inchronically epileptic rats. This type of up-regulation of



Table 2. Main characteristics of GABAA receptor-mediated miniature IPSCs recorded before and after drug perfusion fromhippocampal GCs of Li-saline and Li-pilocarpine rats examined 24–48 h and 3–5 months after SE

Amplitude RT T50% Frequency n(pA) (ms) (ms) (Hz)

Diazepam (DZP) Li-saline Before DZP −35.5 ± 2.4 1.36 ± 0.081 8.0 ± 0.51 0.50 ± 0.0 11After DZP −35.2 ± 2.5 1.64 ± 0.09∗∗ 11.5 ± 0.6∗∗ 0.48 ± 0.06

Li-Pilo 24–48 h Before DZP −46.7 ± 3.9 1.13 ± 0.03 8.7 ± 0.2 0.56 ± 0.1 7After DZP −46.1 ± 4.2 1.30 ± 0.04 10.2 ± 0.4∗∗ 0.55 ± 0.17

Li-Pilo 3–5 months Before DZP −43.3 ± 2.0 1.38 ± 0.09 9.9 ± 0.4 0.54 ± 0.07 11

After DZP −43.8 ± 2.6 1.43 ± 0.08 10.2 ± 0.4 0.54 ± 0.08Flumazenil (FLU) Li-saline Before FLU −35.2 ± 1.6 1.26 ± 0.1 9.7 ± 0.5 0.50 ± 0.07 9

After FLU −35.6 ± 1.6 1.31 ± 0.1 9.9 ± 0.7 0.48 ± 0.06Li-Pilo 24–48 h Before FLU −43.4 ± 3.8 1.32 ± 0.11 10.1 ± 0.4 0.56 ± 0.18 7

After FLU −42.0 ± 3.8 1.29 ± 0.10 8.2 ± 0.3∗∗ 0.55 ± 0.17Li-Pilo 3–5 months Before FLU −45.4 ± 3.2 1.47 ± 0.08 9.3 ± 0.3 0.55 ± 0.05 11

After FLU −46.8 ± 3.2 1.42 ± 0.08 7.7 ± 0.3∗∗ 0.53 ± 0.05

Allopregnanolone (AP) Li-saline Before AP −36.0 ± 2.1 1.28 ± 0.07 9.2 ± 0.2 0.65 ± 0.13 12After AP −36.5 ± 2.4 1.58 ± 0.11∗∗ 12.5 ± 0.4∗∗ 0.58 ± 0.12

Li-Pilo 24–48 h Before AP −43.5 ± 2.8 1.22 ± 0.1 8.8 ± 0.3 0.59 ± 0.09 8After AP −41.5 ± 3.1 1.26 ± 0.1 8.8 ± 0.5 0.57 ± 0.10

Li-Pilo 3–5 months Before AP −56.5 ± 5.8 1.24 ± 0.07 9.0 ± 0.71 0.54 ± 0.12 7After AP −55.8 ± 5.5 1.44 ± 0.08∗∗ 11.0 ± 0.7∗ 0.55 ± 0.13

Mean peak amplitudes, total rise time (RT), half-decay time (T50%) and frequency of occurrence were analysed. Values areexpressed as means ± S.E.M of the number of cells (n). ∗ P < 0.005, ∗∗ P < 0.001, statistically significant difference fromLi-saline rats.

GABAA receptor density in the dentate gyrus has alreadybeen reported both in human MTLE (Loup et al. 2000)and in the kindling (Otis et al. 1994; Nusser et al. 1998),pilocarpine (Fritschy et al. 1999) and kainate (Schwarzeret al. 1997) models. However, a recent study reporteda reduced amplitude of mIPSCs in pilocarpine-treatedrats at 7 days after SE (Cohen et al. 2003). Thus, thepotentiation observed in the present study shortly afterSE is, rather, a postictal consequence of SE and hence anadaptive process reinforcing inhibitory neurotransmissionin response to the hyperexcitability triggered by seizures,as observed in epileptic Li-pilocarpine rats at 3–5 monthsafter SE.

In addition, the frequency of sIPSCs undergoes atwo-fold increase only in Li-pilocarpine rats studied 3–5 months after SE. The absence of an increase in thefrequency of mIPSCs indicates that the inhibitory hyper-activity is due to increased presynaptic neuronal activity.This adaptation is quite delayed compared to the change inamplitude, which was already recorded 24–48 h after SE.

The mean amplitudes and kinetics of both sIPSCs andmIPSCs recorded in the present study are in the samerange as those from previous studies performed on GABAA

receptors from rat granule cells (see for example Cohenet al. 2003). Conversely, the frequency values of IPSCsrecorded here are lower than those reported in moststudies (Otis et al. 1994; Buhl et al. 1996; Poisbeau et al.

1997, 1999; Cohen et al. 2003). These differences maybe linked to the preparation of the slices, which wererelatively thin (250–300 µm) and were not cut at an angle,making it possible to preserve all entorhino-hippocampalconnections. Recordings were also performed at roomtemperature (20–22◦C), while most studies adopted ahigher temperature – usually 33–35◦C – increasing theprobability of neurotransmitter release.

Altered pharmacology of GABAA-mediated inhibitionin lithium–pilocarpine-treated rats

Many studies have characterized the basal pharmacologicalproperties of synaptic GABAA receptors expressed at GCsynapses. They have revealed a high responsiveness tobenzodiazepines and neurosteroid agonists and only aweak or absent sensitivity to zinc in both human (Shumateet al. 1998) and rat GCs (Gibbs et al. 1997; Mtchedlishviliet al. 2001; Cohen et al. 2003). Accordingly, in the pre-sent study, diazepam and allopregnanolone increased thehalf-decay and rise times of mIPSCs in Li-saline rats. Theincreases in the half-decay time probably result from anincrease in the apparent affinity of GABA for the GABAA

receptor. Conversely, increases in the mean rise timeinduced by diazepam or allopregnanolone perfusion wereobserved in this study. However, both drugs had no effecton the mean peak amplitudes or frequencies of mIPSCs.The absence of an effect on amplitude could be explained



Figure 3. Effects of 1 µM diazepam (DZP) on GABAA mIPSC kinetics in Li-saline rats and both groups ofLi-pilocarpine rats (24–28 h and 3–5 months after SE)Left graphs represent cumulative probability plots of all mIPSC half-decay times (T50%) for a single GC before (thinline) and after perfusion of 1 µM DZP (thick line) in Li-saline rats (A) and Li-pilocarpine rats 24–48 h after SE (B) and3–5 months after SE (C). Although diazepam considerably prolonged mIPSCs in Li-saline rats (A, rightward shift ofthe T50% distribution significant at P < 0.01, KS test), it showed a low efficacy (but still significant at P < 0.01, KStest) in modulating mIPSCs shortly after SE (B). This decreased sensitivity of synaptic GABAA receptors to diazepamwas aggravated in epileptic rats. At this time, diazepam was totally unable to modulate these receptors and thisresulted in the superimposition of the cumulative propability curves of mIPSCs recorded in epileptic GCs (C). Thisis confirmed by the progressive decrease in the percentage potentiation of mIPSC T50% (D) and in the total risetime induced by diazepam (E). The effect of diazepam perfusion on T50% mIPSCs is reversible, as illustrated in thewashout experiment performed in a Li-saline rat (F). ∗ P < 0.005, ∗∗ P < 0.001, statistically significant differencesfrom the recording before diazepam perfusion; ++P < 0.01, statistically significant differences between groups.



Figure 4. Effects of flumazenil (FLU, µM) on GABAA mIPSCs kinetics recorded in GCs from Li-saline andboth groups of Li-pilocarpine rats (24–28 h and 3–5 months after SE)Left graphs represent cumulative probability plots of all mIPSC half-decay times (T50%) for a single GC before (thinline) and after the perfusion of flumazenil (thick line) in Li-saline rats (A) and Li-pilocarpine rats 24–48 h after SE(B) and 3–5 months after SE (C). While flumazenil had no effect on T50% in Li-saline rats, it decreased T50% inLi-pilocarpine rats as early as 24–48 h after SE and the effect did not evolve during the course of epileptogenesis.This is confirmed by the strong decrease in T50% (P < 0.01, KS test) induced by flumazenil in both groups ofLi-pilocarpine rats (D). Conversely, flumazenil perfusion did not influence the total rise time in either Li-saline or Li-pilocarpine rats (E). The effect of flumazenil perfusion on T50% mIPSCs was reversible, as illustrated in the washoutexperiment performed in a Li-pilocarpine rat (F). ∗ P < 0.005, ∗∗ P < 0.001, statistically significant differences fromthe recording before flumazenil perfusion; ++.P < 0.01, statistically significant differences between groups



by a saturating quantity of GABA in the synaptic cleft inrelation to the number of synaptic receptors, as reportedpreviously (see review and references in Mody et al. 1994).

Loss of sensitivity to diazepam potentiation of mIPSCsin epileptic GCs. Our data show the appearance ofrefractoriness to diazepam modulation of synaptic GABAA

receptors. At 24–48 h after SE, there is already a largedecrease in the effect of diazepam on potentiation ofmIPSC deactivation time, which demonstrates the rapidloss of sensitivity of GABAA receptors to diazepam. Thesedata are in accordance with a previous study reportingpharmacological alterations of the total GC GABAA

receptor population as early as 45 min after the onset of Li-pilocarpine SE (Kapur & Macdonald, 1997). We confirmhere the same alterations of GABAA receptors at individualsynapses. Moreover, the loss of sensitivity of GABAA

receptors to diazepam is a progressive process duringepileptogenesis, since it is more marked 3–5 months than24–48 h after SE. Our data are in accordance with pre-vious reports in the literature suggesting that GABAA

receptors in epileptic GCs have a decreased sensitivity tobenzodiazepines (Gibbs et al. 1997; Kapur & Macdonald,1997; Shumate et al. 1998; Mtchedlishvili et al. 2001; Joneset al. 2002; Cohen et al. 2003).

Many hypotheses have been put forward to explain therefractoriness to benzodiazepine agonists after prolongedlimbic seizures. Firstly, the benzodiazepine binding sitemay loose its functionality as a result of uncoupling linkedto an alteration in the conformation of GABAA receptors inMTLE (Klein et al. 1995; Lyons et al. 2000). Secondly, manystudies have reported numerous changes in the mRNA andprotein expression of GABAA receptor subunits in humanMTLE (Loup et al. 2000) and various animal models (Riceet al. 1996; Schwarzer et al. 1997; Brooks-Kayal et al. 1998;Fritschy et al. 1999). In general, these studies report adown-regulation of α1 and γ 2 mRNA subunits, both ofwhich confer a high sensitivity to benzodiazepines andlow sensitivity to zinc when incorporated (Jones-Davis& Macdonald, 2003). Simultaneously, there is an up-regulation of α3, α4, α5 and δ subunits (Rice et al. 1996;Brooks-Kayal et al. 1998; Fritschy et al. 1999). GABAA

receptors containing α3, α4, α5 and δ subunits are highlysensitive to zinc and those containing α4 and δ subunitshave no or low affinity to benzodiazepines, respectively(Barnard et al. 1998; Mody, 2001). Therefore, the majorityof changes in subunit expression reported in the literature –mainly the up-regulation ofα4 andδ subunits – induced bypilocarpine SE, are in accordance with the loss of diazepamsensitivity observed in the present study (Brooks-Kayal

et al. 1998). GABAA receptors containing α3 or α5 sub-units have a high affinity for agonists binding at typeII benzodiazepine sites and therefore can be modulatedby agonists with a ‘broad spectrum’ action (Jones-Davis& Macdonald, 2003) such as diazepam, which was usedhere. Consequently, an up-regulation of α3 and α5 sub-units within epileptic GCs is not consistent with a lossof effectiveness of diazepam on GABAA receptor currents.Thus, the many subunit alterations of GABAA receptorsdescribed in MTLE do not fully explain why synapticGABAA receptors of epileptic GCs have lost their sensitivityto benzodiazepines. Thirdly, most of the subunits formingsynaptic GABAA receptors contain consensus sequencesfor various kinases and phosphatases (Kittler & Moss,2003). Seizures are known to modulate the activitiesof kinases (Jope et al. 1992) but it is still not knownwhether post-translational modification can alter thebenzodiazepine sensitivity of GABAA receptors (Jones et al.2002).

Paradoxical effect of flumazenil on mIPSCs of epilepticGCs. Flumazenil is currently described as a selectivecompetitive benzodiazepine antagonist with a high affinityfor the benzodiazepine binding site of GABAA receptors(Barnard et al. 1998). Therefore, flumazenil can inhibit theeffects of both agonists and inverse agonists by displacingthe binding but is devoid of intrinsic activity on GABAtransmission (Li et al. 2001). Moreover, at low doses,flumazenil has no detectable electrophysiological effect butat high doses, it displays an ‘agonist-like’ effect as shownin acutely dissociated CA1 pyramidal cells (Buldakova& Weiss, 1997). Its low efficacy may explain why thisintrinsic activity goes undetected in most studies and whyflumazenil is currently regarded as an antagonist (Weisset al. 2002). Finally, flumazenil displays different effectsdepending on the subunit composition of the GABAA

receptor. Flumazenil blocks the effects of benzodiazepineat receptors containing α1, α2, α3 or α5 subunits but actsas an agonist at receptors containing α4 or α6 subunits(Whittemore et al. 1996; Hauser et al. 1997; Thomson et al.2000).

Under our experimental conditions, we never detectedan agonist-like effect of flumazenil on GCs in Li-salinerats (data not shown). On the other hand, the synapticGABAA receptors of GCs in Li-pilocarpine rats, whileinsensitive to diazepam, develop a strong response toflumazenil perfusion. In fact, flumazenil displayed aninverse agonist-like effect in reducing the half-decay timeof mIPSCs at both 24–48 h and 3–5 months after SE. Aninverse agonist effect of flumazenil was similarly shown



Figure 5. Effects of allopregnanolone (AP, 100 nM) on GABAA mIPSCs kinetics in GCs from Li-saline andboth groups of Li-pilocarpine rats (24–28 h and 3–5 months after SE)Left graphs represent cumulative probability plots of all mIPSC half-decay times (T50%) for a single GC before (thinline) and after the perfusion of AP (thick line) in Li-saline rats (A) and Li-pilocarpine rats 24–48 h after SE (B) and3–5 months after SE (C). AP induced a prolongation of GABAA mIPSCs in both Li-saline and epileptic Li-pilocarpinerats, as illustrated by a rightward shift of T50% for most mIPSCs (A and C, P < 0.01 for both, KS test). Conversely,AP temporarily lost its effect on T50% over the first 2 days after SE, as illustrated by the fact that the plots beforeand after AP totally matched one another (B). This is confirmed by the transient loss of the T50% potentiation (D)and the rise time (E) induced by AP in Li-pilocarpine rats examined 24–48 h after SE. The effect of AP perfusion onT50% mIPSCs is reversible, as illustrated in the washout experiment performed in a Li-saline rat (F). ∗ P < 0.005,∗∗ P < 0.001, statistically significant differences from the recording before AP perfusion; ++P < 0.01, statisticallysignificant differences between groups.



in CA1 pyramidal neurones from control animals (Kinget al. 1985). The effect of flumazenil is not consistentsolely with the up-regulation of α4 and δ subunits becausereceptors incorporating these subunits are insensitiveto benzodiazepines (Barnard et al. 1998; Mody, 2001).Variability of the effects of flumazenil has been reportedin numerous pathophysiological conditions (Sand et al.2000). For example, flumazenil can reduce epilepticactivity in both untreated patients and patients acutelytreated with benzodiazepines (Hart et al. 1991; Shariefet al. 1993) or who have developed a tolerance tobenzodiazepine agonists Polc et al. 1995; Reisner & Pham,1995; Tietz et al. (1999).

The paradoxical effects of flumazenil have led manyauthors to suggest that there may be endogenous ligandsfor benzodiazepine receptors and that flumazenil may actby competing with or displacing these substances fromthe benzodiazepine binding site (Rothstein et al. 1992;Polc et al. 1995; Lugaresi et al. 1998; Wallace et al. 2001).These endogenous compounds have been shown in bothhuman and rat hippocampus (Alho et al. 1989; Ball et al.1989) and their plasma and CSF concentration is increasedin epileptic patients (Ferrarese et al. 1998) and duringidiopathic recurrent stupor episodes (Rothstein et al. 1992;Lugaresi et al. 1998). The present data are not inconsistentwith the presence of endogenous benzodiazepine ligandsbut would need further exploration to determine whetheror not changes in the concentration and properties of theseligands may have occurred in Li-pilocarpine rats.

Finally, despite the extensive reorganization and neuro-nal loss in Li-pilocarpine rats, the accessibility offlumazenil to GABAA receptors at GC synapses doesnot seem to be facilitated or reflect increased diffusionleading to higher local concentrations, since the effectsof 1 and 10 µm flumazenil were identical in both controland epileptic rats. Moreover, at higher concentrations,flumazenil would, rather, act as a positive agonist(Buldakova & Weiss, 1997; Hauser et al. 1997; Thomsonet al. 2000).

Preservation of neurosteroid sensitivity in mIPSCsof epileptic GCs. In our study, the effects ofallopregnanolone on potentiation of GABAA-mediatedcurrents recorded in Li-saline GCs were preserved inepileptic GCs. Conversely, Mtchedlishvili et al. (2001) haveshown that GABAA receptors from GCs lose sensitivity toneurosteroids in rats rendered epileptic by self-sustainedSE. This difference may be linked to model characteristics,the extent of neuronal loss and tissue reorganization or thetype of GABAA receptor population studied, i.e. the whole

population in their study and the synaptic one in thepresent study. Allopregnanolone is one of the most potentendogenous positive modulators of GABAA receptors(Lambert et al. 1996). In addition, allopregnanolone, likepentobarbital, increases the apparent affinity for GABAat GABAA receptors (Lambert et al. 1995). This confersstrong anticonvulsant properties to allopregnanolonethat have been described in rats exposed to GABAA

receptor antagonists, kainic acid or perforant pathstimulation (Lambert et al. 1995; Frye & Scalize, 2000).Thus, the fact that allopregnanolone preserves its potenteffect on mIPSCs in epileptic rats indicates that a largeproportion of synaptic GABAA receptors remain sensitiveto neurosteroids in these animals. Neurosteroids affectGABAA receptors containing most subunits, exceptthose comprising the α4 subunit (Smith et al. 1998a,b).Conversely, the α4β3δ subunit composition seems highlysteroid sensitive (Belelli et al. 2002; Bianchi & Macdonald,2003) and the δ subunit plays a pivotal role in neurosteroidsensitivity of the GABAA receptor (Mihalek et al. 1999;Belelli et al. 2002; Spigelman et al. 2002; Vicini et al. 2002;Stell et al. 2003).

The puzzling observation in the present study is the lackof potentiation of allopregnanolone on GABAA-mediatedcurrents in Li-pilocarpine rats studied 24–48 h after SE.Refractoriness to neurosteroid modulation is only trans-ient since the effect of allopregnanolone is recoveredat 3–5 months after SE. Although changes in subunitcomposition of the GABAA receptor have been reportedfor the most part in chronically epileptic animals, a trans-ient subunit change could render the receptor insensitive tothe neurosteroid ligands. This phenomenon occurs duringpostnatal development (Cooper et al. 1999; Mtchedlishviliet al. 2003). In the dentate gyrus, GABAA receptorsare sensitive to neurosteroids at postnatal day 10 (P10),insensitive at P20 and sensitive again in adult rats (Cooperet al. 1999; Mtchedlishvili et al. 2001, 2003). Concurrently,from P12, there is a down-regulation ofα4 andβ1 subunitsand the δ subunit becomes detectable (Laurie et al. 1992) –a result which correlates well with the relative insensitivityof granule cells of the P20 rats to neurosteroids (Cooperet al. 1999). The early and transient loss of GABAA receptorsensitivity to neurosteroids recorded in the present studyis possibly the result of changes in the brain metabolismof neurosteroids. Indeed, the sensitivity of dentate GCGABAA synapses to neurosteroids varies according to thecompound and reflects local metabolism (Belelli & Herd,2003; Lambert et al. 2003), which could be transientlydisturbed by all the events characterizing SE. Finally, SEmay lead to an overproduction of peripheral and/or end-ogenous neuroactive steroids inducing acute alterations



in GABAA receptor properties and decreasing sensitivityto neurosteroids. These compounds contribute to homeo-stasis during acute stress (Barbaccia et al. 2001), in whichsituation their plasma and brain concentrations increasemarkedly (Purdy et al. 1991). This is also the case forpostictal circulating levels of allopregnanolone in epilepticchildren (Grosso et al. 2003).

Conclusion

This study reveals chronic alterations of synaptic GABAA

receptors that appear very early after SE and lead toaltered GABAA receptor function in GCs of Li-pilocarpinerats. Thus, despite the enhancement of GABAA receptorcurrents in epileptic GCs, changes in their modulationmay compromise the gatekeeper function of the dentategyrus and facilitate seizure generation and propagation.Such plasticity with regard to endogenous modulatorssuch as zinc and neurosteroids favours increased hyper-excitability and susceptibility to seizures. Understandingthe refractoriness of epileptic GCs to benzodiazepinemodulation could be a major breakthrough in the therapyof epileptic seizures. Similarly, a better understandingof the mechanisms underlying the effect of neuroactivesteroids, which increase GABAergic synaptic inhibition,might lead to new therapeutic approaches and their clinicaluse as an alternative therapy to benzodiazepines.

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Acknowledgements

We are very grateful to E. Koning and F. Herzog for skilfultechnical assistance, to J.-L. Rodeau for his helpful contributionto statistical analysis and to J. Gonzalez de Aguilar (Laboratoirede Signalization Moleculaire et Neurodegenerescence) for hishelp in the use of the confocal microscope.


pharmacological plasticity of gabaa receptors at dentate gyrus

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