The GABA-Withdrawal Syndrome: AModelof Local Status ...downloads.hindawi.com/journals/np/2000/968906.pdf · 14 C. SILVA-BARRAT,J. CHAMPAGNATANDC. MENINI At the same dosage, NMDAantagonists

NEURAL PLASTICITY VOL. 7, NOS. 1-2, 2000

The GABA-Withdrawal Syndrome: A Model ofLocal Status Epilepticus

Carmen Silva-Barrat*, Jean Champagnat and Christian Menini

1Laboratoire de GnOtique de la Neurotransmission et des Processus Neurodgdnratifs. UMR 9923,CNRS. 75634 Paris, France," Institut Alfred Fessard. CNRS. 91198 Gifsur gvette, France

SUMMARY

The GABA-withdrawal syndrome (GWS)is a model of local status epilepticus followingthe interruption of a chronic GABA infusioninto the rat somatomotor cortex. GWS ischaracterized by focal epileptic electroenceph-alographic discharges and associated contra-lateral myoclonus. In neocorticai slices obtainedfrom GWS rats, most neurons recorded in theGABA-infused area are pyramidal neuronspresenting bursting properties. The bursts areinduced by white-matter stimulation and/or

intracellular depolarizing current injectionand correlate with a decrease of cellularsensitivity to GABA, caused by its prolongedinfusion. This effect is related to a calciuminflux that may reduce the GABAA receptor-mediated inward current and is responsiblefor the bursting properties. Here we presentevidence for the involvement of calcium- andNMDA-induced currents in burst genesis. Wealso report modulatory effects of noradrenaline

appearing as changes on firing patterns ofbursting and nonbursting cells. Complementaryhistochemical data reveal the existence of alocal noradrenergic hyperinnervation and an

ectopic expression of tyrosine hydroxylasemRNAs in the epileptic zone.

Corresponding author:Laboratoire d’Epilepsie Exp6rimentale, UMR 9923,CNRS, Facult6 Piti6-Salp6tri6re, 91 bd. de l’Hfpital,75634 Paris Cedex 13, France.t61: (33) 01-40-77-98-27, fax: (33) 01-40-77-97-89e-mail [email protected]

INTRODUCTION

For several years, our group in Gif-sur-Yvette(France) worked on a model of reflex epilepsy,the photosensitive epilepsy of Papio papiobaboons (Killam et al. 1967). When thesepredisposed baboons are submitted to intermittentlight stimulation, generalized paroxysmal mani-festations appear in the frontal motor cortex

(Morrell et al., 1969). We had clearly demonstratedthat generalized paroxysmal manifestations originatein the motor cortex (Silva-Barrat et al. 1988),when Simon Brailowsky came to our laboratory.Simon proposed to block the epilepticmanifestations of baboons by means of a chronicinfusion of ],-aminobutyric acid (GABA) into themotor cortex ofthese animals. We performed 4- to7-day infusions by means of osmotic minipumpsand observed the disappearance of epilepticmanifestations. After cessation of the GABAinfusion, however, a rebound of brain excitabilitywas observed, as evidenced by the presence ofepileptogenic discharges localized in the infusedarea (Bmilowsky et al., 1987; 1989). The phenomenonappearing after the interruption of the chronicGABA infusion was named the "GABA-withdrawal syndrome" (GWS). Initially, we

demonstrated that GWS is not the consequence ofthe genetic epileptic predispositon of baboons,and we tested the effects of chronic intracorticalGABA infusions in non-photosensitive animals.

Indeed, we reproduced GWS not only in non-

epileptic baboons but also in normal rats. Afterreturning to Mexico, Simon and his groupreproduced GWS in hippocampal slices of rats

(Garciaugalde et al., 1992). Finally, this phenomenon

(C)Freund & Pettman, U.K., 2000 9

10 C. SILVA-BARRAT, J. CHAMPAGNAT AND C. MENINI

could be considered a new model of focal epilepsy(Brailowsky et al., 1988). Thanks to this discovery, anew series ofepileptic studies has been initiated thatwe want to present briefly as our tribute to Simon.

GWS AS A MODEL OF LOCAL STATUSEPILEPTICUS IN THE RAT: IN VIVO AND

IN VITRO STUDIES.

Both EEG and clinical studies (Brailowsky etal., 1988) have established that GWS is a statusepilepticus resembling the epilepsia partialiscontinua, a partial status epilepticus that wasdescribed in human patients by Kojewnikow(1895). GABA is infused into the motor cortex ofrats for 5 days. Upon cessation of the infusion,an epileptogenic focus appears, characterized bycontinuous EEG discharges at high frequency,localized in the infused area and associated withmyoclonic twitches ofthe contralateral correspondingbody territory. The epileptic manifestations appear20 min after the infusion interruption, persist for48 h on average, and never generalize into tonic-clonic seizures. GWS is an interesting modelresulting from a local manipulation that isassociated with a focal epilepsy. GWS isdifferent from other epilepsy models that are

provoked by topical application of toxic orirritant drugs or even convulsant drugs (such as

penicillin, kainate or pilocarpine) provokingdiffuse abnormalities.

Using conventional intracellular recordingand stimulation techniques, we studied neuronalactivity in the epileptogenic focus in neocorticalslices obtained from rats presenting GWS (Silva-Barrat et al., 1989). The presence of a greatnumber of burst-generating neurons in an areaclose to the GABA-infused site has enabled theanalysis of epileptic-like pattern generation bycomparing bursting and nonbursting cells.Bursting neurons present paroxysmal depolarizingshifts (PDSs) and bursts of action potentials(APs) after synaptic activation by white matter

stimulation (WM) and/or intrinsic bursts of APs

after intracellular injection with a depolarizingcurrent. Nonbursting neurons present neithersynaptic nor intrinsic bursting properties. In thispaper, we will first describe the cellular typesrecorded in the GWS epileptic focus, and we willshow that the bursting cells are desensitized toGABA. We will also present some ionic mechanismsthat are involved in the genesis of the epilepticactivity, and finally discuss some neuromodula-tory mechanisms involved in this activity.

PHYSIOLOGICAL AND MORPHOLOGICALIDENTIFICATION OF NEURONS SHOWING

BURSTING ACTIVITY

Histological observation of slices showed thatthe recorded cells are impaled at the periphery ofthe necrotic zone (0.7 to 1.2 mm) because ofcannula penetration and GABA infusion. Thesoma of biocytine-labeled neurons are localizedin layer V (Fig. 1A). Cells presenting synapticand/or intrinsic bursts have a pyramidal-shapedsoma with a basal dendritic arborization, an

apical dendrite, and an axon from the basal regionof the soma (Fig. 1B) (Silva-Barrat et al., 1994).Synaptic bursts are evoked by suprathresholdwhite matter stimulation. The bursts consist of 3-8 APs riding on the large depolarizing wave orPDS, EPSPs are obtained by decreasing thestimulus intensity to values subthreshold for APelicitation (Fig. 1C). Intrinsic bursts induced bydepolarizing current injection consist of 2-5 APsriding on a slow wave (Fig. 1D). In nonburstingcells, the white-matter stimulation subthresholdfor AP elicitation evokes a synaptic potential thatat the resting membrane potential is mainlydepolarizing. These cell groups are not differentin their resting membrane potential, inputresistance, and AP amplitude (Silva-Barrat et al.,1989; 1992; 1994). Our first results thereforeshowed a large population of cells, most of thempyramidal cells, presenting bursting properties,with respect to those seen in other models ofepilepsy that were provoked by topical or by

GWS: MODEL OF LOCAL STATUS EPILEPTICUS 11

systemic administration of compounds inhibitingGABA transmission (Connors et al., 1982). Our data

suggest that cortical slices retain in vitro the epilepticcharacteristics presented in vivo during the GWS.

A

B

C

J IlOmV

D

IlOmV

1 O0 ms

Fig. 1: Biocytin-labeled bursting neuron. A: photomicrograph of GABA infused site and a biocytin-labeled pyramidalneuron situated in layer V. Nissl-counterstained material (40). B: higher magnification photomicrograph ofneuron. Note the important arborization of basal dendrites, the initial segment of the apical dendrite and thesoma (190). C: paroxysmal depolarization shift induced in this neuron by suprathreshold white matter

stimulation Em =-72mV superposed to an EPSP triggered by subthreshold stimulation (hatched line).D: Intrinsic burst induced in the same neuron by intracellular depolarizing current (bottom).


BURSTING NEURONS ARE DESENSITIZEDTO GABA.

Could an abnormal GABAergic transmissionexplain the epileptic manifestations of the GWS?To answer this question, we studied the effects ofGABA application on bursting cells and comparedthese effects with those obtained after the sameapplication on nonbursting cells. We observed that

GABA at 1.6 tM was able to block APs evokedby synaptic stimulation in nonbursting cellsrecorded in slices from saline infused rats (Fig.2A), whereas the same dose was inefficient onPDSs recorded in slices from GWS rats (Fig. 2B).Similar results were observed during applicationsof isoguvacine, a specific GABA agonist, thusindicating that bursting cells in GWS are indeedtolerant to GABA (Silva-Barrat et al., 1989).

A

50 ms

B

8 4O50 mV

50 ms100 mV

250 ms

Fig. 2: GABA effects on synaptic potentials evoked by white matter stimulation (arrow). A: EPSPs induced in a

saline-infused slice, before and during 1.6tM of GABA (5 min). B: paroxysmal depolarization shift induced

in a slice obtained from a GABA-withdrawal syndrome rat during 1.6, 8, and 40 M of GABA application

(5 min). Note the different time scales. C: Effects of GABA (5 min) on intrinsic bursts induced by intracellular

depolarizing current (8, 100, and 1000tM). Note that intrinsic bursts are more resistant to GABA than to PDSs.


CA2+ IONIC MECHANISMS INVOLVED INBURSTING ACTIVITY

The decrease in GABA efficacy could berelated to a Ca+ influx that may reduce theGABAa receptor-mediated inward current andseems to be responsible for the bursting properties.The role of Ca+ currents and of Ca-dependentprocesses in determining bursting patterns wasstudied by using organic and inorganic antagonistsof Ca channels. The organic Ca antagonistverapamil, as well as the inorganic blockers ofCaz channels Co+, Mg+, and Cdz+, reduce theamplitude of PDSs, block the associated bursts,and also block intrinsic bursts. None of the Ca+

antagonists affect EPSPs induced in bursting ornonbursting cells by synaptic stimulation at lowintensity or the EPSP-single AP sequences evokedin nonbursting cells by stronger stimuli (Silva-Barrat et al., 1992). Therefore the effects of Ca+

blockers are not related to suppression of Ca+-dependent release of transmitters.

In addition, intracellular application of ethyleneglycol-bis-(13-aminoethyl ether)-N,N,N’,N’-tetra-acetic acid (EGTA), a Ca+ chelator, increased theamplitude and duration of PDSs and favored theappearance of additional APs during intrinsic andsynaptically-induced bursts (Silva-Barrat et al.,1992). The involvement of L channels, slowlyinactivating, in bursting activity as suggested inother preparations (Johnston et al., 1980) wouldexplain the high sensitivity to Ca2+ blockers andthe regulatory effects of intracellular Caz+ ions

sensitive to the injection of EGTA.Bursting activities are also affected in conditions

of reduced outward K+ currents. Thus tetraethyl-ammonium (TEA), at doses that do not changeinput resistance, spike duration, or first interspiketime interval, abolish the burst-terminating processand induce plateau-like potentials (up to 500 ms) thatare tetrodoxin-resistant and blocked by the Caz+

antagonists Cd+ and Co+ (Silva-Barrat & Cham-

pagnat, 1995). Therefore, it appears that bursts

during GWS are generated by Ca+-dependentplateau potentials that are terminated by a K+

current that is highly sensitive to TEA.Given that the entry of Ca+ depends not only

on voltage-dependent Ca+ channels but also onreceptor-operated Ca+ channels, we tested theeffects of selective antagonists of NMDAreceptors on bursting activities. Bath applicationsof APH (DL-2amino-7-phosphonoheptanoate)and APV (DL-2amino-5-phosphonovalerate), atdoses of 10 to 50 tM, reversibly reduced theamplitude and duration ofPDSs (Fig. 3).

control

APH

Fig. 3: Effects of DL-2-amino-7-phosphono-heptanoicacid (APH) on paroxysmal depolarizationshifts. Paroxysmal depolarization shift, inducedby white matter stimulation before (control),during (APH), and after (recovery) APHapplication (20tM). Em -74mV.


At the same dosage, NMDA antagonists do notaffect the EPSPs induced by a stimulation at lowintensity in bursting or nonbursting neurons northe firing pattern induced by intracellulardepolarizing current injection in the same cells.This observation suggests that NMDA receptorsplay a specific role in the induction of PDSs

(Silva-Barrat et al., 1992) as similarly shown forepileptiform bursting in the pyramidal hippo-campal neurons (Dingledine et al., 1986). In summary,PDSs result from the cooperation of at least threedifferent currents: (1) the classical EPSP that isinsensitive to NMDA antagonists, (2) a latercomponent (the depolarizing wave) that depends

Fig. 4: Overexpression of tyrosine hydroxylase mRNAs revealed by in situ hybridization in the epileptic focus zoneof a rat presenting GABA-withdrawal syndrome. A: densitometric figure of a brain slice labeled with a

radioactive tyrosine hydroxylase-specific oligonucleotide. B: Magnified photomicrograph (x250) of the

labeled zone, showing neurones with high density of silver grains.

GWS" MODEL OF LOCAL STATUS EPILEPTICUS 15

on NMDA receptor activation, and (3) the Ca2+

voltage-dependent current that is responsible forbursts of APs and sensitive to Ca+ antagonists,as shown above.

NEUROMODULATORY ROLE OFNORADRENALINE IN GWS

Given the preceding results, we wanted toverify if neurotransmitters other than GABA andglutamate are involved in GWS. An immuno-cytochemical study revealed an important labelingof noradrenergic cell bodies and terminals in azone corresponding to the epileptic focus (Aranedaet al., 1994). This zone also presents an ectopicexpression of tyrosine hydroxylase mRNAs,which has been revealed by in situ hybridizationand which could lead to noradrenergic neosynthesisin modified cells (Menini et al., 1992) (Fig. 4), giventhat tyrosine hydroxylase mRNAs are not normallyexpressed in the cerebral cortex of adult rats.

The data presented here lead to the followingquestion: what are the functional consequences ofthe noradrenaline expressed in GWS at the level ofthe epileptic focus? Bath norepinephrine (NE)applications provoked a small cell membranedepolarization associated with a decrease in inputK+ conductance that is not significantly different forbursting and nonbursting cells (Silva-Barrat et al.,1994). In addition, the NE-induced depolarizationreplaces the intrinsic bursts by a regular firing ofsingle APs and causes intrinsic bursts to appearduring previously subthreshold depolarizing currentpulses. These NE-increased activities are abolishedby Ca2+ channels antagonists.

In nonbursting cells recorded from slices ofGWS but not from saline-infused rats, NE promotesthe appearance of bursts after WM stimulation, aswell as during depolarizing current injection. Thus,a threshold current pulse inducing a single APbefore NE application became efficient after NEapplication in inducing bursts of APs (Fig. 5). Thepossibility that these bursts result indirectly from

a

0.2nA

20mv

50ms

b lOmv

20ms

Fig. 5: Norepinephrine (10 tM) promotes burst appearance in nonbursting cells. Response to suprathreshold(0.2nA) intracellular depolarizing current pulse for eliciting a single AP in control (a) and duringnorepinephrine-induced depolarization (b). Bottom: superimposition of enlarged records a and b, taking the

fast hyperpolarization peak as a reference.


the NE-induced depolarization and from theincrease in input resistance has been eliminatedby compensating the NE-induced depolarization.The NE-induced bursts are generated by Caa+-dependent currents because they are transformedinto a regular firing ofAPs by Ca+-channel blockers.Generation of bursts in nonbursting cells has

been found to be related to an increased after-depolarization following the AP (Fig. 5). It istherefore possible that the afterdepolarizationactivates Ca+ currents, which in tum, couldgenerate extra spikes grouped in a burst. NE hasnot been previously reported to induce burstingpatterns in cortical neurons, so we can suggest

C

lOOpVls

Fig. 6: Effects of norepinephrine on paroxysmal depolarization shitts recorded in a bursting cell. A and B:

paroxysmal depolarization shifts recorded in a bursting cell before and atter norepinephrine application,respectively, at the same resting membrane potential. Norepinephrine provoked a decrease in paroxysmaldepolarization shift amplitude and duration, a decrease in the number of Aps, and an increase in the

afterhyperpolarization amplitude. C: Effects of norepinephrine on burst frequency: Note that the shortening

of paroxysmal depolarization shifts and the increase in afterhyperpolarizations facilitates the appearance of

extraburst during norepinephrine application and determines their frequency. D: Electroencephalographicspikes observed in living animals; Note that their frequency corresponds to the frequency of paroxysmaldepolarization shifts, namely, 5 Hz during the peak of the norepinephrine-induced depolarization.


that NE unmasks potential bursting capabilitiesby increasing Ca:/ currents in nonbursting cellsofGWS rats.

An other specific effect of NE observed inbursting cells of GWS rats concerns the after-hyperpolarization (AHP) that follows PDS triggeredby WM stimulation (Fig. 6A). We observed thatAHP increases during NE application, resulting ina 50% decrease in amplitude and duration ofPDSs (Fig. 6B) and an increase of their frequencyup to 5 Hz (Fig. 6C). Such bursts are generated byinteractions between Ca+ currents responsible forburst generation, K+ currents involved in AHPgeneration, and the strength ofthe K+ conductancedecrease associated with the NE-induced depolari-zation. The AHPs after PDSs could be responsiblefor the intervals between EEG spikes observed inliving animals (Fig. 6D).

The data presented above show that NE hasdifferential effects on neurons recorded in theepileptic focus area of GWS rats in comparisonwith those recorded in the normal cortex: NEpromotes appearance of bursts in nonburstingcells and accelerates the postburst repolarizationin bursting cells. Both effects are probably relatedto an increase of GWS-related Ca+ currents. Thishypothesis is supported by anatomical datarevealing the existence of a local noradrenergichyperinervation in the epileptic focus zone.

CONCLUSIONS

The interruption of a chronic intracorticalGABA infusion into the somatomotor cortex ofrats provokes the appearance of a GABA-withdrawal syndrome that is associated with an

epileptic focus localized to the infused site. Atthe periphery of this infusion site, numerousneurons present bursts of APs resulting from atolerance to GABA, correlated to an exaggeratedCa+ entry as a consequence of the prolongedGABA infusion. Such bursts, and by consequencethe epileptic focus, are subject to a noradrenergicmodulation resulting from an ectopic expression

of this neurotransmitter at the level of the GABAinfusion site. As immunocytochemical data(Araneda et al., 1994) and in situ hybridizationstudies (Menini et al., 1992) have revealed that inaddition to noradrenaline, choline acetyltransfer-ase is also overexpressed in the GWS focus area,we suggest that the GWS represents a valid modelof transitory epilepsy for studying the physiologicalconsequences of the reorganization of neuronalnetworks that is due to the expression of abnormalcellular phenotypes in this epileptic focus zone.

ACKNOWLEDGEMENT

This work was partly supported by a grantfrom ECOS, n U95E01

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The GABA-Withdrawal Syndrome: AModelof Local Status ...downloads.hindawi.com/journals/np/2000/968906.pdf · 14 C. SILVA-BARRAT,J. CHAMPAGNATANDC. MENINI At the same dosage, NMDAantagonists