Epilepsy Research (2008) 82, 194—199

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Serotonin depletion effects on the pilocarpinemodel of epilepsy

Euclides Maurício Trindade-Filhoa, Eduardo Ferreira de Castro-Netob,Reinaldo de A. Carvalhob, Eliangela Limab, Fulvio Alexandre Scorzab,Débora Amadob, Maria da Graca Naffah-Mazzacoratti b,∗,Esper Abrão Cavalheirob

a Departamento de Ciências Fisiológicas, Universidade Estadual de Ciências da Saúde de Alagoas (UNCISAL), Alagoas, Brazilb Disciplina de Neurologia Experimental, Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP), SãoPaulo, Brazil

Received 20 September 2007; received in revised form 25 July 2008; accepted 28 August 2008Available online 8 October 2008

KEYWORDSSerotonin depletion;Epilepsy;Pilocarpine;5,7-Dihydroxytryptamine

Summary The monoamine content in cerebral structures has been related to neuronalexcitability and several approaches have been used to study this phenomenon during seizurevulnerability. In the present work, we have described the effects of serotonin (5-HT) depletionafter the administration of 5,7-dihydroxytryptamine (5,7-DHT) into the median raphe nucleus inrats submitted to the pilocarpine model of epilepsy. Susceptibility to pilocarpine-induced statusepilepticus as well as the spontaneous seizure frequency during the chronic period of the modelwas determined. Since the hippocampus is one of the main structures in the development of thisepilepsy model, the 5-HT levels in this region were also determined after drug administration.Sixty-three percent of 5,7-DHT pre-treated rats (15/24) and only 33.4% of those receiving thecontrol solution (9/24) progressed to motor limbic seizures evolving to status epilepticus, fol-lowing the administration of pilocarpine. The frequency of seizures during the chronic period,in epileptic rats that received 5,7-DHT, showed a significant (58%) increase after the treatment,when compared with control group. Our data showed that serotonin may play an important role

on seizure activity which seems to be exerted by its inhibitory action on the expression of overtbehavior seizures departing from© 2008 Elsevier B.V. All rights re

∗ Corresponding author at: Departamento de Neurologia e Neu-rocirurgia, UNIFESP, Rua Botucatu 862, Ed. Leal Prado, São Paulo,Brazil.

E-mail address: naffahmazz.nexp@epm.br(M.d.G. Naffah-Mazzacoratti).

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0920-1211/$ — see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.eplepsyres.2008.08.010

an established focus in the limbic system.served.

ntroduction

he relationship between epileptogenic underlying mecha-isms and the neurotransmitter systems is a two-way roadnd as such it has been investigated during the last decades.n one hand, this relation can be concentrated in veri-

ying the changes induced by the epileptic condition on

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Serotonin depletion and epilepsy

neurotransmitter synthesis, release and content, receptoractivity. On the other hand, the investigation can be aimedat verifying the effect of direct or indirect stimulation orinhibition of a given neurotransmitter system on the estab-lishment or spreading of an epileptic event. In both cases,simple or sophisticated methodologies have been used.In general terms, epileptic discharges and consequentlyepileptic seizures have been associated with an imbalancebetween excitation and inhibition in specific brain circuitries(McNamara, 1994; Schwartzkroin, 1994). This hypothesis hasbeen repeatedly tested in experiments where agonists orantagonists of specific receptor sites (mainly amino acidacidergics) were able to interfere with the occurrence ofan epileptic event (Masukawa et al., 1991).

The first evidence about the role of monoamines inepilepsy was reported by Chen et al. (1954), who reportedlowering of electroconvulsive threshold after monoaminedepletion by reserpine. These results were confirmed byother authors (Wenger et al., 1973). The participation ofmonoamines in epileptic activity seems to be related totheir ability to interfere with the modulation of the neuronalexcitability (Segal, 1975; Watanabe et al., 1998).

The inhibitory effects of noradrenaline were reported intemporal lobe epileptogenesis. When this monoamine lev-els are artificially augmented by blocking its uptake or byelectric stimulation of noradrenaline rich locus coeruleus,there is a significant attenuation of kindling process. On theother hand, depletion of noradrenergic system by injecting6-OHDA has facilitating effects on kindling (McIntyre et al.,1982; Corcoran, 1988). Dopamine (DA) agonist apomorphinehas anticonvulsant action against sound-induced convulsionsin mice (Anzelak et al., 1981) and against photically inducedseizures in baboons (Meldrum, 1991).

The involvement of serotonergic function in epilepsycould represent the new concept needed to solve theproblem of drug-resistance (Löscher and Leppik, 2002).Experimental approaches to investigate the role of 5-HT inseizures have been developed by increasing or decreasingbrain 5-HT content either pharmacologically or by lesioningthe raphe nuclei. The administration of drugs that depleteserotonin or destroy serotonergic terminals, such as 5,7-dihydroxytryptamine (5,7-DHT), has been found to facilitatethe convulsive response in several models of seizure induc-tion (McIntyre et al., 1982; Jobe et al., 1982; Cabral-Filhoet al., 1987), which have shown that serotonergic neuronsof the central nervous system (CNS) are etiologically impor-tant in seizure predisposition of genetically epilepsy-pronerats (GEPR). They have shown that GEPRs have widespreaddeficits in CNS serotonin concentration and turnover rates(Jobe et al., 1982; Jobe et al., 1986). In addition, Daileyet al. (1992) have shown that fluoxetine is an effectiveanticonvulsant drug in GEPR rats, increasing 5-HT levelsin the thalamus. In the pilocarpine model, our group hasshowed increased synthesis and increased utilization rateof hippocampal 5-HT during the first hours of status epilep-ticus (Cavalheiro et al., 1994). Radley and Jacobs (2003)using a 5-HT1A antagonist showed that the cell prolifera-

tion and survival in the dentate gyrus, induced by seizures,occur via 5-HT1A receptor dependent mechanism. How-ever, the blockade of this receptor does not prevent themossy fiber sprouting neither the occurrence of spontaneousseizures.

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Moving from animal to human studies, the latter havendicated that patients with status epilepticus (Verma et al.,984) and febrile convulsion (Giroud et al., 1990) have lowerebrospinal fluid (CSF) levels of 5-hydroxyndolacetic acid,he primary metabolite of serotonin. On the other hand,tudies developed in our laboratory (Naffah-Mazzacorattit al., 1996) have shown increased 5-HT level in spik-ng compared with non-spiking temporal cortex, obtainedrom patients with complex partial seizures, unresponsive tovailable anticonvulsants. In addition, Favale et al. (1995)howed that humans with complex partial seizure present aecrease in seizure frequency, induced by administration ofuoxetine, a 5-HT reuptake inhibitor, used to treat depres-ion. This data confirm previous report of Leader (1992), whohowed that fluoxetine enhances the anticonvulsant effectf phenytoin, cabamazepine and ameltolide. In recent work,obe and Browning (2005) showed that in therapeutics doseshe antidepressants elevate noradrenergic and serotonergicransmission decreasing seizures. However, larger doses canctivate other biological process that may be convulsant.

Systemic administration of pilocarpine, a potent cholin-rgic muscarinic agonist, in rats promotes sequentialehavioral and electrographic changes that can be dividednto three periods: (a) an acute period, which develops intoimbic status epilepticus lasting up to 12 h, (b) a latentsilent) period with a progressive normalization of EEG andehavior, lasting from 4 to 44 days (mean of 15 days), and (c)chronic period characterized by the occurrence of spon-

aneous and recurrent seizures (Turski et al., 1984; Leite etl., 1990).

Altogether, these data suggest that serotonin may playn important role in seizure development, maintenancend control. Accordingly, the present study was aimed attudying the role of serotonin depletion on the pilocarpineodel of epilepsy and two hypotheses were investigated.

irst, is the serotonergic innervations important to theevelopment of pilocarpine-induced status epilepticus? Howan 5,7-DHT modify the acute fase of this model? Second:nto the chronic phase, when the epileptic focus has beenstablished, how can serotonergic denervation modify therequency of seizures? To analyze both paradigms experi-ent 1 and 2 were performed as below described.

aterials and methods

hemicals and drugs

nalytical grade chemicals were used in sample and mobile-phasereparations. Perchloric acid (HClO4), Na2S2O2, EDTA-disodiumalt (titriplex III), citric acid, orthophosphoric acid, hydrochlo-ic acid, sodium chloride, methanol (Lichorov), Na2HPO4, and-mercaptoethanol were purchased from Merck (Darmstadt). Nore-inephrine, dihydroxybenzilamine (DHBA), serotonin (5-HT), pilo-arpine hydrochloride, and scopolamine methylnitrate were pur-hased from Sigma (St. Louis, MO, USA). 5,7-Dihydroxytryptamineas purchased from R. Biochemical International.

nimals

or at least 1 week before the experiments, adult male Wistar rats,andomly selected from the same pool, weighting 220—280 g wereoused in groups of five on a standard light/dark cycle of 12 h (night

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t 7:00 P.M.). Room temperature and humidity were controlledetween 20 and 24 ◦C and 45—55%, respectively. Rat Chow pelletnd water was given ad libitum. The experiments were performedith institutional approval of ethical protocols and all efforts wereade to minimize animal suffering. Moreover, assisted feeding and

ydration were carried out during the initial recovery of the acuteeriod (status epilepticus) to improve the animals’ general physicaltate and survival rate.

rocedure

xperiment 1he aim of this experiment was to test the effect of serotonin deple-ion on the development of pilocarpine-induced status epilepticusacute period of the model). Accordingly, rats were stereotaxi-ally injected into the median raphe nucleus with 0.2 �l of 5,7-DHT0.4 mg ml−1, 5,7-DHT group, n = 24) or control (control group,= 24) solutions. In both cases the injections were performed FORmin at a speed of 0.1 �l/min between 8 and 10 h A.M. One weekfter surgery, all rats received a systemic injection of pilocarpine320 mg kg−1, i.p.). Thirty minutes before pilocarpine, the animalsere pretreated with scopolamine methylnitrate (1 mg kg−1, s.c.)

o limit peripheral cholinergic effects. Following pilocarpine treat-ent the rats were continuously observed for the next 72 h to

nalyze the main characteristics of the acute period of this epilepsyodel.

xperiment 2his experiment was designed to study the effect of serotoninepletion on the frequency and main behavioral characteristics ofhe spontaneous recurrent seizures that occurs during the chroniceriod of the pilocarpine model. Accordingly, 27 rats receivedilocarpine (320 mg kg−1, i.p., plus scopolamine methylnitrate asescribed above and were video-monitored 24 h/day. After theppearance of the first spontaneous seizure (chronic period) (meanf 15 days), 16 rats were stereotaxically injected with 5,7-DHT0.2 �g/�l). The other eleven epileptic rats received a control solu-ion, in the same nucleus. To avoid inflammatory interference oneizure frequency, after a recovery period of 2 weeks, rats wereideo-monitored for an additional period of 3 weeks for detectionf changes in the frequency of spontaneous seizures. In this proce-ure, the seizure frequency of each animal was compared beforend after 5,7-DHT treatment.

urgery procedure

ll rats were anaesthetized with a chloral hydrate/pentobarbitalixture and placed in a stereotaxic frame. A 30-gauge needle was

owered into the median raphe nucleus (AP: −1.6 mm, L: 0.0 mm,

: −2.4 mm, according to the Paxinos and Watson atlas (Paxinos andatson, 1982), and a 0.2 �l of a solution containing 0.4 �g 5,7-DHTissolved in 1% ascorbic acid or the control solution (0.2 ul of 1%scorbic acid) was infused over 2 min at a speed of 0.1 �l/min, evert 8—10 h A.M.

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Table 1 Incidence of pilocarpine-induced status epilepticus (SE)(5,7-DHT) or control solutions into the median raphe nucleus

Group Status epilepticus

Yes No

Control 9 155,7-DHT 15 6

*p < 0.0.5 (�2 test).

E.M. Trindade-Filho et al.

eurotransmitter analysis

n order to control the effect of 5,7-DHT injection into the medianaphe nucleus on hippocampal serotonin content, a different groupf animals (n = 10) were sacrificed at the end of each experimentnd both hipocampi were removed, placed on an ice-chilled plate,eighted, and stored at −80 ◦C until a biochemical assay was per-

ormed. Tissues were ultrasonically homogenized in a 0.1 M solutionf HClO4 containing 0.02% Na2S2O2 and DHBA (as internal standard)t a proportion of 15 �g solution for each milligram of tissue. Theamples were then centrifuged at 11,000 × g at 4 ◦C for 40 min. Theupernatant was filtered and injected into a high-peformance liquidhromatography system (HPLC) and serotonin, noradrenaline andopamine were quantified as previously described by Cavalheiro etl. (1994).

Neurochemical determinations were not performed on the hip-ocampus of animals used in experiments 1 and 2, since it wasreviously demonstrated by Cavalheiro et al. (1994) that importanthanges in monoamine content occur in the hippocampus of rats,uring the acute and chronic periods of the pilocarpine model ofpilepsy.

ata analysis

tatistical differences in hippocampal serotonin levels were evalu-ted using the Student’s t-test. Comparisons of seizure frequencyetween groups were made using the Mann—Witney test and theffects of 5,7-DHT on the acute effects of the pilocarpine were ana-yzed using the chi-square test. A value of p ≤ 0.05 was accepted asignificant.

esults

xperiment 1

mmediately after pilocarpine injection, both 5,7-DHT andontrol solution pretreated rats displayed several behavioralhanges that included an initial period of akinesia followedy ataxic lurching, tremor and masticatory automatism thatersisted for 10—15 min. The latency of these behavioralhanges was similar for both groups. Sixty three percent of,7-DHT pre-treated rats (15/24) and only 33.4% of thoseeceiving the control solution (9/24) progressed to motorimbic seizures evolving to status epilepticus. This condition

ressive behavior normalization during the remaining hoursf observation. Three of the 5,7-DHT pre-treated rats exhib-ted tonic seizures followed by death during the SE periodTable 1). Others, from 5,7-DHT group, died during the silenteriod.

in rats pretreated with injections of 5,7-dihydroxytryptamine

Death Total % Status epilepticus

0 24 333 24 63*

Serotonin depletion and epilepsy

Figure 1 Representation of the number of seizure in thechronic period/3 weeks. A: chronic animals prior to vehicle

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treatment, B: chronic animals after treatment with vehicle solu-tion; C: chronic animals prior to 5,7-DHT treatment; D: chronicanimals after treatment with 5,7-DHT (p < 0.01).

Experiment 2

The development of seizure expression or other behavioralabnormalities before 5,7-DHT or control solution adminis-tration into the median raphe nucleus, followed the patternof serial events previously described for the pilocarpinemodel of epilepsy (for details see Leite et al., 1990).The frequency of spontaneous seizure, analyzed during the3-week pretreatment period showed 3—4 seizures/weekper animal (9.59 ± 3.37 seizures/3 weeks). This frequencyremained unchanged throughout the period of observa-tion for rats that received an injection of control solutioninto the median raphe nucleus (10.63 ± 2.77 seizures/3weeks). On the other hand, epileptic rats that received5,7-DHT in the same structure had a significant increasein their seizure frequency showing 10.5 ± 6.71seizures/3weeks before and 18.25 ± 8.61seizures/3 weeks after 5,7-DHT treatment (p = 0.01). In contrast, behavioral aspects,severity and duration of each individual seizure were notmodified by 5,7-DHT treatment (Fig. 1).

Neurochemical determination

The 5,7-DHT treatment markedly decreased the sero-tonin content in the hippocampal formation to 66.7%(p < 0.05), when compared to control levels. In addition,

the noradrenaline and dopamine concentrations remainedunchanged. These data show that 5,7-DHT, in the concentra-tion employed, was able to reduce the 5-HT concentrationin the hippocampus (Fig. 2).

Figure 2 Hippocampal concentration of 5-HT in ng/mg of wettissue, measured by HPLC method. A: rats vehicle-treated; B:rats 5,7-DHT-treated (p < 0.05).

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iscussion

his study evaluated the effects of 5,7-DHT, a drug thatecreases the content of the neurotransmitter serotonin,n the brain of rats during the acute and chronic periods ofhe pilocarpine model of epilepsy. Pretreatment with 5, 7HT facilitated the induction of status epilepticus inducedy pilocarpine, observed during the acute period of thisxperimental model. As reported by Freitas et al. (2005)he pilocarpine-induced status epilepticus was not able tonduce changes in 5-HT2 receptors density in the hippocam-us, but can increase the Kd values, suggesting that theffinity of 5-HT for 5-HT2 receptor decreases in this tissue.ccording to O’Dell et al. (2000) the 5-HT2 receptor has con-ulsant activity, supporting the idea that during the statuspilepticus a decrease in the binding could protect limbicreas.

When 5,7-DHT was administered to animals in the chronichase, a marked increase in the frequency of spontaneouseizures was also observed. Taken together, these resultsuggest that normal serotonin levels could have an inhibitoryction on seizure activity.

According to Löscher (1999), several drugs, which areble to improve acute epilepsy are less effective on chronicodels. In addition, chronic models are more predictive

f clinical efficacy than acute models. The involvement oferotonergic function in epilepsy pathogenesis could rep-esent the new concept needed to solve the problem ofrug-resistance (Löscher, 2002). In this context, the resultseported here are important since serotonin depletion wors-ned both acute and chronic seizures, confirming the role of-HT in the pathogenesis of epilepsy.

Our results are in accordance with those previ-usly reported for audiogenic seizures (Jobe et al.,986), pentylenetetrazol-induced seizures (Alexander andopeloff, 1970), and electroconvulsive seizures (Kiilliannd Frey, 1973). Dailey et al. (1996) showed that fluox-tine and sertraline, serotonin reuptake inhibitors, haventiconvulsant activity in GEPRs. In addition, Wada et al.1992) showed that 5-HTP (the serotonin precursor) has aotent antiepileptic action against hippocampal and lat-ral geniculate nucleus-kindled seizures, suggesting that theerotonergic system is involved in the suppression of epilep-ic activity in these brain regions.

Ojemann et al. (1983) showed that drugs that blockerotonin reuptake are anticonvulsant in man. In addition,ecent works have shown that anticonvulsant drugs modify-HT levels (Ahmad et al., 2005; Bagdy et al., 2007; Favalet al., 2003). In pilocarpine model of temporal lobe epilepsyernandez et al. (2002) showed that fluoxetine is able toeduces spontaneous seizures.

Naffah-Mazzacoratti et al. (1996) observed an increasen the 5-HT and 5-HIAA content of spiking tissue obtainedrom patients with complex partial seizures unresponsiveo available anticonvulsants. These authors hypothesizedhat these changes could be a compensatory effect, tryingo block the tissue hyperexcitability. In addition, patients

ith myoclonus have low cerebrospinal levels of 5-HIAA,

uggesting that some forms of myoclonus might be relatedo a deficiency of brain serotonin (Pranzatelli et al., 1995).ther studies have also demonstrated that 5-HTP was effec-ive in decreasing the severity of myoclonic seizures in

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everal patients (Van Woert et al., 1977; Thal et al.,980).

The serotonergic innervations of the hippocampus ariserom 5-HT neurons of the median and dorsal raphe locatedn the midbrain (Conrad et al., 1974). Early tracing studiesy anterograde or retrograde axonal transport have demon-trated that these 5-HT afferents arise mostly from theidbrain median raphe nucleus and to a lesser extent from

he central part of raphe nucleus (Azmitia and Segal, 1978).iscreet raphe lesions have indicated that the destruction ofhe median raphe produces a greater decrease in hippocam-al serotonin content than the destruction of the dorsalaphe (Moore and Halaris, 1975).

Our results also showed that 5,7-DHT could be used as anffective tool to decrease the hippocampal serotonin con-ent. After 5,7-HDT administration into the median rapheucleus a decreased level of 5-HT could be observed in theippocampus. However, no alterations in noradrenaline andopamine levels were detected, showing specificity in thereatment employed, although the decrease in serotoninontent in the present experiment was more pronouncedhan that observed by Moore and Halaris (1975).

Early electrophysiological studies have demonstratedn inhibitory effect of 5-HT on hippocampal neuronsOleskevich et al., 1991; Blier et al., 1993; Mongeau et al.,997). The median raphe nucleus projects to the stratumadiatum of CA3, to the polymorphic layer of the den-ate gyrus and to the region between the stratum radiatumnd stratum lacunosum-moleculare of CA1. 5-HT appliedo hippocampal neurons increases potassium conductanceesulting in hyperpolarization and hypoexcitability. Thisnhibition is mimicked by selective 5-HT1A agonist 8-OH-DPATnd is blocked by the selective 5-HT1A antagonist, NAN-190Watanabe et al., 1998; Salgado-Comissuriat and Alkadhi,998).

Increased serotonin content could also be a mechanismy which some antiepileptic drugs exert their actions. Sev-ral antiepileptic drugs such as carbamazepine, zonizamide,amotrigine, loreclezole and valproate, citalopram haveeen shown to increase the release or extracellular con-entration of serotonin as part of their pharmacodynamicction on neuronal tissue. It has been suggested that thisffect contributes to the therapeutic actions of these drugsDailey et al., 1996; Favale et al., 2003).

The hippocampus is one of the main structures related tohe development of the pilocarpine model of epilepsy andhe reduced 5-HT levels in this region may be very impor-ant for seizure control. Nevertheless, we cannot excludehe possible role of 5,7-DHT in decreasing 5-HT levels inther regions, which could also be important for seizureeverity. In summary, our data support the hypothesis of anmportant role of the serotonergic system on epileptic activ-ty, reinforcing previous reports of Hernandez et al. (2002),hich seems to be exerted by its inhibitory action on thexpression of overt behavioral seizures departing from anstablished limbic focus.

cknowledgments

his work was supported by Fundacão de Amparo a Pesquisao Estado de São Paulo (FAPESP), Conselho Nacional deesquisa (CNPq), Programa de Apoio a Núcleo de Excelên-

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ia (PRONEX), Coordenacão de Pessoal de Nível SuperiorCAPES).

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