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Research Report

Anticonvulsant effects of melatonin onpenicillin-induced epileptiform activity in rats

Mehmet Yildirim⁎, Cafer MarangozDepartment of Physiology, Faculty of Medicine, University of Ondokuz Mayis, 55139 Samsun, Turkey

A R T I C L E I N F O

⁎ Corresponding author. Fax: +90 362 457 604E-mail address: mehmetyd@omu.edu.tr (

0006-8993/$ – see front matter © 2006 Elsevidoi:10.1016/j.brainres.2006.04.093

A B S T R A C T

Article history:Accepted 27 April 2006Available online 9 June 2006

In the present study, we investigated the effects of melatonin on penicillin-inducedepileptiform activity in female Wistar rats. The left cerebral cortex was exposed bycraniotomy under urethane anesthesia for the induction of epilepsy by intracorticalmicroinjection of penicillin (200 IU) into the left sensorimotor cortex. The epileptiformactivity was analyzed by electrocorticogram (ECoG). Ten minutes before the penicillininjection, 20, 40 or 80 μg of melatonin was administered intracerebroventricularly and ECoGwas monitored for 1 h. Forty or 80 μg of melatonin significantly increased the latency toepileptiform activity. Furthermore,melatonin significantly decreased the frequency of spikeand spike-wave activity, whereas the amplitude of spikes remained unchanged. Inconclusion, data obtained from the present study suggest that melatonin suppressespenicillin-induced epileptiform activity, and it may be an endogenous anticonvulsant.

© 2006 Elsevier B.V. All rights reserved.

Keywords:MelatoninEpilepsyECoGPenicillinRat

Abbreviations:aCSF, artificial cerebrospinal fluidECoG, electrocorticogramEEG, electroencephalogramGABA, γ-aminobutyric acidNMDA, N-methyl-D-aspartatePTZ, pentylenetetrazol

1. Introduction

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized bythe pineal gland from the amino acid precursor L-tryptophanin all vertebrate species including humans (Lerner et al.,1959). It is secreted into the cerebrospinal fluid andcirculatory system in a circadian pattern (Lynch et al.,1975). Melatonin levels are low during the day and maximalduring the hours of darkness (Snyder et al., 1967). Melatoninplays a role in a variety of physiological and pathophysio-logical processes in the brain including regulation ofbiological rhythms and seasonal reproduction (Arendt,

1.M. Yildirim).

er B.V. All rights reserved

1998), scavenging of free radicals (Ianas et al., 1991) andneuroprotection (Lipartiti et al., 1996; Kilic et al., 2002; Kilicet al., 2005). Melatonin also participates in other physiologicalprocesses including immunomodulation (Guerrero and Reiter,2002).

Epilepsy is a common neurological disorders, affectingalmost 1% of the population (Elger, 2002). Animal modelsfor seizures and epilepsy have played a fundamental role inour understanding of the basic physiological and behavioralchanges associated with human epilepsy (Marangoz et al.,1994; Sarkisian, 2001; Ayyildiz et al., 2006). There are anumber of clinical and experimental studies indicating an

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association between melatonin and epilepsy (Reiter andMorgan, 1972; Reiter et al., 1973; Sandyk et al., 1992;Mevissen and Ebert, 1998; Costa-Lotufo et al., 2002; Stewartand Leung, 2005). However, the role of melatonin in seizuredisorders is controversial. Although most of these studiessuggest that melatonin acts as an endogenous anticonvul-sant (Reiter and Morgan, 1972; Reiter et al., 1973; Mevissenand Ebert, 1998; Costa-Lotufo et al., 2002), others reportedthat this indole may be proconvulsant (Sandyk et al., 1992;Stewart and Leung, 2005). Furthermore, some authorsdemonstrated that pinealectomy leads to seizure activityand facilitates epileptogenesis in some species of rodents;however, these seizures were not reversed by pretreatmentwith melatonin (Rudeen et al., 1980; de Lima et al., 2005).To our knowledge, there is no study related to the effectsof melatonin on the penicillin model of experimentalepilepsy. The aim of this work was to investigate theeffects of melatonin on penicillin-induced epileptiformactivity in Wistar rats.

Fig. 1 – Representative ECoGs at 20–28 min after penicillin admiinjection of other substances. (B) Intracortical injection of penicilAdministration of 1% ethanol did not change the frequency of pewas decreased pretreated 20, 40 or 80 μg melatonin 10 min befo

2. Results

Baseline activities of each animal were recorded before theadministration of melatonin, and it has been confirmed thatnone of the animals had spontaneous epilepsy (Fig. 1).Intracortical injection of penicillin (200 IU) induced epilepti-form activity in the all experimental animals (Fig. 1). However,onset of these activities was delayed in the melatonin-treatedrats (Fig. 2). The mean spike latencies were determinated178 ± 16, 180 ± 29, 293 ± 21, 455 ± 50 and 407 ± 45 s in the rats ofcontrol, ethanol, 20, 40 and 80 μg melatonin groups, respec-tively. The mean spike latencies were the some in the controland ethanol-treated rats, whereas 40 and 80 μg doses ofmelatonin caused significantly increases in the mean spikelatencies compared to the control animals (P < 0.001).

There were no significant differences between the meanspike frequency of control and ethanol rats. However, themean spike frequencies after melatonin administration were

nistration. (A) Baseline ECoG activity before penicillin or thelin (200 IU) induced epileptiform activity on ECoG. (C)nicillin-induced epileptic activity. (D–F) Epileptiform activityre penicillin administration.

Fig. 2 – The effects of melatonin on the latency ofepileptiform activity in rats. The doses of 40 or 80 μgmelatonin significantly increased the latency to epileptiformactivity whereas the low dose of melatonin (20 μg) did notsignificantly change the latency. Ethanol (1%) administrationdid not significantly change the latency to epileptiformactivity compared to control rats. ★★★P < 0.001.

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significantly decreased dose dependently compared with thecontrol animals (Figs. 3A and B). The mean spike frequencieswere 30 ± 2, 28 ± 2, 22 ± 1, 20 ± 2 and 17 ± 1 spike/min at 25 minafter penicillin injection in the control, ethanol, 20, 40 and80 μg melatonin groups, respectively.

The mean spike amplitudes were 1967 ± 103, 1862 ± 131,1882 ± 140, 1920 ± 98 and 1890 ± 125 μV at 25 min afterpenicillin injection in the control, ethanol, 20, 40 and 80 μgmelatonin groups, respectively. There was no significantdifference in the mean spike amplitude between any exper-iment groups.

Intracortical injections of aCSF or 20, 40 and 80 μg doses ofmelatonin did not cause any change of the frequency oramplitude of ECoG activity in non-penicillin-injected animals.

3. Discussion

The results of the present study show thatmelatonin caused adecrease in the spike frequency and an increase in the latencyof the onset of epileptiform activity induced by penicillin. Themean spike frequency was significantly decreased after themelatonin administration compared with controls. On theother hand, melatonin did not effect the amplitude ofepileptiform activity.

The existence of a relationship between melatonin andseizures has been reported. Various studies suggest thatmelatonin is an anticonvulsant substance (Reiter et al., 1973;Mevissen and Ebert, 1998; Costa-Lotufo et al., 2002). It has beenreported that melatonin, at doses of 75 mg/kg or higher,reduced both the susceptibility to seizures and the spread ofseizure activity in the amygdala kindling model of epilepsy inthe rat (Mevissen and Ebert, 1998). In another study, it wasshown that melatonin produced a weak anticonvulsantactivity on pilocarpine-induced seizures, as exhibited by anincrease in latency of the appearance of the first seizure

(Costa-Lotufo et al., 2002). In the study of Bikjdaouene et al.(2003), melatonin (80 mg/kg) given before pentylenetetrazol(PTZ) administration (100 mg/kg, s.c.) significantly increasedthe latency and decreased the duration of the first seizure, andreduced PTZ induced mortality from 87.5% to 25% in rats.Additionally, the anticonvulsant effect of melatonin has beendemonstrated in the different studies of experimental epilep-sy induced by pentylenetetrazol, quinolinate, kainate, gluta-mate, N-methyl-D-aspartate (NMDA), maximal electricalshock and electrically kindled of the amygdala (Albertson etal., 1981; Lapin et al., 1998; Borowicz et al., 1999). The findingthat the exogenousmelatonin is able to suppress epileptiformactivity in penicillin-induced model is consistent with above-mentioned studies.

Although many reports have indicated in anticonvulsanteffect for melatonin, this was not found in all studies. Forexample, there are reports that melatonin may decreaseseizure thresholds and exert proconvulsive activity in humans(Sandyk et al., 1992) and in animals (Stewart and Leung, 2005).It was also reported that blockade of hippocampal Mel1breceptors significantly delays the onset of pilocarpine-inducedseizures and suppresses hippocampal electroencephalogram(EEG) amplitude during the dark phase in Wistar rats (Stewartand Leung, 2005). These workers stated that nocturnalactivation of hippocampal Mel1b receptors depresses GABAA

receptor function in the hippocampus and enhances seizuresusceptibility (Stewart and Leung, 2005). The discrepancybetween this and our findings may be due to differentexperimental conditions.

The idea of involvement of melatonin in seizure activity issupported by studies in pinealectomized animals. It has beenreported that pinealectomy leads to seizure activity in gerbils,which is reversed by treatment with melatonin (Rudeen et al.,1980). In addition, recent studies indicate that the pinealecto-my facilitates the epileptogenic process in the development ofthe epilepsy model induced by pilocarpine and exerts asignificant influence on the amygdala kindling developmentin rats (de Lima et al., 2005; Janjoppi et al., 2006). Clinicalstudies have also indicated a beneficial effect of melatonin onthe seizure activity (Molina-Carballo et al., 1997; Fauteck et al.,1999).

In the present study, we used penicillin to produceepileptiform activity. Penicillin acts as a non-competitiveantagonist of GABAA receptors, binding to the picrotoxin siteat the GABAA receptor complex (Curtis et al., 1972; Macdonaldand Barker, 1977). The mechanisms of anticonvulsant actionsof melatonin are not fully understood. It has been reportedthat the melatonin regulates the GABA–benzodiazapinereceptor complex in rat brain and increases GABA levels(Castroviejo et al., 1986; Rosenstein and Cardinali, 1990). Thismay explain the anticonvulsant effects of melatonin inpenicillin-induced epilepsy.

In conclusion, data obtained from the present studysuggest thatmelatoninmay be an endogenous anticonvulsantsubstance, at least under these experimental conditions, andalso suggest that further investigations are needed to answerthe fundamental role of melatonin in epilepsy. The presentresults indicate that melatonin decreased epileptiform activ-ity at penicillin-induced epilepsy in rats. However, all doses ofmelatonin did not completely prevented epileptic activity. It is

Fig. 3 – (A, B) The effects of melatonin on the mean spike frequency. There were no significant differences between themean spike frequency of control and ethanol-treated rats. The mean spike frequency was significantly decreaseddose-dependently in themelatonin-treated rats comparedwith the control animals. The significant effects appeared between 5and 40 min after penicillin injection. ★P < 0.05; ★★P < 0.01; ★★★P < 0.001.

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perhaps that melatonin could be used as a co-agent withantiepileptic drugs to suppress epileptiform activity inhumans (Molina-Carballo et al., 1997).

4. Experimental procedures

4.1. Animals

Experiments were performed on 63 adult female Wistar ratsweighing 220–280 g. Animals were housed under a 12:12-hlight–dark cycle (light on 7:00 h) and a room temperature of21 ± 2°C. All experiments were performed between 10:00 and16:00 h. They were given free access to food and water. Everyeffort was made to minimize animal suffering and thenumber of animals used. When artificial cerebrospinal fluid(aCSF) was used, it contained the following: NaCl, 124; KCl, 5;KH2PO4, 1.2; CaCl2, 2.4; MgSO4, 1.3; NaHCO3, 26; glucose, 10;HEPES, 10; pH 7.4 when saturated with 95% O2 and 5% CO2. Allexperiments were carried out according to local guidelines forthe care and use of laboratory animals and the guidelines of

the European Community Council for experimental animalcare.

4.2. Chemicals and experiment groups

Melatonin and urethane were purchased from Sigma (St.Louis, MO, USA). Melatonin was dissolved in 1% ethanol andinjected intracerebroventricularly (i.c.v.) 10 min before peni-cillin administration. Sixty-three animals were equally divid-ed into nine experimental groups and chemicals wereadministrated as follows: control animals (1) received: aCSF(4 μl, i.c.v.) and penicillin (200 IU/i.c.); (2) ethanol group: 1%ethanol (4 μl, i.c.v.) and penicillin (200 IU/i.c.); (3) 20 μgmelatonin group: melatonin (20 μg/4 μl, i.c.v.) and penicillin(200 IU/i.c.); (4) 40 μg melatonin group: melatonin (40 μg/4 μl,i.c.v.) and penicillin (200 IU/i.c.); (5) 80 μg melatonin group:melatonin (80 μg/4 μl, i.c.v.) and penicillin (200 IU/i.c.); (6) aCSFgroup: aCSF (4 μl, i.c.v.) alone; (7) 20 μg melatonin group(alone): melatonin (20 μg/4 μl, i.c.v.); (8) 40 μg melatonin group(alone): melatonin (40 μg/4 μl, i.c.v.); and (9) 80 μg melatoningroup (alone): melatonin (80 μg/4 μl, i.c.v.).

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4.3. Surgical procedure and induction of epileptiformactivity

Animals were anesthetized with 1.25 mg/kg urethane intra-peritoneally (i.p.) and additional doses were given as needed.The left cerebral cortex was carefully exposed by craniotomy.After incision of the skull, the head of the animalwas placed ina stereotaxic apparatus (Harvard Instruments, South Natick,MA, USA). Four different corners of the scalp were eachstitched by surgical threads and stretched in order to form aliquid Vaseline pool (37°C). Body temperature was monitoredusing a rectal probe and maintained at 37°C with a homeo-thermic blanket system (Harvard Homoeothermic Blanket,USA). A polyethylene cannula was placed into the right femo-ral artery to monitor blood pressure, which was kept above100 mmHg during the experiments (mean 116 ± 9 mmHg). Allcontact and incision points were infiltrated with procainehydrochloride to minimize possible sources of pain.

The epileptic activity was produced by intracortical injec-tion of 200 IU penicillin G (Marangoz et al., 1994; Ayyildiz et al.,2006). The penicillin (1 μl) was injected into the left sensori-motor cortex, 1 mm beneath the brain surface using aHamilton microsyringe (type 701N, Hamilton Co., Reno, NV,USA). The coordinates used for i.c. injection, with the bregmapoint as the reference, were AP = −2 mm, L = 3 mm.

Melatonin, 1% ethanol and aCSF were injected in the leftcerebral lateral ventricle. Ten minutes before penicillininjection, the microsyringe was inserted 2.5 mm beneath thebrain surface and 4 μl of melatonin, 1% ethanol or aCSF wereinjected stereotactically into the left lateral ventricle. Thecoordinates used for lateral ventricle, with the bregma point asthe reference, were AP = −0.8 mm, L = 1.5 mm. The injectionrate was 4 μl/min and the needle was left in place for 2 minfollowing the infusion.

4.4. Electrophysiological recordings

Two Ag-AgCl ball electrodes were placed over the leftsomatomotor cortex and the common reference electrodebeing fixed on the right pinna (electrode coordinates: firstelectrode, 2 mm lateral to sagittal suture and 1mm anterior tobregma; second electrode, 2 mm lateral to sagittal suture5 mm posterior to bregma). The recordings of electrocortico-gram (ECoG) were carried out using a data acquisition system(PowerLab 4/SP, AD Instruments, Australia). The signals fromthe electrodes were amplified and filtered (0.1–0.50 Hz band-pass) using BioAmp amplifiers (AD Instruments, Australia).After ECoG signal was digitized at a sampling rate of 1024 withthe PowerLab 4/SP. The digitized brain signal was displayedand stored on a personal computer (PC). The frequency andamplitude of epileptic activity was analyzed off line.

4.5. Statistical analyses

Spike frequencies and amplitudes for each animal wereautomatically calculated and measured using the Chartv.5.1.1 (PowerLab software). The latency of epileptiformactivity determined latency for the first spike to appear onthe ECoG. Statistical analyses were carried out by one-wayanalysis of variance (ANOVA), followed by Bonferroni post hoc

test to correct for multiple comparisons of treatments. Dataare expressed as the mean ± SEM. The significance level wasP < 0.05.

Acknowledgments

The authors would like to thank Dr. Haluk Kelestimur forproviding melatonin and Dr. Handan Çamdeviren for thestatistical analysis.

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