Enhanced susceptibility to the GABA antagonist pentylenetetrazole during the latent period following a pilocarpine-induced status epilepticus in rats

Neuropharmacology 60 (2011) 505e512

Contents lists available at ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Enhanced susceptibility to the GABA antagonist pentylenetetrazole during the latent period following a pilocarpine-induced status epilepticus in rats Marta Rattka a, b, Claudia Brandt a, b, Marion Bankstahl a, b, Sonja Bröer a, b, Wolfgang Löscher a, b, * a b

Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany Center for Systems Neuroscience, Hannover, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 October 2010 Received in revised form 1 November 2010 Accepted 2 November 2010

A variety of acute brain insults bear the risk of subsequent development of chronic epilepsy. Enhanced understanding of the brain alterations underlying this process may ultimately lead to interventions that prevent, interrupt or reverse epileptogenesis in people at risk. Various interventions have been evaluated in rat models of symptomatic epilepsy, in which epileptogenesis was induced by status epilepticus (SE) or traumatic brain injury (TBI). Paradoxically, recent data indicated that administration of proconvulsant drugs after TBI or SE exerts antiepileptogenic or disease-modifying effects, although epilepsy is often considered to represent a decrease in seizure threshold. Surprisingly, to our knowledge, it is not known whether alterations in seizure threshold occur during the latent period following SE. This prompted us to study seizure threshold during and after the latent period following SE induced by lithium/pilocarpine in rats. Timed intravenous infusion of the GABAA receptor antagonist pentylenetetrazole (PTZ) was used for this purpose. The duration of the latent period was determined by continuous video/EEG monitoring. Compared to control seizure threshold determined before SE, threshold significantly decreased two days after SE, but returned to pre-SE control thereafter. Moreover, the duration of PTZ-induced seizures was significantly increased throughout the latent period, which ranged from 6 to 10 days after SE. This increased susceptibility to PTZ likely reflects the complex alterations in GABA-mediated transmission that occur during the latent period following SE. The data will allow developing dosing regimens for evaluation of whether treatment with subconvulsant doses of PTZ during the latent period affects the development of epilepsy. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Epilepsy Epileptogenesis Seizure threshold Neuronal hyperexcitability Antiepileptogenesis Disease modification

1. Introduction Epilepsy, one of the most common neurological disorders, often develops after brain insults, such as traumatic brain injury (TBI), brain tumors, CNS infections, and stroke (Dichter, 2009; Jacobs et al., 2009). Typically, the primary brain insult is associated with acute symptomatic seizures, which are followed by a latent (“silent”) period of months to years before onset of spontaneous recurrent seizures that characterize the onset of epilepsy (Walker et al., 2002; Jacobs et al., 2009). The process by which a brain insult induces epilepsy is termed epileptogenesis. It is characterized by numerous molecular and functional alterations, which take

Abbreviations: EEG, electroencephalogram; PTZ, pentylenetetrazole; SE, status epilepticus; TBI, traumatic brain injury. * Corresponding author. Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Bünteweg 17, D-30559 Hannover, Germany. Tel.: þ49 511 856 8721; fax: þ49 511 953 8581. E-mail address: [email protected] (W. Löscher). 0028-3908/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2010.11.005

place during the latent period and ultimately lead to epilepsy (Jacobs et al., 2009; Pitkänen and Lukasiuk, 2009). Although the molecular steps leading to epileptogenesis are not fully understood, one hallmark of epileptogenesis is the development of neuronal hyperexcitability (Pitkänen et al., 2007; Jacobs et al., 2009; Löscher and Brandt, 2010). In rat models of traumatic brain injury, this neuronal hyperexcitability is associated with decreased seizure threshold (Golarai et al., 2001; Zanier et al., 2003; Statler et al., 2008; Echegoyen et al., 2009; Pitkänen et al., 2009), which would be consistent with the old view that epilepsy represents a decrease in seizure threshold (Dudek, 2009). However, based on experiments with the cannabinoid type 1 (CB1) receptor antagonist rimonabant (SR141716A), Armstrong et al. (2009) recently proposed that administration of a proconvulsant drug after an epileptogenic brain insult may provide a prophylaxis for epileptogenesis. Evidence for this apparently paradoxical approach does not only arise from experiments with rimonabant (Chen et al., 2007; Echegoyen et al., 2009) but also from previous studies with the adenosine antagonist caffeine and the a2-receptor antagonist

506

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512

atipamezole, which are acutely proconvulsant but exert diseasemodifying effects in rat models in which epileptogenesis is induced by status epilepticus (SE; Rigoulot et al., 2003; Pitkänen et al., 2004). Induction of SE by electrical stimulation or by convulsants such as pilocarpine and kainate in rats provides widely used models of temporal lobe epilepsy, in which spontaneous recurrent seizures develop after a latent period of 1e4 weeks (Pitkänen et al., 2007). To our knowledge, it is not known whether alterations in seizure threshold occur during the latent period following SE. In the present study, we used timed intravenous (i.v.) pentylenetetrazole (PTZ) infusion to repeatedly determine seizure threshold in individual rats following a pilocarpine-induced SE. PTZ acts predominantly by antagonizing GABAergic inhibition via an effect at the picrotoxin site of the chloride ionophore of the GABAA receptor (Macdonald and Barker, 1977; Pacheco et al., 1981; Ramanjaneyulu and Ticku, 1984). During SE-induced epileptogenesis, complex alterations in GABAergic transmission have been described, including a shift from hyperpolarizing, inhibitory to depolarizing and even excitatory GABA actions that is thought to contribute to the development of neuronal hyperexcitability in the hippocampus (Köhling, 2002; Payne et al., 2003; Blaesse et al., 2009), but also enhanced GABAergic inhibition that may increase network synchronization and thus contribute to epileptogenesis (Khazipov and Holmes, 2003; Cossart et al., 2005; Mann and Mody, 2008), so that the overall consequence for systemically administered GABA antagonists such as PTZ is difficult to anticipate. The present data show that the susceptibility to the convulsant effects of PTZ is increased during the latent period following pilocarpineinduced SE. 2. Materials and methods 2.1. Animals For the present experiments, female Sprague-Dawley rats (Harlan Laboratories, Horst, Netherlands), weighing 200e220 g, were used. Female rats were housed without males in order to keep them acyclic or asynchronous with respect to their estrous cycle. We have recently demonstrated that female Wistar rats do not cycle synchronously or do not cycle at all when housed in this way over several weeks in our laboratory (Kücker et al., 2010). This was confirmed by daily vaginal smears analysis in a group of 44 female Sprague-Dawley rats (Katrin Becker, unpublished data). One may argue that even an irregular estrous cycle exerts an influence on seizure susceptibility (Scharfman and MacLusky, 2006); however, Finn and Gee (1994) reported that the estrous cycle did not affect the PTZ seizure threshold, determined by timed i.v. PTZ infusion in Sprague-Dawley rats. To address the effect of gender on PTZ seizure threshold, the PTZ threshold was also determined in agematched male rats, which were housed in a separate room. Rats of both genders were housed under controlled conditions (ambient temperature 24e25  C, humidity 50e60%, lights on from 6:00 am to 6:00 pm). Animals were adapted to the laboratories for at least one week before being used in the experiments. Food (Altromin 1324 standard diet; Altromin, Lage, Germany) and water were freely available. All animal experiments were carried out in accordance with the European Communities Council Directive of 24. November 1986 (86/609/EEC) and were formally approved by the animal subjects review board of our institution. All efforts were made to minimize both the suffering and the number of animals. 2.2. Induction of status epilepticus by pilocarpine Before induction of SE by pilocarpine, rats received lithium chloride (127 mg/kg per os [to avoid abdominal pain associated with i.p. injection of lithium chloride in rats]; SigmaeAldrich; Steinheim, Germany) 14e16 h and methyl-scopolamine (1 mg/kg i.p.; SigmaeAldrich) 30 min before pilocarpine treatment. Following injection of pilocarpine, all rats were continuously observed for the occurrence of behavioral (limbic and generalized convulsive) seizures. In order to decrease mortality, individual dosing of pilocarpine was performed by ramping up the dose until onset of SE as described previously (Glien et al., 2001). For this purpose, pilocarpine (SigmaeAldrich) was administered i.p. at a dose of 10 mg/kg every 30 min until the onset of an SE, consisting of continuous limbic seizure activity (stereotyped oro-facial movements, salivation, eye-blinking, twitching of vibrissae, Straub tail, stiffened hindlimbs so that animals seem to walk on tiptoes, and reduced responsiveness), which was frequently interrupted by generalized convulsive

seizures. The total number of pilocarpine injections was limited to 5 injections per animal. In a total of 41 rats used for the present experiments with pilocarpine, SE could be induced by this protocol of pilocarpine administration in 23 rats (56%) and mortality was very low (only one of the rats died). The average dose of pilocarpine for inducing SE was 37 mg/kg (range 20e50 mg/kg). The time from the first injection of pilocarpine to onset of SE was 103 min (range 63e166 min). After 90 min of SE, diazepam (10 mg/kg i.p.; Ratiopharm; Ulm, Germany) was injected to terminate the behavioral signs of generalized convulsive SE and thereby reduce mortality. If convulsive activity was not terminated by the first injection, the injection of diazepam was repeated twice at an interval of 10 min. All rats were closely observed during the SE and for several hours after termination of SE for recurrence of seizures. In order to reduce mortality and facilitate recovery after SE, rats were injected once daily with 4 ml saline and fed with baby pep over 3 days on average following the SE. Seizures occurring in some rats (n ¼ 3) during the first 1e3 days of recovery from SE were considered insult-associated symptomatic seizures (Sirven, 2009) and were not further evaluated. About 2e3 weeks before the induction of SE, electrodes were implanted in part of the rats (n ¼ 25) into the right dentate gyrus of the hippocampal formation under anesthesia (chloral hydrate plus local anesthesia with tetracaine and bupivacaine) for recording of the electroencephalogram (EEG). Stereotaxic coordinates in mm relative to bregma according to the atlas of Paxinos and Watson (2007) were: AP 3.9, L 1.7, V 3.5. Furthermore, some rats received cortical screw electrodes in addition to the hippocampal electrode (AP 2.2, L 4.8). During postsurgical recovery, rats were treated for one week with marbofloxacine. Rats (n ¼ 12) that did not develop SE after pilocarpine were used as pilocarpine/ non-SE controls. Furthermore, a group of 19 rats that received all treatments except pilocarpine were used as sham-SE controls. Part of these sham-SE rats (n ¼ 9) were implanted with hippocampal and cortical EEG electrodes as described above for the SE rats. 2.3. Determination of latency to spontaneous seizures after SE Ten rats with EEG electrodes were continuously video/EEG monitored (24 h/day, 7 days/week) for development of spontaneous seizures after SE. Six of these rats were not used for PTZ seizure threshold determination during the latent period, whereas in the other 4 rats the PTZ threshold was determined 2, 6, and 13 days after SE (and 5 days before SE; see below). Continuous video/EEG monitoring was performed for 2e4 weeks after SE. For EEG monitoring, the system consisted of 16 one-channel amplifiers (BioAmp, Axon Instruments, Inc. Foster City, CA) and an analogueedigital converter (PowerLab/800s, ADInstruments Ltd, Hastings, East Sussex, UK). The data were recorded and analyzed with Chart4 or LabChart 6 for windows software (ADInstruments Ltd, Hastings, East Sussex, UK). The sampling rate for EEG recording was 200 Hz. A high pass filter for 0.1 s and a low pass filter for 60 Hz were used. Simultaneously to the EEG recording, digital video recordings were carried out. The video files were connected to the corresponding EEG file using the LabChart 6 software so that a simultaneous analysis of the EEG and video was possible. To allow video recording of seizures during the dark phase, red LEDs were installed over each cage. Rats were housed in clear glass cages (one per cage) to allow optimal video observation. For detection of spontaneous seizures, the EEG recordings were visually analyzed for characteristic ictal events. To evaluate the severity of motor seizure activity during a paroxysmal alteration in the EEG, the corresponding video recording was viewed. For rating of seizure severity of spontaneous seizures, Racine’s scale (1972) was used. In addition to the five seizure stages rated by this grading system, a stage 6 was used to characterize runningebouncing seizures, which were occasionally observed before or after a generalized convulsive seizure. Based on this scale, seizures were subdivided into non-convulsive (stage 1 and 2) and convulsive (stages 3e6). In addition to video/EEG monitoring of spontaneous seizures, all spontaneous seizures observed during handling or other manipulations of the animals in the period after continuous video/EEG monitoring were noted. 2.4. PTZ seizure threshold 2.4.1. Experimental groups For determination of seizure threshold before and after SE, the timed i.v. PTZ infusion seizure test was performed as described in detail recently (Löscher, 2009). This test can be repeatedly performed in the same rats at intervals of 48 h, thus allowing to study alterations in seizure threshold in individual rats (Löscher, 2009). In the present study, the PTZ seizure threshold was determined in two separate experiments. Because we did not know if and when the PTZ threshold would be altered after SE, a total of 7 threshold determinations was performed in the first experiment, i.e., 5 days before SE as well as 2, 4, 6, 9, 15, and 22 days after SE in 11 rats. Ten sham-SE controls and 8 pilocarpine/non-SE controls were used in parallel to the SE rats. Based on the findings of the first experiment, we reduced the number of PTZ threshold determinations from 7 to 4 in the second experiment, and determined the threshold 5 days before SE as well as 2, 6, 13 or 14 days after SE in 6 rats. Nine sham-SE controls and four pilocarpine/non-SE controls were used in parallel to the SE rats. Because data obtained in the two experiments were very similar, they were combined for final analyses. Some rats died during PTZ seizures or data could

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512 not be used because of paravenous infusion, so that final group size was 15 SE rats, 10 pilocarpine/non-SE controls, and 18 sham-SE controls. In several of these rats, the hippocampal or, in some rats, the cortical EEG was recorded during PTZ infusion by using the equipment and recording technique described above for monitoring of spontaneous seizures. The additional cortical recordings were performed because we did not observe paroxysmal EEG alterations at the first myoclonic twitch in hippocampal recordings (see Results). In an additional group of 8 naive rats, we examined whether diazepam, which was used to interrupt SE in the pilocarpine-SE group, affected the PTZ threshold determined 2 days later. These animals received diazepam at the same dose (three times 10 mg/kg at an interval of 10 min) as the SE rats. Furthermore, we determined the PTZ threshold in a group of male rats (n ¼ 78) to allow a comparison with control thresholds in female rats. 2.4.2. Determination of seizure threshold In all rats, the PTZ seizure threshold was determined by infusion of a 0.8% solution of PTZ (in saline) via a 26 G needle into the lateral tail vein of conscious, freely moving (i.e., unrestricted) rats. The needle was secured to the tail vein by a piece of adhesive tape and the animal was permitted to move freely inside a MakrolonÒcage type III. The needle was connected to a syringe by a flexible polyethylene tubing (Kleinfeld Labortechnik, Gehrden, Germany) and the PTZ solution was infused at a constant rate of 1.0 ml/min using an infusion pump (PHD 2000 Infusion, Harvard Apparatus Holliston, Massachusetts). Infusion was terminated immediately following the onset of the first myoclonic twitch, to reduce mortality that is otherwise associated with continued infusion of PTZ. The PTZ seizure threshold was calculated in mg/kg PTZ based on the time needed to induce this seizure, the body weight of the animal, and the rate of infusion and concentration of PTZ in the infusate. Determination of PTZ threshold was always performed at the same time in the morning to avoid any circadian variation in seizure threshold. To exclude that the seizure threshold was affected by spontaneous seizures occurring shortly before PTZ infusion, SE rats were observed for 1 h prior to each PTZ threshold determination. In case that a spontaneous seizure was observed, which occurred in only 2 rats, an interval of 1 h was used before performing the threshold determination. 2.4.3. Scoring of behavioral responses to PTZ In addition to calculating the PTZ dose to the first myoclonic twitch, all seizures or other abnormal behaviors occurring after this endpoint were recorded. For this purpose, rats were closely observed until they resumed normal behavior. Seizures occurring in addition to the myoclonic twitch shortly after termination of PTZ infusion were scored as follows (“score I”): 0, no further seizures; 1, a generalized clonic seizure; 2, a generalized clonic seizure with loss of righting reflexes; 3, a generalized clonic seizure with loss of righting reflexes plus running and bouncing; 4, score 3 plus forelimb tonus. In naive rats, which typically exhibited score 1, the myoclonic twitch and the clonic seizure appearing shortly after this twitch were followed by a period of behavioral suppression, and rats usually resumed normal behavior within 3e5 min after termination of PTZ infusion (see Results). However, this changed after several PTZ threshold determinations or in rats after SE, so that additional convulsive behavior appeared after the first myoclonic twitch and clonic seizure, resulting in an increased duration of convulsive activity before the rats exhibited behavioral suppression and finally returned to normal behavior. This additional abnormal behavior was scored as follows (“score II”): 0, no difference from naive controls with first PTZ threshold determination; 1, sustained myoclonic seizures, stereotyped sniffing, Straub tail for up to 2 min following the first myoclonic and clonic seizure after termination of PTZ infusion; 2, as score 1 but duration for more than 2 min; 3, repeatedly occurring generalized seizures (type 4 or 5 on the Racine scale, Racine, 1972); 4, repeatedly occurring myoclonic and generalized (type 4 or 5) seizures. 2.5. Statistics Differences in PTZ threshold were analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni test. Score data were analyzed by ANOVA for nonparametric data (KruskaleWallis test) followed by the Dunn’s test. Individual comparisons were performed either by Student’s t-test or by the ManneWhitney U-test, depending on whether data were normally distributed or not. A P < 0.05 was considered significant.

3. Results 3.1. Duration of the latent period in the lithiumepilocarpine model used in this study

507

following SE. The average latent period was 7.3  0.62 days. Spontaneous seizures were either non-convulsive focal (stage 1e2) or convulsive (mostly stage 4 or 5) seizures and were associated with paroxysmal alterations in the EEG (Fig. 1D). In four additional rats, the PTZ seizure threshold was determined at 2, 6, and 13 days after SE. Continuous video/EEG recording in these rats indicated that the first spontaneous seizure occurred after 6 and 9 days, respectively, in two rats, while no spontaneous seizure was determined during monitoring for either 2 or 3 weeks in the other two rats. Whether this was a consequence of the intermittent PTZ administration or just due to variation in the length of the latent period has to be studied in a larger group of rats with PTZ administration after SE. 3.2. Control seizure threshold The first PTZ seizure threshold determined in control groups or before SE in the SE group was about 20 mg/kg (19.6  0.49 mg/kg) with low inter-individual variation (Fig. 2). In most rats, the myoclonic twitch (after which PTZ infusion was terminated) was followed within few seconds by a generalized clonic seizure (Figs. 1A and 3A), after which rats exhibited behavioral suppression for some minutes and then rapidly resumed normal behavior (Fig. 3B). For assessing sex-related differences in PTZ seizure threshold, we also determined the threshold in a large group of age-matched male rats, resulting in an average seizure threshold of 19.07  4.68 mg/kg (n ¼ 78), which was not significantly different from the threshold determined in female rats. Because diazepam was used to terminate convulsive SE (and was also used in sham-SE controls), we had to determine whether this affected the PTZ seizure threshold determined 2 days later. Although diazepam is rapidly eliminated in rats (elimination halflife is <2 h), it is metabolized to several active metabolites, including desmethyldiazepam, which may increase its duration of anticonvulsant activity, particularly after administration of high doses as in the present study (Löscher, 2007). When the PTZ threshold was determined in a group of 8 naive (non-SE) rats before and two days after administration of diazepam (three times 10 mg/kg at an interval of 10 min), the threshold did not differ significantly. It was 21.7  1.2 mg/kg before vs. 23.1  0.77 mg/kg two days after diazepam (P ¼ 0.2912), respectively. Thus the interval of two days after diazepam was sufficiently long to exclude any significant anticonvulsant effect of this drug on the PTZ threshold during our experiments in the sham-SE and SE groups of rats. When the PTZ seizure threshold was repeatedly determined for up to 7 times in a group of 18 sham-SE controls, it tended to increase over the course of the experimental period (Fig. 2A). This became statistically significant at the 3rd and 6th threshold determination when compared to the first threshold determination. In addition to this relatively moderate alteration in seizure threshold, the postPTZ behavior following termination of PTZ infusion was altered markedly upon repeated threshold determinations in sham-SE controls (Fig. 3A,B). Thus, beginning with the 5th threshold determination, running-and-bouncing and tonic seizures occurred much more often in addition to generalized clonic seizures than observed during the first four threshold determinations (Fig. 3A). Furthermore, the duration of the post-PTZ behavioral alterations increased in that rats exhibited prolonged myoclonic seizures, stereotyped sniffing, and, in part, generalized convulsive behavior after the first myoclonic twitch and clonic seizure (Fig. 3B). 3.3. Alterations in PTZ seizure threshold after SE

To our knowledge, the latent period for the pilocarpine model with ramping-up dosing design used in this study has not been reported before. In the 6 rats with continuous video/EEG recording after SE, the first spontaneous seizure was determined 6e10 days

Two days following SE, the PTZ seizure threshold significantly decreased compared to pre-SE control (Fig. 2B). No significant difference to pre-SE control was seen at subsequent threshold

508

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512

A

Cortical EEG 5 days before sham-SE M

C/LR

RB

PTZ

1 mV 5s

B

Hippocampal EEG 4 days after sham-SE M

PTZ

C RB FT

1 mV 5s

C

Hippocampal EEG 2 days after SE PTZ

M

C

C/LR

M (for 17 min)

1 mV 5s

D

Hippocampal EEG during a spontaneous seizure

1 mV 10 s Fig. 1. EEG recordings of paroxysmal alterations during PTZ-induced (A, B, C) and spontaneous seizures (D). Data are representative examples of the different groups. Baseline activity recorded shortly before onset of PTZ infusion did not differ between rats. In “A”, the cortical EEG was recorded in a control rat 5 days before sham-SE. After onset of PTZ infusion, it took 37 s to a first myoclonic twitch (“M”), after which PTZ infusion was terminated. The first myoclonic twitch was followed by additional myoclonic twitches and, at 41 s, by a generalized clonic seizure (C) with loss of righting reflexes (LR), which was followed by running/bouncing (RB), after which the rat exhibited behavioral suppression (postictal refractoriness) and then rapidly resumed normal behavior. In “B”, the hippocampal EEG was recorded in a control rat 4 days after sham-SE. After onset of PTZ infusion, it took 53 s to a first myoclonic twitch (“M”), after which PTZ infusion was terminated. The first myoclonic twitch was followed by additional myoclonic twitches and, at 59 s, by a generalized clonic seizure (C), which was interrupted by running/bouncing (RB) and a forelimb tonus (FT). After termination of clonic activity, the rat exhibited behavioral suppression (postictal refractoriness) and then rapidly resumed normal behavior. In “C”, the hippocampal EEG was recorded in a rat 2 days after SE. After onset of PTZ infusion, it took 32 s to a first myoclonic twitch (“M”), after which PTZ infusion was terminated. The first myoclonic twitch was followed by additional myoclonic jerks and, at 38 s, by a generalized clonic seizure (C), which progressed at 42 s to a clonic seizure with loss of righting reflexes (C/LR). After this generalized clonic activity, myoclonic activity was observed for additional 17 min (only the onset is illustrated), during which intermittent spikes were observed in the EEG, before the rat exhibited behavioral suppression (postictal refractoriness) and then returned to normal behavior. Note that, due to a technical problem, the EEG of this rat could not be recorded for the first 7 s after onset of PTZ infusion, which, however, did not affect the calculation of seizure latency. “D” shows paroxysmal EEG alterations recorded from the hippocampus during a spontaneous (stage 5) seizure, which was recorded 10 days after SE. Note that the time scales are different for the different EEG recordings illustrated in the figure.

determinations. However, when data from the SE group were directly compared with those from the sham-SE control group, PTZ threshold was significantly below control values at all post-SE threshold determinations, including the period following the latent period (Fig. 2C). In addition to the lowered PTZ seizure threshold after SE, the duration of behavioral alterations after termination of PTZ infusion was markedly enhanced in the SE group (Fig. 3B). Thus, whereas sham-SE controls rapidly resumed normal behavior at the threshold determinations 2 days and 4 days following sham-SE (Fig. 3B), the post-SE rats exhibited sustained myoclonic seizures and stereotyped sniffing after the initial myoclonic and clonic activity (Figs. 1C and 3B). In individual animals, it took up to w1 h before they resumed normal behavior after termination of PTZ infusion. Interestingly, this increased duration of post-PTZ behavior

was observed up to 9 days following SE, i.e., during the latent period, but not thereafter (Fig. 3B). In contrast to the significant differences between controls and post-SE rats in the duration of behavioral alterations after termination of PTZ infusion (Fig. 3B), no such differences were determined for the additional seizure types (“score I”) occurring immediately after terminating PTZ infusion at the first myoclonic twitch (Fig. 3A). No significant differences in score I were determined in the post-SE group over the course of the experiments, and post-SE rats did not significantly differ in score I from sham-SE controls at any PTZ threshold determination. In contrast to SE rats, PTZ seizure thresholds in 10 rats that received pilocarpine but did not develop SE did not significantly differ from sham-SE controls (not illustrated). Therefore this group was not further analyzed.

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512

Seizure threshold after sham-SE

A

A

509

Post-PTZ behavior (score I) after sham-SE

30 4

*

Seizure severity (score)

PTZ (mg/kg i.v.)

* 20

10

*

Sham-SE SE

*

3

2

1

0

-5 +2 +4 +6 +9 +14 +22 Days before and after sham-SE or SE

0 -5

+2

+4

+6

+9

+14

+22

B

Post-PTZ behavior (score II) after sham-SE

Additional abnormal behavior (score)

Days before and after sham-SE

Seizure threshold after SE

B 25

PTZ (mg/kg i.v.)

20

* 15

Sham-SE SE

2.0

o

*

o

*

o

*

*

1.5

1.0

*

0.5

0.0 -5

10

+2

+4

+6

+9

+14

+22

Days before and after sham-SE or SE

5

0 -5

+2

+4

+6

+9

+14

+22

Days before and after SE

Seizure threshold sham-SE vs. SE

C

sham-SE SE

30

PTZ (mg/kg i.v.)

2.5

*

20

*

*

*

*

*

Fig. 3. Behavioral alterations occurring after termination of PTZ infusion in sham-SE and SE rats. The severity of seizures occurring immediately after the myoclonic twitch (at which PTZ infusion was stopped) was rated by 1e4 (see Methods) and is shown as “score I” (A). During the first threshold determinations, rats typically exhibited generalized clonic seizures without (score 1) or with (score 2) loss of righting reflexes shortly after the myoclonic twitch, but, particularly in sham-SE controls, additional more severe seizures were observed at subsequent threshold determinations. The duration and severity of additional abnormal behavior (seizures, stereotyped sniffing, Straub tail) appearing after the myoclonic twitch and clonic seizure were rated from 1 to 4 (see Methods) and are shown as “score II” (B). Before SE and in sham-SE rats, rats rapidly resumed normal behavior after PTZ seizure threshold determination. However, this changed after several PTZ threshold determinations in sham-SE rats or immediately after SE, so that additional convulsive behavior appeared after the first myoclonic twitch and clonic seizure, resulting in an increased duration of convulsive activity before the rats exhibited behavioral suppression and finally returned to normal behavior. Data are shown as means  SEM of 18 (sham-SE) and 15 (SE) rats, respectively. The significance of differences to control data within each group determined 5 days before sham-SE or SE is indicated by asterisk (P < 0.05), while significant differences between groups are indicated by circle (P < 0.05).

3.4. EEG alterations during PTZ infusion 10

0 -5

+2 +4 +6 +9 +14 +22 Days before and after sham-SE or SE

Fig. 2. PTZ seizure threshold before and after sham-SE (A) or SE (B) as well as comparison between both groups (C). The threshold is indicated in mg/kg PTZ inducing a myoclonic twitch, i.e., the first seizure occurring during PTZ infusion. Data are shown as means  SEM of 18 (sham-SE) and 15 (SE) rats, respectively. In “A” and “B”, the significance of differences to the control threshold determined 5 days before sham-SE or SE is indicated by asterisk (P < 0.05). In “C”, significant differences between the two groups are indicated by asterisk (P < 0.05).

As shown in Fig. 1, the first myoclonic twitch during PTZ infusion was not associated with obvious paroxysmal EEG alterations in cortical (Fig. 1A) or hippocampal (Fig. 1B,C) recordings, but convulsive discharges were observed shortly before and during the clonic seizure that immediately followed the myoclonic twitch at which PTZ infusion was terminated. EEG alterations then progressed into sustained high-amplitude spiking, which was observed both in cortical and hippocampal EEG recordings. Paroxysmal EEG alterations were not qualitatively different between sham-SE and SE rats, but, similar to the behavioral alterations described above (score II; Fig. 3B), convulsive discharges in the EEG lasted much longer following SE compared to control recordings at the PTZ seizure threshold determination.

510

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512

4. Discussion To the best of our knowledge, this is the first study demonstrating reduced seizure threshold during the latent period following SE. Using timed i.v. infusion of the GABAA receptor antagonist, PTZ, for determining seizure threshold, the threshold decreased significantly two days after SE when compared to pre-SE control values. However, when compared to concurrent sham-SE controls, the PTZ threshold was significantly decreased from days 2 to 22 post-SE, i.e., over the whole post-SE period examined. In addition to lowered seizure threshold, the duration of abnormal convulsive and stereotypic behavior following termination of PTZ infusion markedly increased in post-SE rats at 2e9 days following SE, but returned towards control values thereafter. Thus, this parameter of enhanced susceptibility to PTZ paralleled the duration of the latent period before onset of spontaneous seizures. 4.1. Duration of latent period after SE By using continuous video/EEG monitoring for determining the duration of the latent period from SE to the appearance of the first spontaneous epileptic seizures, an average latent period of 7.3 days (range 6e10 days) was determined in the present study, in which we used lithiumepilocarpine and a ramping-up dosing protocol for SE induction, which allows a more individual pilocarpine dosing compared to protocols with fixed bolus doses of this convulsant. A very similar latent period of 7.2 days (range 5e17 days) was determined by Goffin et al. (2007) after injecting a bolus dose of 360 mg/kg pilocarpine in rats. 4.2. Susceptibility to convulsants after SE Only few previous studies examined whether SE results in increased susceptibility to convulsants, but none of these studies determined seizure threshold during the latent period (Wu and Leung, 2001; Nehlig et al., 2002; Zhang et al., 2004; Blanco et al., 2009). In the study of Blanco et al. (2009), rats received 50 mg/kg PTZ s.c. one month after a pilocarpine-induced SE, i.e., in the chronic epileptic phase, resulting in a higher incidence of PTZinduced severe generalized convulsions compared to controls. Furthermore, the latency to PTZ seizures was markedly reduced in the epileptic rats. In contrast to the enhanced susceptibility to PTZ, the incidence or severity of electrically induced seizures did not differ between epileptic rats and controls (Blanco et al., 2009). The enhanced sensitivity to PTZ of epileptic rats from the pilocarpine model resembles previous findings with PTZ in genetically epilepsy-prone rats (GEPR; Browning et al., 1990). In the study of Wu and Leung (2001), SE was induced by kainate and the GABAA receptor antagonist bicuculline was administered 2e3 months later. At this time, paired-pulse inhibition of the population spikes in the dentate gyrus was increased, which was readily blocked by a small dose of bicuculline. However, bicuculline induced paroxysmal spike bursts in kainate-treated rats but not control rats, indicating that the increased inhibition in dentate gyrus was fragile (Wu and Leung, 2001). The authors suggested that this fragile inhibition could explain the seizure susceptibility in patients with TLE. In the study of Nehlig et al. (2002), SE was induced by lithiumepilocarpine in 10-day-old (P10) rats and sensitivity to different convulsants (PTZ, picrotoxin, kainate) was determined 3 months later, i.e., during adulthood. No increased susceptibility to convulsants was obtained but the rats had not developed epilepsy after lithiumepilocarpine induced SE at P10. In apparent contrast, a similar study by Zhang et al. (2004) with lithiumepilocarpine in P10 rats reported increased susceptibility to kainate-induced

seizures three months later, which was associated with alterations in glutamate receptor and transporter gene expression in the hippocampal formation. This discrepancy could be due to a difference in methods, as Nehlig et al. (2002) injected a fixed amount of kainate i.p., whereas Zhang et al. (2004) infused kainate continuously through lateral tail vein. These differences may have permitted Zhang et al. (2004) to assess more subtle differences in seizure onset. In the present study, we used timed i.v. infusion of PTZ as a highly sensitive method for determining alterations in seizure threshold (Löscher, 2009). It has been previously reported that stable seizure thresholds are obtained when intervals between repeated threshold determinations in individual rats are at least 48 h, allowing repeated seizure threshold assessments in the same animal (Pollack and Shen, 1985; Löscher, 2009). However, the present experiments in sham-SE controls showed a tendency to increased seizure threshold upon repeated determination at intervals of at least 48 h. Furthermore, after five threshold determinations in sham-SE rats, seizures became more severe and enduring, indicating a kindling-like effect as previously reported after repeated PTZ seizure threshold determination in dogs (Löscher, 1983). The apparent paradox of an increase in seizure threshold but concomitantly more severe and enduring seizures in response to repeated administration of PTZ in control rats resembles observations from repeated administration of electroconvulsive stimulation in rats and has been explained by increased excitability in two antagonistic systems, one responsible for terminating ictus and postictal refractoriness and the other for kindling-like events, i.e., progressive increase in seizure susceptibility (Löscher and Köhling, 2010). In sham-SE controls, such alterations did not occur before 3e5 PTZ threshold determinations, whereas SE rats exhibited decreased seizure threshold and increased seizure duration and severity at their second PTZ threshold determination, i.e., 2 days after SE, strongly indicating that this reflected development of neuronal hyperexcitability during the latent period. Furthermore, the observation that increased duration and severity of seizures in response to PTZ were only observed during the latent period, but not upon subsequent PTZ injections, indicates that the protocol used for seizure threshold analysis in the present experiments was suitable to disclose functionally meaningful alterations in the latent period after SE. 4.3. Estrous cycle and seizure susceptibility As in our previous studies on pilocarpine-induced epileptogenesis in rats (Glien et al., 2001, 2002; Freichel et al., 2006; Bankstahl and Löscher, 2008; Kuteykin-Teplyakov et al., 2009; Brandt et al., 2010), we used female rats, which may form a bias for the question under investigation, because of the possibility of estrous cycle-dependent variation in seizure susceptibility (Scharfman and MacLusky, 2006). However, as described in the Methods section, female rats were housed without males so that they did not cycle synchronously and thus individual animals within a group were at different cycle phases on a given day of PTZ threshold determination (Kücker et al., 2010; Katrin Becker, unpublished data). Furthermore, more importantly, Finn and Gee (1994) reported that seizure susceptibility to PTZ is not affected by the estrous cycle in Sprague-Dawley rats, and that PTZ seizure threshold is not different between male and female rats, which was confirmed in the present study. The lack of any significant effect of estrous cycle on PTZ seizure threshold reported by Finn and Gee (1994) also explains the low intra- and inter-individual variation in threshold determined in the present study. It is long known that effects of the estrous cycle on seizure susceptibility depend on the

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512

choice of convulsant (Scharfman and MacLusky, 2006). For instance, similar to the lack of any significant alterations in PTZ seizure threshold during the estrous cycle reported by Finn and Gee (1994), we did not determine any effect of the estrous cycle on seizure threshold in the kindling model (Wahnschaffe and Löscher, 1992), whereas cyclic changes were observed in some other seizure models (Woolley and Schwartzkroin, 1998; Scharfman and MacLusky, 2006). Based on these considerations, it is highly unlikely that the lowered PTZ seizure threshold determined after SE in the present experiments was due to estrous cyclicity or an SEinduced deregulation of the cycle as reported by Amado and Cavalheiro (1998). 4.4. Mechanisms underlying enhanced susceptibility to PTZ after SE A likely explanation for the enhanced susceptibility to the GABAA receptor antagonist PTZ during the latent phase of the pilocarpine model are alterations in GABAergic neurotransmission following SE. Numerous studies have examined alterations in the GABA system after SE in rats, showing that complex changes take place in the hippocampal formation and other limbic areas, including (1) loss of GABAergic interneurons, resulting in a decrease of GABA-mediated inhibition and network reorganization (Morimoto et al., 2004); (2) significant alterations in GABAA receptor subunit composition and expression, resulting in functional and pharmacological alterations of these receptors (Coulter, 2001); (3) depolarizing actions of GABA due to higher intracellular chloride concentration (Pathak et al., 2007); and (4) a paradoxical increase in GABA-mediated inhibitory effects, which can be viewed as possible compensatory change but may increase network synchronization and thus contribute to epileptogenesis (Cossart et al., 2005; Ben-Ari, 2006; Mann and Mody, 2008). The spatiotemporal development of these alterations after SE and their individual contribution to neuronal hyperexcitability developing during the latent period after SE is only incompletely understood. Which of these complex alterations in GABA-mediated transmission explain the changes in response to PTZ during the latent period after SE determined in the present experiments cannot be determined by systemic administration of PTZ. 5. Conclusions As shown in the present study, similar to models of TBI (Pitkänen et al., 2009), seizure threshold is decreased during the latent period following SE. Importantly, the latent period after SE was also associated with increased duration of abnormal convulsive and stereotypic behavior in response to PTZ, which was restricted to the latent period, but returned towards control behavior thereafter, which may be a result of endogenous anticonvulsant mechanisms developing in response to the spontaneous seizures that occur after the latent period. Paradoxically, despite reduced seizure threshold in the latent period following TBI (Pitkänen et al., 2009), complex febrile seizures (Armstrong et al., 2009), or SE (present study), administration of subconvulsant doses of proconvulsant drugs such as the CB1 receptor antagonist rimonabant (Chen et al., 2007; Echegoyen et al., 2009) or the a2-receptor antagonist atipamezole (Pitkänen et al., 2004) exert antiepileptogenic or disease-modifying effects in such models. Although more studies will be required to determine what properties of these agents - their proconvulsant nature or their mechanisms of action - are the ones that mediate their antiepileptogenic properties (Armstrong et al., 2009), it is tempting to speculate that subconvulsant doses of PTZ also exert such effects when administered after SE, which will be studied in forthcoming experiments. The rationale for using the GABAA receptor antagonist PTZ for this

511

purpose is that this compound may counteract both the increased network synchronization in response to enhanced GABAergic inhibition and depolarizing GABA-mediated effects following SE. In view of the fact that a large number of preclinical and clinical studies have demonstrated that drugs designed to prevent epileptic seizures, i.e., antiepileptic (anticonvulsant) drugs, do not prevent epileptogenesis after brain insults (Pitkänen and Kubova, 2004; Temkin, 2009; Löscher and Brandt, 2010), there is an urgent need to test new avenues to find the ideal preventative strategy for acquired epilepsy (Jacobs et al., 2009). Acknowledgements We thank P. Demmer, N. Ernst, M. Krüger, and M. Töpfer for skilful technical assistance. The study was supported by a grant (Lo 274/11-1) from the Deutsche Forschungsgemeinschaft (Bonn, Germany). References Amado, D., Cavalheiro, E.A., 1998. Hormonal and gestational parameters in female rats submitted to the pilocarpine model of epilepsy. Epilepsy Res. 32, 266e274. Armstrong, C., Morgan, R.J., Soltesz, I., 2009. Pursuing paradoxical proconvulsant prophylaxis for epileptogenesis. Epilepsia 50, 1657e1669. Bankstahl, J.P., Löscher, W., 2008. Resistance to antiepileptic drugs and expression of P-glycoprotein in two rat models of status epilepticus. Epilepsy Res. 82, 70e85. Ben-Ari, Y., 2006. Seizures beget seizures: the quest for GABA as a key player. Crit. Rev. Neurobiol. 18, 135e144. Blaesse, P., Airaksinen, M.S., Rivera, C., Kaila, K., 2009. Cation-chloride cotransporters and neuronal function. Neuron 61, 820e838. Blanco, M.M., Dos Jr., S.J., Perez-Mendes, P., Kohek, S.R., Cavarsan, C.F., Hummel, M., Albuquerque, C., Mello, L.E., 2009. Assessment of seizure susceptibility in pilocarpine epileptic and nonepileptic Wistar rats and of seizure reinduction with pentylenetetrazole and electroshock models. Epilepsia 50, 824e831. Brandt, C., Nozadze, M., Heuchert, N., Rattka, M., Löscher, W., 2010. Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy. J. Neurosci. 30, 8602e8612. Browning, R.A., Wang, C., Lanker, M.L., Jobe, P.C., 1990. Electroshock- and pentylenetetrazol-induced seizures in genetically epilepsy-prone rats (GEPRs): differences in threshold and pattern. Epilepsy Res. 6, 1e11. Chen, K., Neu, A., Howard, A.L., Foldy, C., Echegoyen, J., Hilgenberg, L., Smith, M., Mackie, K., Soltesz, I., 2007. Prevention of plasticity of endocannabinoid signaling inhibits persistent limbic hyperexcitability caused by developmental seizures. J. Neurosci. 27, 46e58. Cossart, R., Bernard, C., Ben-Ari, Y., 2005. Multiple facets of GABAergic neurons and synapses: multiple fates of GABA signalling in epilepsies. Trends Neurosci. 28, 108e115. Coulter, D.A., 2001. Epilepsy-associated plasticity in gamma-aminobutyric acid receptor expression, function, and inhibitory synaptic properties. Int. Rev. Neurobiol. 45, 237e252. Dichter, M.A., 2009. Emerging concepts in the pathogenesis of epilepsy and epileptogenesis. Arch. Neurol. 66, 443e447. Dudek, F.E., 2009. Commentary: a skeptical view of experimental gene therapy to block epileptogenesis. Neurotherapeutics 6, 319e322. Echegoyen, J., Armstrong, C., Morgan, R.J., Soltesz, I., 2009. Single application of a CB1 receptor antagonist rapidly following head injury prevents long-term hyperexcitability in a rat model. Epilepsy Res. 85, 123e127. Finn, D.A., Gee, K.W., 1994. The estrus cycle, sensitivity to convulsants and the anticonvulsant effect of a neuroactive steroid. J. Pharmacol. Exp. Ther. 271, 164e170. Freichel, C., Potschka, H., Ebert, U., Brandt, C., Löscher, W., 2006. Acute changes in the neuronal expression of gaba and glutamate decarboxylase isoforms in the rat piriform cortex following status epilepticus. Neuroscience 141, 2177e2194. Glien, M., Brandt, C., Potschka, H., Voigt, H., Ebert, U., Löscher, W., 2001. Repeated low-dose treatment of rats with pilocarpine: low mortality but high proportion of rats developing epilepsy. Epilepsy Res. 46, 111e119. Glien, M., Brandt, C., Potschka, H., Löscher, W., 2002. Effects of the novel antiepileptic drug levetiracetam on spontaneous recurrent seizures in the rat pilocarpine model of temporal lobe epilepsy. Epilepsia 43, 350e357. Goffin, K., Nissinen, J., Van Laere, K., Pitkänen, A., 2007. Cyclicity of spontaneous recurrent seizures in pilocarpine model of temporal lobe epilepsy in rat. Exp. Neurol. 205, 501e505. Golarai, G., Greenwood, A.C., Feeney, D.M., Connor, J.A., 2001. Physiological and structural evidence for hippocampal involvement in persistent seizure susceptibility after traumatic brain injury. J. Neurosci. 21, 8523e8537. Jacobs, M.P., Leblanc, G.G., Brooks-Kayal, A., Jensen, F.E., Lowenstein, D.H., Noebels, J.L., Spencer, D.D., Swann, J.W., 2009. Curing epilepsy: progress and future directions. Epilepsy Behav. 14, 438e445.

512

M. Rattka et al. / Neuropharmacology 60 (2011) 505e512

Khazipov, R., Holmes, G.L., 2003. Synchronization of kainate-induced epileptic activity via GABAergic inhibition in the superfused rat hippocampus in vivo. J. Neurosci. 23, 5337e5341. Köhling, R., 2002. Neuroscience. GABA becomes exciting. Science 298, 1350e1351. Kuteykin-Teplyakov, K., Brandt, C., Hoffmann, K., Löscher, W., 2009. Complex timedependent alterations in the brain expression of different drug efflux transporter genes after status epilepticus. Epilepsia 50, 887e897. Kücker, S., Töllner, K., Piechotta, M., Gernert, M., 2010. Kindling as a model of temporal lobe epilepsy induces bilateral changes in spontaneous striatal activity. Neurobiol. Dis. 37, 661e672. Löscher, W., 1983. Alterations in CSF GABA levels and seizure susceptibility developing during repeated administration of pentetrazole in dogs. Effects of yacetylenic GABA, valproic acid and phenobarbital. Neurochem. Int. 5, 405e412. Löscher, W., 2007. The pharmacokinetics of antiepileptic drugs in rats: consequences for maintaining effective drug levels during prolonged drug administration in rat models of epilepsy. Epilepsia 48, 1245e1258. Löscher, W., 2009. Preclinical assessment of proconvulsant drug activity and its relevance for predicting adverse events in humans. Eur. J. Pharmacol. 610, 1e11. Löscher, W., Köhling, R., 2010. Functional, metabolic, and synaptic changes after seizures as potential targets for antiepileptic therapy. Epilepsy Behav 19, 105e113. Löscher, W., Brandt, C., 2010. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research. Pharmacol. Rev 62, 668e700. Macdonald, R.L., Barker, J.L., 1977. Pentylenetetrazol and penicillin are selective antagonists of GABA-mediated post-synaptic inhibition in cultured mammalian neurones. Nature 267, 720e721. Mann, E.O., Mody, I., 2008. The multifaceted role of inhibition in epilepsy: seizuregenesis through excessive GABAergic inhibition in autosomal dominant nocturnal frontal lobe epilepsy. Curr. Opin. Neurol. 21, 155e160. Morimoto, K., Fahnestock, M., Racine, R.J., 2004. Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog. Neurobiol. 73, 1e60. Nehlig, A., Dube, C., Koning, E., 2002. Status epilepticus induced by lithiumepilocarpine in the immature rat does not change the long-term susceptibility to seizures. Epilepsy Res. 51, 189e197. Pacheco, M.F., Fowler, J.C., Partridge, L.D., 1981. The site of action for the convulsant effect of pentylenetetrazol. Comp. Biochem. Physiol. C 68C, 99e102. Pathak, H.R., Weissinger, F., Terunuma, M., Carlson, G.C., Hsu, F.C., Moss, S.J., Coulter, D.A., 2007. Disrupted dentate granule cell chloride regulation enhances synaptic excitability during development of temporal lobe epilepsy. J. Neurosci. 27, 14012e14022. Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed. Elsevier, Amsterdam. Payne, J.A., Rivera, C., Voipio, J., Kaila, K., 2003. Cationechloride co-transporters in neuronal communication, development and trauma. Trends Neurosci. 26,199e206. Pitkänen, A., Narkilahti, S., Bezvenyuk, Z., Haapalinna, A., Nissinen, J., 2004. Atipamezole, an alpha(2)-adrenoceptor antagonist, has disease modifying effects on epileptogenesis in rats. Epilepsy Res. 61, 119e140.

Pitkänen, A., Kubova, H., 2004. Antiepileptic drugs in neuroprotection. Expert Opin. Pharmacother. 5, 777e798. Pitkänen, A., Kharatishvili, I., Karhunen, H., Lukasiuk, K., Immonen, R., Nairismagi, J., Grohn, O., Nissinen, J., 2007. Epileptogenesis in experimental models. Epilepsia 48 (Suppl. 2), 13e20. Pitkänen, A., Immonen, R.J., Grohn, O.H., Kharatishvili, I., 2009. From traumatic brain injury to posttraumatic epilepsy: what animal models tell us about the process and treatment options. Epilepsia 50 (Suppl. 2), 21e29. Pitkänen, A., Lukasiuk, K., 2009. Molecular and cellular basis of epileptogenesis in symptomatic epilepsy. Epilepsy Behav. 14 (Suppl. 1), 16e25. Pollack, G.M., Shen, D.D., 1985. A timed intravenous pentylenetetrazol infusion seizure model for quantitating the anticonvulsant effect of valproic acid in the rat. J. Pharmacol. Methods 13, 135e146. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281e294. Ramanjaneyulu, R., Ticku, M.K., 1984. Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepineeGABA receptoreionophore complex. Eur. J. Pharmacol. 98, 337e345. Rigoulot, M.A., Leroy, C., Koning, E., Ferrandon, A., Nehlig, A., 2003. Prolonged lowdose caffeine exposure protects against hippocampal damage but not against the occurrence of epilepsy in the lithiumepilocarpine model in the rat. Epilepsia 44, 529e535. Scharfman, H.E., MacLusky, N.J., 2006. The influence of gonadal hormones on neuronal excitability, seizures, and epilepsy in the female. Epilepsia 47, 1423e1440. Sirven, J.I., 2009. Not all first seizures are created equally. Epilepsy Curr. 9, 164e165. Statler, K.D., Swank, S., Abildskov, T., Bigler, E.D., White, H.S., 2008. Traumatic brain injury during development reduces minimal clonic seizure thresholds at maturity. Epilepsy Res. 80, 163e170. Temkin, N.R., 2009. Preventing and treating posttraumatic seizures: the human experience. Epilepsia 50 (Suppl. 2), 10e13. Wahnschaffe, U., Löscher, W., 1992. Lack of changes in seizure susceptibility during the estrous cycle in kindled rats. Epilepsy Res. 13, 199e204. Walker, M.C., White, H.S., Sander, J.W., 2002. Disease modification in partial epilepsy. Brain 125, 1937e1950. Woolley, C.S., Schwartzkroin, P.A., 1998. Hormonal effects on the brain. Epilepsia 39 (Suppl. 8), S2eS8. Wu, K., Leung, L.S., 2001. Enhanced but fragile inhibition in the dentate gyrus in vivo in the kainic acid model of temporal lobe epilepsy: a study using current source density analysis. Neuroscience 104, 379e396. Zanier, E.R., Lee, S.M., Vespa, P.M., Giza, C.C., Hovda, D.A., 2003. Increased hippocampal CA3 vulnerability to low-level kainic acid following lateral fluid percussion injury. J. Neurotrauma 20, 409e420. Zhang, G., Raol, Y.S., Hsu, F.C., Brooks-Kayal, A.R., 2004. Long-term alterations in glutamate receptor and transporter expression following early-life seizures are associated with increased seizure susceptibility. J. Neurochem. 88, 91e101.