Therapeutic window of opportunity for the neuroprotective effect of valproate versus the competitive AMPA receptor antagonist NS1209 following status epilepticus in rats

Neuropharmacology 61 (2011) 1033e1047

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Therapeutic window of opportunity for the neuroprotective effect of valproate versus the competitive AMPA receptor antagonist NS1209 following status epilepticus in rats Melanie Langer a, b, Claudia Brandt a, b, Christina Zellinger a, b,1, 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 10 March 2011 Received in revised form 16 June 2011 Accepted 21 June 2011

Epileptogenesis, i.e., the process leading to epilepsy, is a presumed consequence of brain insults including head trauma, stroke, infections, tumors, status epilepticus (SE), and complex febrile seizures. Typically, brain insults produce morphological and functional alterations in the hippocampal formation, including neurodegeneration in CA1, CA3, and, most consistently, the dentate hilus. Most of these alterations develop gradually, over several days, after the insult, providing a therapeutic window of opportunity for neuroprotective agents in the immediate post-injury period. We have previously reported that prolonged (four weeks) treatment with the antiepileptic drug valproate (VPA) after SE prevents hippocampal damage and most of the behavioral alterations that occur after brain insult, but not the development of spontaneously occurring seizures. These data indicated that VPA, although not preventing epilepsy, might be an effective disease-modifying treatment following brain insult. The present study was designed to (1) determine the therapeutic window for the neuroprotective effect of VPA after SE; (2) compare the efficacy of different intermittent i.p. versus continuous i.v. VPA treatment protocols; and (3) compare VPA with the glutamate (AMPA) receptor antagonist NS1209. As in our previous study with VPA, SE was induced by sustained electrical stimulation of the basolateral amygdala in rats and terminated after 4 h by diazepam. In vehicle controls, >90% of the animals developed significant neurodegeneration in the dentate hilus, whereas damage in CA1 and CA3 was more variable. Hilar parvalbumin-expressing interneurons were more sensitive to the effects of seizures than somatostatin-stained hilar interneurons or hilar mossy cells. Among the various VPA treatment protocols, continuous infusion of VPA for 24 immediately following the SE was the most effective neuroprotective treatment, preventing most of the neuronal damage. Infusion with NS1209 for 24 h exhibited similar neuroprotective efficacy. These data demonstrate that short treatment after SE with either VPA or NS1209 is powerfully neuroprotective, and may be disease-modifying treatments following brain insult. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Epilepsy Epileptogenesis Hippocampus Glutamate Seizures Disease-modification

1. Introduction Various brain insults, including traumatic brain injury, encephalitis, stroke, tumors and status epilepticus (SE), can initiate complex morphological and functional alterations that ultimately

lead to epilepsy (Dichter, 2009; Jacobs et al., 2009; Pitkänen and Lukasiuk, 2009; Löscher and Brandt, 2010). Often, this process, termed epileptogenesis, is associated with neuronal damage in the hippocampal formation and parahippocampal areas, as well as psychopathology and cognitive impairment. It is an ongoing debate

Abbreviations: AED, antiepileptic drug; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BLA, basolateral amygdala; EEG, electroencephalogram; GluR 2/3, glutamate (AMPA) receptor 2/3; HDAC, histone deacetylase; NS1209, 8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9-tetrahydro-1H-pyrrolo[3,2-h]-iso-quinoline2,3-dione-3-O-(4-hydroxybutyric acid-2-yl)oxime; SE, status epilepticus; SSSE, self-sustained status epilepticus; TBS, tris-buffered saline; TLE, temporal lobe epilepsy; VPA, valproic acid. * 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). 1 Present address: Institute of Pharmacology, Toxicology & Pharmacy, Ludwig-Maximilians-University, Munich, Germany. 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.06.015

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whether the hippocampal pathology, which is characterized by neurodegeneration in the CA1 and CA3 pyramidal sectors of the hippocampus and the hilus of the dentate gyrus, is involved in the mechanisms leading to epilepsy or is more important for cognitive impairment and behavioral alterations associated with epilepsy (Sloviter, 1994; Pitkänen and Lukasiuk, 2009; Löscher and Brandt, 2010). In a previous study, we reported that the antiepileptic drug (AED) valproate (VPA) is capable of preventing the hippocampal damage, including hilar cell loss, developing after an electrically induced SE in rats (Brandt et al., 2006). This neuroprotective effect did not prevent the development of epilepsy with spontaneous recurrent seizures, but counteracted the development of some of the behavioral alterations associated with epilepsy in the animals (Brandt et al., 2006). Furthermore, based on previous data that pharmacoresistance of epileptic rats in this post-SE model of temporal lobe epilepsy (TLE) is associated with extensive hippocampal damage, whereas AED-responsive rats do not exhibit extensive damage (Volk et al., 2006), we suggested that, although neuroprotection by VPA did not prevent epilepsy, it might prevent the development of AED resistance (Löscher and Brandt, 2010). If so, VPA may offer promise as a prophylactic treatment in patients at risk of developing epilepsy after brain insults. Although speculative, this possibility was a driving force for performing the present study, in which we attempted to identify the therapeutic window for the neuroprotective effect of VPA after SE. In previous experiments, we administered VPA three times daily for 4 weeks, starting immediately after termination of SE by diazepam (Brandt et al., 2006). The aim of the present study was to determine the therapeutic window after SE, i.e., which duration of treatment with VPA is needed for its neuroprotective effect in this model of TLE. Furthermore, because VPA is rapidly eliminated in rats (Löscher, 2007), we wanted to compare the efficacy of intermittent i.p. versus continuous i.v. treatment with VPA. Finally, in order to compare the neuroprotective potential of VPA, we included NS1209 [8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9tetrahydro-1H-pyrrolo[3,2-h]-iso-quinoline-2,3-dione-3-O-(4hydroxybutyric acid-2-yl)oxime], a drug that selectively blocks glutamate receptors of the AMPA (a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid) subtype and that has been shown to exert neuroprotective effects in different brain insult models, including a rat SE model (Nielsen et al., 1999; McCracken et al., 2002;

Pitkänen et al., 2007). During histological analyses of the experiments of the present study, particular attention was paid to degeneration of different subpopulations of hilar neurons, because hilar neuron loss has been suggested to be the common pathological denominator and primary network defect underlying development of a hippocampal seizure “focus” (Sloviter, 1991, 1994; 2008). 2. Materials and methods 2.1. Animals Adult female Sprague-Dawley rats were purchased at a body weight of 200e220 g from either Harlan-Winkelmann (Borchen, Germany) or, after Harlan closed the German colony in 2008, Harlan (Horst, Netherlands). Following arrival, the rats were kept under controlled environmental conditions (22e24  C; 50e60% humidity; 12 h light/dark cycle; light on at 6:00 a.m.) with free access to standard laboratory chow (Altromin 1324 standard diet) and tap water. Female rats were used to allow comparisons of the present study with our previous study with VPA in female Sprague-Dawley rats (Brandt et al., 2006). Furthermore, when different rat strains and genders were compared in the SE model used for the present study, the most consistent findings were obtained in female Sprague-Dawley rats (Brandt et al., 2003). All experiments were done in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize pain or discomfort of the animals used. The experimental protocol is illustrated in Fig. 1. 2.2. Electrode implantation For electrode implantation, rats were anesthetized with chloral hydrate (360 mg/kg i.p.). A Teflon-isolated bipolar stainless steel electrode was stereotaxically implanted into the right anterior basolateral amygdala (BLA) and served as stimulation- and recording-electrode. The stereotaxic coordinates in millimeter relative to bregma according to the atlas of Paxinos and Watson (2007) were: AP, 2.2; L, 4.7; V, 8.3. One screw, placed above the left parietal cortex, served as the indifferent reference electrode. Additional skull screws and dental acrylic cement anchored the entire headset. After surgery, the animals were allowed to recover for a period of four weeks. During postsurgical recovery, rats were treated for one week twice daily with marbofloxacine. 2.3. Electrical induction of a self-sustained status epilepticus About four weeks after electrode implantation, 133 rats were electrically stimulated over a time period of 25 min via the BLA electrode for induction of a selfsustained status epilepticus (SSSE) as previously described (Brandt et al., 2006). For the stimulation, an Accupulser A310C stimulator connected to a Stimulus Isolator A365 (World Precision Instruments, Berlin, Germany) was used. The stimulus consisted of 100 ms trains of 1 ms alternating positive and negative square wave pulses.

Prophylactic treatment with valproate or NS1209 after SE - Experimental protocol -

Induction of SE for 4 h Four weeks

Perfusion 24 hours – 7 days

Prophylactic treatment with VPA, NS1209 or vehicle after SE (for dosing protocols see Table 1) Electrode implantation

Implantation of venous catheter

Daily drug administration Blood sampling for drug analysis in plasma

Six to 16 weeks No treatment

Video/EEG recording of seizures in NS1209treated rats and SE controls

Histological and immunohistological analyses of neuronal damage in the hippocampal formation

Fig. 1. Schematic illustration of the experimental protocol used in this study. Note that venous catheters were only implanted in rats in which VPA or NS1209 were continuously infused after SE.

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047 The train frequency was 2 Hz and the intra-train pulse frequency was 50 Hz. The intensity of the stimulus was 700 mA. In all rats, the EEG was recorded via the BLA electrode during SSSE and up to 20 h after termination of SSSE by diazepam (see below). Four hours after the induction of the SSSE, the rats were injected with diazepam (10 mg/kg i.p.) to terminate the seizure activity. If necessary, the application of this dose of diazepam was repeated twice within 30 min, which was only needed in 12 rats. As previously reported (Brandt et al., 2003), diazepam completely stopped motor and EEG seizure activity in all rats. This differentiates the BLA-stimulationinduced SE from chemically induced SE, which can not be terminated completely by diazepam (Bankstahl and Löscher, 2008). Furthermore, while clinical and EEG seizures typically recur several hours following termination of SE by diazepam in chemical models such as the pilocarpine model (Löscher and Brandt, 2010), this does not occur with BLA-stimulation-induced SE, but only some rats show transient recurrence of low-amplitude discharges in the EEG which are not associated with any convulsive behavioral abnormalities (see Fig. 1F in Brandt et al., 2003). This was also observed in the present study in that the EEG normalized in all rats following injection of diazepam, but some rats (up to 30% per group) exhibited recurrence of low-amplitude discharges, which lasted several hours and were observed both in the diazepam and diazepam plus VPA groups. For treatment with NS1209, SE was not interrupted with diazepam because in preliminary experiments subsequent administration of diazepam and NS1209 induced mortality. Furthermore, NS1209 alone has previously been shown to block electrically induced SE without recurrence of seizures in the EEG when administering the dosing protocol also used in the present study (Pitkänen et al., 2007). In the present study, in which NS1209 was given after 4 h of SE, SE stopped shortly after i.v. injection of NS1209 at a bolus dose of 20 mg/kg (see below), so that it was not necessary to administer diazepam for SE termination in this group. As in the diazepam and diazepam plus VPA groups, NS1209 terminated both the clinical and EEG seizures, so that SE termination was comparable in all treatment groups. One hundred twenty of the 133 stimulated rats developed an SSSE after the BLAstimulation. As previously reported (Brandt et al., 2003), sustained BLA-stimulation induces three different types of SSSE. In the present study, 80 rats (60%) had a generalized convulsive (type III) SSSE and 38 rats (29%) developed a focal SSSE with frequently occurring generalized seizures (type II) (Brandt et al., 2003). Only two rats (1.5%) developed a pure focal SE (type I); these rats were not used for further experiments. The typical paroxysmal EEG alterations occurring during these types of SSSE have been described in detail by us previously (see Fig. 1 in Brandt et al., 2003). Previous experiments by our group have shown that both type II and type III SSSE induce spontaneous recurrent seizures within 2e3 months in >90% of female Sprague-Dawley rats and about the same extent of neuronal damage in the hippocampus (Brandt et al., 2003), so that all rats with these types of SSSE were used for further experiments. None of the rats died during SSSE. Following termination of SSSE, rats received an i.p. injection of 4 ml of 0.9% NaCl to prevent dehydration. Heat lamps were used to minimize the hypothermia associated with administration of VPA after SE. Furthermore, if necessary, rats were hand-fed with baby food (Babydream milk pudding with banana; Rossmann, Großburgwedel, Germany) over the days after SE until they resumed normal feeding behavior. Body weight was measured once daily following SE. 2.4. Drug treatment After termination of a type II or III SSSE, the 118 rats were randomly divided into vehicle and drug treatment groups, so that the severity of SSSE did not significantly differ between groups. Overall, six different treatment protocols were compared, using either intermittent i.p. or continuous i.v. administration (Table 1). For administration of VPA, a commercial aqueous solution of the sodium salt (100 mg/ml; OrfirilÒ, Desitin, Hamburg, Germany) was diluted with distilled water and injected at a volume of 4.64 ml/kg (for 400 mg/kg) or 2.32 ml/kg (for 200 mg/kg) i.p., respectively. For i.v. infusion, the concentration of VPA was adjusted individually (based on body weight) to achieve an infusion rate of 25 mg/kg/h in 1 ml/kg/h (see 2.4.2). NS1209 was kindly provided by Neurosearch A/S (Ballerup, Denmark). It was

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dissolved in 0.1 M NaOH and then diluted with 0.9% NaCl to obtain a concentration of 10 (bolus) or 4.3 mg NS1209/ml. Infusion volume was 1 ml/kg/h. Some of the rats died or had to be killed because of poor general condition during treatment with VPA (8 of 66 rats) or NS1209 (1 of 15 rats) and additional rats had to be excluded because of catheter problems during continuous infusion, so that the overall group size for final analysis of neurodegeneration was 95 rats (Table 1). In the vehicle SE control groups (see below), 2 of 24 rats that received 0.9% NaCl i.p. (instead of VPA) died. 2.4.1. Intermittent treatment with VPA For intermittent i.p. treatment with VPA, we used the dosing protocol recently shown to result in neuroprotection in the BLA-stimulation SE model (Brandt et al., 2006), but with shorter duration of treatment. This dosing protocol had been based on pharmacokinetic studies with VPA in female Sprague-Dawley rats with the aim to maintain effective drug plasma levels (>50 mg/ml) during treatment (Brandt et al., 2006). For this purpose, VPA was administered as a bolus injection of 400 mg/kg i.p. immediately following interruption of SE by diazepam after 4 h of SE. From the next day on, VPA was administered three times per day (7 a.m., 3 p.m. and 11 p.m.) at a dose of 200 mg/kg i.p. over a period of 4 weeks (Brandt et al., 2006). For the present experiments, three groups of rats were treated with this dosing protocol. In the first group (“24 h VPA i.p.”; final group size ¼ 8 rats), only the bolus dose of 400 mg/kg VPA was administered after SE, resulting in effective plasma levels for about 24 h (Brandt et al., 2006). In a second group (“48 h VPA i.p.”; n ¼ 12), the bolus dose (400 mg/kg) was injected at the first day, followed by three times daily 200 mg/kg on the second day following SE. In the third group (“7 d VPA i.p.”; n ¼ 10), rats were treated with the bolus dose on day one, followed by 6 days with three times daily 200 mg/kg. Vehicle SE controls (n ¼ 20) received one i.p. administration of 4.64 ml/kg 0.9% NaCl (i.e., the injection volume of the VPA bolus administration). 2.4.2. Continuous i.v. treatment with VPA Due to the rapid elimination of VPA in rats (Löscher, 2007), intermittent i.p. administration of VPA is associated with large variation of drug levels (Löscher et al., 1989; Brandt et al., 2006). Furthermore, even if plasma VPA levels within or above the “therapeutic range” (40e100 mg/ml) known from patients with epilepsy can be maintained by three times daily i.p. injection of 200 mg/kg in rats (Brandt et al., 2006), this range may be too low to achieve neuroprotection by short term treatment with VPA in rats. Thus, we decided to add groups in which VPA was continuously infused i.v. to maintain drug levels above the therapeutic range known from epilepsy patients. For this purpose, different infusion rates were tested in preliminary experiments, resulting in a final protocol consisting of i.p. injection of a bolus dose of 200 mg/kg, immediately followed by continuous i.v. infusion with 600 mg/kg/d for either 24 h (“24 h VPA i.v.”; n ¼ 12) or 5 days (“5 d VPA i.v.”; n ¼ 10). Since prolonged administration of VPA in rats has been reported to result in enhanced drug elimination (Fisher et al., 1991; Löscher and Hönack, 1995) because VPA induces its own metabolism in this species (Fisher et al., 1991), the infusion rate was increased to 700 mg/kg in the “5 d VPA i.v.” group starting with the second day of infusion (Table 1). A control group (n ¼ 12) received i.v. infusion of 0.9% NaCl for 24 h (Table 1). Continuous infusion was performed through a chronically implanted catheter in the jugular vein in freely moving rats as described in detail recently (Brandt et al., 2010). The catheter was implanted one day before induction of SE (Fig. 1). 2.4.3. Continuous i.v. treatment with NS1209 The dosing protocol used for NS1209 was based on a previous study of Pitkänen et al. (2007) in which treatment with a 20 mg/kg i.v. bolus followed by 5 mg/kg/h infusion for 24 h was neuroprotective against SE-induced hippocampal neurodegeneration. In the present study, the 20 mg/kg i.v. bolus was slowly injected i.v. over a period of 5 min, followed by continuous infusion with 5 mg/kg/h (i.e., 120 mg/kg/day). 2.5. Analysis of plasma levels of VPA To exclude erroneous i.p. injections, blood (0.3e0.5 ml) was sampled by puncture of the retro-orbital vasculature (after local anesthesia of the eye with tetracain)

Table 1 Overview about the different SE treatment groups. An additional group of 23 naive controls was used for comparison with the SE groups. Drug

Route of administration

Duration of treatment after SE

Dosing on day 1

Dosing on day 2 and thereafter

Final group size for histology

Vehicle (0.9% NaCl)

i.p. i.v. infusion i.p.

Once 24 h Once 48 h 7 days 24 h 5 days 24 h

4.64 ml/kg 24 ml/kg/day 400 mg/kg (bolus)

e e e 200 mg/kg t.i.d. 200 mg/kg t.i.d. e 700 mg/kg/day e

20 12 8 12 10 12 10 11

Valproate

i.v. infusion NS1209

i.v. infusion

200 mg/kg i.p. (bolus) plus 25 mg/kg/h (600 mg/kg/day) infusion 20 mg/kg i.v. (bolus) plus 5 mg/kg/h (120 mg/kg/day) infusion

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15e60 min after the bolus VPA injection for drug analysis in plasma. Depending on the treatment protocol, a second and third blood sample was taken during subsequent treatment, including the experiments with continuous i.v. infusion of VPA. VPA plasma concentrations were determined by high pressure liquid chromatography (HPLC) with ultraviolet detection as described previously (Baltes et al., 2007). 2.6. Monitoring of spontaneous seizures after treatment with NS1209 Because Pitkänen et al. (2007) had not determined whether treatment with NS1209 after SE affected the development of spontaneous seizures, 12 NS1209treated rats and 12 NaCl-treated SE controls were used to monitor spontaneous recurrent seizures over one week, starting 10 weeks after treatment. The VPAtreated rats were not monitored for occurrence of seizures, because our previous study had shown that prophylactic treatment with VPA after SE does not prevent or modify the development of spontaneous recurrent seizures (Brandt et al., 2006). The NaCl- and NS1209-treated rats were continuously EEG- and videomonitored for 7 days (24 h/d) by a combined video- and EEG-detection system. For EEG-monitoring, the system consisted of 16 one-channel amplifiers and an analogue-digital converter (PowerLab/800s, ADInstruments Ltd, Sydney, Australia). The data were recorded and analyzed with the Chart4 or LabChart 6 for windows software (ADInstruments). The sampling rate for the EEG-recording was 200 Hz. A high pass filter of 0.1 s and a low pass filter of 60 Hz was used. Simultaneously to the EEG-recording, the rats were video-monitored using four light-sensitive blackewhite cameras (CCD-Kamera-Modul, Conrad Electronic, Hannover, Germany) which allowed video-recording of up to four rats per camera. The cameras were connected to a multiplexer (TVMP-400, Monacor, Bremen, Germany) which converted the signals from the four cameras to a video recorder (Time Lapse recorder, Sanyo TLS-9024P, Monacor, Bremen, Germany). To allow video-recording of seizures during the night, infrared light was used. 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 videorecording was viewed. For rating of seizure severity of spontaneous seizures, a modified Racine’s scale (1972) was used. In addition to seizures observed by video/ EEG-recording, seizures observed during handling, other manipulations of the animals or by direct observation of the rats in their home cages were noted. 2.7. Analysis of neurodegeneration in the hippocampal formation About 6e8 weeks (VPA-treated groups and part of controls) or 16 weeks (NS1209 and other part of controls) after treatment, rats were deeply anesthetized with chloral hydrate and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). In most rats, 6 series of 40 mm-thick transverse (coronal) sections of the brain were cut on a freezing microtome. In one group (24 h VPA i.p.), only two series of sections were cut, so that not all stainings could be performed in this group (see Results). In all groups, one of the series was Nissl-stained with thionin for verifying correct localization of the BLA electrode and visualization of cell loss in the hippocampal formation. The other series were used for immunohistochemical analysis of subpopulations of hilar neurons. As non-SE controls, we used 23 age-matched rats. This control group consisted of 15 naive, non-implanted rats and 8 rats that were implanted with EEG electrodes but not stimulated. Because histological data did not differ between naive and electrode-implanted controls, data from both groups were averaged for comparison with SE rats. 2.7.1. Thionin-stained sections In a first step, thionin-stained sections of the following subregions of the hippocampal formation were visually inspected for damage: CA1, CA3a, CA3c/CA4, and dentate gyrus. For this purpose, four section levels of the hippocampus (2.8, 3.8, 4.8 and 5.8 mm posterior from bregma) were analyzed from each rat, and both the ipsilateral (right) and contralateral hemispheres (with respect to the BLA electrode) were inspected. At 5.8 mm, the dorso-septal and ventro-temporal parts of the hippocampal formation were separately inspected. Severity of neuronal damage in a section was semi-quantitatively assessed by a grading system similar to that previously described by Brandt et al. (2003, 2006) and Pitkänen et al. (2007) as follows: score 0, no obvious damage; score 1, abnormal appearance of the structure without clear evidence of visible neuronal loss; score 2, lesions involving 20e50% of neurons; score 3, lesions involving >50% of neurons. In this respect, it is important to note that neuronal loss must exceed 15%e20% before it is reliably detected by visual inspection (Fujikawa et al., 2000). Visual assessment was conducted blindly with respect to the treatment status of the animal. Compared to the extent of cell loss in areas CA1 and CA3 after SE, which can easily be scored by microscopic examination, loss of neurons in the dentate hilus is more difficult to assess without cell counting. Therefore, in a second step, polymorphic neurons (i.e., mossy cells and interneurons) were counted at four section levels in the dentate hilus of the hippocampal formation. The hilus was defined as the inner border of the granule cell layer and two straight lines connecting the tips of the granule cell layer and the proximal end of the CA3c region. All profiles >8 mm within these borders were counted at a 200 magnification (Axioscope, Carl Zeiss,

Germany). The area of each hilus counted was measured with the Axiovision software (Carl Zeiss, Germany). In addition to calculating average numbers of neurons in the hilus, neuronal densities (neurons per unit area) were calculated, because the area of the hilus often slightly varied from section to section at one section level within the same group of rats, most likely as a result of slight variation in the exact section level per rat. All neuron counts were performed in a blinded fashion. During visual inspection of sections, inter-individual variation in the localization of neuronal damage along the anterioreposterior axis of the hippocampus became evident, so that averaging damage from one or more section levels may form a bias when determining the extent of damage in a group of rats or the effect of neuroprotective drugs on this damage. For instance, in one rat the damage may be present only at 5.8 mm from bregma whereas in another rat damage only occurs in a more anterior section. To deal with this problem, we developed a system that permits a semi-quantitative assessment of the extent of damage (i.e., space over which the lesions extend) across all section levels per animal. For this purpose, the extent of damage was assessed in 13 hippocampal locations per hemisphere in each rat : CA1, CA3 (i.e., CA3a and CA3c), and hilus at 2.8, 3.8, 4.8 and 5.8 mm from bregma plus hilus in the ventro-temporal part of the dentate gyrus at 5.8. For CA1 and CA3, a score 2 or 3 lesion (assessed by the grading system described above) was considered neuronal damage, while neuronal damage in the hilus was assumed if the number of hilar neurons or neuronal density in the respective location of an SE rat was at least 20% below the lowest individual control value in this location. If damage was obvious in a region by this procedure, a score of 1 was given, so that a maximum cumulative score of 26 (13 locations/hemisphere  2 hemispheres) could be obtained per rat. This method also allowed us to determine the incidence of rats without any obvious lesion per group. 2.7.2. Immunohistological staining of hilar neurons Three subpopulations of hilar neurons were separately stained immunohistochemically, i.e., somatostatin- and parvalbumin-positive GABAergic interneurons and glutamatergic mossy cells, using previously published methods (Fujise and Kosaka, 1999; Gernert et al., 2000; Volk et al., 2006). For each staining protocol, three sections (2.8, 3.8, and 4.8 mm posterior from bregma) of each rat were used; parvalbumin staining was also performed for the ventro-temporal part of the hilus (at 5.8 mm). Because of the limitation of sections, it was not possible to perform all stainings in all groups. In short, the sections were thoroughly washed in 0.05 M Trisbuffered saline (TBS; pH 7.6) and incubated in 0.5% H2O2 in order to block endogenous peroxidase activity. Then, the sections were preincubated for 60 min with a blocking solution in TBS containing 2% bovine serum albumin, 0.3% Triton X-100, and 10% serum of the species of the secondary antibody. Then sections were incubated overnight with the primary antibody, rinsed in TBS, and placed for 60 min in biotin-labeled secondary antiserum (1:500). The following primary and secondary antibodies were used for staining of somatostatin- and parvalbumin-positive neurons and glutamatergic mossy cells, respectively. (1) polyclonal rabbit antisomatostatin (1:2000; Bachem, St. Helens, UK), polyclonal pig anti-rabbit (Dako, Hamburg, Germany); (2) monoclonal mouse anti-parvalbumin (1:4000; SigmaeAldrich, Steinheim, Germany), polyclonal rabbit anti-mouse (Dako); (3) polyclonal rabbit anti-glutamate (AMPA) receptor (GluR) 2/3 (1:200; Millipore, Schwalbach, Germany), polyclonal goat anti-rabbit (Dianova, Hamburg, Germany). Sections were then washed again and incubated in horseradish peroxidase-labeled streptavidin (1:375; Dako) for 60 min, followed by the nickel-intensified diaminobenzidine reaction (0.05% 3,3-diaminobenzidine and 0.6% ammonium nickel sulfate; both from SigmaeAldrich) in the presence of 0.01% H2O2 for 15 min. Sections were washed, air-dried, treated with xylol for 1 min, and coverslipped with Entellan (Merck, Darmstadt, Germany). Following staining, the respective neuronal subpopulations were counted in the hilus as described above for thionin-stained neurons. All countings were performed in a blinded fashion. With respect to staining mossy cells by an antibody against GluR 2/3 (Fujise and Kosaka, 1999), it should be noticed that the GluR 2/3 receptor is also expressed by a subset of smaller hilar cells, which presumably represent interneurons (Sloviter et al., 2003). Furthermore, in addition to hilar interneurons, parvalbumin also stains GABAergic basket cells, which are located at the margin of the granule cell layer and the hilus and were not included in counting of parvalbumin-positive hilar cells in this study.

2.8. Statistics Depending on whether or not data were normally distributed, either parametric or nonparametric tests were used for statistical evaluation. Fisher’s exact test was used for calculation of significant differences in the incidence of lesions and spontaneous seizures, and of the “neuroprotection score” (see Results). The significance of differences in numbers and densities of neurons between sham controls and the vehicle and VPA-treated post-SE groups was calculated by analysis of variance (ANOVA), either for parametric or nonparametric data (KruskaleWallis test). Posthoc testing for individual differences was done in a selective manner, comparing only those groups with obvious alterations with respective controls in order to minimize the potential for a-error. Depending on the number of post-hoc comparisons and the type of data, either the Bonferroni test, Dunnett’s test, Dunn’s test,

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047 t-test, or U-test was used for post-hoc testing. All tests used were two-sided; a P < 0.05 was considered significant.

3. Results As described in the Methods section, sustained electrical stimulation of the BLA induced SSSE in the majority (90%) of rats. In the two vehicle (NaCl) SE control groups (Table 1), mortality was low (2 of the 24 rats treated with NaCl i.p. and none of the 12 rats treated with NaCl i.v. died following SSSE). However, as reported previously (Brandt et al., 2006), mortality was greater in the VPAtreated groups (see Methods). All SE rats lost 10e20% body weight during 48 h post-SE (not illustrated). NaCl controls and rats treated over 24 h with either VPA or NS1209 rapidly regained preSE body weight thereafter, whereas body weight gain was delayed in rats treated over several days with VPA. 3.1. Neurodegeneration after SE induced by sustained stimulation of the BLA As reported previously (Brandt et al., 2003; Volk et al., 2006; Bethmann et al., 2008), a 4 h SE induced by sustained electrical stimulation of the basolateral nucleus of the amygdala caused neurodegeneration in the hippocampal formation of both hemispheres. Nineteen of the 20 SE controls (95%) that received i.p. injection of vehicle (saline) instead of drug (Table 1) exhibited

obvious lesions in CA1, CA3 and/or hilus (Fig. 2A). The most consistent damage was neuronal loss in the hilus (Fig. 3), which was observed in all 19 SE controls with hippocampal damage, whereas obvious lesions in CA1 and/or CA3 were observed in only 10 of the 20 rats. Except in a few rats, damage in the hilus or the pyramidal cell layers was not determined throughout all section levels, but some rats exhibited obvious damage at only one section level. The temporal part of the hippocampal formation was equally affected by lesions than the septal part. When we scored lesions in 26 locations of the hippocampal formation in both hemispheres as described in the Methods section, individual scores in SE controls ranged from 1 to 24, demonstrating large variability in hippocampal damage following SE (Fig. 2B). Only 3 of the 20 SE vehicle controls showed overall scores of >20. In the second SE control group, which had received i.v. infusion of 0.9% NaCl for 24 h following SE, 10 of 12 rats (83%) exhibited damage in the hippocampal formation, which was not significantly different from the control group with i.p. saline injection (Fig. 2A). In addition, when scoring lesions in 26 locations, both control groups did not differ significantly (Fig. 2B). When the two control groups were combined for comparison with drug treated groups, the statistical results shown in Fig. 2 remained the same. In general, the two SE control groups also behaved similar in the numerous evaluations of neuronal counts in the hilus described in the following, so that only the SE NaCl i.p. group was used for subsequent comparisons with the various drug treatments.

B

Percent of rats with lesions in CA1, CA3 or hilus

Extent of lesions in CA1, CA3 and hilus

100

12

80

10

*

*

60

Score (max. = 26)

*

40 20

8

4

48

SE

SE

24

SE

h

Score (max. = 10)

2

iv N S12 09 iv

iv

VP A d

5

h 24

li v 24

h

VP A

ip 24

h

N aC

VP A d

7

h

VP A

ip

ip

0

48

*

N SE aC li 24 p h VP SE A 48 ip h VP SE A ip 7 d VP SE A 24 ip h N SE aC li 24 v h VP SE A iv 5 SE d VP 24 A h iv N S1 20 9 iv

*

*

* li p

2

4

N aC

4

6

VP A

6

h

48

h 24

SE

SE

Extent of lesions in hilus 8

SE

Score (max. = 16)

h

N aC

A ip VP A 7 ip d VP SE A i p SE N aC 24 li v h SE VP A SE 5 i 24 d V v PA h N S1 iv 20 9 iv

li p N aC SE

SE

VP

D

Extent of lesions in CA1 and CA3

SE

li p VP A i p h SE VP A 7 i p d VP SE A i p SE N aC 24 li v h V SE PA SE 5 i 24 d V v PA h N S1 iv 20 9 iv

0

8

0

*

*

2

0

C

*

6

24

Percent

A

1037

Fig. 2. Effect of treatments on the incidence and extent of SE-induced lesions in rats. Lesions were assessed in thionin-stained sections in 13 hippocampal locations per hemisphere in each rat: CA1, CA3 (i.e., CA3a and CA3c), and hilus at 2.8, 3.8, 4.8 and 5.8 mm from bregma plus hilus in the ventro-temporal part of the dentate gyrus at 5.8. For CA1 and CA3, a score 2 or 3 lesion (assessed by the grading system described in the Methods section) was considered neuronal damage, while neuronal damage in the hilus was assumed if number of hilar neurons or neuronal density in the respective location of an SE rat was at least 20% below the lowest individual control value in this location. If damage was obvious in a region by this procedure, a score 1 was given, so that a maximum cumulative score of 26 (13 locations/hemisphere  2 hemispheres) could be obtained per rat. “A” illustrates the percent of rats with any lesion (independent of its extent) per group, “B” the overall cumulative score, reflecting the extent of lesions in CA1, CA3 and dentate hilus in the two hemispheres, “C” extent of lesions in CA1 and CA3, and “D” extent of lesions in the dentate hilus. In B, C, and D, data are shown as means and SEM; for group sizes see Table 1. Significance of differences to rats which were treated with NaCl after SE (“SE NaCl ip”) is indicated by asterisk (P < 0.05).

1038

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

Fig. 3. Representative coronal sections of the dentate gyrus in naive controls and rats after SE. A and B are thionin-stained sections, whereas immunohistochemistry was used to stain parvalbumin-expressing interneurons (C,D), somatostatin-expressing interneurons (E,F), or GluR 2/3-expressing mossy cells (G,H), respectively.

When thionin-stained neurons were counted in the hilus of the septal hippocampal formation at 4 section levels (ranging from 2.8 to 5.8 mm from bregma), SE rats had significantly fewer neurons than naive controls in both hemispheres (Fig. 4A,B). This effect was observed at all section levels, so that data were averaged for further analysis (Fig. 4A,B). Average cell loss in the septal parts of the dentate gyrus (illustrated in Fig. 4A,B) was 28% (left hemisphere) and 25% (right hemisphere), respectively. In the ventro-temporal part of the dentate gyrus (at 5.8 mm from bregma), neuronal counts in the hilus were much higher (w600 neurons per section) than in the septal parts (w120 neurons/section; including the dorso-septal part at 5.8 mm), so that data from the septal and temporal parts of the hilus were not averaged, but the temporal part was evaluated separately (Fig. 5). Following SE, neuronal loss in the ventrotemporal part of the hilus was 40% in the left and 35% in the right hemisphere (P < 0.05; Fig. 5A,B).

Immunohistochemical staining of subpopulations of hilar neurons disclosed marked differences in the susceptibility of these subpopulations to damage induced by SE (Figs. 3 and 4). The largest effect was seen for parvalbumin-stained interneurons, which were significantly reduced by 48% (left hilus) and 44% (right hilus) in the septal part of the dentate gyrus, respectively (Fig. 4C,D). In the ventro-temporal part of the hilus, which contained significantly more parvalbumin-stained neurons (w30 neurons per section) than the septal parts (w12 neurons per section), the SE-induced reduction of stained parvalbumin-immunoreactive neurons was 61% (left hilus) and 56% (right hilus), respectively (Fig. 5 E,F). Somatostatin-stained interneurons were less severely affected with average significant decreases of 24% (left hilus) and 28% (right hilus), respectively (Fig. 4E,F). The number of hilar mossy cells, which were stained by an antibody to GluR 2/3, was not significantly reduced by SE, although there was a tendency (about 20%; P ¼ 0.0554) of a reduction versus naive controls (Fig. 4G,H).

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

A

B

Thionin, left hilus

Thionin, right hilus 150

150

*

*

*

100

Neurone [n]

50

*

*

100

*

50

C

Neurons [n]

*

10

*

*

*

5

*

*

iv 20 9

S1

N h

*

*

*

*

5

iv

iv N h

5

SE

SE

24

SE

SE

S1 20 9

VP A d

VP A h

VP A 24

d 7

h SE

48 SE

h

ip

ip VP A

N aC

l

e N ai v

iv

iv N

d 5 24

SE

SE

S1 20 9

VP A

VP A h

d

24

7

h SE

48 SE

iv

ip VP A

ip VP A

N aC l SE

N ai ve

iv

0

E

F

Somatostatin, left 80

Somatostatin, right 60

60

*

40

Neurons [n]

*

20 0

40

*

*

20

40 20

h

5

N

S1 20 9

VP A d

h

24

SE

SE

iv

iv

iv VP A

ip VP A

7

24

d

VP A h

SE

Neurons [n]

60

SE

48 SE

SE

H

GluR 2/3, left 80

ip

l aC N

N ai ve

iv S1 20 9

24

SE

h

5

N

h

d

VP A

VP A

iv

iv

ip 24 SE

SE

SE

48

7

h

d

VP A

VP A

ip

l aC N SE

N ai ve

0

SE

GluR 2/3, right

80 60 40 20

SE

24

h

24

N

h

S1 20 9

VP A

iv

iv

l aC N SE

ai v N

S1 20 9 h 24

SE

SE

24

N

h

SE

N

VP A

iv

iv

l aC

e ai v N

e

0

0

SE

Neurons [n]

iv

iv d 5

10

SE

Neurons [n]

Parvalbumin, right hilus 15

0

Neurons [n]

24

SE

D

Parvalbumin, left hilus 15

G

VP A

ip

VP A SE

SE

7

24

d

h

VP A

ip

ip

SE

SE

SE

SE

24

VP A h

h 24

48

N

N h

5

VP A

ai ve

iv

iv

S1 20 9

VP A d

VP A h

24

SE

7

SE

SE

SE

iv

ip

ip d

h 48

24 SE

VP A

ip

VP A

N aC l

h

VP A

N ai ve

SE

N aC l

0

0

SE

Neurons [n]

1039

Fig. 4. Number of neurons per hilus section in the septal part of the hippocampal formation. Neurons were counted in four sections (at 2.8, 3.8, 4.8 and 5.8 mm from bregma) and the average number of neurons determined in these 4 sections per rat was used for calculating group means (þSEM). See Table 1 for number of rats per group. Data are illustrated for thionin-stained cells (A,B), parvalbumin-stained cells (C,D), somatostatin-stained cells (E,F), and GluR 2/3-stained cells (G,H), respectively. Significance of differences to naive controls (n ¼ 20) is indicated by asterisk (P < 0.05).

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

B

Thionin, right hilus

600

C

iv NS 12 09 iv

iv

h

5 24 SE

D

Thionin, left hilus

1500

VP A d

VP A h

24

SE

SE

7

48

d

h

ip

ip

ip

VP A

aC l

VP A SE

SE

h

24

N S

N

12 09

ai ve

iv

iv d 5 24

SE

SE

SE

7

24

d

h

VP A

VP A

ip

ip

VP A

VP A h

48

24 SE

SE

SE

N

VP A

h

SE

N

iv

0

ip

200

0

aC l

200

*

*

400

N

*

*

400

VP A

800

600

SE

800

Neurons [n]

1000

h

Thionin, left hilus 1000

a iv e

Neurons [n]

A

SE

1040

Thionin, right hilus

1000

* * * * *

*

500

Neurons/mm²

Neurons/mm²

1500

1000

* *

* *

* 500

20

*

*

*

*

Neurons [n]

30

*

10 0

30

*

20

*

iv NS 12 09 iv

iv

h

*

*

*

10

iv 12 09

N S

d 5 SE

24

h

SE

SE

iv

iv

iv

N S

12 09

VP A d SE

24

h

5

h SE

24 SE

7

d

h

VP A

VP A

ip

ip VP A

aC l N

iv

N S

d SE

24

h

5

h SE

12 09

VP A

iv VP A

ip 24 SE

SE

7

d

h

VP A

VP A

N SE SE

48

N

SE

0

iv

0

ip

20

aC l

20

*

*

*

40

48

*

40

60

SE

*

*

ai ve

*

*

*

80

N

60

Neurons/mm²

80

Parvalbumin, right hilus

100

SE

H

Parvalbumin, left hilus

100

iv VP A

iv VP A h

VP A 7

24

d

h SE

SE

SE

24

ip

ip VP A

aC l N SE

N

h

48

d 5 SE

SE

ai ve

iv N S1 20 9 iv

VP A

iv h

VP A 7

24

d

h SE

SE

VP A

ip

ip VP A

aC l N SE

48

N

ai ve

0

ai ve

Neurons/mm²

VP A d

5 SE

Parvalbumin, right hilus 40

40

G

24

24

SE

SE

SE

SE

7

48

d

h

VP A

VP A

ip

ip

ip h

VP A

VP A

aC l 24 SE

F

Parvalbumin, left hilus 50

Neurons [n]

N

ai ve N

SE

h 24 +

SE

E

h

iv

iv N S

d 5 +

24

SE

SE

0

12 09

VP A

iv

ip h

VP A d +

+

7

48

SE

SE

+

24 + SE

VP A

ip

ip

VP A h

VP A

h

na iv

SE

0

Fig. 5. Number of neurons and neuron density (neurons per unit area) per hilus section in the ventro-temporal part of the hippocampal formation. Data are shown as means þ SEM; number of rats per group are shown in Table 1. Data are separately illustrated for thionin-stained neurons (A,B,C,D) and parvalbumin-stained neurons (E,F,G,H), respectively. Significance of differences to naive controls (n ¼ 20) is indicated by asterisk (P < 0.05).

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

When the reduction in neuron numbers in thionin-stained septal sections and immunohistochemically stained septal sections of the dentate gyrus was compared (Fig. 4), the average reduction for left and right hemisphere was 26.5% for thionin-stained sections, and 46%, 26%, and 21% for parvalbumin-, somatostatin-, and GluR 2/3-stained sections, respectively. In this respect, it is important to note that the size of the three immunostained subpopulations of hilar neurons was quite different in controls with about 9% of all immunostained neurons being parvalbuminpositive, 34% somatostatin-positive, and 55% GluR 2/3-positive, respectively, so that the relative reduction of these subpopulations in SE rats contributed differently to the overall reduction of neurons in the hilus. We therefore calculated also the sum of all immunostained neurons in controls and SE rats, resulting in an average percent reduction of immunostained neurons in SE rats of 26.2%, which was very similar to the 26.5% calculated for Nissl-stained hilar neurons. This indicates that the reduction of immunostained neurons reflects loss of these neurons and not just reduced marker expression. The area of the hilus was not significantly altered by SE (not illustrated). When densities (neurons per unit area) of hilar neurons were calculated, SE rats had significantly lower neuronal densities with all staining methods (Fig. 6). Compared to naive controls, average decreases (left/right hilus) following SE were 28% and 31% for thionin-stained neurons, 47% and 51% for parvalbumin, 19% and 24% for somatostatin, and 37% and 42% for GluR 2/3, respectively (Fig. 6). In the ventro-temporal part of the dentate gyrus (at 5.8 mm from bregma), respective decreases were 44% and 43% for thionin-stained neurons (Fig. 5C,D), and 58% and 59% for parvalbumin-stained neurons (Fig. 5G,H), respectively, i.e., the density decreases tended to be more marked in the temporal part of the hilus as already observed for the number of neurons/hilus section (see above). 3.2. Neuroprotective effects of VPA treatment As reported previously (Brandt et al., 2006), adverse effects observed after 400 mg/kg VPA following interruption of SE by diazepam were much more severe than observed in normal rats without SE and were characterized by pronounced decrease of body temperature and prolonged loss of righting reflexes. Even though we tried to counteract hypothermia by the use of heat lamps (see Methods), some of the rats died or had to be euthanized because of poor general condition (see Methods). With respect to the increased toxicity of VPA after SE, it has to be noted that, after induction of an SE, the general condition of most of the rats is severely impaired, including reduced body weight, reduced locomotor activity and sometimes no independent intake of food and water at all. By the time the rats had recovered from SE, no VPAinduced sedation or ataxia could be observed in groups with prolonged treatment, but the only obvious adverse effect was wet-dog shaking. Plasma levels of VPA obtained during treatment are shown in Table 2. Following the bolus dose of 400 mg/kg i.p. in the “24 h VPA i.p.” group and groups with longer intermittent i.p. administration (see Table 1), peak plasma levels of >700 mg/ml were determined within 0.25e1 h after bolus injection, followed by rapid decline of plasma levels to w60 mg/ml after 15 h (Table 2). Trough levels on day 3 were still within the “therapeutic range” (40e100 mg/ml) known from patients with epilepsy, whereas trough levels on day 5 were only 17 mg/ml on average, indicating induced elimination of VPA as previously reported in rats (Fisher et al., 1991; Löscher and Hönack, 1995). During continuous infusion, markedly higher plasma levels could be maintained compared to intermittent i.p. administration, but these levels also clearly declined despite

1041

continuous infusion, again indicating enhanced elimination of VPA during prolonged treatment (Table 2). As shown in Fig. 2A, of the 5 different treatment protocols compared for VPA, only intermittent treatment for 7 days and continuous infusion for 5 days significantly reduced the percent of rats with hippocampal lesions compared to SE controls. The extent of lesions in the hippocampal formation was significantly reduced by continuously infusing VPA for either one or 5 days (Fig. 2B). Infusion for 24 h almost completely prevented the lesions in CA1 and CA3 (Fig. 2C), whereas the lesions in the hilus were reduced by both infusion protocols, but not by any of the intermittent i.p. dosing protocols (Fig. 2D). When thionin-stained hilar cell counts in the septal hippocampal formation were averaged from all four section levels, continuous infusion of VPA was more effective than intermittent i.p. administration to protect neurons from damage (Fig. 4A,B). The only treatment that partially prevented the reduction of parvalbumin-stained interneurons in the hilus was 24 h VPA infusion (Fig. 4D), whereas the reduction of somatostatin-positive interneurons was also prevented by intermittent i.p. administration (Fig. 4E,F). The number of mossy cells did not differ between vehicle and VPA-treated groups (Fig. 4G,H). In the temporal part of the hippocampal formation, the loss of thionin-stained neurons was prevented by all VPA treatment protocols except the 48 h treatment with VPA i.p. (Fig. 5A,B). In contrast, none of the VPA treatment protocols protected parvalbumin-stained interneurons in the temporal hilus (Fig. 5E,F). Data obtained by calculating cell density (neurons per unit area) differed in part from data expressed as neurons per hilus. As described in the Methods section, this was obviously due to the fact that the area of the hilus often slightly varied from section to section at one section level within the same group of rats. In the septal part of the hippocampal formation, only the single i.p. VPA bolus dose counteracted the reduced density of thionin-stained neurons induced by SE (Fig. 6A,B). In apparent contrast, only infusion of VPA for 24 h prevented the reduced density of parvalbuminstained neurons (Fig. 6C,D). Data for density of somatostatinstained neurons (Fig. 6E,F) were similar to those obtained for neurons/hilus (Fig. 4E,F) in that most VPA treatment protocols appeared to protect against reduction of such neurons. Density of mossy cells, which was significantly reduced by SE, was increased by 24 h infusion with VPA, which became significant in the left hilus (Fig. 6G,H). SE-induced reduction of density of hilar neurons in the temporal part of the hippocampal formation was most effectively counteracted by 24 h treatment with VPA (Fig. 5C,D). For parvalbuminstained neurons, reduced density was only partially counteracted in the right (ipsilateral) hilus by two of the VPA treatment protocols (Fig. 5G,H). To allow direct comparison of the neuroprotective effects of VPA with the different treatment protocols and staining methods, these effects are summarized in Table 3. Furthermore, a “neuroprotection score” was calculated from these effects (see Table 3), indicating that continuous infusion of VPA for 24 h was the most effective neuroprotective treatment among the different VPA dosing protocols. 3.3. Neuroprotective effects of NS1209 treatment Compared to the different treatment protocols performed with VPA, only one treatment protocol was chosen for NS1209, which was based on previous experiments with this drug in an SE model (Pitkänen et al., 2007). The only obvious adverse effect of this treatment was moderate sedation. The treatment protocol chosen for NS1209 proved to be highly effective in preventing neuronal damage after SE. It significantly reduced the percent of rats with lesions in the hippocampal

1042

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

A

B

Thionin, left hilus

Thionin, right hilus 400

300

*

* * *

* *

200 100

Neurons/mm²

300

*

* *

* *

200 100

40

40

*

20

*

*

*

10

30

*

*

20

iv 9

N iv

N S1 20 9

A VP 24

SE

SE

SE

h

5

d

h

VP 24

d 7

h

SE

F

Somatostatin, left hilus

Somatostatin, right hilus 250

200

200

*

Neurone/mm²

250

150

iv

iv

A

VP A

ip

ip VP A

N aC l

SE

SE

24

h

48

N S

SE

N ai ve

iv 12 09

A VP d 5

SE

SE

SE

iv

iv h

VP d

24

7

h 48

SE

VP A

ip A

ip VP A

N aC l

SE

N ai ve

0

E

*

100 50

*

150

*

100 50

0

G

iv

h

5 24

SE

SE

N S1 20 9

A VP d

h

VP 24

d 7 SE

SE

H

GluR 2/3 left

iv

iv

A

VP A

ip

ip VP A

h

SE

SE

24

h

48

N S

N aC l

N ai ve

12 09

A VP d 5

SE

SE

iv

iv

iv VP A h

VP 24

d SE

7

h 48

SE

ip A

ip VP A

N aC l SE

N ai ve

0

SE

Neurons/mm²

S1 20

VP d

5

*

*

10

0

GluR 2/3, right 400

300

*

*

200 100

300

*

*

200

*

100

iv 20 9

iv h 24 SE

SE

24

h

N S1

VP A

N aC l SE

N ai ve

iv 20 9 N S1 h

24 SE

SE

24

h

SE

VP A

N aC l

iv

0 N ai ve

0

Neurone/mm²

400

Neurons/mm²

h

Parvalbumin, right hilus 50

30

24

SE

SE

SE

D

Parvalbumin, left hilus 50

Neurons/mm²

Neurons/mm²

C

iv

iv

A

ip

VP A

A

7

24

d

h

VP

VP A h

SE

SE

48

24 SE

ip

ip

l aC N

VP A

N ai ve

h SE

24

SE

SE

SE

A VP

5

d

h 24

iv N S1 20 9 iv

iv

ip

VP A

ip

A d

7

h 48

SE

SE

24 SE

VP

VP A

VP A

h

SE

N aC

l

ip

0

N ai ve

0

h

Neurons/mm²

400

Fig. 6. Neuron density (neurons per unit area) in the septal part of the hippocampal formation. Number of neurons and hilus area were determined in four sections (at 2.8, 3.8, 4.8 and 5.8 mm from bregma) and the average neuron density determined in these 4 sections per rat was used for calculating group means (þSEM). See Table 1 for number of rats per group. Data are illustrated for thionin-stained cells (A,B), parvalbumin-stained cells (C,D), somatostatin-stained cells (E,F), and GluR 2/3-stained cells (G,H), respectively. Significance of differences to naive controls (n ¼ 20) is indicated by asterisk (P < 0.05).

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

1043

Table 2 Plasma concentration of VPA after acute and subchronic treatment (for details see Table 1 and Methods). To reduce the number of blood samples per rat, not all time points were sampled in all animals. In groups with intermittent i.p. injection, samples on days 3 and 5 were taken before drug administration in the morning. Data are shown as means  SEM. n.d. indicates not determined. Treatment protocol

VPA concentration in plasma (mg/ml) Day one

400 mg/kg i.p. bolus on day one; subsequent treatment with three times daily 200 mg/kg i.p. 200 mg/kg i.p. bolus plus i.v. infusion with 600 mg/kg/day on day 1; subsequent infusion with 700 mg/kg/day

Day 3

Day 5

n.d.

73  38 (n ¼ 8)

17  4.4 (n ¼ 6)

321  44 (n ¼ 13)

131  12 (n ¼ 12)

65  7.1 (n ¼ 12)

0.25 h

1h

8h

15 h

18 h

719  38 (n ¼ 38)

781  34 (n ¼ 14)

93  18 (n ¼ 10)

64  8.1 (n ¼ 21)

n.d.

n.d.

n.d.

n.d.

formation (Fig. 2A) and completely prevented lesions in the CA1 and CA3 sectors (Fig. 2C). However, hilar cell loss was not significantly reduced by NS1209, although there was a tendency for such an effect (Fig. 2D). In terms of hilar neuron number, NS1209 prevented the reduction of thionin-stained cells, as well as parvalbumin- and somatostatin-stained neurons, in both septal and temporal parts of the hippocampal formation, except for parvalbumin-stained neurons in the right septal hilus (Figs. 4 and 5). Similarly, although less marked neuroprotective effects of NS1209 were observed for neuronal density (Figs. 5 and 6), NS1209 was as effective as the 24 h infusion of VPA in preventing the reduction in density of parvalbumin-stained neurons in the septal hilus (Fig. 6C,D). Furthermore, NS1209 was the only treatment that counteracted the reduction of parvalbumin-stained neurons in the temporal hilus (Fig. 5E,F). Table 3 gives an overview of the neuroprotective effects of NS1209, indicating that, overall, this drug was as effective as 24 h infusion with VPA. It was therefore important to determine whether NS1209 also prevented or modified the development of epilepsy with spontaneous recurrent seizures after SE. 3.4. Effect of NS1209 on development of spontaneous seizures Ten weeks after treatment, spontaneous recurrent seizures were continuously monitored by video/EEG over a one week period in 12 NaCl-treated controls and 12 NS1209-treated rats. Spontaneous seizures were observed in 9/12 controls (75%) and 6/12 NS1209treated rats (50%), respectively. This difference was not significant (P ¼ 0.4003). Most seizures were partial onset, secondarily generalized motor seizures without any obvious indication that spontaneous seizures in the NS1209 group were less severe compared to controls. Finally, we evaluated whether histological findings in NS1209treated SE rats that did not exhibit spontaneous seizures during video/EEGrecording differ from NS1209-treated SE rats that exhibited seizures. Similarly, histological data from NaCl-treated SE rats with or without spontaneous seizures were compared. No obvious relationship between the individual extent of neuronal damage and the occurrence of spontaneous seizures was found (not illustrated). For instance, NS1209-treated rats without spontaneous seizures during video/EEG-recording had an overall cumulative lesion score (see Methods and Fig. 1 for explanation) of 3.3  1.3 compared to 2.2  1.4 in NS1209-treated rats with seizures (P ¼ 0.5826). 4. Discussion To our knowledge, this is the first study to evaluate which VPA treatment protocol optimally protects against SE-induced neuronal damage in the dentate hilus and pyramidal cell layers of the hippocampal formation, and whether the neuroprotective potential

of VPA and the AMPA receptor antagonist NS1209 differs among major subpopulations of hilar neurons. 4.1. BLA-stimulation-induced SE affects specific subpopulations of hilar neurons Extensive neuronal hilar cell loss without a similarly severe loss of granule cells in the dentate gyrus, also known as “endfolium” sclerosis, is a hallmark of mesial TLE and also occurs in rat models of TLE (Sloviter, 1996, 2008; Morimoto et al., 2004). Because of extensive hilar cell loss and subsequent synaptic and functional reorganization, the dentate gyrus, which is the main gateway to the hippocampus, is thought to be a major source for epileptogenicity in TLE developing after various brain insults, including SE (Ribak et al., 1992; Dalby and Mody, 2001; Dudek and Sutula, 2007). The two main polymorphic cell types in the dentate hilus are glutamatergic spiny hilar cells, the prototype being the excitatory mossy cell, and GABAergic aspiny “fast spiking” cells, the prototype being the local circuit cell or interneuron (Scharfman, 1992). GABAergic interneurons in the hilus can be further subdivided by colocalization of neuropeptides such as somatostatin or calcium-binding proteins such as parvalbumin (Houser, 2007). In the present study, across all hippocampal sections, about 55% of all immunohistochemically stained hilar cells were glutamatergic mossy cells, 34% were somatostatin-stained interneurons and 9% were parvalbumin-stained interneurons, which is in line with previous reports that about half of all hilar neurons are glutamatergic and half are GABAergic, and that the ratio of somatostatin to parvalbumin-stained interneurons is about 3:1 (Buckmaster and Dudek, 1997; Amaral et al., 2007; Houser, 2007). Both mossy cells and interneurons in the dentate hilus are sensitive to damage by SE, but the relative extent of damage depends on the SE model used (cf., Morimoto et al., 2004). Furthermore, decreased density of hilar neurons may result from hilar expansion, which may lead to false conclusions on neuron loss when only neuron density is studied (Morimoto et al., 2004). We therefore determined both neuron density and the total number of neurons per hilus in a section for analysis of effects of SE and neuroprotective treatments. Rats did not exhibit hilar expansion in the SE model used for the present experiments, so that this did not form a bias for analyses of neurodegeneration. In rats with 4 h of SE induced by electrical stimulation of the BLA, the most sensitive hilar neurons were parvalbumin-stained interneurons, whereas somatostatinstained interneurons and mossy cells were clearly less sensitive to damage in this model. In the temporal part of the hippocampal formation, >60% of parvalbumin-stained neurons were undetectable immunocytochemically after SE, whereas about 40% fewer parvalbumin-positive hilar cells were detected in the septal hippocampus, which is consistent with data from other SE models that the temporal part of the hippocampal formation is more sensitive to

/ n.a. n.a. n.a. þ/ n.a. /

0/26 8/12* 5/24* 7/24* 17/26*# 9/24* 17/26*# D N

01 / n.a. þ/þ þ/þ þ/þ /þ þ/þ

D N

/ n.a. þ/þ þ/þ þ/þ / þ/þ / n.a. /þ / / /þ /þ

D N

/ n.a. / / / / þ/þ / n.a. / / þ/þ / þ/þ

D N D

/ þ/þ / / /þ / /

N D N

/ þ/þ / / þ/þ þ/þ þ/þ e e e e þ þ e e e e e þ e þ NaCl 24 h VPA i.p. 48 h VPA i.p. 7 d VPA i.p. 24 h VPA i.v. 5 d VPA i.v. 24 h NS1209 i.v. SE SE SE SE SE SE SE

Septal

Extension of lesions in CA1 and CA3

Extension of lesions in hilus Treatment

/ þ/þ / / /þ / /

/ þ/þ / þ/þ þ/þ þ/þ þ/þ e e e e þ þ þ e e e þ e þ þ

/ n.a. / / /þ / þ/

Somatostatin

Septal Septal

Thionin

Extension of lesions in CA1, CA3 and hilus Percent of rats with lesions

Temporal

Data from cell counting in the hilus Data from scoring (thionin-staining)

Parvalbumin

Temporal

Septal

GluR 2/3 (mossy cells)

Neuroprotection score

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047 Table 3 Overview of neuroprotective effects of the different dosing and administration protocols in the various staining protocols. For actual data, see Figs. 2 and 4e6. Neurodegeneration (or lack of neuroprotection) is indicated by “”, while “þ” indicates a neuroprotective effect of treatment in one (“þ”) or both (“þ/þ”) hemispheres. Because SE did not significantly decrease the number of GluR 2/3-stained neurons in the hilus, neuroprotection could not be evaluated for this variable (indicated by 01). Data from cell counting were separately evaluated for number of neurons per hilus (N) and neuron density (D). From these data, a “neuroprotection score” was calculated, in that for each “þ” a score of 1 was given, resulting in a maximum cumulative score of 26 (data from scoring were not separately calculated from left and right hilus; see Fig. 2). Because not all variables were available for all treatments (indicated by n.a.), the maximum cumulative score was lower for such treatments. Significance of differences in this score to SE NaCl controls is indicated by asterisk (P < 0.05). Significant differences to intermittent i.p. administration of VPA for 2 or 7 days are indicated by rhomb. For group sizes see Table 1.

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damage than the septal part (Pitkänen et al., 2002). Parvalbumincontaining neurons are a subset of GABAergic interneurons, which play a major role in the inhibition of pyramidal and granule cell activity, so that loss of such neurons may critically contribute to the altered function of the hippocampal formation developing during epileptogenesis (Gorter et al., 2001; van Vliet et al., 2004). However, with the methods used in the present paper, it is not possible to know with certainty whether the decreased number of stained parvalbumin-immunoreactive hilar neurons after BLAstimulation-induced SE reflects a loss of these neurons or a loss of parvalbumin expression rather than cell death. By using focal stimulation of the perforant pathway in urethane-anesthetized rats as an SE model, Sloviter and colleagues have previously reported that, although parvalbumin immunocytochemistry and in situ hybridization revealed decreased staining, this apparently was due to altered parvalbumin expression rather than cell death, because substance P receptor-positive interneurons, some of which contained residual parvalbumin immunoreactivity, survived (Sloviter et al., 2003). Whether this is also true for the present SE model in unanesthetized rats is not known, but the fact that the percent reduction of immunostained hilar neurons in SE rats (26.2%) almost exactly corresponded to the percent reduction of thionin-stained hilar neurons (26.5%; see Results) favors the view that the reduction of parvalbumin-immunostained neurons reflects cell death rather than decreased parvalbumin expression. Compared to the consistent loss of hilar neurons, which was observed in the majority (95%) of rats after SE, lesions in CA1 or CA3 were less frequently observed, demonstrating that endfolium sclerosis is the hallmark of this post-SE model of SE. Interestingly, although the BLA was stimulated in only one hemisphere, neuronal damage was observed in both hemispheres after a generalized convulsive (type III) or a mixed focal plus generalized convulsive (type II) SE induced by electrical BLA-stimulation, confirming previous experiments in this model (Brandt et al., 2003; Volk et al., 2006). 4.2. Neuroprotective effect of VPA and NS1209 We have previously reported that intermittent i.p. treatment with VPA, starting immediately after termination of SE by diazepam, completely protects the hippocampal formation from damage in the CA1, CA3 and dentate hilus (Brandt et al., 2006). In this previous study, in which rats were treated with VPA (200 mg/ kg) three times daily for four weeks following BLA-stimulationinduced SE, neurons were stained by thionin in horizontal brain sections. The SE-induced cell loss was prevented in all parts (septal, medial, temporal) of the hippocampal formation. At a first glance, the shorter treatment protocols used for VPA in the present study seemed to be less efficacious in preventing loss of thionin-stained neurons across the hippocampal formation than the four-week protocol used in our previous study, but the data from these two studies cannot be directly compared because coronal rather than horizontal sections were used in the present study, plus a new scoring system, which identifies the localization and extent of lesions across the hippocampal formation more detailed than the system used in our previous study. Furthermore, subpopulations of hilar neurons were not differentiated in our previous study, so that we could not exclude significant neurodegeneration in one of the less prominent populations of hilar neurons, such as parvalbumincontaining neurons. In the present experiments, in which various treatment protocols of VPA were compared after SE, none of these protocols prevented loss of parvalbumin-stained neurons in the temporal part of the hippocampus, where the number and density of these neurons are markedly higher than in septal parts. In contrast to the temporal dentate gyrus, VPA was capable of counteracting loss of parvalbumin-containing in the septal dentate

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

gyrus, indicating that these different parts of the hippocampal formation exhibit a differential response to neuroprotective agents. This, however, was not observed with the AMPA receptor antagonist NS1209, which protected parvalbumin-containing neurons in both the septal and temporal parts of the dentate gyrus. NS1209 was recently reported to exert neuroprotective activity in a rat model, in which SE was induced by sustained electrical stimulation of the lateral amygdala (Pitkänen et al., 2007). When treatment with NS1209 (bolus 20 mg/kg followed by continuous infusion with 5 mg/kg/h for 24 h) was started 2 h after SE onset, neuronal damage was reduced in the hilus and CA1 compared to vehicle-treated controls. However, the same neuroprotective effect was obtained when diazepam was used to interrupt SE after 2 h (Pitkänen et al., 2007), indicating that the reduced neurodegeneration observed with both treatments was due to “initial insult modification” by reducing SE duration rather than a true neuroprotective effect (cf., Löscher and Brandt, 2010). In the present study, we terminated SE after 4 h by either diazepam or NS1209, resulting in neurodegeneration in the diazepam group but significantly reduced neuronal damage in the NS1209 group, thus representing a true neuroprotective effect. Because NS1209 has a very short half-life in rats (Nielsen et al., 1999), we used the bolus injection and continuous infusion protocol reported by Pitkänen et al. (2007) for our experiments. 4.3. Specific neuroprotection or insult modification? With respect to drug effects resulting from “initial insult modification” rather than true neuroprotective or antiepileptogenic effects, it is important to note that a BLA-stimulation-induced SE has the advantage that both the clinical and EEG seizures can be completely stopped by a high dose of diazepam, which is not possible with chemically induced SE, such as the pilocarpine model (Brandt et al., 2003; Bankstahl and Löscher, 2008; Löscher and Brandt, 2010). When the BLA-stimulation-induced SE is terminated after 4 h by diazepam, >90% of the rats develop neurodegeneration in the hippocampus, and epilepsy, while interruption of SE after 3 h by diazepam markedly reduces the percentage of rats developing epilepsy by decreasing the insult (Brandt et al., 2003). In the present study, VPA was given immediately after terminating SE after 4 h by diazepam, so that it is highly likely that the findings in the VPA-treated groups reflected a neuroprotective effect of this drug rather than insult modification. The same is true for NS1209, which was administered without diazepam, because it was as effective as diazepam in terminating SE. An additional argument in favor of a specifically neuroprotective, rather than an insultmodifying, effect of VPA is that all VPA treatment groups received a high initial bolus of VPA immediately after diazepam; however, the neuroprotective efficacy of VPA treatment depended on the subsequent dosing protocol and time window of treatment. This argues against the possibility that the effects of VPA are simply due to decreasing the insult by suppressing neuronal activity that occurs in the immediate post-SE period. 4.4. The treatment protocol is critical for neuroprotective effects of VPA and NS1209 The only VPA treatment protocol which exhibited a similar neuroprotective efficacy as NS1209 was continuous i.v. infusion of VPA for 24 h. The finding that continuous infusion of VPA was more effective than intermittent i.p. treatment is a result of the rapid elimination of this drug in rats, which is increased further by prolonged administration as a result of induction of its own metabolism in this species (Fisher et al., 1991). Unexpectedly, infusion of VPA for 5 days was less effective in protecting neurons from

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damage than infusion for 24 h, indicating (1) a sensitive therapeutic window of opportunity shortly after the brain insult, and (2) that treatment exceeding this window may result in less prominent neuroprotective activity, which is difficult to explain at present. Furthermore, the finding that the briefest treatment (for 24 h) was the most effective indicates that the most critical cell death changes may occur in the immediate post-injury period. In this regard, the cell death in the BLA model seems to proceed quite rapidly, over the first days post-injury. The present finding that the neuroprotective potential of VPA depends on the treatment protocol used for this drug may explain previous equivocal findings in the literature. For instance, Bolanos et al. (1998) treated rats over 40 days following a kainate-induced SE, injecting VPA at a dose of 600 mg/kg i.p. every 12 h for 30 days, followed by 300 mg/kg for 10 days. Lesions in CA1, but not CA3, were prevented by VPA. In apparent contrast, Jessberger et al. (2007), who injected rats VPA twice daily with 150 mg/kg i.p. for 7 dayse5 weeks following a kainate-induced SE, did not find any substantial neuroprotective effect in the hippocampal formation. However, the treatment potently blocked seizure-induced neurogenesis, an effect that appeared to be mainly mediated by inhibiting histone deacetylases (HDAC) and normalizing HDAC-dependent gene expression within the epileptic dentate area (Jessberger et al., 2007). The different neuroprotective potential of VPA between studies may indicate that higher doses (or brain concentrations) of this drug are needed to achieve neuroprotective activity compared to some other effects of this drug, e.g., HDAC inhibition- mediated effects. 4.5. Lack of clear relationship between neuroprotection and antiepileptogenesis We previously reported that the neuroprotective effect of VPA on hippocampal neurons was not associated with any antiepileptogenic effect, i.e., rats developed spontaneous recurrent seizures after SE despite neuroprotection with VPA (Brandt et al., 2006). In theory, this might have been due to the present finding that even continuous infusion with VPA did not protect all susceptible subpopulations of neurons from damage, but particularly parvalbumin-containing interneurons in the temporal part of the dentate gyrus were still affected despite prophylactic treatment with VPA. Because these neurons were protected by NS1209, it was important to determine whether this was associated with an antiepileptogenic effect. In contrast to our expectations, NS1209 did not significantly prevent or modify the development of spontaneous recurrent seizures in our model. In this respect, however, it is important to note that we monitored only one week for seizures, so that sample of time may be inadequate to conclude that no decrease in seizure latency or frequency occurred. 4.6. Mechanisms of neuroprotective activity The two drugs that were compared in the present study act by different mechanisms of action. NS1209 is a competitive AMPA receptor antagonist (Nielsen et al., 1999), which probably explains its potent neuroprotective activity, because glutamate-mediated excitotoxic cell death is critically involved in brain damage induced by SE and other brain insults (Lau and Tymianski, 2010). The mechanism of action of the neuroprotective effect of VPA, which has been demonstrated for a variety of experimentallyinduced insults, is less clear (Löscher, 2002; Vajda, 2002; Bachmann et al., 2005; Rosenberg, 2007; Löscher and Brandt, 2010). VPA regulates a number of factors involved in cell survival pathways, including cyclic adenosine monophosphate (cAMP) responsive element binding protein (CREB), brain-derived neurotrophic factor (BDNF), bcl-2, and mitogen-activated protein (MAP)

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M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047

kinases, which may underlie its neuroprotective and neurotrophic effects (Löscher, 2002; Vajda, 2002; Rogawski and Löscher, 2004; Bachmann et al., 2005; Rosenberg, 2007). VPA has also been shown to activate the cell survival factor, Akt, presumably through inhibition of HDAC (De Sarno et al., 2002). Furthermore, VPA has been shown to suppress the seizure-induced brain expression of cfos and c-jun, which, as transcription factors, are involved in the pathways for apoptosis and necrosis (Szot et al., 2005). 5. Conclusions The present study demonstrates that a relatively short treatment with neuroprotective drugs, including the widely used AED VPA, after SE is capable of markedly reducing or preventing damage in the hippocampal formation. This is possible because most of the neuronal damage induced by SE develops slowly after the insult (over several days), providing a therapeutic window of opportunity for neuroprotective agents (Löscher and Brandt, 2010). The data further demonstrate that the dosing and administration protocol is important for achieving significant neuroprotective effects in this window, particularly because of the rapid drug elimination in rodents compared to humans (Löscher, 2007). The finding that immediate but short treatment with VPA or NS1209 exerts powerful neuroprotective activity after a brain insult has a potential clinical impact, because the long treatment periods after brain insults that have been used with several drugs, including VPA, in clinical trials are associated with several inherent problems, including poor compliance (Temkin, 2009). As demonstrated by the present data with NS1209 and various previous studies with different neuroprotective drugs, including VPA, neuroprotection following brain insults does not seem to provide a promising strategy to prevent epilepsy, but may reduce or prevent other consequences of brain insults, such as cognitive defects or psychopathology (Löscher and Brandt, 2010), provided that sufficiently high drug levels are achieved and maintained in the therapeutic window of opportunity immediately following a brain insult. Furthermore, as discussed in the Introduction and recently by Löscher and Brandt (2010), prevention of hippocampal damage after brain insults by neuroprotective drugs such as VPA, although not preventing the development of spontaneous recurrent seizures, may decrease the risk of pharmacoresistance of such seizures, which will be addressed in future experiments. Acknowledgments The study was supported by a grant (Lo 274/11-1) from the Deutsche Forschungsgemeinschaft (Bonn, Germany) within the Forschergruppe FOR 1103. M. Langer received a “Georg-ChristophLichtenberg-Stipendium” from the Niedersächsisches Ministerium für Kultur und Wissenschaft (Hannover, Germany) and, subsequently, a “Bodelschwingh-Stipendium” from the Gesellschaft für Epilepsieforschung e.V. (Bielefeld, Germany). We thank Dr. Carolyn R. Houser (Department of Neurobiology and Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, USA) for discussion during preparation of the manuscript, Julia Förster, Martina Gramer and Maria Hausknecht for technical assistance, and Neurosearch A/S (Ballerup, Denmark) for providing NS1209. References Amaral, D.G., Scharfman, H.E., Lavenex, P., 2007. The dentate gyrus: fundamental neuroanatomical organization (dentate gyrus for dummies). Prog. Brain Res. 163, 3e22. Bachmann, R.F., Schloesser, R.J., Gould, T.D., Manji, H.K., 2005. Mood stabilizers target cellular plasticity and resilience cascades: implications for the development of novel therapeutics. Mol. Neurobiol. 32, 173e202.

Baltes, S., Fedrowitz, M., Luna-Tortós, C., Potschka, H., Löscher, W., 2007. Valproic acid is not a substrate for P-glycoprotein or multidrug resistance proteins 1 and 2 in a number of in vitro and in vivo transport assays. J. Pharmacol. Exp. Ther. 320, 331e343. 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. Bethmann, K., Fritschy, J.M., Brandt, C., Löscher, W., 2008. Antiepileptic drug resistant rats differ from drug responsive rats in GABAA-receptor subunit expression in a model of temporal lobe epilepsy. Neurobiol. Dis. 31, 169e187. Bolanos, A.R., Sarkisian, M., Yang, Y., Hori, A., Helmers, S.L., Mikati, M., Tandon, P., Stafstrom, C.E., Holmes, G.L., 1998. Comparison of valproate and phenobarbital treatment after status epilepticus in rats. Neurology 51, 41e48. Brandt, C., Glien, M., Potschka, H., Volk, H., Löscher, W., 2003. Epileptogenesis and neuropathology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats. Epilepsy Res. 55, 83e103. Brandt, C., Gastens, A.M., Sun, M.Z., Hausknecht, M., Löscher, W., 2006. Treatment with valproate after status epilepticus: effect on neuronal damage, epileptogenesis, and behavioral alterations in rats. Neuropharmacology 51, 789e804. 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. Buckmaster, P.S., Dudek, F.E., 1997. Neuron loss, granule cell axon reorganization, and functional changes in the dentate gyrus of epileptic kainate-treated rats. J. Comp. Neurol. 385, 385e404. Dalby, N.O., Mody, I., 2001. The process of epileptogenesis: a pathophysiological approach. Curr. Opin. Neurol. 14, 187e192. De Sarno, P., Li, X., Jope, R.S., 2002. Regulation of Akt and glycogen synthase kinase3 beta phosphorylation by sodium valproate and lithium. Neuropharmacology 43, 1158e1164. Dichter, M.A., 2009. Posttraumatic epilepsy: the challenge of translating discoveries in the laboratory to pathways to a cure. Epilepsia 50 (Suppl. 2), 41e45. Dudek, F.E., Sutula, T.P., 2007. Epileptogenesis in the dentate gyrus: a critical perspective. Prog. Brain Res. 163, 755e773. Fisher, J.E., Nau, H., Löscher, W., 1991. Alterations in the renal excretion of valproate and its metabolites after chronic treatment. Epilepsia 32, 146e150. Fujikawa, D.G., Itabashi, H.H., Wu, A., Shinmei, S.S., 2000. Status epilepticus-induced neuronal loss in humans without systemic complications or epilepsy. Epilepsia 41, 981e991. Fujise, N., Kosaka, T., 1999. Mossy cells in the mouse dentate gyrus: identification in the dorsal hilus and their distribution along the dorsoventral axis. Brain Res. 816, 500e511. Gernert, M., Hamann, M., Bennay, M., Löscher, W., Richter, A., 2000. Deficit of striatal parvalbumin-reactive GABAergic interneurons and decreased basal ganglia output in a genetic rodent model of idiopathic paroxysmal dystonia. J. Neurosci. 20, 7052e7058. Gorter, J.A., van Vliet, E.A., Aronica, E., Lopes Da Silva, F.H., 2001. Progression of spontaneous seizures after status epilepticus is associated with mossy fibre sprouting and extensive bilateral loss of hilar parvalbumin and somatostatinimmunoreactive neurons. Eur. J. Neurosci. 13, 657e669. Houser, C.R., 2007. Interneurons of the dentate gyrus: an overview of cell types, terminal fields and neurochemical identity. Prog. Brain Res. 163, 217e232. 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. Jessberger, S., Nakashima, K., Clemenson Jr., G.D., Mejia, E., Mathews, E., Ure, K., Ogawa, S., Sinton, C.M., Gage, F.H., Hsieh, J., 2007. Epigenetic modulation of seizure-induced neurogenesis and cognitive decline. J. Neurosci. 27, 5967e5975. Lau, A., Tymianski, M., 2010. Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch. 460, 525e542. Löscher, W., Fisher, J.E., Nau, H., Hönack, D., 1989. Valproic acid in amygdala-kindled rats: alterations in anticonvulsant efficacy, adverse effects and drug and metabolite levels in various brain regions during chronic treatment. J. Pharmacol. Exp. Ther. 250, 1067e1078. Löscher, W., Hönack, D., 1995. Comparison of anticonvulsant efficacy of valproate during prolonged treatment with one and three daily doses or continuous (“controlled release”) administration in a model of generalized seizures in rats. Epilepsia 36, 929e937. Löscher, W., 2002. Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy. CNS Drugs 16, 669e694. 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., Brandt, C., 2010. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research. Pharmacol. Rev. 62, 668e700. McCracken, E., Fowler, J.H., Dewar, D., Morrison, S., McCulloch, J., 2002. Grey matter and white matter ischemic damage is reduced by the competitive AMPA receptor antagonist, SPD 502. J. Cereb. Blood Flow Metab. 22, 1090e1097. Morimoto, K., Fahnestock, M., Racine, R.J., 2004. Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog. Neurobiol. 73, 1e60. Nielsen, E.O., Varming, T., Mathiesen, C., Jensen, L.H., Moller, A., Gouliaev, A.H., Watjen, F., Drejer, J.,1999. SPD 502: a water-soluble and in vivo long-lasting AMPA antagonist with neuroprotective activity. J Pharmacol. Exp. Ther. 289, 1492e1501. Paxinos, G., Watson, C., 2007. The Rat Brain in Stereotaxic Coordinates, sixth ed. Elsevier, Amsterdam.

M. Langer et al. / Neuropharmacology 61 (2011) 1033e1047 Pitkänen, A., Nissinen, J., Nairismagi, J., Lukasiuk, K., Grohn, O.H., Miettinen, R., Kauppinen, R., 2002. Progression of neuronal damage after status epilepticus and during spontaneous seizures in a rat model of temporal lobe epilepsy. Prog. Brain Res. 135, 67e83. Pitkänen, A., Mathiesen, C., Ronn, L.C., Moller, A., Nissinen, J., 2007. Effect of novel AMPA antagonist, NS1209, on status epilepticus. An experimental study in rat. Epilepsy Res. 74, 45e54. Pitkänen, A., Lukasiuk, K., 2009. Molecular and cellular basis of epileptogenesis in symptomatic epilepsy. Epilepsy Behav. 14 (Suppl. 1), 16e25. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32, 281e294. Ribak, C.E., Gall, C.M., Mody, I., 1992. The Dentate Gyrus and Its Role in Seizures. Elsevier, Amsterdam. Rogawski, M.A., Löscher, W., 2004. The neurobiology of antiepileptic drugs for the treatment of nonepileptic conditions. Nat. Med. 10, 685e692. Rosenberg, G., 2007. The mechanisms of action of valproate in neuropsychiatric disorders: can we see the forest for the trees? CELL MOL. Life Sci. 64, 2090e2103. Scharfman, H.E., 1992. Differentiation of rat dentate neurons by morphology and electrophysiology in hippocampal sclices: granule cells, spiny hilar cells and aspiny “fast-spiking” cells. In: Ribak, C.E., Gall, C.M., Mody, I. (Eds.), The Dentate Gyrus and Its Role in Seizures. Elsevier, Amsterdam, pp. 93e112. Sloviter, R.S., 1991. Permanently altered hippocampal structure, excitability, and inhibition after experimental status epilepticus in the rat: the “dormant basket cell” hypothesis and its possible relevance to temporal lobe epilepsy. Hippocampus 1, 41e66.

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Sloviter, R.S., 1994. The functional organization of the hippocampal dentate gyrus and its relevance to the pathogenesis of temporal lobe epilepsy. Ann. Neurol. 35, 640e654. Sloviter, R.S., 1996. Hippocampal pathology and pathophysiology in temporal lobe epilepsy. Neurologia 11 (Suppl. 4), 29e32. Sloviter, R.S., Zappone, C.A., Harvey, B.D., Bumanglag, A.V., Bender, R.A., Frotscher, M., 2003. “Dormant basket cell” hypothesis revisited: relative vulnerabilities of dentate gyrus mossy cells and inhibitory interneurons after hippocampal status epilepticus in the rat. J. Comp. Neurol. 459, 44e76. Sloviter, R.S., 2008. Hippocampal epileptogenesis in animal models of mesial temporal lobe epilepsy with hippocampal sclerosis: the importance of the “latent period” and other concepts. Epilepsia 49 (Suppl. 9), 85e92. Szot, P., White, S.S., Shen, D.D., Anderson, G.D., 2005. Valproic acid, but not lamotrigine, suppresses seizure-induced c-fos and c-Jun mRNA expression. Brain Res. Mol. Brain Res. 135, 285e289. Temkin, N.R., 2009. Preventing and treating posttraumatic seizures: the human experience. Epilepsia 50 (Suppl. 2), 10e13. Vajda, F.J., 2002. Valproate and neuroprotection. J Clin. Neurosci. 9, 508e514. van Vliet, E.A., Aronica, E., Tolner, E.A., Lopes Da Silva, F.H., Gorter, J.A., 2004. Progression of temporal lobe epilepsy in the rat is associated with immunocytochemical changes in inhibitory interneurons in specific regions of the hippocampal formation. Exp. Neurol. 187, 367e379. Volk, H.A., Arabadzisz, D., Fritschy, J.M., Brandt, C., Bethman, K., Löscher, W., 2006. Antiepileptic drug resistant rats differ from drug responsive rats in hippocampal neurodegeneration and GABAA-receptor ligand-binding in a model of temporal lobe epilepsy. Neurobiol. Dis. 21, 633e646.