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Keywords:

  • Temporal lobe epilepsy;
  • Lithium–pilocarpine;
  • Carisbamate;
  • Neuroprotection;
  • Learning and memory

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Purpose

Administration of carisbamate during status epilepticus (SE) prevents the occurrence of motor seizures in the lithium–pilocarpine model and leads in a subpopulation of rats to spike-and-wave discharges characteristic of absence epilepsy. Widespread neuroprotection accompanied this change in seizure expression. To assess whether these carisbamate-induced changes affected comorbidity, we used a large battery of behavioral tests in rats that had developed temporal lobe or absence-like seizures.

Methods

Lithium–pilocarpine or saline was administered to 60 adult rats. Carisbamate (90 mg/kg) or diazepam and saline was given 1 h after SE onset, and repeated 8 h later and twice daily over 6 more days. Rats were video-monitored for 2 months. Subsequently, locomotor activity, anxiety, and various types of memory were assessed.

Key Findings

In rats with motor seizures, treated or not with carisbamate, all features of behavior were impaired compared to controls. Rats exhibiting absence-like seizures after carisbamate treatment behaved as controls in all paradigms tested along with widespread neuroprotection.

Significance

Carisbamate treatment leading to absence-like instead of temporal lobe seizures impressively prevented behavioral comorbidities reported by patients with epilepsy as the most disabling.

Temporal lobe epilepsy (TLE) is one of the most deleterious forms of epilepsy. Its development is often marked by three steps: an initial insult classically appearing during infancy, a latent period with reorganizations and plasticity, and a chronic epileptic phase (Mathern et al., 1996; Pitkanen & Sutula, 2002). TLE is often refractory to antiepileptic drugs (Semah et al., 1998) and surgery is the only remaining option. A better strategy than acting on seizures would be to intervene before chronic seizure onset and to prevent epilepsy development and improve long-term outcome.

The lithium–pilocarpine model has been widely studied as status epilepticus (SE) model followed by a latent period before expression of chronic epilepsy (Curia et al., 2008). In this model, rats develop lesions affecting mainly the hippocampal formation, amygdala, thalamus, and piriform cortex (Clifford et al., 1987; Persinger et al., 1993), associated with reorganizations such as mossy fiber sprouting (Mello et al., 1993). Behavioral studies reported that lithium–pilocarpine rats have long-term and working memory impairments (Persinger et al., 1993; Detour et al., 2005; Lenck-Santini & Holmes, 2008; Inostroza et al., 2011). Reduced anxiety (Detour et al., 2005; dos Santos et al., 2005; Inostroza et al., 2011) and increased locomotor activity (Persinger & Koren, 1998; Stewart & Leung, 2003) complete the behavioral syndrome.

We recently found that carisbamate has insult- and disease-modifying effects in lithium–pilocarpine rats (Francois et al., 2011). Carisbamate is a compound known to inhibit voltage-gated sodium channels (Liu et al., 2009) and to block action potentials in piriform neurons (Whalley et al., 2009). Administration of carisbamate (90 mg/kg) during the initial insult (i.e., 1 h after SE onset) and repeated over 7 days alleviated insult severity in a rat subpopulation and prevented the occurrence of motor spontaneous recurrent seizures (SRS). In 25–50% of the rats carisbamate treatment led to pharmacosensitive absence-like seizures with spike-and-wave discharges (SWDs) instead of temporal lobe seizures. This subpopulation with SWDs showed reduced brain damage in most limbic regions and limited mossy fiber sprouting (Francois et al., 2011).

Cognitive impairment is the most disabling comorbidity of epilepsy (Fisher et al., 2000) and constitutes one of the benchmarks of the National Institute of Neurological Disorders and Stroke in epilepsy research (Kelley et al., 2009). Here we extended our previous work on early carisbamate-treatment effects on neuronal loss and disease expression to a comprehensive exploration of the behavioral functions known to be severely impaired in TLE patients and lithium–pilocarpine rats. Ten weeks after SE we tested four groups of rats with a broad battery of behavioral tasks: (1) controls not exposed to pilocarpine and carisbamate; (2) lithium–pilocarpine rats that did not receive carisbamate; and two groups of lithium–pilocarpine rats treated with carisbamate, (3) one developing motor seizures; and (4) one absence-like seizures, as previously reported (Francois et al., 2011). We first aimed at completing the knowledge on consequences of lithium–pilocarpine SE in rats. The second goal was to determine if the seizure expression change after early carisbamate administration was associated with behavioral changes.

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Animals

Sixty adult male Sprague–Dawley rats provided by Charles River Breeding Center (L'Abresle, France) were placed in individual cages under controlled conditions (22 ± 1°C, 12/12 h light/dark cycle, lights on at 7:00 a.m.) with water and food ad libitum. Experiments were performed in accordance with the rules of the European Community council directive of November 24, 1986 (86–609) and the French Department of Agriculture (87–848; License #67-97 for AN and 67–215 for JCC). The protocol was approved by the ethical Animal Research Committee Board of the University of Strasbourg (CREMEAS #AL/01/04/03/07). All efforts were made to reduce animal suffering and the number of animals used.

Status epilepticus

Rats were injected with lithium (127 mg/kg; Sigma-Aldrich, Saint-Louis, MO, U.S.A.). About 20 h later they received 1 mg/kg methylscopolamine (Sigma-Aldrich) to limit pilocarpine peripheral effects. SE was induced 30 min later by the injection of pilocarpine (25 mg/kg; Sigma-Aldrich), and rats received a second injection of methylscopolamine (1 mg/kg) 30 min after SE onset. Controls received lithium and saline injections instead of pilocarpine.

Carisbamate treatment

One hour and 9 h after SE onset, rats were randomly administered diazepam (2.5 and 1.25 mg/kg, respectively; Roche, Meylan, France), or carisbamate (90 mg/kg; Johnson & Johnson Research & Development, L.L.C., Raritan, NJ, U.S.A.) dissolved in 45% hydroxypropyl-β-cyclodextrin (Acros Organics, Geel, Belgium). Diazepam allows enhancing survival without modifying SE characteristics at low dose (Morrisett et al., 1987). During the following 6 days, carisbamate-treated groups received two daily carisbamate injections (90 mg/kg). Diazepam-treated rats received saline instead of carisbamate. Five rats from the diazepam–lithium–pilocarpine group died.

The acute behavioral effects of treatments during SE were quantified 2 h after injection of diazepam or carisbamate with an in-house developed scale based on observations ranging from 0 to 4 with 0 = no effect; 1 = reduced seizure severity; 2 = calm with head bobbing; 3 = sedated; 4 = apathetic (Francois et al., 2011).

Epileptogenesis and seizure control

Daily video-recording for 10 h (7:00 a.m.–5:00 p.m.) started 1 week after SE and lasted for 8 weeks. After displaying at least one stage III motor forelimb clonus (Racine, 1972), rats were removed from video-monitoring. Every rat receiving diazepam developed motor SRS. After 8 weeks, rats without motor SRS were considered having absence-like seizures (Francois et al., 2011). In the present study, video-monitoring was favored over electroencephalography (EEG) recording because in a recent comparative study we noticed that latency to the first motor SRS is underestimated at most by 24–72 h by video-monitoring compared to EEG (Francois et al., 2011). Furthermore the large number of animals used (60 rats) did not allow easy EEG recording over a short period of time. In addition, we wanted to avoid any potential infection due to electrode insertion, since rats were kept for many months and exposed to various surroundings including water.

After video-monitoring, four groups were constituted: (1) controls (n = 12), (2) diazepam-treated rats (DZP-TLE, n = 18), (3) carisbamate-treated rats developing motor SRS (CRS-TLE, n = 17), and (4) carisbamate-treated rats displaying absence-like seizures (CRS-ALE, n = 8).

Behavioral studies

Starting at 10 weeks after SE, animals were subjected to a large variety of behavioral tests (Fig. 1). To limit potential seizure effects on behavioral skills, rats were tested at least 2 h after the last motor SRS. Concerning CRS-ALE rats, absence-like seizures might have occurred shortly before and even during testing. However, concentration on a task usually prevents absence seizures from occurring. Moreover, CRS-induced absence-like seizures are more frequent during night than during day time (Francois et al., 2011) when testing was performed.

image

Figure 1. Schematic representation of the time course of the cognitive and behavioral evaluation performed after lithium–pilocarpine SE. The data from the attention test are not reported here. *The results of this test are not included in this article.

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Elevated plus-maze test

Anxiety was assessed with a black acrylic glass classical plus-maze (Cassel et al., 2005). Rats were placed in the maze facing the same closed arm for a unique 5-min video-recorded session, during which the number of entries and time spent in each arm and on the platform were rated. Halogen lights provided a 30 and 10 lux illumination on open and closed arms, respectively. Some rats jumped out but were immediately placed back into the maze. We performed the statistical analysis excluding and including these rats.

Morris water maze

A circular pool (diameter 160 cm, height 60 cm) was half-filled with water (21 ± 1°C) rendered opaque by powdered milk (Traissard et al., 2007). Visual cues were present in the room. A video-tracking system (Ethovision, Noldus Information Technology, Wageningen, The Netherlands) recorded the distance swum and latency to reach the platform. Neon lights provided a 175 lux illumination on the apparatus.

Spatial long-term memory procedure

After a 3-day visible platform habituation to the maze (platform placed 1 cm above water surface and moved every day), the platform was immersed in the southwest quadrant for five consecutive days. Rats were given 4 trials per day from different starting points. The sixth day, the platform was removed for the probe trial and rats were placed in the pool for 60 s. This 5-day procedure is considered to test spatial learning and memory. The probe trial on the sixth day tests the strength of this memory.

Visible platform procedure

Next, rats were tested with a visible platform for 1 day. The platform was placed 1 cm above the water surface in a position different from the previous procedure, and rats were placed in four different starting points as in the long-term memory procedure. This procedure is considered to be sensitive to visual, sensorimotor, and motivation biases.

Spatial working memory procedure

During the nine following days the platform was moved every day and rats were given 2 trials/day to reach the platform from two equidistant starting points. This procedure is considered to test working memory.

Double-H maze

The apparatus was composed of a transparent acrylic glass maze formed by six parallel arms (160 × 20 cm) joined in their middle by a perpendicular-oriented fourth arm (Pol-Bodetto et al., 2011). Each arm was surrounded by 35 cm high walls, the whole shaped as two adjacent Hs (Fig. 4A). The maze was placed 80 cm above the floor in a room containing visual cues. The double-H was half-filled with water (21 ± 1°C) rendered opaque by powdered milk. A platform (diameter 11 cm) was placed 2.5 cm underneath water surface at the end of the northwest (NW) arm (see Fig. 4C). The test consisted of four learning sessions of four trials each day ended by a probe trial on day 5, followed by another set of four learning sessions ended by another probe trial on day 10. The four learning trials were separated by 5 min. During these learning trials, the S arm was closed and rats were placed in the N arm facing the wall to force a strategy based on procedural memory. Rats were given a maximum of 60 s to reach the platform. Latency and distance swum to reach the platform and swim path of rats were video-recorded. For the probe trial, the platform was removed and all arms were open. Rats were placed in the maze for 60 s starting from NE arm to test the strength of procedural memory and the rats' capacity to shift to spatial strategy. Time spent in the NW arm (target arm) was assessed. The calculated chance level corresponded to 7.26 s. Neon lights provided a 190 lux illumination on the maze.

Histologic and immunohistochemical studies

Effect of carisbamate treatment on neuronal loss was assessed with anti-Neuronal Nuclear protein (NeuN). At the end of behavioral procedures, rats received an overdose of pentobarbital (150 mg/kg; CEVA, Libourne, France) and were perfused with paraformaldehyde. Brains were removed, post-fixed, and transferred to a sucrose solution. They were frozen and 40-μm whole brain coronal sections were cut. The NeuN expression was revealed on free-floating sections. These were incubated with a primary mouse monoclonal antibody directed against NeuN (1:2,000; Millipore Corporation, Billerica, MA, U.S.A.) and then a biotinylated horse anti-mouse antibody (1:500; Vector Laboratories International, Burlingame, CA, U.S.A.) using diaminobenzidine tetrahydrochloride (Sigma-Aldrich) for revelation. Effect of carisbamate on mossy fiber sprouting was assessed with Timm staining as detailed previously (Francois et al., 2011).

Quantification

A 100× computerized picture was taken on at least three sections per rat for each structure of interest. For anti-NeuN stained slices, quantifications were performed in the lateral septum (LS, bregma −0.48 mm), dorsolateral striatum (DLStr, bregma −0.48 mm), laterodorsal dorsomedial thalamus (LDDM, bregma −3.24 mm), dorsal hippocampus (dorsal hilus, CA1 and CA3 [dCA1 and dCA3], bregma −3.24 mm), basolateral amygdala (BLA, bregma −3.24 mm), dorsal and ventral piriform cortex (dPIR and vPIR, bregma −3.24 mm), ventral hippocampus (CA1 and CA3 [vCA1 and vCA3], bregma −5.40 mm), and dorsal and ventral entorhinal cortex (dENT and vENT, bregma −5.40 mm).

Neuronal density was assessed by a standardized two-dimensional (2D) grain count module (MCID software; InterFocus Imaging, Cambridge, United Kingdom), as described previously (Linard et al., 2010). Percentage of shrinkage compared to controls was measured for all structures explored: 50% in hilus in all groups, 32% in entorhinal cortex of DZP-TLE rats and 40% in piriform cortex of DZP-TLE rats. This percentage was used to correct for neuronal density.

Mossy fiber sprouting was evaluated in dorsal hippocampus according to the classical criteria established by Cavazos et al. (1991) and described previously in detail (Francois et al., 2011).

Statistical analyses

For most variables, repeated-measures two-way analysis of variance (ANOVA) was used. In contrast, analysis of video monitoring, home-cage activity, beam-walking test, open field, elevated plus-maze, double-H maze, neuronal density, and some parts of the Morris water maze were performed using one-way ANOVA. The post hoc analysis was always performed using the Newman-Keuls test. One-sample Student's t-test was used when a group performance had to be compared to a standard reference (i.e., chance level).

Results

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

Status epilepticus and video-monitored motor SRS

At 2 h after drug injection all rats receiving DZP and some rats exposed to carisbamate reached a 1.6–1.8 behavioral score. Conversely, other carisbamate-treated rats showed almost no seizures, were sedated, or were apathetic (behavioral score of 3–3.5). Over 90% of rats with marked sedation developed absence-like seizures; as previously reported comparing behavioral and EEG analyses, the behavior of the rats during SE was as predictive of epileptic outcome as EEG changes (Francois et al., 2011).

CRS-TLE and DZP-TLE rats were hyperreactive when handled and aggressive, whereas CRS-ALE rats were calm. In addition, CRS-ALE rats expressed behavioral arrest and rhythmic vibrissae twitching easily noticed during long duration absence-like seizures resulting from carisbamate treatment (Francois et al., 2011).

Elevated plus-maze

Concerning the time spent by all rats in open arms (Fig. 2A), ANOVA revealed a group effect (F3,45 = 10.71, p < 0.001). DZP-TLE and CRS-TLE rats spent significantly more time in open arms than control and CRS-ALE rats (p < 0.001). Performance of CRS-ALE rats did not differ from controls. Control and CRS-ALE rats spent a shorter time than chance in open arms (p < 0.001), whereas the time spent by DZP-TLE and CRS-TLE rats was at chance level. Statistical data were identical when all rats or only rats that did not jump out of the maze were included in the analysis.

image

Figure 2. Evaluation of the anxiety in the elevated plus-maze. (A) Time spent in open arms (%) by all rats (plain bars) and only nonjumping rats (dotted bars). (B) Number of entries in open (plain bars) and closed arms (hatched bars). The dotted line represents the chance level (50%). Data represent means ± SEM. *p < 0.01, **p < 0.001, statistically significant differences compared to control and CRS-ALE rats; #p < 0.001, statistically significant difference compared to chance level; $p < 0.001, statistically significant difference between the number of entries in open and closed arms; £p < 0.05, statistically significant difference compared to CRS-ALE rats. Controls n = 12 (no jump: n = 12), DZP-TLE n = 18 (no jump: n = 8), CRS-TLE n = 17 (no jump: n = 5), CRS-ALE n = 8 (no jump: n = 8).

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Regarding the number of entries in open and closed arms performed by all rats (Fig. 2B), ANOVA indicated no significant group effect, but arm effect (F1,45 = 24.58, p < 0.001) and group × arm interaction (F3,45 = 13.66, p < 0.001). Absence of group effect indicated that all rats displayed a similar number of entries in all arms, which reflects similar locomotor activity in all groups. Control and CRS-ALE rats (p < 0.001) entered more closed than open arms, whereas DZP-TLE and CRS-TLE rats performed the same number of entries in all arms.

Morris water maze

Visible platform task

The visible platform test (Fig. 3A) evaluates visual ability of the rats. The ANOVA of the four trials indicated a significant group effect (F3,51 = 7.39, p < 0.001) due to longer distance swum by DZP-TLE (p < 0.05) and CRS-TLE rats (p < 0.005) compared to controls. CRS-ALE rats did not differ from control and DZP-TLE rats. No significant trial effect and group × trial interaction were revealed.

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Figure 3. Evaluation of long-term spatial and working memory in the Morris water maze. (A) Distance swum (m) before reaching the visible platform during the four trials. (B) Distance swum (m) before reaching the hidden platform during learning (5 days of four trials each). (C) Time spent (%) in the target quadrant during the probe trial (60 s). The dotted line represents the chance level (25%). (D) Distance swum (m) before reaching the hidden platform during the first and second trials (9 sessions). *p < 0.001, statistically significant difference compared to controls and CRS-ALE rats. #p < 0.001, statistically significant difference compared to chance level; $p < 0.001, statistically significant difference compared to the respective first trials. Controls n = 12, DZP-TLE n = 18, CRS-TLE n = 17, CRS-ALE n = 8.

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Long-term spatial learning and memory

In the fixed hidden platform paradigm performed over 5 days, spatial learning of platform location was evaluated (Fig. 3B). ANOVA showed that all groups decreased the distance swum over 5 days as confirmed by a significant day effect (F4,204 = 9.53, p < 0.001) and the absence of group × day interaction. The significant day effect was due to decrease of the distance swum between days 1 and 4 (p < 0.05). However the distance swum to reach the platform varied, as confirmed by a significant group effect (F3,51 = 20.22, p < 0.001). Control (254.1 ± 18.5 cm) and CRS-ALE rats (426.8 ± 132.0 cm) swam a shorter distance per trial than DZP-TLE (1013.8 ± 76.0 cm, p < 0.001) and CRS-TLE rats (950.1 ± 86.1 cm, p < 0.001).

Because motivational or motor biases could invalidate spatial learning evaluation, we rated the rats' velocity reflecting both biases. No significant difference was found between groups. The percentage of time spent in thigmotaxis (swimming at <20 cm from walls) could reflect anxiety or misunderstanding of the task. DZP-TLE (27.87 ± 3.94%, p < 0.01) and CRS-TLE rats (28.69 ± 5.22%, p < 0.05) spent more time in thigmotaxis than controls (4.62 ± 1.25%). CRS-ALE rats (16.60 ± 9.52%) did not differ from any group. Video-recorded path tracks indicated that DZP-TLE and CRS-TLE rats often swam in circles.

Twenty-four hours later, during a 60 s probe trial (Fig. 3C), only control and CRS-ALE rats spent more time than chance in the target quadrant containing the platform during learning (p < 0.001). ANOVA of the percentage of time spent in the target quadrant revealed a significant group effect (F3,51 = 24.29, p < 0.001) due to shorter time spent by DZP-TLE and CRS-TLE rats than control and CRS-ALE rats (p < 0.001). No difference was observed between control and CRS-ALE rats.

Spatial working memory

For the mean distance swum during the two trials (Fig. 3D), ANOVA revealed significant group (F3,51 = 9.91, p < 0.001) and trial effects (F1,51 = 30.76, p < 0.001), and group × trial interaction (F3,51 = 5.00, p < 0.005). Only control and CRS-ALE rats decreased their swimming distance in the second trial compared to the first one (p < 0.001). During the first trial no group differed from another, whereas during the second trial DZP-TLE and CRS-TLE rats swam a significantly longer distance than control and CRS-ALE rats (p < 0.01).

Double-H

ANOVA of the distance swum to reach the platform during learning (Fig. 4B) revealed a significant group effect (F3,38 = 15.57, p < 0.001) due to longer distance swum by DZP-TLE (419.1 ± 42.0 cm) and CRS-TLE rats (568.9 ± 58.1 cm) compared to control (228.0 ± 4.5 cm, p < 0.01) and CRS-ALE rats (234.2 ± 16.1 cm, p < 0.005). All groups improved performance similarly throughout learning, since ANOVA revealed a significant day effect (F7,266 = 8.02, p < 0.001) in the absence of group × day interaction.

image

Figure 4. Evaluation of procedural memory in the double-H maze. (A) Picture of the maze. The top of the walls are drawn in black to enhance the contrast. (B) Distance swum (m) before reaching the hidden platform during learning (8 days of four trials each). (C) Drawing of the maze during training and probe trials with the respective starting arms, the target arm and the hidden platform represented. During probe trials the door closing the S arm was removed. (D) Time spent (s) in the target arm during probe trial 1 (60 s) performed on day 5. The dotted line represents the chance level (7.3 s, obtained by calculation). (E) Time spent (s) in the target arm during probe trial 2 (60 s) performed on day 10. *p < 0.05, **p < 0.005, statistically significant differences compared to chance level; #p < 0.05 statistically significant difference compared to control rats. Controls n = 11, DZP-TLE n = 12, CRS-TLE n = 12, CRS-ALE n = 7.

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Probe trials were performed on days 5 and 10. Concerning time spent in the target arm (NW) during probe trial 1 (Fig. 4D), there was no group effect, although only controls (p < 0.05) spent more time than chance in the target arm. During probe trial 2 (Fig. 4E), ANOVA revealed a significant group effect (F3,38 = 4.29, p < 0.05). DZP-TLE and CRS-TLE rats spent significantly less time in the target arm than controls (p < 0.05). Only CRS-ALE and control rats spent more time than chance in the target arm (p < 0.005).

Histology

Neuronal cell counting

Compared to controls, neuronal density was significantly reduced in DZP-TLE rats in dCA1, hilus, BLA, vENT layers III–IV, dPIR and vPIR, and LDDM (Fig. 5A–H). In ventral hippocampus, neuronal density decreased in vCA1 and vCA3 (data not shown; p < 0.05). In DLStr, DZP-TLE rats did not display any significant change in neuronal density.

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Figure 5. Neuronal density in the hippocampus, the amygdala, the entorhinal and piriform cortices, lateral septum, and laterodorsal thalamus. Number of neurons/mm² in dorsal CA1 (dCA1) (A), dorsal CA3 (dCA3) (B), the hilus of the dentate gyrus (C), the basolateral amygdaloid nucleus (BLA) (D), layers III/IV of the ventral entorhinal cortex (vENT) (E), layer III of the ventral piriform cortex (vPIR) (F), the lateral septal nucleus (LS) (G), and in the laterodorsal dorsomedial thalamic nucleus (LDDM) (H). *p < 0.05, **p < 0.01, ***p < 0.005, statistically significant differences compared to control rats; #p < 0.05, ##p < 0.005 statistically significant differences compared to CRS-ALE rats; $p < 0.05, $$p < 0.01, statistically significant differences compared to CRS-TLE rats. Controls n = 6, DZP-TLE n = 8, CRS-TLE n = 7, CRS-ALE n = 4.

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In CRS-TLE rats compared to controls, hilus, vCA1 and vCA3, vENT layers III–IV, dPIR layer II, and vPIR exhibited a reduction in neuronal density as dramatic as DZP-TLE rats. Conversely, in dCA1, BLA, and vPIR, neuronal density was larger than in DZP-TLE rats but remained significantly lower than in controls. In LS and LDDM, neuronal density was no longer different from control levels (Fig. 5G–H).

In CRS-ALE rats, neuronal density returned to control levels in dCA1, BLA, dENT and vENT, dPIR layer III, vPIR layer II, LS, LDDM, and vCA3. In dCA3, hilus, dPIR layer II, vPIR layer III, and vCA1, neuronal density remained below control levels.

Mossy fiber sprouting

DZP-TLE (4.01 ± 0.42, p < 0.005) and CRS-TLE rats (3.48 ± 0.44, p < 0.01) showed higher Timm score than controls (0.77 ± 0.36). CRS-ALE rats (2.07 ± 0.68) reached also a higher score than controls but the difference was not significant (p = 0.09). CRS-ALE rats displayed a lower Timm score than DZP-TLE rats (p < 0.05), but the difference with CRS-TLE rats failed to reach significance (p = 0.07). These results agree with our previous data (Francois et al., 2011).

Discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

In line with previous studies in TLE rats (Persinger et al., 1993; Stafstrom et al., 1993; Letty et al., 1995; Persinger & Koren, 1998; Stewart & Leung, 2003; Detour et al., 2005; dos Santos et al., 2005; Frisch et al., 2007; Lenck-Santini & Holmes, 2008; Inostroza et al., 2011; Langer et al., 2011), we report here that DZP-TLE rats display widespread temporal lesions associated with severe behavioral impairments. These affected basal activity, anxiety, spatial memory, and procedural memory, as reported here for the first time. As shown by Francois et al. (2011) in carisbamate-treated rats starting 1 h after SE onset, CRS-TLE rats exhibited slight neuroprotection, developed motor seizures with behavioral impairments as dramatic as DZP-TLE rats, and CRS-ALE rats with large neuroprotection developed absence-like epilepsy translating into behavioral preservation.

Status epilepticus

Early after carisbamate injection, future CRS-ALE rats displayed weaker SE characteristics (sedation, no motor SRS), whereas future CRS-TLE rats still expressed motor SRS. Electrographic SE severity was alleviated 2 h after drug injection in CRS-ALE compared to DZP-TLE and CRS-TLE rats in which polyepileptiform discharges were frequent. This intergroup difference disappeared at 6 h after treatment (Francois et al., 2011). Total SE durations were not different between groups. Both behavioral and EEG changes point to an insult modification action of carisbamate that contributes to epilepsy expression change.

DZP-TLE rats

Locomotion, novel environment habituation, and anxiety

In accordance with present data (See supplemental data in Appendix S1.1), Persinger and Koren (1998) observed nocturnal hyperactivity in lithium–pilocarpine rats for 25 days post-SE. We still recorded night hyperactivity at 10 weeks post-SE, suggesting more persistent effects. Stewart and Leung (2003) found both diurnal and nocturnal hyperactivity for 8 weeks post-SE, with return to baseline after 12 weeks.

The open field evaluates habituation to a novel environment, and is sometimes used as an anxiety test. In the open field, DZP-TLE, CRS-TLE, and CRS-ALE rats performed as controls (Appendix S1.3). All groups showed habituation, that is, reduced their locomotion over time. There was no clear difference in anxiety levels in this test between epileptic groups and controls, as assessed by the number of center crossings.

In TLE rats, decreased anxiety was reported in the elevated plus-maze (Detour et al., 2005; dos Santos et al., 2005; Brandt et al., 2007; Inostroza et al., 2011; Langer et al., 2011). It usually translates into longer time spent and higher number of entries in open arms. Reduced anxiety was confirmed here in DZP-TLE rats. Lesions in amygdala (Davis, 1992) and ventral hippocampus (McHugh et al., 2004) and inactivation of both ventral hippocampus and LS (Trent & Menard, 2010) have been linked to anxiety deregulations. Extended neuronal loss observed in these regions could underlie the decrease in anxiety in DZP-TLE rats.

Some authors reported “psychosis-like” behavior in lithium–pilocarpine rats because they jumped from the elevated plus-maze. Here, 55% of DZP-TLE and 70% of CRS-TLE rats jumped from the maze, whereas none of control and CRS-ALE rats did so. Whether this behavior reflects psychosis, hyperactivity, or disinhibition is difficult to discriminate.

Spatial working memory

Working memory allows processing items during a few seconds. Earlier studies found impaired working memory in TLE rats in the radial-arm maze (Persinger et al., 1993; Letty et al., 1995; Detour et al., 2005). Here, DZP-TLE rats also showed dramatic spatial working memory alteration in the radial-arm maze. Moreover, for the first time we also found working memory impairment in the Morris water maze.

In the visible platform condition, DZP-TLE rats did not find the platform as directly as controls, suggesting possible vision or motivation impairments. However, high swim velocity of DZP-TLE rats in the Morris water maze and appetency for food reinforcement similar to controls in the radial-arm maze do not suggest motivational problems. Furthermore, visual acuity was normal in an attentional task requiring perfect visual function, with only slight attention deficit in DZP-TLE and CRS-TLE rats (data not shown) that could slightly impact on behavior.

Thigmotaxis behavior has two possible explanations: raised anxiety or task misunderstanding (Whishaw et al., 1995; Devan et al., 1999). In the open field and elevated plus-maze we rather found unchanged or lower anxiety level. Moreover, TLE rats often swam in circles, which is consistent with task misunderstanding by the rat seeking for an exit without learning anything. As a consequence, rats might be unable to process the complete task context.

In the radial-arm maze, rats can use their knowledge of the surrounding spatial configuration (allocentric strategy) or a strategy relying on repetition of angular choices (egocentric strategy). During no trial interruption training, both control and DZP-TLE rats did not show any preference for either strategy. Interrupting a testing session imposes a delay to the working memory system and makes egocentric strategies less efficient. In controls, such interruption led to a shift toward allocentric strategy. However, DZP-TLE rats were unable to make such a shift, indicating their inability to process the spatial configuration (See Appendix S1.5).

Long-term spatial memory

Long-term spatial memory includes learning and remembering a stable spatial representation (e.g., learning the location of a hidden platform in the Morris water maze). Most behavioral studies on TLE rats assessed long-term spatial memory in the Morris water maze (Frisch et al., 2007; Lenck-Santini & Holmes, 2008; Inostroza et al., 2011) and radial-arm maze (Persinger et al., 1993). Our results agree with literature, since during learning DZP-TLE rats swam a longer distance before reaching the platform. Furthermore, during the probe trial, they spent shorter time in the target quadrant than controls. Likewise, in both probe trials of the double-H maze, controls remembered platform location, whereas DZP-TLE rats neither remembered platform location during learning nor learned any automatism. DZP-TLE rats had marked neuronal loss in CA1 and entorhinal cortex, two regions containing place cells (O'Keefe & Dostrovsky, 1971) and grid cells (Hafting et al., 2005), respectively, which are thought to contribute to neuronal coding of spatial information. In addition, firing patterns of hippocampal place cells are altered in lithium–pilocarpine rats (Lenck-Santini & Holmes, 2008), which reinforces the potential role of global hippocampal dysfunction in behavioral impairments of DZP-TLE rats.

Procedural memory

Procedural memory integrates automatisms such as constant itineraries to reach a goal (i.e., go right to reach the platform). It is classically considered as requiring high number of trials and involves the dorsolateral striatum as its substrate (Packard & McGaugh, 1996). To our knowledge, procedural memory has been explored so far neither in TLE patients nor in TLE animal models. This is the first study assessing procedural memory in TLE rats. For this purpose, we used a procedural version of the Morris water maze (Cassel et al., 2007) and a novel testing device, the double-H maze, which is simpler than the Morris water maze (Pol-Bodetto et al., 2011). In both tasks, DZP-TLE rats displayed clear deficits in procedural learning and memory, although the performance of DZP-TLE rats improved over learning trials, particularly in the double-H maze (See Fig. 4 and Appendix S1.4). Because DZP-TLE rats did not display any evidence of damage in dorsolateral striatum, damage to striatum-cooperating structures might be responsible for this deficit.

CRS-TLE and CRS-ALE rats

Altogether, behavioral performance of TLE rats, whether treated or not with carisbamate, was impaired to the same extent. In CRS-TLE rats, neuroprotection was limited compared to CRS-ALE rats, did not prevent the occurrence of TLE, and was insufficient to influence behavioral deficits.

The behavioral performance of CRS-ALE rats was remarkably similar to that of controls, in terms of anxiety, working, long-term spatial, and procedural memory. This preservation could result from large neuroprotection induced by carisbamate and/or from epilepsy type, absence-like instead of temporal lobe seizures. The protection provided by carisbamate in amygdala and ventral hippocampus could participate in prevention of anxiety changes occurring in TLE rats (Davis, 1992; McHugh et al., 2004; Trent & Menard, 2010). Dorsal hippocampus and entorhinal cortex were also protected, potentially contributing to learning scores and memory systems normalization (Morris et al., 1986; Dudchenko, 2004).

In CRS-ALE rats, the main features of absence epilepsy are present, that is, thalamocortical SWDs and response to clinically effective antiepileptic drugs (Francois et al., 2011). The main differences between CRS-ALE rats and genetic models of absence epilepsy are lesions in limbic regions of CRS-ALE rats. Such lesions are not found in genetic models or human absence epilepsy (Niedermeyer, 1996; Danober et al., 1998; Berg et al., 2010). Potential impact of absence epilepsy on behavior has been studied in two genetic strains of absence epilepsy, the Genetic Absence Epilepsy Rat from Strasbourg (GAERS) and the WAG/Rij. Compared to nonepileptic rats, GAERS and/or WAG/Rij rats showed no difference in spontaneous locomotor activity, feeding, social interactions, and avoidance learning, but decreased sucrose consumption, higher anxiety, increased immobility in a forced-swim test, and reduced long-term memory were noted (Coenen et al., 1991; Vergnes et al., 1991; Jones et al., 2008; Sarkisova & van Luijtelaar, 2011).

The limbic system is involved in SWD occurrence. Blood oxygen level–dependent magnetic resonance imaging in WAG/Rij rats (Nersesyan et al., 2004) and cerebral glucose utilization rates in GAERS (Nehlig et al., 1991) increase in thalamocortical structures expressing SWDs but also limbic regions and basal ganglia. Likewise, in childhood absence epilepsy, functional activity increases were recorded in all brain areas (Engel et al., 1985; Ochs et al., 1987). This indicates full participation of limbic regions in absence seizures and interdependence of temporal lobe and thalamocortical structures. The lesions remaining in some limbic regions of CRS-ALE rats (CA3, hilus, piriform, and entorhinal cortex) are likely indicative of functional changes within the limbic system that might no longer regulate the thalamocortical system, allowing the thalamocortical SWDs expression (Onat et al., 2013).

In conclusion, the current study confirms the profound deficits found in rats developing TLE. It also extends previous findings to procedural memory, affected to a dramatic extent. Despite slight neuroprotection, the behavior of TLE-CRS rats was as dramatically impaired as that of DZP-TLE rats. Finally, in CRS-ALE rats, behavioral functions were entirely preserved. These changes resulted from both insult and disease modification. It would be of interest to study further this compound with different patterns of administration to discriminate between the respective effect of insult and epileptogenesis modification and open new avenues in the search of antiepileptogenic compounds.

Acknowledgments

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

This work was supported by the French Ministry of Research, the French Institut National de la Science et de la Recherche Médicale (INSERM U 666), the French Centre National de la Recherche Scienfifique (CNRS-UDS UMR 7364), and the University of Strasbourg. Carisbamate was a gift from Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, NJ, USA.

Disclosure

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information

None of the authors has any conflict of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

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  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure
  8. References
  9. Supporting Information
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epi12219-sup-0001-AppendixS1.pdfapplication/PDF269KAppendix S1. Effects of carisbamate treatment on locomotion, working and procedural memory.

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