N-methyl-d -aspartate receptor NR2B subunit involved in depression-like behaviours in lithium chloride-pilocarpine chronic rat epilepsy model

Epilepsy Research 119 (2016) 77–85

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N-methyl-d-aspartate receptor NR2B subunit involved in depression-like behaviours in lithium chloride-pilocarpine chronic rat epilepsy model Wei-Feng Peng a , Jing Ding a,∗ , Xin Li a , Fan Fan b , Qian-Qian Zhang a , Xin Wang a,c,∗ a

Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai, China Department of Emergency, Zhongshan Hospital, Fudan University, Shanghai, China c The State Key Laboratory of Medical Neurobiology, The Institutes of Brain Science and the Collaborative Innovation Center for Brain Science, Fudan University, Shanghai, China b

a r t i c l e

i n f o

Article history: Received 21 April 2015 Received in revised form 25 August 2015 Accepted 21 September 2015 Available online 25 September 2015 Keywords: Epilepsy Depression Glutamate N-methyl-d-aspartate receptor NR2B subunit

a b s t r a c t Depression is a common comorbidity in patients with epilepsy with unclear mechanisms. This study is to explore the role of glutamate N-methyl-d-aspartate (NMDA) receptor NR1, NR2A and NR2B subunits in epilepsy-associated depression. Lithium chloride (Licl)-pilocarpine chronic rat epilepsy model was established and rats were divided into epilepsy with depression (EWD) and epilepsy without depression (EWND) subgroups based on forced swim test. Expression of NMDA receptor NR1, NR2A and NR2B subunits was measured by western blot and immunofluorescence methods. The immobility time (IMT) was significantly greater in Licl-pilocarpine model group than in Control group, which was also greater in EWD group than in EWND group. No differences of spontaneous recurrent seizure (SRS) counts over two weeks and latency were found between EWD and EWND groups. The number of NeuN positive cells was significantly less in Licl-pilocarpine model group than in Control group, but had no difference between EWD and EWND groups. The ratios of phosphorylated NR1 (p-NR1)/NR1 and p-NR2B/NR2B were significantly greater in the hippocampus in EWD group than in EWND group. Moreover, the expression of p-NR1 and p-NR2B in the CA1 subfield of hippocampus were both greater in Licl-pilocarpine model group than Control group. Selective blockage of NR2B subunit with ifenprodil could alleviate depression-like behaviours of Licl-pilocarpine rat epilepsy model. In conclusion, glutamate NMDA receptor NR2B subunit was involved in promoting depression-like behaviours in the Licl-pilocarpine chronic rat epilepsy model and might be a target for treating epilepsy-associated depression. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Depressive disorders are very common in patients with epilepsy. According to the data from a recent meta analysis, patients with

Abbreviations: DG, dentate gyrus; EWD, epilepsy with depression; EWND, epilepsy without depression; FST, forced swim test; 1 H-MRS, proton magnetic resonance spectroscopy; IMT, immobility time; Licl, lithium chloride; NeuN, neuronal specific nuclear protein; NMDA, N-methyl-d-aspartate; SCT, sucrose consumption test; SE, status epilepticus; SRS, spontaneous recurrent seizures; SucroRate, sucrose consumption rate; TLE, temporal lobe epilepsy. ∗ Corresponding authors at: Department of Neurology, Zhongshan Hospital, Fudan University, 180 Fenglin Road, 200032 Shanghai, China. E-mail addresses: [email protected] (W.-F. Peng), [email protected] (J. Ding), [email protected] (X. Li), [email protected] (F. Fan), [email protected] (Q.-Q. Zhang), [email protected] (X. Wang). http://dx.doi.org/10.1016/j.eplepsyres.2015.09.013 0920-1211/© 2015 Elsevier B.V. All rights reserved.

epilepsy had an overall prevalence of active depression of 23.1% and lifetime depression of 13% (Fiest et al., 2013). Previous clinical data also indicated that the prevalence of depression in epilepsy was higher than most of other neurological and non-nervous diseases, and depression had significantly negative influence on quality of life in patients with epilepsy (Cramer et al., 2003; Ettinger et al., 2004). Recent evidences indicated that there were common neurobiological mechanisms between the two conditions (Harden, 2002; Sankar and Mazarati, 2010). Kanner et al. proposed that there were bidirectional relations between epilepsy and depression, and several pathogenic mechanisms identified in animal models and patients with psychiatric disorders were found to facilitate the occurrence of seizures or process of epileptogenesis in animals (Kanner, 2012a). These mechanisms included: (1) disturbances of neurotransmitters such as serotonin (5-HT) and 5-HT1A receptor in the central nervous system; (2) endocrine

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disturbances such as hyperactivity of hypothalamic-pituitaryadrenal axis; and (3) inflammatory mechanisms (Kanner, 2012b). The common underlying mechanisms may explain the high comorbidity of epilepsy and depression. However, Mazarati et al. (2008) found that depression-like behaviours in Licl-pilocarpine rat chronic epilepsy model were resistant to fluoxetine, a selective serotonin reuptake inhibitor, suggesting epilepsy-associated depression might have other underlying mechanisms beyond alterations of serotonergic pathways. Glutamate is the principal excitatory neurotransmitter in central nervous system. Excessive glutamate release has pathogenic roles in epilepsy which has been established for a long time (Nadler, 2012). In recent years, evidences demonstrated that glutamatergic system might also take part in the pathogenesis of depression (Mitchell and Baker, 2010; Szakacs et al., 2012). Glutamate N-methyl-d-aspartate (NMDA) receptor is an ionotropic glutamate receptor both involved in the normal neuronal function and pathological process of some neurological disorders (Waxman and Lynch, 2005). Antagonists of NMDA receptor, such as MK801 and ketamine, have been shown to have antidepressant properties in animal models and patients with depression (Pittenger et al., 2007). On the basis of these findings, we hypothesised that glutamatergic pathways might be involved in the pathogenesis of comorbidity of epilepsy and depression. There was a primary demonstration in our previous clinical study that glutamate and glutamine level in the right hippocampus significantly increased in patients with epilepsy and moderate depression and was the independent risk factor for depression (Peng et al., 2013). In this study, we employed Licl-pilocarpine chronic rat epilepsy model which were suggested by Mazarati et al. (Mazarati et al., 2009; Pineda et al., 2011) to be served as an animal model of the comorbidity of epilepsy and depression to further explore the role of glutamate NMDA receptor NR1, NR2A and NR2B subunits in epilepsy-associated depression. We found elevated phosphorylations of NR1 and NR2B (p-NR1 and p-NR2B) subunits level in the hippocampus in EWD group compared with EWND group, and in the CA1 subfield of hippocampus in Licl-pilocarpine model group compared with Control group. As NR1 subunit was the necessary subunit of NMDA receptor, blocking NR1 might produce some inevitable side effects. So selective blockage of NR2B subunit with ifenprodil was administered in Licl-pilocarpine chronic rat epilepsy model and changes of depression-like behaviours were observed to further investigate the role of NMDA receptor NR2B subunit in the pathogenesis of epilepsy-associated depression. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (SLRC laboratory Animal Corporation) weighing about 200–250 g were housed for 1 week before experiment. Housing temperature was maintained at 22 ± 1 ◦ C, with 12 h light–12 h dark cycle and humidity of 35–40%. The experiments were done in accordance with the guidelines of the National Institutes of Health and the study was approved by Animal Care and Use of Committee of Zhongshan Hospital, Fudan University, China. 2.2. Establish Licl-pilocarpine chronic rat epilepsy model The Licl-pilocarpine chronic rat epilepsy model was established according to previous studies (Mazarati et al., 2008). Animals received an intraperitoneal (i.p.) injection of lithium chloride (Licl, 127 mg/kg, dissolved in deionised water, Sigma, St. Louis, MO, USA). On the next day (22–24 h after the injection of Licl) animals

were injected i.p. with scoporamine methyl nitrate (1 mg/kg, Sigma) to alleviate peripheral cholinergic effects of pilocarpine, and 30 min later they were injected i.p. with pilocarpine hydrochloride (40 mg/kg, Sigma). Continuous or repetitive limbic seizures started 15–30 min after pilocarpine injection. The stages of seizure degree were classified by the Racine scale (Racine, 1972). After 30 min from the first seizure with equal to or greater than Racine 4th stage, rats were injected i.p. with diazepam (10 mg/kg) to terminate further seizures. Control animals were injected i.p. with the same dose of Licl and scoporamine but used saline instead of pilocarpine. One week after status epilepticus (SE), animals underwent two-week continuous video monitoring for detecting spontaneous seizures. All of the videos were analysed offline. To avoid immediate effects of spontaneous seizures on outcome of behavioural assay, further experiments were performed after verifying no seizures had developed for at least 6 h prior the forced swim test. 2.3. Sucrose consumption test (SCT) This test for anhedonia is on the basis of the innate preference of rodents towards sweets (Pucilowski et al., 1993). All of the rats were habituated with 1% sucrose water for 2 days before beginning the test, and those drank too much or too little sucrose water were removed. On regular days the rats were housed in groups. Every 5 rats were kept in 1 cage. But on the day of SCT test, 1 rat was put in 1 cage to calculate the sucrose and common water consumption of every rat. Water deprivation was carried out for 24 h before the test. Then on the day of test, every cage was supplied with two identical bottles of water, the one was regular water, and the other was 1% sucrose. The test was performed starting from 9:00 to 10:00 AM. On half of the test time, the places of two bottles were exchanged. Regular and sucrose water intakes were calculated 1 h later. Sucrose preference rate (SucroRate) = sucrose consumption/(sucrose consumption + water consumption) × 100%. A low sucrose preference rate was interpreted as an equivalent of the state of anhedonia. The SCT was performed before experiment and every 1 week after injection of pilocarpine. 2.4. Forced swim test (FST) The FST is carried out to test state of despair (Porsolt et al., 1979). A rat was put into a transparent tank filled with water (60 cm height and 30 cm diameter), and the water temperature was about 22–25 ◦ C. Then 5 min of swimming behaviour was videotaped and analysed offline. There are 3 types of swimming behaviours in the modified FST: immobile behaviour, climbing behaviour, and swimming behaviour. The longer time of immobility is indicative of state of despair (Detke et al., 1995). The immobility time (IMT) was recorded for all of the tested rats. As the IMT was an important parameter to define depression-like behaviours in previous studies (Mazarati et al., 2008; Ghasemi et al., 2010), we set the average IMT of control rats add 1 standard deviation as the cutoff of depressionlike behaviours in this study. The FST was performed at the end of SRS observation, that is, 21 days after SE. 2.5. Western blot analysis After 21 days from SE and the FST was completed, half of the rats were transcardially perfused with 4 ◦ C 0.1 M PBS 200 ml rapidly and the hippocampus and frontal lobe of both hemispheres were dissected out on ice, and then stored at −80 ◦ C. The tissues of rat brains were homogenised in RIPA lyisis buffer (Beyotime Institute of Biotechnology, China) and centrifuged at 18,000 × g r.p.m. at 4 ◦ C for 20 min. The supernatant was shifted out and total protein content was measured by BCA protein assay kit (Beyotime). About 50 ␮g proteins from each sample were used for measurement. SDS-PAGE

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and western blotting were performed according to standard procedures. The primary antibodies were rabbit anti-NR1 (120 kDa, 1:1000, Epitomics), rabbit anti-NR1-phospho S889 (120 kDa, 1:500, Epitomics), rabbit anti-NR2A (180 kDa, 1:300, Epitomics), rabbit anti-NR2A-phospho Y1325 (163 kDa, 1:300, Abcam), rabbit antiNR2B (180 kDa, 1:300, Abcam), and rabbit anti-NR2B-phospho S1303 (180 kDa, 1:200, Epitomics). The rabbit anti-␤-actin primary antibody (40 kDa, 1:6000, Epitomics) was used as internal reference. And the anti-rabbit secondary antibody coupled to Odyssey IRDye 800 (Rockland) was used. The signals were scanned and quantified on Odyssey imaging system (LI-COR). The optical density (O.D.) values of each sample were normalised with the corresponding amount of ␤-actin. The ratios of p-NR1/NR1, p-NR2A/NR2A, and p-NR2B/NR2B were calculated for further statistical analysis. 2.6. Immunofluorescence procedure After the FST was completed, another half of the rats were deeply anesthetised and transcardially perfused with 0.1 M phosphate buffer saline (PBS, PH 7.4) followed by 4 ◦ C 4% paraformaldehyde in 0.1 M PB (PH 7.4). The rats were decapitated and their brains were removed and stored in 4% paraformaldehyde at 4 ◦ C for 24 h, then shifted to 20% sucrose in 0.1 M PBS at 4 ◦ C for 48 h, and then to a 30% sucrose solution kept at 4 ◦ C until the rat brain sank. Coronal brain sections (20 ␮m) were cut from the bregma −3.24 mm to −3.96 mm (Watson and Paxions, 2005) and collected in 0.1 M PBS for immunostaining. The free-floating slices were incubated in 3% goat serum in 0.1 M PBS for 30 min at room temperature, then incubated over night at 4 ◦ C in primary antibody diluted with 0.3% triton in 0.1 M PBS (rabbit anti-NR1-phospho S889, 1:200, Epitomics; rabbit anti-NR2B-phospho S1303 (180 kDa, 1:200, Epitomics)). Sections were subsequently incubated with mouse anti-neuronal specific nuclear protein (NeuN, 1:600, Millipore). After rinsing 3 times in 0.1 M PBS, the sections were incubated with the secondary antibodies (anti-rabbit, Alexa 546; anti-mouse, Alexa 488, Molecular Probes, Leiden, Netherlands) for 1–2 h at room temperature. The primary antibody omission controls were conducted to test specificity of the antibodies. All of the sections were mounted in slides and coverslipped with DPX mountant (Sigma, Aldrich, USA). The sections were observed by fluorescence microscopy (Olympus). Photomicrographs of CA1, CA3 and dentate gyrus (DG) subfields of the hippocampus were taken using the 20× magnification of fluorescent microscope with excitation of 546 nm (red) for p-NR1 and p-NR2B subunits staining and 488 nm (green) for NeuN protein. Cell counting was conducted in 2 slices in each rat brain. The positive cells of NeuN and double labelled p-NR1 and NeuN, and p-NR2B and NeuN cells were counted using ImageJ software. The ratios of double labelled p-NR1 and NeuN to total NeuN cells, and p-NR2B and NeuN cells to total NeuN cells were calculated. Data for each group were obtained and a statistical analysis by means of t-test or one-way ANOVA was used with SPSS19.0 software. 2.7. Blockage of NR2B subunit treatment The selective antagonist of NR2B subunit ifenprodil was used. Ifenprodil was dissolved in deionised water and diluted to 3 mg/ml. It was freshly prepared before the experiment. The Licl-pilocarpine chronic rat epilepsy model was established as above described. After 21 days from SE, the rats were randomly assigned to groups with and without treatment of ifenprodil. According to given ifenprodil or not, the Licl-pilocarpine chronic rat epilepsy model was divided into Licl-Pilo + ifenprodil group and Licl-pilocarpine model group, the same with control rats (Control group and Control + ifenprodil group). The treatment groups were i.p. administered ifenprodil (3 mg/kg) 45 min before the FST according to the appropriate dose chosen by the previous study (Ghasemi et al.,

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Table 1 Comparison of IMT, SRS latency and SRS counts over 2 weeks between EWD and EWND groups.

n IMT (s) Latency of SRS (d) SRS counts over 2 weeks

EWD group

EWND group

P value

8 143.30 ± 10.67 11.88 ± 0.88 18.75 ± 8.81

15 43.76 ± 3.96 11.60 ± 0.99 15.53 ± 4.43

0.00 0.86 0.72

EWD, epilepsy with depression; EWND, epilepsy without depression; SRS, spontaneous recurrent seizures. Data were presented as mean ± SEM.

2009). The rats in Licl-pilocarpine model and Control groups were used saline instead of ifenprodil. 2.8. Data analysis All of the data were analysed with the SPSS19.0 software. Quantitative variables were expressed as a mean ± standard error of the mean (SEM). An independent t-test and a one-way ANOVA (Analysis of Variance) method were used to compare means between groups when data conformed to a normal distribution; otherwise the Wilcoxon rank sum test was used. A statistical probability of P < 0.05 was considered to be significant. 3. Results 3.1. Behaviours of spontaneous recurrent seizures (SRS) in the Licl-pilocarpine chronic rat epilepsy model After screening with the SCT, 6 rats were excluded from drinking too much or too little sucrose and a total of 80 rats were included, of which 10 rats were used as Control group and the other 70 rats were adopted to establish Licl-pilocarpine chronic rat epilepsy model. Removing the rats died or without seizures arriving Racine 4th degree, 29 rats that survived the first week after SE were monitored with video surveillance system for 2 weeks (from the day 7 to day 21 after SE). The two-week video tapes were analysed, and SRS counts over 2 weeks and latency to first SRS from the day of SE were recorded. Only spontaneous seizures reached stage 4–5 (rearing and/or rearing and falling) (Racine, 1972) were counted and considered to be chronic epilepsy model. After video analysed, 23 rats met the criteria of chronic epilepsy model and were included in the further study. 3.2. Depression-like behaviours in the Licl-pilocarpine chronic rat epilepsy model The IMT in FST and SucroRate in SCT were compared between Control and Licl-pilocarpine model groups. The IMT in Liclpilocarpine model group was significantly longer than Control group (P < 0.05, see Fig. 1A). The SucroRate in Licl-pilocarpine model group was in declining trend after SE compared with Control group (see Fig. 1B). Based on the severity of behavioural impairment in FST, we divided the Licl-pilocarpine model group into two subgroups: those with IMT more than 77.91 s (the average IMT in Control group was 48.20 ± 29.71 s, according to the standard of depression-like behaviours describing above, so the cutoff of IMT was set as mean + 1 standard deviation [48.20 + 29.71 = 77.91 s]) were classified as having depression-like behaviours (EWD group, n = 8), and those with IMT ≤ 77.91 s were regarded as animals without depression (EWND group, n = 15). The IMT, latency of SRS and total SRS counts over 2 weeks were compared between EWD and EWND groups. The IMT in EWD group was significantly longer than EWND group, while the SRS variables had no differences between EWD and EWND groups (see Table 1).

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the CA1, CA3 and DG subfields of hippocampus in Licl-pilocarpine model group was significantly less than Control group (*P < 0.01, #P < 0.05, see Fig. 2A). No difference of NeuN positive cell number in the CA1, CA3 and DG subfields of hippocampus were found between EWND and EWD groups (P > 0.05, see Fig. 2B and C). 3.4. The expression of p-NR1 and p-NR2B subunits in the hippocampus was greater in EWD group than in EWND group, and also greater in the CA1 subfield of hippocampus in Licl-pilocarpine model group than Control group

Fig. 1. Comparisons of depression-like behaviours between Control (n = 10) and Licl-pilocarpine model (n = 23) groups. (A) The IMT in Licl-pilocarpine model group was significantly longer than Control group, *P < 0.05. (B) The SucroRate in Liclpilocarpine model group was in declining trend after SE compared with Control group. Values are presented as mean ± SEM. IMT – immobility time; SucroRate – sucrose preference rate.

3.3. The number of NeuN positive cells decreased in Licl-pilocarpine model but had no difference between EWND and EWD groups The neuronal loss in the hippocampus was firstly compared between Control and Licl-pilocarpine model groups using NeuN staining. The NeuN protein is specifically expressed in neuronal nuclei. As a marker of neurons in central nervous system, the decreased level of NeuN positive cells reflects neuronal loss in the specific fields. The number of NeuN positive cells in the equal size areas of CA1, CA3, and DG subfields of hippocampus was counted. The results showed that the number of NeuN positive cells in

The ratios of NR1, NR2A and NR2B subunits and their phosphorylations (p-NR1, p-NR2A and p-NR2B) in the hippocampus quantified by the western blot method were firstly compared between Control and Licl-pilocarpine model groups, and no significant differences of p-NR1/NR1, p-NR2A/NR2A and p-NR2B/NR2B ratios in the hippocampus were found (see Fig. 3A, D and G). Then comparisons of p-NR1/NR1, p-NR2A/NR2A and p-NR2B/NR2B ratios in the hippocampus were conducted between EWND and EWD groups. It showed that p-NR1/NR1 (P < 0.01) and p-NR2B/NR2B (P < 0.05) ratios in the hippocampus were both significantly greater in EWD group than in EWND group (see Fig. 3B–C and H–I). But there was still no significant difference of pNR2A/NR2A in the hippocampus between EWND and EWD groups (see Fig. 3E and F). Based on the above positive findings of NR1 and NR2B subunits, the distribution and location of p-NR1 and p-NR2B subunits in the hippocampus was further measured using immunofluorescence method. The p-NR1 and p-NR2B subunits were all extensively distributed in cell bodies and axons of neurons in the hippocampus. When double labelled with NeuN, the ratios of p-NR1 and p-NR2B positive neurons to total NeuN cells in the hippocampus were compared between Control and Licl-pilocarpine model groups, and then between EWND and EWD groups. The results showed that the ratio of p-NR1 positive cells to total NeuN cells in the CA1 subfield of hippocampus was significantly greater in Licl-pilocarpine model group than in Control group, which was also significantly greater in EWD group than in EWND group (see Fig. 4A–C). And the ratio of p-NR2B positive cells to total NeuN cells in CA1 subfield of hippocampus was significantly greater in Licl-pilocarpine model group than in

Fig. 2. (A) The number of NeuN positive cells in the CA1, CA3 and DG subfields of hippocampus in Licl-pilocarpine model group (n = 11) was significantly less than Control group (n = 5), *P < 0.01, #P < 0.05. (B) No difference of NeuN positive cell number in the CA1, CA3 and DG subfields of hippocampus was found between EWND (n = 7) and EWD groups (n = 4). (C) 10× magnification immunofluorescence micrographs showing NeuN positive cells in the CA1, CA3 and DG subfields of hippocampus in Control, EWND and EWD groups. Values are presented as mean ± SEM. DG – dentate gyrus; EWD – epilepsy with depression; EWND – epilepsy without depression; NeuN – neuronal specific nuclear protein.

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Fig. 3. (A, D and G) No significant differences of p-NR1/NR1, p-NR2A/NR2A and p-NR2B/NR2B ratios in the hippocampus were found between Control (n = 5) and Liclpilocarpine (n = 12) groups. (B and C) The ratios of p-NR1/NR1 and (H and I) p-NR2B/NR2B in the hippocampus were both significantly greater in EWD group (n = 4) than in EWND group (n = 8), * P < 0.01, #P < 0.05. (E and F) There was no significant difference of p-NR2A/NR2A in the hippocampus between EWND and EWD groups. Values are presented as mean ± SEM. EWD – epilepsy with depression; EWND – epilepsy without depression.

Control group, which was greater in the CA1 and CA3 subfields of hippocampus in EWD group than in EWND group (see Fig. 4D–F). 3.5. Blockage of NR2B subunit treatment significantly decreased the IMT of Licl-pilocarpine chronic rat epilepsy model Based on the above findings, the NR1 and NR2B subunits of NMDA receptor might be both involved in promoting depressionlike behaviours of the Licl-pilocarpine chronic rat epilepsy model. Considering the NR1 subunit is necessary for NMDA receptor and blocking it might produce some inevitable side effects, the selective antagonist of NR2B subunit (ifenprodil) was used. Forty rats were i.p injected Licl-pilocarpine to induce SE. Sixteen rats became Licl-pilocarpine chronic epilepsy model with depressionlike behaviours (IMT = 92.22 ± 13.44 s) after 21 days from SE according to the dividing standard mentioned above. The IMT was compared between Licl-Pilo + ifenprodil, Licl-pilocarpine model, Control, and Control + ifenprodil groups. The results showed that the IMT was significantly longer in Licl-pilocarpine model group than in Control group (P < 0.05) and significantly shorter in LiclPilo + ifenprodil group than Licl-pilocarpine model group (P < 0.01). There was no statistical difference of the IMT between Control and Control + ifenprodil groups (see Fig. 5). 4. Discussion Although the phenomenon of comorbidity between epilepsy and depression is very common, the underlying mechanisms have not been clear yet. One of the important reasons is the difficulty to establish an ideal animal model of comorbidity of epilepsy and depression. There were many epilepsy models such

as GAERS (Danober et al., 1998), WAG/Rij (Sarkisova et al., 2003) and Long–Evans rat (Huang et al., 2012) demonstrated to have psychiatric symptoms. In this study, we used Licl-pilocarpine chronic rat epilepsy model because it was suggested that the prevalence of comorbidity of epilepsy and depression was higher in temporal lobe epilepsy (TLE) than other epilepsy types (Garcia, 2012). Pilocarpine or Licl-pilocarpine animal epilepsy models had been demonstrated to have depression-like behaviours in some studies (Mazarati et al., 2008; Muller et al., 2009) and were suggested to be served as a model of the comorbidity of epilepsy and depression. However, there was no standard cutoff point of IMT in previous studies, and epilepsy models with depressionlike behaviours were regarded as a whole of the comorbidity of epilepsy and depression models when compared with normal controls (Sarkisova et al., 2003; Jones et al., 2008; Mazarati et al., 2008). In the Licl-pilocarpine chronic rat epilepsy model of our study there were big interindividual differences of IMT in FST, although the IMT was significantly greater in Licl-pilocarpine model group than Control group, which was about twice as long as compared with Control group (Fig. 1A) and in accordance with a study by Pineda et al. (Pineda et al., 2011). Some rats in Licl-pilocarpine model group even had similar IMTs with Control group. Therefore, these rats should be classified as epilepsy without depression group. But how to set an ideal dividing standard was extremely difficult. In the study of Pineda et al. (Pineda et al., 2011), they divided the post-SE rats into two groups based on IMT: moderately depressed group with IMT ≤ 100 s and severe depressive group with IMT > 100 s. According to this standard, the percentage of obviously depressive rats in their study was about 50% (the average IMT in naïve rats was about 60 s, which was about 120 s in the post-SE rats). In our study, the average IMT in naïve rats was about 40 s, which was about

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Fig. 4. (A) The ratio of p-NR1 positive cells to total NeuN cells in the CA1 subfield of hippocampus was significantly greater in Licl-pilocarpine model group (n = 11) than in Control group (n = 5), (B) which was also significantly greater in EWD group (n = 4) than EWND group (n = 7), *P < 0.01, #P < 0.05. (D) The ratio of p-NR2B positive cells to total NeuN cells in CA1 subfield of hippocampus was significantly greater in Licl-pilocarpine model group than in Control group, (E) which was greater in the CA1 and CA3 subfields of hippocampus in EWD group than EWND group, *P < 0.01, #P < 0.05. (C) 20× magnification double-immunofluorescence micrographs showing p-NR1 and NeuN positive cells in the CA1 subfield of hippocampus and (F) p-NR2B and NeuN positive cells in the CA3 subfield of hippocampus in Control, EWND and EWD groups. Values are presented as mean ± SEM. DG – dentate gyrus; EWD – epilepsy with depression; EWND – epilepsy without depression; NeuN – neuronal specific nuclear protein.

80 s in Licl-pilocarpine model group. Considering the differences of experimental environment and procedures, the percentage of obviously depressive rats in our study was 34.6% when the dividing standard was set as 1 average + 1 standard deviation of control IMT (77.91 s) and it was close to the percentage of the study by Pineda et al. But if it was set as 1 average + 2 standard deviation of control IMT, the standard of IMT was 107.62 s and would be greater than the average IMT in Licl-pilocarpine model group. As for the brain region related to depression, some studies indicated that amygdale was also involved in the depressive disorders of patients with epilepsy (Briellmann et al., 2007), but many other studies demonstrated that it was hippocampal atrophy or functional changes in the hippocampus that promoted the evolution of depressive symptoms in patients with epilepsy (Seidenberg et al., 2005; Gilliam et al., 2007; Finegersh et al., 2011). In our previous 1 H-MRS study, we also found that the increased right hippocampal glutamate and glutamine level was an independent risk factor for depressive symptoms in patients with epilepsy (Peng et al., 2013). Therefore, in this study we chose the hippocampus as our studying target.

Fig. 5. The IMT was significantly longer in Licl-pilocarpine model group than in Control group, and significantly shorter in Licl-Pilo + ifenprodil group than Liclpilocarpine model group, *P < 0.01, #P < 0.05. Values are presented as mean ± SEM of 8 rats every group. No statistical difference of IMT between Control and Control + ifenprodil groups. IMT, immobility time.

Glutamate is an important excitatory neurotransmitter in central nervous system which has been demonstrated to take part in the pathogenesis of epilepsy and depression respectively (Mitchell and Baker, 2010; Nadler, 2012). Extracellular glutamate was revealed to be elevated immediately before and during seizures

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(Carlson et al., 1992; During and Spencer, 1993; Neppl et al., 2001), which was about 4–5 fold higher in the seizure originating regions of patients with TLE than other brain regions (Cavus et al., 2005). In addition, glutamatergic changes could also take part in the pathophysiology of primary depression. Some 1 H-MRS studies found increased level of glutamate/glutamine in the frontal lobe, basal ganglia, and occipital lobe (Castillo et al., 2000; Michael et al., 2003; Sanacora et al., 2004) of patients with depression. Our previous 1 H-MRS study showed that glutamate might be involved in the pathogenesis of promoting depressive symptoms in patients with epilepsy (Peng et al., 2013). Glutamate mainly takes effects through its receptors, and NMDA receptor is an important ionotropic glutamate receptor which participates in pathological processes of many neurological diseases including epilepsy (Kalia et al., 2008). Therefore, in this study we further investigated the role of glutamate NMDA receptor NR1, NR2A, and NR2B subunits in the pathogenesis of epilepsy-associated depression. NMDA receptors are primarily composed of three subunits designated NR1, NR2A, and NR2B in the matured brain, of which NR1 is necessary. Glutamate binds to regulatory NR2 subunits. The expression level of NR1 always represents total NMDA receptor level. A study by Toro et al. (Toro et al., 2007) found that NR1 subunit of NMDA receptor was upregulated in the dentate molecular layer of hippocampus in TLE patients with dysphoria and depression, suggesting increased NMDA receptor function was involved in the pathogenesis of comorbidity between TLE and depression. Partly in coincidence with their study, we also found p-NR1 subunit level was not only greater in the CA1 subfield of hippocampus in Liclpilocarpine model group than in Control group, but also greater in EWD group than in EWND group. The key findings of this study were that glutamate NMDA receptor NR2B subunit might be involved in the pathogenesis of epilepsy-associated depression, which implied clinical potential. NR2B subunit is one of the critical subtypes of NMDA receptor NR2 subunit (Nadler, 2012). Increased NR2B expression has been observed in the cortex and hippocampus of patients with TLE (Mathern et al., 1998, 1999), and also in limbic structure of amygdale kindling epilepsy model (Al-Ghoul et al., 1997). Many in vitro and in vivo studies suggested NR2B subunit was associated with epileptogenesis (Moddel et al., 2005; Di Maio et al., 2011). In agreement with these results, we also found the expression of p-NR2B subunit was greater in the CA1 subfield of hippocampus in Licl-pilocarpine model group than in Control group. Moreover, the expression of p-NR2B subunit was also greater in the hippocampus in EWD group than in EWND group, and selective blockage of NMDA receptor NR2B subunit could ameliorate the depression-like behaviour of Licl-pilocarpine chronic rat epilepsy model. Therefore, NR2B subunit pathway was probably the key site both involved in the epileptogenesis and the pathogenesis of epilepsy-associate depression. However, no differences of pNR1/NR1 and p-NR2B/NR2B ratios measured by the western-blot method in the hippocampus were found between Licl-pilocarpine model and Control groups in this study. The reason that we thought might be related to different distributions and functions of the NMDA receptor which locates postsynaptically and extrasynaptically. According to previous studies, the postsynaptic NMDA receptor mostly contains the NR1/NR2A combination and has antiapoptotic activity, whereas the extrasynaptic NMDA receptor is mainly composed of NR1/NR2B type and promotes cell death (Hardingham et al., 2002; Li and Ju, 2012). In a study by Frasca et al. (2011), decreased postsynaptic NR2B subunit and increased extrasynaptic NR2B subunit predicted receptor translocation from synaptic to extrasynaptic sites in the epileptogenesis. So, in our study, although there were no differences of the p-NR1 and pNR2B levels in the hippocampus measured by the western-blot method between Licl-pilocarpine model and Control groups, the

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translocation of these subunits from postsynaptic to extrasynaptic sites might exist. In recent years, antagonists of NMDA receptor were widely used clinically to treat neurological diseases, such as felbamate, riluzole, and memantine (Kalia et al., 2008). The non-competitive antagonist of NMDA receptor named ketamine had been found to have rapid antidepressant effects in animal models and patients with depressive disorders (Garcia et al., 2009; Diazgranados et al., 2010; Li et al., 2010). However, it had limitations in clinical trials due to some unacceptable adverse effects (Mathew et al., 2012). Selective antagonists of NR2B were found to have neuroprotective effects, and the selective NR2B antagonist MK-0657 even had antidepressant properties in patients with treatment-resistant depression (Ibrahim et al., 2012). In our study, it indicated that selective antagonists of NR2B also had antidepressant effects in epilepsyassociated depression. Ifenprodil was proved to have antiepileptic effects in animals with epilepsy either (Hrncic et al., 2009; Di Maio et al., 2012). The antiepileptic effect of ifenprodil was probably related to its neuroprotective effect, as in the study of Frasca et al. reduced hippocampal pyramidal cell loss was observed in the epileptogenesis after administering ifenprodil (Frasca et al., 2011). The findings of this study and previous studies suggested that antagonists of NR2B might become therapeutic pharmacies for patients with comorbidity of epilepsy and depression in the future. The results NeuN staining indicated that there was significant neuronal loss in the hippocampus of Licl-pilocarpine model, but no significant difference of neuronal loss degree between EWND and EWD groups. On one hand, it demonstrated the comparisons of NR1, NR2A and NR2B between EWND and EWD groups was comparable. On the other hand, the negative results of p-NR1/NR1 and p-NR2B/NR2B between Control and Licl-pilocarpine model groups might also be attributed by the neuronal loss of Licl-pilocarpine model group. Neuronal damage or neuronal loss could be found in the pilocarpine epilepsy model from several hours to weeks after SE (Do et al., 2012). The brain regions of olfactory cortex, amygdala, thalamus, hippocampal formation, and neocortex all had histopathological alterations in the animals experiencing SE induced by pilocarpine (Curia et al., 2008). Hippocampal neuronal loss may promote the epileptogenesis, but the specific mechanisms have still not been clear. The SRS latency and SRS counts over two weeks all had no differences between EWD and EWND groups in this study, which excluded the confounding influences of seizure severity on glutamate NMDA receptor NR1, NR2A and NR2B subunits levels. There were some reports about the influences of seizure frequency or severity on depression, and the results had so far been proved inconclusive (Gandy et al., 2012). The results of this study indicated that seizure might not be simply as a stressor to cause depression, there were probably more neurobiological mechanisms between epilepsy and depression. 5. Conclusions In conclusion, our study indicated that glutamate NMDA receptor NR2B subunit was involved in promoting depression-like behaviours in the Licl-pilocarpine chronic rat epilepsy model and might be a target for treating epilepsy-associated depression. Author contributions Wei-Feng Peng carried out the vast majority of the experimental procedures, conducted the majority of the data analysis and interpretation of the data as well as wrote the first draft of the manuscript. Jing Ding instructed the whole experimental procedures, conducted some of the experiments and participated

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in the interpretation of the data and preparing the manuscript. Xin Wang oversaw the design of this study, instructing the data analysis and preparing the manuscript. Xin Li, Fan Fan and Qian-Qian Zhang have been involved in conducting parts of experimental procedures. Grants This work was supported by the National Natural Science Foundation of China (Code: 31271188, 81200999) and Shanghai Municipal Science and Technology Commission China (Code: 13DJ1400301). Conflict of interest None of the authors has any potential financial conflict of interest related to this study. References Al-Ghoul, W.M., Meeker, R.B., Greenwood, R.S., 1997. Amygdala kindling alters N-methyl-d-aspartate receptor subunit messenger RNA expression in the rat supraoptic nucleus. Neuroscience 77, 985–992. Briellmann, R.S., Hopwood, M.J., Jackson, G.D., 2007. Major depression in temporal lobe epilepsy with hippocampalsclerosis: clinical and imaging correlates. J. Neurol. Neurosurg. Psychiatry 78, 1226–1230. Carlson, H., Ronne-Engstrom, E., Ungerstedt, U., Hillered, L., 1992. Seizure related elevations of extracellular amino acids in human focal epilepsy. Neurosci. Lett. 140, 30–32. Castillo, M., Kwock, L., Courvoisie, H., Hooper, S.R., 2000. Proton MR spectroscopy in children with bipolar affective disorder: preliminary observations. AJNR Am. J. Neuroradiol. 21, 832–838. Cavus, I., Kasoff, W.S., Cassaday, M.P., Jacob, R., Gueorguieva, R., Sherwin, R.S., Krystal, J.H., Spencer, D.D., Abi-Saab, W.M., 2005. Extracellular metabolites in the cortex and hippocampus of epileptic patients. Ann. Neurol. 57, 226–235. Cramer, J.A., Blum, D., Reed, M., Fanning, K., 2003. The influence of comorbid depression on quality of life for people with epilepsy. Epilepsy Behav. 4, 515–521. Curia, G., Longo, D., Biagini, G., Jones, R.S., Avoli, M., 2008. The pilocarpine model of temporal lobe epilepsy. J. Neurosci. Methods 172, 143–157. Danober, L., Deransart, C., Depaulis, A., Vergnes, M., Marescaux, C., 1998. Pathophysiological mechanisms of genetic absence epilepsy in the rat. Prog. Neurobiol. 55, 27–57. Detke, M.J., Rickels, M., Lucki, I., 1995. Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology (Berl) 121, 66–72. Di Maio, R., Mastroberardino, P.G., Hu, X., Montero, L., Greenamyre, J.T., 2011. Pilocapine alters NMDA receptor expression and function in hippocampal neurons: NADPH oxidase and ERK1/2 mechanisms. Neurobiol. Dis. 42, 482–495. Di Maio, R., Mastroberardino, P.G., Hu, X., Montero, L.M., Greenamyre, J.T., 2012. Thiol oxidation and altered NR2B/NMDA receptor functions in in vitro and in vivo pilocarpine models: implications for epileptogenesis. Neurobiol. Dis. 49C, 87–98. Diazgranados, N., Ibrahim, L., Brutsche, N.E., Newberg, A., Kronstein, P., Khalife, S., Kammerer, W.A., Quezado, Z., Luckenbaugh, D.A., Salvadore, G., Machado-Vieira, R., Manji, H.K., Zarate, C.J., 2010. A randomized add-on trial of an N-methyl-d-aspartate antagonist in treatment-resistant bipolar depression. Arch. Gen. Psychiatry 67, 793–802. Do, N.A., Dos, S.N., Campos, P.F., Aparecida, T.S., de Moraes, F.E., Langone, F., 2012. Neuronal degeneration and gliosis time-course in the mouse hippocampal formation after pilocarpine-induced status epilepticus. Brain Res. 1470, 98–110. During, M.J., Spencer, D.D., 1993. Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain. Lancet 341, 1607–1610. Ettinger, A., Reed, M., Cramer, J., 2004. Depression and comorbidity in community-based patients with epilepsy or asthma. Neurology 63, 1008–1014. Fiest, K.M., Dykeman, J., Patten, S.B., Wiebe, S., Kaplan, G.G., Maxwell, C.J., Bulloch, A.G., Jette, N., 2013. Depression in epilepsy: a systematic review and meta-analysis. Neurology 80, 590–599. Finegersh, A., Avedissian, C., Shamim, S., Dustin, I., Thompson, P.M., Theodore, W.H., 2011. Bilateral hippocampal atrophy in temporal lobe epilepsy: effect of depressive symptoms and febrile seizures. Epilepsia 52, 689–697. Frasca, A., Aalbers, M., Frigerio, F., Fiordaliso, F., Salio, M., Gobbi, M., Cagnotto, A., Gardoni, F., Battaglia, G.S., Hoogland, G., Di Luca, M., Vezzani, A., 2011. Misplaced NMDA receptors in epileptogenesis contribute to excitotoxicity. Neurobiol. Dis. 43, 507–515. Gandy, M., Sharpe, L., Perry, K.N., 2012. Psychosocial predictors of depression and anxiety in patients with epilepsy: a systematic review. J. Affect. Disord. 140, 222–232.

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