Involvement of the nitrergic system in the ... - Epilepsy & Behavior

Epilepsy & Behavior 41 (2014) 158–163

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Involvement of the nitrergic system in the proconvulsant effect of social isolation stress in male mice Shayan Amiri a,b,1, Armin Shirzadian a,b,1, Arya Haj-Mirzaian a,b, Muhammad Imran-Khan c, Maryam Rahimi Balaei d, Nastaran Kordjazy a,b, Ahmad Reza Dehpour a,b, Shahram Ejtemaei Mehr a,b,⁎ a

Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran International Campus, Tehran University of Medical Sciences, Tehran, Iran d Department of Human Anatomy and Cell Science, College of Medicine, Faculty of Health Science, University of Manitoba, Winnipeg, MB, Canada b c

a r t i c l e

i n f o

Article history: Received 26 July 2014 Revised 26 September 2014 Accepted 29 September 2014 Available online 25 October 2014 Keywords: Social isolation Nitric oxide PTZ-clonic seizure model Mice Stress

a b s t r a c t Social isolation stress (SIS) in adolescence is accompanied by neurobehavioral disturbances and pathophysiological changes in certain regions of the CNS such as the hippocampus. In this study, we tested whether SIS impacts seizure susceptibility in postnatal male mice due to a role of hippocampal nitric oxide (NO). To do this, we used the pentylenetetrazole (PTZ) model of clonic seizures, open-field test, hole-board test, forced swimming test, and plasma corticosterone assay. We aimed to evaluate if 4 weeks of SIS is capable of decreasing seizure threshold along with altering affective and neuroendocrine responses in isolated conditioned (IC) animals in comparison with socially conditioned (SC) animals. In addition, we applied subeffective doses of NO precursor L-arginine (25, 50, and 100 mg/kg) and NOS inhibitors 7-NI (15 and 40 mg/kg), aminoguanidine (50 and 100 mg/kg), and L-NAME (10 and 15 mg/kg) to both IC and SC groups prior to the determination of seizure threshold. Injection of a single dose of all mentioned drugs did not induce changes in seizure threshold of SC mice. On the other hand, L-NAME and 7-NI, but not aminoguanidine, modulated the proconvulsant effect of SIS, while L-arginine augmented the latter effect. We also measured the hippocampal nitrite levels after the administration of the aforementioned drugs. Social isolation stress increased the nitrite levels in comparison with those in SC mice, whereas 7-NI and L-NAME, unlike aminoguanidine, mitigated the effect of SIS. Additionally, L-arginine boosted the effects of SIS on nitrite production. In summary, we showed that SIS enhanced seizure susceptibility in the PTZ model of clonic seizures through the activation of the nitrergic system in the hippocampus. Also, we proved that nNOS, but not iNOS, accounts for these changes following SIS. © 2014 Elsevier Inc. All rights reserved.

1. Introduction People with epilepsy (PWE) experience greater psychosocial challenges compared with the general population, thereby contributing to poor quality of life [1]. Among psychosocial problems, stress and social isolation have been reported as the most determinant factors which affect the severity of epilepsy and social functioning of PWE, respectively [2,3]. Previous studies have reported that social isolation stress (SIS) in the adolescent period induces considerable psychobiological abnormalities, neurobehavioral disturbances, and hypothalamic–pituitary–adrenocortical (HPA) axis malfunction [4,5]. In addition, the social isolation paradigm has been suggested as a reliable animal model for the investigation of neurobehavioral changes in psychiatric disorders similarly seen in humans [6]. Under chronic stress ⁎ Corresponding author at: Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, P.O. Box 13145-784, Tehran, Iran. E-mail addresses: [email protected], [email protected] (S.E. Mehr). 1 Please note that the first two authors are considered as the first author.

http://dx.doi.org/10.1016/j.yebeh.2014.09.080 1525-5050/© 2014 Elsevier Inc. All rights reserved.

circumstances, the neurotoxic action of excitatory neurotransmitters such as glutamate causes an overproduction of nitric oxide (NO) via the excessive activity of nitric oxide synthase (NOS) [7,8]. Nitric oxide contributes to a variety of physiological and pathophysiological processes in the hippocampus (HIPP), such as learning, memory, depression, and seizure susceptibility [9–13]. Among NOS isoforms, both iNOS (inducible NOS) and nNOS (neuronal NOS) have been reported to increase the NO levels in the HIPP in response to stressful paradigms [14,15]. In addition, early stressful life events have negative enduring effects on the HIPP which are relevant to increased susceptibility to seizures in adulthood [16]. Recently, it has been demonstrated that endogenous NO is a key factor for initiation of seizurelike events [17]. In another study, Watanabe et al. showed that elevated NO levels in the murine brain are associated with increased seizure susceptibility in the pentylenetetrazole (PTZ) model of convulsive seizures. They also showed that PTZ-induced convulsive seizure is sensitive to small changes of NO levels in the brain; therefore, it is a valid animal model for the evaluation of epileptic activity [13,18]. Surprisingly, there are few studies about the effects of social isolation,

S. Amiri et al. / Epilepsy & Behavior 41 (2014) 158–163

as a chronic stress model, on seizure vulnerability in animals [19,20]. Therefore, considering the increased activity of NOS by chronic stress in the HIPP (an important part of the limbic system in epileptogenesis) [2,21], we hypothesized that hippocampal NO levels are correlated with seizure vulnerability changes in socially isolated animals. In this study, firstly, we determined whether SIS could induce stress-related behavioral and neuroendocrine changes in animals. Secondly, we investigated the relationship between SIS and seizure susceptibility by using NOS inhibitors such as aminoguanidine (AG) (a specific iNOS inhibitor), 7-nitroindazole (7-NI) (a specific nNOS inhibitor), and NG-nitro-Larginine methyl ester (L-NAME) (a nonspecific NOS inhibitor) as well as the NO precursor, L-arginine (L-arg). Our aim was to determine whether the nitrergic system is involved in seizure susceptibility as an underlying mechanism. 2. Materials and methods 2.1. Animals Male NMRI mice (Pasteur Institute, Tehran, Iran), weighing 10–14 g and in the postnatal stage (PND: 21–25), were used throughout the study. Animals were housed under standard conditions (temperature: 22 ± 2 °C, humidity: 50 ± 10%, 12-h light–dark cycle, and free access to food and water) for 28 days in either of two different conditions: 1) social condition (SC) and 2) isolated condition (IC). Socially conditioned mice were housed in groups (6 mice per cage) in Plexiglas boxes (25 × 25 × 15 cm), and isolated conditioned mice were housed individually in Plexiglas boxes (24 × 17 × 12 cm). Wood shavings were used as bedding for animals. The cages of IC mice were cleaned weekly by the same experimenter to minimize handling and social contact. All procedures in this study were carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH publication # 80-23) and institutional guidelines for animal care and use (Department of Pharmacology, School of Medicine, TUMS). Also, each experimental group contained 6 to 8 animals. 2.2. Drugs and treatments The following drugs were used in this study: pentylenetetrazole (PTZ), L-arginine, NG-nitro-L-arginine methyl ester (L-NAME), aminoguanidine (AG), and 7-nitroindazole (7NI). The drugs were purchased from Sigma, UK. 7-Nitroindazole was suspended in 1% aqueous solution of Tween 80, and all other drugs were dissolved in saline and were administered in the volume of 10-ml/kg mouse weight. To assess clonic seizures in experimental animals, we administered PTZ intravenously (0.5%, i.v.) and all other drugs intraperitoneally (i.p.). Doses of each drug were chosen according to the pilot treatments which were published in our previous studies [22,23]. 2.3. Open-field test (OFT) The open-field test was used to evaluate the locomotion and anxiety behavior of animals in response to SIS. The open-field apparatus was made of white opaque Plexiglas (50 cm × 50 cm × 30 cm) which was dimly illuminated. Each mouse was placed gently on the center square (30 cm × 30 cm), and behaviors were recorded by a camera for 5 min and were analyzed by Ethovision software version 8 (Noldus, Netherlands). The surface of the apparatus was cleaned with 70% ethanol after each experiment. Each animal was used in only one experiment. 2.4. Hole-board test (HBT) The hole-board test was used to evaluate the anxiety of subjects [24]. The apparatus consisted of a white Plexiglas square (50 cm × 50 cm) with 16 equidistant holes (3 cm in diameter) and was positioned

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50 cm above the floor. Mice were placed in the center of the board, and the number of head dips was counted in a 5-min period. The apparatus was cleaned with 70% ethanol after each experiment. Each animal was used in only one experiment. 2.5. Forced swimming test (FST) Mice were individually placed in an open glass cylinder (diameter: 10 cm, height: 25 cm) containing 19 cm of water at 25 ± 1 °C [25,26]. Mice were allowed to swim for 6 min, and the immobility time was recorded during the last 4 min of the test. Immobility behavior was considered when the animal remained floating motionless in the water and made only those movements necessary to keep its head above water. 2.6. Determination of clonic seizure threshold In order to measure the clonic seizure threshold in mice, we used the method that was previously described. Briefly, a winged infusion set (30 gauge) was used to infuse the PTZ (0.5%) at a constant rate of 1 ml/min into the tail vein of the freely moving subject. Infusion was halted when forelimb clonus followed by full clonus of the body (began with running and then loss of righting ability) was observed. The minimal dose of PTZ (mg/kg mouse weight) needed to induce a clonic seizure was considered as the index of seizure threshold. As such, seizure threshold is dependent on the dose and time of PTZ administration [27]. 2.7. Hippocampal nitrite assay To determine the NO levels in the hippocampus, we measured nitrite levels as the result of the NO end product [28]. The animals were decapitated under mild anesthesia, and then the hippocampi were dissected on ice cold surface and immediately immersed in liquid nitrogen. Tissue homogenates were prepared, and nitrite levels were determined by using a colorimetric assay based on the Griess reaction. Initially, each well was loaded with 100-μl samples which were then mixed with 100-μl Griess reagent. Following 10-minute incubation at room temperature, absorbance was measured at 540 nm in an automated plate reader. Concentration of nitrite was determined by reference to a standard curve of sodium nitrite (Sigma, USA) and normalized to the weight of each sample. 2.8. Corticosterone assay To assess HPA axis activity in mice, we measured basal and postacute (after 60 min) stress levels of corticosterone (CORT) in plasma using the method which was previously described [29]. Blood samples were centrifuged at 3000 g for 10 min at 4 °C, and plasma samples were then stored at −20 °C until the assay day. We evaluated the HPA axis response to acute stress by applying the forced swim stress which is known as a strong stressor for rodents [30]. Corticosterone concentrations were measured by a commercial ELISA kit (Biospes, China). 2.9. Statistical analysis Comparison between the groups was analyzed using t-test and oneway ANOVA followed by Tukey's post hoc test. p b 0.05 was considered statistically significant. The factors were housing [social condition (SC) and isolation condition (IC)] and treatments [control: no treatment and treatment: drug-administered animals by a single i.p. injection] for all assessments except CORT values. For the evaluation of CORT, treatment factor was the same as mentioned above, and in the case of housing factors, subjects were divided into four groups: 1) unstressed SC, 2) SC + stress (SC mice which experienced the FST prior to plasma collection), 3) unstressed IC, and 4) IC + stress.

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3. Results

3.2. Effects of social isolation on seizure threshold

3.1. Effects of social isolation stress on depressive-like behaviors

Social isolation stress induced a significant decrease in the seizure threshold of IC mice, whereas the same results were not obtained in SC animals. t-Test revealed a significant difference between IC mice and SC mice in relation to the seizure threshold (Fig. 2) (p b 0.0001).

In the OFT, SIS increased the distance moved in IC mice compared with SC animals (p b 0.01, Fig. 1A). Additionally, in comparison with SC mice, a significant decrease in the time spent in the central zone (30 × 30) was observed in IC mice (p b 0.001, Fig. 1B). In the HBT, the number of head-dipping was significantly more decreased in stressed animals than in SC mice (p b 0.001, Fig. 1C). Additionally, SIS induced depressive signs such as despair behavior in the FST, in which a significant increase in immobility time was observed in comparison with SC mice (p b 0.001, Fig. 1D). The HPA axis response in IC + stress was significantly different from that in SC + stress (p b .001, Fig. 1E). No significant difference was observed in corticosterone levels of unstressed IC mice compared with unstressed SC animals (p N 0.05, Fig. 1E).

3.3. Proconvulsant effect of social isolation was modulated by nNOS inhibitor, 7-NI Firstly, seizure threshold was determined for the subeffective doses of each drug in SC animals. Fig. 3A shows that the administration of L-NAME (10 and 15 mg/kg, i.p.) as a nonspecific NOS inhibitor, 7-NI (40 mg/kg, i.p.) as a selective nNOS inhibitor, and AG (50 and 100 mg/kg, i.p.) as a selective iNOS inhibitor had no effect on the seizure threshold of SC mice [F 7, 52 = 8.207, p N 0.05].

Fig. 1. Effects of different housing conditions, social condition (SC) and isolated condition (IC), on distance moved (A) and time spent in the central zone (B) in the open-field test. Number of head-dipping in the hole-board test (C). Duration of immobility time in the FST (D). Plasma corticosterone levels after acute stress (E). Values are expressed as the mean ± S.E.M. from 6 to 8 animals and were analyzed using t-test and one-way ANOVA followed by Tukey's post hoc test. **p b 0.01 and ***p b 0.001 compared with the SC group. ###p b 0.001 compared with the IC stressed group. @@@p b 0.001 compared with the SC stressed group.


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which were compared with Tween 80 1%-treated mice as the control group. 3.4. Effect of L-arginine on the proconvulsant effect of social isolation

Fig. 2. Effects of different housing conditions, social condition (SC) and isolated condition (IC), on clonic seizure threshold in the PTZ model of seizures. Values are expressed as the mean ± S.E.M. from 8 animals and were analyzed using t-test and one-way ANOVA followed by Tukey's post hoc test. ****p b 0.0001 compared with the SC group.

Secondly, the effects of subeffective doses of the aforementioned drugs on the seizure threshold of IC mice were determined. The result shows that the administration of L-NAME (15 mg/kg, i.p.) and 7-NI (40 mg/kg, i.p.) significantly reversed the proconvulsant effect of SIS on IC mice [F 7, 54 = 6.924, p b 0.01 and p b 0.001] (Fig. 3B). In addition, a significant difference was observed between the IC mice which were treated separately with L-NAME (p b 0.01) or 7-NI (p b 0.001). However, the administration of AG (50 and 100 mg/kg, i.p.) did not alter the seizure threshold in IC animals (p N 0.05). The administration of saline or Tween 80 1% (45 and 30 min before the test, respectively) had no effect on the seizure threshold of both IC and SC mice (Fig. 3B, p N 0.05). All experimental groups were compared with salineadministered mice as the control group except for 7-NI-treated animals

L-Arginine (25, 50, and 100 mg/kg, i.p.) as a NO precursor was administered to both IC and SC mice. According to Fig. 4, our data showed that L-arg (25, 50, and 100 mg/kg) had no effect on the seizure threshold of SC animals [F 3, 26 = 0.668, p N 0.05]. The administration of L-arg (50 mg/kg, i.p.) significantly decreased the seizure threshold of IC animals in comparison with saline-injected IC mice [F 3, 27 = 8.112, p b 0.01]. However, L-arg at the dose of 25 mg/kg had no effect on the seizure threshold of IC mice (p N 0.05). Consequently, we considered 50 mg/kg as a subeffective dose of L-arg. Taken together, it seems that the NO precursor augments the proconvulsant effect of SIS on IC mice. Additionally, our data showed that saline injection (45 min before the test) had no effect on the seizure threshold of either experimental group (p N 0.05, Fig. 4).

3.5. Hippocampal NO levels: in the absence and presence of NOS inhibitors/ NO precursor t-Test analysis showed that IC mice had higher levels of nitrite levels in the HIPP (p b 0.0001) in comparison with SC mice (Fig. 5). The effects of NOS inhibitors or NO precursor administration on hippocampal nitrite levels were also investigated. Our data revealed that L-NAME (15 mg/kg, i.p.) and 7-NI (40 mg/kg, i.p.) administration in IC mice significantly decreased the nitrite levels in the HIPP compared with saline/ Tween 80-injected IC animals [F 5, 42 = 27.04, p b 0.01 and p b 0.001, respectively] (Fig. 6B). However, the administration of AG (100 mg/kg, i.p.) to IC mice had no effect on the nitrite levels of the HIPP (p N 0.05). Also, the administration of L-arg (100 mg/kg, i.p.) boosted the levels of nitrite in the HIPP [F 5, 42 = 27.04, p b 0.001]. On the other hand, none of these treatments altered the hippocampal nitrite level in SC animals (p N 0.05) (Fig. 6A). 4. Discussion In this study, we showed that SIS, as an acknowledged model of chronic stress, enhanced the seizure susceptibility in the PTZ model of seizures. Our findings suggest that SIS increased the tone of NO partly in the HIPP, which, in turn, resulted in the decreased seizure threshold in stressed animals. However, inhibition of the NO pathway (especially constitutive NOS) plays a determinant role in reversing the seizure threshold to a normal range, whereas iNOS seemed to have no effect. Applying the animal models of stress provides conditions to evaluate the impact of stress on behavior because of close and relevant

Fig. 3. Effects of NOS antagonists on the proconvulsant effect of IC in the PTZ model of clonic seizures. Effects of L-NAME (LNM) (10 and 15 mg/kg, i.p.), aminoguanidine (AG) (50 and 100 mg/kg, i.p.), and 7-nitroindazole (7-NI) (15 and 40 mg/kg, i.p.) on the seizure threshold in SC (A) and IC (B) animals. Values are expressed as the mean ± S.E.M. from 7 to 8 animals and were analyzed using one-way ANOVA followed by Tukey's post hoc test. **p b 0.01 compared with the IC saline-treated group. ###p b 0.001 compared with the IC Tween 80 1%-treated group.

Fig. 4. Effects of the NO precursor on the proconvulsant effect of IC in the PTZ model of clonic seizures. Effects of L-arginine (L-arg) (25, 50, and 100 mg/kg, i.p.) on the seizure threshold in SC (A) and IC (B) animals. Values are expressed as the mean ± S.E.M. from 7 to 8 animals and were analyzed using one-way ANOVA followed by Tukey's post hoc test. **p b 0.01 compared with the IC saline-treated group.

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Fig. 5. Effects of SC/IC on the hippocampal nitrite level. Values are expressed as the mean ± S.E.M. from 8 animals and were analyzed using t-test. ####p b 0.0001 compared with the SC group.

pathophysiological similarities to humans [31]. In the first step, we showed that 28 days of SIS in the adolescence period induced anxiety and depressive-like behaviors in adult mice. As stated above, early-life social isolation is accompanied by considerable behavioral and neurochemical changes in adulthood. In the current study, altered openfield activity of IC animals (hyperlocomotion and decrease in the time spent in the central area) and decreased exploration (number of headdipping) in the hole-board test indicate that SIS induced anxiety and behavioral changes in animals. Previous studies have demonstrated that socially isolated rodents exhibit anxiogenic-like behaviors and hyperlocomotor activity in novel environments [4,32–34]. Taking the FST as a valid model to investigate the despair behavior in animal

Fig. 6. Effects of NOS antagonists and the NO precursor on the hippocampal nitrite level in SC (A) and IC (B) animals. Effects of L-NAME (LNM) (15 mg/kg, i.p.), aminoguanidine (AG) (100 mg/kg, i.p.), 7-nitroindazole (7-NI) (40 mg/kg, i.p.), and L-arginine (L-arg) (100 mg/kg, i.p.) on the hippocampal nitrite level in IC animals. Values are expressed as the mean ± S.E.M. from 8 animals and were analyzed using one-way ANOVA followed by Tukey's post hoc test. **p b 0.01 and @@@p b 0.001 compared with the IC saline-treated group. ###p b 0.001 compared with the IC Tween 80 1%-treated group.

models of depression [35], we found that our results are consistent with previous studies which have reported that SIS is capable of producing depressive-like behaviors in animals [36,37]. There are also lines of evidence indicating that SIS alters the normal responses to acute stress by dysregulation of HPA axis activity [38]. Hypothalamic–pituitary– adrenocortical axis dysregulation by SIS leads to the amplification of responses to acute stress via massive release of CORT. However, the HPA axis is capable of regulating the plasma CORT levels and reverses them to the basal state after an hour [39]. In our study, 1 h after the FST, plasma levels of CORT in IC mice were higher than basal levels when compared with SC mice, indicating the inability of the HPA axis to manage negative feedback in response to acute stress. This result demonstrated that elevated levels of CORT in IC animals were due to heightened sensitivity of HPA axis to environmental challenges and agrees with that found by Lukkes et al. who reported that impaired glucocorticoid negative feedback processes in social isolation fail to regulate HPA axis functionality. Taken together, in the first step, we showed the deleterious impact of SIS on the HPA axis activity and the behavioral profile of IC animals which was previously reported by other studies. In the second step, we focused on the relationship between SIS and the nitrergic system as an underpinning mechanism in seizure vulnerability. Regarding the deleterious effects of stress on the HIPP, which plays a pivotal role in epileptogenesis, SIS increased the NO levels in the HIPP. There are data which indicate that exposure to chronic stress such as SIS is related to overexpression of nNOS in the hippocampus through glutamate excitotoxicity, which results in NO overproduction [14,40]. Additionally, Zlatković et al. have shown that SIS induces the upregulation of nNOS (but not iNOS), which increases the NO levels in the HIPP [41]. We used the continuous IV infusion of PTZ as a sensitive method to produce clonic seizures [18]. According to Watanabe et al., PTZ triggers motor seizures via overproduction of NO by nNOS, and also the background levels of NO play an important role in seizure susceptibility [13,42]. According to our findings, subeffective doses of L-NAME and 7NI (but not AG) reduced the levels of NO in the HIPP, while the administration of a subeffective dose of L-arg (50 mg/kg) increased the NO production in IC mice (Fig. 3). Considering the elevated levels of NO in the HIPP, we found that SIS dramatically decreased the seizure threshold in IC mice. However, the treatment with subeffective doses of L-NAME and 7NI (and not AG) showed anticonvulsant properties in IC mice (Fig. 3). With respect to our results, the administration of subeffective doses of L-arg worsened the seizure susceptibility even more than SIS, whereas the same results were not observed in SC mice. Despite the different reports which suggest dual proconvulsant and anticonvulsant properties for NO [23,43], we found that SIS induced the overproduction of NO in the HIPP (data not shown) of IC mice, and it consequently boosted the seizure susceptibility to PTZ. Previous studies which have investigated the effects of chronic stress are in agreement with our findings that the chronic stress increases seizure risk in animals. Matsumoto et al. reported that long-term social isolation for seven weeks increased seizure susceptibility to picrotoxin in mice [20], and more recently, another study by Russo et al. illustrated that chronic mild stress in combination with PTZ kindling can be used as a model of epilepsy and mood disorder comorbidity [44]. However, our results are inconsistent with previous observations by Chadda et al. who reported that SIS increased seizure susceptibility to bicuculline and not PTZ [19]. This discordance can be explained by differences in our experiment designs, as Chadda et al. reared adult rats for ten days whereas we housed adolescent mice in an isolated condition for 28 days. In another study, Salzberg et al. demonstrated that maternal stress (PND: 2–14) increases seizure risk in later life of both genders, mostly females, while our study focused on the adolescence period (PND: 21–28) in male mice [16]. There are different studies which have investigated the role of NOS isoforms in seizure vulnerability [12, 45]. Although there is evidence which indicates the involvement of iNOS in seizure susceptibility [46], the majority of data confirm the role of nNOS in proportion to increased risk of seizure [12,14,27]. We


showed that nNOS is responsible for the elevated levels of NO in the HIPP since the administration of 7NI (40 mg/kg) decreased both NO levels in the HIPP and seizure vulnerability, while the administration of AG (50 mg/kg) had no effect. Furthermore, background levels of NO are involved in determining the seizure threshold in IC mice, since increased amounts of NO in the HIPP by the administration of L-arg (50 mg/kg) correlated with the decreased seizure threshold in IC animals. We also suggest that SIS in adolescent mice may be considered as a novel model of comorbidity for psychiatric–neurologic disorders in which impairments that are related to disorders such as anxiety, depressive-like behaviors [4–6], aggressiveness [47], decreased seizure threshold [19,20], reduced GABA and serotonin transmission [48,49], and cognitive disturbances [50] can be seen. However, there are additional studies that should be done in regard to this animal model, which is beyond the scope of this study. In conclusion, SIS increased the NO tone mostly in the HIPP and enhanced seizure susceptibility in the PTZ seizure model. In addition, proconvulsant properties of SIS were modulated by subeffective doses of L-NAME and 7-NI, while L-arg administration aggravated the proconvulsant effect of SIS in IC animals. Acknowledgment The authors would like to thank Mr. E. Piryousefi and Prof. M. GhaziKhansari for their collaborations on this study. References [1] McCagh J, Fisk JE, Baker GA. Epilepsy, psychosocial and cognitive functioning. Epilepsy Res 2009;86:1–14. [2] Maguire J, Salpekar JA. Stress, seizures, and hypothalamic–pituitary–adrenal axis targets for the treatment of epilepsy. Epilepsy Behav 2013;26:352–62. [3] Charyton C, Elliott JO, Lu B, Moore JL. The impact of social support on health related quality of life in persons with epilepsy. Epilepsy Behav 2009;16:640–5. [4] Fone KC, Porkess MV. Behavioural and neurochemical effects of post-weaning social isolation in rodents—relevance to developmental neuropsychiatric disorders. Neurosci Biobehav Rev 2008;32:1087–102. [5] Marsden CA, King MV, Fone KC. Influence of social isolation in the rat on serotonergic function and memory—relevance to models of schizophrenia and the role of 5-HT6 receptors. Neuropharmacology 2011;61:400–7. [6] Koob GF, Ehlers CL, Kupfer DJ. Animal models of depression. Boston: Birkhauser; 1989. [7] Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87:315–424. [8] Olivenza R, Moro MA, Lizasoain I, Lorenzo P, Fernández AP, Rodrigo J, et al. Chronic stress induces the expression of inducible nitric oxide synthase in rat brain cortex. J Neurochem 2000;74:785–91. [9] Hölscher C. Nitric oxide, the enigmatic neuronal messenger: its role in synaptic plasticity. Trends Neurosci 1997;20:298–303. [10] Zhou Q-G, Zhu L-J, Chen C, Wu H-Y, Luo C-X, Chang L, et al. Hippocampal neuronal nitric oxide synthase mediates the stress-related depressive behaviors of glucocorticoids by downregulating glucocorticoid receptor. J Neurosci 2011;31:7579–90. [11] Hu Y, Wu D-L, Luo C-X, Zhu L-J, Zhang J, Wu H-Y, et al. Hippocampal nitric oxide contributes to sex difference in affective behaviors. Proc Natl Acad Sci 2012;109: 14224–9. [12] Banach M, Piskorska B, Czuczwar S. Nitric oxide, epileptic seizures, and action of antiepileptic drugs. CNS Neurol Disord Drug Targets 2011;10:808–19. [13] Watanabe M, Miyai A, Danjo S, Nakamura Y, Itoh K. The threshold of pentylenetetrazole-induced convulsive seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains. Exp Neurol 2013;247:645–52. [14] Zhou QG, Hu Y, Hua Y, Hu M, Luo CX, Han X, et al. Neuronal nitric oxide synthase contributes to chronic stress‐induced depression by suppressing hippocampal neurogenesis. J Neurochem 2007;103:1843–54. [15] Harvey BH, Oosthuizen F, Brand L, Wegener G, Stein DJ. Stress–restress evokes sustained iNOS activity and altered GABA levels and NMDA receptors in rat hippocampus. Psychopharmacology (Berl) 2004;175:494–502. [16] Salzberg M, Kumar G, Supit L, Jones NC, Morris MJ, Rees S, et al. Early postnatal stress confers enduring vulnerability to limbic epileptogenesis. Epilepsia 2007;48:2079–85. [17] Kovács R, Rabanus A, Otáhal J, Patzak A, Kardos J, Albus K, et al. Endogenous nitric oxide is a key promoting factor for initiation of seizure-like events in hippocampal and entorhinal cortex slices. J Neurosci 2009;29:8565–77. [18] Löscher W, Hönack D, Fassbender CP, Nolting B. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazole seizure models. Epilepsy Res 1991;8:171–89. [19] Chadda R, Devaud LL. Sex differences in effects of mild chronic stress on seizure risk and GABA A receptors in rats. Pharmacol Biochem Behav 2004;78:495–504.

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