NK1 receptors antagonism of dorsal hippocampus counteract the anxiogenic-like effects induced by pilocarpine in non-convulsive Wistar rats

Behavioural Brain Research 265 (2014) 53–60

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NK1 receptors antagonism of dorsal hippocampus counteract the anxiogenic-like effects induced by pilocarpine in non-convulsive Wistar rats Filipe Silveira Duarte a , Alexandre Ademar Hoeller b,f , Marcelo Duzzioni c , Elaine Cristina Gavioli d , Newton Sabino Canteras e , Thereza Christina Monteiro De Lima f,∗ a

Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife, PE 50670-901, Brazil Postgraduate Program in Medical Science, Center of Health Sciences, University Hospital, Federal University of Santa Catarina, Florianópolis, SC 88040-970, Brazil c Institute of Biological Sciences and Health, Federal University of Alagoas, Maceió, AL 57020-720, Brazil d Department of Biophysics and Pharmacology, Federal University of Rio Grande do Norte, Natal, RN 59072-970, Brazil e Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-000, Brazil f Department of Pharmacology, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, SC 88049-900, Brazil b

h i g h l i g h t s • NK1 receptor antagonist’s FK888 did not alter emotional responses in CA1 or LSD. • Infusion of FK888 into CA1 inhibits long-term anxiogenic responses induced by PILO. • PILO changes anxiogenic responses modulated by SP-NK1 receptor signaling in CA1 .

a r t i c l e

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Article history: Received 3 October 2013 Received in revised form 28 January 2014 Accepted 31 January 2014 Available online 7 February 2014 Keywords: Pilocarpine Anxiety NK1 receptor Dorsal hippocampus Lateral septum Elevated plus-maze

a b s t r a c t Recent evidence supports a role for the substance P (SP) in the control of anxiety and epilepsy disorders. Aversive stimuli alter SP levels and SP immunoreactivity in limbic regions, suggesting that changes in SP-NK1 receptor signaling may modulate the neuronal excitability involved in seizures and anxiogenesis. The involvement of NK1 receptors of the dorsal hippocampus and lateral septum in the anxiogenic-like effects induced by a single injection of pilocarpine (PILO) was examined in non-convulsive rats evaluated in the elevated plus-maze (EPM). Male Wistar rats were systemically injected with methyl-scopolamine (1 mg/kg) followed 30 min later by saline or PILO (350 mg/kg) and only rats that did not present status epilepticus were used. One month later, vehicle or FK888 (100 pmol) – an NK1 receptor antagonist – were infused in the dorsal hippocampus or the lateral septum of the rats and then behaviorally evaluated in the EPM. Previous treatment with PILO decreased the time spent in and the frequency of entries in the open arms of the EPM, besides altering risk-assessment behaviors such as the number of unprotected headdipping, protected stretch-attend postures and the frequency of open-arms end activity, showing thus a long-lasting anxiogenic-like profile. FK888 did not show any effect per se but inhibited the anxiogenic responses induced by PILO when injected into the dorsal hippocampus, but not into the lateral septum. Our data suggest that SP-NK1 receptor signaling of the dorsal hippocampus is involved in the anxiogeniclike profile induced by PILO in rats evaluated in the EPM test. © 2014 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author at: Department of Pharmacology, Center of Biological Sciences, Federal University of Santa Catarina, Campus Universitario, Trindade, Florianopolis, SC 88049-900, Brazil. Tel.: +55 48 3721 9491x225/3721 2476; fax: +55 48 3721 9813. E-mail addresses: [email protected], t de [email protected] (T.C.M. De Lima). 0166-4328/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2014.01.050

Depression and anxiety disorders are major comorbidities in epilepsy [1–6]. Several animal models demonstrated that the status epilepticus (SE) observed in epileptic animals induces aggressive, depressive and anxiety-like behaviors [7–14] supporting the existence of a shared causation of epilepsy and affective disorders.

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Substance P (SP) – a member of the neurokinin family – exhibits an excitatory outline in the brain and has been implicated in a variety of physiological processes including the regulation of stress, anxiety and depression responses [15–20]. The exposure of animals to aversive and stressful stimuli alter the SP levels or SP immunoreactivity (SP-ir) in several brain regions – such as lateral septum, dentate gyrus, periaqueductal gray, hypothalamic nuclei, nucleus accumbens and amygdala – involved in the regulation of mood and affective behaviors [21–26]. Blocking SP transmission by NK1 antagonists or genetic disruption attenuates the effects of stress and induces anxiolytic-like effects in animal models [20,27–29] – effects that can be associated with an attenuation of neuroendocrine stress responses [26]. SP also exhibits a critical role in the control of neuronal excitability and maintenance of the SE [30–33]. In vivo studies have shown that there is a strong correlation between SP levels and the propensity, severity and duration of seizures [31,32]. An increase in preprotachykinin-A (PPT-A) mRNA and SP-ir in the CA1 , CA3 , and dentate gyrus of the hippocampus was observed after SE induced by the injection of lithium-pilocarpine or the stimulation of the perforant path. The intrahippocampal injection of SP triggers SE and induces hippocampal damage whereas the SP receptor antagonists block both the development and maintenance of the self-sustaining SE [32]. Over the last years, our research group showed that a single systemic administration of subconvulsant doses of pilocarpine (PILO, 75–350 mg/kg) – a non-selective cholinergic agonist – triggers anxious-like behaviors in rats evaluated in the EPM test 24 h up to three months after the treatment, suggesting that the anxiogenesis is not totally dependent of convulsive episodes [34–36]. In addition, we showed that the septo-hippocampal pathway is critically involved in these anxiogenic responses [36]. In the current work, we assume the hypothesis that the SP-NK1 receptor pathway may modulate neuronal hyper-excitability involved in anxiogenesis in non-convulsive rats. Thus, changes in the SP signaling in the dorsal hippocampus and lateral septum – areas richly innervated by SP-NK1 receptor-containing neurons and widely affected by stress conditions and seizures – would be expected to interfere with the anxiety-related behavioral alterations in these animals.

We here investigated the potential participation of the NK1 receptors of the CA1 region of the dorsal hippocampus and lateral septum in the anxiogenesis induced by PILO treatment in non-convulsive rats. 2. Material and methods 2.1. Animals Male Wistar rats, 2.5 months old, were housed in groups of four animals per cage and kept in a room with controlled temperature (22 ± 2 ◦ C) and a 12-h light/dark cycle (lights on at 07:00 a.m.) with food and water ad libitum, except during the experiments. Rats were allowed to adapt to the laboratory conditions for one week before the experiments. All experiments were conducted in accordance with international standards of animal welfare recommended by the Brazilian Society of Neuroscience and Behavior (Act, 1992) and the experimental protocols were approved by the Animal Care and Use Committee of the Federal University of Santa Catarina (#23080.001156/2001-50/UFSC). The minimum number of animals (7–10 animals per group) and duration of observation required to obtain consistent data were used. 2.2. Drugs and treatment schedule Methyl-scopolamine bromide (a muscarinic receptor antagonist; RBI, USA) was given subcutaneously and used to prevent the peripheral cholinomimetic effects elicited by PILO. Pilocarpine hydrochloride (a non-selective muscarinic receptor agonist; Merck SA, Brazil) was injected intraperitoneally afterward. FK888 (a selective NK1 receptor antagonist; Fujisawa Pharmaceutical Co., Osaka, Japan) was previously prepared as a stock solution (10−3 M) in ethanol and diluted in phosphate-buffered saline (PBS, pH = 7.4; Sigma, USA) immediately before the experiments. Methylscopolamine and PILO were dissolved in saline (NaCl 0.9%). All doses were taken from previous studies [27,28,34,36–39]. Rats received methyl-scopolamine (1 mg/kg) followed 30 min later by a single systemic injection of saline or PILO (350 mg/kg).

Fig. 1. Photomicrographs (left) and schematic drawings (right), based on the atlas of Paxinos and Watson (1986), of coronal sections from 4.2 mm and 0.2 mm posterior to bregma of the rat brain showing the microinjection sites (black dotted circles) of PBS or FK888 (100 pmol) into the CA1 region of the dorsal hippocampus (A) or lateral septum (B) of male Wistar rats.

F.S. Duarte et al. / Behavioural Brain Research 265 (2014) 53–60

Only rats that did not develop any behavioral features of the SE (i.e. akinesia, facial automatisms, limbic seizures consisting of forelimb clonus with rearing, salivation, masticatory jaw movements and falls) or spontaneous recurrent seizures (SRS) were used in the present study. Rats were injected one month after with PBS or FK888 (100 pmol), followed 5 min later by the behavioral evaluation in the EPM test. 2.3. Surgical process Twenty-five days after the systemic treatment with PILO or saline, animals were anesthetized with ketamine (100 mg/kg; Francotar® , Eurofarma Lab. Ltda, SP, Brazil) and xylazine (20 mg/kg;

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Anasedan® , Vetbrands Labs., SP, Brazil) and positioned in a stereotaxic frame. Stainless-steel 22-gauge cannulae were stereotaxically implanted in the lateral septum (coordinates: AP, −0.2 mm from bregma; L, ±1.0 mm from the midline; DV, −2.8 mm from the skull surface) or in the CA1 dorsal hippocampus (coordinates: AP, −4.2 mm from bregma; L, ±2.6 mm from the midline; DV, −2.0 mm from the skull) according to the atlas of Paxinos and Watson [40]. Cannulae were fixed with polyacrylic cement and anchored to the skull with stainless-steel screws. A stylet was introduced into the guide cannula to prevent possible obstruction. Five days after surgery, animals were gently held and a removable injector was inserted into the guide cannula, extending 1 mm beyond the tip guide. The injector was linked to a 5-␮L

Fig. 2. (A) Outline of the treatments and experimental schedule: methylscopolamine (M-Scop, 1 mg/kg), saline (Sal, 0.9%), pilocarpine (Pilo, 350 mg/kg), phosphate-buffered saline (PBS) and FK888 (100 pmol). (B) Effect of central microinjection of NK1 receptor antagonist FK888 into the CA1 dorsal hippocampus on behavioral parameters of rats evaluated in the elevated plus-maze test. Each value represents the mean ± S.E.M. of 8–10 animals per group. *p < 0.05 or **p < 0.01 as compared to saline/PBS group; ## p < 0.01 as compared to PILO/PBS group (all comparisons were made by two-way ANOVA followed by Student Newman–Keuls’ test).

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Hamilton syringe and volumes of 0.3 ␮L of PBS or FK888 were administered over a 30-s period. The injector remained in the guide cannula for 30 s after the infusion to allow spreading of the substance. 2.4. Apparatus 2.4.1. Elevated plus-maze (EPM) The EPM was made of clear Plexiglas and consisted of two opposed open arms (50 cm × 10 cm) and two opposed closed arms (50 cm × 10 cm × 30 cm) mounted at an angle of 90◦ , all facing a central platform (10 cm × 10 cm) elevated 50 cm from the floor. To prevent falls, the open arms were surrounded by a 1-cm high acrylic rim. The apparatus was placed in a small closed room lit by a 15-W red light, which provided 3 lux in both the open and closed arms. Each rat was used only once and was placed individually on the central platform facing a closed arm. The frequency of entries into either open or closed arms as well as the time spent in each arm type were recorded (in seconds) for 5 min [41]. Ethological parameters such as unprotected head-dipping (uHD), open-arms end activity (OAEA), protected stretch-attend postures (pSAP), and rearing were also recorded to increase the sensitivity of the test [42,43]. 2.5. Histological analysis At the end of the experiments, rats were sacrificed by choral hydrate overdose and intracardially perfused with 0.9% (w/v) saline followed by 10% (v/v) formalin. Brains were removed, post-fixed for 24 h in the same formalin solution and subsequently in 30% (w/v) sucrose–formalin solution until sectioning. Brains were then frozen and cut on a cryostat (CM1850; Leica® , Germany) in the transverse plane in sections of 30 ␮m and mounted on glass microscope slides. Sections were stained with cresyl violet to localize the injection sites. Data from animals that presented a cannula placement in the wrong location (not in the lateral septum or CA1 dorsal hippocampus) were excluded from the statistical analysis (less than 5%). 2.6. Statistical analysis All values are expressed as means ± S.E.M. Data of experiments 1 and 2 were analyzed by two-way ANOVA (pretreatment × treatment) followed by the Student Newman–Keuls’ post hoc test for multiple comparisons. Differences were considered significant at P ≥ 0.05. All tests were performed using the software Statistica® , version 6.0, and graphs were drawn with the software GraphPad Prism® , version 4.0. 3. Results 3.1. Histological verification The microinjection sites in the CA1 region of the dorsal hippocampus (panel A) or lateral septum (panel B) are illustrated in Fig. 1. 3.2. Elevated plus-maze As seen in Figs. 2 and 3, the two-way ANOVA showed a significant effect for the pretreatment and the interaction between treatment and pretreatment factors. A decrease in the time spent in (Fig. 2, F(3,31) = 4.42, P < 0.05 and Fig. 3, F(3,31) = 10.59, P < 0.01) and frequency of entries into the open arms (Fig. 2, F(3,31) = 6.74, P < 0.01 and Fig. 3, F(3,31) = 12.03, P < 0.01), number of unprotected head-dipping (Fig. 2, F(3,31) = 11.52, P < 0.01 and Fig. 3, F(3,31) = 17.69, P < 0.001), and open-arms end activity (Fig. 2, F(3,31) = 15.63,

P < 0.001 and Fig. 3, F(3,31) = 29.56, P < 0.001) in the EPM were found in rats evaluated one month after pretreatment with PILO. These exploration-related alterations in the open arms associated with an increase in the number of protected stretched-attend postures (Fig. 2, F(3,31) = 22.64, P < 0.0001 and Fig. 3, F(3,31) = 12.50; P < 0.001) indicates an anxiogenic-like effect caused by PILO treatment. The number of entries into enclosed arms as well as of rearings was unaffected by the previous treatment (P > 0.05). Injection of FK888 into the CA1 region did not alter per se the parameters reported in the EPM when compared to the control group, since the two-way ANOVA did not reveal effects in the treatment factor (P > 0.05; Fig. 2). However, FK888 injection blocked the anxiogenic-like effect elicited by PILO treatment on time spent in (Fig. 2, F(3,31) = 4.10, P < 0.05) and frequency of entries into the open arms (Fig. 2, F(3,31) = 4.33, P < 0.05), and the number of protected stretch-attend postures (Fig. 2, F(3,31) = 9.09, P < 0.001). Moreover, injection of FK888 reversed the decrease in unprotected headdipping and open-arms end activity induced by PILO. Injection of FK888 into the lateral septum did not alter per se the parameters registered in the EPM when compared to the control group (P > 0.05; Fig. 3). FK888 was not able to block the anxiogeniclike effects induced by PILO in rats evaluated in the EPM, since the two-way ANOVA did not reveal effects in the interaction of pretreatment × treatment factors (P > 0.05; Fig. 3).

4. Discussion The present work aimed to analyze the long-lasting anxiogeniclike effects induced by PILO in non-convulsive rats and its modulation by NK1 receptors of the CA1 region of dorsal hippocampus and lateral septum. Rats previously treated with PILO (350 mg/kg) – that did not develop neither motor SE nor SRS one month after treatment – were injected with the NK1 -receptor antagonist FK888 into the CA1 region of the dorsal hippocampus or lateral septum and were behaviorally evaluated in the EPM test. Our results showed that a single systemic injection of PILO triggered long term emotionally-related behavior changes in non-convulsive rats when evaluated one month later in the EPM, with no motor disturbances in the maze. An anxiogenic-like effect induced by PILO treatment was largely marked by a decrease in the time spent in and the frequency of entries into the open arms besides a decrease in the number of unprotected head-dipping and open-arms end activity, indicating a sensitization of the emotional responses, in agreement with our previous studies [34–36]. Similar anxiogenic-like effects were observed in non-convulsive rats evaluated in the elevated T-maze and open-field tests, and no alterations were observed when aversive memory formation was evaluated in the step-down avoidance apparatus (test sessions at 5 s, 1.5 h and 24 h after the training) or in the elevated T-maze test 1 month after treatment [34]. Here, we also showed that the injection of FK888 inhibited the anxiogenic-like responses induced by PILO when injected into the CA1 region of the dorsal hippocampus but not into the lateral septum suggesting an involvement of the NK1 receptor of the dorsal hippocampus in the anxiogenic-like effects induced by PILO treatment. Importantly, no behavioral changes were observed when FK888 was injected out of the targeted site (data not shown), suggesting that the effects produced by NK1 antagonist are regionspecific. The PILO model of epilepsy has been a useful approach for studying the neural mechanisms and the cascade of molecular events involved in the physiopathogenesis of the temporal lobe epilepsy [37–39,44]. In this model, the systemic administration of PILO (at doses higher than 300 mg/kg) promotes sequential behavioral and electrographic alterations characterized by three distinct phases: an acute phase that gradually built up into limbic SE, followed by

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Fig. 3. (A) Outline of the treatments and experimental schedule: methylscopolamine (M-Scop, 1 mg/kg), saline (Sal, 0.9%), pilocarpine (Pilo, 350 mg/kg), phosphate-buffered saline (PBS) and FK888 (100 pmol). (B) Effect of central microinjection of NK1 receptor antagonist FK888 into the lateral septum on behavioral parameters of rats evaluated in the elevated plus-maze test. Each value represents the mean ± S.E.M. of 7–9 animals per group. *p < 0.05 or **p < 0.01 as compared to saline/PBS group; ## p < 0.01 as compared to PILO/PBS group (all comparisons were made by two-way ANOVA followed by Student Newman–Keuls’ test).

a silent phase, and afterwards by a chronic phase with SRS (for review see [45]). Nevertheless, contradictory results regarding the anxiety- and fear-related behavior changes were found in animals submitted to the PILO model: anxiogenic-like effects in the openfield and light-dark tests [12,46], anxiolytic-like effects in the EPM, open-field and contextual fear conditioning [34,35,47,48], and no alterations in anxiety/fear responses in the EPM, open-field and light-dark tests [12,46,49]. A significant number of animals submitted to the PILO model do not develop SE [34,36,50–55] and are usually eliminated from the experiments. Although it has been hypothesized that the affective symptoms associated with epilepsy are secondary to seizures [56], we recently showed the first evidence that a single non-convulsant

dose of PILO induces a long-lasting increase in anxiety-related responses in rats evaluated in the EPM test [34–36]. The anxiogeniclike effects induced by PILO may last up to three months after treatment, suggesting that there are chronic plastic changes in the rat’s brain and that anxiogenesis depends neither on motor SE nor on SRS, but rather on a deep alteration of molecular biology, which leads to affective impairments. Several studies have shown the participation of the hippocampus in the regulation of hormonal and behavioral responses to stress [51,57–60] as well as in controlling the hyperexcitability involved in seizures regulation in adult rodents [37,61,62]. Although the hippocampus may present functional differences in the dorsal and ventral subregions [63], with the first involved in

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memory processes and the latter in defensive behaviors [64,65], the CA1 region of the dorsal hippocampus was chosen in our study due to two key reasons: first, its sensitivity to epilepsy [12,48], although previous studies have shown neither damage nor degeneration in the hippocampal formation of non-convulsive animals [12,66]; second, the injection of substance P into the ventral hippocampus, but not dorsal, failed to produce behavioral changes in rats evaluated in the EPM and OF tests [67]. Moreover, several regions of the hippocampus – including the strata oriens and radiatum of the CA1 region – are innervated by SP-containing axon terminals [68]. However, further experiments are necessary to evaluate the involvement of the NK1 receptor in the ventral hippocampus in long-lasting anxiogenic-like effects observed in non-convulsive rats to completely discard its participation in this phenomenon. A widespread damage in the brain – which includes the hippocampal formation – was previously observed in the PILO model [44,69]. Thus, concerning the role of the hippocampus in the regulation of anxiety responses, it could be argued that the anxiogenic-like effects found in non-convulsive animals may be supported by a hippocampal damage [64]. In this regard, Müller and colleagues [12] showed that neither damage nor degeneration in the hippocampus were observed in mice which did not develop SE up to five months after PILO treatment. In line with these data, Navarro Mora and colleagues (2009) did not observe any evident signs of neuronal suffering or degeneration in non-convulsive rats after the injection of PILO although the animals presented recurrent spontaneous seizures about 10 months after the treatment. Therefore, the increased emotionality observed in non-convulsive animals in the EPM test five weeks after PILO administration by Müller and colleagues may not be attributed to damage or degeneration in the hippocampus of the rats. Similar to the hippocampus, the lateral septum is directly involved in seizures induced by high doses of PILO or by the combination of lithium and PILO [70,71] as well as in anxiety (for review see [72]). Moreover, the lateral septum is known to contain a high density of NK1 receptors [73] and is considered an important site involved in the mediation of the anxiogenic-like responses induced by SP [28]. Most of the lateral septum neurons receive excitatory hippocampal input and project massively into hypothalamic areas [74,75]. The septum receives a SP fiber innervation [76–78] that derives from several hypothalamic nuclei and the tegmental area as well as from the intrinsic septal cells [75,79], which are densely distributed to regions of the nucleus known to provide GABAergic inputs to hypothalamic circuits mediating defensive rage [80,81]. In addition, injured neurons (mainly interneurons) were found in the hippocampus, piriform cortex, amygdala, and other limbic structures of rats treated with PILO [82]. Since the lateral septum receives a strong projection from the hippocampus and its neurons – particularly SPergic neurons – are involved in the modulation of experimental anxiety and are vulnerable to seizures, their functional properties are probably altered in both anxiety and epilepsy situations. At this point, it would be important to know the circumstances (aversive and/or convulsive) in which NK1 -SP transmission is actually altered in these brain structures. SP excites neurons by suppressing an inwardly rectifying potassium current and by suppressing the M- and N-type calcium currents [83–86] leading to a reduction of Ca2+ -activated potassium currents and resulting in after-hyperpolarization [87]. SP also enhances glutamate currents mediated by NMDA receptors [88] as well as glutamate release from hippocampal slices [32]. Thus, by regulating the activity of hippocampal interneurons and considering that this neuropeptide exhibits a critical role in the control of neuronal excitability involved in seizures [30–32] and anxiety [27–29], we presumed that long-lasting changes in the SP-NK1 receptor signaling pathway might be occurring in

non-convulsive animals, promoting deep anxiety-related alterations in rats. Of particular interest, previous data provide the evidence of the participation of hippocampal pathways in the modulation of the anxiogenic-like effects of a single systemic injection of PILO at subconvulsant doses, despite the tonic inhibition of this pathway revealed by lidocaine treatment [36]. Thus, the present data support the idea of a possible increase of endogenous tonus in the SP-NK1 receptor signaling within the hippocampus induced by PILO. This event may trigger anxious-like behavior in rats evaluated one month after the previous treatment with PILO. To some extent, this may be due to a local disinhibition of GABA neurons [35]. In conclusion, our results suggest a pivotal role for the SP-NK1 receptor signaling pathway in the long-lasting adaptive changes that occur in the CA1 region of the dorsal hippocampus of rats that do not convulse after PILO treatment, supporting the idea of the excitatory role of SP in the anxiogenesis observed in the PILO model. Acknowledgments This work was supported by CNPq, FACEPE, CAPES/PNPD, National Institute of Translational Neuroscience (INNT) and NENASC project (PRONEX Program CNPq/FAPESC) which provided research grants to FS Duarte, AA Hoeller, M Duzzioni, EC Gavioli, NS Canteras and TCM De Lima. References [1] Caplan R, Siddarth P, Gurbani S, Hanson R, Sankar R, Shields WD. Depression and anxiety disorders in pediatric epilepsy. Epilepsia 2005;46:720–30. [2] Kanner AM, Balabanov A. Depression and epilepsy: how closely related are they? Neurology 2002;58:S27–39. [3] Ekinci O, Titus JB, Rodopman AA, Berkem M, Trevathan E. Depression and anxiety in children and adolescents with epilepsy: prevalence, risk factors, and treatment. Epilepsy Behav 2009;14:8–18. [4] LaFrance WC, Kanner AM, Hermann B. Psychiatric comorbidities in epilepsy. Int Rev Neurobiol 2008;83:347–83. [5] Plioplys S. Depression in children and adolescents with epilepsy. Epilepsy Behav 2003;4(Suppl. 3):S39–45. [6] Stefanello S, Marín-Léon L, Fernandes PT, Li LM, Botega NJ. Depression and anxiety in a community sample with epilepsy in Brazil. Arq Neuropsiquiatr 2011;69:342–8. [7] Vergnes M, Marescaux C, Boehrer A, Depaulis A. Are rats with genetic absence epilepsy behaviorally impaired? Epilepsy Res 1991;9:97–104. [8] Marescaux C, Vergnes M, Depaulis A. Genetic absence epilepsy in rats from Strasbourg—a review. J Neural Transm Suppl 1992;35:37–69. [9] Sarkisova KY, Midzianovskaia IS, Kulikov MA. Depressive-like behavioral alterations and c-fos expression in the dopaminergic brain regions in WAG/Rij rats with genetic absence epilepsy. Behav Brain Res 2003;144:211–26. [10] Mesquita AR, Tavares HB, Silva R, Sousa N. Febrile convulsions in developing rats induce a hyperanxious phenotype later in life. Epilepsy Behav 2006;9:401–6. [11] Midzyanovskaya IS, Shatskova AB, Sarkisova KY, van Luijtelaar G, Tuomisto L, Kuznetsova GD. Convulsive and nonconvulsive epilepsy in rats: effects on behavioral response to novelty stress. Epilepsy Behav 2005;6:543–51. [12] Müller CJ, Bankstahl M, Gröticke I, Löscher W. Pilocarpine vs. lithiumpilocarpine for induction of status epilepticus in mice: development of spontaneous seizures, behavioral alterations and neuronal damage. Eur J Pharmacol 2009;619:15–24. [13] Bouilleret V, Hogan RE, Velakoulis D, Salzberg MR, Wang L, Egan GF, et al. Morphometric abnormalities and hyperanxiety in genetically epileptic rats: a model of psychiatric comorbidity? Neuroimage 2009;45:267–74. [14] Jones NC, Salzberg MR, Kumar G, Couper A, Morris MJ, O’Brien TJ. Elevated anxiety and depressive-like behavior in a rat model of genetic generalized epilepsy suggesting common causation. Exp Neurol 2008;209:254–60. [15] File SE. NKP608, an NK1 receptor antagonist, has an anxiolytic action in the social interaction test in rats. Psychopharmacology (Berl) 2000;152:105–9. [16] Bilkei-Gorzo A, Racz I, Michel K, Zimmer A. Diminished anxiety- and depression-related behaviors in mice with selective deletion of the Tac1 gene. J Neurosci 2002;22:10046–52. [17] Czéh B, Fuchs E, Simon M. NK1 receptor antagonists under investigation for the treatment of affective disorders. Expert Opin Investig Drugs 2006;15:479–86. [18] Kramer MS, Cutler N, Feighner J, Shrivastava R, Carman J, Sramek JJ, et al. Distinct mechanism for antidepressant activity by blockade of central substance P receptors. Science 1998;281:1640–5. [19] Rupniak NM, Carlson EJ, Webb JK, Harrison T, Porsolt RD, Roux S, et al. Comparison of the phenotype of NK1R−/− mice with pharmacological blockade of the substance P (NK1 ) receptor in assays for antidepressant and anxiolytic drugs. Behav Pharmacol 2001;12:497–508.

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