Expression of neuropeptide Y1 receptors in the ...

Epilepsy & Behavior 37 (2014) 175–183

Contents lists available at ScienceDirect

Epilepsy & Behavior journal homepage: www.elsevier.com/locate/yebeh

Expression of neuropeptide Y1 receptors in the amygdala and hippocampus and anxiety-like behavior associated with Ammon's horn sclerosis following intrahippocampal kainate injection in C57BL/6J mice Elaine K. O'Loughlin, Janelle M.P. Pakan 1, Kieran W. McDermott, Deniz Yilmazer-Hanke ⁎ Department of Anatomy and Neuroscience, University College Cork, Cork, Ireland

a r t i c l e

i n f o

Article history: Received 9 June 2014 Revised 24 June 2014 Accepted 26 June 2014 Available online 20 July 2014 Keywords: Temporal lobe epilepsy Amygdala Anxiety Neuropeptide Y1 receptor Kainic acid Hippocampal sclerosis Comorbidities

a b s t r a c t Damage to the amygdala is often linked to Ammon's horn sclerosis (AHS) in surgical specimens of patients suffering from temporal lobe epilepsy (TLE). Moreover, amygdalar pathology is thought to contribute to the development of anxiety symptoms frequently found in TLE. The neuropeptide Y (NPY) Y1 receptor is critical in the regulation of anxiety-related behavior and epileptiform activity in TLE. Therefore, intrahippocampal kainate (KA) injection was performed to induce AHS-associated TLE and to investigate behavioral and cytoarchitectural changes that occur in the amygdala related to Y1 receptor expression. Status epilepticus was induced by intrahippocampal KA injection in C57BL/6J mice. Anxiety-like behavior was assessed using the elevated plus maze (EPM). Pathology of hippocampus and amygdala (volume loss and gliosis) was examined in KA-injected and saline-injected controls. Y1 receptor expression was measured using immunohistochemistry and ELISA. Animal injected with KA showed increased anxiety-like behaviors and reduced risk assessment in the EPM test compared with saline-injected controls. In the ipsilateral hippocampus of KA-injected animals, CA1 ablation, granule cell dispersion, and volume reduction were accompanied by astrogliosis indicating the development of AHS. In the amygdala, a significant decrease in the volume of nuclei and numbers of neurons was observed in the ipsilateral lateral, basolateral, and central amygdalar nuclei, which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor-expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsilateral and contralateral granule cell layer of the dentate gyrus, and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. Our results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Moreover, intrahippocampal KA injection can induce amygdalar damage suggesting that AHS-associated amygdala damage may contribute to behavioral alterations seen in patients with TLE. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Temporal lobe epilepsy (TLE) represents one of the most common and treatment-resistant forms of epilepsy and is associated with pathological alterations in the hippocampus and amygdala [1]. A common finding in TLE is Ammon's horn sclerosis (AHS), which includes loss of pyramidal neurons in the CA1 sector (Sommer's sector), granule cell dispersion, and gliosis in the hippocampal formation [2,3]. Patients

Abbreviations: AHS, Ammon's horn sclerosis; BLA, basolateral nucleus of amygdala; Ce, central nucleus of amygdala; DDE, dysphoric disorder epilepsy; DG, dentate gyrus; EPM, elevated plus maze; GFAP, glial fibrillary acidic protein; KA, kainic acid; LA, lateral nucleus of amygdala; NPY, neuropeptide Y; TLE, temporal lobe epilepsy. ⁎ Corresponding author at: Department of Biomedical Sciences, Creighton University, School of Medicine, Criss II, Rm 314B, 2500 California Plaza, Omaha, NE 68178, USA. Tel.: +1 402 280 2965; fax: +1 402 280 2690. E-mail address: [email protected] (D. Yilmazer-Hanke). 1 Current address: Centre for Integrative Physiology, University of Edinburgh, UK.

http://dx.doi.org/10.1016/j.yebeh.2014.06.033 1525-5050/© 2014 Elsevier Inc. All rights reserved.

with TLE often experience affective symptoms, and anxiety disorders are twice as frequent in patients with epilepsy in comparison with the general population [4,5]. These comorbid symptoms have been related to pathological alterations in the hippocampus and amygdala [6]. In recent years, the neuropeptide Y (NPY) system has stimulated much interest because of its involvement in epilepsy and emotional disorders. Neuropeptide Y is a highly conserved 36-amino acid neuropeptide that is expressed by specific neuronal populations and mediates its effects through G-protein-coupled receptors abundantly expressed in the brain [7,8]. In particular, hippocampal and amygdalar NPY receptors are implicated in anxiety and seizure activity [9,10]. Neuropeptide Y administration produces anxiolytic responses via Y1 receptors in vivo, and administration of a Y1 receptor antagonist increases anxiety behavior in rodents [11]. The Y1 subtype of the NPY receptor is considered to have proconvulsant effects because application of Y1 receptor antagonist reduces seizures induced by systemic or intrahippocampal delivery of kainic acid (KA) [12]. However, NPY administration into the lateral ventricles or the hippocampus in vivo using a cannula and in vitro on

176

E.K. O'Loughlin et al. / Epilepsy & Behavior 37 (2014) 175–183

hippocampal slices markedly reduces epileptic activity, probably through the activation of Y2 or Y5 receptors [13–15]. Here, we used a mouse model of TLE to investigate anatomical alterations that occur in the amygdala downstream of intrahippocampal KA injection because many aspects of TLE can be reproduced in this model including the development of AHS [16,17]. Temporal lobe epilepsy models, in which KA is delivered systemically, have been extensively characterized, but to our knowledge, little is known about downstream effects of intrahippocampal KA injection on the cytoarchitecture of the amygdala and whether this may impact on behavior. Patients with TLE often experience affective symptoms with anxiety being at the forefront, which prompted us to investigate specific spontaneous anxietylike behavior. We then carried out volumetric stereology of limbic structures to examine pathological changes after a single intrahippocampal KA injection. Finally, we examined Y1 receptor expression not only in the hippocampus but also in amygdala nuclei, which has not been shown before in this model. 2. Materials and methods 2.1. Induction of status epilepticus All procedures were carried out in accordance with Republic of Ireland Department of Health and Children licenses approved by the Institutional Animal Care and Use Committee and complied with the European Council Directive of November 24th, 1986 (86/609/EEC). Mice were held under a twelve-hour light/dark cycle (06:00–18:00). Water and food were provided ad libitum. Surgeries were carried out on adult inbred male C57BL/6J mice (Biological Services Unit, University College Cork). Mice were anesthetized intraperitoneally (i.p.) with a cocktail of 37.5 mg/kg ketamine, 3 mg/kg xylazine, and 7.7 mg/kg acepromazine (per body weight). Kainic acid (Sigma-Aldrich) dissolved in saline (0.2 μg in 50 nl) was injected into the right dorsal hippocampus (coordinates according to [16,18]; from bregma: anteroposterior (AP), −1.8 mm; mediolateral (ML), 1.6 mm; dorsoventral (DV), −1.8 mm) using a microsyringe (Hamilton, Reno, Nevada) with a 26-gauge needle. Control animals received 0.9% NaCl under the same surgical conditions. Postsurgery, individually housed mice were placed in their cages on a heating pad, and 1-ml 0.9% NaCl was administered subcutaneously (s.c.) to maintain hydration. For postoperative analgesia, carprofen (5 mg/kg) was injected, and diazepam 4 mg/kg s.c. was administered to mice that developed convulsive status epilepticus following intrahippocampal KA injection. 2.2. Seizure monitoring Behavioral alterations were extensively monitored for 2 weeks postsurgery. Further qualitative monitoring was continued up to three months postsurgery to observe chronic epileptic activity. Seizure activity was scored according to a modified Racine scale [19] based on the following criteria: score 0 — no behavioral alterations; score 1 — immobility, mouth and facial movements, and facial clonus; score 2 — head nodding, forelimb and/or tail extension with rigid posture; score 3 — forelimb clonus and repetitive movements; score 4 — rearing and forelimb clonus with rearing; score 5 — rearing and falling and jumping; and score 6 — severe tonic–clonic seizures. Animals that were recorded to have a minimum score of 2 (head nodding, forelimb and/or tail extension, and intermittent rigidity in posture) were included in this study. 2.3. Elevated plus maze (EPM) The elevated plus maze (EPM) test was performed two months after stereotaxic surgery. The EPM was made up of two protected (closed arms with sidewalls, 30 × 5 × 25 cm) and two unprotected arms (open platforms, 30 × 5 cm) connected by a central stage (5 × 5 cm). After cleaning the apparatus, mice were placed individually on the

central platform facing a closed arm. Each animal was allowed to explore the platform for 5 min. The number of entries (all paws) to and time spent on different locations were recorded using custom-made software. Relative open-arm variables were calculated as the percentage of time/entries to all arms (both open and closed arms). If a spontaneous seizure occurred during the tests, the test was suspended until 1 h after the animal was placed back to its home cage.

2.4. ELISA for Y1 receptor Saline- and KA-injected animals were terminally anesthetized four weeks after behavioral experiments. Brains were removed from the skull, and brain regions were dissected out. Tissue was snap frozen and stored at − 80 °C. Y1 receptor levels were determined using a commercially available ELISA kit (USCN, Life Science Inc.) per manufacturer's instructions in 96-well plate format. Protein concentrations were measured using a Bradford assay (Sigma-Aldrich, Wicklow, Ireland). A volume of 100 μl of standards, blank, and samples (in triplicate) was incubated in sealed plates for 2 h at 37 °C. Liquid was removed, and plates were incubated for 1 h at 37 °C with 100 μl of detection reagent A (1:100). After washing the plates, 100 μl of detection reagent B (1:100) was added to each well. Plates were incubated for 30 min at 37 °C. Following washing, plates were incubated with 90-μl substrate solution for 20 min at 37 °C in the dark room. The reaction was stopped with 50 μl of stop solution, and Y1 receptor concentrations were measured using a plate reader at optical density of 450 nm. 2.5. Histology Histological analyses were conducted in a blinded manner with respect to treatment. Animals were deeply anesthetized and transcardially perfused with 0.05-M phosphate-buffered saline followed by 4% paraformaldehyde in 0.05-M phosphate-buffered saline three months postsurgery. Brains were removed from the skull and postfixed in 4% paraformaldehyde overnight. Coronal 50-μm thick sections (onein-four series) were sectioned using a vibratome (Vibratome Series 100 Sectioning System, Ted Pella). The first and second series of sections were used for immunohistochemistry. The third series was Nisslstained with 0.1% cresyl violet to identify structural alterations and neuronal damage. Processed sections were dehydrated, cleared in histolene, and coverslipped using DPX (Sigma-Aldrich, Wicklow, Ireland). Photomicrographs were taken with 10 ×, 40 ×, or 60 × oil objectives using Olympus DP70 High-Resolution Color Digital Imaging System and cellSens entry camera software™ and adjusted in Adobe Photoshop 6.0. 2.6. Hippocampal and amygdalar volumes The volumes of brain regions were assessed according to the Cavalieri principle [20]. Hippocampal subfields and amygdalar nuclei were delineated in Nissl-stained sections using a mouse atlas [21]. Consecutive sections [section sampling fraction (ssf) of 1:4, sections 200 μm apart from each other] were sampled in salineand KA-injected animals. Volumes were calculated using the following formula: V = ∑ P i · a(p) · d, where P i is the sum of hit point counts in all sections, a(p) is the area associated with each point (0.073 mm2), and d is the section spacing (200 μm) (whereby ssf = 4 and section thickness = 50 μm). In the hippocampus, the volumes of the granule cell layer, CA1 and CA3 regions, and the hilus were measured in consecutive sections commencing at − 1.22 mm caudal to bregma according to the mouse atlas. In the amygdala, the volumes of the lateral (LA), basolateral (BLA), and central (Ce) nuclei were measured in consecutive sections, commencing at bregma − 0.70 mm to −2.30 mm according to the mouse atlas [21].


2.7. Quantification of amygdalar neurons

3. Results

Total numbers of neurons were estimated in Nissl-stained amygdalar nuclei using the optical dissector method [20]. Neurons were distinguished from astrocytes and oligodendrocytes based on cell and nuclear size, amount of nuclear heterochromatin, and organization of nucleolus [22]. Six sections per animal (1 in 4 series; 200 μm apart) throughout the rostrocaudal extent of the amygdala were used in saline- (n = 7) and KA-injected (n = 9) animals [bregma −0.70 mm to −2.30 mm] [21]. An Olympus BX 40 microscope (100× objective) was used to count the neurons. An unbiased counting frame with inclusion and exclusion lines was superimposed, and neurons that lay between the boundaries were counted. The focal plane was moved down the z-axis using a digimatic micrometer (Mitutoyo) that was attached to the stage of the microscope to measure the z-axis depth. The following equation was used: E(N) = Nv × Vref, as previously described in detail elsewhere [23].

3.1. Heightened anxiety-like behavior of KA-injected mice in the EPM

177

The KA-injected animals (n = 16) showed a significant increase (df = 30, p b 0.001) in the time spent in “safe” closed arms of the EPM compared with saline-injected controls (n = 16; Fig. 1A). In addition, the relative time spent on open versus all arms of the maze was reduced in KA-injected mice compatible with heightened anxiety (Fig. 1B; df = 30, p b 0.01). The number of entries to the closed arms showed a trend towards a significant decrease in KA-injected animals (df = 30, p = 0.1; Fig. 1C). The relative number of entries to open arms versus all arms also decreased significantly in KA-injected animals compared with controls (Fig. 1D; df = 30, p b 0.05). In the center stage of the EPM, thought to be important for risk assessment [24], KA-injected animals spent three times less time in the center (Fig. 1A; df = 30, p b 0.001), and the number of entries also decreased in KA-injected animals compared with controls (Fig. 1C; df = 30, p b 0.01).

2.8. Y1 receptor immunohistochemistry and analysis 3.2. KA induces volumetric alterations indicative of AHS The first series of sections were processed for free-floating immunohistochemistry using specific antisera against Y1 receptor (1:100) (ab73897 rabbit polyclonal antimouse Y1 antibody; Abcam, Cambridge, UK). Bound antibody was detected using a biotinylated secondary antibody and the avidin–biotin–peroxidase complex (ABC) method (Vector Laboratories, Peterborough, UK). Immunohistochemical staining was visualized using 3′,3′-diaminobenzidine (Sigma-Aldrich, Wicklow, Ireland) as a chromogen. Total numbers of Y1 receptor-positive neurons were estimated using the optical dissector method [20] with an Olympus BX 40 microscope at a 60× objective as described above. Consecutive sections per animal (1 in 4 series; 200 μm apart) throughout the rostrocaudal extent of the amygdala, including the BLA, LA, and Ce nuclei [bregma −0.70 mm to −2.30 mm], were analyzed [21]. Hippocampal analysis was performed by selecting consecutive sections that included the granule cell layer of the dentate gyrus (DG) and CA1 and CA3 layers of Ammon's horn [bregma −1.34 mm to −2.46 mm] [21]. An unbiased counting frame with inclusion and exclusion lines was superimposed, and positively stained neurons that lay between the boundaries were counted. The focal plane was moved down the z-axis using a digimatic micrometer (Mitutoyo).

2.9. Glial scaring Glial fibrillary acidic protein (GFAP) staining was used as an indication of glial scar formation. Free-floating sections of the second series were incubated with antisera against GFAP (1:1000) (ab53554 goat polyclonal antimouse GFAP antibody; Abcam, Cambridge, UK). Antibody staining was detected as described above. Semiquantitative scoring of GFAP staining was performed as follows: 0 = none, 1 = very little, 2 = faint, 3 = moderate, and 4 = intense staining. Glial fibrillary acidic protein scoring was analyzed to indicate the pathology of amygdala in KA-injected animals in comparison with saline-injected controls.

2.10. Statistics Statistical analyses were carried out using GraphPad Prism software version 5.01 or SPSS version 21.0. Data were represented as mean values ± the standard error of the mean (S.E.M.). Student's t-test was used to compare parametric data of two groups by using a Welch's approximation where appropriate. Nonparametric correlations were performed using the Spearman rank test. Two-tailed p-values ≤ 0.05 were considered significant.

Alterations in the hippocampal cytoarchitecture of KA-injected mice indicated the development of AHS, also called hippocampal sclerosis (Figs. 2A–D). In KA-injected animals (n = 9), the CA1 sector exhibited severe neuronal loss, whereas in saline-injected animals (n = 7), layers of the CA1 and CA3 sectors and the dentate gyrus appeared intact. Volumetric analysis in ipsilateral hippocampal subfields in KA-injected mice showed a significant decrease in the CA1 (df = 14, p b 0.05) but not in the CA3 region (df = 12, p = 0.1), whereas the volume of the hilus of the DG was significantly increased compared with that of the saline-injected mice (df = 14, p = 0.01; Fig. 2E). Contralaterally, there were no significant alterations in the CA1 or CA3 fields, but the hilus volume was slightly increased in KA-injected animals (df = 12, p = 0.01), suggesting that damage was not unilateral (Fig. 2F). Moreover, severe ipsilateral and mild contralateral granule cell dispersion indicative of AHS was seen after KA administration (Figs. 2C and D), although the increase in granule cell layer volumes was not significant in comparison with that of controls (Fig. 2G; ipsilateral: 0.03 ± 0.004 vs. 0.04 ± 0.01 mm3; contralateral: 0.02 ± 0.002 vs. 0.03 ± 0.07 mm3). Analysis of whole hippocampus volumes studied to assess global hippocampal damage after KA administration (Fig. 2G) revealed a significant reduction in the ipsilateral hippocampus of KA-injected animals compared with controls (df = 14, p b 0.05) but no changes in the contralateral hippocampus (Fig. 2H). 3.3. Histopathology as indicated by astrogliosis in hippocampus of KA-injected animals Glial fibrillary acidic protein immunohistochemistry (Figs. 2I–L) revealed reactive astrocytes in ipsilateral and contralateral hippocampi of some KA-injected animals (n = 8), which was less noticeable in saline-injected mice (n = 6). When hippocampal volumes were correlated with the GFAP score, there was a difference between ipsilateral and contralateral sides of saline-injected animals only (Fig. 2M). Ipsilaterally, no correlation was observed between GFAP score and hippocampal volumes. Contralaterally, there was a correlation between total volume (mm3) and the GFAP score in saline-injected animals (r = 0.8, p ≤ 0.05) but not in KA-injected animals (Fig. 2N). 3.4. Unilateral KA hippocampal injection induces alterations in the amygdala Cresyl violet staining was lighter in the ipsilateral amygdala of KAinjected mice compared with the contralateral side or the amygdala of saline-injected controls (Figs. 3A–D), resembling the human epileptic

178


Fig. 1. Anxiety-like behavior observed in kainic acid-injected animals in comparison with saline controls by use of the elevated plus maze. A: Time spent (s) in arms of maze. B: Relative time spent (s) in the open arm vs. the open plus closed arms of maze. C: Number of entries into each arm of the maze. D: Relative number of entries into the open vs. the open plus closed arms of maze. Saline- (n = 17) and kainic acid (KA)-injected (n = 16) animals. *p b 0.05, **p b 0.01, and ***p b 0.001 compared with the saline-injected animals (Student t-test).

amygdala [25,26]. Volumetric analyses of amygdala nuclei (lateral, LA; basolateral, BLA; and central, Ce nuclei) revealed a significant reduction in the ipsilateral LA (df = 13, p = 0.01), BLA (df = 13, p b 0.05), and Ce (df = 13, p b 0.01) nuclei of KA-injected animals (Fig. 3E). Contralateral nuclei remained unaffected by intrahippocampal injection (Fig. 3F). Total amygdala volumes were significantly decreased only ipsilaterally (df = 13, p b 0.05), supporting unilateral volume loss downstream intrahippocampal KA injection (Fig. 3G). 3.5. Astrogliosis is present in both ipsilateral and contralateral amygdalae after KA injection Intrahippocampal KA injection (n = 8) induced astrogliosis in the ipsilateral and contralateral amygdala as identified by increased GFAP immunoreactivity (Fig. 3J-K), but some saline-injected animals (n = 6) also showed mild astrogliosis (Fig. 3H). When we assessed the correlation between total volume (mm3) and GFAP score, no difference was observed in either ipsilateral or contralateral amygdalae of saline- or KA-injected animals (Figs. 3L-M). 3.6. Y1 receptor expression is decreased in the hippocampus of epileptic animals There was a significant reduction in the number of Y1 receptorimmunopositive neurons in the CA1 (df = 13, p b 0.01) and CA3 (df = 13, p b 0.05) subfields of the ipsilateral hippocampus of KA-injected animals (n = 9) compared with controls (n = 7) (Fig. 4E). This decrease in Y1 receptor expression was also evident in the contralateral CA1 region with significant loss in KA-injected animals (df = 13, p b 0.05), but no changes were seen in the contralateral CA3 region (Fig. 4F; df = 13, p = 0.3). Numbers of Y1 receptor-immunopositive neurons were reduced considerably in the granule cell layer of the ipsilateral DG in KA-injected animals (Fig. 4G; df = 13, p b 0.01). Analyses using ELISA showed a significant decrease in Y1 receptor protein levels in KA-injected animals

(n = 16) compared with saline-injected controls (n = 17) (Fig. 4H; df = 30, p b 0.01). 3.7. Decrease in amygdalar Y1 receptors following intrahippocampal KA administration Numbers of Y1 receptor-immunoreactive neurons were only decreased significantly in the ipsilateral Ce of KA-injected animals (n = 9) compared with controls (n = 7) (Fig. 5E; LA: df = 13, p = 0.17; BLA: df = 13, p = 0.14; Ce: df = 13, p b 0.01). No decrease in Y1 receptor-expressing neurons was found in the contralateral amygdala (Fig. 5F). Counts of Y1 receptor-immunopositive neurons in the whole amygdala also showed a significant decrease in the ipsilateral (df = 31, p b 0.05) but not in the contralateral amygdala of KA-injected animals compared with controls (Fig. 5I). However, Y1 receptor protein levels measured in the ELISA did not differ between the amygdala of KA-injected animals (n = 16) and that of controls (n = 17) (Fig. 5J; df = 31, p = 0.19). In addition, total neuronal cell counts performed using the Nissl stain showed a significant decrease in all three ipsilateral nuclei (Fig. 5G; LA: df = 13, p b 0.05; BLA: df = 13, p b 0.01; Ce: df = 13, p b 0.0001). Contralaterally, there was no change in neuronal cell numbers (Fig. 5H). 4. Discussion Psychiatric comorbidity and increased anxiety in patients with TLE are thought to result from pathology in mesial temporal brain areas. Because changes in the amygdala, an important brain center for emotions, are more severe in the presence of AHS [25], we performed unilateral intrahippocampal KA injections to model AHS-associated TLE. Mice injected with KA showed an increase in anxiety-like behavior, a decrease in hippocampal and amygdalar volumes with variable astrogliosis, severe AHS, and an overall loss of amygdalar neurons in all three nuclei studied four months after treatment. This was accompanied by a decrease in the expression of the anxiolytic but proconvulsant


179

Fig. 2. Volumetric changes (A–H) and gliosis (I–M) hippocampus in KA- and saline-injected animals. A–D: Nissl-stained sections of the hippocampal formation showing the ipsilateral (left panels) and contralateral (right panels) subfields. E–H: Bar graph summarizing changes in stereological estimates of volumes. Ipsilaterally, there was a volume loss in CA1 and CA3 subfields, but the volume of the hilus was increased after KA injection (E). Contralaterally, the hilar volume was also increased in the KA-injected group with no changes in other subfields (F). The granule cell layer showed a trend towards an increased volume in KA-injected mice indicative of TLE-characteristic “granule cell dispersion” (G). Whole volume measurements on ipsilateral and contralateral sides of the hippocampus revealed a significant decrease on the ipsilateral side in KA-injected animals. Contralateral hippocampal volume was not affected (H). Saline- (n = 7) and KA-injected (n = 9) animals. Statistical significances: *p b 0.05 compared with the saline-injected animals (Student t-test). I–L: Astroglial pathology as indicated by immunoreactivity for glial fibrillary acidic protein (GFAP). Inset in (K) shows reactive astrocytes in the dentate gyrus at higher magnification. M–N: Dot graphs demonstrate scoring of GFAP pathology against total hippocampal volume (mm3) in KA-injected animals compared with saline-injected controls. No correlations were observed between GFAP scores and hippocampal volumes except for the contralateral hippocampus of saline-injected mice (Spearman rank correlation test). Saline- (n = 6) and KA-injected (n = 8) animals. Abbreviations: CA1, CA1 subfield of the hippocampus; CA3, CA3 subfield of the hippocampus; g, granule cell layer; h, hilus; m, molecular layer. Arrows depict areas of structural damage. A–D: Scale bar = 500 μm; I–L: scale bar = 200 μm.

Y1 receptor in several hippocampal sectors and the Ce of the amygdala, compatible with a compensatory downregulation of Y1 in TLE.

amygdala damage and AHS through intrahippocampal KA injection adds to the understanding of mechanisms that lead to amygdala pathology in patients suffering from AHS-associated TLE.

4.1. Intrahippocampal KA injection mimicked AHS-associated TLE 4.2. Increased anxiety in TLE model Systemic KA application replicates several characteristics seen in patients with TLE, such as progression of the disorder, pathological findings, and seizure semiology [27]. However, the severity and range of damage in brain regions can vary considerably following peripheral KA delivery. In the present study, intrahippocampal KA injections were performed and downstream effects on the amygdala were investigated because amygdala damage is often present along with hippocampal damage in patients with TLE [25,28,29]. In KA-injected mice, we found typical features of AHS including granule cell dispersion, ablation of the CA1 region, astrogliosis, and a reduction in hippocampal volume [16–18]. Thus, our approach allowed us to investigate amygdala damage associated with AHS-like hippocampal pathology as seen in the majority of patients with TLE. In addition, TLE in humans associated with AHS may be a different entity than lesion-associated TLE without AHS in terms of disease genesis [30,31]. Therefore, simultaneous induction of

Patients with TLE often suffer from anxiety symptoms [4–6,32]. This conforms to the heightened anxiety-like behavior in the EPM test seen in our inbred male C57BL/6J mice, which were tested two months after KA injection into the hippocampus to model the chronic phase of TLE [18]. However, the increase in anxiety was subtle partly because of a concomitant decrease in visits to the center of the EPM in KAinjected mice. This may indicate a reduced risk assessment, which is a defensive behavior closely related to fear and anxiety [6]. Similarly, enhanced defensive behaviors were observed in epileptic cats following KA injection into the dorsal hippocampus (for review, see [33]). In outbred female NMRI mice examined in the same TLE model, anxiety-like behavior was not altered in the open-field, EPM, and light–dark box paradigms [33]. In addition to such species and sex differences already noted, our C57BL/6J mice developed convulsive status epilepticus and

180


Fig. 3. Changes in amygdala volume (A–H) and gliosis (I–M) in the in KA- and saline-injected animals. A–D: Nissl-stained sections showing the nuclei of the amygdala. E–G: Bar graphs demonstrate volumes of amygdala nuclei. On the ipsilateral side, the absolute volumes (E) were significantly reduced in KA-injected animals, with the central nucleus being most profoundly affected. Contralaterally, volumes of amygdala nuclei were not altered in KA-injected animals for absolute volume measurements (F). Whole volume measurements of the amygdala also showed a significant reduction on the ipsilateral side in KA-injected animals compared with saline-injected animals (G). Saline- (n = 7) and KA-injected (n = 9) animals. Statistical significances: *p b 0.05 and **p b 0.01 compared with the saline-injected animals (Student t-test). H–K: GFAP-stained sections demonstrating glial scar pathology. Inset in image (M) shows reactive astrocytes in a magnified area of the basolateral nucleus. L–M: Dot graphs indicate scoring of GFAP immunoreactivity against total amygdala volume (mm3). There was no correlation between GFAP scores and total amygdalar volumes in the KA- or saline-injected group on either side (Spearman rank correlation test). Saline- (n = 6) and KA-injected (n = 8) animals. Abbreviations: LA, lateral amygdaloid nucleus; BLA, basolateral amygdaloid nucleus; Ce, central amygdaloid nucleus. A–D: Scale bar = 500 μm; H–K: scale bar = 200 μm.

structural changes in the contralateral hippocampus following intrahippocampal KA injection. This differs from the nonconvulsive status epilepticus and structural changes limited to the ipsilateral hippocampus found in the latter study, suggesting more widespread brain pathology in our animals. In the pilocarpine model, increased anxiety-like behavior in most behavioral paradigms studied was detected [34]. 4.3. Amygdala neuropathology and its implications for affective symptoms The hippocampus and amygdala are both involved in anxietyrelated behavior and generation of seizures, but kindling of the amygdala is more efficient than kindling of the hippocampus to increase emotional reactivity [35]. Magnetic resonance imaging in patients shows that amygdala atrophy is among the most common pathological features of TLE [28,36,37], whereby a reduced volume associated with neuronal loss was also evident in the epilepsy model described here.

Amygdalar volume loss can range from 10% to 57% in patients with TLE [28]. Using volumetric stereology, we found that the volume of the ipsilateral amygdala is significantly decreased by almost half in KA-injected animals in comparison with saline controls. More specifically, the volume loss recorded ranged from 29% to 51% in the LA, BLA, and Ce nuclei and was often accompanied by varying degrees of gliosis as also seen in patients with TLE [25,26,31]. Together with the increased anxiety-like/defensive behaviors seen in KA-injected animals, the reduction in amygdala volume may be suggestive of a potential cause and effect. Consistent with this finding, amygdalar volume loss is associated with dysphoric disorder epilepsy (DDE), which correlates with psychopathological features of comorbidity [38]. In patients with DDE, those with a smaller amygdala volume had more pronounced comorbidity, e.g., anxiety, social withdrawal, and irritability. Likewise, patients with TLE presenting with ictal fear showed a reduced amygdalar volume associated with cellular damage compared with patients with TLE without a history of fear [39].


181

Fig. 4. Y1 receptor expression in hippocampus in KA- and saline-injected animals. A–D: Left (A, C) and right (B, D) panels showing Y1 immunoreactivity in the ipsilateral and contralateral hippocampal subfields, respectively. E–G: Analysis of the number of Y1-immunopositive neurons indicated a reduction in the ipsilateral CA1 and CA3 subfields (E), ipsilateral granule cell layer (F), and contralateral CA1 subfield (G). Saline- (n = 7) and KA-injected (n = 9) animals. H: ELISA displays decrease in Y1 receptor expression in hippocampal tissue. Saline- (n = 17) and KA-injected (n = 16) animals. *p b 0.05 and **p b 0.01 compared with the saline-injected animals (Student t-test). Arrows depict Y1-immunopositive cells. A–D: Scale bar = 200 μm.

4.4. The involvement of the Y1 receptor in the kainic acid model and its implications in anxiety The plasticity of the NPY system is thought to act as an endogenous system that counteracts hyperexcitability [40,41]. During epileptogenesis, downregulation of the Y1 receptor is accompanied by an upregulation of Y2 receptor expression in the hippocampus [42,43]. Our results, showing a considerable reduction in the number of Y1 receptor-expressing neurons in ipsilateral hippocampal sectors together with decreased hippocampal Y1 protein levels, are consistent with these findings. The loss of Y1-immunopositive neurons as well as the reduction in Y1 protein levels was more prominent in the hippocampus than in the amygdala. However, the impact of hippocampal Y1 receptors on the anxiety-like behavior of KA-injected animals may be limited

because overexpression of Y1 receptors in the hippocampus only has a mild anxiolytic effect [41]. In addition, we found a reduction in Y1 receptor-expressing neurons in the contralateral hilus, supporting the extension of damage to the contralateral side as also seen in our volumetric analyses. Hippocampal Y1 receptors are mainly expressed postsynaptically and also on hilar NPY neurons [9]. Thus, our findings are in agreement with a loss of hilar NPY/somatostatin-containing neurons found in TLE [44]. In the amygdala, the Y1 receptor is expressed by numerous BLA excitatory pyramidal neurons, most of which express calcineurin, and by GABAergic BLA interneurons [45]. Moreover, a small population of Y2positive neurons in the LA and BLA expresses Y1, but NPY-containing neurons in these nuclei lack the Y1 receptor [46–48]. The neuronal cell types mediating Y1 receptor effects in the Ce are less clear. Binding to

182


Fig. 5. Y1 receptor expression in amygdala in KA- and saline-injected animals. A–D: Y1 receptor immunoreactivity in amygdala nuclei. E–F: Bar graphs demonstrating numbers of Y1 receptor-positive cells in individual nuclei of the amygdala. There was a significant loss of Y1 receptor-expressing neurons in the Ce of KA-injected animals in comparison with salineinjected controls (E). However, no changes were found in the number of Y1 receptor-positive cells in contralateral amygdala nuclei (F). Saline- (n = 7) and KA-injected (n = 9) animals. G–H: Total number of neurons quantified in Nissl-stained sections. In the ipsilateral amygdala, the total number of neurons was significantly reduced in all three nuclei in KA-injected animals (G), but the contralateral amygdala remained unaffected in comparison with saline-injected animals (H). Saline- (n = 7) and KA-injected (n = 9) animals. I: There was a trend for a decrease in the total number of Y1 receptor-positive cells in the ipsilateral amygdala of KA-injected animals, but no changes were observed in the contralateral amygdala. J: Likewise, the reduction in Y1 protein levels in the whole amygdala (ELISA) did not reach significance level in KA-injected animals. Saline- (n = 17) and KA-injected (n = 16) animals. *p b 0.05, **p b 0.01, and ***p b 0.001 compared with the saline-injected animals (Student t-test). A–D: Scale bar = 200 μm.

Y1 receptors is moderate in the La, BLA, and Ce of rodents and humans [47,49], but stimulation of Y1 receptors in the BLA [50] and Ce [51,52] has consistent anxiolytic effects. As the Ce is the motor output center of the amygdala for regulating anxiety- and fear-related responses, the significant decrease in the number of Y1 receptor-containing neurons in the ipsilateral Ce following intrahippocampal KA injection is compatible with the increase in anxiety-like behaviors recorded in this study. In addition, the ipsilateral Ce showed the highest total neuronal cell loss among the amygdalar nuclei studied here, suggesting that the Ce may also be affected in TLE. In patients with TLE, changes in Y1 receptors in the Ce and their clinical relevance are largely unknown, but the Ce is usually not removed by surgical treatment in patients with intractable TLE because of its close topographical relationship to the basal forebrain [25,26].

4.5. Conclusions In summary, we studied amygdala damage after unilateral intrahippocampal KA injection and found significant amygdalar pathology three months after epilepsy induction including gliosis, volume reduction, and neuronal cell loss. We also showed that KA-injected mice exhibited increased anxiety-like behavior, which may be directly linked with the observed amygdala damage. Furthermore, KA-injected mice showed a significant decrease in Y1 receptor expression in the hippocampus and the Ce of the amygdala, a region implicated in the regulation of emotional behaviors. These data add to the understanding of amygdala pathology in TLE and its involvement in comorbid anxiety symptoms.

Acknowledgments Ms T. Foley is acknowledged for training provided. This project was funded by the Health Research Board (HRB), Ireland (Project HRA_POR/ 2010/159). Disclosures The authors declare no conflict of interest. References [1] Engel Jr J. Introduction to temporal lobe epilepsy. Epilepsy Res 1996;26:141–50. [2] Sano K, Malamud N. Clinical significance of sclerosis of the cornu ammonis: ictal psychic phenomena. AMA Arch Neurol Psychiatry 1953;70:40–53. [3] Hatanpaa KJ, Raisanen JM, Herndon E, Burns DK, Foong C, Habib AA, et al. Hippocampal sclerosis in dementia, epilepsy, and ischemic injury: differential vulnerability of hippocampal subfields. J Neuropathol Exp Neurol 2014;73:136–42. [4] Kimiskidis VK, Valeta T. Epilepsy and anxiety: epidemiology, classification, aetiology, and treatment. Epileptic Disord 2012;14:248–56. [5] Vazquez B, Devinsky O. Epilepsy and anxiety. Epilepsy Behav 2003;4(Suppl. 4): S20–5. [6] Hamid H, Ettinger AB, Mula M. Anxiety symptoms in epilepsy: salient issues for future research. Epilepsy Behav 2011;22:63–8. [7] Parker RM, Herzog H. Regional distribution of Y-receptor subtype mRNAs in rat brain. Eur J Neurosci 1999;11:1431–48. [8] Silva AP, Cavadas C, Grouzmann E. Neuropeptide Y and its receptors as potential therapeutic drug targets. Clin Chim Acta 2002;326:3–25. [9] Eva C, Serra M, Mele P, Panzica G, Oberto A. Physiology and gene regulation of the brain NPY Y1 receptor. Front Neuroendocrinol 2006;27:308–39. [10] Frisch C, Hanke J, Kleineruschkamp S, Roske S, Kaaden S, Elger CE, et al. Positive correlation between the density of neuropeptide Y positive neurons in the amygdala and parameters of self-reported anxiety and depression in mesiotemporal lobe epilepsy patients. Biol Psychiatry 2009;66:433–40.

E.K. O'Loughlin et al. / Epilepsy & Behavior 37 (2014) 175–183 [11] Kask A, Harro J, von Horsten S, Redrobe JP, Dumont Y, Quirion R. The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y. Neurosci Biobehav Rev 2002;26:259–83. [12] Gariboldi M, Conti M, Cavaleri D, Samanin R, Vezzani A. Anticonvulsant properties of BIBP3226, a non-peptide selective antagonist at neuropeptide Y Y1 receptors. Eur J Neurosci 1998;10:757–9. [13] Baraban SC. Antiepileptic actions of neuropeptide Y in the mouse hippocampus require Y5 receptors. Epilepsia 2002;43(Suppl. 5):9–13. [14] Klapstein GJ, Colmers WF. Neuropeptide Y suppresses epileptiform activity in rat hippocampus in vitro. J Neurophysiol 1997;78:1651–61. [15] Woldbye DP, Madsen TM, Larsen PJ, Mikkelsen JD, Bolwig TG. Neuropeptide Y inhibits hippocampal seizures and wet dog shakes. Brain Res 1996;737:162–8. [16] Bouilleret V, Ridoux V, Depaulis A, Marescaux C, Nehlig A, Le Gal La Salle G. Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy. Neuroscience 1999;89:717–29. [17] Riban V, Bouilleret V, Pham-Le BT, Fritschy JM, Marescaux C, Depaulis A. Evolution of hippocampal epileptic activity during the development of hippocampal sclerosis in a mouse model of temporal lobe epilepsy. Neuroscience 2002;112:101–11. [18] Bouilleret V, Nehlig A, Marescaux C, Namer IJ. Magnetic resonance imaging followup of progressive hippocampal changes in a mouse model of mesial temporal lobe epilepsy. Epilepsia 2000;41:642–50. [19] Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol 1972;32:281–94. [20] Gundersen HJ, Jensen EB. The efficiency of systematic sampling in stereology and its prediction. J Microsc 1987;147:229–63. [21] Franklin KBJ, Paxinos G. The mouse brain in stereotaxic coordinates. 3rd ed. Amsterdam: Academic Press; 2008. [22] Chareyron LJ, Banta Lavenex P, Amaral DG, Lavenex P. Stereological analysis of the rat and monkey amygdala. J Comp Neurol 2011;519:3218–39. [23] Yilmazer-Hanke DM, Faber-Zuschratter H, Linke R, Schwegler H. Contribution of amygdala neurons containing peptides and calcium-binding proteins to fearpotentiated startle and exploration-related anxiety in inbred Roman high- and low-avoidance rats. Eur J Neurosci 2002;15:1206–18. [24] Rodgers RJ, Dalvi A. Anxiety, defence and the elevated plus-maze. Neurosci Biobehav Rev 1997;21:801–10. [25] Yilmazer-Hanke DM, Wolf HK, Schramm J, Elger CE, Wiestler OD, Blumcke I. Subregional pathology of the amygdala complex and entorhinal region in surgical specimens from patients with pharmacoresistant temporal lobe epilepsy. J Neuropathol Exp Neurol 2000;59:907–20. [26] Yilmazer-Hanke DM, Faber-Zuschratter H, Blumcke I, Bickel M, Becker A, Mawrin C, et al. Axo-somatic inhibition of projection neurons in the lateral nucleus of amygdala in human temporal lobe epilepsy: an ultrastructural study. Exp Brain Res 2007;177:384–99. [27] Sperk G, Lassmann H, Baran H, Kish SJ, Seitelberger F, Hornykiewicz O. Kainic acid induced seizures: neurochemical and histopathological changes. Neuroscience 1983;10:1301–15. [28] Pitkänen A, Tuunanen J, Kälviäinen R, Partanen K, Salmenperä T. Amygdala damage in experimental and human temporal lobe epilepsy. Epilepsy Res 1998;32:233–53. [29] Hudson LP, Munoz DG, Miller L, McLachlan RS, Girvin JP, Blume WT. Amygdaloid sclerosis in temporal lobe epilepsy. Ann Neurol 1993;33:622–31. [30] Mathern GW, Adelson PD, Cahan LD, Leite JP. Hippocampal neuron damage in human epilepsy: Meyer's hypothesis revisited. Prog Brain Res 2002;135:237–51. [31] Faber-Zuschratter H, Huttmann K, Steinhauser C, Becker A, Schramm J, Okafo U, et al. Ultrastructural and functional characterization of satellitosis in the human lateral amygdala associated with Ammon's horn sclerosis. Acta Neuropathol 2009;117:545–55. [32] Lanteaume L, Bartolomei F, Bastien-Toniazzo M. How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy. Epilepsy Behav 2009;15:218–24.

183

[33] Gröticke I, Hoffmann K, Löscher W. Behavioral alterations in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainate. Exp Neurol 2008;213:71–83. [34] Gröticke I, Hoffmann K, Löscher W. Behavioral alterations in the pilocarpine model of temporal lobe epilepsy in mice. Exp Neurol 2007;207:329–49. [35] Depaulis A, Helfer V, Deransart C, Marescaux C. Anxiogenic-like consequences in animal models of complex partial seizures. Neurosci Biobehav Rev 1997;21:767–74. [36] Guerreiro C, Cendes F, Li LM, Jones-Gotman M, Andermann F, Dubeau F, et al. Clinical patterns of patients with temporal lobe epilepsy and pure amygdalar atrophy. Epilepsia 1999;40:453–61. [37] Kalviainen R, Salmenpera T, Partanen K, Vainio P, Riekkinen Sr P, Pitkanen A. MRI volumetry and T2 relaxometry of the amygdala in newly diagnosed and chronic temporal lobe epilepsy. Epilepsy Res 1997;28:39–50. [38] van Elst LT, Groffmann M, Ebert D, Schulze-Bonhage A. Amygdala volume loss in patients with dysphoric disorder of epilepsy. Epilepsy Behav 2009;16:105–12. [39] Cendes F, Andermann F, Gloor P, Gambardella A, Lopes-Cendes I, Watson C, et al. Relationship between atrophy of the amygdala and ictal fear in temporal lobe epilepsy. Brain 1994;117(Pt 4):739–46. [40] Richichi C, Lin EJ, Stefanin D, Colella D, Ravizza T, Grignaschi G, et al. Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. J Neurosci 2004;24:3051–9. [41] Olesen MV, Christiansen SH, Gotzsche CR, Nikitidou L, Kokaia M, Woldbye DP. Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice. J Neurosci Res 2012;90:498–507. [42] Furtinger S, Pirker S, Czech T, Baumgartner C, Ransmayr G, Sperk G. Plasticity of Y1 and Y2 receptors and neuropeptide Y fibers in patients with temporal lobe epilepsy. J Neurosci 2001;21:5804–12. [43] Gobbi M, Gariboldi M, Piwko C, Hoyer D, Sperk G, Vezzani A. Distinct changes in peptide YY binding to, and mRNA levels of, Y1 and Y2 receptors in the rat hippocampus associated with kindling epileptogenesis. J Neurochem 1998;70:1615–22. [44] Sloviter RS. Permanently altered hippocampal structure, excitability, and inhibition after experimental status epilepticus in the rat: the “dormant basket cell” hypothesis and its possible relevance to temporal lobe epilepsy. Hippocampus 1991;1:41–66. [45] Leitermann RJ, Sajdyk TJ, Urban JH. Cell-specific expression of calcineurin immunoreactivity within the rat basolateral amygdala complex and colocalization with the neuropeptide Y Y1 receptor. J Chem Neuroanat 2012;45:50–6. [46] Caberlotto L, Fuxe K, Hurd YL. Characterization of NPY mRNA-expressing cells in the human brain: co-localization with Y2 but not Y1 mRNA in the cerebral cortex, hippocampus, amygdala, and striatum. J Chem Neuroanat 2000;20:327–37. [47] Larsen PJ, Sheikh SP, Jakobsen CR, Schwartz TW, Mikkelsen JD. Regional distribution of putative NPY Y1 receptors and neurons expressing Y1 mRNA in forebrain areas of the rat central nervous system. Eur J Neurosci 1993;5:1622–37. [48] Stanić D, Mulder J, Watanabe M, Hökfelt T. Characterization of NPY Y2 receptor protein expression in the mouse brain. II. Coexistence with NPY, the Y1 receptor, and other neurotransmitter-related molecules. J Comp Neurol 2011;519:1219–57. [49] Yilmazer-Hanke D. Amygdala. In: Paxinos G, Mai JK, editors. The human nervous system. 3rd ed. London: Academic Press-Elsevier; 2012. p. 759–834. [50] Molosh AI, Sajdyk TJ, Truitt WA, Zhu W, Oxford GS, Shekhar A. NPY Y1 receptors differentially modulate GABAA and NMDA receptors via divergent signal-transduction pathways to reduce excitability of amygdala neurons. Neuropsychopharmacology 2013;38:1352–64. [51] Heilig M, McLeod S, Brot M, Heinrichs SC, Menzaghi F, Koob GF, et al. Anxiolytic-like action of neuropeptide Y: mediation by Y1 receptors in amygdala, and dissociation from food intake effects. Neuropsychopharmacology 1993;8:357–63. [52] Taksande BG, Kotagale NR, Gawande DY, Bharne AP, Chopde CT, Kokare DM. Neuropeptide Y in the central nucleus of amygdala regulates the anxiolytic effect of agmatine in rats. Eur Neuropsychopharmacol 2014;24:955–63.