Experimental Neurology 267 (2015) 123–134

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PI3Kγ deficiency enhances seizures severity and associated outcomes in a mouse model of convulsions induced by intrahippocampal injection of pilocarpine Isabel Vieira de Assis Lima a, Alline Cristina Campos b, Aline Silva Miranda b, Érica Leandro Marciano Vieira b, Flávia Amaral-Martins a, Juliana Priscila Vago c, Rebeca Priscila de Melo Santos a, Lirlândia Pires Sousa c, Luciene Bruno Vieira a, Mauro Martins Teixeira d, Bernd L. Fiebich f, Márcio Flávio Dutra Moraes e, Antonio Lucio Teixeira b, Antonio Carlos Pinheiro de Oliveira a,⁎ a

Department of Pharmacology, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901, Belo Horizonte, Brazil Department of Internal Medicine, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901, Belo Horizonte, Brazil Department of Clinical and Toxicological Analysis, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901, Belo Horizonte, Brazil d Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901, Belo Horizonte, Brazil e Department of Biophysics and Physiology, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901, Belo Horizonte, Brazil f Department of Psychiatry, University of Freiburg Medical School, Hauptstr. 5, D-79104 Freiburg, Germany b c

a r t i c l e

i n f o

Article history: Received 18 June 2014 Revised 22 January 2015 Accepted 18 February 2015 Available online 5 March 2015 Keywords: Phosphatidylinositol 3-kinase Seizures Cytokines Neurodegeneration Neurotrophins Microglia Glutamate Pilocarpine

a b s t r a c t Phosphatidylinositol 3-kinase (PI3K) is an enzyme involved in different pathophysiological processes, including neurological disorders. However, its role in seizures and postictal outcomes is still not fully understood. We investigated the role of PI3Kγ on seizures, production of neurotrophic and inflammatory mediators, expression of a marker for microglia, neuronal death and hippocampal neurogenesis in mice (WT and PI3Kγ−/−) subjected to intrahippocampal microinjection of pilocarpine. PI3Kγ−/− mice presented a more severe status epilepticus (SE) than WT mice. In hippocampal synaptosomes, genetic or pharmacological blockade of PI3Kγ enhanced the release of glutamate and the cytosolic calcium concentration induced by KCl. There was an enhanced neuronal death and a decrease in the doublecortin positive cells in the dentate gyrus of PI3Kγ−/− animals after the induction of SE. Levels of BDNF were significantly increased in the hippocampus of WT and PI3Kγ−/− mice, although in the prefrontal cortex, only PI3Kγ−/− animals showed significant increase in the levels of this neurotrophic factor. Pilocarpine increased hippocampal microglial immunolabeling in both groups, albeit in the prelimbic, medial and motor regions of the prefrontal cortex this increase was observed only in PI3Kγ−/− mice. Regarding the levels of inflammatory mediators, pilocarpine injection increased interleukin (IL) 6 in the hippocampus of WT and PI3Kγ−/− animals and in the prefrontal cortex of PI3Kγ−/− animals 24 h after the stimulus. Levels of TNFα were enhanced in the hippocampus and prefrontal cortex of only PI3Kγ−/− mice at this time point. On the other hand, PI3Kγ deletion impaired the increase in IL-10 in the hippocampus induced by pilocarpine. In conclusion, the lack of PI3Kγ revealed a deleterious effect in an animal model of convulsions induced by pilocarpine, suggesting that this enzyme may play a protective role in seizures and pathological outcomes associated with this condition. © 2015 Elsevier Inc. All rights reserved.

Introduction

Abbreviations: BDNF, brain-derived neurotrophic factor; DCX, doublecortin; DG, dentate gyrus; EAAT3, excitatory amino acid transporter 3; FJC, Fluoro-Jade® C; GLT1, glutamate transporters type 1; IFN-γ, interferon γ; IL, interleukin; NGF, nerve growth factor; PI3K, phosphatidylinositol 3-kinase; SE, status epilepticus; TNFα, tumor necrosis factor α. ⁎ Corresponding author at: Department of Pharmacology, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil. Fax: +55 31 3409 2695. E-mail address: [email protected] (A.C.P. de Oliveira).

http://dx.doi.org/10.1016/j.expneurol.2015.02.021 0014-4886/© 2015 Elsevier Inc. All rights reserved.

Status epilepticus (SE) is characterized by prolonged seizures, which represents a life-threatening condition. This severe condition is associated not only with short-term consequences, but different studies have also demonstrated that a significant percentage of the patients developed epilepsy after SE (Aicardi and Chevrie, 1970; Lowenstein, 1999; Verity et al., 1993). A plethora of events may occur after the occurrence of SE, such as neuroinflammation, neuronal cell death, altered production of neurotrophic factors and neurogenesis (Scharfman and Gray, 2007; Simonato

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et al., 2006; Vezzani, 2005). In epileptic patients, these pathological outcomes are associated with epileptogenesis and long-term behavioral alterations (Pitkanen and Sutula, 2002). The pathophysiology of the seizures, as well as the postictal complications, is not fully understood. In addition, the epileptogenic process is not controlled by antiepileptic drugs which are refractory in thirty percent of the patients (Sander, 2003). Therefore, it would be important to investigate molecular pathways that might act as regulators of these complex events raising the possibility of identifying new and more effective pharmacological targets. Phosphatidylinositol 3-kinase (PI3K) is a key regulator of a plethora of actions in physiological and pathological conditions. The PI3K family is divided into three classes according to their structure and lipid substrate specificity, namely class I, class II and class III, from which the class I PI3K is the most studied. Class IA consists of heterodimers of a catalytic p110 subunit (p110α, p110β or p110δ) bound to a particular regulatory subunit (p85α, p55α, p50α, p85β or p55γ) (Marone et al., 2008). Different stimuli can activate PI3K, which, in turn, phosphorylates phosphatidylinositol 4,5-bisphosphate to generate phosphatidylinositol3,4,5-trisphosphate. This latter molecule binds to the pleckstrin homology domain of one of the Akt (also known as protein kinase B) isoforms and facilitates the phosphorylation of Akt1, Akt2 or Akt3 at Thr308/309/305 and Ser273/474/472 residues, respectively, by the phosphatidylinositoldependent kinases 1 and 2 (Laine et al., 2002). Previous studies have shown an increased Akt phosphorylation in the hippocampus of patients with temporal lobe epilepsy (Shinoda et al., 2004) and in the cortex of rats injected with kainic acid into the basolateral nucleus of the amygdala (Henshall et al., 2002). However, the role of PI3K in seizures, as well as in postictal events, is still not fully understood. It is suggested that the mechanisms of action of anticonvulsive drugs might be dependent on PI3K activation. Lee et al. (2005) demonstrated that carbamazepine increased the activation of excitatory amino acid transporter 3 (EAAT3) in transfected oocytes and in C6 glioma cells that constitutively express this transporter. LY294002, a non-selective PI3K inhibitor, inhibits the activation of the transporter and the glutamate uptake in this model (Lee et al., 2005). Moreover, anticonvulsant drugs activate or modulate Akt expression. For instance, valproic acid activates Akt (Creson et al., 2009), and phenytoin treatment increases Akt expression in rats (Mariotti et al., 2010). However, the relevance of these findings for the anticonvulsant effect of these drugs has not been established yet. PI3K are also involved in the production of inflammatory mediators by central nervous system cells (de Oliveira et al., 2008, 2012; Zhao et al., 2014). Therefore, considering the key role of inflammation in seizures and postictal events, it is possible to argue that PI3K could be involved in the development of these pathological conditions. In contrast to the other isoforms, which are ubiquitously expressed, PI3Kγ expression is more restricted to the brain, immune, hematopoietic and cardiovascular systems (Ruckle et al., 2006). This might indicate that this enzyme presents a key role in brain physiology and pathology. For example, PI3Kγ has been implicated in the long-term depression and behavioral flexibility (Kim et al., 2011). PI3Kγ deficiency has been shown to reduce the blood–brain barrier leakage and tissue damage, and to improve neurological outcome in an animal model of stroke (Jin et al., 2011). Thus, in the present study, we aimed to evaluate the role of PI3Kγ in seizures and associated outcomes induced by a convulsive stimulus. Material and methods Animals and surgical procedures All experiments were performed in accordance with the Brazilian Institutional Ethics Committee (CEUA), protocol number 068/11. Every effort was made to avoid unnecessary pain or stress to the animals. Adult male C57Bl/6 and PI3Kγ−/− mice (25–30 g), aged ten to twelve weeks,

were obtained from Animal Care Facilities of the Institute of Biological Sciences, Federal University of Minas Gerais (ICB-UFMG), Belo Horizonte, Brazil. Animals were anesthetized with ketamine:xylazine (80 mg/kg:8 mg/kg, i.p. Syntec®, Cotia, Brazil), placed in a stereotaxic apparatus (Insight®, Ribeirão Preto, SP, Brazil), and implanted with guide cannula in both hippocampi. Stereotaxic coordinates for injection were: AP: − 1.9 mm, ML: ± 1.5 mm, DV: − 2.3 mm, bregma as reference, according to Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates, Second Edition, 2001 (Paxinos and Franklin, 2001). Animals were allowed to recover for 5 days before the intrahippocampal administrations of pilocarpine through the cannulae. Microinjection procedures We used a 10 μL syringe (Hamilton, Sigma, Reno, NV, USA) connected to a pump (Insight®, Ribeirão Preto, SP, Brazil) adjusted to a flow of 0.2 μL/minute (min). After the end of microinjection, the syringe remained connected to the cannula for approximately 2 min to avoid reflux of the drug. All animals were gently restrained during the injection procedures. The experimental group received pilocarpine (Sigma-Aldrich, St. Louis, MO, USA) through the cannulae at different doses [5, 10, 20 and 40 μg/0.2 μL/site (corresponding to concentrations of 25, 50, 100 and 200 μg/μL, respectively)] while the control group received saline. Ninety minutes after status epilepticus (SE) onset, animals received a single i.p. injection of diazepam (10 mg/kg; Hipolabor, Belo Horizonte, Brazil). Working dose was defined in this dose response curve experiment, and then all following experiments were performed using the dose of 20 μg/0.2 μL/site of pilocarpine. Evaluation of Akt protein activation by western blotting Hippocampal tissues of the animals were carefully dissected 30 min or 24 h after the SE, homogenized in a lysis buffer (1% Triton X-100; 100 mM Tris/HCl, pH 8.0; 10% glycerol; 5 mM EDTA; 200 mM NaCl; 1 mM DTT; 1 mM PMSF; 25 mM NaF; 2.5 µg/ml leupeptin; 5 µg/ml aprotinin; and 1 mM sodium orthovanadate) and stored in − 80 °C until the beginning of the analysis. Protein concentration was determined by using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). 60 μg of protein samples were separated on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. After blocking in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, membranes were incubated overnight at 4 °C with primary antibodies against anti-pAktSer473 (1:1000; Cell Signaling Technology, Danvers, MA, USA) and anti-β-actin (1:5000; SigmaAldrich, St. Louis, MO, USA). Following three washes with TBST, membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (1:3,000). Finally, membranes were incubated with enhanced chemiluminescence ECL-Plus (GE Healthcare, Piscataway, NJ, USA). The optical densities of detected bands were quantified using the ImageJ software. The results were normalized to the quantity of β-actin in each sample lane. Seizure scoring Immediately after saline or pilocarpine injection, we recorded the seizures of all animals with a camera (Sony DCR-SR68, Brazil) up to 90 min after SE onset. Behavioral assessment was made from the videotape analysis by an investigator blind to the genotype and treatments. We used a set of acrylic cages that allowed the simultaneous observation of ten animals at the same time. The seizures computed were of those who achieved a score of at least 3 according to Racine's scale (Racine, 1972). This scale categorizes five stages of severity, and it is based on the behavioral repertoire of the animals during a seizure, including “mouth and facial movements” (intensity score 1); “head nodding” (score 2); “forelimb clonus” (score 3); seizures characterized by rearing (score 4); and seizures characterized by rearing and falling

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(score 5). The following parameters were analyzed: percentage of mortality, percentage of SE (which included animals that achieved at least score 3 according to Racine's scale), latency to reach the seizures and number of seizures with scores 4 and 5.

by 4% paraformaldehyde. The brains were dissected out and sectioned on a cryostat.

Preparation of hippocampal brain synaptosomes

Fluoro-Jade® C (FJC, Millipore, Billerica, MA, USA) staining was performed as follows: brain tissues (hippocampal slices, 30 μm) were mounted on glass and immersed in basic alcohol solution (1% sodium hydroxide in 80% ethanol) for 5 min, followed by 2 min in 70% ethanol and 2 min in distilled water. After this step, slides were transferred to a solution of 0.06% potassium permanganate for 20 min and were gently shaken on a rotating platform. Slides were rinsed three times for 2 min in distilled water and were then transferred to the FJC staining solution and gently shaken for 20 min. The 0.0001% working solution of FJC was prepared by adding 1 ml of stock FJC solution (0.01%) to 99 ml of 0.1% acetic acid in distilled water. After staining, sections were rinsed two times (1 min) in distilled water. The tissue sections were dried and coverslipped with DPX (Sigma-Aldrich, St. Louis, MO, USA).

Adult C57Bl/6 and PI3Kγ−/− mice (25–30 g) were decapitated and had their hippocampus removed and homogenized in 1:10 (w/v) 0.32 M sucrose solution containing dithiothreitol (0.25 mM; Molecular Probes, Eugene, OR, USA) and EDTA (1 mM; Sigma-Aldrich, St. Louis, MO, USA). Homogenates were then submitted to low-speed centrifugation (1000 × g × 10 min) and synaptosomes were purified from the supernatant by discontinuous Percoll-density gradient centrifugation [Sigma-Aldrich, St. Louis, MO, USA (Dunkley et al., 1988)]. The isolated nerve terminals were resuspended in Krebs–Ringer–HEPES solution (KRH; 124 mM NaCl, 4 mM KCl, 1.2 mM MgSO4, 10 mM glucose, 25 mM HEPES, pH 7.4) with no added CaCl2, to a concentration of approximately 10 mg/ml. For glutamate release measurement, aliquots of 30 μl were prepared and kept on ice until use. For measurement of cytosolic Ca2+ concentration, aliquots of 200 μl were prepared and kept on ice until loaded with fura-2 AM (stock solution 1 mM in DMSO; SigmaAldrich, St. Louis, MO, USA) for 30 min. Measurement of continuous glutamate release Glutamate release was measured by the fluorescence increase due to the production of NADPH in the presence of glutamate dehydrogenase Type II (Sigma-Aldrich, St. Louis, MO, USA) and NADP+ (Nicholls et al., 1987) (modified to microplates). In brief: the reaction medium containing a mixture of synaptosomes (approximately 30 μg of protein/well), CaCl2 (1 mM) and NADP+ (1 mM; Sigma-Aldrich, St. Louis, MO, USA) in KRH was transferred to Elisa microplates (300 μl/well) attached to a spectrofluorimeter (Synergy 2, Winooski, USA). After 3 min, glutamate dehydrogenase (35 units per well; Sigma-Aldrich, St. Louis, MO, USA) was added and the reading was restarted until the fluorescence reached balance (approximately 10 min). Subsequent additions of the PI3Kγ inhibitor AS605240 (1, 10 and 100 nM, − 15 min) and/or KCl (33 mM) were made as indicated in the figure legends. Calibration curves were done in parallel by adding known amounts of glutamate (5 units; Sigma-Aldrich, St. Louis, MO, USA) to the reaction medium. The experimental data were expressed as nmol of glutamate released per mg of protein. The experiments were performed at 37 °C for 30–50 min with excitation wavelength of 360 nm and emission of 450 nm. Measurements of cytosolic Ca2+ concentration [Ca2+]i Fura-2 AM (Sigma-Aldrich, St. Louis, MO, USA) measurements of cytosolic calcium concentration [Ca2+]i in synaptosomes were performed in Biotek spectroflurimeter according to Grynkiewicz et al. (1985). CaCl2 was added to the synaptosomal suspension at the beginning of each fluorimetric assay (1.0 mM final concentration). Synaptosomes were stirred throughout the experiment in a plate maintained at 35 °C. Drugs were added to the synaptosomal suspension 10 min prior to membrane depolarization with KCl (33 mM). Baseline (240 s) measurements were obtained before addition of drugs. At the end of each experiment sodium dodecyl sulfate (SDS) 10% (0.1% final) was added to obtain Rmax followed by 3.0 M Tris + 400.0 mM EGTA (pH 8.6) for Rmin, as described by (Grynkiewicz et al., 1985). Perfusion and sectioning of brain tissue Twenty-four (for Fluoro-Jade C and Iba-1 analysis) or seventy-two (for doublecortin analysis) hours after the injection of pilocarpine, animals were anesthetized with ketamine:xylazine (80:8 mg/kg) and then transcardially perfused with phosphate buffered saline followed

Fluoro-Jade C staining

Cytokine and neurotrophins (BDNF and NGF) measurement by ELISA and CBA Hippocampal and prefrontal cortex tissues of the animals were carefully dissected 24 h post-SE and homogenized in a PBS-buffer extraction solution containing a protease inhibitor cocktail (NaCl 0.4 M, Tween 20 0.05%, BSA 0.5%, PMSF 0.1 mM, benzethonium chloride 0.1 mM, EDTA 10 mM, aprotinin 20 UI, all diluted in PBS). Lysates were centrifuged and quantified using the Bradford assay reagent from Bio-Rad (Hercules, CA, USA). Cytokine levels were determined using a mouse Th1/ Th2/Th17 cytometric bead array kit (CBA; BD Biosciences, San Diego, CA, USA) and analyzed on a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The following cytokines were measured: interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17A (IL-17A), interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα). The concentration of the neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were determined by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Minneapolis, MN, USA) in accordance with manufacturer's instructions. Immunofluorescence analysis of Iba-1 expression Immunofluorescence staining was performed on free-floating 30 μm sections pretreated with citrate buffer for 30 min at 70 °C for antigen recovery. Nonspecific binding was blocked for 30 min in blocking solution (4% bovine serum albumin and 0.5% Triton-X 100 in TBS). After rinsing, sections were incubated with primary antibody (anti-Iba-1, 1:400; Wako Chemicals, Osaka, Japan) in blocking solution at 4 °C overnight. The tissue sections were then incubated for 1 h with secondary antibody Alexa Fluor 594 (1:1000; Invitrogen, Carlsbad, CA, USA) in solution and then rinsed in TBS. The tissue sections were dried, mounted on slides and coverslipped with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). Immunohistochemistry of doublecortin (DCX)-positive cells Immunohistochemistry staining was performed on free-floating 30 μm sections pretreated with citrate buffer for 30 min at 70 °C to antigen recovery. Nonspecific binding was blocked for 30 min in blocking solution (0.1% bovine serum albumin and 0.2% Triton-X 100 in TBS). After rinsing, sections were incubated with primary antibody (antidoublecourtin, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in blocking solution at 4 °C overnight. The tissue sections were then incubated for 1 h with corresponding biotinylated secondary antibody (1:1000), followed by incubation in avidin–biotin–peroxidase complex (Elite ABC kit: Vector Laboratories, Peterborough, UK). Peroxidase enzyme activity was revealed by using 3, 30-diaminobenzidine

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tetrahydrochloride (DAB; 0.06% in TB, pH 7.4) as the chromogen and 0.01% H2O2 as the substrate. The tissue sections were dried, mounted on slides and coverslipped with the mounting medium Entellan (Sigma-Aldrich, St. Louis, MO, USA). Image analysis The sections were photographed with a digital camera attached to a Zeiss microscope (Axio Imager A2, Carl Zeiss), and the images were analyzed by using MacBiophotonics ImageJ software (NIH, Bethesda MD, USA). In order to count equal areas (mm2) in every subject, a mask was applied in anatomical areas of interest to ensure that density measurements were representative of the analyzed areas. Using ImageJ software, the photomicrographs were converted to grayscale images, and a black and white mask was generated. Pixel intensity (pixels/mm2) from FJC-positive cells was calculated for the three different anatomical areas of the hippocampus, cornus ammonis 1 (CA1), dentate gyrus (DG) and CA3. Immunoreactivity of microglia using anti-Iba-1 antibody was also calculated in the three hippocampal areas, as well as in the prefrontal cortex: pre-limbic, medial, motor, ventrolateral and insular areas (Supplementary data — Fig. 1). Since the injections were performed at −1.9 mm (AP) and to avoid the influence of the physical damage near the site of the injection, we used the sections between − 1.7 to 1.8 mm and 2.1 to −2.2 mm AP from bregma to perform the analysis. Seven to nine sections for each brain hemisphere were used for the analysis. After the quantification of each section, the mean value of both sides of each animal was determined. This mean was used as the representative value for the animal. The total number of DCX+ cells was evaluated in the dentate gyrus at 20 × magnification. Positive cells were quantified in subgranular and granular layers of the dentate gyrus in 8 coronal sections per animal (Campos et al., 2013, 2014; Palazuelos et al., 2012). DCX positive cells were normalized to the hippocampus area determined with ×10 objective. The number of positive cells was estimated by calculating the total hippocampal volume as determined by the sum of the areas of the sampled sections (8 coronal slices) multiplied by the distances between them (series of hippocampal sections located between 1.3 mm and 2.5 mm posterior to bregma - Paxinos and Franklin, 2001). Doublecortin immunoreactivity was quantified manually by using a computerized image analysis system (Axiovision software, Carl Zeiss Microscopy, Thornwood, NY). Only cells that expressed DCX in cell body were considered as positively labeled.

(supplementary data), seventy-five percent of the animals developed SE with a bilateral injection of pilocarpine at the dose of 20 μg/site, albeit no mortality was observed. The dose of 40 μg/site induced more than eighty-seven percent of SE; however, mortality was also observed. In the following experiments, we used the dose of 20 μg/site, since no mortality was observed and an increased or decreased percentage of seizures could be detected. Since a variation from 62.5 to 75% of animals developing seizures could be observed in the experiments using 20 μg/site of pilocarpine, all the comparisons between the groups were made using the animals stimulated in the same experiment.

Activation of Akt in the hippocampus induced by pilocarpine We first evaluated whether pilocarpine injection would activate PI3K, as revealed by Akt phosphorylation. Pilocarpine increased the immunoreactivity of the phosphorylated form of Akt in the hippocampus at 30 min after the injection (Figs. 1A–B). Moreover, a sustained phosphorylation of Akt was observed 24 h after the convulsive stimulus (Figs. 1A–B). However, phosphorylation of Akt was not induced by pilocarpine in PI3Kγ−/− mice (Figs. 1A–B).

PI3Kγ deficiency enhances the seizures induced by pilocarpine Considering that Akt phosphorylation was increased in the hippocampus after a convulsive stimulus, we next evaluated whether PI3Kγ deficiency could alter the seizures' severity induced by pilocarpine. We first observed that 62.5% and 76.5% of the WT and PI3Kγ−/− mice developed seizures with 20 μg/site of pilocarpine, respectively. Moreover, the latency for the PI3Kγ−/− mice to develop the seizures was shorter (Fig. 2A). PI3Kγ−/− mice also revealed an enhanced number of seizure scores 4 and 5 (Fig. 2B).

Statistical analysis Statistical analyses were performed using Prism 5 software (GraphPad Software, La Jolla, CA, USA). Unpaired t test was used to analyze seizure scoring. Data from FJC, microglia and doublecortin staining, cytokines and neurotrophic factor levels and Akt phosphorylation were analyzed by using a two-way analysis of variance (ANOVA) followed by a post-hoc analysis with a Bonferroni post-test. One-way ANOVA was used to analyze the data of synaptosomes. Values of p b 0.05 were set as statistically significant. Values of the effect of genotype, treatment and interactions are described in Supplementary data — Table 3. Results Standardization of the animal model of seizures induced by intrahippocampal injection of pilocarpine in C57Bl/6 Due to the high mortality induced by intraperitoneal injection of pilocarpine described in the literature (Curia et al., 2008), we have standardized the model of seizures induced by intrahippocampal injection of pilocarpine in mice, as previously demonstrated in rats (Castro et al., 2011; Furtado Mde et al., 2002). As shown in Table 1

Fig. 1. Immunoreactivity of p-Akt in the hippocampus of WT and PI3Kγ−/− mice 30 min and 24 h after administration of pilocarpine. A — Quantitative densitometric analysis of phospho-Akt normalized to actin loading control. B — Representative images of the western blots. Results are shown as mean ± SEM of 5 animals per group. *p b 0.05 with respect to saline control.

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Fig. 2. A — Latency and B — severity of SE in WT and PI3Kγ−/− animals. Results are shown as mean ± SEM of 9–11 animals per group. *p b 0.05 with respect to WT control.

PI3Kγ blockade enhances glutamate release and cytosolic calcium in hippocampal synaptosomes In order to investigate whether the enhanced seizures presented by PI3Kγ−/− mice could be due to an enhanced glutamate release in the absence of the enzyme, we investigated the release of this neurotransmitter induced by KCl using synaptosomes prepared from hippocampal tissues. The release of glutamate by hippocampal synaptosomes from PI3Kγ−/− mice was higher in comparison with the WT preparation. In addition, AS605240, a selective PI3Kγ inhibitor, enhanced the release of glutamate in a concentration dependent manner after depolarization with KCl, albeit the compound alone did not alter this parameter (data not shown) (Fig. 3A). In addition, cytosolic calcium concentrations were also increased in hippocampal synaptosomes from PI3Kγ−/− mice and from WT treated with AS605240 (Fig. 3B). PI3Kγ deficiency enhances neuronal death in the hippocampus induced by pilocarpine Intrahippocampal injection of pilocarpine promoted neuronal death in WT and PI3Kγ−/− mice in CA1 (Figs. 4A–B), DG (Figs. 4C–D) and CA3 (Figs. 4E–F) of the hippocampus. Importantly, there was a difference between the genotypes in the CA1 region (p b 0.05), indicating that the neuronal cell death was higher in PI3Kγ−/− mice. The differences between the genotypes observed in DG (p = 0.11) and CA3 regions (p = 0.14) did not reach statistical significance. BDNF levels are altered in WT and PI3Kγ−/− mice injected with pilocarpine: differential regulation in prefrontal cortex We next investigated whether the production of neurotrophins were altered 24 h after the convulsive stimulus. Levels of BDNF increased in the hippocampus of both WT and PI3Kγ−/− mice after SE

Fig. 3. A — Release of glutamate and B — concentration of cytosolic calcium by hippocampal synaptosomes of PI3Kγ−/− mice and synaptosomes from WT mice incubated with the selective PI3Kγ inhibitor AS605240 (1, 10 or 100 nM) induced by KCl. Results are shown as mean ± SEM of 3–6 independent experiments. *p b 0.05, **p b 0.01; ***p b 0.0001 with respect to WT control.

(Fig. 5A). On the other hand, BDNF levels in prefrontal cortex were increased only in PI3Kγ−/− mice (Fig. 5B). Levels of NGF were not changed in the hippocampus (Fig. 5C) and prefrontal cortex (Fig. 5D) at this time point.

Pilocarpine induces a differential pattern of microglia activation in the hippocampus and prefrontal cortex of WT and PI3Kγ−/− mice Different studies have demonstrated that seizures are strictly associated with microglia activation and neuroinflammatory responses (Shapiro et al., 2008). Therefore, we further evaluated the effect of pilocarpine injection to alter Iba-1 immunofluorescence, a molecule that is expressed specifically in microglia and that is upregulated during microglia activation. Intrahippocampal injection of pilocarpine increased the number of Iba-1+ cells in WT and PI3Kγ−/− mice 24 h after the convulsive stimulus in CA1 (Figs. 6A–B), DG (Figs. 6C–D) and CA3 (Figs. 6E–F) of the hippocampus. On the other hand, in the prefrontal cortex, we observed an increase in Iba-1+ cells in the prelimbic (Figs. 7A–B), medial (Figs. 7C–D) and motor (Figs. 7E–F) regions of PI3Kγ−/− mice, but not in WT mice. Ventrolateral (Fig. 7G — Supplementary data) and insular agranular (Fig. 7H — Supplementary data) regions did not reveal any difference between the pilocarpine and saline groups. However, microglia activation, which can be observed by the enlargement of the cell body, as well as by retraction and thickening of their processes, can be observed in the hippocampus and prefrontal cortex of both WT and PI3Kγ−/− mice.

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Fig. 4. A, C, E — Fluoro-Jade C staining density in the hippocampus (CA1, DG, CA3) of WT and PI3Kγ−/− animals 24 h after pilocarpine (black bars) or saline (white bars) injection. Results are shown as mean ± SEM of 4–6 animals per group. *p b 0.05; **p b 0.01; ***p b 0.0001 with respect to saline control. B, D, F — Representative photomicrographies of the hippocampus (CA1, DG, CA3) stained with Fluoro-Jade C in WT mice and PI3Kγ−/−. I, II, III and IV represent the groups WT + saline, WT + pilocarpine, PI3Kγ−/− + saline and PI3Kγ−/− + pilocarpine, respectively.

PI3Kγ deficiency differently alters the production of cytokines induced by pilocarpine In order to evaluate whether pilocarpine injection alters the production of inflammatory mediators, we performed CBA analysis to evaluate the content of IL-2, IL-4, IL-6, IL-10, IL-17A, TNFα and IFNγ in the hippocampus and prefrontal cortex. Pilocarpine increased the levels of IL-6 in the hippocampus of both WT and PI3Kγ−/− mice (Fig. 8A) 24 h after the injection. However, prefrontal cortex levels of IL-6 were increased only in PI3Kγ−/− mice (Fig. 8B). TNFα levels increased in the hippocampus

(Fig. 8C) and prefrontal cortex (Fig. 8D) of PI3Kγ−/−, but not WT mice, at this time point. On the other hand, levels of IL-10 were increased in the hippocampus only in WT mice (Fig. 8E). In the prefrontal cortex, although a tendency towards reduction is observed in the levels of IL-10 in pilocarpine-treated WT mice in comparison with saline group, no statistical difference was observed (Fig. 8F). The cytokines IL-2, IL-4, IFNγ and IL-17A were not altered by the injection of pilocarpine (Tables 2A and B, supplementary data). The alterations in the levels of all cytokines in the hippocampus and prefrontal cortex are presented in Table 2C, supplementary data.

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Fig. 5. Levels of BDNF and NGF in hippocampus (A, C) and prefrontal cortex (B, D) of WT and PI3Kγ−/− animals 24 h after pilocarpine (black bars) or saline (white bars) injection. Results are shown as mean ± SEM of 4–5 animals for BDNF and 7–9 animals for NGF per group. *p b 0.05; **p b 0.001 with respect to saline control.

Pilocarpine injection reduces the DCX+ cells in the dentate gyrus of PI3Kγ−/− mice Another phenomenon that might be altered by seizures is adult neurogenesis. To further investigate the effect of an intrahippocampal injection of pilocarpine, we evaluated the number of DCX+ cells in the dentate gyrus 72 h after the convulsive stimulus. As demonstrated in Figs. 9A–B, pilocarpine reduced the number of DCX+ cells in PI3Kγ−/−, but not in WT mice.

Discussion PI3K is involved in various pathological conditions. Therefore, we investigated whether the deficiency of PI3Kγ could interfere with seizures and postictal events. We demonstrated that ablation of PI3Kγ−/− enhanced the pathological pattern in an animal model of seizures induced by pilocarpine. SE induced Akt phosphorylation, a kinase that is regulated by PI3K, 30 min and 24 h after pilocarpine injection in WT, but not PI3Kγ−/− mice. Activation of Akt has been previously demonstrated in the hippocampus of patients with TLE and in the cortex of rats that received a kainic acid injection in the amygdala (Henshall et al., 2002; Shinoda et al., 2004). PI3Kγ is the only member of the family that can be activated by G protein coupled receptors. Increased phosphorylation of Akt can be a result of M1 muscarinic receptors activation by pilocarpine, as activation of this receptor leads to activation of PI3Kγ (Murga et al., 1998). Activation of muscarinic receptors by pilocarpine in glutamatergic neurons can also increase the release of glutamate, leading to Akt activation through action on NMDA or metabotropic glutamate receptors (Sanchez-Perez et al., 2006; Sutton and Chandler, 2002; Tokuda et al., 2011). This prompt activation of Akt induced by NMDA receptor activation has been shown to mediate neuroprotective effects (Wang et al.,

2012). Therefore, one can speculate that activation of Akt induced by pilocarpine might be a protective reflex to avoid neuronal cell death induced by the convulsive stimulus. PI3Kγ deficiency reduced the latency to develop SE and enhanced the severity of the seizures. By using hippocampal synaptosomes from PI3Kγ−/− mice, we demonstrated an enhanced release of glutamate induced by KCl in comparison with the synaptosomes from WT mice. Due to the limited number of available PI3Kγ selective inhibitors and because of the lack of information regarding their pharmacokinetics in vivo, we used an in vitro approach to investigate whether pharmacological PI3Kγ inhibition would mimic the effect of a genetic deletion of the enzyme. Pharmacological inhibition of PI3Kγ in synaptosomes from WT mice also enhanced glutamate release induced by KCl in comparison with the controls. In addition, genetic or pharmacological blockade of PI3Kγ increased the cytosolic calcium concentration, an event that would be important to enhance the release of neurotransmitters. This effect could contribute to the increased susceptibility of PI3Kγ−/− mice to seizures, although other mechanisms might also play a role. For instance, mutations in the group II metabotropic glutamate receptor gene increased neuronal excitability by preventing PI3K activation (Howlett et al., 2008). In astrocytes culture medium enriched with growth factors, incubation with PI3K or Akt inhibitors reduce the expression of glutamate transporters type 1 (GLT1) (Wu et al., 2010). In neuronal enriched cultures, wortmannin, a PI3K inhibitor, reduced cellular membrane expression of GLT1 and excitatory amino-acid carrier 1 (EAAC1) (Guillet et al., 2005). Altogether, activation of the PI3K/Akt pathway could increase glutamate uptake and reduce the release of this neurotransmitter, thus reducing the activation of its receptors and consequently the seizures. We also observed neuronal death in WT and PI3Kγ−/− mice in three hippocampal regions (CA1, DG, CA3). In the CA1 region, neuronal cell death was more intense in PI3Kγ−/− in comparison with WT mice. This higher cell death might be due to the more severe seizures in

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Fig. 6. A, C, E — Expression of Iba-1+ cells in hippocampus (CA1, DG and CA3) of WT and PI3Kγ−/− animals 24 h after pilocarpine (black bars) or saline (white bars) injection. Results are shown as mean ± SEM of 4–6 animals per group. *p b 0.05; **p b 0.001, ***p b 0.0001 with respect to saline control. B, D, F — Representative photomicrographies of the hippocampus (CA1, DG, CA3) stained with Iba-1 in WT and PI3Kγ−/− mice. I, II, III and IV represent the groups WT + saline, WT + pilocarpine, PI3Kγ−/− + saline and PI3Kγ−/− + pilocarpine, respectively.

knockout animals. In addition, the pro-survival role of PI3K in various cells, including neurons, is well established (Brunet et al., 2001; Datta et al., 1999), a role that could be reduced in the absence of the enzyme. Neurotrophic factors promote neuronal survival, proliferation and differentiation (Hagg, 2005; Simonato et al., 2006). Hippocampal BDNF was increased 24 h after injection of pilocarpine in both WT and PI3Kγ−/− mice, while cortical levels were increased only in PI3Kγ−/− mice. This may suggest that an initial activation in the hippocampus leads to a recruitment of other areas, and that an increase in BDNF levels would be an attempt to repair the damaged tissue. This putative role of BDNF in the cortex in the absence of PI3Kγ requires further investigation.

SE triggers microglia activation and production of different inflammatory mediators (Shapiro et al., 2008; Vezzani et al., 2011, 2013). We showed that pilocarpine injection increased microglia immunolabeling and activation in the hippocampus of both WT and PI3Kγ−/− mice. We did not observe any difference in the pattern of microglia activation in the hippocampus of both WT and PI3Kγ−/− mice, although an increased neuronal cell death in the hippocampus was observed in knockout animals. It is possible that even lower neuronal death, as observed in WT mice, is sufficient to induce high staining of microglia. Importantly, the morphology of microglial cells does not always parallel their biochemical changes (Nakamura et al., 1999). Even considering that the morphology of microglia is similar in both groups, it is possible that cells from

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Fig. 7. A, C, E — Expression of Iba-1+ cells in prefrontal cortex (prelimbic, medial and motor) of WT and PI3Kγ−/− animals 24 h after pilocarpine (black bars) or saline (white bars) injection. Results are shown as mean ± SEM of 4–6 animals per group *p b 0.05 with respect to saline control. B, D, F — Representative photomicrographies of the prelimbic, medial and motor regions of the prefrontal region stained with Iba-1 in WT and PI3Kγ−/− mice. I, II, III and IV represent the groups WT + saline, WT + pilocarpine, PI3Kγ−/− + saline and PI3Kγ−/− + pilocarpine, respectively.

knockout mice are responsible for a differential production of inflammatory mediators. Of note, a more pronounced immunofluorescence indicative of microglia activation was observed in cortical regions of knockout animals. This might be due to a more abundant recruitment for these brain regions, which is in line with increased severity of crises in PI3Kγ−/− mice. We also investigated the effect of the convulsive stimulus in the levels of the pro-inflammatory cytokines IL-6 and TNFα, and in the

anti-inflammatory cytokine IL-10. We observed an increase of hippocampal IL-6 levels 24 h after pilocarpine injection in both WT and PI3Kγ−/− mice; however, there was an increase of cortical levels only in PI3Kγ−/− mice. Elevated levels of IL-6 have been reported in human patients both in the postictal state (Bauer et al., 2009; Peltola et al., 2000) and interictal stage (Hulkkonen et al., 2004; Nowak et al., 2011). A significant increase in hippocampal and cortical TNFα was observed only in PI3Kγ−/− mice 24 h after pilocarpine injection. Few

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Fig. 8. Levels of cytokines in hippocampus (A, C, E) and prefrontal cortex (B, D, F) of WT and PI3Kγ−/− animals 24 h after pilocarpine (black bars) or saline (white bars) injection. Results are shown as mean ± SEM of 4–6 animals per group. *p b 0.05; **p b 0.001 with respect to saline control.

studies have shown a rapid increase in basal levels of this cytokine mRNA in rodent brain, and a return to baseline levels within a few days (De Simoni et al., 2000; Dhote et al., 2007). It is possible that TNFα is increased immediately after convulsive stimulus, returning to basal levels after few hours in WT mice (i.e. 24 h), although the high levels could be maintained in PI3Kγ−/− mice. We observed that IL-10 was increased only in the hippocampus of the WT, but not in knockout mice after pilocarpine injection, suggesting that PI3Kγ might favor an anti-inflammatory response. Godukhin et al. (2009) demonstrated that intrahippocampal injections of IL-10 suppressed the development of focal ictal discharges. This indicates a protective role of this cytokine in the early stages of seizures, an effect that could be lost in the absence of PI3Kγ. Induction of seizures is accompanied by a significant increase in neurogenesis in the subgranular zone of the dentate gyrus (Arisi and Garcia-Cairasco, 2007; Parent et al., 1997). Convulsive stimuli may increase the number of DCX positive cells as early as three days after seizures (Gu et al., 2010; Shapiro et al., 2007a). At this time point, DCX positive newborn cells present basal dendrites phenotype that may form aberrant synapsis in the hilus, contributing to reverberant circuits and epileptogenesis (Shapiro and Ribak, 2006; Shapiro et al., 2007b). In

the present study, we did not observe any increase in DCX positive cells in WT mice treated with pilocarpine. This discrepant result might be due to different convulsive stimuli, route of administration and animal species used in the studies. Moreover, in the present study, we did not use an unbiased stereological method to quantify DCX positive cells, that is a limitation of the approach. Most studies evaluate DCX expression after 3 to 4 weeks after seizures, since a marked increase on hippocampal proliferation after pharmacological induction of seizures in rodents is only detected after 7–9 days after the insult (Gu et al., 2010; Jessberger et al., 2005; Kim et al., 2012; Walter et al., 2007). Interestingly, PI3Kγ−/− animals exhibited a significant decrease in the number of hippocampal DCX+ cells after pilocarpine. This event would suggest an important role of PI3Kγ on neuroblast adult hippocampal neurogenesis. It has been shown that PI3K/Akt signaling regulates proliferation and inhibits differentiation of adult neural progenitors (Peltier et al., 2007). Inhibition of PI3K-p110α also interferes with cell proliferation, survival and neuronal differentiation through mTORC2 activation (Wahane et al., 2014). Besides the possible role of PI3Kγ in neurogenesis, the reduced number of DCX+ cells in PI3Kγ−/− mice could also reflect the increased death of these cells.

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Fig. 9. A — Expression of DCX+ cells in the dentate gyrus of the hippocampal formation of WT and PI3Kγ−/− mice 72 h after pilocarpine (black bars) or saline (white bars) injection. Results are shown as mean ± SEM of 4–5 animals per group. *p b 0.05 with respect to saline control. B — Representative photomicrographies (20× magnification) of the dentate gyrus of the hippocampal formation stained with DCX in WT and PI3Kγ−/− mice.

Although pathological findings observed in PI3Kγ absence could be due to enhanced glutamate release, more severe seizures and excitotoxic effects of pilocarpine, the changes following seizures could also be attributed to the absence of the enzyme. The absence of PI3Kγ could impair protecting mechanisms triggered by an excitotoxic stimulus. Moreover, absence of the enzyme during development could induce compensatory mechanisms responsible for some of the observed effects (Fung-Leung, 2013; Roller et al., 2012; Russo et al., 2011). In conclusion, PI3Kγ seems to confer protective effects during the development of seizures and postictal period, what could be important to avoid the development of long-term pathological effects. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.expneurol.2015.02.021. Acknowledgments This work was funded by Programa Primeiros Projetos, number CBBAPQ-04389-10, Programa Pesquisador Mineiro, number PPM-00372-13, and Programa de Apoio a Núcleos Emergentes de Pesquisa, number CBBAPQ-04625-10, all from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG); and Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, number 479254/2013-3. MFDM, LPS, MMT and ALT are recipients of CNPq fellowships. References Aicardi, J., Chevrie, J.J., 1970. Convulsive status epilepticus in infants and children. A study of 239 cases. Epilepsia 11, 187–197. Arisi, G.M., Garcia-Cairasco, N., 2007. Doublecortin-positive newly born granule cells of hippocampus have abnormal apical dendritic morphology in the pilocarpine model of temporal lobe epilepsy. Brain Res. 1165, 126–134.

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