[1] 17w Vagus nerve stimulation inhibits seizure activity and protects blood-brain barrier integrity in kindled rats with cortical dysplasia

[1] 252w Aims: This study investigates the effects of vagus nerve stimulation (VNS) on seizure severity and bloodbrain barrier (BBB) integrity in kindled rats with cortical dysplasia (CD). Main methods: Pregnant rats were exposed to 145 cGy of gamma-irradiation on day 17 of pregnancy. In offsprings, kindling was induced by giving subconvulsive doses of pentylenetetrazole. Left VNS was performed for 48 h at output currents of 0.5 or 1 mA. Horseradish peroxidase (HRP) was used to study the BBB permeability. Immunohistochemistry for occludin and P-glycoprotein (P-gp) was also performed. Key findings: Kindled rats with CD exhibited seizures with mean Racine's scores of 3.57 ± 1.2 during video EEG recording. Kindled animals with CD receiving VNS at 0.5 and 1.0 mA did not exhibit either clinical or electrophysiological signs of seizure. Immunostaining for occludin, a tight junction protein, in hippocampus remained relatively intact in all groups. VNS-treated and -untreated kindled animals with CD revealed intense immunostaining for P-gp in hippocampal formation (P b 0.01). Electron microscopic observations revealed frequent transport vesicles containing electron-dense HRP reaction products in the cytoplasm of brain capillary endothelial cells in both cerebral cortex and hippocampus of kindled animals with CD. Those which were exposed to 1 mA VNS were observed to have brain capillary endothelial cells largely devoid of HRP reaction products in both cerebral cortex and hippocampus. Significance: The results of this study suggest that VNS therapy at 1 mA inhibits seizure activity and protects BBB integrity by limiting the enhancement of transcellular pathway in kindled animals with CD.

[1] 130w Cortical dysplasia (CD) is one of the neuronal migration disorders of the brain, which is characterized by dislamination in the neocortex, abnormal spatial orientation of the neurons, and cytoskeletal and functional abnormalities of neuronal cells (Mischel et al., 1995;Guerrini and Barba, 2010). Animal models of CD present a variety of specific pathological features observed in patients with CD including double cortex (Lee et al., 1997), microdysgenesis (Amano et al., 1996), type I lissencephaly (Hirotsune et al., 1998), granule cell dispersion (Wenzel et al., 2001), polymicrogyria (Jacobs et al., 1999), nodular heterotopia (Baraban and Schwartzkroin, 1995), and abnormal cortical lamination with heterotopic neurons (Marin-Padilla et al., 2003). The experimental models of CD induced by methylazoxymethanol acetate or in-utero irradiation are suggested to exhibit the greatest similarity to human CD (Wong, 2009).

[2] 101w Blood-brain barrier (BBB) is constituted primarily by brain capillary endothelial cells and regulates the brain homeostasis. Recent studies indicated that the integrity of BBB becomes vulnerable or is impaired in cortical dysplastic lesions as a result of alterations in BBB characteristics (Marchi et al., 2006;Gürses et al., 2009). P-glycoprotein (P-gp), a multidrug efflux transporter at the BBB, plays an important role in protecting the brain from toxic substances. Human and animal studies have documented overexpression of P-gp in the cells of the neurovascular unit in the epileptic foci (Sisodiya et al., 1999;Marchi et al., 2006;Langer et al., 2007;Aronica et al., 2012).

[3] 34w Increased expression of P-gp may limit the central distribution of drugs which results in pharmacoresistance to therapeutic medication of brain diseases such as epilepsy (Seegers et al., 2002;Löscher et al., 2011;Aronica et al., 2012).

[4] 153w Epilepsy surgery represents an alternative treatment option to drug therapy for patients with CD, since in 20% of the operated adults and 50% of the operated children, surgery yielded histologically proven focal CD in spite of a negative magnetic resonance imaging (Bast et al., 2006;Lüders and Schuele, 2006). However, the number of patients with CD who are medically and surgically refractory remains to be quite high. On the other hand, vagus nerve stimulation (VNS) has emerged as an effective alternative treatment modality for epileptic patients who are resistant to medical and surgical treatment. In this regard, animal models may represent a means to elucidate the mechanism of action of VNS by which it prevents or reduces seizure activity (Dedeurwaerdere et al., 2006;Aalbers et al., 2011;Raedt et al., 2011). Moreover, a recent study indicated that application of VNS decreased BBB permeability to FITC-dextran after traumatic brain injury in BALB/c mice (Lopez et al., 2012).

[5] 72w In light of the aforementioned literature, we hypothesized that VNS might exert a positive effect on seizures in the setting of CD and in this regard alterations in BBB characteristics may constitute a basis in deducing a reasonable mechanistic interpretation. Therefore, the aim of the present study was to investigate whether VNS alters the severity of kindled seizure and/or exerts protective effects on BBB integrity during seizures in kindled rats with CD.

[1] 298w Approximately 20 s following the last dose of PTZ injection, behavioral seizures started typically from hippocampi in all animals but one and were scaled according to Racine's scale. PTZ induced severe seizures in kindled rats with CD under no-VNS condition, and these animals exhibited seizures with Racine's scores of 3.57 ± 1.2 (range: 2-5; median: 4). VNS at 1 mA completely suppressed seizures in all kindled animals with CD (P b 0.01). Among the kindled rats with CD treated with 0.5 mA VNS, only one rat exhibited seizure activity with Racine's score of 2 and the rest of the animals showed no clinical and electrophysiological seizures. Seizure activity was also verified with video EEG recordings and no seizures were recorded from animals but one in 0.5 mA VNS group. EEG recordings showed good build-up with high frequency activity at the beginning of the seizures and continued to become clinically generalized tonic-clonic convulsions (Fig. 1A). The average seizure duration in all animals was 26.6 ± 3.2 min. If the duration of the spikes lasted longer than 5-6 s, the rats had intermittent short lasting clonic or tonic jerks; if longer than 20 s, seizures turned into generalized tonic-clonic convulsions. During 1 h recording, electrophysiological seizure activity continued with gradual decrease and occasional interictal discharges towards the end. In kindled rats with CD under no-VNS condition, 7 out of 8 rats died. However, in the 0.5 and 1.0 mA VNS-treated groups of kindled animals with CD, no behavioral changes or EEG pattern alterations were observed following the last dose of PTZ except for one rat in 0.5 mA group which exhibited interictal epileptogenic spikes despite the absence of a seizure (Fig. 1B). On the other hand, neither ictal nor interictal discharges were observed in 1 mA VNS-treated group (Fig. 1C).

[2] 103w Occludin immunostaining remained essentially unchanged in the hippocampal microvessels of rats in all experimental groups and the relative intensity of occludin immunoreactivity did not differ significantly among the experimental groups (Figs. 2A-E). In kindled animals with CD, P-gp immunostaining in the wall of the capillaries in hippocampus increased noticeably and in these animals the relative intensity of P-gp immunoreactivity was found to be significantly increased compared to animals with CD and controls (P b 0.01). VNS treatment at 0.5 or 1 mA to kindled animals with CD did not lead to a significant alteration in the relative intensity of P-gp immunoreactivity (Figs. 3A-E).

[3] 210w Macroscopic observations of the pattern of HRP extravasation in Vibratome sections of brains following the last dose of PTZ in VNS-treated and -untreated kindled animals with CD are shown in Figs. 4A-C. Ultrastructurally, HRP reaction product was observed neither in the cytoplasm of endothelial cells nor in the interendothelial clefts of brain capillaries in the cerebral cortex and hippocampus (Figs. 5A and B) of in utero irradiated rats. However, frequent caveolar vesicles containing HRP reaction products in the cytoplasm of endothelial cells in the cerebral cortex and hippocampus were observed in epileptic seizures induced by the last dose of PTZ in kindled animals with CD (Figs. 5C and D). In these animals astrocytic endfeet around the brain capillaries exhibited normal ultrastructure and did not reveal any signs of swelling. While 0.5 mA VNS therapy did not yield a marked alteration in the frequency of HRP reaction products in the cytoplasm of endothelial cells in both cerebral cortex and hippocampus, the endothelial cells were observed to be largely devoid of vesicles containing the tracer upon 1 mA VNS treatment in kindled rats with CD (Figs. 5E-H). TJs between adjacent endothelial cells were ultrastructurally intact and no sign of passage of HRP reaction product through TJs was noted in all experimental groups.

[1] 89w The present study reveals the influence of VNS on seizure severity and BBB integrity during seizures induced by kindling with subconvulsive doses of PTZ in rats with CD. Our data demonstrate for the first time that VNS at the output current of 1 mA, but not at 0.5 mA exerted overall protective effects on BBB disrupted by PTZ-induced seizures in these animals. The occurrence of seizures in kindled animals with CD was also noted to be strikingly prevented by VNS at output currents of both 0.5 and 1 mA.

[2] 497w It has been demonstrated that VNS successfully prevents the emergence of seizures or reduces their frequency and severity in a variety of clinical and experimental settings (Ben-Menachem et al., 1995;Krahl et al., 2000;Ben-Menachem, 2002). In children with refractory epilepsy, a decrease in the duration and severity of seizures was observed by VNS therapy (Shahwan et al., 2009). In our experiments, long lasting clinical and electrophysiological seizures were evoked by PTZ-kindling in rats with CD, however, upon VNS treatment at an output current of 0.5 mA, none but one of these rats exhibited seizures and all showed series of interictal of spikes in EEG recordings. Moreover, no seizure activity was observed in the EEGs of kindled rats with CD with VNS at 1 mA. Our findings agree with those of earlier studies which demonstrated that VNS reduced seizure severity in amygdala-kindled animals (Fernandez-Guardiola et al., 1999;Dedeurwaerdere et al., 2006). Conversely, acute or prolonged application of VNS did not suppress absence seizures in genetic absence epilepsy rats from Strasbourg (GAERS) (Dedeurwaerdere et al., 2004). An explanation for this contradiction might be the differences in the strains of rats and/or in the VNS protocols used in the mentioned studies. In the meantime, the majority of patients (50-82%) who are candidates for surgery remain seizure-free after tuberectomy. In the recent years, technical advances in the localization of the epileptogenic zone have lead to a 63% of Engel class I status after surgery in comparison to a previous 52%. In medically refractory patients who were not suitable for surgery, VNS therapy has proved efficacy in reducing seizure frequency in more than 50-67% of cases (Elliott et al., 2009;Moavero et al., 2010). In our study, rats with CD were shown to be seizure-free after VNS both clinically and with electron microscopic findings at the cellular level. The beneficial effects of VNS in preventing from seizures have been mechanistically attributed to modulations in brain noradrenergic pathways. In this context, it was shown that VNS (at 1.0 or 2.0 mA) increased norepinephrine (NE) levels in both the cerebral cortex and hippocampus in rats which suggested that increased brain levels of NE contribute to the antiepileptic effects of VNS (Krahl et al., 1998(Krahl et al., , 2000;;Roosevelt et al., 2006). Bilateral damage to the locus coeruleus by neurotoxin 6-hydroxydopamine in an animal model inhibited the seizure-suppressing effects of VNS (Krahl et al., 1998(Krahl et al., , 2000)). VNS was shown to increase NE efflux in the basolateral amygdala, and electrical stimulation of the locus coeruleus has been reported to suppress epileptiform activity produced by stimulation of the amygdala (Jimenez-Rivera et al., 1987;Hassert et al., 2004). VNS has also been associated with increased cerebrospinal fluid levels of glycine and γ-aminobutyric acid and enhanced dopaminergic and serotoninergic activity, all of which may have suppressive effects on seizure activity (Hammond et al., 1992;Ben-Menachem et al., 1995). In spite of the above-mentioned literature data, the exact mechanism(s) of suppression of acute or chronic seizures by VNS is not fully understood.

[3] 143w In our study although VNS provided a protective effect on BBB integrity and suppressed the seizure activity in kindled animals with CD, no influence of the treatment was observed on the immunoreactivity of P-gp. The multidrug resistance gene 1 (MDR1) is known to play the most important role in pharmacoresistant epilepsies (Sisodiya et al., 1999;Lazarowski et al., 2004;Volk et al., 2004). The expression of MDR1 mRNA in brain specimens from patients with intractable focal epilepsy has been reported to be higher than those from normal brains, and this applies in particular to capillary endothelial cells and astrocytes in epileptogenic brain tissue (Sisodiya et al., 1999;Lazarowski et al., 2004;Volk et al., 2004;Aronica et al., 2012). In this regard, our findings provide substantial immunohistochemical evidence that the beneficial effects of VNS in chronic epilepsy may be related to mechanisms other than modulations in P-gp expression.

[4] 143w Disorganization of parenchymal blood vessels in the brain and focal BBB leakage was reported to be topographically associated with reactive gliogenesis in rats with CD (Fan et al., 2008). Moreover, the observation of abnormal vessel morphology, serum albumin leakage along with increased P-gp expression in the barrier type endothelial cells of the heterotopic hippocampus in methylazoxymethanol acetatetreated rats implied that changes in the BBB function may contribute critically to the determination of brain uptake and distribution of P-gp substrates in epileptic tissues of developmental malformations (Marchi et al., 2006). In our study, the abundance of pinocytotic "caveolar" vesicles containing HRP reaction products in the cytoplasm of endothelial cells in the capillaries of the cerebral cortex and hippocampus of rats with CD exposed to PTZ-induced seizures indicates increased transcytotic activity which was attenuated with 1 mA VNS therapy after the last dose PTZ injection.

[5] 171w VNS treatment can provide protective effects on BBB integrity through several mechanisms. Recent studies indicate that VNS prevents an increase in BBB permeability and intestinal dysfunction to FITC-dextran after experimental traumatic brain injury (Bansal et al., 2010;Lopez et al., 2012). A significant decrease in NE concentration in brain could make cerebral blood vessels more vulnerable to the decreased cerebral blood flow in ischemic brain edema (Kobayashi et al., 1990). The increase in extracellular NE levels in the brain by NE reuptake inhibitors can play a role in combating brain inflammation by reducing expression of chemokines (O'Sullivan et al., 2010). It is also reported that VNS application increased the serum levels of corticosterone (De Herdt et al., 2009). In addition, corticosterone induction has been shown to increase electrical resistance and to improve integrity of TJ proteins zonula occludens-1, occludin and claudin-5 (Calabria et al., 2006). Despite the aforementioned literature, the exact mechanisms of the protective effects of VNS on BBB integrity during kindling seizures in the setting of CD are still unclear.

[6] 278w We did not observe any open TJs during PTZ-induced seizures in kindled animals with CD regardless of the VNS application. It is well-known that decreased occludin expression is associated with decreased electrical resistance of TJs and increased BBB permeability through paracellular route. However, we did not observe any alterations in occludin immunoreactivity in the wall of the brain capillaries following the last dose of PTZ in kindled rats with CD treated or untreated with VNS. On the other hand, despite the lack of any alteration in occludin immunoreactivity, a question on whether a relocation of the protein, for instance, to the cytosolic compartment which may lead to enhanced BBB permeability can also be raised. However, in our electron microscopic evaluations, we observed that TJs between adjacent endothelial cells were ultrastructurally intact and no sign of paracellular passage of HRP reaction products was noted. Therefore, occludin redistribution which would allow for paracellular permeability does not seem to be functional in this setting. The mechanisms leading to enhanced barrier function in chronic epilepsy may also account for our data regarding the preservation of expression patterns of occludin along with intact ultrastructural appearance of TJs constituting BBB (Kasantikul et al., 1983). In various experimental settings, increased caveolar activity along with ultrastructurally unaffected TJs has been interpreted as an enhancement of transcellular pathway in the absence of alterations in immunoreactivity of TJ proteins (Armulik et al., 2010;Martins et al., 2012). Thus, our above-mentioned ultrastructural and immunohistochemical data may lead us to the conclusion that transcellular rather than paracellular pathway is responsible for the alterations in BBB that occur during both induction and VNS therapy of PTZ kindling seizures in rats with CD.

[1] 125w Our data on seizure activity assessed by EEG recording and Racine scoring showed that VNS treatment at both 0.5 and 1 mA markedly suppressed PTZ-induced seizures in kindled rats with CD, which implies the therapeutic potential of VNS application in experimentally-induced seizures in animals with CD. Our electron microscopic observations regarding the frequent caveolar vesicles containing HRP reaction products in the cytoplasm of brain capillary endothelial cells along with intact TJs advocated the predominance of transcytotic activity rather than paracellular pathway as the mechanism of BBB disruption in epileptic seizures in kindled rats with CD. The paucity of transport vesicles containing HRP reaction products upon VNS treatment at 1 mA suggests the overall protective action of the application on BBB integrity in this experimental setting.

[1] 293w The experimental procedures were conducted in time-pregnant Wistar albino rats (n = 15) and their litters of both sexes. The female rats were caged for 24 h with the male rat and mating was confirmed by the presence of a vaginal plug and spermatozoa in vaginal smear. The day on which insemination was detected was determined as the embryonic day (E) 0. In order to induce CD, the animals were treated according to a protocol that was previously described (Roper et al., 1995;Kaya et al., 2008;Gürses et al., 2009). On E17, the pregnant rats were exposed to gamma-irradiation. For this purpose, the animals were anesthetized with chloral hydrate (360 mg/kg, i.p), and were placed in groups of five, each in prone position on a wooden board. A polystyrene phantom was put under the board to achieve an acceptable backscatter. A nominal single dose of 145 cGy to mid-plane of the abdominal area was delivered by 6 × 27 cm posterior field using Co-60 tele-therapy unit (Alcyon II, General Electric, France). After irradiation, rats were taken back to the animal facility and cared for routinely until birth. After parturition, litters were maintained with their mothers for one month until weaning and then were allowed to live in cages with free access to food and water until the age of two months. The experimental protocols used in this study were approved by the Local Ethics Committee for Animal Experimentation of Istanbul University (2010/109). The litters were randomly divided into experimental groups consisting of 8 rats; CD plus pentylenetetrazole (PTZ) kindling, CD plus PTZ kindling plus 0.5 mA VNS and CD plus PTZ kindling plus 1 mA VNS, while in-utero unirradiated litters served as controls. Separate subsets of experimental groups were used for each experimental procedure.

[2] 140w Fifteen-day-old in-utero irradiated rats received a subconvulsive dose of 30 mg/kg PTZ (Sigma, St. Louis, USA) dissolved in saline, intraperitoneally three times a week for 45 days to induce chemical kindling according to a protocol that was previously modified and published by our group (Kaya et al., 2008;Gürses et al., 2009). After each PTZ injection, the convulsive behavior was observed for 30 min and classified into the following stages as described by Racine: 0-no behavioral changes; 1-facial movements, ear and whisker twitching; 2-myoclonic convulsions without rearing; 3-myoclonic convulsions with rearing; 4-clonic convulsion with loss of posture; and 5generalized clonic-tonic seizures (Racine, 1972). The animals receiving repeated injections of subconvulsive dose of PTZ were considered as "kindled" at the end of 45 days. Immunohistochemistry and electron microscopy procedures mentioned below were performed at 30 min after the last injection of PTZ.

[3] 194w Animals were anesthetized with chloral hydrate (360 mg/kg, i.p) and the scalp was incised along the midline and opened from back to front by following the center of middle line. All stereotaxic coordinates were established according to the atlas of Paxinos and Watson under a stereotaxic frame (Stereotact, World Precision Instruments, Inc., USA). Two stainless steel bipolar electrodes (inner and outer diameter: 200 and 260 μm; A-M Systems, Inc. #791900, Carlsborg WA, USA) were implanted stereotactically into the left and right hippocampi through 0.6 mm wide burr holes that were made by drilled and they were secured with dental acrylic (coordinates: posterior 2.0 mm and lateral to mid-line ± 2.0 mm from bregma, 3.2 mm below dura). Two stainless steel screws for supporting and 1 stainless steel screw for reference electrode (coordinates: anterior 3.0 mm, right lateral to mid-line 1.0 mm from bregma) were placed on the skull and dental acrylic was used to secure. A mini-USB connector was used to connect electrodes to an EEG recording system (Micromed S.p.A., Italy). EEG and video recording were continuously and synchronously monitored while the rats were let to move freely in their cages for 1 h.

[4] 295w Four days before the administration of the last dose of PTZ, a VNS electrode (Cyberonics Inc., Houston, Texas, USA, Lot # 545452QTYS) was implanted around the left vagus nerve and carotid artery of each rat. For this purpose, rats were anesthetized with chloral hydrate (360 mg/kg i.p) and an incision was made on the midline of the ventral neck. The skin and muscles were meticulously separated and bipolar electrodes (Cyberonics Inc., Houston, Texas, USA) were wrapped around the left carotid artery and vagus nerve which were isolated together under a surgical microscope. After suturing to the underlying muscle, the electrodes were tunneled subcutaneously toward an incision made in the back and were then connected to the stimulator (Pulse generator; 6.9 mm× 52.2 mm× 51.6 mm and weight 15 g) which was placed in a dorsal pocket made under the back skin and wiped with iodine and marbofloxacin. Then, rats were given fluid replacement to ease recovery and placed individually into small cages. A general antibiosis with marboflaxin (subcutaneous; 2 mg/kg/day) was performed to the animals for 5 days following surgery. One day after the operation, the impedance of the stimulating electrodes was tested in animals receiving electrical VNS while awake with a device that provided intermittent electrical stimulation (VNS Therapy Demipulse, Model 103 Generator, Cyberonics Inc., Houston, Texas, USA) using the diagnostic setting on a handheld-computer (NeuroCybernetic Prosthesis VNS Therapy Software Model 250, Cyberonics Inc., Houston, Texas, USA) and programming wand (NeuroCybernetic Prosthesis device; WAND Model 201, Cyberonics Inc., Webster, Texas, USA). After a two-day recovery, the stimulator was turned on for 48 h in kindled rats with CD and programmed with outputs at 0.5 mA or 1 mA intensity, 20 Hz frequency, 0.5 ms pulse width and duty cycle 14 s on/5 min off.

[5] 159w To demonstrate the immunostaining of tight junction (TJ) protein occludin and P-glycoprotein (P-gp) in the brain sections, rats were anesthetized with the overdose of chloral hydrate (700 mg/kg, ip) at the end of all experimental procedures. A bolus transcardial perfusion with 75 ml saline was administered at a pressure of 110 mm Hg for 15 s, followed by 200 ml fixative (4% paraformaldehyde in phosphate buffer; pH: 7.4) for 10 min. After the perfusion, brains were removed, immersed in the same fixative, kept for 24 h at 4 °C, and then embedded in paraffin. Three-μm thick sections were deparaffinized and incubated with protease (1 mg/ml; Sigma, USA) for 10 min for occludin or heated in tris-EDTA buffer (pH: 8.0) in a microwave oven for 15 min for P-gp to achieve antigen retrieval. Endogenous peroxidase activity was quenched using 0.3% hydrogen peroxide for 20 min. A nonspecific blocking reagent (Ultra-V-Block, Lab Vision, Westinghouse, CA) was used to prevent nonspecific binding.

[6] 222w Polyclonal rabbit anti-occludin (1/50, 2 h; Zymed, CA) and monoclonal mouse anti-P-gp (1/50, 4 °C overnight; C219; ABCAM, Cambridge, UK) antibodies were used as primary antibodies followed by biotinylated goat anti-polyvalent secondary antibody. After rinsing, streptavidin-peroxidase complex was applied and aminoethyl carbazole (AEC) chromogen was used. The sections were counterstained with Mayer's hematoxylin to enhance nuclear staining. For negative controls, adjacent sections were processed following the same steps with the exception of the primary antibodies. The relative intensity of immunoreactivity was evaluated as previously described (Greenway et al., 2004). Briefly, under 100× magnification, at least 20 images containing an average of 100 capillaries were obtained from hippocampus of each animal by means of a digital camera (Nikon, Coolpix 4500) attached to a light microscope by 2 observers blinded to experimental groups. The images were then saved in Photoshop (Version 12.0, Adobe Systems Incorporated) and AEC staining in the wall of the hippocampal capillaries was carefully selected. The images were then imported to the Image-ProPlus 6.0 software (Media Cybernetics Inc.) and the sum of the intensity values of the pixels of all selected objects in the images of individual rats was divided into the total number of the evaluated capillaries of the same animal. The relative intensity of immunoreactivity was determined for each group and the obtained data were processed for statistical analysis.

[7] 40w Data were presented as mean ± SEM. Group differences were determined by ANOVA followed by Tukey's test as required using a computer program (SPSS 11.0 ver.). In all cases, differences between the means were considered significant if P b 0.05.

[1] 256w A polyethylene catheter was inserted into the femoral vein of animals under chloral hydrate anesthesia (100 mg/kg). Six hours later, the rats were recovered from anesthesia and the animals in the experimental groups received an intravenous injection of horseradish peroxidase (HRP; type II, Sigma Chemical Co., St. Louis, MO; 200 mg/kg body weight in 0.2 ml saline) by femoral vein to ultrastructurally assess the integrity of BBB. HRP was allowed to circulate for 20 min and then animals were perfused transcardially with saline (75 ml) followed by 200 ml fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH: 7.4). Afterwards, animals were kept at 4 °C overnight and brains were removed. Coronal sections of 50-μm thickness were cut with a Vibratome and incubated in a solution of 0.05% 3.3′-diaminobenzidine in 0.05 M Tris-HCl buffer (pH: 7.6) containing 0.01% H 2 O 2 for 30 min to obtain HRP-reaction products. For ultrastructural observation, samples from the cerebral cortex and hippocampus regions of the Vibratome sections were post-fixed in 1% osmium tetroxide for 1 h, dehydrated in ethanol and embedded in Epon. Then, ultrathin sections (60 nm) were examined under a transmission electron microscope (JEOL, 1011, Japan) equipped with a CCD camera (MegaView III, Soft Imaging System, GmBH, Germany). During the electron microscopic procedures, en block staining of the samples with uranyl acetate and staining of the ultrathin sections on grids with uranyl acetate and lead citrate were omitted in an attempt to avoid misinterpretation of the possible artifactual precipitates as HRP reaction products.