Effects of genistein on cognitive dysfunction and hippocampal synaptic plasticity impairment in an ovariectomized rat kainic acid model of seizure

European Journal of Pharmacology 786 (2016) 1–9

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Behavioural pharmacology

Effects of genistein on cognitive dysfunction and hippocampal synaptic plasticity impairment in an ovariectomized rat kainic acid model of seizure Mehdi Khodamoradi a, Majid Asadi-Shekaari a, Saeed Esmaeili-Mahani b, Khadije Esmaeilpour a, Vahid Sheibani a,n a b

Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran Department of Biology, Faculty of Science, Shahid Bahonar University, Kerman, Iran

art ic l e i nf o

a b s t r a c t

Article history: Received 8 December 2015 Received in revised form 21 April 2016 Accepted 23 May 2016 Available online 24 May 2016

The major objective of this study was to investigate the probable effects of genistein (one of the most important soy phytoestrogens-SPEs) on seizure-induced cognitive dysfunction, hippocampal early longterm potentiation (E-LTP) impairment and morphological damage to CA1 neurons in ovariectomized (OVX) rats. Three weeks after ovariectomy, cannulae were implanted over the left lateral ventricle. After a 7-day recovery period, animals were injected by genistein (0.5 or 5 mg/kg) or vehicle during four consecutive days, each 24 h. One h after the last treatment, kainic acid (KA) or vehicle was perfused into the left lateral ventricle to induce generalized tonic-clonic seizures. Finally, 7 days later, spatial learning and memory of animals were examined using the Morris water maze (MWM) task, hippocampal E-LTP was assessed using in-vivo field potential recordings and the morphology of hippocampal CA1 area was examined using Fluoro-Jade C staining. KA-induced generalized seizures resulted in spatial learning and memory impairment, E-LTP deficit and CA1 cell injury. Seizure-induced abnormalities improved partially only by the lower dose of genistein (0.5 mg/kg). However, genistein at the higher dose (5 mg/kg) did not have any beneficial effects. Also, genistein did not affect seizure activity. It is concluded that genistein may have partially preventive effects against seizure-induced cognitive impairment in OVX rats. Also, it seems that such effects of genistein are correlated with its beneficial effects on hippocampal synaptic plasticity and morphology. & 2016 Elsevier B.V. All rights reserved.

Keywords: Genistein Seizure Spatial memory Long-term potentiation Neurodegeneration

1. Introduction Seizure can induce consciousness impairment, cognitive dysfunction, synaptic reorganization and hippocampal neurodegeneration (Arthuis et al., 2009; Artinian et al., 2015; Ben-Ari, 2001; Chan et al., 2004; Nadler et al., 1978). Hippocampal synaptic plasticity is involved in memory formation (Neves et al., 2008) and seizure-induced hippocampal damage, especially in the CA1 area, impairs memory (Kotloski et al., 2002; Ramírez-Munguía et al., 2003). Intracerebroventricular (i.c.v.) administration of kainic acid (KA, an analog of glutamate) is a standard experimental model which results in severe limbic seizures and hippocampal neuronal damage (Nadler et al., 1978). There are various antiepileptic drugs to control seizure; n

Corresponding author. E-mail addresses: [email protected], [email protected] (V. Sheibani).

http://dx.doi.org/10.1016/j.ejphar.2016.05.028 0014-2999/& 2016 Elsevier B.V. All rights reserved.

however, some types of seizures are drug-resistant and some antiepileptic drugs have adverse effects (Perucca and Gilliam, 2012; Regesta and Tanganelli, 1999). It seems that further investigations are needed on alternative therapies, like herbal compounds. Soy phytoestrogens (SPEs), like genistein, are herbal compounds that bind to estrogen receptors and therefore mimic the actions of estrogen (Molteni et al., 1995). It is stated that SPEs can be used as an alternative therapy in postmenopausal women (Molteni et al., 1995; Soni et al., 2014) instead of estrogen replacement therapy which has more adverse effects (Beral, 2003; Grady et al., 1995). It is worth pointing out that ovariectomized (OX) rat is an experimental model of postmenopausal women which can be used to study the effects of the herbal medicine or other experimental designs in the absence of ovarian hormones (Gallo et al., 2005; Kalu, 1991). Various studies have investigated the effects of estrogen on seizure (Nicoletti et al., 1985; Velísková et al., 2000; Woolley, 2000). It has been reported that estrogen has neuroprotective

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Fig. 1. The Overview and timeline of the experimental protocol used in this study. Three weeks after ovariectomy, cannulae were implanted over the left lateral ventricle. After a 7-day post-operative recovery period, animals were injected by genistein (0.5 or 5 mg/kg) or vehicle (DMSO) during four consecutive days, each 24 h. One h after the last treatment, KA or vehicle (PBS) was injected into the left lateral ventricle. Finally, the Morris water maze test, hippocampal field excitatory post-synaptic potential recordings or morphological experiments were conducted 7 days later. Gen: genistein, KA: kainic acid, MWM: Morris water maze, fEPSPEs: field excitatory post-synaptic potentials, w: week, d: day, h: hour.

effects against seizure (Velísková et al., 2000); however, many studies have shown the proconvulsant effects of estrogen (Nicoletti et al., 1985; Woolley, 2000). But, a few studies have focused on the effects of genistein on seizure. This study therefore set out to assess the effect of genistein on cognitive impairment, hippocampal synaptic plasticity deficit and CA1 neurodegeneration following KA-induced seizure in OVX rats.

2. Materials and methods 2.1. Animals Female Wistar rats (200–250 g in weight) were used in this study. Animals were maintained under controlled conditions including temperature of 23 71 °C, 12-h light-dark cycle (lights on: 07:00–19:00 h) and free access to food and water. The present study was approved by the Regional Ethics Committee of Kerman Neuroscience Research Center, Kerman, Iran (Ethics Code: EC/ KNRC/91-36) and the experimental procedures had been conducted in accordance with the Guide for the Care and Use of Laboratory Animals. 2.2. Ovariectomy Ovariectomy was done according to a previous study (Saadati et al., 2015). Rats were anesthetized with ketamine (60 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and the ovaries were removed under a laparotomy surgery. 2.3. Cannula implantation After a 3-week recovery period, ovariectomized rats were again anesthetized with ketamine and xylazine and placed in a stereotaxic frame. The scalp was cut, a hole was drilled in the skull and a unilateral stainless steel (21-gauge) guide cannula was inserted into the brain so that its tip was placed 1 mm over the left lateral ventricle (AP ¼0.9 mm; ML¼1.3 mm; DV ¼ 3.5 mm) (Paxinos and Watson, 1998). Skull screws and dental cement were used for securing of the guide cannulae so that the skull over the right hippocampus remained uncovered. 2.4. Treatment and seizure induction After a 7-day post-operative recovery period, animals were randomized into five main groups. Before KA administration, animals were intraperitoneally (i.p.) injected for four consecutive days, each 24 h, by genistein (G-6649; Sigma, St. Louis MO, USA) at two doses; 0.5 mg/kg for lower dose genistein (LDG-KA) groups and 5 mg/kg for higher dose genistein (HDG-KA) groups (Qian et al., 2012; Rodríguez-Landa et al., 2009). The DMSO-KA groups received i.p. injection of dimethyl sulfoxide (DMSO) as vehicle of

genistein. The animals of the KA and PBS groups just received i.c.v. injection of KA or PBS, respectively. Each of the five main groups was divided into 3 subgroups for behavioral (n¼ 7–8), electrophysiological (n ¼7–8) or histological (n¼ 4–5) studies. One h after the last injection of genistein or DMSO, kainic acid (KA) (K-0250; Sigma, St. Louis Mom USA) (0.5 mg/1 ml) (Li et al., 2010; Nadler et al., 1978) was dissolved in 0.1 M phosphate buffered saline (PBS) and perfused slowly into the left lateral ventricle during 1 min Injection was conducted through pre-implanted guide cannulae and using a microinjection cannula which was connected to a 10-ml Hamilton syringe. Microinjection cannula protruded a further 1 mm longer than to the tip of the guide cannula to perfuse KA into the ventricle. Seizure stages were scored as follows: 1- mouth and facial movements, 2- head nodding, 3- forelimb clonus, 4- rearing, 5rearing and falling (Racine, 1972). The seizure activity of animals were recorded for 3–4 h and only those rats which showed the fifth stage of the scale were selected for behavioral, electrophysiological or histological studies which were conducted 7 days later (Fig. 1). It has been shown that neuronal damage happens at this time following i.c.v. administration of KA (Li et al., 2010). It should be noted that we also conducted a separate study for the same groups (n¼ 7 in each group) to examine the effects of KA and genistein administration on seizure activity (seizure stages and latency to onset of seizures). 2.5. Morris water maze (MWM) 2.5.1. Spatial learning The MWM included a metallic circular tank, 160 cm in diameter, 80 cm height, and filled with water to a depth of 40 cm. The tank was divided into 4 quadrants, and a platform (10 cm in diameter) was placed in the center of the training quadrant at  1.5 cm beneath the surface of water. Various posters were placed on the wall around the tank as spatial navigation cues and a smart video tracing system (Noldus Ethovisions system, version 5, USA) was used to record the performance of animals. Learning acquisition consisted of three blocks which were separated by 30-min resting periods and each block included four consecutive trials with 60 s duration and 60 s inter-trial intervals. In each trial, rats were randomly released into water from the center of one of the quadrants of the maze and allowed to swim for 60 s If the animals found the platform during 60 s, the trial would finish; otherwise, rats were helped by the experimenter to find the platform. Rats were allowed to rest on the platform for 30 s and then to rest at their cages for 20–30 s The time and path length to find the hidden platform were recorded to measure spatial learning (Hajali et al., 2012; Saadati et al., 2015). 2.5.2. Spatial short-term memory retention To examine spatial short-term memory, a single probe trial was conducted 2 h after the last training trial. During the probe test,

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the time spent and traveled distance in the target quadrant were recorded to measure spatial short-term memory retention. Following the probe trial, rats completed a visible platform test to determine whether seizures have interference with sensory and motor coordination/motivation or not. A visible platform (which covered with aluminum foil) was placed at  2 cm over the surface of water and the scape latency to find the visible platform was evaluated (Hajali et al., 2012; Saadati et al., 2015). 2.6. Electrophysiology Field excitatory post-synaptic potentials (fEPSPEs) recordings were conducted according to previous studies (Hajali et al., 2015; Saadati et al., 2014). Animals were anesthetized with urethane (1.2 g/kg) and placed in a stereotaxic apparatus. To insert stimulating and recording electrodes, two holes were drilled in the skull over the right hippocampus in accordance with the Paxinos and Watson atlas (Paxinos and Watson, 1998). During the experiment, rectal temperature was maintained at 36.57 0.5 °C (Harvard Apparatus). A stainless steel concentric bipolar stimulating electrode (0.125 mm diameter, Advent, UK) was inserted in the Schaffer collateral pathway (AP ¼3 mm; ML ¼3.5 mm; DV ¼ 2.8–3 mm), and the other similar (recording) electrode was placed in the stratum radiatum of the ipsilateral CA1 area (AP¼ 4.1; ML¼ 3 mm; DV ¼2.5 mm). The stimulating and recording electrodes were connected to a stimulator and an amplifier, respectively. By stimulating the Schaffer collateral pathway and recording of responses from stratum radiatum of CA1 area, the maximum of fEPSP slope was obtained. A 30-min stabilization period was considered and, then, the input-output (I-O) curve was plotted by gradually increasing the stimulus intensity (input) and recording the fEPSPEs (output). A differential amplifier was used to amplify and filter field potential recordings (1 Hz to 3 KHz band pass filters). The intensity of stimulation for baseline recording was calibrated to provide a response magnitude of 50% of maximum slope by giving a test stimulus every 10 s for 20 min Also, paired-pulse facilitation (PPF) ratios were examined in those rats which were then considered for LTP induction. The evaluation of PPFs was conducted by applying ten sequential paired-pulses at 20, 50, 70 and 100 ms interstimulus intervals to the Schaffer collateral pathway at a frequency of 0.1 Hz (10 s interval). The second fEPSP slopes were divided by the first fEPSP slopes (fEPSP2/fEPSP1) to evaluate fEPSP slope ratios at all interstimulus intervals. The induction of early long-term potentiation (E-LTP) was then examined by delivering a train of high frequency stimulation (HFS: 10 pulses at 400 Hz/7 s repeated for 70 s). Afterwards, by applying a test stimulus every 10 s, the maintenance of LTP was evaluated for 2 h. As shown in Fig. 6, each time point represents the average slope of fEPSPEs values from every ten sequential traces (Hajali et al., 2015; Saadati et al., 2014). Neurotrace software (version 9) and Electromodule 12 were used for stimulation and recording and Potentalize software was used to analyze E-LTP. All of them were obtained from the Science Beam Institute, Tehran, Iran. 2.7. Histology Animals were killed and transcardially perfused with 100 ML normal saline followed by 100 ML fixative solution (glutaraldehyde 1.25% and paraformaldehyde 4% in 0.1 M PBS at pH ¼7.4) for 1 h. Then, the brain was removed and immediately washed in normal saline, and fixed in paraformaldehyde 4% for 48 h at room temperature. After that, the brain tissues were dehydrated with ascending ethanol series, cleared with xylene and embedded in paraffin. Then, 10 mm coronal sections were prepared and mounted on gelatin coated slides for Fluoro-Jade C staining. The Fluoro-Jade

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C staining protocol was the same as the protocol of Schmued and co-workers (Schmued et al., 2005). Briefly, the gelatin coated slides were immersed in descending ethanol series and then transferred to 0.06% potassium permanganate solution, 0.0001% solution of Fluoro-Jade C (Histo-Chem Inc.; Jefferson, AR), and 0.0001% solution of DAPI (DAPI; Sigma, St. Louis MO, USA), respectively. Slides were then rinsed in distilled water, dried in oven at 50 °C, cleared with xylene and coverslipped with DPX mountant (DPX; Sigma, St. Louis MO, USA) (Schmued et al., 2005). The number of pyramidal neurons of the hippocampal CA1 area was calculated in three fields (medial, middle and lateral parts) (Fig. 8E) at  400 magnifications. An average number of cells from these three fields were considered as cell counts from each animal (Asadi-Shekaari et al., 2010; Schmued et al., 2005). The double labeled sections were examined using a fluorescent microscope. Double exposure using ultraviolet and blue light excitation revealed blue DAPI labeled and green Fluoro-Jade C positive neurons (Schmued et al., 2005). 2.8. Data analysis Seizure stages and latency to onset of seizures were analyzed based on one-way analysis of ANOVA and Tukey's post hoc tests. The two-way analysis of variance (ANOVA) was used to analyze escape latency and path length to find the hidden platform and repeated measurement was used to analyze differences between the groups (groups and blocks as the factors). The swim speed and memory performance (the time spent and traveled distance in the target quadrant) were analyzed using the one-way analysis of variance followed by Tukey's post hoc multiple comparison test. Overall differences in LTP time points between the groups (group and time as the factors) were analyzed based on repeated measurement and single time points between the groups were analyzed based on one-way analysis of ANOVA. The significant differences in time points were analyzed using Tukey's post hoc multiple comparison test. Also, the number of cells was analyzed using one-way analysis of ANOVA and Tukey's post hoc tests. Data are presented as mean 7 standard error of the mean (S.E.M.) and P values less than 0.05 was considered statistically significant.

3. Results 3.1. Seizure activity Seizure activity was not different between the groups. The animals of the PBS group did not show seizure activity, but in other groups KA induced non-convulsive (stages 1 and 2) and convulsive (stages 3–5) seizures. Analysis indicated no significant difference between the groups (except the PBS group) and genistein and DMSO administration did not have significant effects on seizure activity. It is worth pointing out that genistein in the HDGKA group partially increased seizure stages and decreased latency to onset of seizures and in the LDG-KA group partially decreased seizure stages, but they were not significantly different compared with the KA group (P 40.05). Additionally, spontaneous seizure activity was not seen during the next 7 days (Table 1). 3.2. Morris water maze 3.2.1. Spatial learning Learning acquisition was confirmed by reduction in both the swimming path length and escape latency to find the hidden platform during training blocks (Fig. 2). The repeated measures ANOVA indicated that the path length (Fig. 2A) and escape latency (Fig. 2B) of animals of the KA group significantly increased in block

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Table 1. Seizure stages and latency to onset of seizures. Groups

Seizure stage

Seizure latency (s)

PBS KA DMSO-KA HDG-KA LDG-KA

No seizure activity 4.42 7 0.29 4.4 7 0.4 4.85 7 0.14 3.85 7 0.34

No seizure activity 103.28 7 6.01 95.8 7 6.07 86.717 6.23 105.577 8.24

The Comparison of seizure stages and latency to onset of seizures between the groups. The PBS group did not show any seizure activity. One way analysis of variance (ANOVA) showed no significant difference between other groups. Data are presented as mean7 S.E.M.

Fig. 2. The effects of genistein on path length (A) and escape latency (B) to find the hidden platform in the MWM test following KA administration. Animals of the KA group traveled significantly more distance and time to find the hidden platform at all three blocks than those of the PBS group, which indicates spatial learning impairment following i.c.v. injection of KA. Genistein only at the lower dose (in the LDG-KA group) significantly decreased the path length and escape latency to find the hidden platform in comparison with the KA group. The effects of the higher dose genistein (in the HDG-KA group) and DMSO (in the DMSO-KA group) were not significantly different compared with the KA group. (*) P o 0.05, (**) P o 0.01, (***) P o 0.001 in comparison with the PBS group. (#) P o 0.05, (##) P o 0.01 in comparison with the KA group.

1 (P o0.001), block 2 (P o0.01 for distance and P o0.001 for escape latency), and block 3 (P o0.001) in comparison with the PBS group, which indicated spatial learning impairment following KAinduced generalized seizures. Genistein, only at the lower dose (in the LDG-KA group), decreased the path length (Fig. 2A) and escape latency (Fig. 2B), to find the hidden platform, compared with that of the KA group. The mean path length which traveled by the LDGKA group in block 1 (P o0.05) and block 3 (Po 0.01) decreased in comparison with the KA group (Fig. 2A). Also the mean escape latency of the LDG-KA group in block 3 (P o 0.01) decreased compared with the KA group (Fig. 2B). Interestingly, a higher dose of genistein (5 mg/kg in the HDG-KA group) increased both the path length (Fig. 2A) and escape latency (Fig. 2B) to find the hidden platform than those of the KA group; however, its effect was not significant. Also, DMSO (in the DMSO-KA group) did not have any effects compared with the KA group (Fig. 2A and B).

Fig. 3. The effects of genistein on spatial memory in the MWM test following KA administration. Animals of the KA group spent significantly less time, distance (A), and the number of crossings (B) in the target quadrant than those of the PBS group, which indicated spatial memory impairment following i.c.v. injection of KA. Genistein only at the lower dose (in the LDG-KA group) significantly increased the traveled distance and time spent and the number of crossings in the target quadrant than that of the KA group. The effects of the higher dose of genistein (in the HDG-KA group) and DMSO (in the DMSO-KA group) were not significant compared with the KA group. (*) P o 0.05, (**) P o0.01, (***) P o 0.001 in comparison with the PBS group. (#) P o 0.05 in comparison with the KA group.

3.2.2. Spatial short-term memory A probe test was conducted 2 h after the last training trial and the mean percentage (%) of the traveled distance and time and the number of crossings in the target quadrant were analyzed to assess spatial short-term memory retention. Tukey’s test following one-way analysis of ANOVA indicated that animals of the KA group spent significantly less time (P o0.001), distance (Po0.01) (Fig. 3A), and the number of crossings (Fig. 3B) in the target quadrant (P o0.05) compared with the PBS group, which indicated spatial short-term memory impairment. Genistein, similar to the results of spatial learning, only at the lower dose (0.5 mg/kg in the LDG-KA group) ameliorated spatial short-term memory deficit. Animals of the LDG-KA group spent significantly more time (Po0.05), distance (P o0.05) (Fig. 3A) and the number of crossings (Fig. 3B) in the target quadrant (Po0.05) than those of the KA group. Also, the animals of the higher dose of genistein group (5 mg/kg in the HDG-KA group) spent less time and distance in the target quadrant than those of the KA group, but they were not significant (Fig. 3A and B). Genistein vehicle in the DMSO-KA group did not have significant effects on KA-induced memory impairment (Fig. 3A and B). 3.2.3. Latency to find the visible platform and swimming speed There was no difference in escape latency to find the visible platform and swimming speed between the groups (P 40.05) (Table 2). KA-induced seizures and genistein administration did

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Table 2. Swimming speed and latency to find the visible platform. Groups

Swimming speed (cm/s)

Escape latency (s)

PBS KA DMSO-KA HDG-KA LDG-KA

20.03 7 2.31 20.6 7 1.81 19.647 2.51 21.157 2.83 18.86 7 3.19

19.85 72.76 25 75.46 24.66 76.38 19 74.09 21 74.3

The Comparison of swimming speed and latency to find the visible platform in MWM. One way analysis of variance (ANOVA) indicated no significant difference between the groups. Data are expressed as mean 7S.E.M.

not have any effects on the visible test and swimming speed. Therefore, visual and motor functions were not significantly different between the groups. It is worth pointing out that since DMSO administration did not have any effects in the behavioral study and also in order to minimize killing of animals, the electrophysiological and histological studies were not conducted for the DMSO-KA group. 3.3. Electrophysiology 3.3.1. CA1 basal synaptic transmission and PPF relationship Input-output (I-O) curve, which was the changes in the slope of the fEPSPEs in response to increasing stimulus intensities, was plotted to examine whether administration of KA and genistein can affect basal synaptic transmission or not. The alterations of I-O relationship between the groups were not statistically significant (P 40.05) (Fig. 4). Furthermore, we assessed the effects of KA and genistein on neurotransmitter release from synapses of the hippocampal CA1 area at 20, 50, 70 and 100 ms interstimulus intervals. There was no significant difference in PPF relationship between the groups (P40.05) (Fig. 5). 3.3.2. Early long-term potentiation (E-LTP) The induction of E-LTP was determined by increasing fEPSPEs slope and recordings were continued for 2 h as LTP maintenance. The repeated measures ANOVA indicated an overall significant decrease in LTP induction and maintenance in the KA group in

Fig. 5. The effects of seizures and/or genistein administration on neurotransmitter release from synapses of the hippocampal CA1 area at various interstimulus intervals (20, 50, 70 and 100 ms). There was no significant difference in PPF relationship between the groups (P 40.05).

comparison with the PBS group (P o0.001) (Fig. 6). This overall reduction improved by genistein only at the lower dose (0.5 mg/kg in the LDG-KA group) compared with the KA group (P o0.05). The HDG-KA group did not have significant difference compared with the KA group (P 40.05) (Fig. 6). The increase of the mean fEPSPEs slope immediately after applying HFS, as E-LTP induction, significantly reduced (P o0.001) in the KA group (146.21 75.73% of baseline) in comparison with the PBS group (208.22 79.69% of baseline) (Fig. 6). This mean fEPSPEs slope significantly increased in the LDG-KA group (182.64 75.42% of baseline) (P o0.05); however, was not significantly different in the HDG-KA group (142.6775.16 of baseline) compared with the KA group (P 40.05) (Fig. 6). Additionally, the PBS (174.767 13.31% of baseline) (P o0.001) and LDG-KA (142.037 5.28% of baseline) (Po0.05) groups showed a sustained increase of the mean fEPSPEs slope 2 h after applying HFS than those of the KA group (110.81 74.07% of baseline). But, this slope decreased approximately near the baseline in the HDGKA group (109.7776.22% of baseline) and was not significantly different in comparison with the KA group (P 40.05) (Fig. 6). 3.4. Histology As expected, the results of histology study were consistent with behavioral and electrophysiological experiments. The percentage of degenerated neurons were significantly decreased by genistein in the LDG-KA group (14% were degenerated) in comparison with the KA group (37% were degenerated) (Po 0.01). But, the higher dose of genistein in the HDG-KA group (35% were degenerated) did not have any significant effects compared with the KA group (P4 0.05) (Figs. 7 and 8).

4. Discussion

Fig. 4. Input-output curve which was the changes in the slope of the fEPSPEs in response to increasing stimulus intensities was plotted to examine whether the induction of seizures and/or genistein administration can affect basal synaptic transmission or not. The alterations of the relation of I-O between the groups were not statistically significant (P 40.05). Note that, arbitrary units are stimulus intensities so that 1 is the intensity which evoked minimum of responses and 9 is the intensity which evoked maximum of responses.

In the current study we examined the probable effects of genistein on cognitive impairment, hippocampal early long-term potentiation (E-LTP) impairment and morphological damage to CA1 neurons induced by seizure in ovariectomized rats. Genistein only at the lower dose partially prevented KA-induced such abnormalities, but at the higher dose did have any beneficial effects. Furthermore, genistein did not have any significant effects on seizure activity. Our results are compatible with previous studies where seizure induces cognitive dysfunction and neurodegeneration and soy phytoestrogens (SPEs) have beneficial neurobehavioral and neuroprotective effects (Alonso et al., 2010; Arthuis et al., 2009; Ben-

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Fig. 6. The effects of genistein on LTP impairment induced by KA administration. Seizure induction in the KA group resulted in significant reduction of LTP induction and maintenance compared with the PBS group (Po 0.001). Genistein at the lower dose (in the LDG-KA) significantly increased LTP induction and maintenance (Po 0.05), but at the higher dose (in the HDG-KA group) did not have any beneficial effect in comparison with the KA group. Each time point (the mean 7 S.E.M.) in the graph is the average of normalized fEPSPEs slope from every ten sequential traces. Calibrations (500 mv/5 ms) were used throughout the recordings. (*) P o0.05, (***) P o 0.001 in comparison with the KA group (at both 5 and 120 min after applying HFS).

Fig. 7. Effects of genistein on KA-induced neurodegeneration in the pyramidal neurons of hippocampal CA1 area. Seizures induced intense neurodegeneration in the KA group compared with the PBS group (P o 0.001). The percentage of degenerated neurons was significantly decreased by genistein at the lower dose (0.5 mg/kg in the LDG-KA) in comparison with the KA group (Po 0.01). But, the higher dose of genistein (5 mg/kg in the HDG-KA group) did not have any significant effects compared with the KA group (P4 0.05). (**) P o 0.01, (***) P o0.001 in comparison with the PBS group. (###) P o 0.001 in comparison with the KA group.

Ari, 2001; Chan et al., 2004; Molteni et al., 1995; Pan et al., 2000; Soni et al., 2014). Previous studies have shown the negative effects of seizure on hippocampal neurons, synaptic connections (Ben-Ari, 2001; Kotloski et al., 2002; Nadler et al., 1978), memory (Chan et al., 2004; Chauvière et al., 2009) and LTP in the hippocampus (Palizvan et al., 2001; Zhang et al., 2010; Sgobio et al., 2010). Seizure destroys actin filaments in dendritic spines and induces memory problems (Wong and Guo, 2013; Zeng et al., 2007). Seizure also promotes neuroinflammation which induces the activation of microglia and astrocytes (Choi et al., 2009; Shapiro et al., 2008). It is well-known that neuroinflammation results in cognitive dysfunction (Lee et al., 2008) and LTP deficit in the hippocampus (Min et al., 2009). Furthermore, seizure induces oxidative stress (Patel and Li, 2003). Expression of free radicals and modulation of the antioxidative defense systems can result in LTP deficit (Pellmar et al., 1991) and learning and memory impairment (Fukui et al., 2001). LTP in the hippocampus, as an experimental model of memory and synaptic plasticity, has been shown to be

impaired by seizure (Palizvan et al., 2001; Sgobio et al., 2010; Zhang et al., 2010). It has also been reported that pentylenetetrazole kindling in male rats impairs LTP of fEPSP slope in the hippocampal CA1 area at both 48–144 h and 30–33 days after kindling (Palizvan et al., 2001). Similar to our results, another study showed that seizure impairs basal hippocampal synaptic transmission and neurotransmitter release from presynaptic CA1 terminals of Bassoon mutant epileptic mice (Sgobio et al., 2010). It should be noted that there is a correlation between hippocampal LTP impairment and hippocampal-dependent memory dysfunction (Sgobio et al., 2010). These studies indicate that cognitive dysfunction, LTP deficit and neuronal damage by seizure in our study are consistent with previous studies and can be explained by such mechanisms. On the other hand, genistein and other SPEs similar to 17βestradiol have neuroprotective effects (Azcoitia et al., 2006; Qian et al., 2012) and promote neuronal viability and proliferation (Pan et al., 2012; Perez-Martin et al., 2005). Genistein enhances the expression of neurotrophins such as brain-derived neurotrophic factor (Pan et al., 2012, 1999) which is an important factor for hippocampal LTP (Ying et al., 2002) and memory formation (Alonso et al., 2002). It is well-known that genistein is an antioxidant agent which has neuroprotective effects and improves memory via reduction of both oxidative stress and neuroinflammation (Menze et al., 2015; Verdrengh et al., 2003). It has also been shown that genistein similar to estradiol improves spatial memory in aged OVX female rats (Alonso et al., 2010). In the present study, genistein partially prevented seizure-induced cognitive impairment, LTP deficit and neuronal damage. It is possible that the above-mentioned mechanisms are involved in the effects of genistein against seizure in our work. Interestingly, we showed the beneficial effects of genistein on LTP; however, genistein is known as a LTP inhibitor (O’Dell et al., 1991). Genistein is a protein tyrosine kinase (PTK) inhibitor and PTK is one of the key factors for LTP induction (O’Dell et al., 1991). It is worth noting that we performed the extracellular recordings 7 days, but not immediately, after the last genistein administration. Therefore, it can be suggested that genistein may have beneficial neuroplasticity effects indirectly through modulating the destructive effects of seizure on hippocampal LTP, but not directly on LTP itself. It is important to note that genistein may have dose dependent

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Fig. 8. Photomicrographs of the merged double-labeled (DAPI and Fluoro-Jade C) neurons of the hippocampal CA1 area. The CA1 of the PBS group (A) were intact; however, CA1 of the animals of the KA group (B) had neuronal damage. Animals of the LDG-KA group (C) had less cell injury, but animals of the HDG-KA group (D) also showed similar neuronal damage compared with the KA group. Red arrows point to normal DAPI positive pyramidal neurons and white arrows point to degenerated Fluoro-Jade C positive neurons. Scale bars: 50 mm. (E) Illustration of the three separate fields (medial, middle and lateral parts) in a Toluidine-blue stained micrograph. An average number of cells from these three fields were considered as cell counts from each animal. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

effects (Alonso et al., 2010; Azcoitia et al., 2006). For example, it has been shown that an acute administration of genistein at high dose (10 mg/kg), but not at low doses (0.1 and 1 mg/kg), prevents hilar neuronal loss in the dentate gyrus of OVX rats against systemic administration of KA (Azcoitia et al., 2006). In the present study genistein only at lower dose partially prevented seizureinduced abnormalities. However, at higher dose partially exacerbated seizure activity and cognitive impairment induced by seizure. It is possible that genistein affects seizure activity and seizure-induced abnormalities differently depending on various factors similar to its endogenous counterpart 17beta-estradiol.

Estradiol benzoate is a neuroprotective agent against seizure-induced neuronal damage (Velísková et al., 2000); however, it has mainly proconvulsant effects (Nicoletti et al., 1985; Woolley, 2000). It is believed that the effect of estrogens on seizure depends on various factors including estrogen dose, hormonal status, sex and seizure model (Velísková, 2006). Thus, the effect of genistein on seizure may also depend on similar factors. 5. Conclusion Taken together, these results indicate that seizure induces cognitive dysfunction, hippocampal LTP impairment and neuronal

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damage and genistein may have partially preventive effects against such destructive effects of seizure. Although the study demonstrates that genistein may improve cognitive impairment via modulating the negative effects of seizure on hippocampal synaptic plasticity and morphology. Using confocal microscopy to improve pictures of DAPI labeled and Fluoro-Jade positive neurons and conducting experiments to assess neuroglia activation and neuroinflammation were considered as limitations of our work. These techniques will be considered for our future studies.

Conflict of interest The authors have no conflict of interest.

Acknowledgements This original research is a part of PhD thesis of the first author. We would like to acknowledge the Kerman Neuroscience Research Center, Kerman, Iran for financial support (KNRC/91-36).

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