GPER1 Modulates Synaptic Plasticity During the Development of Temporal Lobe Epilepsy in Rats

Xian Zhang1 · Yang Yang1,2 · Li Guo2 · Jinyu Zhou3 · Jianguo Niu1,2 · Peng Wang1 · Yuanyuan Qiang1 · Kunmei Liu1 · Yujun Wen1,2 · Lianxiang Zhang1,2 · Feng Wang1,4

Abstract

G-protein coupled estrogen receptor 1 (GPER1) is a novel type of estrogen receptor. Several studies have shown that it has an anti-inflammatory action,which plays an important role in remyelination and cognitive ability adjustment. However, whether it is involved in the development of temporal lobe epilepsy (TLE) is still unknown. The present study established a TLE model by intraperitoneal injection of lithium chloride (3 mmol/kg) and pilocarpine (50 mg/kg) in rats to study the effect of GPER1 in the synaptic plasticity during the development of temporal lobe epilepsy. A microinjection cannula was implanted into the lateral ventricle region of rats via a stereotaxic instrument. G-1 is the specific GPER1 agonist and G15 is the specific GPER1 antagonist. The G1 or G15 and Dimethyl sulfoxide were injected into the rat brains in the intervention groups and control group, respectively. After G1 intervention, the learning and memory abilities and hippocampal neuron damage in epileptic rats were significantly improved, while G15 weakened the neuroprotective effect of GPER1. Meanwhile, G1 controlled the abnormal formation of hippocampal mossy fiber sprouting caused by seizures, and participated in the regulation of synaptic plasticity by reducing the expression of Synapsin I and increasing the expression of gephyrin. Inhibitory synapse gephyrin may play a significant role in synaptic plasticity.

Keywords Temporal lobe epilepsy · GPER1 · G1 · G15 · Synaptic plasticity

Introduction

Epilepsy is a common central nervous system (CNS) disorder and is often accompanied by a self-limiting abnormal discharge [1]. It is associated with neurobiological, cognitive, psychological, and sociological consequences [2]. The cognitive function of patients with epilepsy is lower than that of normal individuals, including impaired memory, reduced attention, and mental decline [3]. Currently, antiepileptic drugs used in clinical can control seizures and prevent and reduce the risk of recurrence. However, they cannot change the underlying pathological process and long-term prognosis of epilepsy [4]. Epilepsy persists and often develops into chronic refractory epilepsy, causing significant damage to patients’ physical and psychological health [3]. Therefore, there is an urgent need to find an endogenous material intervention pathway to manage epilepsy.
G-protein coupled estrogen receptor 1 (GPER1), discovered in the 1990 s, is a novel estrogen receptor. In the early days, it was also called G protein-coupled receptor 30 (GPR30). The structure of GPER1 comprises a seven-transmembrane polypeptide chain. Like other G-protein coupled receptors (GPCRs), GPER1 is expressed on the endoplasmic reticulum, plasma membrane and other intracellular structures [5]. In recent years, several studies have shown that GPER1 has an anti-inflammatory action [6], which plays an important role in demyelination [7] and cognitive ability regulation in the onset and progression of epilepsy. Moreover, GPER1 may mediate the epileptogenic effect of estrogens by changing the oxidative or anti-oxidative parameters in the brain [8]. In addition, GPER1 may be related to the susceptibility and severity of seizures. GPER1 plays a neuroprotective role in status epilepticus, and might be a candidate target for epilepsy therapy [9]. Therefore, the effect of GPER1 in the development of epilepsy is worth further exploration.
Recently, the agonist G1 and the inhibitor G15 have been widely used in the study of GPER1. Both G1 and G15 are synthetic small molecule protein compounds. Their chemical structure characteristics are similar [10]. Therefore, the combination of G1 and GPER1 can cause changes in its molecular conformation and the polarity of oxygen atoms [3]. All the agonist ligands of GPER1 will activate the oxygen atom after binding to it, while antagonistic ligands including the G15 and G36 do not [11]. Besides, as a selective agonist of GPER1, G1 has a more substantial targeting effect on GPER1 than ERα and ERβ. G1 protected neurons by regulating autophagy [12] in vitro and in vivo. The use of G1 in the acute phase reduced the ischemic damage of hippocampal neurons in aged female rats [13]. G15 is a tetrahydro-3 H-cytochrome C-quinoline analog with a higher affinity for GPER1 and is a selective inhibitor of GPER1. Related studies have shown that G15 can inhibit the proliferation of rat uterine epidermal cells induced by estrogen [14].
Epilepsy often leads to impairment of cognitive abilities represented by learning and memory, and synaptic plasticity is the structural basis of learning and memory [15]. Mossy fiber sprouting (MFS) is the main change of synaptic plasticity in the hippocampus after temporal lobe epilepsy (TLE) [16]. Related studies have shown that MFS can be observed in animals’ hippocampus and patients with epilepsy [17]. Epilepsy can cause new dendrites of granular cells in the dentate gyrus (DG) site to participate in the formation of abnormal neural circuits. The neurons’ conduction entering the hippocampal site is restricted [18], further aggravating the symptoms.
At present, most studies on neuronal damage and synaptic plasticity changes after epilepsy focus on the chronic phase, and there are few long-term dynamic studies. Besides, there are few reports about the expression of GPER1 in the development of epilepsy and its role in neuronal damage. Therefore, we conducted this study to explore the possible involvement of GPER1 in the development of epilepsy.

Materials and Methods

Subjects

This research used 72 SPF male Sprague Dawley rats (200–250 g in weight) purchased from Laboratory Animal Center of Ningxia Medical University, animal certificate number (SCXK (Ningxia) 2015-001). Animals were given regular gavage in the morning and free food and water during feeding. All experiments were performed in compliance with Ningxia’s regulations and guidelines for the use of animals in research, and the study was approved by the Laboratory Animal Ethical and Welfare Committee of Laboratory Animal Center, Ningxia Medical University (Approval No. IACUC-NYLAC-2018-115).

Drugs

Lithium chloride and pilocarpine were purchased from Sigma-Aldrich Co., USA, and GPER1 selective agonist G1 and selective antagonist G15 were purchased from APE×BIO Technology Co., USA. The solutions of G1 and G15 were prepared in organic solvent dimethyl sulfoxide (DMSO, concentration 0.5%) and administered as intraperitoneal injections. All behavioral experiments and drug injections were carried out from 8 a.m. to 10 a.m. Doses and timings of injections used in the current investigation were adopted from the previous and pilot experiments carried out in our laboratory (Zuo et al., [9]).

Model Establishment

In order to compare and emphasize the effects of G1 and G15 under the same conditions, we established epileptic rats with DMSO intervention as a control group instead of normal rats. According to the number of days after modeling, the rats were randomly divided into 9 groups, each with 8 rats, grouped as follows: 2 days of administration: epilepsy control rats given DMSO (DMSO 2d group), epileptic rats receiving G1 intervention (G1 2d group), epileptic rats with G15 intervention (G15 2d group); 7 days of administration: epilepsy control rats with DMSO (DMSO 7d group), epilepsy rats with G1 intervention (G1 7d group) and receiving G15 intervention epileptic rats (G15 7d group); 28 days of administration: epilepsy control rats given DMSO (DMSO 28d group), epileptic rats receiving G1 intervention (G1 28d group) and epileptic rats receiving G15 intervention (G15 28d group).
The lithium chloride-pilocarpine TLE model was prepared by the following methods. The rats in each group were intraperitoneally injected with lithium chloride solution (3 mmol/kg,),) and then intraperitoneally injected with pilocarpine solution (50 mg/kg),) 16 h later. After modeling, we refer to the Racine seizure criteria [19] to determine whether the model is successful or not. In the epileptic group, IV–V seizures lasted for more than one time, and 1 h was selected as the successful model of rats. After meeting the modeling criteria, a 10% chloral hydrate solution was used to terminate the seizures.

Positioning Tube and Drug Delivery in the Lateral Ventricle

After anesthesia, fix the mouse on the stereotaxic instrument. Next, according to the rat brain’s stereotactic map, the starting point of the anterior fontanelle and the injection point is marked as 1.0 mm backward and 1.5 mm from the side. Rotate the torsion arm to position the trocar near the surface of the meninges and into the brain. The needle depth is 4 mm below the meninges.
All rats in the 9 groups required surgery. The intervention began on the first day after the successful establishment of the model. The drug dose of the control group was 0.5% DMSO solution 5 µg/rat/day, and the drug dose of G1 solution was 5 µg/rat/day; the drug dose in the G15 group was 5 µg/rat/day. The continuous injection time of the model animals were 2 days, 7 days, and 28 days, respectively.

Morris Water Maze (MWM) Test

The 28d group was selected, and the MWM experiment was started 21 days after modeling. The MWM was divided into two parts. The first part was the positioning and navigation experiment. This part lasted for 6 days, the first 5 days were training, and the 6th day was testing. The underwater platform was fixed in the same position. Each day, the rats were released into the water from the middle of the four quadrants of the maze with their backs facing the wall of the box. If the rat climbed on the platform within 60 s, the time it took to find the platform was the escape latency. If the platform could not be reached, the rat was guided to the underwater platform for 15 s, and the escape latency was recorded as 60 s. The second part was the space exploration experiment. After the positioning and navigation experiment, the underwater platform was removed on the 7th, the quadrant farthest from the underwater platform was drained, and the number of mice passing through the platform in 60 s was recorded.

Nissl Staining

The tissues were dehydrated and embedded, and then paraffin sectioned, with a thickness of 5 μm. Five slices/group were selected and were deparaffinized with xylene and dewaxed with gradient alcohol to render the tissues transparent. The slices were put in tar purple Nissl staining solution, the dyeing vat was placed in a 56 °C incubator for 1 h, and then, the slices were placed in a deionized water rinse. Each slice in turn, was dipped in the special differentiation solution for Nissl staining and observed under the microscope to control the differentiation time. It is better to make the background close to colorless during observation. After differentiation, the sections were rinsed with ddH2O for 10 min, dehydrated with gradient alcohol and transparent xylene, and mounted with neutral gum. The sealed slices were placed in a fume hood to dry, and the slices were observed under a microscope, each group had 5 slices, and each slice contained 5 fields of view.

Timm Staining

After anesthesia, the rats were perfused with 150 ml normal saline for 5–8 min, then injected with 100 ml 1% Na2S and 100 ml 4% PFA, and finally perfused with 50 ml 1% Na2S. The removed brain tissue was placed in 4% PFA and fixed overnight and then dehydrated in 30% sucrose PBS for 48 h. The 30 μm thick sections from the septal hippocampus (2.8–4.0 mm from the point posterior to the bregma) were obtained with a cryostat. Subsequently, the slices were stained for Timm staining every 100 μm at the following concentrations: A solution (protective gel), B solution (citrate buffer solution), and C solution (reducing agent) were stirred evenly, and D solution (silver nitrate solution) was added in dark conditions. The mixed Timm dye solution was shaken at 26 ℃ for 150 min. After dyeing, the slices were washed in deionized water. Finally, the slices were dried and pasted with neutral gum.

Golgi Staining

A Golgi staining kit (M057, Shanghai Gefan Biotechnology Co., Ltd., CN) was used for staining. The brain tissue with the thickness of approximately 3 mm in each group was fixed in a 10% formaldehyde solution for 24 h. The fixed tissue blocks were stained in potassium dichromate solution at 37 ℃ for 3 days and then washed with distilled water for 2 min. The tissues were stained in silver nitrate solution at 37 ℃ for 3 days and washed with d dH2O. After dyeing, it was dehydrated with alcohol and xylene and embedded in paraffin. A microtome was used for cutting the paraffin sections with a thickness of 10–30 μm, followed by xylene deparaffinization, neutral resin sealing, and microscopic analysis.

Immunohistochemical Staining

The continuous paraffin-embedded sections with a thickness of about 4 μm were dewaxed and rehydrated, and then endogenous peroxide quenching, blocking, primary antibody culture, and detection were carried out. Finally, 3 fields of vision per section were selected to observe the Cornu Ammonis (CA) 1, CA3, and DG regions of the hippocampus under a 20× objective lens. Six paraffin sections were taken from each group and analyzed using Image J image analysis software.

Western Blot Analysis

Total proteins from the brains were prepared and extracted. Protein concentration was measured using the BCA Protein Assay Kit (KGP902, KeyGEN, Nanjing, China). Protein separation was performed by SDS-PAGE, followed by electro-transfer, antigen and antibody reaction, color rendering, and exposure. Importantly, in immunodetection, the membranes were probed with primary antibodies: GPER1 (1:250, ab39742, Abcam, UK), Synapsin I (1:5,000, ab64581, Abcam, UK), PSD95 (1:2,000, ab18258 Abcam, UK), gephyrin (1:2,000, ab228674, Abcam, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000, ab37168, Abcam, UK) at 4 °C overnight. Finally, the protein bands were analyzed using Image J software, and the gray value of the internal reference GAPDH was compared to analyze the results and calculate the relative percentages.

Statistical Analysis

Data are expressed as the mean ± SD. Statistical analysis and data plotting were performed via one-way analysis of variance (ANOVA) using SPSS23.0 software and GraphPad Prism 8. Values of P < 0.05 were considered statistically significant.

Results

G1 Intervention Successfully Increased the Expression of GPER1 in the Hippocampus in Rats Per Group

Immunohistochemistry analysis and western blot results were used to test the expression of GPER1 in the hippocampus of rats per group after intervention in Fig. 1a and b. On day 2, compared to the DMSO group, the G1 expression was significantly increased in the CA1 and CA3 regions (P < 0.05), compared to that of the DMSO in the G1 group, while its G15 expression was significantly reduced (P < 0.05). Moreover, the expressions of the G1 and G1 in Fig. S.1 are consistent with the results in Fig. 1a and b. On the contrary, on day 7, compared to the DMSO group, G1 expression in the 7d group was significantly increased in the CA1 region (P < 0.05). The GPER1 expression was significantly lower in the CA1 and CA3 regions (P < 0.05). On day 28, the expressions of G1 and G15 in the CA1 region were significantly increased compared to that in the DMSO group (P < 0.05, Fig. 1a and b, Fig. S.1). Western blot results verified this trend at the molecular biology level, which indicated that G1 successfully increased the expression of GPER1 in the hippocampus of rats per group (Fig. 1c and d).

G1 Ameliorated the Impairment of Learning and Memory in Epileptic Rats

The Morris water maze (MWM) was used to detect the changes in learning and memory ability of rats after the intervention. In the acute phase of epilepsy, the rats manifested with weight loss, inability to consume food, and motionlessness, and could not bear the water maze, which requires a specific physical strength in Fig. 2a. Nest, we tested the changes in rats’ learning and memory abilities after the training for 1 to 5 days. The result showed that compared to the DMSO group, the G1 group required less time to find the platform with the extension of training time, and the G15 group required a longer time to find the platform (Fig. 2b). In the positioning and navigation experiment, on day 28, the time required to find the platform in 60 s was significantly shorter in the G1 group than that of the DMSO group (P < 0.01), which implied that the learning ability of the rats improved after G1 intervention. In comparison, the time taken by the rats in G15 group rats was significantly prolonged compared to that of the DMSO group (P < 0.05), further indicating that G1 had a memory improvement effect (Fig. 2c). In the space exploration experiment, compared to that of the DMSO group, the number of crossing the platform within 60 s in G1 group rats was significantly higher (P < 0.05), while G15 group rats were lower, showing that G1 intervention could improve the memory ability of rats (Fig. 2d).

G1 Repaired Damage to Hippocampal Neurons in Epileptic Rats

The results of immunohistochemistry staining in Fig. 3 and S2 showed that at day 2, day 7, and day 28, the damage of pyramidal cells and granular cells of the hippocampus after modeling in CA1, region CA3 and DG regions were improved in the rats in the G1 group compared to the DMSO group in rats. The rats’ damage in hippocampal neurons worsened in the G15 group, indicating that GPER1 could play a neuroprotective role in hippocampal neuron damage caused by epilepsy.

G1 Ameliorated the Formation of Hippocampal MFS and Damage to Neuronal Axons and Dendrites Caused by Epilepsy in Rats

Timm staining and Golgi staining were used to detect hippocampal MFS changes and hippocampal neuron axon dendrites. The concentration of Zn2+ in the hippocampal moss fibers of the brain is the highest. To test the moss fibers’ sprouting, Timm staining solution was used to mark the distribution of Zn2+. Our previous experiments showed that MFS was not found in the early stages of epilepsy. We chose to test the 7d and 28d groups to observe MFS changes after the intervention. As shown as in Fig. 4a and b, on the 7d,the G1 group showed only spotted silver-stained or no Timm particles, while the DMSO group showed flaky Timm silverstained particles mean, and the G15 group showed striped silver-stained particles (P < 0.05). On the 28d, there were no silver staining or spot-like silver staining particles were seen in the G1 28d group compared to the DMSO group (P < 0.05), and there exited nearly continuous ribbon-like silver staining particles in the G15 28d group compared to the DMSO group (P < 0.05). These results indicated that the G1 intervention could significantly ameliorated the abnormal sprouting of mossy fibers in epileptic rats, and blocked abnormal epileptic discharge, and ultimately achieved the goal of reducing seizures.
Golgi staining is one of the most effective methods to study the normal and abnormal morphology of neurons and glial cells and detect tiny morphological changes in the nerve axons and dendrites. The results showed that on day 28, the G1 28d group had denser neurons, extended axons, and dense and orderly dendritic networks in the CA1 region compared to the DMSO group, while the G15 group had fewer dendrites and fewer networks. In the CA3 region, the axons were longer, the dendritic network recovered, and the density increased in the G1 group compared to the DMSO group. On the contrary, in the G15 28d group, the dendrites were sparse, and the axons were shortened in the G15 group. In the DG region, the G15 group’s neuron density was sparse in the G15 group compared to the DMSO group. (Fig. 4c). These results indicated that after G1 intervention, the neuronal axons and dendrites damaged by epilepsy were repaired, and the normal functioning of neurons was restored, resulting in the improvement of symptoms of epilepsy.

G1 Reduces the Expression Level of Synapsin I and Increases Gephyrin Expression to Participate in the Regulation of Synaptic Plasticity

To explore the effect of G1 intervention on the regulation of hippocampal synaptic plasticity, we used immunohistochemistry and western blot to detect the changes in the expression of synapse-related proteins, including postsynaptic density (PSD) 95, Synapsin I, and gephyrin.PSD is the structural basis of postsynaptic signal transduction and integration. PSD95 is the most basic synaptic protein, and it is also the area where multiple proteins interact. It is also one of the marker proteins of regular synaptic function changes, and its changes can reflect synaptic plasticity to a certain extent [20, 21]. Synaptic marker proteins are mainly expressed in the CA3 region. PSD95 positive staining was distributed in the CA3 region of the hippocampus in rats per group, mainly expressing in the transparent layer. On day 2, the PSD95 expression in the hippocampus in the G1 group was lower (P < 0.05) than the DMSO group. The PSD95 expression in the hippocampus in the G15 group was significantly higher in the G15 group than in the G1 group. On day 7, the PSD95 expression in the G1 group was significantly lower than that of the DMSO group (P < 0.05), and the expression of PSD95 in the G15 group was significantly lower than in the G1group (P < 0.05). On day 28, the expression of PSD95 in the G1 group and G15 group was significantly lower than that of the DMSO group (P < 0.05), and PSD95 expression in the G15 group was significantly higher than that of the G1 28d group (P < 0.01, Fig. 5a, b). Western blot results showed that compared to the DMSO group, the relative expressions of PSD95 in the G1 and G15 groups at day 2 was not statistically significant. PSD95 expression at day 7 was significantly lower in the G1 and G15 groups than in the DMSO group (P < 0.01). On day 28, the expression of PSD95 in the G1 group was significantly lower than that of the DMSO group, and there was no significant change in the G15 group (P < 0.05, Fig. 6a and b).
Synapsin I is a specific phosphoprotein located on the presynaptic membrane of nerve terminals in the central and peripheral nervous systems and is a critical protein in the regulatory network of neurotransmitter release [22]. Studies [23] have shown that Synapsin I have a strong affinity for Ca2+ and plays a crucial role in releasing calcium-dependent neurotransmitters. Simultaneously, it can be used as a specific marker of the presynaptic terminal to detect synapses’ density and distribution [24]. In this study, immunohistochemistry analysis at day 2 showed that the Synapsin I was mainly expressed in the transparent layer in the CA3 region. The expression of Synapsin I in the hippocampus of the G1 group was significantly reduced compared to that of the DMSO group (P < 0.01), while its expression in the G15 group was substantially lower than that of the DMSO group (P < 0.05). On day 7, compared to the Synapsin I
expression in the DMSO group, the expression in the G1 group was significantly decreased (P < 0.05), while it was increased considerably in the G15 group (P < 0.05). On day 28, the Synapsin I expression in the hippocampus in the G1 group was significantly lower than that of in the DMSO group (P < 0.05), while its expression in the G15 group was significantly increased compared to that of the DMSO 28d group(P < 0.05). The expression of Synapsin I in the G15 group was also significantly increased compared to that of the G1 group (P < 0.05, Fig. 5c, d). Western blot results showed at the day 2 that compared to the DMSO group, the relative expression of Synapsin I was significantly decreased in the G1 2d group (P < 0.01) and significantly reduced in the G15 group (P < 0.05). Compared to the G1 group, the relative expression of Synapsin I in the G15 group was significantly increased (P < 0.05). On day 7, compared to the DMSO group, the relative expression of Synapsin I in the G1 group was significantly decreased (P < 0.05). On day 28, compared to the G1 group, the relative expression of Synapsin I in the G15 group was significantly increased (P < 0.01). Compared to the DMSO group, the relative expression of Synapsin I was significantly decreased in the G1 group (P < 0.05, Fig. 6c and d).
Gephyrin is a lean protein located on the inhibitory postsynaptic membrane. In mammals, gephyrin is mainly expressed in the CNS [25, 26] and may be involved in the formation and maintenance of partial inhibitory synaptic plasticity. In our study, immunohistochemical analysis showed that gephyrin immunopositive staining was mainly located in the cytoplasm. On day 2, compared to the gephyrin expression in the DMSO group, its expression in the hippocampus of the G1 group was significantly reduced (P < 0.05) and was significantly increased (P < 0.05) in the G15 group. On day 7, compared to that of the DMSO group, gephyrin expression in the G1 group was significantly increased (P < 0.05), while that of the G15 group was significantly reduced (P < 0.05). Simultaneously, compared to that of the G1 group, gephyrin expression in the G15 group was decreased significantly (P < 0.05). On day 28, compared to that of the DMSO group, the gephyrin expression in the G1 group was significantly reduced (P < 0.05), while the expression was significantly increased in the G15 group (P < 0.05, Fig. 5e and f). On day 2, the western blot results showed that compared to the DMSO group, the relative expression of gephyrin in the G1 2d and G15 groups was not statistically significant. On day 2, compared to the DMSO group, the relative expression of gephyrin was significantly increased in the G1 group (P < 0.01) and significantly decreased in the G15 group (P < 0.05), and compared to the G1 group, the relative expression of gephyrin in the G15 group was significantly decreased (P < 0.05). On day 28, Compared to the DMSO group, gephyrin’s relative expression in the G1group was also significantly reduced (P < 0.05, Fig. 6e and f).

Discussion

Epidemiological data show that the current prevalence of epilepsy is 5% in China, and the incidence rate of male is higher than that of female [27] TLE is the most common type of refractory epilepsy [28]. In addition, the hippocampal sclerosis TLE has a high incidence rate in adults [29]. Hippocampal sclerosis as an important pathogenic factor of epilepsy has attracted more and more attention [30]. .Due to drug resistance and repeated seizures, the clinical effect of epilepsy is poor. In recent years, GPER1 has an antiinflammatory action,which plays an important role in the onset and progression of epilepsy [2]. In our study, we found that GPER1 modulated the synaptic plasticity during the development of temporal lobe epilepsy in rats.
The synaptic plasticity, which acted as the basis of learning and memory, can reflect the regulatory effect of GPER1 in rats with temporal lobe epilepsy. Synaptic plasticity can be divided into functional and structural plasticity [31]. It can also be divided into short-term and long-term plasticity in time history [32]. The former manifests as an enhancement, inhibition, and alienation, while the latter manifests as long-term potentiation (LTP) and long-term depression (LTD) [33]. Synapses can be divided into electrical and chemical synapses due to their different presenting signals [34]. Therefore, the changes in membrane potential can reflect changes in electrical synapses.We used the synaptic proteins’ expression to assess the synaptic plasticity.
Previous studies have shown that short-term treatment with both the GPER1 agonist G1 and estradiol in ovariectomized rats could enhance spatial recognition memory [35]. Our results showed that the time required for G1 intervention rats to find an underwater platform was significantly reduced within 60 s, indicating that the learning ability of rats after G1 intervention was improved. Simultaneously, after the medium was removed, for the G1 group, the number of times a rat crossed the area where the underwater platform was located increased significantly, indicating that G1 can improve the memory ability of epileptic rats. Neurons are the basic functional unit of the CNS. The Nissl body is one of the characteristic structures in neurons. The Nissl body is located at the neuronal cell bodies and dendrites and has basophilic properties [36]. The results showed that at day 2, day 7, and day 28 after modeling, the deterioration of pyramidal cells and granular cells in each region of the hippocampus was increasing in the rats treatment with the GPER1 agonist G1 compared to the DMSO group, and the damage of hippocampal neurons in rats increased treatment with GPER1 antagonist G15. In addition, we found that GPER1 inhibitor G15 promoted epilepsy in rats. And the degree of epileptic seizures were more severe in the G15 intervention group, this result indicated that GPER1 played a neuroprotective effect in the hippocampal neuron damage caused by epilepsy.
Relevant studies have shown that the loss of hippocampal neurons and the degree of hippocampal MFS are near related to cognitive function impairment [37]. The MFS of the granule cells of the hippocampus’s DG region may be the structural basis for the decline in learning and memory and cognitive dysfunction after epilepsy [38]. Timm staining results showed that after G1 intervention, the abnormal sprouting of hippocampal mossy fibers in rats caused by epilepsy was significantly controlled, and the degree of MFS in rats with G15 intervention was more severe than that in the rats treated with DMSO. Meanwhile, the expression of Synapsin I decreased on the seventh day after G1 intervention in rats. Synapsin I can regulate the release of neurotransmitters and play an essential role in synaptic plasticity. It is recognized as a specific marker of the axonal end. It directly reflects the density and distribution of synapses [39]. A decrease in its level indicates that the synapse’s remodeling is reversed and that the normal state of the synaptic network is maintained. Besides, Golgi staining results showed that the density and length of dendrites were significantly improved after G1 intervention, especially in the CA3 region, while the axonal and dendritic damage in the rats administered G15 was more serious. Our results implyed that GPER1 has a repairing effect on the neurons damaged after epilepsy. The expression level of PSD95 reflects whether the basic function of synapses is normal [40]. Our results showed that the expression level of PSD95 was decreased in the G1 intervention group, while the expression of PSD95 was increased in the G15 intervention group.
In this study, G1 intervention resulted in the decrease of PSD95 expression, suggesting that the G1 was involved in the restoration of synaptic structure and function after repairing the damage. Our results showed that in the early stage of epilepsy, the expression of gephyrin in the G1 group was lower than that of the DMSO group, while the expression of gephyrin in the G1 group was higher than that of the DMSO control group on day 7. On day 28, the gephyrin expression in the G1 intervention group declined fter modeling. This result indicated that G1 could participate in the rescue of normal neuronal function by promoting gephyrin expression.
In summary, we found that GPER1 selective agonist G1 significantly ameliorated the learning and memory impairment of epileptic rats. Furthermore, G1 eased the hippocampal neuron damage caused by epilepsy, while G15 reduced the protective effect of GPER1 on the hippocampal neurons after epilepsy. Meanwhile, G1 ameliorated the formation of MFS in the hippocampus of epileptic rats. Besides, G1 may participate in synaptic plasticity regulation by reducing the Synapsin I expression and increasing the gephyrin expression.

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