Insulin-like growth factor-1 (IGF-1) is known to promote neurogenesis and survival. However, recent studies have suggested that IGF-1 regulates neuronal firing and excitatory neurotransmission. In the present study, focusing on temporal lobe epilepsy, we found that IGF-1 levels and IGF-1 receptor activation are increased in human epileptogenic tissues, and pilocarpine- and pentylenetetrazole-treated rat models. Using an acute model of seizures, we showed that lateral cerebroventricular infusion of IGF-1 elevates IGF-1 receptor (IGF-1R) signalling before pilocarpine application had proconvulsant effects. In vivo electroencephalogram recordings and power spectrogram analysis of local field potential revealed that IGF-1 promotes epileptiform activities. This effect is diminished by co-application of an IGF-1R inhibitor. In an in vitro electrophysiological study, we demonstrated that IGF-1 enhancement of excitatory neurotransmission and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor- and N-methyl-D-aspartate receptor-mediated currents is inhibited by IGF-1R inhibitor. Finally, activation of extracellular signal-related kinase (ERK)-1/2 and protein kinase B (Akt) in seizures in rats is increased by exogenous IGF-1 and diminished by picropodophyllin. A behavioural study reveals that the ERK1/2 or Akt inhibitor attenuates seizure activity. These results indicate that increased IGF-1 levels after recurrent hippocampal neuronal firings might, in turn, promote seizure activity via IGF-1R-dependent mechanisms. The present study presents a previously unappreciated role of IGF-1R in the development of seizure activity.

CLINICAL PERSPECTIVES

  • IGF-1 is usually considered to be a potential neuroprotectant. However, recent studies have demonstrated that IGF-1 has increased the excitability of neurons and extended the growth cone, which may be related to the formation of abnormal neural circuits.

  • The abnormal neuronal networks are the basis for epileptic seizures and the development of epilepsy, and thus IGF-1 may play an important role in this process.

  • The present study found that IGF-1 expression increased in patients with epilepsy and two epileptic rat models, and we indicated that IGF-1 increased the excitability of the hippocampus and promoted seizure activity via IGF-1R-mediated signalling, which suggests that IGF-1R may represent a new target in the search for antiepileptic strategies.

INTRODUCTION

Epilepsy is a common neurological condition, for which the representative feature is recurrent and unprovoked neuronal firings [14]. Many lesions in the brain can lead to epilepsy, e.g. infection, tumours, stroke and traumatic injury [5]. However, the mechanisms underlying the pathogenesis of epilepsy are not clear. Insulin-like growth factor-1 (IGF-1) functions as a neurotrophic factor that plays an important role in childhood growth, dwarfism and cancer [6]. In the central nervous system, IGF-1 increases brain growth and myelination, maintains neuronal survival and promotes functional recovery after excitotoxic lesions [79]. Transport of IGF-1 into the central nervous system across the blood–brain barrier is activity dependent [10].

However, the function of IGF-1 may not always be beneficial in the pathophysiology of epilepsy. IGF-1 has been shown to increase progenitor cell proliferation in the dentate gyrus of the hippocampus [11], whereas altered neurogenesis is also present in patients with intractable temporal lobe epilepsy (TLE) [12]. In addition, the IGF-1 receptor (IGF-1R) shares common signalling pathways with the brain-derived neurotrophic factor (BDNF) receptor, tropomyosin receptor kinase B (TrkB), which involve activation of the serine–threonine kinase (protein kinase B or Akt) and extracellular signal-regulated protein kinases (ERKs) [11,13]. Studies have demonstrated that conditional deletion of TrkB or anti-nerve growth factor (NGF) immunoglobulin G prevents epileptic activities in animal models of epilepsy [14,15]. Erythropoietin (EPO) activates the phosphoinositide 3-kinase (PI3K) and ERK1/2 pathways in cultured hippocampal neurons and modulated calcium influx in kainate-induced seizures [16]. On the other hand, the application of glutamate increases phospho-Akt and disrupts the glutamate-mediated homoeostasis of neuronal excitability [17]. IGF-1 and downstream signalling from PI3K/Akt are essential for the regulation of membrane expansion at the nerve growth cone and related neurite outgrowth [18,19], which may promote neural circuits and network reorganization, and increase the excitability in the epileptic brain. Moreover, as IGF-1 also controls calcium ion channels, it is likely that it directly affects the excitation of hippocampal neurons [20,21].

Therefore, we propose that IGF-1 may play a role in pilocarpine- and pentylenetetrazole (PTZ)-induced seizure activities. In the present study we first determined the expression pattern of IGF-1 and its receptor IGF-1R in patients with epilepsy and in two rat models. Next, we investigated the role of IGF-1 and IGF-1R in seizure activities in two animal models and in neuronal excitatory neurotransmission through in vivo and in vitro electrophysiological studies. We finally examined the role of the IGF-1-signalling molecules ERK and Akt in animal behaviours. Our study demonstrated that IGF-1 promotes seizure activity via IGF-1R-mediated signalling.

MATERIALS AND METHODS

Human participants

All the temporal neocortical samples were selected at random from our brain tissue bank, which consisted of more than 300 tissues from people with epilepsy and 60 control tissues [22,23]. The study was performed with the formal consent of the patients or their lineal relatives for the use of data and brain tissues. The research protocol complied with the Declaration of Helsinki of the World Medical Association and the guidelines for the conduct of research involving human participants, as established by the National Institutes of Health and the Committee on Human Research at Chongqing Medical University, Chongqing, China.

Some 24 patients (13 males and 11 females with mean age 29.79±1.71 years, range 14–47 years; mean course 10.29±0.98 years, range 3–19 years) with medically intractable TLE demonstrated typical clinical manifestations and characteristic electroencephalograms (EEGs), and their diagnosis of epilepsy was in accordance with the 2001 International Classification of Epileptic Seizures by the International League Against Epilepsy. The patients were screened for the presence or a family history of a psychiatric disease or a neurodegenerative disease. All patients participated in a presurgical clinical evaluation, including a detailed history and neurological examination, neuropsychological testing, brain magnetic resonance imaging (MRI) and 24-h EEG or video-EEG. All patients were resistant to the maximal tolerable doses of at least two antiepileptic drugs (AEDs), including valproic acid, carbamazepine, phenytoin, phenobarbital, topiramate, oxcarbazepine, clonazepam, lamotrigine and gabapentin (Table 1). Control temporal neocortical samples (12: 6 males and 6 females with mean age of 27.42±2.79 years, range 15–45 years), with no histological changes, were obtained from patients who underwent therapeutic surgical resection after increased intracranial pressure due to head trauma without exposure to seizure or AEDs (Table 2). There were no statistically significant differences in age and sex between the TLE patients and the control participants (P > 0.05).

Table 1
Clinical characteristics of the patients with TLE

Abbreviations: AEDs taken before the operation: CBZ, carbamazepine; GBP, gabapentin; LTG, lamotrigine; OXC, oxcarbazepine; PB, phenobarbital; PHT, phenytoin; TPM, topamax; VPA, valproic acid. F, female; g, gliosis; LTN, left temporal neocortex; M, male; nd, neuron degeneration; nl, neuron loss; RTN, right temporal neocortex.

PatientsSex (M/F)Age (years)Course (years)AEDs before surgeryResection tissuePathology
14 VPA, PB, CBZ, PHT RTN g, nd 
47 14 VPA,PB,LTG,PHT RTN g, nd 
23 VPA, CBZ, TPM, GBP RTN g, nl, nd 
30 18 VPA, PB, CBZ, TPM LTN g, nl, nd 
34 10 VPA, CBZ, TPM, LTG RTN g, nl 
36 10 VPA, CBZ, PB RTN g, nl 
31 12 CBZ, VPA, TPM, RTN g, nl, nd 
24 CBZ, VPA, TPM, PHT LTN g, nd 
25 CBZ, VPA, TPM LTN g, nd 
10 26 CBZ, VPA, TPM, OXC LTN 
11 31 12 PB, VPA, TPM, LTG RTN g, nl, nd 
12 21 VPA, TPM, PB RTN g, nd 
13 22 VPA, TPM, LTG LTN g, nl 
14 30 VPA, CBZ, TPM, GBP LTN g, nl 
15 34 VPA, TPM, OXC LTN g, nd 
16 34 19 VPA, PHT, LTG RTN g, nd 
17 35 18 VPA, TPM, LTG, OXC RTN g, nd 
18 22 15 CBZ, TPM, CZP LTN g, nd 
19 39 17 CBZ, VPA, TPM RTN g, nd 
20 40 15 CBZ, TPM, VPA, LTG RTN g, nl 
21 45 10 CBZ, VPA, TPM, LTG LTN g, nl 
22 17 PB, CBZ, PHT, LTG LTN g, nl 
23 23 10 PB, CBZ, PHT, LTG LTN g, nl 
24 32 PHT, PB, CZP, LTG RTN g, nl 
PatientsSex (M/F)Age (years)Course (years)AEDs before surgeryResection tissuePathology
14 VPA, PB, CBZ, PHT RTN g, nd 
47 14 VPA,PB,LTG,PHT RTN g, nd 
23 VPA, CBZ, TPM, GBP RTN g, nl, nd 
30 18 VPA, PB, CBZ, TPM LTN g, nl, nd 
34 10 VPA, CBZ, TPM, LTG RTN g, nl 
36 10 VPA, CBZ, PB RTN g, nl 
31 12 CBZ, VPA, TPM, RTN g, nl, nd 
24 CBZ, VPA, TPM, PHT LTN g, nd 
25 CBZ, VPA, TPM LTN g, nd 
10 26 CBZ, VPA, TPM, OXC LTN 
11 31 12 PB, VPA, TPM, LTG RTN g, nl, nd 
12 21 VPA, TPM, PB RTN g, nd 
13 22 VPA, TPM, LTG LTN g, nl 
14 30 VPA, CBZ, TPM, GBP LTN g, nl 
15 34 VPA, TPM, OXC LTN g, nd 
16 34 19 VPA, PHT, LTG RTN g, nd 
17 35 18 VPA, TPM, LTG, OXC RTN g, nd 
18 22 15 CBZ, TPM, CZP LTN g, nd 
19 39 17 CBZ, VPA, TPM RTN g, nd 
20 40 15 CBZ, TPM, VPA, LTG RTN g, nl 
21 45 10 CBZ, VPA, TPM, LTG LTN g, nl 
22 17 PB, CBZ, PHT, LTG LTN g, nl 
23 23 10 PB, CBZ, PHT, LTG LTN g, nl 
24 32 PHT, PB, CZP, LTG RTN g, nl 
Table 2
Clinical characteristics of the control group

Abbreviations: F, female; LTN, left temporal neocortex; M, male; n, normal; RTN, right temporal neocortex.

PatientGenderAge (years)Aetiology/diagnosisResection tissueAdjacent tissue pathology
15 Trauma RTN 
18 Trauma RTN 
25 Trauma LTN 
28 Trauma LTN 
35 Trauma RTN 
45 Trauma RTN 
18 Trauma LTN 
21 Trauma RTN 
24 Trauma LTN 
10 25 Trauma RTN 
11 32 Trauma RTN 
12 43 Trauma LTN 
PatientGenderAge (years)Aetiology/diagnosisResection tissueAdjacent tissue pathology
15 Trauma RTN 
18 Trauma RTN 
25 Trauma LTN 
28 Trauma LTN 
35 Trauma RTN 
45 Trauma RTN 
18 Trauma LTN 
21 Trauma RTN 
24 Trauma LTN 
10 25 Trauma RTN 
11 32 Trauma RTN 
12 43 Trauma LTN 

Experimental animals

All animal experiments were performed in accordance with the Animal Research Committee of the Chongqing Medical University and were formally approved by the Chinese Animal Welfare Act for the use and care of laboratory animals. Young male Sprague Dawley rats weighing 200–220 g were obtained from the Laboratory Animal Center of Chongqing Medical University. The rats were housed in individual cages under controlled conditions (ambient temperature 24–25°C, humidity 50–60%, lights on from 8:00 h to 20:00 h), as well as free access to food and water.

Animal models of pilocarpine- and PTZ-induced seizures

The pilocarpine model reproduces most of the clinical and neuropathological features of TLE and has been widely used for study. The rats were administered lithium chloride (127 mg/kg, intraperitoneally; Sigma) 20 h, and atropine sulphate (1 mg/kg, intraperitoneally) 30 min, before the first pilocarpine administration, respectively. After 30 min of the initial dose (35 mg/kg, intraperitoneally; Sigma), pilocarpine was given repeatedly (10 mg/kg, intraperitoneally) every 10 min until the rats developed seizures. The total number of pilocarpine injections was limited to five per animal. After 60 min of status epilepticus, diazepam (10 mg/kg intraperitoneally) was administered to terminate the convulsive seizures. The seizure activities were scored according to Racine's standard criteria [24]. Control rats were treated with saline in an identical manner.

PTZ is an antagonist of γ-aminobutyric acid (GABA) receptors, which induces a minimal clonic seizure (MCS) or a generalized tonic–clonic seizure (GTCS). An appropriate single-dose (60 mg/kg, intraperitoneally) of PTZ induces only a transient GTCS (about dozens of seconds) with no significant pathological changes or spontaneous epilepsy.

Tissue preparation

For both rat and human brain tissues, one portion of the samples obtained from each participant was immediately placed in a cryovial, which was soaked in buffered diethylpyrocarbonate (1:1000) for 24 h, and then stored in liquid nitrogen for quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting study.

Determination of IGF-1 protein levels

IGF-1 protein levels from human neocortical and rat hippocampal tissues were measured using ELISA kits (human and rats), according to the manufacturer's directions (R&D Systems, Inc.). The absorbance was measured using a microplate reader (Bio-Rad Laboratories) and measured spectrophotometrically at 450 nm.

Quantitative RT-PCR

IGF-1 mRNA levels were analysed using qRT-PCR. Total RNA was extracted using the TRIzol reagent (Takara Bio) according to the manufacturer's protocol. Total RNA was purified and then cDNA synthesis was performed using a PrimeScript RT reagent kit and gDNA Eraser (Takara Bio). RT-PCR was performed using a SYBR Premix Ex Taq Kit (Takara Bio) with the iQ5 RT-PCR detection system (Bio-Rad Laboratories). The threshold cycle (CT) to determine the relative expression of IGF-1 relative to actin and relative quantity (RQ) were calculated using the 2-△△C(T) method of Racine [25].

Western blotting

Tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer with protease and phosphatase inhibitors (Sigma) and concentrations of total protein were determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Primary antibodies against phosphorylated (p)-IGF-1R, IGF-1R, pERK1/2, ERK1/2, pAkt and Akt (1:500–1000) were purchased from Cell Signaling Technology, Inc. Antibody against pIGF-1R (#3024) detects pIGF-1R β subunits at Tyr1135/1136. Blot intensities were calculated using the Quantity One software version 4.6.2 (Bio-Rad Laboratories).

Experimental intervention

Human recombinant IGF-1 (rhIGF-1, SinoBio Co.) was injected intracerebroventricularly (10 μg/5 μl saline) [26]. The selective IGF-1R inhibitor picropodophyllin, which inhibits tyrosine phosphorylation of the IGF-1R without notable toxic effect [27], was administered (20 mg/kg, intraperitoneally) 1 h before chemically induced seizures. The PI3K inhibitor LY294002 (10 mM, 5 μl) or the ERK inhibitor U0126 (5 mM, 5 μl) was injected intraventricularly [28]. All inhibitors were purchased from Selleck Chemicals.

In vivo multichannel EEG recording and local field potential analysis

Rats were anaesthetized with chloral hydrate (350 mg/kg, intraperitoneally), placed in a stereotaxic device, and ground screws were secured to the anterior cranium and above the cerebellum. A microwire array (a 2×8 array of platinum–iridium alloy wire, each 25 μm in diameter) was surgically implanted into the right dorsal hippocampus [anteroposterior (AP) −3.6, mediolateral (ML) 2.8, dorsoventral (DV) −3.5] as described by Paxinos and Watson [28]. At the same time, a microinjection cannula was implanted into the left lateral ventricle (AP −1.5, ML 1.0, DV −3.5) and fixed with dental cement on the skull. Five rats per group were allowed to recover for several days before advancing the electrophysiological recording. Local field potentials (LFPs) were preamplified (1000×), filtered (0.1–1000 Hz) and digitized at 4 kHz using a OmniPlex D Neural Data Acquisition System (Plexon). The direct current-coupled headstages were used for neurophysiological recordings, and all recordings were referenced to two of the implanted screws. NeuroExplorer v4.0 (Plexon) was used for analysis of the LFPs and the power spectrogram.

In vitro electrophysiology

Hippocampal slices were prepared as described previously. In brief, male Sprague Dawley rats (14–16 days old) were anaesthetized with isoflurane and decapitated. The brain was quickly removed and placed into ice-cold dissection solution (22 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 1 mM NaH2PO4, 3 mM KCl, 7 mM MgCl2, 0.5 mM CaCl2, pH 7.4). Hippocampal slices were made using a Leica VP 1200S and allowed to recover in artificial cerebrospinal fluid (ACSF: 124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM glucose, 2.5 mM CaCl2 and 1.3 mM MgCl2, saturated with 95% O2/5% CO2, pH 7.4) at room temperature for 1 h.

Recordings were performed on pyramidal neurons in the hippocampal CA1 area. A patch pipette (of resistance 2–4 MΩ) was filled with intracellular solution containing: 130 mM Cs-MeSO4, 10 mM Hepes, 10 mM CsCl, 4 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 5 mM NMG, 5 mM MgATP, 0.5 mM Na2GTP, 0.2 mM EGTA and 5 mM QX-314 (pH 7.2–7.4). The bathing solution was ACSF. Picrotoxin (100 μM) and tetrodotoxin (1 μM) were added to record the miniature excitatory postsynaptic current (mEPSC). Evoked currents were generated with a 40-μs pulse (0.1 Hz) from a stimulation isolation unit controlled by an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) generator. The 50% maximum stimulation intensity was chosen for final recording. A bipolar stimulation electrode (FHC) was positioned in Schaeffer collaterals. Evoked AMPA receptor (AMPAR)/N-methyl-D-aspartate receptor (NMDAR) currents were recorded at two holding potentials. At −70 mV, currents were collected and the peak amplitude was identified as the AMPAR-mediated currents. Neurons were then held at +40 mV, and the amplitude of the evoked excitatory postsynaptic current (EPSC) 50 ms post-stimulus was identified as the NMDAR-mediated currents. The AMPA:NMDA ratio was calculated using the AMPAR-mediated currents relative to the NMDAR-mediated currents. Paired pulses were delivered at a 50-ms interval when cells were held at −70 mV. Ten traces were averaged and the resultant mean values were used for analysis. For all whole-cell recordings, access resistance (Ra) was <25 MΩ and cells were rejected if Ra or membrane resistance (Rm) changed >20% over the course of the experiment.

Signals were acquired using a MultiClamp 700B amplifier (Axon) and then recorded with pClamp 9.2 software (Molecular Devices). All recordings were digitized at 10 kHz and filtered at 2 kHz. A Mini Analysis program (Synaptosoft) was used to analyse synaptic activity. Individual synaptic events, with fast onset and exponential decay kinetics, were captured using threshold detectors in Mini Analysis software.

Statistical analysis

All population measurements are specified as means± S.E.M.s. Analyses of significance were carried out using Student's t-test or ANOVA for comparing two groups or at multiple time points. Fisher's exact test was used to compare two groups of two categorical variables. P < 0.05 was considered statistically significant. Statistical analysis was performed using the software package SPSS 17.0.

RESULTS

IGF-1 expression is increased in patients with TLE and in rat models

As shown in Figures 1(A) and 1(B), IGF-1 mRNA and protein expression in the temporal neocortex were significantly increased in 24 patients with TLE over those in 12 control participants (n=12). The IGF-1 mRNA relative quantitative value was 2.461±0.309 vs 1.191±0.098 (Student's t-test: F=13.941, P=0.001). In addition, IGF-1 protein was 311.584±20.785 in TLE patients, which was 235.75±14.592 in the control group (Student's t-test: F=5.871, P=0.021). To investigate whether IGF-1 expression was altered in rats of the seizure model, we performed a dynamic analysis of IGF-1 mRNA and protein levels from dissected ipsi- and contra-lateral hippocampus at 6 h, 24 h, 7 days, 21 days, and 60 days after pilocarpine-induced seizures (n=5), respectively. As shown in Figures 1(C) and 1(D), IGF-1 mRNA and protein expression remained significantly high during acute seizures, the initial stage of spontaneous seizures and recurrent spontaneous attacks, and fell back during the latent period, compared with the control (ANOVA, P < 0.05). Consistently, the activation of IGF-1R as measured by pIGF-1R was also increased in TLE patients at 6 h, 24 h, 7 days, 21 days and 60 days after pilocarpine-induced seizures compared with controls (n=5), respectively (Figures 1E and 1F, ANOVA, P < 0.05). These results suggest that up-regulation of IGF-1 after seizures activates the IGF-1R. To validate the effect of seizures on IGF-1 and IGF-1R expression, we measured IGF-1 and activation of IGF-1R levels in another model, PTZ-induced GTCS. As shown in Figures 1(G) and 1(H), IGF-1 protein levels (14.84±0.75 and 14.052±0.907 at 6 h and 24 h after seizures vs 8.932±0.513 in control group, F=18.73 and 23.858, respectively, P < 0.01) and the pIGF-1R relative quantitative value (2.142±0.217 and 1.985±0.208 at 6 h and 24 h after seizures vs 1.098±0.021 in control group, F=18.73 and 23.858, respectively, P < 0.05) were significantly increased in the hippocampus after induction of seizures by PTZ.

Expression of IGF-1 and its receptor (IGF-1R) in patients with TLE and animal models

Figure 1
Expression of IGF-1 and its receptor (IGF-1R) in patients with TLE and animal models

(A, B) The expression of (A) IGF-1 mRNA and (B) protein in the temporal neocortex of control (n=12) and TLE patients (n=24) by qRT-PCR and ELISA, respectively (*P < 0.05, compared with control, ANOVA). (C, D) Dynamic changes of (C) rat IGF-1 mRNA and (D) protein in the hippocampus of pilocarpine-induced rat model of seizure (*P < 0.05, compared with control, n=5, ANOVA). (E) Representative immunoblots of phosphorylated IGF-1R and total IGF-1R in TLE (top). The pIGF-1R relative to total IGF-1R was significantly increased compared with control (bottom, *P < 0.05). (F) Representative immunoblots of pIGF-1R (relative to total IGF-1R) in the hippocampus of pilocarpine-treated rats (*P < 0.05 compared with control, n=5, ANOVA). (G) IGF-1 protein level and (H) its receptor expression in the hippocampus of rats after PTZ injection (60 mg/kg intraperitoneally) (*P < 0.05 compared with control, n=5, ANOVA).

Figure 1
Expression of IGF-1 and its receptor (IGF-1R) in patients with TLE and animal models

(A, B) The expression of (A) IGF-1 mRNA and (B) protein in the temporal neocortex of control (n=12) and TLE patients (n=24) by qRT-PCR and ELISA, respectively (*P < 0.05, compared with control, ANOVA). (C, D) Dynamic changes of (C) rat IGF-1 mRNA and (D) protein in the hippocampus of pilocarpine-induced rat model of seizure (*P < 0.05, compared with control, n=5, ANOVA). (E) Representative immunoblots of phosphorylated IGF-1R and total IGF-1R in TLE (top). The pIGF-1R relative to total IGF-1R was significantly increased compared with control (bottom, *P < 0.05). (F) Representative immunoblots of pIGF-1R (relative to total IGF-1R) in the hippocampus of pilocarpine-treated rats (*P < 0.05 compared with control, n=5, ANOVA). (G) IGF-1 protein level and (H) its receptor expression in the hippocampus of rats after PTZ injection (60 mg/kg intraperitoneally) (*P < 0.05 compared with control, n=5, ANOVA).

Enhancement of hippocampal excitatory and seizure activity by IGF-1 is blocked by the IGF-1R inhibitor

The IGF-1R inhibitor picropodophyllin has been used in vivo to inhibit malignant cell growth without affecting insulin receptor function. Thus, we tested whether exogenous IGF-1 may affect seizure activities by intracerebroventricular injection and whether this effect is mediated by IGF-1R using intraperitoneal application of picropodophyllin when rats were awake and freely moving. After the first injection of pilocarpine (35 mg/kg, intraperitoneally), 10/13 (77%) rats in the IGF-1 group, 6/13 (46%) in the vector control and 2/15 (13%) in the picropodophyllin group reached grade IV and above attacks as measured by Racine's score. Pilocarpine was given repeatedly (10 mg/kg, intraperitoneally) every 10 min until the rats developed seizures. The total number of pilocarpine injections was limited to five per animal. The last, 13/13 (100%) rats in the IGF-1 group, 10/13 (76.92%) in the vector control and 6/15 (40%) in the picropodophyllin group reached grade IV and above attacks as measured by Racine's score. On the other hand, the latency of seizures means the time from injection of pilocarpine to the first onset of class IV or V seizures.

To determine whether IGF-1 and IGF-1R may play a role in epileptiform discharge by multichannel recording in vivo, we first observed IGF-1 and its receptor antagonist picropodophyllin on neuronal firing activity in conscious rats. As shown in Figures 2(A) and 2(B), compared with control, the epileptiform discharge occurred earlier and more clustered in IGF-1-treated animals, but was relatively delayed and sparse in the picropodophyllin-treated group. Representative power spectrum analysis demonstrated that seizure activity was enhanced by IGF-1 and inhibited by the IGF-1R antagonist picropodophyllin (Figure 2C). The duration of discharges and burst numbers in these three groups are summarized in Figures 2(D) and 2(E), which show that IGF-1 significantly increased discharge duration (34.4±2.502 min) and burst numbers (32.4±1.077 times/min) within 1 h of the pilocarpine injection, whereas picropodophyllin (8.08±1.114 min and 3.44±0.584 times/min, respectively) showed an opposite effect (n=5 in each group, compared with control 23.0±2.0 min and 15.6±1.077 times/min, ANOVA, P < 0.01). A behavioural study showed that, compared with the control, IGF-1 significantly decreased onset latency of seizures and increased maximum Racine's scores, whereas picropodophyllin showed an opposite effect (Figures 2F and 2G; ANOVA, P < 0.05). These results indicate that IGF-1 increased seizure susceptibility and severity whereas the IGF-1 inhibitor picropodophyllin exhibited the opposite effect.

Effects of IGF-1 inhibitor picropodophyllin on seizure activities in a pilocarpine-induced rat model of seizure

Figure 2
Effects of IGF-1 inhibitor picropodophyllin on seizure activities in a pilocarpine-induced rat model of seizure

(A) Representative traces of in vivo EEG in IGF-1 (top), control (middle) and picropodophyllin (PPP; bottom). (B) Representative EEG traces in rats treated with IGF-1 taken from (A): (a) outbreak of epileptiform activity; (b, c) ripple rhythms (100–200 Hz) above 3 Hz background activities. (C) Power spectrograms in rats treated with saline (control), IGF-1 and picropodophyllin. (D, E) Summary of (D) the epileptiform discharge duration and (E) burst numbers from intracranial microelectrode LFP recordings (*P < 0.05, P < 0.01, compared with control, n=5 each, ANOVA). (F, G) Behavioural assessment showed (F) the onset latency of seizures and (G) maximum Racine's score in rats treated with saline (control), IGF-1 or picropodophyllin before pilocarpine injection (35 mg/kg, n=13 in control; n=15 in picropodophyllin or IGF-1 group) (*P < 0.05, P < 0.01, compared with control, ANOVA).

Figure 2
Effects of IGF-1 inhibitor picropodophyllin on seizure activities in a pilocarpine-induced rat model of seizure

(A) Representative traces of in vivo EEG in IGF-1 (top), control (middle) and picropodophyllin (PPP; bottom). (B) Representative EEG traces in rats treated with IGF-1 taken from (A): (a) outbreak of epileptiform activity; (b, c) ripple rhythms (100–200 Hz) above 3 Hz background activities. (C) Power spectrograms in rats treated with saline (control), IGF-1 and picropodophyllin. (D, E) Summary of (D) the epileptiform discharge duration and (E) burst numbers from intracranial microelectrode LFP recordings (*P < 0.05, P < 0.01, compared with control, n=5 each, ANOVA). (F, G) Behavioural assessment showed (F) the onset latency of seizures and (G) maximum Racine's score in rats treated with saline (control), IGF-1 or picropodophyllin before pilocarpine injection (35 mg/kg, n=13 in control; n=15 in picropodophyllin or IGF-1 group) (*P < 0.05, P < 0.01, compared with control, ANOVA).

To validate IGF-1 enhancement of seizure activity in a pilocarpine-induced model, and to determine if this effect is mediated by IGF-1R, we further tested the effects of IGF-1 and picropodophyllin in a PTZ-induced rat model. In vivo EEG recording and power spectrogram analysis showed that IGF-1 application led to shortened onset latency (68.2±3.153 s) and extended the duration of the discharge time (146.6±3.32 s), whereas picropodophyllin (119.8±4.81 s and 93.4±3.641 s) displayed the opposite effect to IGF-1, compared with the control group (89.4±3.472 s and 120.25±4.789 s; ANOVA, P < 0.05). The IGF-1 effect was attenuated by co-application of IGF-1 and picropodophyllin (Figures 3A and 3B). Although co-application of IGF-1 and picropodophyllin did not significantly alter onset latency compared with IGF-1 alone (ANOVA, P > 0.05), the corresponding burst numbers were significantly reduced (Figures 3C and 3D; ANOVA, P < 0.05). Consistent with the electrophysiological findings, the behavioural study showed that picropodophyllin with IGF-1 significantly attenuated the IGF-1 effect on duration of seizures without influencing onset latency (Figures 3E and 3F). These results demonstrate that exogenous IGF-1 accelerated neuronal firing and seizure activity, which was prevented by IGF-1R inhibitor, suggesting that IGF-1 and IGF-1R may play important roles in the development of seizure activity.

Effects of IGF-1 and the IGF-1R inhibitor picropodophyllin on seizure activities in the PTZ-induced rat model of seizure

Figure 3
Effects of IGF-1 and the IGF-1R inhibitor picropodophyllin on seizure activities in the PTZ-induced rat model of seizure

(A) Representative intracranial LFP recordings and (B) power spectrogram in rats treated with saline (control), IGF-1, picropodophyllin (PPP) and PPP+IGF-1. (C, D) Summary of (C) the epileptiform discharge latency and (D) burst numbers from EEG recording (n=5 in each, *P < 0.05, P < 0.01 compared with control, ANOVA). (D) Onset latency and (E) duration of seizures in rats treated with saline (control), IGF-1, PPP or PPP+IGF-1 (*P < 0.05, P < 0.01, compared with control, n=10 per group, ANOVA).

Figure 3
Effects of IGF-1 and the IGF-1R inhibitor picropodophyllin on seizure activities in the PTZ-induced rat model of seizure

(A) Representative intracranial LFP recordings and (B) power spectrogram in rats treated with saline (control), IGF-1, picropodophyllin (PPP) and PPP+IGF-1. (C, D) Summary of (C) the epileptiform discharge latency and (D) burst numbers from EEG recording (n=5 in each, *P < 0.05, P < 0.01 compared with control, ANOVA). (D) Onset latency and (E) duration of seizures in rats treated with saline (control), IGF-1, PPP or PPP+IGF-1 (*P < 0.05, P < 0.01, compared with control, n=10 per group, ANOVA).

IGF-1R mediates IGF-1 effect on excitatory neurotransmission

To test the effect of IGF-1 on neuronal and circuit excitability and related neurotransmission, in vitro electrophysiological studies were performed in pyramidal neurons of the CA1 area. Bathing application of IGF-1 (100 ng/ml, 10 min) alone led to significantly increased amplitude and frequency of the mEPSCs when compared with the controls (n=9 cells/5 mice, P < 0.05), which were diminished after co-application of picropodophyllin (5 μM, 1 min) with IGF-1 (Figure 4A; n=9 cells/5 mice, ANOVA, P > 0.05). In a different set of cells, we measured the effect of picropodophyllin alone on mEPSCs. Picropodophyllin (5 μM), 15-min perfusion, resulted in significantly decreased mEPSC amplitude and frequency (Figure 4B; n=9 cells/5 mice, ANOVA, P < 0.05). The effect of IGF-1 or picropodophyllin on mEPSCs could not be washed out during the 20 min of recording (data not shown). These results indicate that the IGF-1 effect on excitatory neurotransmission was also mediated by IGF-1R.

Effect of IGF-1 and picropodophyllin on mEPSCs in CA1 pyramidal neurons

Figure 4
Effect of IGF-1 and picropodophyllin on mEPSCs in CA1 pyramidal neurons

(A) Representative traces (top) and cumulative distributions of amplitudes and inter-event intervals of mEPSCs (bottom) before and 10 min after IGF-1 application (100 ng/ml), and 10 min after IGF-1R inhibitor application [picropodophyllin (PPP), 5 μM, nine cells/five mice]. (B) Representative traces (top) and cumulative distributions of amplitudes and inter-event intervals of mEPSCs (bottom) before and 10 min after picropodophyllin application (9 cells/5 mice). (C, D) Sample traces (top) and summary graph (bottom) of AMPAR- and NMDAR-mediated currents (*P < 0.05, ANOVA, n=5). (E) Representative traces (left) and summary (right) of PPR (paired-pulse ratio, n=5 in each).

Figure 4
Effect of IGF-1 and picropodophyllin on mEPSCs in CA1 pyramidal neurons

(A) Representative traces (top) and cumulative distributions of amplitudes and inter-event intervals of mEPSCs (bottom) before and 10 min after IGF-1 application (100 ng/ml), and 10 min after IGF-1R inhibitor application [picropodophyllin (PPP), 5 μM, nine cells/five mice]. (B) Representative traces (top) and cumulative distributions of amplitudes and inter-event intervals of mEPSCs (bottom) before and 10 min after picropodophyllin application (9 cells/5 mice). (C, D) Sample traces (top) and summary graph (bottom) of AMPAR- and NMDAR-mediated currents (*P < 0.05, ANOVA, n=5). (E) Representative traces (left) and summary (right) of PPR (paired-pulse ratio, n=5 in each).

To dissect potential mechanisms of IGF-1 regulation of excitatory neurotransmission further, we tested IGF-1 on AMPAR- and NMDAR-mediated currents. As shown in Figure 4(C), bathing application of IGF-1 for 10 min significantly increased both AMPA- and NMDA-mediated currents (n=5, ANOVA, P < 0.05). The effect of IGF-1 on the AMPAR current was attenuated by co-application of picropodophyllin and IGF-1 (n=5, P < 0.05). In a different set of cells, picropodophyllin significantly reduced both AMPAR- and NMDAR-mediated currents (Figure 4D; n=5, ANOVA, P < 0.05). AMPAR:NMDAR ratios were 0.82±0.15 in control, 1.41±0.21 in IGF-1, 1.19±0.18 in IGF-1 with picropodophyllin and 0.69±0.12 in picropodophyllin alone, respectively (n=3 in each, data not shown in Figures). The difference in the AMPAR:NMDAR ratio was significant between control and IGF-1, IGF-1 and IGF-1 with picropodophyllin, and control and picropodophyllin (ANOVA, P < 0.05). These results indicate that the IGF-1R-mediated IGF-1 effect was preferentially on the AMPAR, suggesting that AMPAR may be a potential target of IGF-1. However, IGF-1 did not significantly alter paired-pulse ratios (Figure 4E; ANOVA, n=5), suggesting that IGF-1 enhancement of synaptic plasticity might not be mediated by a presynaptic mechanism.

Activation of ERK1/2 and Akt is enhanced by IGF-1 and diminished by the IGF-1R inhibitor in a rat model of seizure

IGF-1 exerts its function through PI3K/Akt and mitogen-activated protein kinase (MAPK) kinase (MEK)–ERK signalling [2931]. To test if these mechanisms are involved in IGF-1 regulation of seizure activity, we measured expression of ERK1/2 and Akt in the hippocampus of the pilocarpine-induced rat model of seizure. As shown in Figures 5(A) and 5(B), pERK1/2 and pAkt were significantly increased at 6 h and 24 h after pilocarpine-induced seizures. The increased pERK1/2 was maintained at significantly high levels for up to 60 days (n=5, ANOVA, P < 0.05), except in the silent stage (7 days), whereas pAkt returned to normal levels after 24 h of seizures. These results indicate that ERK1/2 and Akt are differentially regulated in seizures.

Activation of ERK1/2 and Akt is regulated by IGF-1 and picropodophyllin in the hippocampus of the pilocarpine-induced rat model of seizure

Figure 5
Activation of ERK1/2 and Akt is regulated by IGF-1 and picropodophyllin in the hippocampus of the pilocarpine-induced rat model of seizure

(A, B) Representative immunoblots (top) and corresponding densitometric analysis (bottom) of (A) pERK1/2 relative to ERK1/2 and (B) pAkt relative to total Akt (*P < 0.05, compared with the control (ANOVA, n=5). (CE) Sample Western blots and summary of (C) pIGF-1R relative to total IGF-1R, (D) pERK1/2 relative to total ERK1/2 and (E) pAkt relative to total Akt in pilocarpine-induced seizure alone (pilo) and in the presence of IGF-1 (pilo+IGF-1) or picropodophyllin (pilo+PPP). *P < 0.05, compared with control at 6 h after seizures; #P < 0.05, compared with control at 24 h after seizures (ANOVA, n=4 in each).

Figure 5
Activation of ERK1/2 and Akt is regulated by IGF-1 and picropodophyllin in the hippocampus of the pilocarpine-induced rat model of seizure

(A, B) Representative immunoblots (top) and corresponding densitometric analysis (bottom) of (A) pERK1/2 relative to ERK1/2 and (B) pAkt relative to total Akt (*P < 0.05, compared with the control (ANOVA, n=5). (CE) Sample Western blots and summary of (C) pIGF-1R relative to total IGF-1R, (D) pERK1/2 relative to total ERK1/2 and (E) pAkt relative to total Akt in pilocarpine-induced seizure alone (pilo) and in the presence of IGF-1 (pilo+IGF-1) or picropodophyllin (pilo+PPP). *P < 0.05, compared with control at 6 h after seizures; #P < 0.05, compared with control at 24 h after seizures (ANOVA, n=4 in each).

To test whether IGF-1 regulation of downstream signalling is mediated by IGF-1R, activated expression of IGF-1R, ERK1/2 and Akt was measured in the hippocampus of seizure rats after IGF-1 and picropodophyllin treatment. As shown in Figures 5(C)–5(E), 6 and 24 h of seizures led to activation of IGF-1R, ERK1/2 and Akt, respectively. Their activation was enhanced in the presence of IGF-1, which was inhibited when picropodophyllin was co-applied with IGF-1. These results indicate that intraventricular infusion of IGF-1 results in activation of the IGF-1R and downstream signalling molecules ERK1/2 and Akt in the hippocampus.

Effect of inhibitor of ERK (U0126) and Akt (LY294002) on PTZ-induced seizure activities

Figure 6
Effect of inhibitor of ERK (U0126) and Akt (LY294002) on PTZ-induced seizure activities

(A) Seizure onset latency and (B) duration in rats treated with LY294002 and U0126 alone or in combination. LY294002 extended seizure latency and U0126 shortened seizure duration (*P < 0.05, compared with control, ANOVA, n=10). Combined usage of these two inhibitors did not result in additional effects. (C) The ERK inhibitor U0126 reduced the percentage of PTZ-induced GTCSs (*P < 0.05, compared with the control, Fisher's exact test, n=10).

Figure 6
Effect of inhibitor of ERK (U0126) and Akt (LY294002) on PTZ-induced seizure activities

(A) Seizure onset latency and (B) duration in rats treated with LY294002 and U0126 alone or in combination. LY294002 extended seizure latency and U0126 shortened seizure duration (*P < 0.05, compared with control, ANOVA, n=10). Combined usage of these two inhibitors did not result in additional effects. (C) The ERK inhibitor U0126 reduced the percentage of PTZ-induced GTCSs (*P < 0.05, compared with the control, Fisher's exact test, n=10).

ERK1/2 or Akt inhibitor attenuates behavioural activities of seizures

U0126 and LY294002 are selective inhibitors of ERK and Akt, respectively. For further identification of the involvement of IGF-1 signalling in epilepsy, we tested the effect of these inhibitors on seizure activity. Rats received an intracerebroventricular injection infusion of U0126 (5 mM/5 μl) and/or LY294002 (10 mM/5 μl) at 24 h and 60 min before PTZ induced the GTCSs [32,33]. As shown in Figure 6 the Akt inhibitor LY294002, but not the ERK1/2 inhibitor U0126, significantly prolonged onset latency, whereas U0126 but not LY294002 effectively shortened the duration of seizures and reduced the percentage of GTCSs. Combined usage of these two inhibitors did not result in synergistic effects. These results indicate that ERK1/2 and Akt contribute to onset latency and severity of PTZ-induced seizure activity, respectively, suggesting that they may play different roles in seizure activity.

DISCUSSION

The major finding of this study is that IGF-1 and activated IGF-1R are consistently increased in patients with epilepsy and animal models of seizure. IGF-1R mediates IGF-1 promotion of seizure activity as well as excitatory neurotransmission. Moreover, IGF-1 downstream signalling of ERK1/2 and Akt may be involved in IGF-1 regulating the changes of epileptic behaviour and electrophysiology. This study provides direct evidence that, in addition to its well-known neuroprotective effect, IGF-1 facilitates neuronal firing through IGF-1R-dependent mechanisms, suggesting its role in epileptic activity.

In the present study, there was increased expression of IGF-1 in patients with epilepsy, which was replicated in two animal models of acute seizures induced by chemical convulsants, indicating that up-regulation of IGF-1 and activation of IGF-1R may be a result of recurrent neuronal firings. Neuronal hyperexcitability together with glutamate release result in intracellular calcium accumulation [3133], which may be associated with deregulation of synaptotagmin10 (syt10), a calcium sensor that is responsible for IGF-1 exocytosis [34]. It is of interest that expression of syt10, but not other synaptotagmins, is induced in the cortex and the dentate granule cells in an animal model of epilepsy [34]. Studies have shown that activation of the cAMP-response element-binding protein (CREB) is involved in epileptic activities; moreover, one of the target genes of CREB is IGF-1 [29,31]. It is therefore conceivable that IGF-1 transcription is also enhanced after neuronal hyperexcitability.

Studies have revealed that neurotrophins such as NGF and BDNF, and their receptor TrkB, play an important role in limbic seizures [30]. These neurotrophins are consistently increased as a consequence of seizures [35]. TrkB-null mice show a modest inhibition of the development of the kindling model [36]. Consistently, the present study demonstrates that exogenous IGF-1 significantly increases seizure susceptibility and severity in pilocarpine- and PTZ-induced models. However, this result is in contrast to what has been found by Miltiadous et al. [37,38]. In their study, intrahippocampal injection of IGF-1 led to decreased seizure severity and neurodegeneration at a cellular level. Similar discrepancies have been found with respect to BDNF, long-term infusion of which into the hippocampus results in inhibition of kindling development and reduced duration of seizures. One possible mechanism is that local high concentrations of IGF-1 may down-regulate IGF-1R expression, as a 6-day infusion of BDNF results in a decrease in the TrkB receptor by 80% [39]. In our experiment, however, IGF-1R proves to be activated 24 h after intraventricular infusion (see Figure 5C), although the total level of IGF-1R remains unchanged (data not shown). In support of IGF-1 function in neuronal excitability, our in vitro study has shown that IGF-1 application rapidly increases mEPSC frequency and amplitude as well as NMDAR- and AMPAR-mediated, evoked EPSCs. Importantly, these effects are blocked by the IGF-1R inhibitor picropodophyllin. These results demonstrate that activation of IGF-1R promotes neuronal hyperexcitability through APMAR- and NMDAR-mediated mechanisms.

IGF-1 induced phosphorylation of ERK1/2 and Akt promotes cell proliferation and neuronal survival [40]. PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone, and neurite outgrowth and neuronal migration in the brain. However, IGF-1 signalling may play an important role in epilepsy. In the subgranular layer of the dentate gyrus of pilocarpine-induced epilepsy, increased expression of IGF-1 and activation of MAPK correlate with progenitor cell proliferation, a common feature of epileptic seizures [11]. IGF-1 increases fast excitatory postsynaptic potential- (fEPSP-) and AMPAR-mediated synaptic transmission though IGF-1R and PI3K/Akt signalling [41]. Therefore, IGF-1 and downstream signalling may be a double-edged sword in epileptic seizures. IGF-1 is neuroprotective and promotes survival and cognitive function, although it facilitates neuronal firing and epileptic activity.

In the present study, both ERK and Akt are rapidly activated after kindling, with the former maintained at a significantly higher level throughout the chronic stage (up to 60 days measured), whereas the latter returns to normal level at the silent stage (7 days and after, see Figures 5A and 5B), which is consistent with increased expression of IGF-1 in both patients with epilepsy and animal models. Exogenous IGF-1 leads to increased activation of IGF-1R, ERK1/2 and Akt, which is blocked by the IGF-1R inhibitor picropodophyllin, suggesting that the IGF-1 effect on neuronal hyperexcitability is mediated by IGF-1R-dependent mechanisms (see Figures 5C–5E). Behavioural studies show further that the major effect of Akt is on onset latency, whereas that of ERK1/2 is on percentage of GTCSs (see Figure 6). These findings, together with timely dependent expression patterns in epilepsy development, suggest that Akt and ERK1/2 may play different roles in acute epileptic seizures. One of the major targets of Akt is the mammalian target of rapamycin (mTOR) which is responsible for microtubule assembly, protein synthesis and mitochondrial function; this is intensively investigated and known to contribute to the onset of seizures and development of epilepsy [42]. ERK1/2, on the other hand, can regulate AMPAR and potassium voltage-gated channel subfamily member 2 (Kv4.2) activity which directly control neuronal excitability [41]; this is consistent with our finding that IGF-1 increased the AMPAR:NMDAR ratio. In addition, nuclear translocation of ERK1/2 triggers related gene expression which may contribute to neuronal hyperexcitability [43]. Of note, ERK1/2 and Akt are not the only type of IGF-1 downstream signalling, e.g. Akt can be activated by neurotrophins and ras signalling [44], whereas ERK1/2 can be activated by neurotransmitters, and calcium-transient and calcium/calmodulin-dependent kinases [45]. Nevertheless, the present study does provide direct evidence that IGF-1R and its mediators ERK1/2 and Akt play important roles in epileptic seizures.

The present study demonstrates that inhibition of IGF-1R rapidly attenuates pilocarpine- and PTZ-induced seizure activities. In our in vitro electrophysiological study, we demonstrated that IGF-1 enhancement of excitatory neurotransmission and APMAR- and NMDAR-mediated currents is inhibited by the IGF-1R inhibitor. On the other hand, in vivo EEG recordings and power spectrogram analysis of LFPs revealed that IGF-1 promotes epileptic activities, and this effect is diminished by co-application of an IGF-1R inhibitor. The present study provides a previously unappreciated role of IGF-1 via IGF-1R and their downstream signalling pathway in seizure activities.

AUTHOR CONTRIBUTION

Guohui Jiang designed the trial and wrote the manuscript. Xuefeng Wang designed the trial and was responsible for obtaining approval of the Institutional Ethics Committee of the Chongqing Medical University. Guohui Jiang, Wei Wang, Qingqing Cao, Juan Gu and Xiujuan Mi carried out the trials. Kewei Wang did the protocol of statistical analysis and drew the figures. Guojun Chen collected and analysed the in vivo and in vitro electrophysiology data. All authors contributed to the preparation of the manuscript and approved the contributions. Guohui Jiang and Wei Wang contributed equally to this work.

We sincerely appreciate Xuanwu Hospital, Beijing Tiantan Hospital of the Capital University of Medical Sciences and Daping Hospital of the Third Military Medical University for supplying the surgery samples. In addition, we would like to thank all the patients and their families for their participation in this study.

FUNDING

This work was supported by funding from the National Natural Science Foundation of China [30870877, 81071039 and 81171197].

Abbreviations

     
  • ACSF

    artificial cerebrospinal fluid

  •  
  • AED

    antiepileptic drug

  •  
  • Akt

    protein kinase B

  •  
  • AMPAR

    α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor

  •  
  • AP

    anteroposterior

  •  
  • BDNF

    brain-derived neurotrophic factor

  •  
  • CREB

    cAMP-response element-binding protein

  •  
  • EEG

    electroencephalogram

  •  
  • ELISA

    enzyme-linked immunosorbent assay

  •  
  • EPSC

    evoked excitatory postsynaptic current

  •  
  • ERK

    extracellular signal-related kinase

  •  
  • GTCS

    generalized tonic–clonic seizure

  •  
  • IGF-1

    insulin-like growth factor-1

  •  
  • IGF-1R

    IGF-1 receptor

  •  
  • LFP

    local field potential

  •  
  • MCS

    minimal clonic seizure

  •  
  • MEK

    mitogen-activated protein kinase (MAPK) kinase

  •  
  • mEPSC

    miniature excitatory postsynaptic current

  •  
  • ML

    mediolateral

  •  
  • NGF

    nerve growth factor

  •  
  • NMDAR

    N-methyl-D-aspartate receptor

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PTZ

    pentylenetetrazole

  •  
  • qRT-PCR

    quantitative real-time PCR

  •  
  • rhIGF-1

    human recombinant IGF-1

  •  
  • syt10

    synaptotagmin10

  •  
  • TLE

    temporal lobe epilepsy

  •  
  • TrkB

    tropomyosin receptor kinase B

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