Animals
Sprague-Dawley (SD) rats (male and female, n = 48, P14) were obtained from the Animal Experimental Center of Zhengzhou University. All experimental procedures complied with the Committee on the Ethics of Animal Experiments of Zhengzhou University (Permit No: SCXK 2010-0002).
Primary neuronal culture
Hippocampal neurons were isolated from the brains of neonatal SD rats (male and female, n = 12) based on described previously[9]. Immunofluorescence staining for microtubule-associated protein 2 (MAP2, 1:200, Abcam, Cambridge, MA, USA) was used to test the purity of the hippocampal neuronal population. The cells were maintained for 14 days before recordings.
Generation of experimental FS in vivo
Experimental FS model were generated in rats using hot water bath immersion as previously described[3].The seizures were scored according to the Racine scale[10]. Animals exhibiting stage 3 seizures (forelimb clonus) were regarded as exhibiting seizure behavior. CXCR4 antagonist AMD3100 (5 mg/ml, Selleckchem Co, Houston, USA) was administrated by i.c.v in rats.
Western blot analysis
The isolated hippocampal neurons maintained on 14 day were collected, and the protein content was determined using a BCA Protein Quantitation Kit (Thermo Fisher, USA) following the instructions. β-actin was used as a control for DPP4, CXCL12 and CXCR4, then the blots were incubated overnight with primary antibodies against DPP4 (1:2000, ab28340, Abcam, Cambridge, Massachusetts, USA), CXCL12 (1:1000, ab9797, Abcam, Cambridge, Massachusetts, USA) and CXCR4 (1:1000, Abs143375, Shanghai, China). The data were analyzed using Image J software (National Institutes of Health, MD, USA).
Enzyme-linked immunosorbent assay( ELISA)
Hippocampal tissues of the rats from the control, FS and FS+ AMD 3100 groups were immediately collected to measure the GABA according to ELISA kits (GABA: MyBioSource; San Diego, CA) according to the manufacturer’s instructions. A VICTOR™ X5 Multilabel Plate Reader (PerkinElmer, USA) was used to analyze the data.
Electroencephalogram (EEG) recording
The rats were deeply anesthetized with 10% chloral hydrate (i.p., 300 mg/kg). Using stereotaxic guidance, the rats were surgically implanted with an injection guide cannula and recording electrodes, as previously described[11, 12]. Rats from the FS+ AMD 3100 group were administered AMD 3100 (5mg/ml, i.c.v). EEG changes were recorded for a period (15-20 min) in the rats with febrile convulsions induced by the required conditions. The recorded EEG was exported, and the data were analyzed in LabChart 7.2(AD Instruments).
siRNA assay
siRNA targeting DPP4 and its nonspecific oligonucleotide were synthesized by GenePharma Co. (Shanghai, China) according to previous report[3]. Neurons were maintained on 10-14 days and transfected with siRNA mediated DPP4 (5'-GCUGAUAGCGCGAGAACUUTT-3'/5'-AAGUUCUCGCGCUAUCAGCTT) following to the manual instructions.
Electrophysiological recordings
The spontaneous inhibitory postsynaptic currents (sIPSCs) in the cultured hippocampal neurons (10-14 DIV) were recorded using the whole cell patch technique. The patch pipette (3–6 MΩ) was filled with internal solutions containing 145 mM CsCl, 5 mM NaCl, 10 mM HEPES, 5 mM EGTA, 4 mM Na2ATP, 0.3 mM Na2GTP and 5 mM QX-314. D (-)-2-amino-5-phosphonopentanoic acid (D-APV, 50 μM; Tocris Cookson, Ellisville, MO), 6-cyano-7-nitroquinoxaline-2, and 3-dione (CNQX, 10 μM; Tocris Cookson) were diluted immediately prior to use and acutely applied by bath superfusion (PH:7.20, osmolarity: 300–310 mosm/liter). The temperature (39.5°C) was monitored using a small thermistor placed directly in the recording chamber with continuous perfusion of a standard external solution at a rate of 2 ml/min[3]. Sita and CXCL12(10 nM, PeproTech, USA) were added into perfusion bath before recording. The currents were measured using a patch-clamp amplifier (Axon 700B, Axon Instruments, Foster City, CA, USA) and analyzed using Clampfit software (Axon Instruments).
Statistical analysis
Data are expressed as the mean ± SEM. Statistical analysis was performed with the GraphPad Prism software (version 6). Two-tailed unpaired Student’s t-test was used to examine significant differences between the two groups. Comparisons involving multiple groups were examined by one-way ANOVA. EEG data were analyzed using LabChart 7.2. Values of p<0.05 were considered statistically significant.