Animals and drug treatments
Aged 6~7 weeks male C57BL/6 mice, which were purchased from model animal research center of Nanjing University (Nanjing, China), were used in the experiments. All mice were housed in a constant temperature and humidity chamber at 25 °C, and sufficient food and water were administered daily. No more than 5 adult mice per cage were subjected to a 12-hour light/dark cycle under standard conditions. All the mice were guaranteed to be hygienic. The animal experiments were carried out in accordance with the “Guidelines for the Care and Use of Laboratory Animals” promulgated by Nanchang University.
LPS (Sigma, L-2880) was dissolved in sterile, endotoxin-free 0.9% (m/v) sodium chloride (NaCl) solution (saline) and was injected intraperitoneally (i.p.) into mice at a dosage of 0.5 mg/kg (bodyweight) to stimulate subclinical infection without causing significant inflammation and other significant damage to the animal [30]. After 10-day (once daily) administration of LPS, the mice were subjected to behavioral tests and electrophysiological or other experiments.
Metformin (Sigma) was dissolved in 0.9% (m/v) NaCl solution (vehicle) and was i.p. injected into mice at a dosage of 200 mg/kg (bodyweight) based on previous studies [31]. After 10-day LPS treatment and following behavioral tests, the mice were administrated daily with Metformin or its vehicle for another 10 days before another behavioral tests and electrophysiological or other experiments.
Behavioral tests
For forced swim test (FST), mice were individually placed in a 2-liter (L) beaker containing clean water (23-25 °C). The depth of water is 20 cm to prevent the animal from touching the bottom with the tail or hind legs. Mice behaviors were videotaped from the side for 6 min under red light. The immobility time of the last 4 minutes was recorded by an observer blinded to animal treatments. Immobility was defined as the time that animals remained floating or stationary.
For tail suspension test (TST), mice were suspended individually about 50 cm above the floor by fixing the tail with a medical tape on the test box hook. Mice behaviors were videotaped from the side for 6 min under red light. The last 4 minutes of rest time was recorded by an observer blinded to animal treatment. Immobility was defined as the time when animals stopped struggling.
Electrophysiological recording
Preparation of hippocampal slices as described previously [32]. The mice were anesthetized with ketamine/xylazine (Sigma, 100/20 mg/kg, respectively, ip), brain was quickly removed in ice-cold partial sucrose solution (PSS) containing (in mM): (80 NaCl, 3.5 KCl, 4.5 MgSO4, 0.5 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, 10 D-(+)-Glucose, 90 Sucrose). Coronal hippocampal slices (300 µm) were cut in ice-cold PSS using a VT-1000S vibratome (Leica, Germany). Then transferred hippocampal slices into traditional artificial cerebrospinal fluid (ACSF) (in mM): (124 NaCl, 2.5 KCl, 2 MgSO4, 2.5 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 10 D-(+)-Glucose) were incubated for 30 min at 34 °C, and 1 hour at room temperature (25 ± 1 °C) before recording. All solutions contained saturated 95% O2 / 5% CO2 (vol / vol).
Slices were transferred into a recording chamber perfused with a 2-3 ml/min ACSF at 32–34 °C. CA1 pyramidal neurons were observed using an upright microscope equipped with an infrared sensitive CCD camera (C2400-75, Hamamatsu) and a 40 ´ soaking lens (Axioskop 2 Plus, Zeiss). Borosilicate pipette were filled with an intracellular solution with a resistance of 3–5 MΩ. Whole-cell patch clamp recording were made using the Multiclamp 700B amplifier, Digidata 1440A interface digitization, pClamp 10.5 software acquisition at 2 kHz.
For mEPSC recording, adding 1 μM TTX and 20 μM bicuculline to ACSF to block action potential and inhibitory synaptic transmission, respectively. The pipettes were filled with potassium gluconate intracellular solution containing (in mM): 130 K-gluconate, 5 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.1 Na-GTP, 5 QX-314 (pH 7.30, 293 mOsm). The hippocampal CA1 pyramidal neurons were held at −70 mV recorded by whole-cell patch clamp method in the voltage-clamp mode.
For electrical properties of hippocampal CA1 pyramidal neurons detect, adding 20 μM bicuculline 50 µM DL-AP5 and20 µM DNQX to ACSF blocked excitatory and inhibitory synaptic transmission. The pipette solution containing (in mM): 130 K-gluconate, 5 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.1 Na-GTP (pH 7.30, 294 mOsm). The whole-cell patch clamp recording method was used to record in the current clamp mode, and action potential was recorded by injecting depolarizing pulses (from 0 to 350 pA) at a step of 50 pA per 10 s.
For paired-pulse ratios recording, adding 20 μM bicuculline and 5 µM CGP52432 blocked inhibitory synaptic transmission. The Pipettes were filled with potassium gluconate intracellular solution containing (in mM): 130 K-gluconate, 5 NaCl, 1 MgCl2, 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.1 Na-GTP, 5 QX-314 (pH 7.30, 293 mOsm). EPSCs were stimulated by a paired stimulus of the same size at intervals of 100, 50, and 25 ms in SC-CA1 pathway. The ratio value is calculated as [(p2 / p1) × 100], where p2 and p1 are the amplitudes of the EPSCs induced by the second pulse and the first pulse, respectively.
Brain morphological analysis
Mice were anesthetized by intraperitoneal injection of 0.7% sodium pentobarbital. After 5-10 min of perfusion of saline, the mice were perfused with pre-cold paraformaldehyde for 5-10 min, brain was weighed and fixed in 4% PFA at 4 °C overnight. Slices (50 µm) were cut in ice-cold PBS using a VT-1000S vibratome (Leica, Germany). After washing with 0.01 M PBS, Sections were attached to a gelatin-coated slide and air-dried at room temperature. Samples were incubated in a cresol purple solution, heated by water bath in 60 ° C for 3-10 min. Then washing with Milli-Q water for 3 times, samples were put into 75%, 95% and 100% ethanol for 1 min respectively. Samples were immersed in xylene for 10 min, sealed with a neutral resin overnight, and photographed with a fluorescence microscope (FN1,Nikon).
Statistical analysis
The data were statistically analyzed and graphed using the Graphpad Prism 6.0 software. Electrophysiological data were analyzed using Clampfit and Mini-Analysis software. Images were edited using Photoshop CS3. The experimental data were expressed as Min to Max or Mean ± SEM. Student's t-tests were used to compare the two groups of data. One-way ANOVA was used to compare three or more groups of data. Two-way ANOVA was used in electrophysiological studies when more than two parameters were analyzed. * p < 0.05; ** p < 0.01; *** p < 0.001.