Animal and Drug Administration
C57BL/6 mice (Male, 14 ~ 18 g, 4 ~ 5 weeks old) were provided by the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine (SHUTCM, Shanghai). All experiments on animals were performed according to the protocol approved by Animal Care and Use Committee of SHUTCM and all animals received humane care (Ethical approval no. SZY201610005). Animals were acclimatized for 2 weeks before the formal behavior experiments.
Fourteen mice were injected intraperitoneally (i.p.) with ASIV (25 mg/kg, 40% 1,2-Propanediol + 5% Ethanol + 1% Polyethylene glycol in phosphate buffer saline solution) for five weeks, while fourteen mice were administered with solvent served as the control. All of the twenty-eight mice were subjected into behavioral tests. Another forty-four C57BL/6 mice of the same source were with only injection for further experiments; twenty-two mice received an i.p. injection of ASIV (25 mg/kg) daily for two weeks, while twenty-two mice were administered with solvent served as the control.
For LTD induction in vitro experiment, eight C57BL/6 mice (male, 12 ~ 17 g, 3 ~ 4 weeks old) were provided by SPF biotechnology Co., Ltd (Beijing, China). Four of them were i.p. administered with ASIV (25 mg/kg) for two weeks, while four mice were administered with solvent served as the control.
EGR-1+/− mice maintained on the C57BL/6 background was obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Experiments were carried out on male EGR-1−/− (EGR-1 KO) mice and their EGR-1+/+ littermates (male, 14 ~ 18 g, 4 ~ 5 weeks old) that were derived from EGR-1+/− × EGR-1+/− breeding at the same time. Fourteen EGR-1 KO mice were injected intraperitoneally (i.p.) with ASIV (25 mg/kg) for five weeks, while fourteen EGR-1 KO mice were administered with solvent served as the control. For LTD induction in vitro experiment, three EGR-1 KO mice (12–17 g, 3 ~ 4 weeks old) were i.p. administered with ASIV (25 mg/kg) for two weeks, while four mice were administered with solvent served as the control.
All mice were housed at room temperature (25 ± 1 °C) under a 12 h light / 12 h dark cycle, and fed with food and drank with water ad libitum freely. All behavioral tests were conducted in the light phase between 12:00 a.m. and 18:00 p.m. In order to avoid experimental deviation, all behavioral observers were blinded to the administration of the experimental mice. All animal experiments were conducted in accordance with the guidelines for Laboratory Animal care and Use Committee of SHUTCM and National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978).
Behavioral Tests
Radial-arm Maze Test (RAMT): The apparatus for radial-arm maze (RAM) (Mobiledatum Inc, Shanghai, China) consists of eight equally spaced arms (length 30 cm, height 15 cm, width 6 cm) radiating from a central maze hub (diameter 12 cm). It was made of opaque plexiglass with manually operated doors lead from the hub of the central maze to each arm. RAMT was conducted in accordance with the procedure described previously, with minor modification [27]. As shown in Fig. 1A, the mice were acclimatized to the RAM for 4 days before administrated with ASIV on day 1. Then, they were trained to explore the RAM with four arms placed with 50 mg bait (sugar: regular chow = 1:1) from day 2 to day 6. At the time of a week's administration, the mice were placed in the octagonal arena at the beginning of the experiment. The experiment ended when the mice explored the maze for 5 min or visited four baited arms. At the time of two weeks' administration, the test was conducted again. The maze was wiped down with 10% ethanol between each run to reduce olfactory cues. During these days, mice were given semi-food diet feeding. In these two tests, reference and working memory errors and the time required to complete the tasks were recorded and analyzed.
Shuttle-box test (SBT)
SBT was performed following RAMT on day 22. The chamber (Mobiledatum Inc, Shanghai, China) is divided into two equal compartments connected by a gate. A light is switched on alternately in the two compartments for conditioned stimulus. The test was conducted following a procedure described by Cheng et al [28]. Briefly, each mouse was allowed to adapt to the chambers for 4 min before the formal test. At the beginning of the experiment, the mice were placed in a compartment of the shuttle-box and back to the gate. Each mouse was given 30 consecutive trials at intervals of 20 s (light 5 s; interval, 3 s; 0.2 mA electric shock, 5 s; interval, 7 s) for five consecutive days. The active avoidance response was recorded automatically if the mouse moved to another compartment during conditioned stimulus. At the day 35 of continuous ASIV administration, all animals received the same experimental protocol to assess memory consolidation.
Neurotransmitter Analysis
After all behavioral tests were completed; the mice as well as those administered with ASIV only for two weeks were sacrificed after anesthetized with 1.5% pentobarbitalum. Immediately, the hippocampus of mice was dissected on ice, frozen rapidly in liquid nitrogen and stored at − 80 ºC until analysis. The content of GABA in mouse hippocampus was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as reported previously [29].
Western Blotting Analysis
The hippocampus of mice was homogenized, sectioned and analyzed by Western blotting. Totally thirty micrograms of protein were separated from each sample by 10% SDS-PAGE electrophoresis. After transferred to polyvinylidene fluoride membrane (GE Healthcare, Little Chalfont, Buckinghamshire, UK), the proteins were incubated with respective primary antibodies against glutamate decarboxylase 2 (GAD65) (cat: ab26113), EGR-1 (cat: ab194357), BDNF (cat: ab88901), TrkB (cat: ab101696) and GAPDH (cat: ab181602) provided by Abcam (Cambridge, MA, USA). Sequentially, the proteins were further incubated with secondary antibodies as described previously [16]. The protein bands were visualized by ECL reagent kit (cat: RPN2232, GE Healthcare) and quantified with Gel-Pro analyzer software (Media Cybernetics, L.P., MD, Rockville, USA).
Quantitative PCR
Total RNAs from mouse hippocampus were extracted using Trizol reagent (Life Technologies, Waltham, MA, USA). Afterwards, the RNA samples were reverse-transcripted into cDNA with Revert Aid First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA). The synthesized cDNA was used as templates for quantitative real-time PCR with Universal SYBR Green/ROX qPCR Master Mix (Roche, Basel, Switzerland) on ABI ViiA7 quantitative real-time PCR platform (Applied Biosystems, Foster City, CA, USA). Quantification of the target gene was conducted by comparative Ct method with GAPDH as a reference gene. Primers used were listed as following: for EGR-1, 5’-AGCAGCGCCTTCAATCCTCA-3’, 5’-TCTCCACCATCGCCTTCTCA-3’; for GAPDH, 5’-ATGTGTCCGTCGTGGATCTGA-3’, 5’-ATGCCTGCTTCACCACCTTCT-3’.
Electrophysiological Recording
LTD induction in vitro
After continous i.p. injection of ASIV (25 mg/kg) for two weeks from 4 weeks old, the mice’s brain were removed, and three brain slices (350 µm thick) were obtained from each mouse, covering the transverse ventral hippocampus. Slices were incubated at room temperature for at least 90 min in artificial cerebrospinal fluid (ACSF; containing 24 mmol/L NaCl, 26 mmol/L NaHCO3, 2 mmol/L KCl, 2 mmol/L CaCl2, 2 mmol/L MgSO4, 1.25 mmol/L NaH2PO4 and 10 mmol/L D-glucose) and ventilated with a mixture of 95% oxygen and 5% carbon dioxide. Subsequently, the slices were transferred to the recording chamber and inflated ACSF at a flow of 2 mL/min rate at (22 ± 2) °C. After the temperature (33 ± 1) °C and perfusion rate, field excitatory post synaptic potentials (fEPSPs) were recorded. A bipolar stimulus electrode was placed on Schaffer’s collateral/commissural bundle in CA3 hippocampus, and a recording glass micropipette (1–3 MΩ, filled with 3 mol/L NaCl) was placed on the radiator in CA1 hippocampus. AxoClamp 700B amplifier filter was set at 3 kHz, while the sampling frequency was set at 20 kHz (Digidata 1440A, GE MDS, NY, USA). Meanwhile, the data were collected and stored with Clampex 10.3 (GE MDS company). Current intensity adjusted to 60–70% of maximal fEPSP (base lines stimulation). Low frequency paired pulse stimulations (LFPS; 25 millisecond paired pulse interval, 1 Hz, monophasic quare pulses same as the baseline) induced LTD for 15 min. Immediately after each single stimulus for 60 s, the fEPSPs reaction was recorded for 60 min. Throughout the course the stimulation intensity is maintained constant. And throughout the recording period bicuculline (10 µmol/L) was included in the continuous perfusion (ACSF). The slope of fEPSPs was analyzed using Clampfit (GE MDS Company) and standardized with the average baseline response. The average changes of fEPSPs in 5 min after pre-stimulus baseline conditions were used to compare with the average response time of the first 30–35 min after stimulation conditions for statistical purpose.
Input-output (I/O) function in vivo
I/O curve reflects the relationship between the amplitude of fEPSP and the intensity of stimulation, which is used to evaluate the synaptic potency [30]. After continous i.p. injection of ASIV (25 mg/kg) for two weeks from 6 weeks old, the mice were anesthetized with 25% urahtane (1 mL/100 g) and fixed on the stereotaxic device (Narishige Ins, Tokyo, Japan). The location parameters of granular cell layer in DG area were 2.0 mm after anterior fontanel, 1.4 mm near midline, 1.5 mm deep under dura mater; and 3.8 mm after anterior fontanel, 3.0 mm near midline, 1.5 mm deep under dura mater. In the front fontanel, recording electrode and stimulating electrode (Nihon Kohden, Tokyo, Japan) were almost in a straight line. According to the above positioning parameters, drill holes in the skull with a skull drill (Minimo, Tokyo, Japan), then insert recording electrodes into the granular cell layer of DG area, and insert stimulating electrodes into performant pathway. The reference electrodes were clamped on the scalp. With 0.1 mA (0.1-2.0 mA) as a step, the stimulation current was changed systematically to produce I/O curve. Three responses at each current level were averaged, and the population spike (PS) amplitude was examined. The signals were transferred through the amplifier (Axon, CA, USA), and filtered by PCLab202 software (Microsignalstar, Beijing, China).
Electron Microscopy
After deeply anesthetized with 20% urethane, mice were perfused with 2.5% glutaraldehyde in 0.1 M phosphate (pH 7.4) transcardially. The brains were dissected and post-fixed in 2.5% glutaraldehyde for 24 h. Sequentially, they were incubated with 2% osmium tetroxide, dehydrated with series acetone and embedded with epoxy resin. The 60 nm-thick ultrathin sections were obtained on an ultra-microtome (ultracut UCT, Leica, Wetzlar, Germany) and observed by transmission electron microscope (Tecnai G2 Spirit BioTWIN, Hongkong, China).
Statistical analysis
All data were performed through Graphpad Prism 6 (Graphpad, La Jolla, CA). The difference of measurement data and numeration data were detected by unpaired student t-test or two-factor analysis of variance (ANOVA) and Mann-Whitney U test, respectively. Error bars represented the standard error of the mean (S.E.M.). P-value less than 0.05 was regarded as a significant difference.