In this study, male Thy1-egfp mice purchased from Jackson Labratories (Bar Harbor, ME, USA) were used. All animals were fed in a standard mouse chow and water ad libitum, housed in the animal center of Shandong University with a temperature and humidity-controlled environment, and under a 12:12 light: dark cycle. All procedures were approved by the Administration Committee of Experimental Animals, Shandong Province and Shandong University.
According to the data from previous study  in establishing the relationship between loss of righting reflex and effective dose (ED50 and ED100), the anesthetizing and sedative dosage of etomidate were set as 20 mg/kg, i.p. and 8 mg/kg, i.p. respectively in aged (18-month-old) mice. At the same time, aged (18-month-old) mice received equal volume of physiological saline (i.p.) were designated for control. To avoid hypothermia or hypoxia, we monitored the body temperature and heart rate by pulse oximetry sensor (MouseOx, Starr Life Sciences Corp., USA). After general anesthetics, mice were allowed to recovery in a temperature controlled (35 ± 1 °C) acrylic chamber flushed with medical air (30% O2, 70% air).
Behavioral function test
Behavioral function test (12 mice per group) including Morris water maze (MWM) test and novel object recognition (NOR) test were performed according to previous study .
In detail, MWM test is divided into three sessions and lasts 8 days (4 trails per day). Before the test, a black plastic pool (100 cm in diameter and 40 cm in height) was filled with water (adding white food color and controlling temperature at 20 ± 1 °C). At day 1 and day 2 (visible platform training), we placed platform 0.5 cm above the water surface and placed mice in the opposite quadrant to platform quadrant. At day 3 to day 7 (hidden platform training), we transfer platform to the opposite quadrant compared to visible platform test and filled water to let water surface 0.5 cm over platform, then mice were placed in the opposite quadrant to platform quadrant. From day 1 to day 7, mice were allowed to swim and find platform for escaping from water in 60 seconds per trail. If mice fail to find platform in 60 seconds, we lead them swimming to the platform by a stick and allow them stay in the platform for 10 seconds. At day 8 (transfer test), we remove platform from pool and placed mice in the opposite quadrant to platform quadrant and allow mice swimming in the pool for 90 seconds. During each trail, swim path of mice were recorded and average swimming speed (m/sec) and time (s) required to reach the platform were calculated by ANY-maze (Global Biotech Inc., NJ, USA).
Novel object recognition test is divided into 3 sessions in an open field (50 cm in length, 50 cm in width, 40 cm in height) and lasts for 3 days. At day 1 (habituation session), mice were placed in the center of open field and allowed to adapt the context for 10 min. At day 2 (acquisition session), mice were placed in the center of open field with two identical objects in it and allowed to recognize objects for 5 min. At day 3 (testing phase), we first replace one of the object with a novel object different in shape and color and then placed mice in the center of open field and allowed them to recognize objects for 5 min. We recorded the whole test by ANY-MAZE software (Global Biotech Inc., NJ, USA) and further analyzed total exploration time (s) and the discrimination index (DI, DI = (time exploring the novel object- time exploring the familiar object)/total exploration time).
The patch clamp whole-cell recording were applied to pyramidal neurons in DG region and EPSC and IPSC were detected and analyzed according to the protocol from previous study . In brief, 350 um-thick brain slices were prepared in pre-cold oxygenated (95% O2/ 5% CO2 for more than 2 hours) artificial cerebrospinal fluid (high-sucrose version of aCSF: 87mM NaCl, 2.5mM KCl, 7mM MgCl2, 1.25mM NaH2PO4, 25mM NaHCO3, and 25mM glucose, 75mM sucrose, pH 7.3). Then, transferred brain slices into a culture chamber and recovered for 2 hours, perfusing the chamber with oxygenated aCSF (120mM NaCl, 3mM KCl, 4mM MgCl2, 1mM NaH2PO4, 26mM NaHCO3, and 10mM glucose, pH 7.3) and maintained temperature between 32-34 °C. After that, we finally transferred brain slice into recording chamber (perfused with aCSF and maintained temperature between 32-34 °C) and pyramidal neurons in DG region and EPSC and IPSC were detected by micropipette were filled with working buffer (1mM MgCl2, 0.2mM EGTA, 4mM Mg-ATP, 0.3mM Na-GTP, 125mM Cs- methanesulfonate, 5mM CsCl, 10mM phosphocreatine and 5mM QX314; pH 7.3, 285 mOsm), under an upright microscope equipped with a 40× water-immersion lens (Axioskop 2 Plus, Zeiss). The sEPSC ( -60mV) and sIPSC (0mV) data were obtained by MultiClamp 700B amplifier and 1440A digitizer (Molecular Device, USA). Then, mEPSC ( -60mV) and mIPSC (0mV) data were obtained in the presence of 1 μum TTX (MCE, China).
Magnetic-activated cell-sorting (MACS)
To assess the response state of microglia and astrocyte in hippocampus, we isolated astrocyte by using magnetic-activated cell-sorting. First, hippocampus were separated from mice and immediately incubated with digestion buffer (300μg/ml DNasel (Sigma-Aldrich, USA) and 1mg/ml papain (MCE, China) at 37 °C for 30 minutes. After that, we centrifuged lyses at 300 g for 15 minutes and collected cell pellets for microglia and astrocyte isolation according to the manufacturer’s instructions of CD11b (Microglia) MicroBeads and Glast- MicroBeads kit (Miltenyi Biotech, Germany) respectively.
Flow cytometry was used to confirm the purity of microglia and astrocyte after MACS and the number of microglia in hippocampus after PLX3397 chow. The hippocampus separated from mice were immediately incubated with digestion buffer (300ug/ml DNasel (Sigma-Aldrich, USA) and 1mg/ml papain (MCE, China) at 37 °C for 30 minutes. After that, we centrifuged lyses at 300 g for 15 minutes and collected cell pellets. The cell pellets together with ones obtained after MACS then were suspended with phosphate buffer saline (PBS) buffer and incubated with corresponding antibody (anti-CD11b-pe (Miltenyi Biotech, Germany) and anti-CD45-APC (Miltenyi Biotech, Germany) for microglia; anti-GLAST-APC (Miltenyi Biotech, Germany) for astrocyte) and prepared for flow cytometry assay by Attune NxT system (BD Biosciences, USA).
Quantitative reverse transcription (RT) quantitative PCR
As previously described , total RNA from hippocampus was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed under standard conditions using the PrimeScriptTM RT Reagent Kit (Takara, Tokyo, Japan). After that, the gene level was detected using the StepOnePlusTM System (Applied Biosystems, Carlsbad, CA, USA) with SYBR Premix Ex Taq™ II (Takara) and the appropriate primers (Table S1) and Gapdh was used as the internal control.
Adeno-associated virus (AAV) stereotaxic injection and CNO treatment
Designer receptors exclusively activated by designer drugs (DREADDs) application were performed as previous study described . Chemogenetical virus AAV2/9-Gfap-hM4D(Gi)-mCherry-WPRE-pA (titer, 1.72 × 1013 vg/mL) was purchased from HANBIO Technology (Shanghai, China) and injected into the hippocampus of mice under anesthetic state which is induced by isoflurane (1.3%) for as much time as it takes and fixed on a stereotaxic apparatus (RWD, Shenzhen, China). The injected coordinate are AP, –2.00 mm; ML, ± 1.6 mm; DV, –2.0 mm; and AP, –3.00 mm, ML, ± 2.6 mm; and DV, –3.2 mm. To inhibit astrocyte, CNO (Clozapine N-oxide, 1 mg/kg/day, i.p., (MCE, Shanghai, China)) or saline (equal volume to CNO, i.p.) were injected into mice according to a former study .
In order to evaluate the role of microglia on astrocytic activation, we eliminated microglia by feeding mice with PLX3397 according to previous study [26, 27]. Taking the patency (over 95% depletion efficiency) into consideration, we begin to provide PLX3397 (290mg/kg, Xietong Pharmaceutical Bio-engineering Co., JS, China) enriched chow for mice 7 days before the timepoint which microglia elimination is needed. Control mice was set by providing standard chow.
To activate the microglia after etomidate sedative application, we injected LPS (i.p.) into mice according to previous study . In detail, LPS (Sigma-Aldrich, MO, USA) was dissolved by saline injection at a concentration of 0.1mg/ml and injected in mice at a dosage of 0.5mg/kg body weight. Control mice was set by injecting with saline.
Data were shown as mean ± standard errors of the mean (SEM) and analyzed by GraphPad Prism software (version 8.0.1, CA, USA). Two-tailed unpaired Student’s t test was used for comparing the difference between two groups, and two-way repeated-measures ANOVA with Bonferroni post hoc analysis was used for the Morris water maze test. Values with p < 0.05 were accepted as significant.