PMID: 32047423 PMCID: PMC6997293 DOI: 10.3389/fncel.2020.00004
Materials and Methods
Animals
All experiments were approved by the relevant Committees of Chungnam National University, Daejeon, South Korea (CNU-01135). C57BL/6J mice were maintained in a specific pathogen-free (SPF) room maintained at 22°C, with a 12 h light/dark cycle, and fed ad libitum. Animals received anesthesia during the light cycle. This research adheres to the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines.

Anesthesia
PND 16/17 mice were randomly divided into three groups: control, sevoflurane, and sevoflurane plus rapamycin groups. Mice in the sevoflurane and sevoflurane plus rapamycin groups were placed in a 1-l plastic chamber and exposed to a constant flow of fresh gas [fraction of inspired oxygen (FiO2) 0.4, 4 L/min] containing 2.5% sevoflurane for 2 h. Full recovery was confirmed 30 min after discontinuing sevoflurane. Control mice were treated identically but without sevoflurane. The anesthesia chamber was placed in a 36°C water bath to maintain a constant temperature. Carbon dioxide and sevoflurane were monitored using an S/5 compact anesthetic monitor and a mCAiO gas analyzer module (Datex-Ohmeda, Helsinki, Finland).

Rapamycin Treatment
Rapamycin (LC Laboratories, Woburn, MA, USA) was reconstituted in ethanol at a concentration 10 μg/μl and then diluted in 5% Tween-80 (Sigma–Aldrich, St. Louis, MO, USA) and 5% PEG-400 (Sigma–Aldrich, St. Louis, MO, USA), as described (Chen et al., 2009). Mice in the sevoflurane plus rapamycin group were each administered three intraperitoneal injections of rapamycin (5 mg/kg) at 24 h intervals prior to sevoflurane exposure, whereas mice in the control and sevoflurane groups were injected with an identical volume of vehicle.

Western blotting
Whole-brain samples were obtained from the mice 24 h after sevoflurane exposure. Mice were exposed to carbon dioxide before brain extraction, and each whole brain was homogenized with a tissue grinder in RIPA lysis buffer [ELPIS-BIOTECH, Daejeon, South Korea, 100 mM Tris–hydrochloride (pH 8.5), 200 mM NaCl, 5 mM EDTA, and 0.2% sodium dodecyl sulfate], containing phosphatase and protease inhibitor cocktails (Sigma–Aldrich). After centrifuging the homogenized samples at 12,000× g for 15 min at 4°C, the supernatants were decanted and their protein concentrations were measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Samples (20 μg) were electrophoresed on SDS PAGE gels, and transferred to nitrocellulose membranes (pore size, 0.2 μm; Amersham Protran®, GE Healthcare, Buckinghamshire, UK) at 200 mA for 2 h. The membranes were blocked for 1 h with Tris-buffered saline-Tween 20 [10 mM Tris–hydrochloride (pH 7.6), 150 mM NaCl, and 0.1% Tween 20], containing 3% bovine serum albumin (BSA), followed by incubation with primary antibodies and the appropriate secondary antibodies coupled to horseradish peroxidase. Specific antibody-labeled proteins were detected using the enhanced chemiluminescence system (WEST-ZOL plus; iNtRON BioTechnology, Seongnam, South Korea). Primary antibodies included antibodies to phospho-mTOR(S2448), mTOR (Cell Signaling Technology, Danvers, MA, USA), postsynaptic density 90 (PSD95; Neuromab, Davis, CA, USA), GAD65 (Abcam, Cambridge, UK), NDUFB8 (a mitochondrial complex I subunit; Santa Cruz Biotechnology, Santa Cruz, TX, USA), COX4 (a mitochondrial complex IV subunit; Novus Biologicals, Centennial, CO, USA) and actin (Santa Cruz Biotechnology, Santa Cruz, TX, USA). Antibodies against GluA1 (1193) and GluA2 (1195) have been described previously (Kim et al., 2009).

Oxygen Consumption Rate
Mitochondria were isolated from brain tissues 24 h after sevoflurane exposure, as previously described (Chung et al., 2017a). Each brain was homogenized in a mitochondrial isolation buffer [70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1 mM EGTA, and 0.5% (w/v) fatty acid–free BSA (pH 7.2)] with a Teflon-glass homogenizer (Thomas Fisher Scientific, Swedesboro, NJ, USA). After centrifugation at 600× g for 10 min at 4°C and at 17,000× g for 10 min at 4°C, the mitochondrial fraction was resuspended in a mitochondrial isolation buffer. Protein concentration was measured by the Bradford assay (Bio-Rad), and 20 μg aliquots of protein were diluted with 50 μl mitochondrial assay solution [70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, 1 mM EGTA, 0.2% (w/v) fatty acid–free BSA, 10 mM succinate, and 2 μM rotenone (pH 7.2)] and seeded in an XF-24 plate (Seahorse Bioscience, North Billerica, MA, USA). The plates were centrifuged at 2,000× g for 20 min at 4°C using a swinging bucket microplate adaptor (Eppendorf, Hamburg, Germany); 450 μl mitochondrial assay buffer was added to each plate, and the plates were maintained at 37°C for 8–10 min. Each plate was transferred to a Seahorse XF-24 extracellular flux analyzer (Seahorse Bioscience) and the oxygen consumption rate (OCR) was measured at five stages: stage I (basal level); stage II, following the addition of adenosine diphosphate (ADP); stage III, following the addition of oligomycin, a mitochondrial oxidative phosphorylation (OXPHOS) complex 5 inhibitor; stage IV, following the addition of carbonyl cyanide m-chlorophenyl hydrazine (CCCP), a mitochondrial OXPHOS complex 4 inhibitor; and stage V, following the addition of antimycin A, a mitochondrial OXPHOS complex 3 inhibitor. OCR was automatically calculated and recorded using Seahorse XF-24 software (Seahorse Bioscience).

Electrophysiology
Whole-cell voltage-clamp recordings of pyramidal neurons in the CA1 region of the hippocampus were obtained as described (Chung et al., 2015a). Twenty-four hours after exposure to sevoflurane or fresh gas, sagittal slices of the hippocampus (300 μm) were prepared in ice-cold dissection buffer (212 mM sucrose, 25 mM NaHCO3, 5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 2 mM sodium pyruvate, 1.2 mM sodium ascorbate, 3.5 mM MgCl2, and 0.5 mM CaCl2) aerated with 95% O2/5% CO2, using a VT1200S vibratome (Leica, Arrau, Switzerland). Slices were transferred immediately to a 32°C chamber containing artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 25 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM d-glucose, 1.3 mM MgCl2, and 2.5 mM CaCl2, continuously aerated with 95% O2/5% CO2) and incubated for 30 min. Glass capillaries were filled with two kinds of internal solutions. For miniature excitatory postsynaptic current (mEPSC) recordings, the glass capillaries were filled with an internal solution containing 117 mM CsMeSO4, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-adenosine triphosphate (ATP), 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA; for miniature inhibitory postsynaptic current (mIPSC) recordings, the glass capillaries were filled with an internal solution containing 115 mM CsCl, 10 mM tetraethylammonium chloride, 8 mM NaCl, 10 mM HEPES, 5 mM QX-314-Cl, 4 mM Mg-ATP, 0.3 mM Na-guanosine triphosphate, and 10 mM EGTA. Whole-cell recordings were performed under visual control (BX50WI; Olympus, Tokyo, Japan) with a multi clamp 700A amplifier (Molecular Devices, San Jose, CA, USA). Data were acquired with Clampex 9.2 (Molecular Devices, San Jose, CA, USA) and analyzed using Clampfit 9 software (Molecular Devices, San Jose, CA, USA).

Statistical Analysis
The sample size was determined based on previous experience or as previously described (Chung et al., 2015b, 2017b). All statistical analyses were performed using R statistical software (3.1.2: R Core Team, Austria). All continuous variables were tested to determine whether they met conditions of normality and homogeneity of variance. One-way ANOVA with post hoc Tukey HSD test was performed when both conditions were met, Welch’s ANOVA with post hoc Tukey HSD test was performed when homogeneity of variance was unmet, and the Kruskal–Wallis test with post hoc Dunn’s test was performed if normality was unmet. P < 0.05 was considered statistically significant. Statistical results are presented as Supplementary Statistics.