20230808-AI coding-1st round/214 – Wen 2021.txt

PMID: 33791908 DOI: 10.1007/s11064-021-03301-5

Methods
Animals
The C57BL/6 WT mice and heterozygous Fmr1 KO (HET) mice were purchased from the Jackson Laboratory. Mice were housed in a temperature-controlled and humidity-controlled room with a 12:12 h light: dark cycle and provided with ad libitum access to water and food. All neonatal offspring used in this study were a result of mating WT male mice with Fmr1 HET female mice. Samples from toe clipping on postnatal day 5 were sent to Transnetyx for genotyping. All protocols involving mice were approved by the Animal Care and Use Committee at the Johns Hopkins University and were conducted in accordance with the NIH guidelines for care and use of animals.

Isoflurane Exposure In vivo
P7 mice were randomly divided into two experimental groups: an isoflurane exposure group and a control group. All mice in the isoflurane exposure group underwent an induction period, in which they were exposed with 3% isoflurane (Baxter Healthcare, Cooperation, Deerfield, IL, USA) for 3 min or until loss of righting reflex, whichever was first. The isoflurane group mice were exposed to 1.5% isoflurane carried in 50% oxygen continuously for 4 h via a nosecone designed to minimize rebreathing of exhaled gases. For the control group, mice were separated from dams and exposed to room air for 4 h. During all exposures, mice were placed under a heat lamp and monitored for skin temperature, oxygen saturation, heart rate, and oxygen saturation (MouseOx, Starr Life Sciences, Oakmont, PA). Mice were returned to their home cage dam upon regaining righting reflex.

Primary Neuron Culture
Primary neurons were isolated from the dissected cortex of P0-P1 mice as described previously [63]. Due to the timing of tissue harvest, the genotype of each neonatal mouse was unknown so the brain tissue of each mouse was collected separately. Tail samples of the mice were sent to Transnetyx for genotyping to select WT and KO cells needed for the follow-up experiments. Neonatal mice were decapitated, and the heads were placed in 75% ethanol, then transferred to cold Hank's Balanced Salt Solution (HBSS) without calcium and magnesium (Gibco, Carlsbad, CA, USA). The brains were collected after the skin and skull were removed. The cortex was isolated under a dissecting microscope, and the meninges were removed completely. Following the instruction of the papain kit (Worthington Biochemical CoRapa, Lakewood, NJ, USA), the cortex was digested in 20 units/ml papain and 0.005% DNAase at 37℃ for 30 minutes and albumin-ovomucoid inhibitor solution was added to stop the digestion. Tissue was further dissociated through gentle repeated pipetting. After allowing undissociated tissue to settle to the bottom of the tube, the supernatant liquid was collected and centrifugated at 1000 rpm for 5 minutes. Then, the cell pellet was resuspended in neurobasal medium (Gibco, Carlsbad, CA, USA) supplemented by 2% B27 (Gibco, Carlsbad, CA, USA), 0.5mM GlutaMax (Gibco, Carlsbad, CA, USA), and Penicillin-Streptomycin (100 U/mL) (Gibco, Carlsbad, CA, USA). After counting and adjusting the cell density, cells were plated at a density of 16×104 cells/ml in 24-well plates with 12mm glass coverslips coated with 0.05mg/ml Poly-D- Lysine (Corning, NY, USA). Cells were incubated in a humidified atmosphere maintained at 37°C, 5% CO2/95% air, and half of the media was changed every 2 days.

Isoflurane Exposure In vitro
At 5DIV, the cell-coated plates were randomly divided into three groups: control group (CON), isoflurane group (ISO), and isoflurane with 100nM rapamycin group (ISO+Rapa). Rapamycin (Sigma- Aldrich Inc, St. Louism, MO, USA) dissolved in DMSO was added to ISO+Rapa group 1 h before isoflurane exposure, bringing the final concentration of rapamycin and DMSO in the culture medium to 100 nM and 0.1% respectively. The same volume of DMSO was added to both the CON and ISO groups. Cell-coated plates for the ISO and ISO+Rapa groups were placed in humidified, sealable chamber, a 15 min equilibration period was performed, in which 1.8% Isoflurane (Baxter Healthcare, Cooperation, Deerfield, IL, USA) in the carrier gas (5% CO2, 21% O2 and 74%N2) was continuously delivered. After this 15 min equilibration, the chamber was tightly sealed containing the 1.8% isoflurane in carrier gas and was placed in a 37 ℃ incubator for 4 h. For the CON group, the same procedure was repeated except cells only received the carrier gas for 4 h. For medium changes, fresh drug was added to maintain the appropriate concentration of vehicle and rapamycin.

Immunofluorescence Staining and Imaging
At P60, mice were anesthetized with isoflurane and transcardially perfused with cold PBS for brain tissue collection. Samples were blinded with code for further analysis. After postfixation with 4% paraformaldehyde (PFA) overnight and dehydration with 30% sucrose for 3–4 days, coronal sections containing the dentate gyrus from the hippocampus were obtained using a microtome. Sections were 50μm thickness, and after collection, they were stored in antifreeze media at − 20℃. For immunohistochemistry, sections were rinsed 3 times with PBS for 5 min and incubated in blocking solution (5% donkey serum with 0.1% Triton X-100 in PBS) for 1 hour at room temperature. Sections were incubated with primary antibodies at 4 ℃ overnight: rabbit anti-Synapsin-1 (1:200, EMD Millipore, Burlington, MA, USA), rabbit anti-PSD-95 (1:200, EMD Millipore, Burlington, MA, USA), rabbit anti-Gephyrin (1:200, Abcam, Cambridge, MA, USA), rabbit anti-pAKT (1:50, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pS6 (1:1000, Fisher Scientific, Hampton, NH, USA), rabbit anti-pmTOR (1:50, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-parvalbumin (PV) (1:1000, Swant, Marly, Fribourg, Switzerland). After another 3 washes in PBS, sections were incubated for 2 hours at room temperature with the secondary antibodies Alex Fluor488 anti‐rabbit antibody (1:200,

Jackson ImmunoResearch, West Grove, PA, USA), Cy5 anti-mouse antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), and 4′, 6-diamidino-2-phenylindole (DAPI, 1:5000). After a final 3 more washes with PBS, sections were mounted on slides with 2.5% PVA/DABCO Mounting Media.

At 12DIV, cells on coverslips were fixed with 4% PFA for 20 min at room temperature. After washing with PBS 3 times, neurons were incubated in blocking solution (5% donkey serum with 0.1% Triton X-100 in PBS) for 1 h at room temperature. Then, the neurons were incubated with primary antibodies at 4 ℃ overnight: rabbit anti-Synapsin-1 (1:200, EMD Millipore, Burlington, MA, USA ), rabbit anti-PSD-95 (1:250, EMD Millipore, Burlington, MA, USA), rabbit anti-Gephyrin (1:800, Abcam, Cambridge, MA, USA), rabbit anti-pAKT (1:50, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-pS6 (1:1000, Fisher Scientific, Hampton, NH, USA), rabbit anti-pmTOR (1:50, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-MAP2 (1:200, Abcam, Cambridge, MA, USA ). After 3 washes with PBS, neurons were incubated for 2 h at room temperature with secondary antibodies Alexa Fluor488 anti‐rabbit antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), Cy5 anti-mouse antibody (1:200, Jackson ImmunoResearch, West Grove, PA, USA), and 4′, 6-diamidino-2-phenylindole (DAPI, 1:5000). Following 3 more washes with PBS, coverslips were mounted on slides with 2.5% PVA/DABCO Mounting Media. Mounted coverslips were labeling in code to facilitate blinding of further experimentation.

Imaging and Analyzing
For evaluating synaptogenesis in vivo, 5 sections representing different coronal level of the dentate gyrus were picked randomly from each animal. For each section, 3 images were randomly taken in the dentate gyrus defined by DAPI staining by an experimenter blind to condition. For evaluating synaptogenesis in vitro, 5 neurons were picked randomly from the 4 quadrants and the center of each coverslip. The image of each dendrite segment defined by MAP2 immunolabeling was taken 20 μm apart from the nucleus defined by DAPI immunolabeling. Representative images were taken using a 63 × 1.0 N.A. objective with an additional 5.0x magnification lens under a Leica SP8 confocal microscope (Leica, Wetzlar, Germany), and the settings were consistent for each capture. Synaptic puncta were quantified using ImageJ software (NIH, Bethesda, MD, USA). For evaluating the activation of mTOR signaling in vivo, 5 sections representing different coronal level of the dentate gyrus were picked randomly from each animal by an investigator blind to condition. Images of the dentate gyrus were taken using a 20 × 1.0 N.A. objective with an additional 0.75x magnification lens on a Leica SP8 confocal microscope (Leica, Wetzlar, Germany). For evaluating the activation of mTOR signaling in vitro, 5 fields were picked randomly from the 4 quadrants and center of each coverslip by an investigator blind to condition, and images were taken with a 20 × 1.0 N.A. objective. Cell counts to determine the proportion of cells positive for markers being analyzed were conducted using ImageJ software (NIH, Bethesda, MD, USA). All imaging and analysis were conducted by an investigator blind to the conditions.

Statistical Analysis
Results were expressed as mean± SEM. Data were analyzed by GraphPad Prism 8 (GraphPad, San Diego, CA, USA). Data were analyzed using two-way analysis of variance (ANOVA) with Tukey’s test for multiple comparisons. Statistical significance was set a priori at p <0.05.