Chemicals
Romidepsin (FK228, Depsipeptide, Selleck Cat#S3020, USA) is a potent HDAC1 and HDAC2 inhibitor; TSA (Trichostatin A, Selleck Cat#S1045, USA) is a non-selective HDAC inhibitor that inhibits multiple types of HDACs; SCA (Santacruzamate A, CAY10683, Selleck Cat#S7595, USA) is a potent and selective HDAC2 inhibitor. The selectivity of SCA for HDAC2 is 3600 times higher than other HDACs.
Animals
C57BL/6J mice (Cat#n000013c57, RRID: MGI: 5657312) were purchased from Jiangsu Ji Cui Yao Kang Biotechnology Co., Ltd (China). All mice were raised under the pathogen-free conditions with constant temperature and humidity. The mice could eat and drink freely in 12h circadian rhythm (light time was 7:00am-7:00pm). The animal experiment was approved by the institutional animal care and use Committee (IACUC) of School of medicine, Southeast University, China (Approval ID: syxk-2010.4987). The date of birth was P0. The experiment was carried out in male mice at P0 (postnatal day 0), P8, P15, P30 and P60.
Primary Neuron Culture
As previously described, hippocampal neurons were isolated from E16.5-E18.5 mice fetus (16) and they were cultivated in 6-well plates or on glass coverslips coated with 100μg/mL poly-D-lysine and placed in 24-well plates (Sigma-Aldrich, USA). The cells were cultured in NeurobasalTM medium with glucose (Sigma-Aldrich, USA), glutamine and B27 (Thermo Fisher Scientific, USA). After 3 days, half of the medium was replaced with fresh medium. Knockdown of HDAC1 in some neurons was realized by infection with LV-hdac1-RNAi lentivirus (Shanghai Jikai Gene Technology Co., Ltd, China)) at 0 DIV. OX18 or the isotype control antibody (Abcam, USA) was added to some primary cultured neurons at 3 DIV.
Real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from the hippocampal tissue of mouse and reverse transcribed into cDNA by HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech, China). The primers for RT-qPCR analysis were used as previously described (13). Real-time PCR was performed in a premix for reaction (Chamq SYBR qPCR main mixture, Vazyme Biotech, China) and by a Real-time PCR system (Applied Biosystems, USA). The expression of β-actin was used as the internal control for normalize the expression of the target gene. All reactions were repeated three times. The results were demonstrated as the fold changes compared to the control group.
Western Blotting Analysis
RIPA lysis buffer was used to lyse the cell sample (Sangon Biotech, China). The extracted protein was quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and denatured in water bath at 100℃. Proteins were separated by SDS-PAGE gel and transferred to the polyvinylidene fluoride membrane. Blocking of the non-specific binding was realized by incubating the membrane in 5% BSA or 10% non-fat milk (Amresco, USA) solution. The membrane was incubated with different primary antibodies (anti-β-actin, 1:1000, Sigma-Aldrich, USA; anti-HDAC1, 1:1000, Cell Signaling Technology, USA; anti-HDAC2, 1:1000, Abcam, UK) at 4℃ overnight and with peroxidase bound secondary antibody (Sigma-Aldrich, USA) at room temperature for another hour. ECL chemiluminescence solution (Thermo Fisher Scientific, USA) was used to detect protein expression by chemiluminescence imaging system (Tanon, China). The Densitometry analysis of each band was conducted using ImageJ software (USA).
Cell immunofluorescence Staining
The coverslips in 24-well plates were washed in PBS to remove the medium. Then, the cells were fixed in 4% paraformaldehyde solution (Sigma-Aldrich, USA) for 20 minutes and permeabilized with 0.25% Triton X-100 (Sigma-Aldrich, USA) for 15 minutes. The coverslips were blocked with 5% BSA solution for 2 hours and incubated with primary antibody (mouse anti-microtubule-associated protein 2 (MAP2), 1:200, Abcam, USA ) at 4℃ overnight in a wet box. After that, the cells were washed and incubated with the secondary antibody (Alexa Fluor 546 or Alexa Fluor 488 conjugated goat anti-mouse IgG (H+L), 1:500, Thermo Fisher Scientific, USA) for 2 hours at room temperature. Finally, the coverslips were mounted with glycerin and observed under fluorescence microscope (Olympus FluoView FV 1000, Japan).
Sholl Analysis
The morphologies of neurons were analyzed by Sholl analysis. Several concentric circles were superimposed on the neuron cell images. By calculating the number of branches intersecting with each concentric circle, the branching patterns of dendrites in different regions are obtained, so as to quantitatively characterize the metamorphic properties of imaged nerves.
Statistical analysis
Statistical analysis was performed by Graphpad prism 8.0.1 software (USA). Univariate analysis of variance was used for comparison the means between multiple groups and Student t-test was used for comparison the means between the two groups. Values are presented as mean±SEM. P<0.05 was considered statistically significant.